Case: 19-2156 Document: 19 Page: 1 Filed: 07/26/2019
No. 2019-2156
UNITED STATES COURT OF APPEALS FOR THE FEDERAL CIRCUIT
GENENTECH, INC., Plaintiff-Appellant, CITY OF HOPE, Plaintiff, v. AMGEN INC., Defendant-Appellee.
On Appeal from the United States District Court for the District of Delaware, No. 1:18-cv-00924-CFC, Judge Colm F. Connolly
NON-CONFIDENTIAL BRIEF FOR PLAINTIFF-APPELLANT GENENTECH, INC.
ROBERT J. GUNTHER JR. WILLIAM F. LEE WILMER CUTLER PICKERING ANDREW J. DANFORD HALE AND DORR LLP WILMER CUTLER PICKERING 7 World Trade Center HALE AND DORR LLP 250 Greenwich Street 60 State Street New York, NY 10007 Boston, MA 02109 (212) 230-8800 (617) 526-6000
DARALYN J. DURIE Attorneys for Plaintiff-Appellant DURIE TANGRI LLP Genentech, Inc. 217 Leidesdorff Street San Francisco, CA 94111 (415) 362-6666
July 26, 2019
Case: 19-2156 Document: 19 Page: 2 Filed: 07/26/2019
CERTIFICATE OF INTEREST
Counsel for Plaintiff-Appellant Genentech, Inc. certifies the following:
1. The full name of every party or amicus represented by me is:
Genentech, Inc.
2. The names of the real party in interest represented by me are:
Not applicable.
3. All parent corporations and any publicly held companies that own 10 percent or more of the stock of the party or amicus curiae represented by me are:
Genentech, Inc. is a wholly-owned subsidiary of Roche Holdings Inc. Roche Holdings Inc.’s ultimate parent, Roche Holdings Ltd, is a publicly held Swiss corporation traded on the Swiss Stock Exchange. Upon information and belief, more than 10% of Roche Holdings Ltd’s voting shares are held either directly or indirectly by Novartis AG, a publicly held Swiss corporation.
4. The names of all law firms and the partners or associates that appeared for the party or amicus now represented by me in the trial court or agency or are expected to appear in this court (and who have not or will not enter an appearance in this case) are:
WILMER CUTLER PICKERING HALE AND DORR LLP: Timothy Cook, Robert J. Gunther Jr., William F. Lee, Jason H. Liss, Mark D. McBriar, Helena Million-Perez, Kevin S. Prussia, Rana Sawaya, Nancy L. Schroeder
DURIE TANGRI LLP: Adam R. Brausa, Daralyn J. Durie, Eneda Hoxha, Eric C. Wiener
MCCARTER & ENGLISH, LLP: Alexandra M. Joyce, Michael P. Kelly, Daniel M. Silver
WILLIAMS & CONNOLLY LLP: David I. Berl, Thomas S. Fletcher, Paul B. Gaffney, Teagan J. Gregory, Kathryn S. Kayali, Charles L. McCloud, Jonathan S. Sidhu, Kyle E. Thomason
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5. The title and number of any case known to counsel to be pending in this or any other court or agency that will directly affect or be directly affected by this court’s decision in the pending appeal:
Genentech, Inc. v. Amgen Inc., No. 18-cv-924-CFC (D. Del.)
Dated: July 26, 2019 /s/ William F. Lee WILLIAM F. LEE WILMER CUTLER PICKERING HALE AND DORR LLP 60 State Street Boston, MA 02109 (617) 526-6000
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TABLE OF CONTENTS
Page CERTIFICATE OF INTEREST ...... i
TABLE OF AUTHORITIES ...... vi
STATEMENT OF RELATED CASES ...... 1
JURISDICTIONAL STATEMENT ...... 1
INTRODUCTION ...... 1
STATEMENT OF ISSUE ON APPEAL ...... 5
STATEMENT OF THE CASE ...... 5
A. Genentech’s Development Of Herceptin And The Dosing Patents...... 5
B. Amgen’s Biosimilar Drug Kanjinti ...... 10
C. Amgen’s Representations Regarding The Timing Of Its Launch Of Kanjinti ...... 11
D. District Court Proceedings ...... 14
SUMMARY OF THE ARGUMENT ...... 18
STANDARD OF REVIEW ...... 22
ARGUMENT ...... 24
I. THE DISTRICT COURT’S DETERMINATION OF NO IRREPARABLE HARM RESTED ON LEGAL ERRORS ...... 24
A. The District Court Adopted An Erroneous Legal Standard To Conclude That Genentech Could Not Establish Irreparable Harm Because Of The Timing Of Its Motion ...... 24
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1. The district court legally erred in inferring that Genentech will not suffer irreparable harm because it waited to seek preliminary injunctive relief until Amgen affirmatively decided to launch ...... 25
2. Nothing in the BPCIA’s text or purpose required Genentech to seek a preliminary injunction before Amgen decided that it was going to launch its infringing product ...... 31
B. The District Court Improperly Created And Applied A Categorical Rule That Licenses To Third Parties For Future Entry Dates Demonstrated A Lack Of Irreparable Harm From Present Infringement ...... 34
1. The district court erred in adopting a categorical rule that licensing of future activity negates irreparable harm from present infringement ...... 35
2. The district court erred in treating Genentech’s settlement agreements as establishing that money damages are an adequate remedy for Amgen’s infringement ...... 38
3. The district court erred in concluding, without evidentiary support, that the harm to Genentech from Amgen’s launch would be quantifiable ...... 41
4. Unless reversed, the district court’s decision will seriously impede future settlement of biosimilars litigation ...... 44
C. Genentech Will Suffer Irreparable Harm Due To Amgen’s Infringement ...... 44
1. Genentech will suffer irreparable harm ...... 44
a. Price erosion ...... 45
b. Lost market share ...... 47
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c. Effect on other products ...... 48
d. Reputational harm ...... 49
2. Genentech’s irreparable harm is connected to Amgen’s infringement ...... 50
II. THE REMAINING EQUITABLE FACTORS OVERWHELMINGLY FAVOR INJUNCTIVE RELIEF ...... 51
A. Genentech Is Likely To Succeed On The Merits ...... 51
B. The Balance Of Hardships Favors Genentech ...... 54
C. The Public Interest Is Served By Enforcing Genentech’s Patent Rights To Encourage Innovation ...... 56
CONCLUSION ...... 57
ADDENDUM
CERTIFICATE OF SERVICE
CERTIFICATE OF COMPLIANCE
CONFIDENTIAL MATERIAL OMITTED
The confidential information that has been deleted on pages 1-4, 8-16, 19-22, 25, 29-30, 32, 34-36, 38-39, 41, 43, 45, 47, 50-52, 54-55, and 57 and Addendum pages Appx8-9 describes highly confidential, competitively sensitive information relating to the Herceptin biosimilars market including market entry, forecast planning, competitive intelligence, and the terms of third-party license agreements.
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TABLE OF AUTHORITIES
CASES
Page(s) Abbott Laboratories v. Andrx Pharmaceuticals, Inc., 452 F.3d 1331 (Fed. Cir. 2006) ...... 23
Abbott Laboratories v. Sandoz, Inc., 500 F. Supp. 2d 807 (N.D. Ill. 2007) ...... 37
Abbott Laboratories v. Sandoz, Inc., 544 F.3d 1341 (Fed. Cir. 2008) ...... 22, 45, 47 Acumed LLC v. Stryker Corp., 551 F.3d 1323 (Fed. Cir. 2008) ...... 36, 39
Allergan, Inc. v. Barr Laboratories, Inc., 501 F. App’x 965 (Fed. Cir. 2013) (nonprecedential) ...... 53 Amgen Inc. v. Apotex Inc., 827 F.3d 1052 (Fed. Cir. 2016) ...... 34 Apple, Inc. v. Samsung Electronics Co., No. 11-cv-01846-LHK, 2011 WL 7036077 (N.D. Cal. Dec. 2, 2011) ...... 26 Apple, Inc. v. Samsung Electronics Co., 678 F.3d 1314 (Fed. Cir. 2012) ...... 26 Apple Inc. v. Samsung Electronics Co., 735 F.3d 1352 (Fed. Cir. 2013) ...... passim Apple Inc. v. Samsung Electronics Co., 809 F.3d 633 (Fed. Cir. 2015) ...... 50, 54 AstraZeneca LP v. Apotex, Inc., 633 F.3d 1042 (Fed. Cir. 2010) ...... 21, 37, 43, 47 Bimbo Bakeries USA, Inc. v. Botticella, 613 F.3d 102 (3d Cir. 2010) ...... 24
Bio-Technology General Corp. v. Genentech, Inc., 80 F.3d 1553 (Fed. Cir. 1996) ...... 49
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BMEF San Diego, L.L.C. v. Gray East Village San Diego L.L.C., No. 9963-VCN, 2014 WL 4923722 (Del. Ch. Sept. 30, 2014) ...... 27
Celltrion, Inc. v. Genentech, Inc., IPR2017-01139 (P.T.A.B. Oct. 3, 2018) ...... 8
Celltrion, Inc. v. Genentech, Inc., IPR2017-01140 (P.T.A.B. Oct. 3, 2018) ...... 8
Celsis In Vitro, Inc. v. CellzDirect, Inc., 664 F.3d 922 (Fed. Cir. 2012) ...... 22, 45, 55, 56
Cordance Corp. v. Amazon.com, Inc., No. 06-491-MPT, 2010 WL 3155505 (D. Del. July 23, 2010) ...... 38
Cordis Corp. v. Boston Scientific Corp., Nos. Civ.A. 03-027-SLR, Civ.A. 03-283-SLR, 2003 WL 22843072 (D. Del. Nov. 21, 2003) ...... 38 Cordis Corp. v. Medtronic, Inc., 780 F.2d 991 (Fed. Cir. 1985) ...... 33 Douglas Dynamics, LLC v. Buyers Products Co., 717 F.3d 1336 (Fed. Cir. 2013) ...... 50 eBay, Inc. v. MercExchange, L.L.C., 547 U.S. 388 (2006) ...... 20, 35 Graceway Pharmaceuticals, LLC v. Perrigo Co., 722 F. Supp. 2d 566 (D.N.J. 2010) ...... 27, 28 High Tech Medical Instrumentation, Inc. v. New Image Industries, Inc., 49 F.3d 1551 (Fed. Cir. 1995) ...... 26, 37, 38, 39
Hoffmann-La Roche Inc. v. Cobalt Pharmaceuticals Inc., Nos. 07-4539 (SRC)(MAS), 07-4540(SRC)(MAS), 08- 4054(SRC)(MAS), 2010 WL 4687839 (D.N.J. Nov. 10, 2010) ...... 46 Hospira, Inc. v. Genentech, Inc., IPR2017-00804 (P.T.A.B. Oct. 3, 2018) ...... 8, 9 Hospira, Inc. v. Genentech, Inc., IPR2017-00805 (P.T.A.B. Oct. 3, 2018) ...... 8
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IGT v. Aristocrat Technologies, Inc., 646 F. App’x 1015 (Fed. Cir. 2014) (nonprecedential) ...... 33
Illinois Tool Works, Inc. v. Grip-Pak, Inc., 906 F.2d 679 (Fed. Cir. 1990) ...... 39
Immunomedics, Inc. v. Venvio Select Advisor LLC, No. 17-176-LPS, 2017 WL 822800 (D. Del. Mar. 2, 2017) ...... 27
Indivior Inc. v. Dr. Reddy’s Laboratories S.A., No. 17-7111 (KM) (CLW), 2018 WL 3496643 (D.N.J. July 20, 2018) ...... 28
Integra Lifesciences Corp. v. Hyperbranch Medical Technology, Inc., No. 15–819-LPS-CJB, 2016 WL 4770244 (D. Del. Aug. 12, 2016) ...... 28
King Pharmaceuticals, Inc. v. Sandoz, Inc., No. 08-5974 (GEB-DEA), 2010 WL 1957640 (D.N.J. May 27, 2010) ...... 42 Kos Pharmaceuticals, Inc. v. Andrx Corp., 369 F.3d 700 (3d Cir. 2004) ...... 54 Metalcraft of Mayville, Inc. v. Toro Co., 848 F.3d 1358 (Fed. Cir. 2017) ...... 56 Murata Machinery USA v. Daifuku Co., 830 F.3d 1357 (Fed. Cir. 2016) ...... 23 Natural Resources Defense Council Inc. v. Ross, 2019 WL 2173792 (Fed. Cir. May 20, 2019) ...... 5 Nichia Corp. v. Everlight Americas, Inc., 855 F.3d 1328 (Fed. Cir. 2017) ...... 37 Nutrition 21 v. United States, 930 F.2d 867 (Fed. Cir. 1991) ...... 18, 25 Oxford Immunotec Ltd. v. Qiagen, Inc., 271 F. Supp. 3d 358, 366-367 (D. Mass. 2017) ...... 53 PPG Industries, Inc. v. Guardian Industries Corp., 75 F.3d 1558 (Fed. Cir. 1996) ...... 52
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Pfizer, Inc. v. Teva Pharmaceuticals, USA, Inc., 429 F.3d 1364 (Fed. Cir. 2005) ...... 27, 55
Polymer Technologies, Inc. v. Bridwell, 103 F.3d 970 (Fed. Cir. 1996) ...... 27, 37, 46
Purdue Pharma L.P. v. Boehringer Ingelheim GmbH, 237 F.3d 1359 (Fed. Cir. 2001) ...... 47, 52
Ranbaxy Laboratories Ltd. v. Abbott Laboratories, Nos. 04 C 8078, 05 C 1490, 2005 WL 3050608 (N.D. Ill. Nov. 10, 2005) ...... 28 Reilly v. City of Harrisburg, 858 F.3d 173 (3d Cir. 2017) ...... 23 Robert Bosch LLC v. Pylon Manufacturing Corp., 659 F.3d 1142 (Fed. Cir. 2011) ...... 54 Sandoz Inc. v. Amgen Inc., 137 S. Ct. 1664 (2017) ...... 34 Sanofi-Synthelabo v. Apotex, Inc., 470 F.3d 1368 (Fed. Cir. 2006) ...... passim
T.J. Smith & Nephew Ltd. v. Consolidated Medical Equipment, Inc., 645 F. Supp. 206 (N.D.N.Y. 1986) ...... 27 T.J. Smith & Nephew Ltd. v. Consolidated Medical Equipment, Inc., 821 F.2d 646 (Fed. Cir. 1987) ...... 26
Tinnus Enterprises, LLC v. Telebrands Corp., 846 F.3d 1190 (Fed. Cir. 2017) ...... 22, 23
Trading Technologies International, Inc. v. eSpeed, Inc., No. 04 C 5312, 2008 WL 4531371 (N.D. Ill. May 22, 2008) ...... 40 Transcontinental Gas Pipe Line Co. v. Permanent Easements, 907 F.3d 725 (3d Cir. 2018) ...... 23 Trebo Manufacturing, Inc. v. Firefly Equipment, LLC, 748 F.3d 1159 (Fed. Cir. 2014) ...... 23
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Winter v. Natural Resources Defense Council, Inc., 555 U.S. 7 (2008) ...... 33
STATUTES 28 U.S.C. § 1292(a)(1) ...... 1 § 1292(c)(1) ...... 1 § 1331 ...... 1 § 1338(a) ...... 1
42 U.S.C. § 262(l)(8) ...... passim § 262(l)(8)(A)...... 31, 34 § 262(l)(8)(B) ...... 32 § 262(l)(9)(A)...... 32
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STATEMENT OF RELATED CASES
This is an interlocutory appeal following the denial of a preliminary injunction in Genentech, Inc. v. Amgen Inc., No. 18-cv-924-CFC (D. Del.). That case remains pending before the district court. Otherwise, counsel is aware of no case in this or any other court that would affect or be affected by the outcome of this appeal.
JURISDICTIONAL STATEMENT
This is an appeal from the denial of a preliminary injunction in a patent- infringement action. The district court had jurisdiction under 28 U.S.C. §§ 1331 and 1338(a). The district court denied Genentech’s motion for a preliminary injunction on July 18, 2019. Appx11. Genentech filed its notice of appeal the following day, on July 19, 2019. Appx4885-4904. This Court has jurisdiction under 28 U.S.C. § 1292(a)(1) and (c)(1).
INTRODUCTION
Genentech holds three patents on the most prescribed dosing regimen for its blockbuster drug Herceptin®. As of last Thursday, July 18, 2019—when the district court issued the order being appealed here—Herceptin was the only commercially available therapy using these patented inventions. Had the preliminary injunction been granted, it would have remained such until
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, when the first licensed Herceptin biosimilar would have launched as a result of a settlement.
The district court refused a preliminary injunction. Notably, the district court did not address whether Genentech was likely to succeed on the merits, although the evidence overwhelmingly favors Genentech: Amgen conceded that its biosimilar version of Herceptin, Kanjinti, . And although Amgen challenges the patents’ validity (via attorney argument unsupported by any expert testimony), it makes essentially the same arguments that were rejected in final written decisions in recent IPR proceedings under a less demanding standard of proof. Nor did the district court address the balance of hardships. Instead, the district court premised its denial of a preliminary injunction entirely on the theory that Genentech would not be irreparably harmed by Amgen’s launch. It made two legal errors in doing so.
First, the district court incorrectly held that Genentech could not show irreparable harm because of its timing in seeking injunctive relief. But Amgen only made the decision to launch at risk on , and Genentech moved for a preliminary injunction days later. Importantly, Genentech’s motion was filed before Amgen’s intended launch, which was to occur beginning on
. Although a patentee’s claim to irreparable harm may fairly be questioned where it has been suffering the alleged irreparable harm for an extended period of
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The district court’s opinion suggests that Genentech should have sought an injunction upon receipt of Amgen’s notice of commercial marketing pursuant to 42
U.S.C. § 262(l)(8) on May 15, 2018, but Kanjinti was not even approved by the
FDA until over a year later, on June 13, 2019, and
.
Nor would it have made sense for Genentech to move for a preliminary injunction when Amgen received FDA approval on June 13, 2019. Even thereafter, Amgen continued to represent that it had not decided whether to launch at risk, and it insisted that disputes about a potential launch “may not be ripe” depending on Amgen’s ultimate launch decision and timing. Appx1277(31:4-11)
(emphasis added). Genentech filed emergency motions for a temporary restraining order and a preliminary injunction in the district court just days after Amgen made its launch decision. Had Genentech filed materially earlier under the circumstances, its motion would have been premature. The district court’s insistence that Genentech must not have faced irreparable harm because it did not seek a preliminary injunction before Amgen received FDA approval and before it made a launch decision was legal error and an abuse of discretion.
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Second, the district court erroneously held that Amgen’s July 2019 launch would not irreparably harm Genentech because Genentech had settled litigation with other biosimilar manufacturers by licensing them to enter the market in the future, even though those agreements allowed Genentech to maintain its market exclusivity until then. The court applied a categorical rule that any license—even one that only licensed a party to enter the market in the future, where the licensor negotiated — demonstrates the absence of irreparable harm by present infringement in a market where the patentee is the only party practicing the invention. That bright-line rule makes no sense and violates eBay and this Court’s precedents. If anything,
Genentech’s decision to negotiate for shows an inability to place a monetary value on Genentech’s market exclusivity. The district court’s conclusion that Genentech forfeited its right to obtain an injunction would mean, as a practical matter, that innovators cannot settle pending cases without losing their right to exclude other entrants in any subsequent litigation. This too is a legal error requiring reversal.
Given these errors in the district court’s analysis, coupled with Genentech’s strong showing on the merits, Genentech is entitled to an injunction that bars
Amgen from launching its biosimilar product at least pending trial, which is scheduled for December 9, 2019. The district court’s denial of such relief was an
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abuse of discretion and should be reversed or, in the alternative, remanded with instructions to consider the availability of a preliminary injunction without regard to the timing of the filing or the fact that Genentech settled with other entrants.
Should the Court grant Genentech’s pending motion under Fed. R. App. P. 8 and subsequently remand for the district court for further consideration, Genentech respectfully requests that this Court order that the injunction pending appeal continue through the district court’s determination on remand. See Nat. Res. Def.
Council Inc. v. Ross, 2019 WL 2173792, at *3 (Fed. Cir. May 20, 2019)
(nonprecedential).
STATEMENT OF ISSUE ON APPEAL
Whether the district court committed legal errors and abused its discretion in denying Genentech’s motion for a preliminary injunction.
STATEMENT OF THE CASE
A. Genentech’s Development Of Herceptin And The Dosing Patents Roughly 25-30% of women diagnosed with breast cancer each year have
“HER2-positive” cancer, which causes them to overexpress human epidermal growth factor 2 (“HER2”). Appx1492(¶19). HER2-positive breast cancer is particularly aggressive: In the 1990s, before there were any targeted treatments, it was “associated with poor prognosis,” with cancer progressing and spreading
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quickly and patients with advanced disease having a life expectancy of only 18 months. Appx1492(¶20).
Starting in the 1980s, Genentech developed Herceptin, which fundamentally changed the treatment of HER2-positive breast cancer. Herceptin’s active ingredient is “trastuzumab,” a monoclonal antibody that targets and arrests the growth of HER2-positive cancers. Appx1492-1493(¶21). Following FDA approval in 1998, Herceptin was hailed as a revolution—demonstrating for the first time that solid tumors could be treated with a targeted therapy. Appx1493(¶22).
Since then, Herceptin has extended and, in early breast cancer, saved the lives of hundreds of thousands of patients. Appx1467-1468(¶12); Appx1493-1494(¶¶23-
24). Indeed, due to Genentech’s research, HER2-positive breast cancer has gone from having the worst prognosis of any breast cancer to one of the best.
Appx1466-1467(¶¶6-9). Herceptin is now the standard of care for HER2-positive breast cancer. Appx1467-1468(¶¶9-12).
Genentech’s work did not stop with Herceptin’s approval in 1998.
Genentech continued to invest billions of dollars and countless hours of research in the intervening two decades to improve therapeutic options for HER-2 positive patients. Appx1467(¶7). This investment of resources was high risk and included investing in clinical trials to extend the use of Herceptin from advanced (i.e., metastatic) breast cancer to early breast cancer patients, who could be given the
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drug in a curative setting following surgery (referred to as “adjuvant” therapy).
Appx1467(¶8).
Of particular relevance here, Genentech researchers successfully developed new dosing regimens that make Herceptin more convenient for early breast cancer patients by extending the intervals between clinical visits from one week to three weeks. Appx1494(¶25). Specifically, Genentech researchers discovered how to administer the drug in ways that allow for longer intervals between doses while still maintaining efficacy, including a method of administering a larger initial dose of 8 mg/kg trastuzumab followed by subsequent doses of 6 mg/kg every three weeks. Appx1494(¶26). This 8/6 three-weekly dosing regimen is claimed in U.S.
Patent Nos. 6,627,196 (“the ’196 patent”), 7,371,379 (“the ’379 patent”), and
10,160,811 (“the ’811 patent”) (collectively, “the dosing patents”), and recited in each of the claims asserted in this case. Appx1494-1496(¶¶27-30).1
The Herceptin label includes the patented dosing regimen as one of two options for adjuvant treatment, instructing administration of an “[i]nitial dose of
8 mg/kg over 90 minutes IV infusion, then 6 mg/kg over 30-90 minutes IV
1 The asserted claims include claims 11 and 22 of the ’196 patent, claims 11 and 21 of the ’379 patent, and claims 6 and 7 of the ’811 patent. See Appx49(’196 patent claims 11 & 22); Appx87-88(’379 patent claims 11 & 21); Appx139(’811 patent claims 6 & 7).
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As between the two options for adjuvant treatment, the 8/6 three-weekly dosing regimen is most often prescribed. Appx1496-1497(¶32); Appx2325;
Appx2335. According to Amgen’s own documents, the claimed dosing regimen constitutes up to of the market. Appx2499; Appx1696-1697(198:21-199:1);
Appx1699-1702(205:16-208:24); Appx1827.
Biosimilar applicants recognized the significance of Genentech’s patents on these inventions and challenged two of the dosing patents—the ’196 and ’379 patents—through inter partes review. The Patent Trial and Appeal Board (PTAB) rejected these challenges in October 2018, and the appeals to this Court have been dismissed. Final Written Decision, Hospira, Inc. v. Genentech, Inc., IPR2017-
00804 (P.T.A.B. Oct. 3, 2018) (’196 patent); Final Written Decision, Celltrion,
Inc. v. Genentech, Inc., IPR2017-01139 (P.T.A.B. Oct. 3, 2018) (’196 patent);
Final Written Decision, Hospira, Inc. v. Genentech, Inc., IPR2017-00805
(P.T.A.B. Oct. 3, 2018) (’379 patent); Final Written Decision, Celltrion, Inc. v.
Genentech, Inc., IPR2017-01140 (P.T.A.B. Oct. 3, 2018) (’379 patent). In each of its final written decisions, the PTAB found that the petitioners did not show, by a preponderance of the evidence, that the challenged claims were invalid. The
PTAB explained that the “Petitioners have not pointed to any prior art reference
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Final Written Decision at 23, 25, Hospira, Inc. v. Genentech, Inc., IPR2017-00804.
The third dosing patent, the ’811, issued after the IPRs concluded and is narrower than the other dosing patents; its notice of allowance references the IPR final written decisions. Appx1231; Appx89-139.
In order to settle various litigations and IPRs brought by other biosimilar applicants, Genentech has entered into settlement agreements authorizing them to enter the market on agreed-to future dates, with the earliest entry date allowed under these settlements being . in connection with those agreements, because Genentech recognized that the value of its hard-earned market exclusivity, protected in part by the dosing patents, could not be quantified or adequately compensated by a . As of last Thursday,
July 18, when the district court refused to enter a TRO or a preliminary injunction,
Genentech’s Herceptin was the only product on the market utilizing Genentech’s dosing patents.
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B. Amgen’s Biosimilar Drug Kanjinti
Amgen developed a biosimilar version of Herceptin called Kanjinti. Long before the FDA approved the product, Amgen sent Genentech a Notice of
Commercial Marketing under 42 U.S.C. § 262(l)(8) on May 15, 2018. At that time, . After the PTAB upheld the ’196 and ’379 patents’ validity in the IPRs in October 2018, Amgen asked the FDA
. Appx1830; Appx1832. But Amgen subsequently determined that a
Appx1837; Appx1694-1695(179:5-14, 179:24-180:4);
Appx1705(27:19-28:11); Appx1676-1678(234:5-24; 239:10-240:10). Even thereafter, Amgen continued to consider
. Appx4074 (“
”). And throughout this time, FDA approval was not guaranteed; indeed, Amgen’s initial application was not approved, and, following receipt of a complete response letter from the FDA in May 2018, Amgen was
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Appx3770(¶3).
The FDA ultimately approved Amgen’s Kanjinti biosimilar on June 13,
2019 with a label materially identical to Herceptin’s. Appx1583; see also
Appx1687 (Amgen press release stating that FDA “has approved KANJINTI™
(trastuzumab-anns) for all approved indications of the reference product, Herceptin
(trastuzumab)”). Given that both labels recite Genentech’s patented 8/6 three- weekly dosing regimens, Amgen the dosing patents.
Appx3759.
C. Amgen’s Representations Regarding The Timing Of Its Launch Of Kanjinti
When Amgen gave Genentech its Notice of Commercial Marketing of
Kanjinti in May 2018, Amgen was precluded from launching not only by the statutory 180-day waiting period, see 42 U.S.C. § 262(l)(8), but also because it had not received FDA approval. Indeed, Amgen did not receive FDA approval until over a year later, on June 13, 2019. Unsurprisingly, given the complexities of preparing for a launch, Amgen began those preparations well before FDA approval, and targeted being ready to launch by July 2019. But Amgen’s actual launch timing remained undetermined: in a deposition on June 26-27, 2019, Molly
Benson—
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Appx4838(353:12-19) (objection omitted).
Appx4835(98:1-16).
Amgen’s other witnesses also testified that
. See, e.g., Appx4841 (May 24, 2019
Hall Dep. Tr. at 72:9-14) (“Q.
.” (objection omitted; emphasis added)); Appx4844 (April 30, 2019 Yant Dep. Tr. at 62:19-62:23) (“Q.
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).
Amgen’s counsel likewise represented to Genentech and the district court that no decision had been made. Even after receiving FDA approval, Amgen explained that, notwithstanding its launch planning, an actual launch could still be as much as a year away, putting it well after the scheduled December 2019 trial date. Appx1277 (June 18, 2019 Hearing Tr. at 30:3-8) (“[I]f we were to launch in two months versus six months versus a year, the company would still need to make the preparation to be in a position to launch[.]” (emphasis added)). And in view of these uncertainties, Amgen argued that disputes relating to a potential launch “may not be ripe” because “the circumstances from Genentech’s standpoint and from the market standpoint may be very different in those different scenarios.” Appx1277(31:4-11; 30:3-8) (emphasis added). Indeed,
Amgen’s counsel flatly told the district court that that it had not determined whether it would actually launch Kanjinti at risk:
Part of the problem is we have not made that ultimate decision yet because we have not launched yet. We have not launched yet. That’s what I’m saying. Those decisions are ongoing.
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Appx1289(78:22-25) (emphasis added). Amgen then reiterated in a brief filed the following week that its launch decision was “something that has not occurred.”
Appx1299 (emphasis added).
Amgen did not make the decision to launch until And, when Amgen made that decision, it decided to launch not within two months, or six months, or a year, but just later, on . Appx3770(¶5). Amgen did not tell
Genentech that it had decided to launch within ; Genentech learned about
Amgen’s forthcoming launch through its own market intelligence and immediately brought a motion for a preliminary injunction in the district court on July 10, five days before Amgen’s intended launch date.
D. District Court Proceedings
Genentech filed this patent infringement action against Amgen on June 21,
2018, shortly after receiving Amgen’s original Notice of Commercial Marketing.
Fact discovery closed on June 10, 2019; the parties are now in the midst of expert discovery, with trial currently scheduled for December 9, 2019. Appx1001-1004;
Appx1011-1023; Appx1233-1235.
During the week of July 8, 2019, Genentech for the first time received concrete market intelligence that Amgen was planning an imminent launch of
Kanjinti. On July 10, 2019— days after Amgen had decided to launch, and
days before the intended launch—Genentech filed emergency motions
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Appx1324-1325; Appx1332-1333. Genentech addressed each of the four factors for injunctive relief, presenting the expert declaration of Dr. Susan Tannenbaum on infringement and the fact declaration of Christy Oliger and expert declaration of
Dr. Anupam Jena outlining the irreparable harm that Genentech would suffer if
Amgen launched its infringing Kanjinti product. Specifically, Genentech explained that Kanjinti infringed the dosing patents, and that its launch would cause, among other things, erosion to the price of Herceptin and a significant loss of market share, consequences that Amgen itself has argued elsewhere could not be remedied by money damages. Appx1931-1935. The district court entered a standstill order that same day. Appx3738-3739.
Amgen filed an opposition on July 15, but did not provide any supporting expert declarations, even though the deadline for expert reports was only 10 days later. Appx3746, Appx1234. Amgen . Appx3759. With respect to invalidity, Amgen largely rehashed the same arguments that had been presented in the IPRs, addressed to art that was cumulative of the art that the
PTAB had already considered. In support of those arguments, Amgen provided only attorney argument without any supporting expert testimony. Nor did Amgen dispute that price erosion and loss of market share suffice to demonstrate irreparable harm—likely because Amgen itself has relied upon price erosion and
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Appx1931-1935. Genentech filed its reply the following day, providing the supplemental declaration of Ms. Oliger and the expert declaration of Dr. George
Grass addressing the patents’ validity. Appx4720-4789.
On July 18, 2019, the district court entered an order denying Genentech’s motions. Appx11. The district court ruled that Genentech had not shown that
Amgen’s launch would cause it irreparable harm, for two reasons. First, the court asserted that Genentech’s failure to move for an injunction earlier negated a finding of irreparable harm, even though Amgen had not yet launched Kanjinti and despite the fact that Amgen consistently represented—even after the FDA’s approval on June 13, 2019—that it had not decided whether to launch at risk.
Appx6-7. Second, the court found that Genentech’s agreement that other companies could enter the market “in ” meant that Genentech had an adequate remedy at law, because any damages accruing before trial “should be quantifiable.” Appx8-9. The court cited no evidence for that conclusion, nor did it address Genentech’s evidence that price erosion and loss of market share were not compensable by money damages—a showing Amgen had not disputed.
The district court did not consider Genentech’s likelihood of success on the merits or the balance of hardships. It considered the public interest only in a footnote, which stated a concern that, because the Kanjinti label (like the Herceptin
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label) contains “two recited methods … that are free of any allegations of infringement,” an injunction would “‘depriv[e] the public of access to a large number of non-infringing features.’” Appx9-10 n.7 (quoting Apple Inc. v.
Samsung Elecs. Co., 735 F.3d 1352, 1372-1373 (Fed. Cir. 2013)). The court did not address the fact that Genentech’s Herceptin product, which is already in the market and can supply all public need, is likewise indicated for those “two recited methods,” such that Kanjinti does not fill any market need currently unmet by
Herceptin.
Very shortly after receiving the district court’s order, Genentech advised
Amgen that it planned to appeal to this Court and to ask the district court and, if necessary, this Court for an interim injunction pending appeal. Appx4938-
1439(25:7-26:15); Appx4945(32:2-10). After meeting and conferring with Amgen the following morning (Friday, July 19), Genentech filed its notice of appeal and a motion for injunction pending appeal under Federal Rule of Civil Procedure 62(d);
Genentech alternatively sought a short 14-day injunction to enable Genentech to ask this Court to enter an injunction pending appeal under Federal Rule of
Appellate Procedure 8. Appx4885-4904; ECF No. 9.
After holding a telephonic hearing, the district court denied Genentech’s
Rule 62(d) motion at 4:45 p.m. that same day (Friday, July 19). Appx4964-
4966(51:4-53:22). Genentech filed its motion in this Court for an injunction
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pending appeal under Federal Rule of Appellate Procedure 8 at 6:30 p.m. that day;
Genentech also asked for the Court to expedite briefing on the motion and on the appeal. ECF No. 9. On Monday morning, July 22, 2019, this Court accepted
Genentech’s motion for filing and ordered Amgen to respond to it by Monday, July
29, 2019. ECF No. 10.
Genentech is filing this opening brief on the merits on an expedited basis, within one week of its notice of appeal.
SUMMARY OF THE ARGUMENT
The district court’s determination that Genentech will suffer no irreparable harm was based on two legal errors that are inconsistent with precedent governing preliminary injunctive relief and that, unless reversed, will make it extremely difficult for innovator companies to settle biosimilar litigation under the framework of the Biologics Price Competition and Innovation Act (BPCIA).
First, the district court was wrong to conclude that Genentech would not suffer irreparable harm from Amgen’s launch because it filed its preliminary injunction request after Amgen had decided to launch an infringing product, but before the launch itself. Appx5-6. The timing of an injunction motion is logically relevant to irreparable harm to the extent that a party’s delay demonstrates that it is willing to tolerate that harm by taking no action to prevent it. See Nutrition 21 v.
United States, 930 F.2d 867, 872 (Fed. Cir. 1991). But this Court has never held
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Appx1289(78:22-25). Genentech filed its motion as soon as it learned that Amgen would launch at risk; that Amgen’s launch was merely days away has nothing to do with Genentech’s irreparable harm and everything to do with the timing of
Amgen’s decision-making.
To the extent the district court viewed the BPCIA as requiring Genentech to act sooner, that was error. A notice under the relevant statutory section may be a necessary condition to moving for a preliminary injunction, but it does not require a reference-product sponsor like Genentech to seek a preliminary injunction immediately upon receipt of a notice of commercial marketing when, under the circumstances, such a motion would be premature. This case illustrates why a contrary rule is unworkable: between issuing its (l)(8) notice in May 2018 and
FDA approval in June 2019, Amgen was forced to resubmit its application to the
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FDA and
. Appx3770(¶3); Appx1830; Appx1832; Appx1837-1839. It would make no sense to require Genentech to seek a preliminary injunction on the basis of patents that had been ; had Genentech moved for a preliminary injunction upon service of the (l)(8) notice, it would have had to withdraw it when Amgen
.
Second, the district court’s reliance on Genentech’s license agreements to find a lack of irreparable harm, without considering the significant differences between Genentech’s settlement agreements allowing other parties to enter the market in the future and Amgen’s present infringement, amounts to a categorical rule that such settlements negate irreparable harm. Apple Inc. v. Samsung Elecs.
Co., 735 F.3d 1352, 1370 (Fed. Cir. 2013) (“Apple III”). Such a rule is contrary to the Supreme Court’s decision in eBay, Inc. v. MercExchange, L.L.C., 547 U.S.
388, 393 (2006), which held that licensing of the patented invention does not preclude injunctive relief. Genentech’s settlement agreements do not show that monetary damages could compensate for infringement. On the contrary,
Genentech’s decision to license other market entrants in the future without
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, rather than accepting an earlier entry date coupled with a
only confirms that it cannot put a on its lawful exclusivity. See
AstraZeneca LP v. Apotex, Inc., 633 F.3d 1042, 1061-62 (Fed. Cir. 2010)
(affirming preliminary injunction against unlicensed market entrant even though a competitor was licensed to enter in the future, because harm after the licensed entry would be impossible to calculate). Finally, unlike the companies with which
Genentech settled, Amgen is uniquely positioned to do damage in the Herceptin market because of its relationships with oncology clinics, its experience with payers in the oncology market, and its established track record as a successful biologic manufacturer. Appx1477-1478(¶¶53-54); Appx1396-1397(¶59).
The district court’s conclusion that under these circumstances Genentech’s harms from Amgen’s launch would be quantifiable finds no support in the record.
There was simply no evidence supporting that proposition—the only evidence was
Genentech’s evidence showing the exact opposite. E.g., Appx1399-1402(¶¶65-
72); see Sanofi-Synthelabo v. Apotex, Inc., 470 F.3d 1368, 1382 (Fed. Cir. 2006).
Once the district court’s errors are corrected, the remaining record evidence establishes Genentech’s entitlement to a preliminary injunction. First, as
Genentech’s expert and fact declarants testified—without any rebuttal from
Amgen—Kanjinti’s launch will erode the price of Genentech’s Herceptin product and cause Genentech to lose market share. It will also cause collateral harm to
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Genentech’s reputation. This Court routinely finds that such harm is irreparable.
E.g., Abbott Labs. v. Sandoz, Inc., 544 F.3d 1341, 1361-1362 (Fed. Cir. 2008).
Second, the record evidence shows that Genentech is likely to succeed on the merits because Amgen did not dispute and merely rehashed, without evidence, invalidity arguments previously rejected by the PTAB under a lower standard of proof. Third, the balance of hardships likewise weighs in Genentech’s favor because any harm Amgen might suffer from an interim injunction pending trial is entirely self-inflicted due to Amgen’s decision to launch at risk while knowing of Genentech’s infringement claims. Celsis In Vitro, Inc. v. CellzDirect,
Inc., 664 F.3d 922, 931 (Fed. Cir. 2012). Finally, the public interest favors enforcing Genentech’s patent rights, especially where Amgen does not contest
or that Genentech is capable of supplying the market.
The district court’s denial of a preliminary injunction should be reversed, or at the very least vacated and remanded.
STANDARD OF REVIEW
In determining whether to grant a preliminary injunction, courts consider four factors: “(1) the likelihood of the patentee’s success on the merits;
(2) irreparable harm if the injunction is not granted; (3) the balance of hardships between the parties; and (4) the public interest.” Tinnus Enters., LLC v.
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Telebrands Corp., 846 F.3d 1190, 1202 (Fed. Cir. 2017); accord Transcontinental
Gas Pipe Line Co. v. Permanent Easements, 907 F.3d 725, 732 (3d Cir. 2018).
This Court generally reviews preliminary injunction decisions under the law of the regional circuit because “‘[t]he grant, denial, or modification of a preliminary injunction … is not unique to patent law.’” Tinnus Enters., 846 F.3d at 1202-1203 (alterations in original) (quoting Trebo Mfg., Inc. v. Firefly Equip.,
LLC, 748 F.3d 1159, 1163 (Fed. Cir. 2014)). “‘However, the Federal Circuit has itself built a body of precedent applying the general preliminary injunction considerations to a large number of factually variant patent cases, and gives dominant effect to Federal Circuit precedent insofar as it reflects considerations specific to patent issues.’” Id. at 1203 (quoting Murata Mach. USA v. Daifuku Co.,
830 F.3d 1357, 1363 (Fed. Cir. 2016)).
Both this Court and the Third Circuit review the grant or denial of a preliminary injunction for an abuse of discretion, which occurs when “the court made a clear error of judgment in weighing relevant factors or exercised its discretion based upon an error of law or clearly erroneous factual findings.”
Tinnus Enters., 846 F.3d at 1203 (quoting Abbott Labs. v. Andrx Pharm., Inc., 452
F.3d 1331, 1335 (Fed. Cir. 2006)); see Reilly v. City of Harrisburg, 858 F.3d 173,
176 (3d Cir. 2017) (“‘When reviewing a district court’s [denial] of a preliminary injunction, we review the court’s findings of fact for clear error, its conclusions of
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law de novo, and the ultimate decision … for an abuse of discretion.’” (alterations in original) (quoting Bimbo Bakeries USA, Inc. v. Botticella, 613 F.3d 102, 109 (3d
Cir. 2010))).
ARGUMENT
I. THE DISTRICT COURT’S DETERMINATION OF NO IRREPARABLE HARM RESTED ON LEGAL ERRORS
A. The District Court Adopted An Erroneous Legal Standard To Conclude That Genentech Could Not Establish Irreparable Harm Because Of The Timing Of Its Motion
The district court’s conclusion that Genentech could not show irreparable harm because of when it filed its motion reflects a misunderstanding of this Court’s precedent on the proper timing of a preliminary injunction request and would require plaintiffs like Genentech to file unnecessary and premature motions in order to preserve their rights to exclude. When a party is suffering harm, waiting to file for a preliminary injunction may belie a later claim that that harm was truly irreparable. But that is not the situation here. Far from sitting idly by while
Amgen captured its market, Genentech moved promptly, indeed with great speed, when it found out that Amgen had decided to launch. Amgen itself acknowledged that disputes might not be ripe prior to that point. There is no requirement for a patentee like Genentech to file a motion to enjoin sales that the infringer itself insists it may not make, and the district court abused its discretion in holding otherwise.
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1. The district court legally erred in inferring that Genentech will not suffer irreparable harm because it waited to seek preliminary injunctive relief until Amgen affirmatively decided to launch
Amgen repeatedly represented, including in open court in late June 2019, that it had not decided whether to launch at risk, much less when it would do so.
Genentech took Amgen at its word. See supra pp. 11-14. Once Genentech learned that Amgen had decided to launch, it moved for a preliminary injunction immediately. Genentech filed its preliminary injunction motion only days after Amgen made its decision to launch and days before Amgen’s intended launch date. At the time Genentech filed its motion, Genentech had not yet suffered any harm from Kanjinti and would not have suffered any harm had its motions been granted.
Notwithstanding this record, the district court found that Genentech could not establish irreparable harm because of Genentech’s supposed “undue delay” in seeking an injunction. Appx7. Although a patentee’s claim to irreparable harm may be undermined where it has long suffered the alleged irreparable harm without complaint, such an inference cannot be drawn when the patentee moves before the harm has even begun. See Nutrition 21, 930 F.2d at 872 (“Finally, that Nutrition
21 delayed for a substantial period of time before seeking a preliminary injunction at least suggests that the status quo does not irreparably damage Nutrition 21.”).
This Court has never found undue delay where the patentee filed for preliminary
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injunctive relief before the irreparable harm even began, simply because there was a possibility that irreparable harm might later take place.
Rather, this Court has found that undue delay defeats an irreparable-harm showing when a patentee who is actually suffering the harm of infringement does not seek injunctive relief for a lengthy period. For example, in Apple, Inc. v.
Samsung Electronics Co., 678 F.3d 1314, 1325-1326 (Fed. Cir. 2012), this Court affirmed the conclusion that a patentee’s decision to wait to seek an injunction until one year after the launch of the most recent generation of infringing products weighed against irreparable harm. Id. at 1325-1326; Apple, Inc. v. Samsung Elecs.,
Co., 2011 WL 7036077, at *22 (N.D. Cal. Dec. 2, 2011). Other cases considering a patentee’s delay in moving for injunctive relief likewise involved lengthy periods when the supposedly harmful infringement was actually happening. See High
Tech Med. Instrumentation, Inc. v. New Image Indus., Inc., 49 F.3d 1551, 1557
(Fed. Cir. 1995) (seventeen-month delay in seeking preliminary injunction after issuance of reexamination certificate weighed against a finding of irreparable harm when infringing product had been on the market for two years); T.J. Smith &
Nephew Ltd. v. Consolidated Med. Equip., Inc., 821 F.2d 646, 648 (Fed. Cir. 1987)
(fifteen-month delay in seeking preliminary injunction after reissuance of patent weighs against finding irreparable harm where infringing product had been on the
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market for three years) (affirming T.J. Smith & Nephew Ltd. v. Consolidated Med.
Equip., Inc., 645 F. Supp. 206 (N.D.N.Y. 1986)).
The cases the district court relied on actually support Genentech’s position that a patentee who moves for an injunction before the irreparably harmful conduct occurs may show irreparable harm from imminent infringement—indeed, the successful patentees in those cases waited far longer than Genentech. In Pfizer,
Inc. v. Teva Pharmaceuticals, USA, Inc., 429 F.3d 1364 (Fed. Cir. 2005), this
Court affirmed the district court’s finding that the plaintiff had not unduly delayed in filing suit two months after the launch of the infringer’s generic product and nearly two years after receiving notice of the defendant’s paragraph IV certification. Id. at 1371, 1382. The Court in Polymer Technologies, Inc. v.
Bridwell, 103 F.3d 970 (Fed. Cir. 1996), determined that filing suit four months after the commencement of infringing activity was a “short lapse of time” and did not negate irreparable harm. Id. at 976.2
2 The cases Amgen cited in its opposition to Genentech’s motion for a preliminary injunction are even farther afield. Immunomedics, Inc. v. Venvio Select Advisor LLC, 2017 WL 822800 (D. Del. Mar. 2, 2017), did not involve patent infringement, but rather held that the plaintiff’s delay in seeking to enjoin a shareholder vote from replacing the board weighed against a finding of irreparable harm where it had known about the alleged violations of securities laws on that board’s watch for six months and had already delayed the annual meeting twice. Id. at *3. BMEF San Diego, L.L.C. v. Gray East Village San Diego L.L.C., 2014 WL 4923722 (Del. Ch. Sept. 30, 2014), involved a demand to expedite a trial in a dispute that was “largely about cash.” Id. at *1-2. In Graceway Pharms. LLC v. Perrigo Co., 722 F. Supp. 2d 566 (D.N.J. 2010), the court initially faulted the
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Indeed, preliminary injunctions are routinely allowed after FDA approval and even after launch. See, e.g., Indivior Inc. v. Dr. Reddy’s Labs. S.A., 2018 WL
3496643, at *4, *11-14 (D.N.J. July 20, 2018) (finding irreparable harm where plaintiff filed its preliminary injunction and TRO motions “on an emergent basis”
“[u]pon learning of DRL’s plans to launch the ANDA product ‘at risk’”), rev’d on other grounds, 752 F. App’x 1024 (Fed. Cir. 2018) (nonprecedential); Integra
Lifesciences Corp. v. Hyperbranch Med. Tech., Inc., 2016 WL 4770244, at *8-9
(D. Del. Aug. 12, 2016) (recommending that the delay analysis should focus on the time the irreparable harm was to begin because “if a patentee is arguing that an accused infringer should be enjoined because a particular kind of infringing act is causing it irreparable harm, then the patentee would not seem to be properly motivated to seek an injunction until the infringer actually started to (or was about to) commit that particular infringing act”) (collecting cases); Ranbaxy Labs. Ltd. v.
Abbott Labs., 2005 WL 3050608, at *29 (N.D. Ill. Nov. 10, 2005) (no undue delay in seeking preliminary injunction a year after FDA approval in ANDA litigation despite being “on notice” of generic infringers for two years, because patent holder
plaintiffs for failing to inform the defendant of its intention to file suit upon the issuance of its patent, but changed its mind on a fuller record because “both parties could have acted more diligently.” Id. at 569-570. It ultimately found no prejudice to the defendant in part because “Graceway brought suit (although not its TRO motion) prior to Nycomed’s launch.” Id. at 570. None of these cases stands for the proposition that a plaintiff acts too late even though the defendant has not yet begun to take the irreparably harmful action.
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Abbott Labs. v. Andrx Pharms., Inc., 473 F.3d 1196 (Fed. Cir. 2007).
In view of this precedent, the district court was wrong to determine that the timing of Genentech’s motion defeated Genentech’s showing of irreparable harm.
It is not as though Genentech tolerated any harm and then later sought to call it irreparable; Genentech acted before the harm to be enjoined even began. Indeed, the record here is unequivocal: Genentech filed its motion for a preliminary injunction less than four weeks after Kanjinti was approved, within days of learning (not from Amgen, but through market intelligence) that Amgen was planning to launch; and days before Amgen’s intended launch date. Nothing in that conduct suggests that the harm Genentech would suffer from a launch was anything short of irreparable. It merely demonstrates that, because a preliminary injunction is an “extraordinary remedy,” Genentech concluded it was inappropriate to burden the district court with a request for a preliminary injunction unless it was clearly necessary.
The consequence of the district court’s rule reveals its unsoundness. Were the district court correct, patent owners would be obliged to move for preliminary injunctions when the fact of a launch, its timing, the exact nature of the product, and the commercial implications are still unknown. It is far from clear that an
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The district court observed that Genentech had stated “as recently as May
16, 2019[,] that it was not seeking a preliminary injunction.” Appx7 n.6. In fact,
Genentech said, “We’re not presently seeking injunctive relief” (Appx1246(26:3-
4)), and it made that statement before the FDA had even approved Kanjinti. See supra pp. 11-14 (discussing statements made in June hearings and depositions).
Genentech’s truthful statement that it was not seeking a preliminary injunction in
May when it was premature to do so should not foreclose it from filing a prompt motion when Amgen changed the game by making a launch decision, nor is it any evidence of a lack of irreparable harm once Amgen decided to launch.3
Any prejudice to Amgen from the timing of Genentech’s motion is entirely a function of its own decision to make a decision to launch a later. The district
3 Amgen’s decision to wait until to make a launch decision, and then to decide to launch just a later, is in stark contrast to what happened in a case involving another defendant’s biosimilar version of Herceptin last year. The district court observed that the parties in that other case were able to “avoid hurried motion practice” (Appx7 n.6), but that was because that defendant, unlike Amgen, made a decision to launch months in advance of the actual launch date and informed the court and Genentech of that decision, which allowed the parties and the court to address the disputed issues in an orderly manner. Appx4948- 4950(35:25-37:24). In the end, Genentech never filed a motion for preliminary injunction against that other biosimilar defendant because the parties settled the litigation.
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court committed legal error and abused its discretion in holding that it instead reflected a lack of irreparable harm on Genentech’s part.
2. Nothing in the BPCIA’s text or purpose required Genentech to seek a preliminary injunction before Amgen decided that it was going to launch its infringing product
The district court also erred in stating that Genentech’s actions were
“contrary to the spirit and purpose” of the Biologics Price Competition and
Innovation Act of 2009, Pub. L. No. 111-148, §§ 7001 et seq., 124 Stat. 804
(codified in relevant part at, 42 U.S.C. § 262(l)(8)). Appx7. The district court seemed to suggest that the BPCIA required Genentech to move for a preliminary injunction when it received Amgen’s notice of commercial marketing pursuant to
42 U.S.C. § 262(l)(8)(A) in May 2018. Id. To the extent the district court took such a view, it was legally erroneous.
The biosimilar applicant’s notice of commercial marketing pursuant to
§ 262(l)(8)(A) will generally be a necessary condition to bringing a motion for a preliminary injunction. Indeed, the biosimilar product cannot come to market for at least 180 days after providing notice under § 262(l)(8)(A), and a motion for a preliminary injunction thus would not be appropriate until a biosimilar applicant provides such notice. But nothing in § 262(l)(8) requires a reference-product sponsor like Genentech to seek a preliminary injunction immediately upon receipt of a notice of commercial marketing when the circumstances do not warrant it.
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The purpose of § 262(l)(8) is to make additional relief available4—not to require the reference-product sponsor to seek relief before it may even be ripe to do so.
The facts of this case illustrate why it makes no sense to require reference- product sponsors to seek preliminary injunctive relief upon receipt of an applicant’s § 262(l)(8) notice of commercial marketing. Amgen issued that notice on May 15, 2018. But Kanjinti was not approved until 13 months later, on June
13, 2019. During that time, Amgen resubmitted its entire application due to its receipt of a complete response letter from FDA and
. Appx3770(¶3); Appx1675-1678(232:9-24, 234:5-24,
239:10-240:10); Appx1694-1695(179:5-14, 179:24-180:4); Appx1698(202:3-19);
Appx1830; Appx1832; Appx1837-1839. Even after
. Appx4074.
4 For example, the reference-product sponsor may seek a preliminary injunction or declaratory relief for additional patents after receiving a notice of commercial marketing. See 42 U.S.C. § 262(l)(8)(B) (preliminary injunction); id. § 262(l)(9)(A) (declaratory judgment).
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Had Genentech moved for a preliminary injunction on the dosing patents when Amgen filed its § 262(l)(8) notice in May 2018 (or even 180 days after that),
Amgen would no doubt have argued the motion was premature because the label was subject to change and its launch uncertain. As this Court has explained, “[i]t is not enough to show a ‘possibility’ of harm, as ‘a possibility of irreparable harm is inconsistent with [the] characterization of injunctive relief as an extraordinary remedy that may only be awarded upon a clear showing that plaintiff is entitled to such relief.’” IGT v. Aristocrat Techs., Inc., 646 F. App’x 1015, 1018 (Fed. Cir.
2014) (nonprecedential) (quoting Winter v. Nat. Res. Def. Council, Inc., 555 U.S.
7, 22 (2008)). Instead, “[a] movant seeking a preliminary injunction must show that ‘irreparable injury is likely in the absence of an injunction.’” Id. (quoting
Winter, 555 U.S. at 22); see also Cordis Corp. v. Medtronic, Inc., 780 F.2d 991,
996 (Fed. Cir. 1985) (“A preliminary injunction will not issue simply to prevent a mere possibility of injury, even where prospective injury is great.”). A blanket rule requiring reference-product sponsors to file, and the courts to address, motions for preliminary injunction during a period when changes in the biosimilar product would potentially moot any need for such relief would waste court and party resources, and is contrary to the requirement of an imminent, irreparable injury necessary to obtain a preliminary injunction.
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The district court quoted this Court’s statements in Amgen Inc. v. Apotex
Inc., 827 F.3d 1052 (Fed. Cir. 2016), to suggest that the BPCIA requires filing a motion for preliminary injunction upon receiving the biosimilar applicant’s notice of commercial marketing. Appx7. But that case was based on this Court’s understanding at the time that a notice of commercial marketing could not be provided prior to FDA approval. 827 F.3d at 1062. The Supreme Court’s subsequent decision in Sandoz Inc. v. Amgen Inc., 137 S. Ct. 1664, 1677-1678
(2017), alters that analysis by holding that a notice of commercial marketing may be provided prior to FDA approval such that the applicant’s actual commercial marketing is not imminent at the time that it provides notice under § 262(l)(8)(A).
Accordingly, nothing in the letter of the BPCIA or its “spirit and purpose”
(Appx7) required Genentech to move for a preliminary injunction before it did: as soon as it knew that Amgen had decided to launch.
B. The District Court Improperly Created And Applied A Categorical Rule That Licenses To Third Parties For Future Entry Dates Demonstrated A Lack Of Irreparable Harm From Present Infringement
The district court further erred in holding that Genentech’s settlement agreements allowing other defendants to enter the market in the future negated any showing of irreparable harm by Amgen’s launch. The district court concluded that the fact that Genentech “granted licenses” for the dosing patents in which it “approved competitors entering the market in a ”
- 34 - Case: 19-2156 Document: 19 Page: 46 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED somehow meant that Genentech had an adequate remedy at law for Amgen’s market entry now. Appx8-9. That was legal error and, unless reversed, would create a rule that would seriously impede settlements of biosimilar litigation.
1. The district court erred in adopting a categorical rule that licensing of future activity negates irreparable harm from present infringement
The district court adopted a categorical rule that settling a case against another entrant—even where the settlement is expressly for — makes it impossible to establish irreparable harm and obtain injunctive relief, even where the settlement only permits entry at a future date. The district court’s analysis is contrary to the Supreme Court’s explicit rejection of the proposition that a patentee’s willingness to license its patents suffices by itself to demonstrate a lack of irreparable harm. eBay, 547 U.S. at 393 (2006) (“To the extent that the
District Court adopted such a categorical rule, then, its analysis cannot be squared with the principles of equity adopted by Congress.”). It is also contrary to Apple
III, where this Court reversed the denial of a preliminary injunction because “the district court’s focus on Apple’s past licensing practices, without exploring any relevant differences from the current situation, hints at a categorical rule that
Apple’s willingness to license its patents precludes the issuance of an injunction.”
735 F.3d at 1370. That is exactly the same error that the district court made here, and it compels the same result.
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This Court has explained that “[a] plaintiff’s past willingness to license is not sufficient per se to establish lack of irreparable harm if a new infringer were licensed,” and that “[a]dding a new competitor to the market may create an irreparable harm that the prior licenses did not.” Acumed LLC v. Stryker Corp.,
551 F.3d 1323, 1328-29 (Fed. Cir. 2008). That is why, when considering whether infringement causes irreparable harm, “[t]he identity of the past licensees, the experience in the market since the licenses were granted, and the identity of the new infringer” are all factors for the court to consider, in addition to the bare fact of the grant of a license. Id. By failing to consider those factors—all of which demonstrate the sharp difference between Genentech’s negotiated future licenses and the present license to Amgen that the district court forced onto Genentech here—the district court committed legal error. Apple III, 735 F.3d at 1370.
Had the district court made the proper comparison, the conclusion is inescapable that Genentech’s settlement with other applicants who agreed not to enter the market before in no way shows that Amgen’s entry now would not cause irreparable harm. Genentech has not licensed any entry for the period between now and . The litigation settlements show, if anything, that Genentech cannot put a on exclusivity.
Genentech is not aware of any case in which a settlement allowing entry in the future has been found to defeat irreparable harm in the present. In facts, courts
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have found the exact opposite. See AstraZeneca, 633 F.3d at 1061-63 (affirming preliminary injunction against unlicensed market entrant even though a competitor was licensed to enter in the future, because harm after the licensed entry would be impossible to calculate); Abbott Labs. v. Sandoz, Inc., 500 F. Supp. 2d 807, 843
(N.D. Ill. 2007) (settlement agreements allowing two specific generic drugmakers to enter the market the following year did not mean that patent holder gave up its right to exclude generics in the present), aff’d, 544 F.3d 1341 (Fed. Cir. 2008); cf.
Nichia Corp. v. Everlight Ams., Inc., 855 F.3d 1328, 1343-1344 (Fed. Cir. 2017)
(no irreparable harm where past “licenses changed the market by making available ‘multiple low-priced non-infringing alternatives’” (emphasis added)).
Certainly none of the cases cited by the district court supports its proposition that licenses for future entry foreclose finding irreparable harm from an earlier entry. In Polymer Technologies, 103 F.3d at 974, there were no licenses at issue; that case merely cited the second case the district court relied upon, High Tech
Medical Instrumentation, Inc. v. New Image Industries, Inc., 49 F.3d 1551, 1557
(Fed. Cir. 1995), for the proposition that a party’s licensing history can be a factor.
High Tech, in turn, considered the patentee’s “apparent willingness to grant a
[royalty-bearing] license under its patent” not to a third party, but to the defendant itself—a factor that unsurprisingly weighed against injunctive relief and that is undisputedly not present here. Id. High Tech is further distinguishable because the
- 37 - Case: 19-2156 Document: 19 Page: 49 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED patentee was a non-practicing entity—“the lack of commercial activity by the patentee” was a “significant factor” in the analysis. Id. at 1556. The cases Amgen cited in its opposition to Genentech’s motion for a preliminary injunction are likewise inapposite. See Cordis Corp. v. Boston Sci. Corp., 2003 WL 22843072, at
*2 (D. Del. Nov. 21, 2003) (plaintiff had licensed major competitors who had competing (though non-infringing) products on the market), aff’d, 99 F. App’x 928
(Fed. Cir. 2004) (nonprecedential); Cordance Corp. v. Amazon.com, Inc., 2010
WL 3155505, at *4 (D. Del. July 23, 2010) (plaintiff had licensed its patent to a nonprofit seeking to make an open-access platform available to anyone).
As Genentech has never agreed to license any competitor to enter the market before , the settlement agreements here in no way suggest that
Genentech would not be irreparably harmed by Amgen’s entry now. Indeed, as is discussed below, the harm from Amgen’s unlicensed entry will continue past the licensed entry of other competitors.
2. The district court erred in treating Genentech’s settlement agreements as establishing that money damages are an adequate remedy for Amgen’s infringement
Genentech’s willingness to allow other defendants to enter the market after
, does not suggest that money damages from Amgen adequately compensate for Amgen’s infringement in July 2019. License agreements may be considered as a factor in the irreparable-harm analysis where
- 38 - Case: 19-2156 Document: 19 Page: 50 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED they show that the patentee is less concerned with maintaining its exclusivity and is willing to trade it for cash. See Acumed, 551 F.3d at 1328 (“While the fact that a patentee has previously chosen to license the patent may indicate that a reasonable royalty does compensate for an infringement, that is but one factor for the district court to consider.”). But the logic behind that rule is inapplicable here for several reasons.
First, none of the licenses at issue here provide a for the right to practice Genentech’s dosing patents, much less a
from Amgen’s infringement. Genentech bargained for . The fact that Genentech did not put a on a license is further evidence that Amgen’s unlicensed entry will cause irreparable harm and cannot be adequately remedied by money. See, e.g., High Tech, 49 F.3d at 1557 (“[T]he evidence shows that HTMI offered a license to New Image, so it is clear that HTMI is willing to forgo its patent rights for compensation” (emphasis added)); Illinois Tool Works, Inc. v. Grip-Pak, Inc., 906 F.2d 679, 683 (Fed. Cir.
1990) (“ITW also argues that its grant of a license to Owens-Illinois, Inc. at a 4% royalty should not evidence an absence of irreparable harm, but does not tell us, nor can we discern, how any ‘lost sales’ attributable to Grip-Pak would be any less compensable in dollars than are the ‘lost sales’ attributable to Owens–Illinois, Inc.”
(emphasis added)).
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Second, Genentech entered into those agreements to settle patent disputes.
That Genentech was willing to trade some future exclusivity for certainty does not speak to whether money damages could adequately compensate for Amgen’s infringement now. See Trading Techs. Int’l, Inc. v. eSpeed, Inc., 2008 WL
4531371, at *4 (N.D. Ill. May 22, 2008) (finding, in the context of a permanent injunction, that the inadequate-remedy-at-law factor favored plaintiff where its past licenses “were negotiated in exchange for the parties’ agreement to settle, rather than litigate”), aff’d, 595 F.3d 1340 (Fed. Cir. 2010).
Third, compared to the companies with which Genentech has settled,
Amgen is an especially formidable competitive threat. See Apple III, 735 F.3d at
1370 (“the district court erred by failing to consider” the differences between
Samsung and the other entities Apple had licensed). Here, Amgen is in a unique position to negotiate in the oncology market due to its multiple long-selling oncology products—including Neupogen and Neulasta—which have given it access to oncology clinics and experience with payers related to oncology products. Appx1477-1478(¶¶53-54); Appx1396-1397(¶59). Amgen has an established track record as a successful biologic manufacturer, and a reputation that will establish its credibility with oncology group purchasing organizations as a reliable, high-quality supplier of a biosimilar, which is not straightforward to manufacture. Appx1477-1478(¶54); Appx1396-1397(¶59). Furthermore, Kanjinti
- 40 - Case: 19-2156 Document: 19 Page: 52 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED is the only trastuzumab biosimilar to have transition data (i.e., data about switching from Herceptin to Kanjinti) as part of its clinical trial, a fact Amgen plans to advertise. Appx1477-1478(¶54); Appx1690; Appx2492-2493; Appx2496;
Appx2574. For all these reasons, Amgen has a competitive advantage relative to other potential biosimilar entrants, enhancing the price-erosion and lost-market- share harms described below—which cannot be remedied by money damages. Id.
Genentech therefore has a particular interest in keeping Amgen off the market, as the harms from Amgen’s infringement will be especially acute.
3. The district court erred in concluding, without evidentiary support, that the harm to Genentech from Amgen’s launch would be quantifiable
The district court further relied on the fact of the licenses to find that “any potential damages for sales in the next four months should be quantifiable.”
Appx9. The court cited no evidence for that proposition; in fact, the evidence is to the contrary, as Amgen itself has acknowledged when it has been the innovator seeking injunctive relief.
There is no evidence supporting the court’s speculation that such harm
“should be” quantifiable. Appx9. As discussed above, the settlement agreements the district court referred to do not contain and so do not place a monetary value on Genentech’s patent rights. The district court cited no other evidence of quantifiability, and it did not explain how such
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damages would be calculated.5 Amgen asserted this point in the district court with nothing more than a single, unsupported sentence of attorney argument.
Appx3756. There was no basis to accept that conclusory argument in the face of undisputed evidence of irreparable harm presented by Genentech in the form of, inter alia, lost market share and price erosion. See infra pp. 44-51.
Genentech, on the other hand, introduced evidence showing that money damages for sales of Herceptin would be difficult to quantify in the months between now and trial. Genentech’s responses to Amgen’s entry will be multi- faceted and complex, and the specific effects of Amgen’s activity—particularly those of price erosion and lost market share—will be difficult to unravel from other market conditions. Appx1399-1402(¶¶65-72); see Sanofi-Synthelabo, 470 F.3d at
1382 (“complex pricing scheme” for prescription drugs means additional entrants have potential to irreversibly erode prices in unpredictable ways). Indeed, Amgen itself has acknowledged these harms when it was the innovator seeking to exclude other market entrants. See Appx1922-1923; Appx1932-1934; Appx1939-1940.
Moreover, to the extent the district court assumed that the only harm that matters is harm to Genentech prior to the entry of other biosimilars, it was wrong.
5 The district court’s citation to King Pharmaceuticals, Inc. v. Sandoz, Inc., 2010 WL 1957640 (D.N.J. May 27, 2010), does not substitute for a determination that the damages in this case would be quantifiable. There, the court faulted the movant’s expert’s testimony for being conclusory. Id. at *5. Here, the district court did not even consider Genentech’s evidence of irreparable harm.
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Genentech presented detailed evidence, supported by expert testimony, that the irreparable harm from Amgen’s entry would continue even after other biosimilars came onto the market, because it would then become more difficult to untangle the harms caused by Amgen’s actions from those other entrants. Appx1401-
1402(¶¶70-72); Appx1411-1413(¶¶98-100). Thus, far from ending a period of irreparable harm, the date marks the day when the harm due to
Amgen’s unlicensed activity becomes even harder to quantify and even harder to compensate with money damages. See AstraZeneca, 633 F.3d at 1062 (affirming preliminary injunction because “to reliably calculate the economic harm” the patentee would suffer after the future licensed entry of another competitor, “the court would need data reflecting a market including only” the patentee and the licensed entrant, but the infringing entry of the defendant would prevent that from occurring, rendering those damages “complete speculation”). The district court’s opinion ignored this evidence entirely.
The district court’s unsupported finding that Genentech’s harm was quantifiable was therefore clearly erroneous and based on its legal error regarding the relevance of the license agreements, and its denial of Genentech’s preliminary injunction on this ground was an abuse of discretion.
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4. Unless reversed, the district court’s decision will seriously impede future settlement of biosimilars litigation
The district court’s decision to hold these settlement agreements against
Genentech would make litigation settlements in the pharmaceutical area much more difficult. Unless reversed, the district court’s rule bars a patent owner from obtaining injunctive relief where it has licensed other parties, irrespective of when those parties are licensed to enter the market, who the parties are, and whether the licenses reflect a willingness of the patent holder to trade exclusivity for money.
Such a rule would mean that a patentee could never settle litigation by permitting licensed entry, even at a future date, without giving up the opportunity to seek to enjoin immediate entry by anyone else who chooses to launch at risk.
C. Genentech Will Suffer Irreparable Harm Due To Amgen’s Infringement
The district court did not even address Genentech’s copious evidence of irreparable harm. But once the district court’s errors are corrected, the only reasonable conclusion from the evidence presented is that Genentech will suffer irreparable harm from Amgen’s launch. Indeed, Amgen did not dispute that
Genentech will suffer price erosion and lost market share.
1. Genentech will suffer irreparable harm
By launching at risk, Amgen will cause incalculable harm to Genentech.
Amgen will be the only Herceptin biosimilar on the market; absent an injunction,
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Amgen’s infringement will cause Genentech to suffer price erosion, lost market share, harm to other products, and damage to Genentech’s reputation. See, e.g.,
Abbott Labs., 544 F.3d at 1361-62 (price erosion, loss or market position, loss of goodwill, and loss of research and development are examples of irreparable harm).
Indeed, Amgen is well aware of these types of injury, many of which it has contended will irreparably harm its own innovative products in other litigation.
See Appx1922 (“Price erosion alone is sufficient to establish irreparable harm.”);
Appx1933-1934 (conceding that an offer of discounts or rebates by a biosimilar maker in the oncology market “will irreparably harm” the reference-product sponsor by causing price erosion); Appx1932 (“Courts have repeatedly held that the steep loss of market share and revenue … caused by the introduction of a generic drug constitute irreparable harm justifying the entry of injunctive relief.”);
Appx1939-1940 (“[L]oss of market share …[is an] accepted form[] of irreparable harm.”).
a. Price erosion
As Amgen does not dispute, its launch of Kanjinti will cause price erosion by forcing Genentech to decrease the amount it can charge for Herceptin. E.g.,
Celsis, 664 F.3d at 930 (collecting cases wherein price erosion constitutes irreparable harm). Specifically, Amgen expects to set Kanjinti’s net price at a
discount to Herceptin through 2028. Appx1741; Appx1396(¶58).
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To maintain customers, Genentech will have to lower its effective net price for
Herceptin, add rebates, and adjust contracts, resulting in a significant loss of revenue. Appx1477(¶¶48-52); Appx1396(¶58).
That price erosion will be irreversible because Genentech will not be able to recoup loss with future, higher prices or reduced discounts following a final judgment of Amgen’s infringement. Appx1411-1413(¶¶99-101); see Sanofi-
Synthelabo, 470 F.3d at 1382 (irreparable harm due to “irreversible price erosion”); see also Polymer Techs., 103 F.3d at 976 (“Requiring purchasers to pay higher prices after years of paying lower prices to infringers is not a reliable business option.”). The harms from Amgen’s entry would continue even if Amgen were later removed from the market because Genentech would be unable to raise prices to pre-entry levels. Appx1411-1413(¶¶99-101).
As described above, the specific harm to Genentech as a result of price erosion is difficult to quantify, because Genentech will have numerous complex responses to Amgen’s entry, and the specific effects of Amgen’s activity will be difficult to unravel from other market conditions. Appx1399-1400(¶¶65-67); see
Sanofi-Synthelabo, 470 F.3d at 1382; Hoffmann-La Roche Inc. v. Cobalt Pharms.,
Inc., 2010 WL 4687839, at *12-13 (D.N.J. Nov. 10, 2010). This is so even after other trastuzumab biosimilars enter the market, as isolating the impact of Kanjinti
- 46 - Case: 19-2156 Document: 19 Page: 58 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED as opposed to other biosimilars on the price of Herceptin will be even more complex. Appx1401-1402(¶¶70-72); AstraZeneca, 633 F.3d at 1061-1062.
b. Lost market share
Amgen’s launch of Kanjinti undisputedly will reduce Genentech’s market share, another form of irreparable harm. See, e.g., Abbott Labs., 544 F.3d at 1361-
1362; Purdue Pharma L.P. v. Boehringer Ingelheim GmbH, 237 F.3d 1359, 1368
(Fed. Cir. 2001). Kanjinti is a direct competitor of Herceptin, and Amgen itself forecasts that it will capture of the trastuzumab market (all of which
Genentech currently holds). Appx1741.
It will be difficult for Genentech to recapture market share from Amgen. As
Amgen’s own documents confirm,
Appx1751-1752. Even if Amgen were later removed from the market,
Genentech is unlikely to recapture its pre-entry market share. Appx1411(¶98).
Kanjinti’s launch is expected to prime the marketplace for other biosimilar entrants as well as for any subsequent re-launch of Kanjinti. Appx1401(¶69). Once other trastuzumab biosimilars enter the market, Genentech will continue to be irreparably harmed because the percentage of the loss of market share attributable to Amgen will become even harder to quantify or to fully remedy. Appx1401-
1402(¶¶70-72).
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c. Effect on other products
The irreparable injuries that Genentech will suffer as a result of Amgen’s launch of its infringing product are not limited only to Herceptin but extend to other Genentech products as well.
First, Amgen’s biosimilar launch would likely have an incalculable but material negative effect on the market for Genentech’s Perjeta and Kadcyla breast- cancer therapies. Perjeta is approved for use with Herceptin and they are thought to have synergistic effects. Appx1467(¶11); Appx1469-1470(¶23). Kadcyla is used for certain patients who have already been treated with Herceptin and chemotherapy and has been newly approved as an alternative to Herceptin for some patients. Appx1467(¶11); Appx1470(¶25). Should Amgen launch Kanjinti, the price differential between Kanjinti and Perjeta or Kadcyla will be far higher than the differential between Kanjinti and Herceptin, which may undermine payers’ and providers’ view of the incremental benefits of these treatments over
Kanjinti. Furthermore, Amgen’s marketing efforts to encourage providers to prescribe Kanjinti will detract from Genentech’s efforts to focus providers on the benefits of Perjeta and Kadcyla, each of which is at an important inflection point in its lifecycle. Appx1406-1409(¶¶85-89, 93). Those negative impacts defy calculation.
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Second, Amgen’s launch would likely cause lost sales and price erosion for two other Genentech biologic drugs, Avastin and Rituxan, which are facing threats of biosimilar competition. For example, the availability of Kanjinti would increase pressure for Genentech to provide price concessions for Avastin and Rituxan.
Appx1410-1411(¶96); Appx1476(¶47); Appx1478(¶¶57, 63). These harms are difficult to quantify and therefore irreparable.
Third, Genentech is a research-based company, and Amgen’s launch will hinder Genentech’s ability to fund research and development for new therapies.
Appx1469(¶20); Appx1419-1420(¶¶117-119). The consequences of those lost research opportunities are impossible to know or quantify and are thus irreparable.
See Bio-Technology Gen. Corp. v. Genentech, Inc., 80 F.3d 1553, 1566 (Fed. Cir.
1996) (affirming irreparable harm based in part on reductions to research and development spending).
d. Reputational harm
Amgen’s pretrial launch will irreparably injure Genentech even after
Genentech earns a judgment at trial. Indeed, as Amgen itself has recognized, once a biosimilar competitor has launched, the patentee cannot enforce its patents by removing the biosimilar product from the market without suffering reputational harm for being “portrayed as taking a medicine off the market.” Appx1934-1935;
Appx1413-1414(¶¶102-104); see also Appx1923-1924. That reputational injury
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See Douglas Dynamics, LLC v. Buyers Prods. Co., 717 F.3d 1336, 1344-1345
(Fed. Cir. 2013) (harm to “perception in the marketplace by customers … and distributors” is irreparable).
2. Genentech’s irreparable harm is connected to Amgen’s infringement
Amgen’s own actions confirm the nexus between its infringing inducement of the three-weekly dosing regimen claims and the irreparable harm to Genentech.
Apple Inc. v. Samsung Elecs. Co., 809 F.3d 633, 640 (Fed. Cir. 2015) (“Apple IV”)
(explaining that there must be “‘some connection’ between the harm alleged and the infringing acts”). As described above, two of the dosing patents were upheld as valid in IPRs. After these decisions, Amgen attempted to
. Appx1694-1695(179:5-14,
179:24-180:4). This course of conduct, driven by Amgen’s understanding of market demand, is overwhelming proof of nexus. See Apple IV, 809 F.3d at 643
(market demand for infringing features and infringer’s belief that infringing features were driver of sales “establishes a causal nexus”).
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The patented dosing regimens account for most of the indications on the
Herceptin label and are used in connection with a substantial majority of Herceptin prescriptions. Appx1496-1497(¶32); Appx2325; Appx2335. Amgen’s own calculations confirm that this dosing regimen accounts for up to of Herceptin prescriptions. Appx1827; Appx2499; Appx1696-1697(198:21-199:1); Appx1699-
1702(205:16-208:24). Thus, there is unquestionably a nexus between Amgen’s infringement and Genentech’s irreparable harm.
II. THE REMAINING EQUITABLE FACTORS OVERWHELMINGLY FAVOR INJUNCTIVE RELIEF
The district court did not reach the remaining factors bearing on Genentech’s preliminary injunction request, except a cursory discussion of the public interest in a footnote. The only reasonable conclusion is that all factors overwhelmingly support granting injunctive relief.
A. Genentech Is Likely To Succeed On The Merits
The only evidence presented to the district court shows that Genentech is likely to succeed on the merits of showing that Amgen infringes valid claims of the dosing patents.
Amgen did not dispute in opposition to Genentech’s motion and expert declaration of Dr. Susan Tannenbaum outlining on an element-by- element basis Amgen’s infringement of the dosing patent claims. Appx3759 (“
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.”); see also Appx1500-1507(¶¶41-63).
As to validity, at the preliminary injunction phase, “the very existence of the patent satisfies [the patentee’s] burden on validity.” Purdue Pharma, 237 F.3d at
1365. Amgen was therefore required to show “evidence of invalidity [that] is sufficiently persuasive that it is likely to overcome the presumption of patent validity.” PPG Indus., Inc. v. Guardian Indus. Corp., 75 F.3d 1558, 1566 (Fed.
Cir. 1996). Amgen barely even tried to carry this burden; it presented no expert evidence and merely recycled art and arguments already rejected by the PTAB under a lower standard of proof.
After a full trial on the merits, a three-judge panel of the PTAB expressly rejected the primary invalidity arguments Amgen made in the district court.6 For example: Amgen argued that “long established modeling tools were available to estimate Herceptin dosing at higher levels and longer intervals” (Appx3760,
Appx3762), but the PTAB found that the relative novelty of using antibodies to treat cancer and the “inherent uncertainty associated with using mathematical models to predict the pharmacokinetic behavior of antibodies” refuted that
6 With one exception, Amgen relied on the same prior art that was already considered and rejected by the PTAB. The one additional reference raised by Amgen, like the prior art considered in the IPRs, only discloses weekly dosing and is therefore, at best, cumulative. Appx3760-3761; Appx4510-4535.
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argument. Appx3994, Appx4004. Amgen argued that a skilled artisan would have relied upon “trivial mathematics” to predict that a once-every-three-week dosing regimen would work (Appx3762), but the PTAB found that the prior art did not support the conclusion that a simple, linear model could be used. Appx3996. And
Amgen argued that “there was no correlation between shed HER2 antigen levels and response to Herceptin treatment” (Appx3763), but the PTAB found that the prior art “highlight[ed] the uncertainty caused by the presence of shed antigens on the pharmacokinetics of trastuzumab.” Appx3997. Amgen cannot demonstrate a likelihood of success when its arguments have been conclusively rejected by the
PTAB in multiple IPR proceedings. See Oxford Immunotec Ltd. v. Qiagen, Inc.,
271 F. Supp. 3d 358, 366-367 (D. Mass. 2017).
Furthermore, Amgen had ample opportunity to develop and present expert testimony but simply chose not to. There are five expert declarations—from three oncologists and two pharmacokineticists—relied on by the PTAB that have been publicly available since 2017, and Amgen’s invalidity expert reports were due less than two weeks after it opposed Genentech’s preliminary injunction motion. In a case like this involving a complex technology, expert testimony is critical to proof of obviousness. See, e.g., Allergan, Inc. v. Barr Labs., Inc., 501 F. App’x 965, 972
(Fed. Cir. 2013) (nonprecedential). Amgen instead chose to rely on conclusory assertions. As a result, the only expert testimony before the district court was that
- 53 - Case: 19-2156 Document: 19 Page: 65 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED of Genentech’s expert, Dr. George Grass, who explained that the experience and data available to skilled artisans failed to establish a reasonable expectation of success regarding the patented dosing regimens. See Appx4759-4768(¶¶53-77).
Amgen cannot raise a substantial question of invalidity based upon attorney argument alone.
In sum, where Amgen has not carried its burden on invalidity and
, this factor weighs strongly in favor of Genentech.
B. The Balance Of Hardships Favors Genentech
The balance of hardships factor “assesses the relative effect of granting or denying an injunction on the parties.” Apple IV, 809 F.3d at 645; accord Kos
Pharms., Inc. v. Andrx Corp., 369 F.3d 700, 727 (3d Cir. 2004). This factor likewise favors Genentech.
If Genentech’s request for an injunction is denied, it will be “requir[ed] to compete against its own patented invention, with the resultant [irreparable] harms.”
Robert Bosch LLC v. Pylon Mfg. Corp., 659 F.3d 1142, 1156 (Fed. Cir. 2011). If
Amgen is allowed to continue its launch, Genentech will suffer price erosion and loss of market share, with no possibility that it can be made whole after trial.
By comparison, Amgen itself has acknowledged that it will not suffer harm by delaying a launch. Amgen’s own projections state it
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(Appx1977), and no other trastuzumab biosimilar will launch before
(Appx1476(¶45)).
To the extent Amgen asserts that having to stop its launch will harm its reputation, “an alleged infringer’s loss of market share and customer relationships, without more, does not rise to the level necessary to overcome the loss of exclusivity experienced by a patent owner due to infringing conduct.” Pfizer, 429
F.3d at 1382. Genentech informed Amgen that it intended to continue to seek preliminary injunctive relief—including an injunction pending resolution of this appeal—immediately after the district court denied Genentech’s motions for a temporary restraining order and preliminary injunction. Appx4945(32:5-10);
Appx4950(37:6-15). Amgen nevertheless issued a press release several hours later announcing that Kanjinti is now available in the United States. ECF No. 9, Ex. 1.
Any harm attributable to Amgen’s decision to proceed with launch in the face of
Genentech’s injunction request is self-inflicted and does not favor Amgen under the balance of hardships. Celsis, 664 F.3d at 931 (district court did not err in finding balance of harms in patent holder’s favor where “the preliminary record suggests that [infringer’s] losses were the result of its own calculated risk in selling a product with knowledge of Celsis’ patent”); Sanofi-Synthelabo, 470 F.3d at 1383
(balance of hardships favored the patent holder where the harms asserted by the infringing party “were the result of its own calculated risk to launch its product
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pre-judgment”). Indeed, Amgen itself has argued that where, as here, the infringer acts at risk, any hardship attributable to the infringer of ceasing its conduct cannot support the infringer under the balance of hardships. Appx4875-4878 (16-19 & n.10).
C. The Public Interest Is Served By Enforcing Genentech’s Patent Rights To Encourage Innovation
The last factor for the Court to consider is the public interest. In the preliminary-injunction context, this factor focuses “on whether a critical public interest would be injured by the grant of injunctive relief.” Metalcraft of Mayville,
Inc. v. Toro Co., 848 F.3d 1358, 1369 (Fed. Cir. 2017). Here, the public interest is best served by “the enforcement of [Genentech’s] patent rights.” Celsis, 664 F.3d at 931-932; Appx1416-1421(¶¶111-124). As this Court has explained,
“investment in drug research and development must be encouraged and protected by the exclusionary rights conveyed in valid patents.” Celsis, 664 F.3d at 931; see also Sanofi-Synthelabo, 470 F.3d at 1383-1384.
Amgen did not dispute that the public interest favors the enforcement of patent rights to encourage innovation—a position that, once again, Amgen itself has taken in other cases. See, e.g., Appx1924-1925 ((“The Public Has a Strong
Interest in a Robust Patent System that Maintains the Incentives for Pharmaceutical
Innovation”); Appx1935 ((“There is a strong public interest in encouraging investment in the research and development to create novel biological therapeutics
- 56 - Case: 19-2156 Document: 19 Page: 68 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED that treat human disease. The fact that a copyist may sell at a lower price does not override this important public interest.”). Nor does Amgen contend that an injunction will adversely affect patient care. Rather, Amgen argued, and the district court agreed in a footnote, that an injunction would deprive the public of access to non-infringing uses of Kanjinti. Appx3765; Appx9-10 n.7 (quoting
Apple III, 735 F.3d at 1372-1373). But by Amgen’s own estimation, of patient use is covered by Genentech’s patents. Appx1827. And if Amgen were truly concerned with public access to non-infringing uses of Kanjinti, it could have tried to eliminate the infringing indications from its label—
See supra pp. 10-11. Moreover, neither Amgen nor the district court has pointed to any patient need for the non-infringing uses that cannot already be supplied by Herceptin, which Genentech is fully able to supply.
CONCLUSION
Genentech respectfully requests that the Court reverse the district court’s denial of a preliminary injunction, or alternatively vacate and remand for further consideration under the correct legal standards. Should the Court grant
Genentech’s pending motion under Fed. R. App. P. 8 and subsequently remand for the district court for further consideration, Genentech respectfully requests that this
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Court order that the injunction pending appeal continue through the district court’s determination on remand.
Respectfully submitted,
/s/ William F. Lee ROBERT J. GUNTHER JR. WILLIAM F. LEE WILMER CUTLER PICKERING ANDREW J. DANFORD HALE AND DORR LLP WILMER CUTLER PICKERING 7 World Trade Center HALE AND DORR LLP 250 Greenwich Street 60 State Street New York, NY 10007 Boston, MA 02109 (212) 230-8800 (617) 526-6000
DARALYN J. DURIE Attorneys for Plaintiff-Appellant DURIE TANGRI LLP Genentech, Inc. 217 Leidesdorff Street San Francisco, CA 94111 (415) 362-6666
July 26, 2019
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ADDENDUM
Case: 19-2156 Document: 19 Page: 71 Filed: 07/26/2019
TABLE OF CONTENTS
Page(s)
Memorandum Opinion on Genentech's Motion for Temorary Restraining Order and Motion for Preliminary Injunction, Dkt. 299 (July 18, 2019) [REDACTED] ...... Appx1-10
Order Denying Genentech's Motion for Temorary Restraining Order and Motion for Preliminary Injunction, Dkt. 300 (July 18, 2019) ...... Appx11
U.S. Patent No. 6,627,196 ...... Appx12-49
U.S. Patent No. 7,371,379 ...... Appx50-88
U.S. Patent No. 10,160,811 ...... Appx89-139
- i - Case: 19-2156 Document: 19 Page: 72 Filed: 07/26/2019
IN THE UNITED STATES DISTRICT COURT
FOR THE DISTRICT OF DELAWARE
GENENTECH, INC. and CITY OF HOPE,
Plaintiffs,
V. Civ. No. 18-924-CFC
AMGEN INC.,
Defendant.
Michael P. Kelly, Daniel M. Silver, MCCARTER &ENGLISH, LLP, Wilmington, Delaware; William F. Lee, Lisa J. Pirozzolo, Emily R. Whelan, Kevin S. Prussia, Andrew J. Danford, WILMER CUTLER PICKERING HALE AND DORR LLP, Boston, Massachusetts; Robert J. Gunther Jr., WILMER CUTLER PICKERING HALE AND DORR LLP, New York, New York; Daralyn J. Durie, Adam R. Brausa, DURIE TANGRI LLP, San Francisco, California. Counsel for PlaintifJGenentech, Inc.
Neal C. Belgam, Eve H. Ormerod, SMITH KATZENSTEIN & JENKINS LLP, Wilmington, Delaware; Michelle Rhyu, Susan Krumplitsch, Daniel Knauss, COOLEY LLP, Palo Alto, California; Orion Armon, COOLEY LLP, Broomfield, Colorado; Eamonn Gardner, COOLEY LLP, San Diego, California. Counsel for Defendant Amgen Inc.
MEMORANDUM OPINION
July 18, 2019
Appx1 Case: 19-2156 Document: 19 Page: 73 Filed: 07/26/2019
CONNOLLY, UNITEDSATES DISTRICT JUDGE
This action arises under the Biologics Price Competition and Innovation Act of 2009 (BPCIA), Pub. L. No. 111-148, §§ 7001-7003, 124 Stat. 119, 804-21
(2010) (codified as amended at 42 U.S.C. § 262, 35 U.S.C. § 271(e), 28 U.S.C. §
220l(b), 21 U.S.C. § 355 et seq.). Plaintiffs Genentech, Inc. and City of Hope have sued Defendant Amgen Inc. based on Amgen' s submission of a Biologics
License Application (BLA) for approval to market Kanjinti, a biosimilar of
Genentech' s drug product Herceptin.
On May 15, 2018, Amgen served Genentech a Notice of Commercial
Marketing pursuant to § 262(/)(8)(A) of the BPCIA. Kanjinti was approved by the
FDA on June 13, 2019. Four weeks later, on July 10, 2019, Genentech moved for a temporary restraining order and preliminary injunction to prevent Amgen from commercially launching, marketing, or selling Kanjinti until the Court renders a decision on the merits of Genentech' s patent infringement claims following trial, and until the Court of Appeals for the Federal Circuit has adjudicated any appeal of that decision. D.I. 273; D.I. 274. That same day, I arranged an emergency teleconference with the parties and orally ordered a standstill until I received
Amgen's response to Genentech's motions and had an opportunity to consider
Appx2 Case: 19-2156 Document: 19 Page: 74 Filed: 07/26/2019
fully the issues and rule on the merits. For the foregoing reasons, I will deny
Genentech' s motions for a temporary restraining order and preliminary injunction.
I. BACKGROUND
The non-proprietary names for Herceptin and Kanjinti are respectively trastuzumab and trastuzumab-anns. 1 For purposes of a trial scheduled for
December 2019, the parties are litigating ten patents which cover: (i) the trastuzumab antibody itself (the Composition Patent)2; (ii) techniques for identifying patients who might benefit from trastuzumab therapy (the HER2
3 Diagnostic Patents) ; (2) various aspects of cell culture, purification, and antibody manufacturing purification (the Manufacturing Patents)4; and (3) methods of administration (the Dosing Patents). D.I. 44; D.I. 60 at 2-3; D.I. 75. Genentech's motions seek relief based on claims in the three Dosing Patents: U.S. Patent Nos.
6,627,196 (the "#196 patent"), 7,371,379 (the "#379 patent") and 10,160,811 (the
"#811 patent"). All three patents relate to methods of treating cancer with a
1 The FDA employs a "naming convention" pursuant to which it gives a "core name" to the reference product (in this case, trastuzumab) and adds for each biosimilar a "distinguishing suffix that is devoid of meaning and composed of four lowercase letters ... attached with a hyphen to the core name" (in this case, "-anns"). 2 U.S. Patent No. 6,407,213 claims the trastuzumab antibody. 3 The HER2 Diagnostic Patents at issue are U.S. Patent Nos. 7,993,834 and 8,076,066. 4 The Manufacturing Patents at issue are U.S. Patent Nos. 6,620,918; 8,512,983; 8,574,869; and 9,714,293. 2
Appx3 Case: 19-2156 Document: 19 Page: 75 Filed: 07/26/2019
specific dosage regimen: intravenous ("IV") administration of an initial 8 mg/kg dose followed by one or more 6 mg/kg doses separated by three weeks. D.I. 279-1,
Ex. 1, CL 11; Ex. 2, CL 11; Ex. 3, CL 6. The #379 patent further recites coadministration with a chemotherapy agent. Id., Ex. 2, CL 6. The #811 patent further recites treatment of breast cancer. Id., Ex. 3, CL 11.
IT. LEGALSTANDARDS
A preliminary injunction is "a drastic and extraordinary remedy that is not to be routinely granted." Intel Corp. v. ULSI Sys. Tech., Inc., 995 F.2d 1566, 1568
(Fed. Cir. 1993). To obtain such extraordinary relief, the moving party must prove that: ( 1) it has a reasonable likelihood of success on the merits; (2) it would suffer irreparable harm in the absence of an injunction; (3) the balance of hardships tips in its favor; and (4) an injunction would have a favorable impact on the public interest. Amazon.com, Inc. v. Barnesandnoble.com, Inc., 239 F.3d 1343, 1350
(Fed. Cir. 2001). "These factors, taken individually, are not dispositive; rather, the district court must weigh and measure each factor against the other factors and against the form and magnitude of the relief requested." Hybritech Inc. v. Abbott
Lab., 849 F.2d 1446, 1451 (Fed. Cir. 1988). The grant or denial of a preliminary injunction is within the sound discretion of the district court. Polymer Tech., Inc. v. Bridwell, 103 F.3d 970, 973 (Fed. Cir. 1996).
3
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The standards for a preliminary injunction also apply to a motion for a
temporary restraining order when, as here, the opposing party has notice of the
motion. See Takeda Pharm. USA, Inc. v. W.-Ward Pharm. Corp., 2014 WL
5088690, at* 1 (D. Del. Oct. 9, 2014). Accordingly, Genentech's motion for a
temporary restraining order rises and falls with its motion for a preliminary
injunction.
III. DISCUSSION
"Central to the movant' s burden are the likelihood of success and irreparable
harm factors." Sofamor Danek Grp., Inc. v. DePuy-Motech, Inc., 74 F.3d 1216,
1219 (Fed. Cir. 1996). "A court may decline to issue a preliminary injunction if
the movant does not prove either of these factors." Jeneric/Pentron, Inc. v. Dillon
Co., 205 F.3d 1377, 1380 (Fed. Cir. 2000). Here, I am denying the motion for
preliminary injunction, because Genentech has failed to establish irreparable harm.
A patentee's undue delay in seeking a preliminary injunction "negates the
idea of irreparability." Pfizer, Inc. v. Teva Pharm., USA, Inc., 429 F.3d 1364, 1382
(Fed. Cir. 2005); Polymer Tech., 103 F.3d at 974 (same). Genentech has known of
Amgen's intent to market Kanjinti since Amgen served its 180-day Notice of
Commercial Marketing on May 15, 2018. In addition, Genentech received
information through discovery that made clear Amgen' s plan to launch its
4
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marketing ofKanjinti in July 2019. Specifically, in February 2019, Amgen produced to Genentech documents showing that it filed a "resubmission" to the
FDA in December 2018.5 Given the known six-month regulatory timeline for the
FDA to consider the resubmission (see D.I. 289-1, Ex. 11 at 4), Genentech would have understood at the time that the FDA would act on the resubmission by the end of June 2019. In April 2019, Amgen produced documents with its launch plan redactions removed, thus enabling Genentech to see that Amgen planned to launch in July 2019. Id. at Ex. 12. From late April through mid-June, five Amgen witnesses testified during depositions that Amgen was preparing to be ready to launch Kanjinti in July 2019. D.I. 289-1, Ex. 14 at 66:12-67:3, 83 :9-12; Ex. 15 at
40:20-23; Ex. 16 at 79:6-10, 81 :3-6; Ex. 17 at 18:5-1 0; and Ex. 18 at 32: 11-33: 18.
The FDA approved Kanjinti on June 13, 2019. But Genentech did not file its motion for a preliminary injunction until July 10, 2019-fourteen months after receiving the Notice of Commercial Marketing, three months after receiving a fairly specific launch date, and almost one month after Amgen had FDA approval to launch Kanjinti.
5 A resubmission is "a submission by the biologics license applicant or supplement applicant of all materials needed to fully address all deficiencies identified in the complete response letter." 21 C.F.R. § 600.3. 5
Appx6 Case: 19-2156 Document: 19 Page: 78 Filed: 07/26/2019
Genentech' s actions are also contrary to the spirit and purpose of the
BPCIA. As the Federal Circuit explained, the 180-day period triggered by the notice of commercial marketing "gives the parties and the district court the time for adjudicating such matters without the reliability-reducing rush that would attend requests for relief against immediate market entry that could cause irreparable injury." Amgen Inc. v. Apotex Inc., 827 F.3d 1052, 1063 (Fed. Cir. 2016), cert. denied, 137 S. Ct. 591 (2016). Thus, the 180-day period is designed to prevent exactly the circumstances that Genentech has engineered in this case-a "race to court for immediate relief to avoid irreparable harm from market entry, and ... the hurried motion practice that (8)(A) is designed to replace."6 Id. at 1065.
Genentech's undue delay in requesting a preliminary injunction, particularly in light of relevant provisions under the BPCIA, should be sufficient by itself to deny the motion. Nevertheless, a finding of no irreparable harm is also supported by the fact that Genentech has engaged in a pattern and practice of licensing the
Dosage Patents.
6 Notably, Genentech demonstrated its ability to avoid hurried motion practice in a related case involving the same patents and the same reference product but a different defendant. With Amgen, however, Genentech represented to the Court as recently as May 16, 2019 that it was not seeking a preliminary injunction. See D.I. 289-1, Ex. 19 at 26:1-4. 6
Appx7 Case: 19-2156 Document: 19 Page: 79 Filed: 07/26/2019 CONFIDENTIAL MATERIAL FILED UNDER SEAL REDACTED
An injunction is a form of equitable relief and, therefore, available only when there is no adequate remedy at law. See N. Cal. Power Agency v. Grace
Geothermal Corp., 105 S.Ct. 459,459 (1984) ("A party seeking an injunction from a federal court must invariably show that it does not have an adequate remedy at law."); Coca-Cola Bottling Co. ofShreveport, Inc. v. Coca-Cola Co., 769 F. Supp.
671, 713 (D. Del. 1991) ("The Court may only invoke its equity powers when there is no adequate remedy at law."). Thus, to establish irreparable harm, the movant must "clearly establish[] that monetary damages could not suffice." Abbott Labs. v.
Andrx Pharm., Inc., 452 F.3d 1331, 1348 (Fed. Cir. 2006). The fact that "movants have engaged in a pattern of granting licenses under the patent" makes it
"reasonable to expect that invasion of the patent right can be recompensed with a royalty rather than with an injunction." Polymer Tech., 103 F.3d at 974; see also
High Tech Med. Instrumentation, Inc. v. New Image Indus., Inc., 49 F.3d 1551,
1557 (Fed. Cir. 1995) (offering to license the patent "suggests that any injury suffered by [the patentee] would be compensable in damages"). Here, Genentech granted for the Dosing Patents to Mylan, Celltrion, and Pfizer that allow a biosimilar to enter the market in
. D.I. 291-1, Exs. 37-39. In other words, Genentech has been able to place a value on the patents and has approved competitors entering the
7
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in the next four months should be quantifiable. See King Pharm., Inc. v. Sandoz,
Inc., 2010 WL 1957640, at *6 (D.N.J. May 17, 2010) (denying a preliminary
injunction where any changes to the market from the non-movant's entry should be
easy to calculate given the short period time).
The "absence of irreparable harm ... ma[kes] unnecessary a consideration of
... [the] likelihood of success in proving infringement." Ill. Tool Works, Inc. v.
Grip-Pak, Inc., 906 F.2d 679, 682 n.3 (Fed. Cir. 1990); Polymer Tech., 103 F.3d at
97 4 ("[T]he district court did not err by focusing its analysis solely on irreparable
harm in denying [the movant's] motion."). Due to the hurried nature of this
particular motion practice, I will not take additional time to set forth my analysis
with respect to other preliminary injunction factors.7 Genentech has failed to
7 I will briefly make note of considerations under the fourth factor that also weigh in favor of denying the motion for a preliminary injunction. "[A]lthough there exists a public interest in protecting rights secured by valid patents, the focus of the district court's public interest analysis should be whether there exists some critical public interest that would be injured by the grant of preliminary relief." Hybritech Inc., 849 F.2d at 1458. For pharmaceutical drugs that prolong and save lives, there is a critical public interest in affordable access to those drugs. Genentech itself acknowledges this public interest by stating that it is "committed to ensuring patient access by providing Herceptin free of charge to patients who are uninsured or cannot afford treatment." D.I. 275 at 18. In that context, I note that Genentech' s exclusivity based on the Composition Patent expired on June 18, 2019, and only two of the four indications on the Kanjinti label allegedly infringe the Dosing Patents, meaning there are two recited methods of using Kanjinti that 8
Appx9 Case: 19-2156 Document: 19 Page: 81 Filed: 07/26/2019
establish irreparable harm and therefore its motions for preliminary and temporary injunctive relief must be denied.
IV. CONCLUSION
For the foregoing reasons, I will deny Genentech' s motions for a temporary restraining order and preliminary injunction (D.I. 273; D.I. 274). The standstill ordered on July 10, 2019 is lifted.
The Court will issue an Order consistent with this Memorandum Opinion.
are free of any allegations of infringement. "[T]he prospect that an injunction would have the effect of depriving the public of access to a large number of non infringing features," weighs against granting an injunction. Apple Inc. v. Samsung Elecs. Co. Ltd., 735 F.3d 1352, 1372-73 (Fed. Cir. 2013). 9
Appx10 Case 1:18-cv-00924-CFCCase: 19-2156 Document Document: 300 19 Filed Page: 07/18/19 82 Filed:Page 07/26/20191 of 1 PageID #: 23481
Appx11 Case: 19-2156 Document: 19 Page: 83 Filed: 07/26/2019
111111 1111111111111111111111111111111111111111111111111111111111111 US006627196B 1
(12) United States Patent (10) Patent No.: US 6,627,196 Bl Baughman et al. (45) Date of Patent: Sep.30,2003
(54) DOSAGES FOR TREATMENT WITH ANTI 5,770,195 A 6/1998 Hudziak et a!. ERBB2 ANTIBODIES 5,772,997 A 6/1998 Hudziak et a!. 5,776,427 A 7/1998 Thorpe eta!. (75) Inventors: Sharon A. Baughman, Ventura, CA 5,783,186 A 7/1998 Arakawa et a!. (US); Steven Shak, Burlingame, CA 5,801,005 A 9/1998 Cheever et a!. 5,821,337 A 10/1998 Carteret a!. (US) 5,824,311 A 10/1998 Greene eta!. 5,834,229 A 11/1998 Vandlen et a!. (73) Assignee: Genentech, Inc., South San Francisco, 5,837,243 A 11/1998 Deo eta!. CA(US) 5,837,523 A 11/1998 Greene eta!. 5,840,525 A 11/1998 Vandlen et a!. ( *) Notice: Subject to any disclaimer, the term of this 5,846,538 A 12/1998 Cheever et a!. patent is extended or adjusted under 35 5,856,110 A 1!1999 Vandlen et a!. U.S.C. 154(b) by 0 days. 5,859,206 A 1!1999 Vandlen et a!. 5,869,445 A 2/1999 Cheever et a!. 5,876,712 A 3/1999 Cheever et a!. (21) Appl. No.: 09/648,067 5,877,305 A 3/1999 Huston eta!. (22) Filed: Aug. 25, 2000 5,908,835 A 6/1999 Bissery 5,910,486 A 6/1999 Curiel eta!. Related U.S. Application Data 5,922,845 A 7/1999 Deo eta!. 5,939,531 A 8/1999 Wels eta!. ( 60) Provisional application No. 60/213,822, filed on Jun. 23, 5,968,511 A 10/1999 Akita eta!. 2000, and provisional application No. 60/151,018, filed on Aug. 27, 1999. 5,977,322 A 11/1999 Marks eta!. 5,985,553 A 11/1999 King eta!. 6,015,567 A 1!2000 Hudziak et a!. (51) Int. Cl? ...... A61K 39/395 6,028,059 A 2/2000 Curiel eta!. (52) U.S. Cl...... 424/138.1; 424/131.1; 6,054,297 A 4/2000 Carteret a!. 424/133.1; 424/134.1; 424/135.1; 424/136.1; 6,054,561 A 4/2000 Ring 424/137.1; 424/139.1; 424/141.1; 424/142.1; 6,096,873 A 8/2000 Schaefer et a!. 424/143.1; 424/144.1; 424/145.1; 424/146.1; 6,123,939 A 9/2000 Shawver et a!. 424/147.1; 424/150.1; 424/151.1; 424/152.1; 6,165,464 A 12/2000 Hudziak et a!. 424/153.1; 424/154.1; 424/155.1; 424/156.1; 6,333,348 B1 * 12/2001 Vogel eta!...... 514/449 424/158.1; 424/172.1; 424/174.1 FOREIGN PATENT DOCUMENTS (58) Field of Search ...... 424/130.1, 138.1, 424/141.1, 142.1, 152.1, 155.1, 131.1, 133.1, EP 0003089 A1 7/1979 EP 0599274 A1 6/1994 134.1, 135.1, 136.1, 137.1, 139.1, 143.1, EP 616812 A1 9/1994 144.1, 145.1, 146.1, 147.1, 150.1, 151.1, EP 0711565 B1 8/1998 153.1, 154.1, 156.1, 158.1, 172.1, 174.1 JP 3-240498 10/1991 JP 5-117165 5/1993 (56) References Cited JP 5-170667 7/1993 JP 5-213775 8/1993 U.S. PATENT DOCUMENTS JP 5-317084 12/1993 4,676,980 A 6/1987 Segal eta!. JP 95006982 B2 1!1995 4,753,894 A 6/1988 Frankel et a!. JP 7-59588 3/1995 4,816,567 A 3/1989 Cabilly et a!. JP 2761543 B2 6/1998 4,935,341 A 6/1990 Bargmann et a!. JP 2895105 B2 5/1999 4,943,533 A 7/1990 Mendelsohn et a!. (List continued on next page.) 4,968,603 A 11/1990 Slamon eta!. 4,975,278 A 12/1990 Senter eta!. OTHER PUBLICATIONS 5,169,774 A 12/1992 Frankel et a!. 5,183,884 A 2/1993 Kraus eta!. Wantanabe et al., ASCO, Vol. 17, abstract 702, May 15-18, 5,288,477 A 2/1994 Bacus 1998 Annual Meeting.* 5,359,046 A 10/1994 Capon eta!. 5,367,060 A 11/1994 Vandlen et a!. (List continued on next page.) 5,401,638 A 3/1995 Carney eta!. 5,464,751 A 11/1995 Greene eta!. Primary Examiner-Anthony C. Caputa 5,480,968 A 1!1996 Kraus eta!. Assistant Examiner-Anne L. Holleran 5,578,482 A 11/1996 Lippman et a!. (74) Attorney, Agent, or Firm-Wendy M. Lee 5,604,107 A 2/1997 Carney eta!. 5,641,869 A 6/1997 Vandlen et a!. (57) ABSTRACT 5,663,144 A 9/1997 Greene eta!. 5,677,171 A 10/1997 Hudziak et a!. The present invention concerns the treatment of disorders 5,705,157 A 1!1998 Greene characterized by the overexpression of ErbB2. More 5,720,937 A 2/1998 Hudziak et a!. specifically, the invention concerns the treatment of human 5,720,954 A 2/1998 Hudziak et a!. patients susceptible to or diagnosed with cancer overex 5,725,856 A 3/1998 Hudziak et a!. pressing ErbB2 with anti-ErbB2 antibody. 5,726,023 A 3/1998 Cheever et a!. 5,728,687 A 3/1998 Bissery 5,747,261 A 5/1998 King eta!. 33 Claims, 5 Drawing Sheets
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US 6,627,196 Bl Page 2
FOREIGN PATENT DOCUMENTS Pegram et al., "Phase II study of receptor-enhanced chemosensitivity using recombinant humanized wo wo 89/06692 7/1989 2 anti-p185HER /neu monoclonal antibody plus cisplatin in wo wo 90/14357 11/1990 wo wo 91/00360 1!1991 patients with HER2/neu-overexpressing metastatic breast wo wo 92/10573 6/1992 cancer refractory to chemotherapy treatment" Journal of wo wo 92/20373 11/1992 Clin Oneal 16(8):2659-2671 (Aug. 1998). wo wo 92/20798 11/1992 Slamon et al., "Use of chemotherapy plus a monoclonal wo wo 93/12220 6/1993 antibody against HER2 for metastatic breast cancer that wo wo 93/21232 10/1993 overexpresses HER2" New England J. of Medicine wo wo 93/21319 10/1993 344(11):783-792 (Mar. 15, 2001). wo wo 94/00136 1!1994 wo wo 94/04690 3/1994 Verma et al., "Efficacy and safety of three-weekly herceptin wo wo 94/22478 10/1994 with paclitaxel in women with her2-positive metastatic wo wo 94/28127 12/1994 breast cancer: preliminary results of a phase II trial" Euro wo wo 95/16051 6/1995 pean Journal of Cancer (abstract only) 37:S146 (2001). wo wo 95/17507 6/1995 Vogel et al., "First-Line Herceptin Monotherapy in Meta wo wo 95/28485 10/1995 static Breast Cancer" Oncology 61(Suppl. 2):37-42 (2001). wo wo 96/18409 6/1996 Washington et al., "A population pharmacokinetic (PK) wo wo 96/27011 9/1996 wo wo 97/04801 2/1997 model for trastuzumab (T) following weekly dosing" Clin wo wo 97/20858 6/1997 Pharmacal Ther (abstract only) 71:P12 (2002). wo wo 97/27848 8/1997 Arteaga et al., "p185c-erbB-2 Signaling Enhances Cisplatin wo wo 97/35885 10/1997 induced Cytotoxicity in Human Breast Carcinoma Cells: wo wo 97/38731 10/1997 Association Between an Oncogenic Receptor Tyrosine wo wo 98/02541 1!1998 Kinase and Drug-induced DNA Repair" Cancer Research wo wo 98/17797 4/1998 54(14):3758-3765 (Jul. 15, 1994). wo wo 98/45479 10/1998 wo wo 99/31140 6/1999 Bacus et al., "Differentiation of Cultured Human Breast wo wo 99/48527 9/1999 Cancer Cells (AU-565 and MCF-7) Associated With Loss wo wo 00/61185 10/2000 of Cell Surface HER-2/neu Antigen" Molecular Carcino genesis 3(6):350--362 (1990). 01HER PUBLICATIONS Bacus et al., "Tumor-inhibitory Monoclonal Antibodies to Carbonell Castellon et al., "Efficacy and safety of 3-weekly the HER-2/Neu Receptor Induce Differentiation of Human Herceptin (H) monotherapy in women with HER2-positive Breast Cancer Cells" Cancer Research 52(9):2580-2589 metastatic breast cancer (MBC): preliminary data from a (May 1, 1992). phase II study" Proc Am Soc Clin Oneal (Abstract #73 from Baselga et al., "Anti HER2 Humanized Monoclonal Anti the 2002 ASCO Meeting) 21:19a (2002). body (MAb) Alone and in Combination with Chemotherapy Carbonell et al., "Efficacy and safety of 3-weekly Herceptin Against Human Breast Carcinoma Xenografts" Proceedings monotherapy in women with HER2-positive metastatic of ASC0-13th Annual Meeting (Abstract #53), Dallas, TX breast cancer: preliminary data from a phase II study" (Oral 13:63 (Mar. 1994). presentation at the 38th Annual Meeting of the American Baselga et al., "HER2 overexpression and paclitaxel sensi Society of Clinical Oncology, May 18-21, 2002 in Orland, tivity in breast cancer: therapeutic implications" Oncology Florida). 11(3 Suppl 2):43-48 (Mar. 1997). Cobleigh et al., "Multinational study of the efficacy and Baselga et al., "Monoclonal antibodies directed against safety of humanized anti-HER2 monoclonal antibody in growth factor receptors enhance the efficacy of chemothera women who have HER2--{)verexpressing metastatic breast peutic agents" Annals of Oncology (abstract #010) S(Suppl. cancer that has progressed after chemotherapy for metastatic 5) (1994). disease" Journal of Clinical Oncology 17(9):2639-2648 Baselga et al., "Phase II Study of Weekly Intravenous (Sep. 1999). Recombinant Humanized Anti-p185HER2 Monoclonal Anti Gelman et al., "Pharmacokinetics and safety of Herceptin body in Patients With HER2/neu-Overexpressing Meta when administered every 3 weeks to women with metastatic static Breast Cancer" J. Clin. Oneal. 14(3):737-744 (Mar. breast cancer" (Oral presentation at the 37th Annual Meeting 1996). of the American Society of Clinical Oncology, May 12-15, Baselga et al., "Receptor Blockade With Monoclonal Anti 2001 in San Francisco, CA). bodies as Anti-Cancer Therapy" Pharmac. Ther. Harris et al., "A population pharmacokinetic (PK) model for 64:127-154 (1994). Herceptin (H) and implications for clinical dosing" (Oral Baselga et al., "Recombinant Humanized Anti-HER2 Anti presentation at the 38th Annual Meeting of the American body (Herceptin) Enchances the Antitumor Activity of Pacli Society of Clinical Oncology, May 18-21, 2002 in Orlando, taxel and Doxorubicin against HER2/neu Overexpressing Florida). Human Breast Cancer Xenografts" Cancer Research Harris et al., "A population pharmacokinetic (PK) model for 58:2825-2831 (Jul. 1998). trastuzumab (Herceptin) and implications for clinical dos Carter et al., "Humanization of an anti-p185HER2 antibody ing" Proc Am Soc Clin Oneal (Abstract #488) 21:123a for human cancer therapy" Proc. Natl. Acad. Sci. (2002). 89:4285-4289 (1992). Leyland-Janes et al., "Pharmacokinetics of Herceptin Chothia and Lesk, "Canonical structures for the hypervari administered with paclitaxel every three weeks" Breast able regions of immunoglobulins" J. Mol. Bioi. Cancer Res Treat (abstract only) 64:124 (2000). 196(4):901-917 (1987).
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D'souza et al., "Overexpression of ERBB2 in human mam Ilgen et al., "Characterization of anti-HER/2 antibodies mary epithelial cells signals inhibition of transcription of the which inhibit the growth of breast tumor cells in vitro" E-cadherin gene" Proc. Natl. Acad. Sci. USA Proceedings of the American Association for Cancer 91(15):7202-7206 (Jul. 19, 1994). Research (abstract #3209) 37:470 (Mar. 1996). De Sautes et al., "Radiolabeled Antibody Targeting of the Jones et al., "Replacing the Complementarity-determining HER-2/neu Oncoprotein" Cancer Research 52:1916-1923 Regions in a Human Antibody with Those From a Mouse" (1992). Nature 321:522-525 (May 29, 1986). DiFiore et al., "erbB-2 is a potent oncogene when overex Kaspryzk et al., "Therapy of an Animal Model of Human pressed in NIH/3T3 cells" Science 237( 4811):178-182 (Jul. Gastric Cancer Using a Combination of Anti-erbB-2 Mono 10, 1987). clonal Antibodies" Cancer Research 52(10):2771-2776 Drebin et al., "Down-Modulation of an Oncogene Protein (May 15, 1992). Product and Reversion of the Transformed Phenotype by Kotts et al., "Differential Growth Inhibition of Human Monoclonal Antibodies" Cell 41(3):695-706 (Jul. 1985). Carcinoma Cells Exposed to Monoclonal Antibodies Drebin et al., "Inhibition of tumor growth by a monoclonal Directed against the Extracellular Domain of the HER2/ antibody reactive with an oncogene-encoded tumor anti ERBB2 Protoocogene" In Vitro (Abstract 1n176) 26(3):59A gen" Proc. Natl. Acad. Sci. 83:9129-9133 (1986). (1990). Drebin et al., "Monoclonal antibodies reactive with distinct Kumar et al., "Regulation of Phosphorylation of the domains of the neu oncogene-encoded p185 molecule exert c--erbB-2/HER2 Gene Product by a Monoclonal Antibody synergistic anti-tumor effects in vivo" Oncogene 2:273-277 and Serum Growth Factor(s) in Human Mammary Carci (1988). noma Cells" Molecular & Cellular Biology 11(2):979-986 (Feb. 1991). Drebin et al., "Monoclonal Antibodies Specific for the neu Oncogene Product Directly Mediate Anti-tumor Effects In Lewis et al., "Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies" Cancer Vivo" Oncogene 2(4):387-394 (1988). Immunol. Immunother. 37:255-263 (1993). Fendly et al., "Characterization of Murine Monoclonal Anti Lewis et al., "Growth Regulation of Human Breast and bodies Reactive to Either the Human Epidermal Growth Ovarian Tumor Cells by Heregulin: Evidence for the Factor Receptor or HER2/neu Gene Product" Cancer Requirement of ErbB2 as a Critical Component in Mediat Research 50:1550-1558 (Mar. 1, 1990). ing Heregulin Responsiveness" Cancer Research Fleiss, JL Statistical Methods for Rates and Proportions, 56:1457-1465 (Mar. 15, 1996). 2nd edition, New York, NY:Wiley pp. 13-17 (1981). Maier et al., "Requirements for the Internalization of a Gemzar (gemcitabine HCL), "Product Information-PDR" Murine Monoclonal Antibody Directed againt the HER-2/ (2000). neu Gene Product c-erbB-2" Cancer Research Goldenberg, M., "Trastuzumab, a Recombinant DNA-De 51(19):5361-5369 (Oct. 1, 1991). rived Humanized Monoclonal Antibody, a Novel Agent for Masui et al., "Growth Inhibition of Human Tumor Cells in the Treatment of Metastatic Breast Cancer" Clinical Thera Athymic Mice by Anti-Epidermal Growth Factor Receptor peutics 21(2):309-318 (1999). Monoclonal Antibodies" Cancer Research 44(3): 1002-1007 Green et al., "Preclinical Evaluation of WR-151327: An (Mar. 1984). Orally Active Chemotherapy Protector" Cancer Research Masuko et al., "A murine Monoclonal Antibody That Rec 54(3):738-741 (Feb. 1, 1994). ognizes an Extracellular Domain of the Human c-erbB-2 Guy et al., "Expression of the neu protooncogene in the Protooncogene Product" JpnJ. Cancer Res. 80:10-14 (Jan. mammary epithelium of transgenic mice induces metastatic 1989). disease" Proc. Natl. Acad. Sci. USA 89(22):10578-10582 McCann et al., "c-erbB-2 Oncoprotein Expression in Pri (Nov. 15, 1992). mary Human Tumors" Cancer 65(1):88-92 (Jan. 1, 1990). Hancock et al., "A Monoclonal Antibody against the McKenzie et al., "Generation and characterization of mono c--erbB-2 Protein Enhances the Cytotoxicity of cis-Diam clonal antibodies specific for the human neu oncogene minedichloroplatinum against Human Breast and Ovarian product, p185" Oncogene 4:543-548 (1989). Tumor Cell Lines" Cancer Research 51:4575-4580 (Sep. 1, Mendelsohn et al., "Receptor blockade and chemotherapy: a 1981). new approach to combination cancer therapy" Annals of Harwerth et al., "Monoclonal Antibodies against the Extra Oncology (abstract #040) 7(Suppl. 1):22 (1996). cellular Domain of the erbB-2 Receptor Function as Partial Myers et al., "Biological Effects of MonoclonalAntireceptor Ligand Agonists" Journal of Biological Chemistry Antibodies Reactive with neu Oncogene Product, p185neu" 267(21):15160-15167 (Jul. 25, 1992). Methods in Enzymology 198:277-290 (1991). Hudziak et al., "Increased expression of the putative growth Nakamura et al., "Strain specificity and binding affinity 2 factor receptor p185HER causes transformation and tumori requirements of neutralizing monoclonal antibodies to the genesis of NIH 3T3 cells" Proc. Natl. Acad. Sci. C4 domain of gp120 from human immunodeficiency virus 84(20):7159-7163 (Oct. 1987). type 1" Journal of Virology 67(10):6179-6191 (Oct. 1993). Hudziak et al., "p185HER2 Monoclonal Antibody Has Anti Norton, L., "Evolving concepts in the system drug therapy proliferative Effects In Vitro and Sensitizes Human Breast of breast cancer" Seminars in Oncology 24(4 Suppl Tumor Cells to Tumor Necrosis Factor" Molecular & Cel 10):S10-3-S10-10 (Aug. 1997). lular Biology 9(3):1165-1172 (Mar. 1989). Pegram et al., "Inhibitory effects of combinations of HER-2/ Hynes and Stern, "The biology of erbB-2/neu/HER-2 and neu antibody and chemotherapeutic agents used for treat its role in cancer" Biochimica et Biophysica Acta ment of human breast cancers" Oncogene 18:2241-2251 1198(2-3):165-184 (Dec. 30, 1994). (1999).
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Pegram et al., "Phase II Study of Receptor-Enhanced Singal et al., "Combination therapy with probucol prevents Chemosensitivity Using Recombinant Humanized adriamycin-induced cardiomyopathy" Journal ofMolecular 2 Anti-p185HER /neu Monoclonal Antibody Plus Cisplatin in & Cellular Cardiology 27(4):1055-1063 (Apr. 1995). Patients With HER2/neu-Overexpressing Metastatic Breast Slamon et al., "Human Breast Cancer: Correlation of Cancer Refractory to Chemotherapy Treatment" Journal of Relapse and Survival with Amplification of the HER-2/neu Clinical Oncology 16(8):2659-2671 (1998). Oncogene" Science 235:177-182 (Jan. 9, 1987). Pietras et al., "Antibody to HER-2/neu receptor blocks DNA Slamon et al., "Studies of the HER-2/neu Proto-oncogene repair after cisplatin in human breast and ovarian cancer in Human Breast and Ovarian Cancer" Science 244:707-712 cells" Oncogene 9:1829-1838 (1994). (May 12, 1989). Presta et al., "Humanization of an Antibody Directed Against IgE" J. Immunol. 151(5):2623-2632 (Sep. 1, 1993). Sliwkowski et al., "A humanized monoclonal antibody for Raefsky et al., "Phase II Trial of Docetaxel and Herceptin as the treatment of HER2 overexpression breast cancer" Pro First--{)r Second-Line Chemotherapy for Women with Meta ceedings of the American Association for Cancer Research static Breast Cancer Whose Tumors Overexpress HER2" (abstract only) 37:625-626 (Mar. 1996). Proceedings of ASCO (Abstract #523) 18:137a (1999). Sliwkowski et al., "Coexpression of erbB2 and erbB3 Pro Ravdin and Chamness, "The c-erbB-2 proto-oncogene as a teins Reconstitutes a High Affinity Receptor for Heregulin" prognostic and predictive marker in breast cancer: a para Journal of Biological Chemistry 269(20):14661-14665 digm for the development of other macromolecular mark (May 20, 1994). ers-a review" Gene 159(1):19-27 (Jun. 14, 1995). Stancovski et al., "Mechanistic aspects of the opposing Renz et al., "Structural requirements for adhesion of soluble effects of monoclonal antibodies to the ERBB2 receptor on recombinant murine vascular cell adhesion molecule-1 to tumor growth," Proc. Natl. Acad. Sci. USA a4~1" Journal of Cell Biology 125(6):1395-1406 (Jun. 88(19):8691-8695 (Oct. 1, 1991). 1994). Stevenson et al., "A chimeric antibody with dual Fe regions Riechmann et al., "Reshaping Human Antibodies for (bisFabFc) prepared by manipulations at the IgG hinge" Therapy" Nature 332:323-327 (Mar. 24, 1988). Anti-Cancer Drug Design 3(4):219-230 (1989). Rodeck et al., "Interactions between growth factor receptors Suresh et al., "Bispecific Monoclonal Antibodies from and corresponding monoclonal antibodies in human tumors" Hybrid Hybridomas" Methods in Enzymology 121:210--228 J. Cellular Biochem. 35(4):315-320 (1987). (1986). Sarup et al., "Characterization of an Anti-P185HER2 Mono Tagliabue et al., "Selection of monoclonal antibodies which clonal Antibody that Stimulates Receptor Function and 2 Inhibits Tumor Cell Growth" Growth Regulation 1:72-82 induce internalization and phosphorylation of p185HER and (1991). growth inhibition of cells with HER2/NEU gene amplifica Schlom, J., "Monoclonal Antibodies: They're More and tion" International Journal of Cancer 47(6):933-937 (Apr. Less Than You Think" Molecular Foundations of Oncology, 1, 1991). Broder, S. ed., Baltimore, MD:Williams & Wilkins, Chapter Verhoeyen et al., "Reshaping Human Antibodies: Grafting 6, pps. 95-134 (1991). an Antilysozyme Activity" Science 239:1534-1536 (Mar. Scott et al., "p185HER 2 Signal Transduction in Breast Cancer 25, 1988). Cells" Journal of Biological Chemistry Vitetta and Uhr, "Monoclonal Antibodies as Agonists: An 266(22):14300-14305 (Aug. 5, 1991). Expanded Role For Their Use in Cancer Therapy" Cancer Seifert et al. "Dexrazoxane in the prevention of doxorubi Research 54(20):5301-5309 (Oct. 15, 1994). cin-induced cardiotoxicity" Annals of Pharmacotherapy Wolff et al., "Monoclonal antibody homodimers: enhanced 28(9):1063-1072 (Sep. 1994). antitumor activity in nude mice" Cancer Research Semba et al., "A v-erbB-related protooncogene, c-erbB-2, 53(11):2560-2565 (1993). is distinct from the c-erb-1/epidermal growth factor-recep Xu et al., "Antibody-induced growth inhibition is mediated tor gene and is amplified in a human salivary gland adeno through immunochemically and functionally distinct carcinoma" Proc. Natl. Acad. Sci. USA 82:6497-6501 epitopes on the extracellular domain of the c-erbB-2 (1985). (HER-2/neu) gene product p185" International Journal of Shawver et al., "Ligand-like Effects Induced by Anti Cancer 53(3):401-408 (Feb. 1, 1993). c--erbB-2 Antibodies Do Not Correlate with and Are Not Required for Growth Inhibition of Human Carcinoma Cells" Yamamoto et al., "Similarity of protein encoded by the Cancer Research 54(5):1367-1373 (Mar. 1, 1994). human c-erb-B-2 gene to epidermal growth factor recep Shepard et al., "Monoclonal Antibody Therapy of Human tor" (GenBank accession number X03363) (Mar. 30, 1995). Cancer: Taking the HER2 Protooncogene to the Clinic" J. Yamamoto et al., "Similarity of protein encoded by the Clin. Immunol. 11(3):117-127 (1991). human c-erb-B-2 gene to epidermal growth factor recep Sims et al., "A Humanized CD18 Antibody Can Block tor" Nature 319:230--234 (1986). Function Without Cell Destruction" The Journal of Immu Zhang et al., "Shared antigenic epitopes and pathobiological nology 151(4):2296-2308 (Aug. 1993). functions of anti-p185her2/neu monoclonal antibodies" Singal and Iliskovic, "Doxorubicin-induced cardiomyopa Experimental and Molecular Pathology 67:15-25 (1999). thy" New EnglandJ. ofMedicine 339(13):900--905 (Sep. 24, 1998). * cited by examiner
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U.S. Patent Sep.30,2003 Sheet 1 of 5 US 6,627,196 Bl
3H4 aa 541-599 405 aa 529-625 7C2 aa 22-53 7F3 aa 22-53
100 200 300 400 500 600 I I I I I I ~7C2 7F3 . , , 1 645 22I 248
3H4
541 599
. 405 I 561 625 500 600 100 200 300 400 I I . I
3H4 epitope (SEQ ID N0:8} 58 residues
VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVAR I . I 541 599
4D5 epitope (SEQ ID N0:9) 64 residues
LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQP I I 625 561
FIG._1
1 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPA 38 SPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFL 75 QDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDN 112 YALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEI 149 LKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLID 186 TNRSRA
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U.S. Patent Sep.30,2003 Sheet 2 of 5 US 6,627,196 Bl
D• 1.0 •• ('I') •• :::=- •• E b 0') ::::t 0 - ('I') -c 0 :.;::: co 0...... •• c •• -(]) • 1.0 () • C\1 c: 0 D• () . ••. E •. .:£ ::J . (]) .... 0 (]) (]) C\1 (J) ?; ..!: 0') ::J ....0 ._ 1.0 .,-- c: . co .• (]) . ~c: 0. . 0 . .,-- f? .• 1.0
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Appx17 HOSPIRA EX. 1001 Page 6 Case: 19-2156Document:19Page:89Filed:07/26/2019 3000~------~------~------~------~------~------~------~ •d \Jl • ~ • ·0· • E25_4 mg_kg_IV ~ ...... 2500 -l I ...... []--IV_1_mg_kg_anti-H ER2 ~ ···-6.···IV _2_mg_kg_anti-HER2 =...... ···0··· IV_4 mg_kg_anti-HER2 e-- SC_2_mg_kg_anti-HER2 2000~ I 'JJ. ~ '? ~ Appx18 ~= ...... cN 1500 , --- 8 # # Cell Mean for # # TUMOR VOL # (mm3) , # 'JJ. # = 1000 # ~ , .....~ # ~
, 0...... , ,' Ul , ,' 500 I ------1 ---.... 2~...' ,'' HOSPIRA EX. 1001 HOSPIRA ~ e rJ'l
-..a-.. a-.. N -..""-l 1--" FIG._ 4A -5oo..J.__-----r-----.---.-----.--,------.------' \0
Page 7 a-.. DAY6 DAY 10 DAY21 DAY24 DAY28 DAY31 ~ 1--" Case: 19-2156Document:19Page:90Filed:07/26/2019 •d 10000.------.------.------.------.------.------.------~\Jl • ~ ~ - -o-. E25_4 mg_kg_IV ...... ~ -a- IV_1_mg_kg_anti-H ER2 =...... ···~···IV_2_mg_kg_anti-HER2 ···0··· IV_4 mg_kg_anti-HER2
SC_2_mg_kg_anti-HER2 'JJ. ,* o------o ~ ,* '? 1000 ," " ~ Appx19 ," p ~= # N # c # # 8 # Cell Mean for , # TUMOR VOL , # (mm3) .... # ...... d 'JJ. ~= .....~ ~
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···············~
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U.S. Patent Sep.30,2003 Sheet 5 of 5 US 6,627,196 Bl
VARIABLE LIGHT 1 10 20 30 40 2C4 DTVMTQSHKIMSTSVGDRVSITC [KASQDVSIGVA----] WYQQRP ** **** * * * 574 DIQMTQSPSSLSASVGDRVTITC [KASQDVSIGVA----] WYQQKP * * **** hum ki DIQMTQSPSSLSASVGDRVTITC [RASQSVSTSSYSYMH] WYQQKP
50 60 70 80 2C4 GQSPKLLIY [SASYRYT] GVPDRFTGSGSGTDFTFTISSVQA ** * * * * * 574 GKAPKLLIY [SASYRYT] GVPSRFSGSGSGTDFTLTISSLQP * **** hum ki GKAPKLLIY [AASSLES] GVPSRFSGSGSGTDFTLTISSLQP
90 100 2C4 EDLAVYYC [QQYYIYPYT] FGGGTKLEIK (SEQ ID N0:10) * * * * 574 EDFATYYC [QQYYIYPYT] FGQGTKVEIK (SEQ ID N0:12) *** hum ki EDFATYYC [QQYNSLPYT] FGQGTKVEIK (SEQ ID NO: 14) FIG._5A VARIABLE HEAVY 1 10 20 30 40 2C4 EVQLQQSGPELVKPGTSVKISCKAS [GFTFTDYTMD] WVKQS ** ** * * *** ** * 574 EVQLVESGGGLVQPGGSLRLSCAAS [GFTFTDYTMD] WVRQA ** * * humiii EVQLVESGGGSVQPGGSLRLSCAAS [GFTFSSYAMS] WVRQA
50 60 70 80 2C4 HGKSLEWIG [DVNPNSGGSIYNQRFKG] KASLTVDRSSRIVYM * * ** *** * **** * 574 PGKGLEWVA [DVNPNSGGSIYNQRFKG] RFTLSVDRSKNTLYL * ****** ** **** * * * humiii PGKGLEWVS [VISGDGGSTYYADSVKG] RFTISRDDSKNTLYL
90 100 110 2C4 ELRSLTFEDTAVYYCAR [NLGPSFYFDY] WGQGTTLVTSS (SEQ ID NO:ll) *** ** * 574 QMNSLRAEDTAVYYCAR [NLGPSFYFDY] WGQGTLVTVSS (SEQ ID N0:13) ** *** humiii QMNSLRAEDTAVYYCAR [GRGGGS--DY] WGQGTLVTVSS (SEQ ID N0:15) FIG._5B
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US 6,627,196 Bl 1 2 DOSAGES FOR TREATMENT WITH ANTI inhibit the tumorigenic growth of neu-transformed NIH-3T3 ERBB2 ANTIBODIES cells as well as rat neuroblastoma cells (from which the neu oncogene was initially isolated) implanted into nude mice. RELATED APPLICATIONS Drebin et al. in Oncogene 2:387-394 (1988) discuss the 5 production of a panel of antibodies against the rat neu gene This application is a non-provisional application filed product. All of the antibodies were found to exert a cyto under 37 CFR 1.53(b)(l), claiming priority under 35 USC static effect on the growth of neu-transformed cells sus- 119(e) to provisional application No. 60/151,018, filed Aug. pended in soft agar. Antibodies of the IgM, IgG2a and IgG2b 27, 1999 and No. 60/213,822, filed Jun. 23, 2000, the isotypes were able to mediate significant in vitro lysis of contents of which are incorporated herein by reference. neu-transformed cells in the presence of complement, 10 whereas none of the antibodies were able to mediate high FIELD OF THE INVENTION levels of antibody-dependent cellular cytotoxicity (ADCC) of the neu-transformed cells. Drebin et al. Oncogene The present invention concerns the treatment of disorders 2:273-277 (1988) report that mixtures of antibodies reactive characterized by the overexpression of ErbB2 or disorders with two distinct regions on the pl85 molecule result in expressing epidermal growth factor receptor (EGFR), com 15 synergistic anti-tumor effects on neu-transformed NIH-3T3 prising administering to a human or animal presenting the cells implanted into nude mice. Biological effects of anti-neu disorders a therapeutically effective amount of an antibody antibodies are reviewed in Myers et al., Meth. Enzyrn. that binds ErbB2. More specifically, the invention concerns 198:277-290 (1991). See also W094/22478 published Oct. the treatment of human patients susceptible to or diagnosed 13, 1994. with cancer overexpressing ErbB2 or expressing EGFR, 20 Hudziak et al., Mol. Cell. Bioi. 9(3):1165-1172 (1989) where the treatment is with an anti-ErbB2 antibody admin describe the generation of a panel of anti-ErbB2 antibodies istered by front loading the dose of antibody during treat which were characterized using the human breast tumor cell ment by intravenous and/or subcutaneous administration. line SKBR3. Relative cell proliferation of the SKBR3 cells The invention optionally includes treatment of cancer in a following exposure to the antibodies was determined by human patient with a combination of an anti-ErbB2 antibody 25 crystal violet staining of the monolayers after 72 hours. and a chemotherapeutic agent, such as, but not limited to, a Using this assay, maximum inhibition was obtained with the taxoid. The taxoid may be, but is not limited to paclitaxel or antibody called 4D5 which inhibited cellular proliferation by docetaxel. The invention further includes treatment of can 56%. Other antibodies in the panel,including 7C2 and 7F3, cer in a human patient with a combination of anti-ErbB2 reduced cellular proliferation to a lesser extent in this assay. antibody and a chemotherapeutic agent, such as, but not 30 Hudziak et al. conclude that the effect of the 4D5 antibody limited to, an anthracycline derivative. Optionally, treatment on SKBR3 cells was cytostatic rather than cytotoxic, since with a combination of anti-ErbB2 and an anthracycline SKBR3 cells resumed growth at a nearly normal rate fol derivative includes treatment with an effective amount of a lowing removal of the antibody from the medium. The cardioprotectant. The present invention further concerns antibody 4D5 was further found to sensitize p 185 infrequent dosing of anti-ErbB2 antibodies. 35 -overexpressing breast tumor cell lines to the cytotoxic effects of TNF-a. See also W089/06692 published Jul. 27, BACKGROUND OF THE INVENTION 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. Cancer Research Proto-oncogenes that encode growth factors and growth 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A factor receptors have been identified to play important roles 40 (1990); Sarup et al. Growth Regulation 1:72-82 (1991); in the pathogenesis of various human malignancies, includ Shepard et al. J. Clin. Irnrnunol. 11(3):117-127 (1991); ing breast cancer. It has been found that the human ErbB2 Kumar et al. Mol. Cell. Bioi. 11(2):979-986 (1991); Lewis gene (erbB2, also known as her2, or c-erbB-2), which et al. Cancer Irnrnunol. Irnrnunother. 37:255-263 (1993); encodes a 185-kd transmembrane glycoprotein receptor Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. 2 (p185HER ) related to the epidermal growth factor receptor 45 Cancer Research 54:5301-5309 (1994); Sliwkowski et al.J. (EGFR), is overexpressed in about 25% to 30% of human Bioi. Chern. 269(20): 14661-14665 (1994); Scott et al. J. breast cancer (Slamon et al., Science 235:177-182 [1987]; Bioi. Chern. 266:14300--5 (1991); and D'souza et al. Proc. Slamon et al., Science 244:707-712 [1989]). Natl. Acad. Sci.91:7202-7206 (1994). Several lines of evidence support a direct role for ErbB2 Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe in the pathogenesis and clinical aggressiveness of ErbB2- 50 two antibodies which were selected for their reactivity on overexpressing tumors. The introduction of ErbB2 into the lung adenocarcinoma cell line (Calu-3) which overex non-neoplastic cells has been shown to cause their malignant presses ErbB2. One of the antibodies, called MGR3, was transformation (Hudziak et al., Proc. Natl. Acad. Sci. USA found to internalize, induce phosphorylation of ErbB2, and 84:7159-7163 [1987]; DiFiore et al., Science 237:78-182 inhibit tumor cell growth in vitro. [1987]). Transgenic mice that express HER2 were found to 55 McKenzie et al. Oncogene 4:543-548 (1989) generated a develop mammary tumors (Guy et al., Proc. Natl. A cad. Sci. panel of anti-ErbB2 antibodies with varying epitope USA 89:10578-10582 [1992]). specificities, including the antibody designated TAl. This Antibodies directed against human erbB2 protein prod TAl antibody was found to induce accelerated endocytosis ucts and proteins encoded by the rat equivalent of the erbB2 of ErbB2 (see Maier et al. Cancer Res. 51:5361-5369 gene (neu) have been described. Drebin et al., Cell 60 [1991]). Bacus et al. Molecular Carcinogenesis 3:350--362 41:695-706 (1985) refer to an IgG2a monoclonal antibody (1990) reported that the TAl antibody induced maturation of which is directed against the rat neu gene product. This the breast cancer celllinesAU-565 (which overexpresses the antibody called 7.16.4 causes down-modulation of cell sur erbB2 gene) and MCF-7 (which does not). Inhibition of face pl85 expression on B104-l-l cells (NIH-3T3 cells growth and acquisition of a mature phenotype in these cells transfected with the neu proto-oncogene) a inhibits colony 65 was found to be associated with reduced levels of ErbB2 formation of these cells. In Drebin et al. PNAS (USA) receptor at the cell surface and transient increased levels in 83:9129-9133 (1986), the 7.16.4 antibody was shown to the cytoplasm.
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US 6,627,196 Bl 3 4 Stancovski et al. PNAS (USA) 88:8691-8695 (1991) disease that involves axillary lymph nodes (Slamon et al., generated a panel of anti-ErbB2 antibodies, injected them [1987] and [19891 supra; Ravdin and Chamness, Gene i.p. into nude mice and evaluated their effect on tumor 159:19-27 [1995]; and Hynes and Stern, Biochim Biophys growth of murine fibroblasts transformed by overexpression Acta 1198:165-184 [1994]), and has been linked to sensi- of the erbB2 gene. Various levels of tumor inhibition were 5 tivity and/or resistance to hormone therapy and chemothera detected for four of the antibodies, but one of the antibodies peutic regimens, including CMF (cyclophosphamide, (N28) consistently stimulated tumor growth. Monoclonal methotrexate, and fiuoruracil) and anthracyclines (Baselga antibody N28 induced significant phosphorylation of the et al., Oncology 11 (3 Suppl 1):43-48 [1997]). However, ErbB2 receptor, whereas the other four antibodies generally despite the association of ErbB2 overexpression with poor displayed low or no phosphorylation-inducing activity. The 10 prognosis, the odds of HER2-positive patients responding effect of the anti-ErbB2 antibodies on proliferation of clinically to treatment with taxanes were greater than three SKBR3 cells was also assessed. In this SKBR3 cell prolif times those of HER2-negative patients (Ibid). rhuMab eration assay, two of the antibodies (N12 and N29) caused HER2 was shown to enhance the activity of paclitaxel a reduction in cell proliferation relative to control. The (TAXOL®) and doxorubicin against breast cancer ability of the various antibodies to induce cell lysis in vitro 15 xenografts in nude mice injected with BT-474 human breast via complement-dependent cytotoxicity (CDC) and adenocarcinoma cells, which express high levels of HER2 antibody-mediated cell-dependent cytotoxicity (ADCC) was (Baselga et al., Breast Cancer, Proceedings of ASCO, Vol. assessed, with the authors of this paper concluding that the 13, Abstract 53 [1994]). inhibitory function of the antibodies was not attributed significantly to CDC or ADCC. 20 SUMMARY OF THE INVENTION Bacus et al. Cancer Research 52:2580-2589 (1992) fur ther characterized the antibodies described in Bacus et al. The present invention concerns the discovery that an early (1990) and Stancovski et al. of the preceding paragraphs. attainment of an efficacious target trough serum concentra Extending the i.p. studies of Stancovski et al., the effect of tion by providing an initial dose or doses of anti-ErbB2 antibodies followed by subsequent doses of equal or smaller the antibodies after i.v. injection into nude mice harboring 25 mouse fibroblasts overexpressing human ErbB2 was amounts of antibody (greater front loading) is more effica assessed. As observed in their earlier work, N28 accelerated cious than conventional treatments. The efficacious target tumor growth, whereas N12 and N29 significantly inhibited trough serum concentration is reached in 4 weeks or less, growth of the ErbB2-expressing cells. Partial tumor inhibi preferably 3 weeks or less, more preferably 2 weeks or less, and most preferably 1 week or less, including 1 day or less. tion was also observed with the N24 antibody. Bacus et al. 30 also tested the ability of the antibodies to promote a mature The target serum concentration is thereafter maintained by phenotype in the human breast cancer cell lines AU-565 and the administration of maintenance doses of equal or smaller MDA-MB453 (which overexpress-ErbB2) as well as amounts for the remainder of the treatment regimen or until MCF-7 (containing low levels of the receptor). Bacus et al. suppression of disease symptoms is achieved. saw a correlation between tumor inhibition in vivo and 35 The invention further concerns a method for the treatment cellular differentiation; the tumor-stimulatory antibody N28 of a human patient susceptible to or diagnosed with a had no effect on differentiation, and the tumor inhibitory disorder characterized by overexpression of ErbB2 receptor action of the N12, N29 and N24 antibodies correlated with comprising administering a therapeutically effective amount the extent of differentiation they induced. of an anti-ErbB2 antibody subcutaneously. Preferably, the Xu et a. Int. J. Cancer 53:401-408 (1993) evaluated a 40 initial dose (or doses) as well as the subsequent maintenance panel of anti-ErbB2 antibodies for their epitope binding dose or doses are administered subcutaneously. Optionally, specificities, as well as their ability to inhibit anchorage where the patient's tolerance to the anti-ErbB2 antibody is independent and anchorage-dependent growth of SKBR3 unknown, the initial dose is administered by intravenous cells (by individual antibodies and in combinations), modu infusion, followed by subcutaneous administration of the late cell-surface ErbB2, and inhibit ligand stimulated 45 maintenance doses if the patient's tolerance for the antibody anchorage-independent growth. See also W094/00136 pub is acceptable. lished Jan 6, 1994 and Kasprzyk et al. Cancer Research According to the invention, the method of treatment 52:2771-2776 (1992) concerning anti-ErbB2 antibody com involves administration of an initial dose of anti-ErbB2 binations. Other anti-ErbB2 antibodies are discussed in antibody of more than approximately 4 mg/kg, preferably Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver 50 more than approximately 5 mg/kg. The maximum initial et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. dose or a subsequent dose does not exceed 50 mg/kg, Cancer Res. 54:3758-3765 (1994); and Harwerth et al. J. preferably does not exceed 40 mg/kg, and more preferably Bioi. Chern. 267:15160--15167 (1992). does not exceed 30 mg/kg. Administration is by intravenous A recombinant humanized anti-ErbB2 monoclonal anti or subcutaneous administration, preferably intravenous infu- body (a humanized version of the murine anti-ErbB2 anti ss sian or bolus injection, or more preferably subcutaneous body 4D5, referred to as rhuMAb HER2, HERCEPTIN®, or bolus injection. The initial dose may be one or more HERCEPTIN® anti-ErbB2 antibody) has been clinically administrations of drug sufficient to reach the target trough active in patients with ErbB2-overexpressing metastatic serum concentration in 4 weeks or less, preferably 3 weeks breast cancers that had received extensive prior anti-cancer or less, more preferably 2 weeks or less, and most preferably therapy (Baselga et al., J. Clin Oneal. 14:737-744 [1996]). 60 1 week or less, including one day or less. The recommended initial loading dose for HERCEPTIN®O According to the invention, the initial dose or doses is/are is 4 mg/kg administered as a 90-minute infusion. The followed by subsequent doses of equal or smaller amounts recommended weekly maintenance dose is 2 mg/kg and can of antibody at intervals sufficiently close to maintain the be administered as a 30-minute infusion if the initial loading trough serum concentration of antibody at or above an dose is well tolerated. 65 efficacious target level. Preferably, an initial dose or subse ErbB2 overexpression is commonly regarded as a predic quent dose does not exceed 50 mg/kg, and each subsequent tor of a poor prognosis, especially in patients with primary dose is at least 0.01 mg/kg. Preferably the amount of drug
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US 6,627,196 Bl 5 6 administered is sufficient to maintain the target trough serum carcinoma, salivary gland carcinoma, kidney cancer, liver concentration such that the interval between administration cancer, prostate cancer, vulval cancer, thyroid cancer, cycles is at least one week. Preferably the trough serum hepatic carcinoma and various types of head and neck concentration does not exceed 2500 ,ug!ml and does not fall cancer. The method of the invention may further comprise below 0.01 ,ug/ml during treatment. The front loading drug 5 administration of a chemotherapeutic agent other than an treatment method of the invention has the advantage of anthracycline, e.g. doxorubicin or epirubicin. The chemo increased efficacy by reaching a target serum drug concen therapeutic agent preferably is a taxoid, such as TAXOL® tration early in treatment. The subcutaneous delivery of (paclitaxel) or a TAXOL® derivative. maintenance doses according to the invention has the advan Preferred anti-ErbB2 antibodies bind the extracellular tage of being convenient for the patient and health care 10 domain of the ErbB2 receptor, and preferably bind o the professionals, reducing time and costs for drug treatment. epitope 4D5 or 3H4 within the ErbB2 extracellular domain Preferably, the initial dose (or the last dose within an initial sequence. More preferably, the antibody is the antibody dose series) is separated in time from the first subsequent 4D5, most preferably in a humanized form. Other preferred dose by 4 weeks or less, preferably 3 weeks or less, more ErbB2-binding antibodies include, but are not limited to, preferably 3 weeks or less, most preferably 1 week or less. 15 antibodies 7C2, 7F3, and 2C4, preferably in a humanized form. In an embodiment of the invention, the initial dose of anti-ErbB2 is 6 mg!kg, 8 mg/kg, or 12 mg/kg delivered by The method of the present invention is particularly suit intravenous or subcutaneous administration, such as intra able for the treatment of breast or ovarian cancer, charac venous infusion or subcutaneous bolus injection. The sub terized by the overexpression of the ErbB2 receptor. sequent maintenance doses are 2 mg/kg delivered once per 20 The present application also provides a method of therapy week by intravenous infusion, intravenous bolus injection, involving infrequent dosing of an anti-ErbB2 antibody. In subcutaneous infusion, or subcutaneous bolus injection. The particular, the invention provides a method for the treatment choice of delivery method for the initial and maintenance of cancer (e.g. cancer characterized by overexpression of the doses is made according to the ability of the animal or ErbB2 receptor) in a human patient comprising administer human patient to tolerate introduction of the antibody into 25 ing to the patient a first dose of an anti-ErbB2 antibody the body. Where the antibody is well-tolerated, the time of followed by at least one subsequent dose of the antibody, infusion may be reduced. The choice of delivery method as wherein the first dose and subsequent dose are separated disclosed for this embodiment applies to all drug delivery from each other in time by at least about two weeks (e.g. regimens contemplated according to the invention. from about two weeks to about two months), and optionally In another embodiment, the invention includes an initial 30 at least about three weeks (e.g. from about three weeks to dose of 12 mg/kg anti-ErbB2 antibody, followed by subse about six weeks). For instance, the antibody may be admin quent maintenance doses of 6 mg!kg once per 3 weeks. istered about every three weeks, about two to about 20 times, e.g. about six times. The first dose and subsequent dose may In still another embodiment, the invention includes an each be from about 2 mg/kg to about 16 mg!kg; e.g. from initial dose of 8 mg/kg anti-ErbB2 antibody, followed by 6 35 about 4 mg!kg to about 12 mg/kg; and optionally from about mg/kg once per 3 weeks. 6 mg/kg to about 12 mg!kg. Generally, two or more subse In yet another embodiment, the invention includes an quent doses (e.g. from about two to about ten subsequent initial dose of 8 mg/kg anti-ErbB2 antibody, followed by doses) of the antibody are administered to the patient, and subsequent maintenance doses of8 mg/kg once per week or those subsequent doses are preferably separated from each 8 mg/kg once every 2 to 3 weeks. 40 other in time by at least about two weeks (e.g. from about In another embodiment, the invention includes initial two weeks to about two months), and optionally at least doses of at least 1 mg/kg, preferably 4 mg!kg, anti-ErbB2 about three weeks (e.g. from about three weeks to about six antibody on each of days 1, 2 and 3, followed by subsequent weeks). The two or more subsequent doses may each be maintenance doses of 6 mg/kg once per 3 weeks. from about 2 mg/kg to about 16 mg/kg; or from about 4 In another embodiment, the invention includes an initial 45 mg/kg to about 12 mg!kg; or from about 6 mg/kg to about dose of 4 mg/kg anti-ErbB2 antibody, followed by subse 12 mg!kg. The invention additionally provides an article of quent maintenance doses of 2 mg/kg twice per week, manufacture, comprising a container, a composition within wherein the maintenance doses are separated by 3 days. the container comprising an anti-ErbB2 antibody, and a In still another embodiment, the invention includes a package insert containing instructions to administer the cycle of dosing in which delivery of anti-ErbB2 antibody is 50 antibody according to such methods. 2-3 times per week for 3 weeks. In one embodiment of the The presently described dosing protocols may be applied invention, each dose is approximately 25 mg!kg or less for to other anti-ErbB antibodies such as anti-epidermal growth a human patient, preferably approximately 10 mg/kg or less. factor receptor (EGFR), anti-ErbB3 and anti-ErbB4 anti This 3 week cycle is preferably repeated as necessary to bodies. Thus, the invention provides a method for the achieve suppression of disease symptoms. 55 treatment of cancer in a human patient, comprising admin In another embodiment, the invention includes a cycle of istering an effective amount of an anti-ErbB antibody to the dosing in which delivery of anti-ErbB2 antibody is daily for human patient, the method comprising administering to the 5 days. According to the invention, the cycle is preferably patient an initial dose of at least approximately 5 mg/kg of repeated as necessary to achieve suppression of disease the anti-ErbB antibody; and administering to the patient a symptoms. 60 plurality of subsequent doses of the antibody in an amount The disorder preferably is a benign or malignant tumor that is approximately the same or less than the initial dose. characterized by the overexpression of the ErbB2 receptor, Alternatively, or additionally, the invention pertains to a e.g. a cancer, such as, breast cancer, squamous cell cancer, method for the treatment of cancer in a human patient small-cell lung cancer, non-small cell lung cancer, gas comprising administering to the patient a first dose of an trointestinal cancer, pancreatic cancer, glioblastoma, cervi 65 anti-ErbB antibody followed by at least one subsequent dose cal cancer, ovarian cancer, liver cancer, bladder cancer, of the antibody, wherein the first dose and subsequent dose hepatoma, colon cancer, colorectal cancer, endometrial are separated from each other in time by at least about two
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US 6,627,196 Bl 7 8 weeks. The invention additionally provides an article of BRIEF DESCRIPTION OF THE DRAWINGS manufacture, comprising a container, a composition within FIG. 1 shows epitope-mapping of the extracellular the container comprising an anti-ErbB antibody, and a package insert containing instructions to administer the domain of ErbB2 as determined by truncation mutant analy sis and site-directed mutagenesis (Nakamura et al. J. of antibody according to such methods. 5 In another aspect, the invention concerns an article of Virology 67 (10):6179-6191 [October 1993]; Renz etJ. Cell manufacture, comprising a container, a composition within Bioi. 125(6):1395-1406 [June 1994]). The anti-proliferative the container comprising an anti-ErbB2 antibody, optionally MAbs 4D5 and 3H4 b bind adjacent to the transmembrane a label on or associated with the container that indicates that domain. The various ErbB2-ECD truncations or point muta tions were prepared from eDNA using polymerase chain the composition can be used for treating a condition char 10 acterized by overexpression of ErbB2 receptor, and a pack reaction technology. The ErbB2 mutants were expressed as age insert containing instructions to avoid the use of gD fusion proteins in a mammalian expression plasmid. This anthracycline-type chemotherapeutics in combination with expression plasmid uses the cytomegalovirus promoter/ the composition. According to the invention, the package enhancer with SV40 termination and polyadenylation sig insert further includes instructions to administer the anti nals located downstream of the inserted eDNA Plasmid ErbB2 antibody at an initial dose of 5 mg/kg followed by the 15 DNA was transfected into 293S cells. One day following same or smaller subsequent dose or doses. In another transfection, the cells were metabolically labeled overnight embodiment of the invention, the package insert further in methionine and cysteine-free, low glucose DMEM con includes instructions to administer the anti-ErbB2 antibody taining 1% dialyzed fetal bovine serum and 25 ,uCi each of subcutaneously for at least one of the doses, preferably for 35S methionine and 35S cysteine. Supernatants were har- all of the subsequent doses following the initial dose, most 20 vested either the ErbB2 MAbs or control antibodies were preferably for all doses. added to the supernatant and incubated 2-4 hours at 4 o C. In a further aspect, the invention provides a method of The complexes were precipitated, applied to a 10-20% treating ErbB2 expressing cancer in a human patient com Tricine SDS gradient gel and electrophoresed at 100 V. The prising administering to the patient effective amounts of an gel was electroblotted onto a membrane and analyzed by anti-ErbB2 antibody and a chemotherapeutic agent. In one 25 autoradiography. SEQ ID NOs:8 and 9 depict the 3H4 and embodiment of the invention, the chemotherapeutic agent is a taxoid including, but not limited to, paclitaxel and doc 4D5 epitopes, respectively. etaxel. In another embodiment, the chemotherapeutic agent FIG. 2 depicts with underlining the amino acid sequence is an anthracyline derivative including, but not limited to, of Domain 1 of ErbB2 (SEQ ID NO: 1). Bold amino acids indicate the location of the epitope recognized by MAbs 7C2 doxorubicin or epirubicin. In still another embodiment of the 30 invention, treatment with an anti-ErbB2 antibody and an and 7F3 as determined by deletion mapping, i.e. the "7C2/ anthracycline derivative further includes administration of a 7F3 epitope" (SEQ ID N0:2). cardioprotectant to the patient. In still another embodiment, FIG. 3 is a graph of anti-ErbB2 antibody an anthracycline derivative is not administered to the patient (HERCEPTIN®) trough serum concentration (ug/ml, mean with the anti-ErbB2 antibody. One or more additional che- 35 ±SE, dark circles) by week from week 2 through week 36 for motherapeutic agents may also be administered to the ErbB2 overexpressing patients treated with HERCEPTIN® patient. The cancer is preferably characterized by overex anti-ErbB2 antibody at 4 mg/kg initial dose, followed by 2 pression of ErbB2. mg/kg weekly. The number of patients at each time point is The invention further provides an article of manufacture represented by "n" (white squares). comprising a container, a composition within the container 40 FIG. 4A is a linear plot of tumor volume changes over comprising an anti-ErbB2 antibody and a package insert time in mice treated with HERCEPTIN® anti-ErbB2 anti instructing the user of the composition to administer the body. FIG. 4B is a semi-logarithmic plot of the same data as anti-ErbB2 antibody composition and a chemotherapeutic in FIG. 4A such that the variation in tumor volume for the agent to a patient. In another embodiment, the chemothera treated animals is observed more readily. peutic agent is other than an anthracycline, and is preferably 45 FIGS. SA and SB depict alignments of the amino acid a taxoid, such as TAXOL®. In still another embodiment, the sequences of the variable light (VL)(FIG. SA) and variable chemotherapeutic agent is an anthracycline, including but heavy (VH) (FIG. SB) domains of murine monoclonal anti not limited to, doxorubicin or epirubicin. In yet another body 2C4 (SEQ ID Nos. 10 and 11, respectively); VL and VH embodiment, the chemotherapeutic agent is an anthracycline domains of humanized Fab version 574 (SEQ ID Nos. 12 and the package insert further instructs the user to administer 50 and 13, respectively), and human VL and VH consensus a cardioprotectant. frameworks (hum Kl, light kappa subgroup I; humiii, heavy The methods and compositions of the invention comprise subgroup III) (SEQ ID Nos. 14 and 15, respectively). an anti-ErbB2 antibody and include a humanized anti-ErbB2 Asterisks identify differences between humanized Fab ver antibody. Thus, the invention further pertains to a compo sion 574 and murine monoclonal antibody 2C4 or between sition comprising an antibody that binds ErbB2 and the use 55 humanized Fab version 574 and the human framework. of the antibody for treating ErbB2 expressing cancer, e.g., Complementarity Determining Regions (CDRs) are in ErbB2 overexpressing cancer, in a human. The invention brackets. Humanized Fab version 574, with the changes also pertains to the use of the antibody for treating EGFR ArgH71 Val, AspH73Arg and IleH69Leu, appears to have expressing cancer. Preferably the antibody is a monoclonal binding restored to that of the original chimeric 2C4 Fab antibody 4D5, e.g., humanized 4D5 (and preferably 60 fragment. Additional FR and/or CDR residues, such as L2, huMAb4D5-8 (HERCEPTIN® anti-ErbB2 antibody); or L54, L55, L56, H35 and/or H48, may be modified (e.g. monoclonal antibody 2C4, e.g., humanized 2C4. The anti substituted as follows-IleL2Thr; ArgL54Leu; TyrL55Glu; body may be an intact antibody (e.g., an intact IgG, ThrL56Ser; AspH35Ser; and ValH48Ile) in order to further
antibody) or an antibody fragment (e.g., a Fab, F(ab')2 , refine or enhance binding of the humanized antibody. diabody, and the like). The variable light chain and variable 65 Alternatively, or additionally, the humanized antibody may heavy chain regions of humanized anti-ErbB2 antibody 2C4 be affinity matured in order to further improve or refine its are shown in FIGS. SA and SB. affinity and/or other biological activities.
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US 6,627,196 Bl 9 10 DETAILED DESCRIPTION OF 1HE epitope is shown in FIG. 1, and includes residues from about PREFERRED EMBODIMENTS 541 to about 599, inclusive, in the amino acid sequence of ErbB2 extracellular domain. I. Definitions The "epitope 7C2/7F3" is the region at theN-terminus of An "ErbB receptor" is a receptor protein tyrosine kinase 5 the extracellular domain of ErbB2 to which the 7C2 and/or which belongs to the ErbB receptor family and includes 7F3 antibodies (each deposited with the ATCC, see below) EGFR, HER2, ErbB3 and ErbB4 receptors as well as bind. To screen for antibodies which bind to the 7C2/7F3 TEGFR (U.S. Pat. No. 5,708,156) and other members of this epitope, a routine cross-blocking assay such as that family to be identified in the future. The ErbB receptor will described in Antibodies, A Laboratory Manual, Cold Spring generally comprise an extracellular domain, which may bind 10 Harbor Laboratory, Ed Harlow and David Lane (1988), can an ErbB ligand; a lipophilic transmembrane domain; a be performed. Alternatively, epitope mapping can be per conserved intracellular tyrosine kinase domain; and a formed to establish whether the antibody binds to the carboxyl-terminal signaling domain harboring several 7C2/7F3 epitope on ErbB2 (i.e. any one or more of residues tyrosine residues which can be phosphorylated. The ErbB in the region from about residue 22 to about residue 53 of receptor may be a native sequence ErbB receptor or an 15 ErbB2; SEQ 1D N0:2). amino acid sequence variant thereof. Preferably the ErbB The term "induces cell death" or "capable of inducing cell receptor is native sequence human ErbB receptor. death" refers to the ability of the antibody to make a viable The terms "ErbB1", "epidermal growth factor receptor" cell become nonviable. The "cell" here is one which and "EGFR" are used interchangeably herein and refer to expresses the ErbB2 receptor, especially where the cell native sequence EGFR as disclosed, for example, in Car 20 overexpresses the ErbB2 receptor. A cell which "overex penter et al.Ann. Rev. Biochem. 56:881-914 (1987), includ presses" ErbB2 has significantly higher than normal ErbB2 ing variants thereof (e.g. a deletion mutant EGFR as in levels compared to a noncancerous cell of the same tissue Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 type. Preferably, the cell is a cancer cell, e.g. a breast, refers to the gene encoding the EGFR protein product. ovarian, stomach, endometrial, salivary gland, lung, kidney, Examples of antibodies which bind to EGFR include MAb 25 colon, thyroid, pancreatic or bladder cell. In vitro, the cell 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRL may be a SKBR3, BT474, Calu 3, MDA-MB-453, MDA HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC MB-361 or SKOV3 cell. Cell death in vitro may be deter CRL8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) mined in the absence of complement and immune effector and variants thereof, such as chimerized 225 (C225) and cells to distinguish cell death induced by antibody dependent reshaped human 225 (H225) (see, WO 96/40210, Imclone 30 cellular cytotoxicity (ADCC) or complement dependent Systems Inc.). cytotoxicity (CDC). Thus, the assay for cell death may be "ErbB3" and"HER3" refer to the receptor polypeptide as performed using heat inactivated serum (i.e. in the absence disclosed, for example, in U.S. Pat. Nos. 5,183,884 and of complement) and in the absence of immune effector cells. 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 To determine whether the antibody is able to induce cell (1989), including variants thereof. Examples of antibodies 35 death, loss of membrane integrity as evaluated by uptake of which bind HER3 are described in U.S. Pat. No. 5,968,511 propidium iodide (PI), trypan blue (see Moore et al. Cyto (Akita and Sliwkowski), e.g. the 8B8 antibody (ATCC HB technology 17:1-11 [1995]) or 7AAD can be assessed 12070) or a humanized variant thereof. relative to untreated cells. Preferred cell death-inducing The terms "ErbB4" and "HER4" herein refer to the antibodies are those which induce PI uptake in the "PI receptor polypeptide as disclosed, for example, in EP Pat 40 uptake assay in BT474 cells". Appln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. The phrase "induces apoptosis" or "capable of inducing USA, 90:1746-1750 (1993); and Plowman et al., Nature, apoptosis" refers to the ability of the antibody to induce 366:473-475 (1993), including variants thereof such as the programmed cell death as determined by binding of annexin HER4 isoforms disclosed in WO 99/19488. V, fragmentation of DNA, cell shrinkage, dilation of endo The terms "HER2", "ErbB2" "c-Erb-B2" are used inter- 45 plasmic reticulum, cell fragmentation, and/or formation of changeably. Unless indicated otherwise, the terms "ErbB2" membrane vesicles (called apoptotic bodies). The cell is one "c-Erb-B2" and "HER2" when used herein refer to the which overexpresses the ErbB2 receptor. Preferably the human protein, and "erbB2," "c-erb-B2," and "her2" refer to "cell" is a tumor cell, e.g. a breast, ovarian, stomach, human gene. The human erbB2 gene and ErbB2 protein are, endometrial, salivary gland, lung, kidney, colon, thyroid, for example, described in Semba et al., PNAS (USA) 50 pancreatic or bladder cell. In vitro, the cell may be a SKBR3, 82:6497-650 (1985)and Yamamoto et al. Nature BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or 319:230-234 (1986) (Genebank accession number SKOV3 cell. Various methods are available for evaluating X03363). ErbB2 comprises four domains (Domains 1-4). the cellular events associated with apoptosis. For example, The "epitope 4D5" is the region in the extracellular phosphatidyl serine (PS) translocation can be measured by domain of ErbB2 to which the antibody 4D5 (ATCC CRL 55 annexin binding; DNA fragmentation can be evaluated 10463) binds. This epitope is close to the transmembrane through DNA laddering as disclosed-in the example herein; region of ErbB2. To screen for antibodies which bind to the and nuclear/chromatin condensation-along with DNA frag 4D5 epitope, a routine cross-blocking assay such as that mentation can be evaluated by any increase in hypodiploid described in Antibodies, A Laboratory Manual, Cold Spring cells. Preferably, the antibody which induces apoptosis is Harbor Laboratory, Ed Harlow and David Lane (1988), can 60 one which results in about 2 to 50 fold, preferably about 5 be performed. Alternatively, epitope mapping can be per to 50 fold, and most preferably about 10 to 50 fold, induction formed (see FIG. 1) to assess whether the antibody binds to of annexin binding relative to untreated cell in an "annexin the 4D5 epitope of ErbB2 (i.e. any one or more residues in binding assay using BT474 cells" (see below). the region from about residue 529, e.g. about residue 561 to Sometimes the pro-apoptotic antibody will be one which about residue 625, inclusive). 65 blocks HRG binding/activation of the ErbB2/ErbB3 com The "epitope 3H4" is the region in the extracellular plex (e.g. 7F3 antibody). In other situations, the antibody is domain of ErbB2 to which the antibody 3H4 binds. This one which does not significantly block activation of the
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US 6,627,196 Bl 11 12 ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2). Further, However, the variability is not evenly distributed throughout the antibody may be one like 7C2 which, while inducing the variable domains of antibodies. It is concentrated in three apoptosis, does not induce a large reduction in the percent of segments called complementarity determining regions cells inS phase (e.g. one which only induces about 0-10% (CDRs) or hypervariable regions both in the light-chain and reduction in the percent of these cells relative to control). 5 the heavy-chain variable domains. The more highly con The antibody of interest may be one like 7C2 which binds served portions of variable domains are called the frame specifically to human ErbB2 and does not significantly work region (FR). The variable domains of native heavy and cross-react with other proteins such as those encoded by the light chains each comprise four FR regions, largely adopting erbB1, erbB3 and/or erbB4 genes. Sometimes, the antibody a ~-sheet configuration, connected by three CDRs, which may not significantly cross-react with the rat neu protein, 10 form loops connecting, and in some cases forming part of, e.g., as described in Schecter et al. Nature 312:513 (1984) the ~-sheet structure. The CDRs in each chain are held and Drebin et al., Nature 312:545-548 (1984). In such together in close proximity by the FRs and, with the CDRs embodiments, the extent of binding of the antibody to these from the other chain, contribute to the formation of the proteins (e.g., cell surface binding to endogenous receptor) antigen-binding site of antibodies (see Kabat et al., NIH will be less than about 10% as determined by fluorescence 15 Publ. No. 91-3242, Vol. I, pages 647-669 [1991]). The activated cell sorting (FACS) analysis or radioimmunopre constant domains involved directly in binding an antibody to cipitation (RIA). an antigen, but exhibit various effector functions, such as "Heregulin" (HRG) when used herein refers to a polypep participation of the antibody in antibody dependent cellular tide which activates the ErbB2-ErbB3 and ErbB2-ErbB4 cytotoxicity. protein complexes (i.e. induces phosphorylation of tyrosine 20 Papain digestion of antibodies produces two identical residues in the complex upon binding thereto). Various antigen-binding fragments, called "Fab" fragments, each heregulin polypeptides encompassed by this term are dis with a single antigen-binding site, and a residual "Fe" closed in Holmes et al., Science, 256:1205-1210 (1992); fragment, whose name reflects its ability to crystallize WO 92/20798; Wen et al., Mol. Cell. Bioi., 14(3): readily. Pepsin treatment yields an F(ab')2 fragment that has 1909-1919 (1994); Nature, 362:312-318 (1993), for 25 two antigen-combining sites and is still capable of cross example. The term includes biologically active fragments linking antigen. and/or variants of a naturally occurring HRG polypeptide, "Fv" is the minimum antibody fragment which contains a such as an EGF-like domain fragment thereof (e.g. complete antigen-recognition and -binding site. This region HRG~1177-244) consists of a dimer of one heavy- and one light-chain 30 The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 variable domain in tight, non-covalent association. It is in protein complex" are noncovalently associated oligomers of this configuration that the three CDRs of each variable the ErbB2 receptor and the ErbB3 receptor or ErbB4 domain interact to define an antigen-binding site on the receptor, respectively. The complexes form when a cell surface of the VH-VL dimer. Collectively, the six CDRs expressing both of these receptors is exposed to HRG and confer antigen-binding specificity to the antibody. However, 35 can be isolated by immunoprecipitation and analyzed by even a single variable domain (or half of an Fv comprising SDS-PAGE as described in Sliwkowski et al., J. Bioi. only three CDRs specific for an antigen) has the ability to Chern., 269(20):14661-14665 (1994). recognize and bind antigen, although at a lower affinity than "Antibodies" (Abs) and "immunoglobulins" (Igs) are the entire binding site. The Fab fragment also contains the constant domain of glycoproteins having the same structural characteristics. 40 While antibodies exhibit binding specificity to a specific the light chain and the first constant domain (CH1) of the antigen, immunoglobulins include both antibodies and other heavy chain. Fab' fragments differ from Fab fragments by antibody-like molecules which lack antigen specificity. the addition of a few residues at the carboxy terminus of the Polypeptides of the latter kind are, for example, produced at heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation low levels by the lymph system and at increased levels by 45 myelomas. herein for Fab' in which the cysteine residue(s) of the "Native antibodies" and "native immunoglobulins" are constant domains bear a free thiol group. F(ab')2 antibody usually heterotetrameric glycoproteins of about 150,000 fragments originally were produced as pairs of Fab' frag daltons, composed of two identical light (L) chains and two ments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. identical heavy (H) chains. Each light chain is linked to a 50 heavy chain by one covalent disulfide bond, while the The "light chains" of antibodies (immunoglobulins) from number of disulfide linkages varies among the heavy chains any vertebrate species can be assigned to one of two clearly of different immunoglobulin isotypes. Each heavy and light distinct types, called kappa (K) and lambda (/c), based on the chain also has regularly spaced intrachain disulfide bridges. amino acid sequences of their constant domains. Each heavy chain has at one end a variable domain (VH) 55 Depending on the amino acid sequence of the constant followed by a number of constant domains. Each light chain domain of their heavy chains, immunoglobulins can be has a variable domain at one end (VL) and a constant domain assigned to different classes. There are five major classes of at its other end; the constant domain of the light chain is immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several aligned with the first constant domain of the heavy chain, of these may be further divided into subclasses (isotypes), and the light-chain variable domain is aligned with the 60 e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy variable domain of the heavy chain. Particular amino acid chain constant domains that correspond to the different residues are believed to form an interface between the light classes of immunoglobulins are called a, ll, E, y, and fA, and heavy-chain variable domains. respectively. The subunit structures and three-dimensional The term "variable" refers to the fact that certain portions configurations of different classes of immunoglobulins are of the variable domains differ extensively in sequence 65 well known. among antibodies and are used in the binding and specificity The term "antibody" is use d in the broadest sense and of each particular antibody for its particular antigen. specifically covers intact monoclonal antibodies, polyclonal
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US 6,627,196 Bl 13 14 antibodies, multispecific antibodies (e.g. bispecific antibody performance. In general, the humanized antibody antibodies) formed from at least two intact antibodies, and will comprise substantially all of at least one, and typically antibody fragments so long as they exhibit the desired two, variable domains, in which all or substantially all of the biological activity. CDRs correspond to those of a non-human inmmunoglobu "Antibody fragments" comprise a portion of an intact 5 lin and all or substantially all of the FRs are those of a human antibody, preferably the antigen binding or variable region immunoglobulin sequence. The humanized antibody opti- of the intact antibody. Examples of antibody fragments mally also will comprise at least a portion of an immuno include Fab, Fab', F(ab')2 , and Fv fragments; diabodies; globulin constant region (Fe), typically that of a human linear antibodies (Zapata et al., Protein Eng. 8(10): immunoglobulin. For further details, see Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 1057-1062 [1995]); single-chain antibody molecules; and 10 multispecific antibodies formed from antibody fragments. 332:323-329 (1988); and Presta, Curr. Op. Struct. Bioi., The term "monoclonal antibody" as used herein refers to 2:593-596 (1992). The humanized antibody includes a PRI an antibody obtained from a population of substantially MATIZED™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by homogeneous antibodies, i.e., the individual antibodies immunizing macaque monkeys with the antigen of interest. comprising the population are identical except for possible 15 naturally occurring mutations that may be present in minor "Single-chain Fv" or "sFv" antibody fragments comprise amounts. Monoclonal antibodies are highly specific, being the VHand VL domains of antibody, wherein these domains directed against a single antigenic site. Furthermore, in are present in a single polypeptide chain. Preferably, the Fv contrast to conventional (polyclonal) antibody preparations polypeptide further comprises a polypeptide linker between the VHand VL domains which enables the sFv to form the which typically include different antibodies directed against 20 different determinants (epitopes), each monoclonal antibody desired structure for antigen binding. For a review of sFv see is directed against a single determinant on the antigen. In Pluckthun in The Pharmacology of Monoclonal Antibodies, addition to their specificity, the monoclonal antibodies are vol. 113, Rosenburg and Moore eds., Springer-Verlag, New advantageous in that they are synthesized by the hybridoma York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments culture, uncontaminated by other immunoglobulins. The 25 modifier "monoclonal" indicates the character of the anti with two antigen-binding sites, which fragments comprise a body as being obtained from a substantially homogeneous heavy-chain variable domain (VH) connected to a light population of antibodies, and is not to be construed as chain variable domain (VL) in the same polypeptide chain requiring production of the antibody by any particular (VH-VL). By using a linker that is too short to allow pairing method. For example, the monoclonal antibodies to be used 30 between the two domains on the same chain, the domains are in accordance with the present invention may be made by the forced to pair with the complementary domains of another hybridoma method first described by Kohler et al., Nature, chain and create two antigen-binding sites. Diabodies are 256:495 (1975), or may be made by recombinant DNA described more fully in, for example, EP 404,097; WO methods (see, e.g., U.S. Pat. No. 4,816,567). The "mono 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, clonal antibodies" may also be isolated from phage antibody 35 90:6444-6448 (1993). libraries using the techniques described in Clackson et al., An "isolated" antibody is one which has been identified Nature, 352:624-628 (1991) and Marks et al., J. Mol. Bioi., and separated and/or recovered from a component of its 222:581-597 (1991), for example natural environment. Contaminant components of its natural The monoclonal antibodies herein specifically include environment are materials which would interfere with diag "chimeric" antibodies (immunoglobulins) in which portion 40 nostic or therapeutic uses for the antibody, and may include of the heavy and/or light chain is identical with or homolo enzymes, hormones, and other proteinaceous or nonpro- gous to corresponding sequences in antibodies derived from teinaceous solutes. In preferred embodiments, the antibody a particular species or belonging to a particular antibody will be purified (1) to greater than 95% by weight of class or subclass, while the remainder of the chain(s) is antibody as determined by the Lowry method, and most identical with or homologous to corresponding sequences in 45 preferably more than 99% by weight, (2) to a degree antibodies derived from another species or belonging to sufficient to obtain at least 15 residues of N-terminal or another antibody class or subclass, as well as fragments of internal amino acid sequence by use of a spinning cup such antibodies, so long as they exhibit the desired biologi- sequenator, or (3) to homogeneity by SDS-PAGE under cal activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. reducing or nonreducing conditions using Coomassie blue Natl. Acad. Sci. USA, 81:6851-6855 [1984]). 50 or, preferably, silver stain. Isolated antibody includes the "Humanized" forms of non-human (e.g., murine) anti antibody in situ within recombinant cells since at least one bodies are chimeric immunoglobulins, immunoglobulin component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be pre chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which pared by at least one purification step. contain minimal sequence derived from non-human immu- 55 As used herein, the term "salvage receptor binding noglobulin. For the most part, humanized antibodies are epitope" refers to an epitope of the Fe region of an IgG human immunoglobulins (recipient antibody) in which resi molecule (e.g., IgG 1 , IgG2 , IgG3 , or IgG4 ) that is respon dues from a complementarity determining region (CDR) of sible for increasing the in vivo serum half-life of the IgG the recipient are replaced by residues from a CDR of a molecule. non-human species (donor antibody) such as mouse, rat or 60 "Treatment" refers to both therapeutic treatment and rabbit having the desired specificity, affinity, and capacity. In prophylactic or preventative measures. Those in need of some instances, framework region (FR) residues of the treatment include those already with the disorder as well as human immunoglobulin are replaced by corresponding non those in which the disorder is to be prevented. human residues. Furthermore, humanized antibodies may "Mammal" for purposes of treatment refers to any animal comprise residues which are found neither in the recipient 65 classified as a mammal, including humans, domestic and antibody nor in the imported CDR or framework sequences. farm animals, and zoo, sports, or pet animals, such as dogs, These modifications are made to further refine and maximize horses, cats, cows, etc. Preferably, the mammal is human.
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US 6,627,196 Bl 15 16 A "disorder" is any condition that would benefit from rodorubicin, streptonigrin, streptozocin, tubercidin, treatment with the anti-ErbB2 antibody. This includes ubenimex, zinostatin, zorubicin; anti-metabolites such as chronic and acute disorders or diseases including those methotrexate and 5-fiuorouracil (5-FU); folic acid analogues pathological conditions which predispose the mammal to the such as denopterin, methotrexate, pteropterin, trimetrexate; disorder in question. Non-limiting examples of disorders to 5 purine analogs such as fiudarabine, 6-mercaptopurine, be treated herein include benign and malignant tumors; thiamiprine, thioguanine; pyrimidine analogs such as leukemias and lymphoid malignancies; neuronal, glial, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, astrocytal, hypothalamic and other glandular, macrophagal, dideoxyuridine, doxifiuridine, enocitabine, fioxuridine, epithelial, stromal and blastocoelic disorders; and 5-FU; androgens such as calusterone, dromostanolone inflammatory, angiogenic and immunologic disorders. propionate, epitiostanol, mepitiostane, testolactone; anti 10 The term "therapeutically effective amount" is used to adrenals such as aminoglutethimide, mitotane, trilostane; refer to an amount having antiproliferative effect. Preferably, folic acid replenisher such as frolinic acid; aceglatone; the therapeutically effective amount has apoptotic activity, aldophosphamide glycoside; aminolevulinic acid; amsa or is capable of inducing cell death, and preferably death of crine; bestrabucil; bisantrene; edatraxate; defofamine; benign or malignant tumor cells, in particular cancer cells. demecolcine; diaziquone; elfornithine; elliptinium acetate; 15 etoglucid; gallium nitrate; hydroxyurea; lentinan; Efficacy can be measured in conventional ways, depending lonidamine; mitoguazone; mitoxantrone; mopidamol; nitra on the condition to be treated. For cancer therapy, efficacy crine; pentostatin; phenamet; pirarubicin; podophyllinic can, for example, be measured by assessing the time to acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; disease progression (TTP), or determining the response rates sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, (RR) (see Example 1, below). Therapeutically effective 20 2',2"-trichlorotriethylamine; urethan; vindesine; dacarba amount also refers to a target serum concentration, such as zine; mannomustine; mitobronitol; mitolactol; pipobroman; a trough serum concentration, that has been shown to be gacytosine; arabinoside ("Ara-C"); cyclophosphamide; effective in suppressing disease symptoms when maintained thiotepa; taxanes, e.g. paclitaxel (TAXOL®, Bristol-Myers for a period of time. Squibb Oncology, Princeton, N.J.) and docetaxel The terms "cancer" and "cancerous" refer to or describe 25 (TAXOTERE®, Rh6ne-Poulenc Rorer, Antony, France); the physiological condition in mammals that is typically chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; characterized by unregulated cell growth. Examples of can methotrexate; platinum analogs such as cisplatin and carbo cer include, but are not limited to, carcinoma, lymphoma, platin; vinblastine; platinum; etoposide (VP-16); ifosfamide; blastoma, sarcoma, and leukemia. More particular examples mitomycin C; mitoxantrone; vincristine; vinorelbine; navel- of such cancers include squamous cell cancer, small-cell 30 bine; novantrone; teniposide; daunomycin; aminopterin; lung cancer, non-small cell lung cancer, gastrointestinal xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS cancer, pancreatic cancer, glioblastoma, cervical cancer, 2000; difiuoromethylornithine (DMFO); retinoic acid; espe ovarian cancer, liver cancer, bladder cancer, hepatoma, ramicins; capecitabine; and pharmaceutically acceptable breast cancer, colon cancer, colorectal cancer, endometrial salts, acids or derivatives of any of the above. Also included carcinoma, salivary gland carcinoma, kidney cancer, pros 35 in this definition are anti-hormonal agents that act to regulate tate cancer, vulval cancer, thyroid cancer, hepatic carcinoma or inhibit hormone action on tumors such as anti-estrogens and various types of head and neck cancer. including for example tamoxifen, raloxifene, aromatase The term "cytotoxic agent" as used herein refers to a inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, substance that inhibits or prevents the function of cells keoxifene, LY 117018, onapristone, and toremifene and/or causes destruction of cells. The term is intended to 40 (Fareston); and anti-androgens such as fiutamide, 131 125 90 186 include radioactive isotopes (e.g. 1 , 1 , Y and Re ), nilutamide, bicalutamide, leuprolide, and goserelin; and chemotherapeutic agents, and toxins such as enzymatically pharmaceutically acceptable salts, acids or derivatives of active toxins of bacterial, fungal, plant or animal origin, or any of the above. fragments thereof. A "growth inhibitory agent" when used herein refers to a A "chemotherapeutic agent" is a chemical compound 45 compound or composition which inhibits growth of a cell, useful in the treatment of cancer. Examples of chemothera especially an ErbB2-overexpressing cancer cell either in peutic agents include alkylating agents such as thiotepa and vitro or in vivo. Thus, the growth inhibitory agent is one cyclosphosphamide (CYTOXAN™); alkyl sulfonates such which significantly reduces the percentage of ErbB2 over as busulfan, improsulfan and piposulfan; aziridines such as expressing cells in S phase. Examples of growth inhibitory benzodopa, carboquone, meturedopa, and uredopa; ethylen 50 agents include agents that block cell cycle progression (at a imines and methylamelamines including altretamine, place other than S phase), such as agents that induce G1 triethylenemelamine, triety lenephosphoramide, triethy 1- arrest and M-phase arrest. Classical M-phase blockers enethiophosphaoramide and trimethylolomelamine; nitro include the vincas (vincristine and vinblastine), TAXOL®, gen mustards such as chlorambucil, chlornaphazine, and topo II inhibitors such as doxorubicin, epirubicin, cholophosphamide, estramustine, ifosfamide, 55 daunorubicin, etoposide, and bleomycin. Those agents that mechlorethamine, mechlorethamine oxide hydrochloride, arrest G1 also spill over into S-phase arrest, for example, melphalan, novembichin, phenesterine, prednimustine, DNA alkylating agents such as tamoxifen, prednisone, trofosfamide, uracil mustard; nitrosureas such as dacarbazine, mechlorethamine, cisplatin, methotrexate, carmustine, chlorozotocin, fotemustine, lomustine, 5-fiuorouracil, and ara-C. Further information can be found nimustine, ranimustine; antibiotics such as aclacinomysins, 60 in The Molecular Basis of Cancer, Mendelsohn and Israel, actinomycin, authramycin, azaserine, bleomycins, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, cactinomycin, calicheamicin, carabicin, carminomycin, and antineoplastic drugs" by Murakami et al. (W B Saun carzinophilin, chromomycins, dactinomycin, daunorubicin, ders: Philadelphia, 1995), especially p. 13. The 4D5 anti detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, body (and functional equivalents thereof) can also be epirubicin, esorubicin, idarubicin, marcellomycin, 65 employed for this purpose. mitomycins, mycophenolic acid, nogalamycin, olivomycins, "Doxorubicin" is an athracycline antibiotic. The full peplomycin, potfiromycin, puromycin, quelamycin, chemical name of doxorubicin is (SS-cis)-10-[(3-amino-2,
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US 6,627,196 Bl 17 18 3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy ]-7 ,8,9,10- A "liposome" is a small vesicle composed of various te tr ahydro -6,8,11-trihydroxy-8-(hydroxy ace ty 1)-1- types of lipids, phospholipids and/or surfactant which is methoxy-5,12-naphthacenedione. useful for delivery of a drug (such as the anti-ErbB2 The term "cytokine" is a generic term for proteins antibodies disclosed herein and, optionally, a chemothera released by one cell population which act on another cell as 5 peutic agent) to a mammal. The components of the liposome intercellular mediators. Examples of such cytokines are are commonly arranged in a bilayer formation, similar to the lymphokines, monokines, and traditional polypeptide hor lipid arrangement of biological membranes. mones. Included among the cytokines are growth hormone The term "package insert" is used to refer to instructions such as human growth hormone, N-methionyl human customarily included in commercial packages of therapeutic growth hormone, and bovine growth hormone; parathyroid 10 products, that contain information about the indications, hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; usage, dosage, administration, contraindications and/or glycoprotein hormones such as follicle stimulating hormone warnings concerning the use of such therapeutic products. (FSH), thyroid stimulating hormone (TSH), and luteinizing The term "serum concentration," "serum drug hormone (LH); hepatic growth factor; fibroblast growth concentration," or "serum HERCEPTIN® anti-ErbB2 anti factor; prolactin; placental lactogen; tumor necrosis factor-a 15 body concentration" refers to the concentration of a drug, and ~; mullerian-inhibiting substance; mouse gonadotropin such as HERCEPTIN® anti-ErbB2 antibody, in the blood associated peptide; inhibin; activin; vascular endothelial serum of an animal or human patient being treated with the growth factor; integrin; thrombopoietin (TPO); nerve drug. Serum concentration of HERCEPTIN® anti-ErbB2 growth factors such as NGF-~; platelet-growth factor; trans antibody, for example, is preferably determined by immu forming growth factors (TGFs) such as TGF-a and TGF-~; 20 noassay. Preferably, the immunoassay is an ELISA accord insulin-like growth factor-! and -II; erythropoietin (EPO); ing to the procedure disclosed herein. osteoinductive factors; interferons such as interferon-a, -~, and -y; colony stimulating factors (CSFs) such as The term "peak serum concentration" refers to the maxi macrophage-CSF (M -CSF); granulocyte-macrophage-CSF mal serum drug concentration shortly after delivery of the drug into the animal or human patient, after the drug has (GM-CSF); and granulocyte-CSF (G-CSF); interleukins 25 (ILs) such as IL-l, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, been distributed throughout the blood system, but before IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as significant tissue distribution, metabolism or excretion of drug by the body has occurred. TNF-a or TNF-~; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine The term "trough serum concentration" refers to the serum drug concentration at a time after delivery of a includes proteins from natural sources or from recombinant 30 cell culture and biologically active equivalents of the native previous dose and immediately prior to delivery of the next sequence cytokines. subsequent dose of drug in a series of doses. Generally, the The term "prodrug" as used in this application refers to a trough serum concentration is a minimum sustained effica precursor or derivative form of a pharmaceutically active cious drug concentration in the series of drug administra substance that is less cytotoxic to tumor cells compared to 35 tions. Also, the trough serum concentration is frequently the parent drug and is capable of being enzymatically targeted as a minimum serum concentration for efficacy activated or converted into the more active parent form. See, because it represents the serum concentration at which e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Bio another dose of drug is to be administered as part of the chemical Society Transactions, 14, pp. 375-382, 615th treatment regimen. If the delivery of drug is by intravenous Meeting Belfast (1986) and Stella et al., "Prodrugs: A 40 administration, the trough serum concentration is most pref Chemical Approach to Targeted Drug Delivery," Directed erably attained within 1 day of a front loading initial drug Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana delivery. If the delivery of drug is by subcutaneous Press (1985). The prodrugs of this invention include, but are administration, the peak serum concentration is preferably not limited to, phosphate-containing prodrugs, attained in 3 days or less. According to the invention, the thiophosphate-containing prodrugs, sulfate-containing 45 trough serum concentration is preferably attained in 4 weeks prodrugs, peptide-containing prodrugs, D-amino acid or less, preferably 3 weeks or less, more preferably 2 weeks modified prodrugs, glycosylated prodrugs, ~-lactam or less, most preferably in 1 week or less, including 1 day or containing prodrugs, optionally substituted less using any of the drug delivery methods disclosed herein. phenoxyacetamide-containing prodrugs or optionally sub The term "intravenous infusion" refers to introduction of stituted phenylacetamide-containing prodrugs, 50 a drug into the vein of an animal or human patient over a 5-fiuorocytosine and other 5-fiuorouridine prodrugs which period of time greater than approximately 5 minutes, pref can be converted into the more active cytotoxic free drug. erably between approximately 30 to 90 minutes, although, Examples of cytotoxic drugs that can be derivatized into a according to the invention, intravenous infusion is altern a prodrug form for use in this invention include, but are not tively administered for 10 hours or less. limited to, those chemotherapeutic agents described above. 55 The term "intravenous bolus" or "intravenous push" By "solid phase" is meant a non-aqueous matrix to which refers to drug administration into a vein of an animal or the antibodies used in accordance with the present invention human such that the body receives the drug in approximately can adhere. Examples of solid phases encompassed herein 15 minutes or less, preferably 5 minutes or less. include those formed partially or entirely of glass (e.g., The term "subcutaneous administration" refers to intro controlled pore glass), polysaccharides (e.g., agarose), 60 duction of a drug under the skin of an animal or human polyacrylamides, polystyrene, polyvinyl alcohol and sili patient, preferable within a pocket between the skin and cones. In certain embodiments, depending on the context, underlying tissue, by relatively slow, sustained delivery the solid phase can comprise the well of an assay plate; in from a drug receptacle. The pocket may be created by others it is a purification column (e.g.,an affinity chroma pinching or drawing the skin up and away from underlying tography column). This term also includes a discontinuous 65 tissue. solid phase of discrete particles, such as those described in The term "subcutaneous infusion" refers to introduction U.S. Pat. No. 4,275,149. of a drug under the skin of an animal or human patient,
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US 6,627,196 Bl 19 20 preferably within a pocket between the skin and underlying extracellular domain of ErbB2 or a portion thereof, contain tissue, by relatively slow, sustained delivery from a drug ing the desired epitope. Alternatively, cells expressing receptacle for a period of time including, but not limited to, ErbB2 at their cell surface (e.g. NIH-3T3 cells transformed 30 minutes or less, or 90 minutes or less. Optionally, the to overexpress ErbB2; or a carcinoma cell line such as infusion may be made by subcutaneous implantation of a 5 SKBR3 cells, see Stancovski et al., PNAS (USA) drug delivery pump implanted under the skin of the animal 88:8691-8695 [1991]) can be used to generate antibodies. or human patient, wherein the pump delivers a predeter Other forms of ErbB2 useful for generating antibodies will mined amount of drug for a predetermined period of time, be apparent to those skilled in the art. such as 30 minutes, 90 minutes, or a time period spanning (i) Polyclonal Antibodies Polyclonal antibodies are preferably raised in animals by the length of the treatment regimen. 10 multiple subcutaneous (sc) or intraperitoneal (ip) injections The term "subcutaneous bolus" refers to drug adminis of the relevant antigen and an adjuvant. It may be useful to tration beneath the skin of an animal or human patient, conjugate the relevant antigen to a protein that is immuno where bolus drug delivery is preferably less than approxi genic in the species to be immunized, e.g., keyhole limpet mately 15 minutes, more preferably less than 5 minutes, and hemocyanin, serum albumin, bovine thyroglobulin, or soy- most preferably less than 60 seconds. Administration is 15 bean trypsin inhibitor using a bifunctional or derivatizing preferably within a pocket between the skin and underlying agent, for example, maleimidobenzoyl sulfosuccinimide tissue, where the pocket is created, for example,- by pinch ester (conjugation through cysteine residues), ing or drawing the skin up and away from underlying tissue. N -hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12 , or The term "front loading" when referring to drug admin 1 1 istration is meant to describe an initially higher dose fol 20 R N=C=NR, where Rand R are different alkyl groups. Animals are immunized against the antigen, immuno lowed by the same or lower doses at intervals. The initial genic conjugates, or derivatives by combining, e.g., 100 jAg higher dose or doses are meant to more rapidly increase the or 5 jAg of the protein or conjugate (for rabbits or mice, animal or human patient's serum drug concentration to an respectively) with 3 volumes of Freund's complete adjuvant efficacious target serum concentration. According to the 25 and injecting the solution intradermally at multiple sites. present invention, front loading is achieved by an initial dose One month later the animals are boosted with 1/s to 1/w the or doses delivered over three weeks or less that causes the original amount of peptide or conjugate in Freund's com animal's or patient's serum concentration to reach a target plete adjuvant by subcutaneous injection at multiple sites. serum trough concentration. Preferably, the initial front Seven to 14 days later the animals are bled and the serum is loading dose or series of doses is administered in two weeks assayed for antibody titer. Animals are boosted until the titer or less, more preferably in 1 week or less, including 1 day 30 plateaus. Preferably, the animal is boosted with the conju or less. Most preferably, where the initial dose is a single gate of the same antigen, but conjugated to a different dose and is not followed by a subsequent maintenance dose protein and/or through a different cross-linking reagent. for at least 1 week, the initial dose is administered in 1 day Conjugates also can be made in recombinant cell culture as or less. Where the initial dose is a series of doses, each dose protein fusions. Also, aggregating agents such as alum are is separated by at least 3 hours, but not more than 3 weeks 35 suitably used to enhance the immune response. or less, preferably 2 weeks or less, more preferably 1 week (ii) Monoclonal Antibodies or less, most preferably 1 day or less. To avoid adverse Monoclonal antibodies are obtained from a population of immune reaction to an antibody drug such as an anti-ErbB2 substantially homogeneous antibodies, i.e., the individual antibody (e.g., HERCEPTIN® anti-ErbB2 antibody) in an antibodies comprising the population are identical except for animal or patient who has not previously been treated with 40 possible naturally occurring mutations that may be present the antibody, it may be preferable to deliver initial doses of in minor amounts. Thus, the modifier "monoclonal" indi the antibody by intravenous infusion. The present invention cates the character of the antibody as not being a mixture of includes front loading drug delivery of initial and mainte discrete antibodies. nance doses by infusion or bolus administration, intrave- For example, the monoclonal antibodies may be made nously or subcutaneously. 45 using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant Published information related to anti-ErbB2 antibodies DNA methods (U.S. Pat. No. 4,816,567). includes the following issued patents and published appli In the hybridoma method, a mouse or other appropriate cations: PCT/US89/00051,published Jan. 5,1989; PCT/ host animal, such as a hamster, is immunized as hereinabove US90/02697, published May 18,1990; EU 0474727 issued 50 described to elicit lymphocytes that produce or are capable Jul. 23, 1997; DE 69031120.6, issued Jul. 23, 1997; PCT/ of producing antibodies that will specifically bind to the US97/18385, published Oct. 9 1997; SA 97/9185, issued protein used for immunization. Alternatively, lymphocytes Oct.14, 1997; U.S. Pat. No. 5,677,171,issued Oct.14, 1997; may be immunized in vitro. Lymphocytes then are fused U.S. Pat. No. 5,720,937, issued Feb. 24, 1998; U.S. Pat. No. with myeloma cells using a suitable fusing agent, such as 5,720,954, issued Feb. 24, 1998; U.S. Pat. No. 5,725,856, 55 polyethylene glycol, to form a hybridoma cell (Goding, issued Mar. 10, 1998; U.S. Pat. No. 5,770,195, issued Jun. Monoclonal Antibodies: Principles and Practice, pp.59-103 23, 1998; U.S. Pat. No. 5,772,997, issued Jun. 30, 1998; [Academic Press, 1986]). PCT/US98/2626, published Dec. 10, 1998; and PCT/US99/ The hybridoma cells thus prepared are seeded and grown 06673, published Mar. 26, 1999, each of which patents and in a suitable culture medium that preferably contains one or publications is herein incorporated by reference in its 60 more substances that inhibit the growth or survival of the entirety. unfused, parental myeloma cells. For example, if the paren tal myeloma cells lack the enzyme hypoxanthine guanine II. Production of anti-ErbB2 Antibodies phosphoribosyl transferase (HGPRT or HPRT), the culture A description follows as to exemplary techniques for the medium for the hybridomas typically will include production of the antibodies used in accordance with the 65 hypoxanthine, aminopterin, and thymidine (HAT medium), present invention. The ErbB2 antigen to be used for pro which substances prevent the growth of HGPRT-deficient duction of antibodies may be, e.g., a soluble form of the cells.
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US 6,627,196 Bl 21 22 Preferred myeloma cells are those that fuse efficiently, Nuc. Acids. Res., 21:2265-2266 [1993]). Thus, these tech support stable high-level production of antibody by the niques are viable alternatives to traditional monoclonal selected antibody-producing cells, and are sensitive to a antibody hybridoma techniques for isolation of monoclonal medium such as HAT medium. Among these, preferred antibodies. myeloma cell lines are murine myeloma lines, such as those 5 The DNA also may be modified, for example, by substi- derived from MOPC-21 and MPC- 11 mouse tumors avail tuting the coding sequence for human heavy- and light-chain able from the Salk Institute Cell Distribution Center, San constant domains in place of the homologous murine Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells avail sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. able from the American Type Culture Collection, Rockville, Natl. Acad. Sci. USA, 81:6851 [1984]), or by covalently Md. USA. Human myeloma and mouse-human heteromy 10 joining to the immunoglobulin coding sequence all or part of eloma cell lines also have been described for the production the coding sequence for a non-immunoglobulin polypeptide. of human monoclonal antibodies (Kozbor, J. Immunol., Typically such non-immunoglobulin polypeptides are 133:3001 (1984); Brodeur et al., Monoclonal Antibody Pro substituted for the constant domains of an antibody, or they duction Techniques and Applications, pp. 51-63 [Marcel are substituted for the variable domains of one antigen- Dekker, Inc., New York, 1987]). 15 combining site of an antibody to create a chimeric bivalent Culture medium in which hybridoma cells are growing is antibody comprising one antigen-combining site having assayed for production of monoclonal antibodies directed specificity for an antigen and another antigen-combining site against the antigen. Preferably, the binding specificity of having specificity for a different antigen. monoclonal antibodies produced by hybridoma cells-is (iii) Humanized and Human Antibodies determined by immunoprecipitation or by an in vitro binding 20 Methods for humanizing non-human antibodies are well assay, such as radioimmunoassay (RIA) or enzyme-linked known in the art. Preferably, a humanized antibody has one immunoabsorbent assay (ELISA). or more amino acid residues introduced into it from a source The binding affinity of the monoclonal antibody can, for which is non-human. These non-human amino acid residues example, be determined by the Scatchard analysis of Mun are often referred to as "import" residues, which are typi- son et al., Anal. Biochem., 107:220 (1980). 25 cally taken from an "import"variable domain. Humanization After hybridoma cells are identified that produce antibod can be essentially performed following the method of Winter ies of the desired specificity, affinity, and/or activity, the and co-workers (Jones et al., Nature, 321:522-525 (1986); clones may be subcloned by limiting dilution procedures and Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen grown by standard methods (Goding, Monoclonal Antibod et al., Science 239:1534-1536 [1988]), by substituting ies: Principles and Practice, pp.59-103 [Academic Press, 30 rodent CDRs or CDR sequences for the corresponding 1986]). Suitable culture media for this purpose include, for sequences of a human antibody. Accordingly, such "human- example, D-M EM or RPMI-1640 medium. In addition, the ized" antibodies are chimeric antibodies (U.S. Pat. No. hybridoma cells may be grown in vivo as ascites tumors in 4,816,567) wherein substantially less than an intact human an animal. variable domain has been substituted by the corresponding The monoclonal antibodies secreted by the subclones are 35 sequence from a non-human species. In practice, humanized suitably separated from the culture medium, ascites fluid, or antibodies are typically human antibodies in which some serum by conventional imnmunoglobulin purification pro CDR residues and possibly some FR residues are substituted cedures such as, for example, protein A-Sepharose, by residues from analogous sites in rodent antibodies. hydroxylapatite chromatography, gel electrophoresis, The choice of human variable domains, both light and dialysis, or affinity chromatography. 40 heavy, to be used in making the humanized antibodies is DNA encoding the monoclonal antibodies is readily iso very important to reduce antigenicity. According to the lated and sequenced using conventional procedures (e.g., by so-called "best-fit" method, the sequence of the variable using oligonucleotide probes that are capable of binding domain of a rodent antibody is screened against the entire specifically to genes encoding the heavy and light chains of library of known human variable-domain sequences. The murine antibodies). The hybridoma cells serve as a preferred 45 human sequence which is closest to that of the rodent is then source of such DNA Once isolated, the DNA may be placed accepted as the human framework region (FR) for the into expression vectors, which are then transfected into host humanized antibody (Sims et al., J. Immunol., 151:2296 cells such as E. coli cells, simian COS cells, Chinese (1993); Chothia et al., J. Mol. Bioi., 196:901 [1987]). Hamster Ovary (CHO) cells, or myeloma cells that do not Another method uses a particular framework region derived otherwise produce immunoglobulin protein, to obtain the 50 from the consensus sequence of all human antibodies of a synthesis of monoclonal antibodies in the recombinant host particular subgroup of light or heavy chains. The same cells. Review articles on recombinant expression in bacteria framework may be used for several different humanized of DNA encoding the antibody include Skerra et al., Curr. antibodies (Carteret al., Proc. Natl. A cad. Sci. USA, 89:4285 Opinion in Immunol., 5:256-262 (1993) and Pluckthun, (1992); Presta et al., J. Immnol., 151:2623 [1993]). Immunol. Revs., 130:151-188 (1992). 55 It is further important that antibodies be humanized with In a further embodiment, antibodies or antibody frag retention of high affinity for the antigen and other favorable ments can be isolated from antibody phage libraries gener biological properties. To achieve this goal, according to a ated using the techniques described in McCafferty et al., preferred method, humanized antibodies are prepared by a Nature, 348:552-554 (1990). Clackson et al., Nature, process of analysis of the parental sequences and various 352:624-628 (1991) and Marks et al., J. Mol. Bioi., 60 conceptual humanized products using three-dimensional 222:581-597 (1991) describe the isolation of murine and models of the parental and humanized sequences. Three- human antibodies, respectively, using phage libraries. Sub dimensional immunoglobulin models are commonly avail sequent publications describe the production of high affinity able and are familiar to those skilled in the art. Computer (nM range) human antibodies by chain shuffling (Marks et programs are available which illustrate and display probable al., Bio/Technology, 10:779-783 [1992]), as well as combi- 65 three-dimensional conformational structures of selected can natorial infection and in vivo recombination as a strategy for didate immunoglobulin sequences. Inspection of these dis- constructing very large phage libraries (Waterhouse et al., plays permits analysis of the likely role of the residues in the
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US 6,627,196 Bl 23 24 functioning of the candidate immunoglobulin sequence, i.e., Methods for making bispecific antibodies are known in the analysis of residues that influence the ability of the the art. Traditional production of full length bispecific anti candidate immunoglobulin to bind its antigen. In this way, bodies is based on the coexpression of two immunoglobulin FR residues can be selected and combined from the recipient heavy chain-light chain pairs, where the two chains have and import sequences so that the desired antibody 5 different specificities (Millstein et al., Nature, 305:537-539 characteristic, such as increased affinity for the target [1983]). Because of the random assortment of immunoglo antigen(s), is achieved. In general, the CDR residues are bulin heavy and light chains, these hybridomas (quadromas) directly and most substantially involved in influencing anti produce a potential mixture of 10 different antibody gen binding. molecules, of which only one has the correct bispecific Alternatively, it is now possible to produce transgenic 10 structure. Purification of the correct molecule, which is animals (e.g., mice) that are capable, upon immunization, of usually done by affinity chromatography steps, is rather producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For cumbersome, and the product yields are low. Similar pro example, it has been described that the homozygous deletion cedures are disclosed in WO 93/08829, and in Traunecker et of the antibody heavy-chain joining region (JH) gene in al., EMBO J., 10:3655-3659 (1991). chimeric and germ-line mutant mice results in complete 15 According to a different approach, antibody variable inhibition of endogenous antibody production. Transfer of domains with the desired binding specificities (antibody the human germ-line immunoglobulin gene array in such antigen combining sites) are fused to immunoglobulin con germ-line mutant mice will result in the production of stant domain sequences. The fusion preferably is with an human antibodies upon antigen challenge. See, e.g., Jakobo immunoglobulin heavy chain constant domain, comprising vits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); 20 at least part of the hinge, CH2, and CH3 regions. It is Jakobovits et al., Nature, 362:255-258 (1993); Brugger preferred to have the first heavy-chain constant region mann et al., Year in Immuno., 7:33 (1993). Human antibod- (CH1) containing the site necessary for light chain binding, ies can also be derived from phage-display libraries present in at least one of the fusions. DNAs encoding the (Hoogenboom et al.,J. Mol. Bioi., 227:381 (1991); Marks et immunoglobulin heavy chain fusions and, if desired, the al., J. Mol. Bioi., 222:581-597 [1991]). 25 immunoglobulin light chain, are inserted into separate (iv) Antibody Fragments expression vectors, and are co-transfected into a suitable Various techniques have been developed for the produc host organism. This provides for great flexibility in adjusting tion of antibody fragments. Traditionally, these fragments the mutual proportions of the three polypeptide fragments in were derived via proteolytic digestion of intact antibodies embodiments when unequal ratios of the three polypeptide (see, e.g., Morimoto et al. , Journal of Biochemical and 30 chains used in the construction provide the optimum yields. Biophysical Methods 24:107-117(1992) and Brennan et al., It is, however, possible to insert the coding sequences for Science, 229:81 [1985]). However, these fragments can now two or all three polypeptide chains in one expression vector be produced directly by recombinant host cells. For when the expression of at least two polypeptide chains in example, the antibody fragments can be isolated from the equal ratios results in high yields or when the ratios are of antibody phage libraries discussed above. Alternatively, 35 no particular significance. Fab'-SH fragments can be directly recovered from E. coli In a preferred embodiment of this approach, the bispecific
and chemically coupled to form F(ab')2 fragments (Carteret antibodies are composed of a hybrid immunoglobulin heavy al., Bio/Technology 10: 163-167 [1992]). According to chain with a first binding specificity in one arm, and a hybrid
another approach, F(ab')2 fragments can be isolated directly immunoglobulin heavy chain-light chain pair (providing a from recombinant host cell culture. Other techniques for the 40 second binding specificity) in the other arm. It was found production of antibody fragments will be apparent to the that this asymmetric structure facilitates the separation of the skilled practitioner. In other embodiments, the antibody of desired bispecific compound from unwanted immunoglobu choice is a single chain Fv fragment (scFv). See WO lin chain combinations, as the presence of an immunoglo 93/16185. bulin light chain in only one half of the bispecific molecule (v) Bispecific Antibodies 45 provides for a facile way of separation. This approach is Bispecific antibodies are antibodies that have binding disclosed in WO 94/04690. For further details of generating specificities for at least two different epitopes. Exemplary bispecific antibodies see, for example, Suresh et al., Methods bispecific antibodies may bind to two different epitopes of in Enzymology, 121:210 (1986). the ErbB2 protein. For example, one arm may bind an According to another approach described in W096/ epitope in Domain 1 of ErbB2 such as the 7C2/7F3 epitope, 50 27011, the interface between a pair of antibody molecules the other may bind a different ErbB2 epitope, e.g. the 4D5 can be engineered to maximize the percentage of het epitope. Other such antibodies may combine an ErbB2 erodimers which are recovered from recombinant cell cul binding site with binding site(s) for EGFR, ErbB3 and/or ture. The preferred interface comprises at least a part of the ErbB4. Alternatively, an anti-ErbB2 arm may be combined CH3 domain of an antibody constant domain. In this method, with an arm which binds to a triggering molecule on a 55 one or more small amino acid side chains from the interface leukocyte such as aT-cell receptor molecule (e.g. CD2 or of the first antibody molecule are replaced with larger side CD3), or Fe receptors for IgG (FcyR), such as FcyRI chains (e.g. tyrosine or tryptophan). Compensatory "cavi (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus ties" of identical or similar size to the large side chain(s) are cellular defense mechanisms to the ErbB2-expressing cell. created on the interface of the second antibody molecule by Bispecific antibodies may also be used to localize cytotoxic 60 replacing large amino acid side chains with smaller ones agents to cells which express ErbB2. These antibodies (e.g. alanine or threonine). This provides a mechanism for possess an ErbB2-binding arm and an arm which binds the increasing the yield of the heterodimer over other unwanted cytotoxic agent (e.g. saporin, anti-interferon-a, vinca end-products such as homodimers. alkaloid, ricin A chain, methotrexate or radioactive isotope Bispecific antibodies include cross-linked or "heterocon- hapten). Bispecific antibodies can be prepared as full length 65 jugate" antibodies. For example, one of the antibodies in the
antibodies or antibody fragments (e.g. F( ab')2 bispecific heteroconjugate can be coupled to avidin, the other to biotin. antibodies). Such antibodies have, for example, been proposed to target
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US 6,627,196 Bl 25 26 immune system cells to unwanted cells (U.S. Pat. No. 7 AAD uptake is assessed relative to control. The preferred 4,676,980), and for treatment of HIV infection (WO assay is the "PI uptake assay using BT474 cells". According 91!00360, WO 92/200373, and EP 03089). Heteroconjugate to this assay, BT474 cells (which can be obtained from the antibodies may be made using any convenient cross-linking American Type Culture Collection [Rockville, Md.]) are methods. Suitable cross-linking agents are well known in the 5 cultured in Dulbecco's Modified Eagle Medium (D-MEM) art, and are disclosed in U.S. Pat. No. 4,676,980, along with :Ham's F-12 (50:50) supplemented with 10% heat- a number of cross-linking techniques. inactivated FBS (Hyclone) and 2 mM L-glutamine. (Thus, Techniques for generating bispecific antibodies from anti the assay is performed in the absence of complement and body fragments have also been described in the literature. immune effector cells). The BT474 cells are seeded at a For example, bispecific antibodies can be prepared using 6 10 density of 3x10 per dish in 100x20 mm dishes and allowed chemical linkage. Brennan et al., Science, 229: 81 (1985) to attach overnight. The medium is then removed and describe a procedure wherein intact antibodies are pro replaced with fresh medium alone or medium containing 10 teolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol com ,ug!ml of the appropriate MAb. The cells are incubated for a plexing agent sodium arsenite to stabilize vicinal dithiols 3 day time period. Following each treatment, monolayers are and prevent intermolecular disulfide formation. The Fab' 15 washed with PBS and detached by trypsinization. Cells are fragments generated are then converted to thionitrobenzoate then centrifuged at 1200 rpm for 5 minutes at 4° C., the 2 (TNB) derivatives. One of the Fab'-TNB derivatives is then pellet resuspended in 3 ml ice cold Ca + binding buffer (10 reconverted to the Fab'-thiol by reduction with mercaptoet mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaC12 ) and hylamine and is mixed with an equimolar amount of the aliquoted into 35 mm strainer-capped 12x75 tubes (1 ml per other Fab'-TNB derivative to form the bispecific antibody. 20 tube, 3 tubes per treatment group) for removal of cell The bispecific antibodies produced can be used as agents for clumps. Tubes then receive PI (10 ,ug/ml). Samples may be the selective immobilization of enzymes. analyzed using a FACSCAN™ flow cytometer and FAC Recent progress has facilitated the direct recovery of SCONVERT™ CellQuest software (Becton Dickinson). Fab'-SH fragments from E. coli, which can be chemically Those antibodies which induce statistically significant levels coupled to form bispecific antibodies. Shalaby et al.,J. Exp. 25 of cell death as determined by PI uptake are selected. Med., 175: 217-225 (1992) describe the production of a In order to select for antibodies which induce apoptosis, fully humanized bispecific antibody F(ab')2 molecule. Each an "annexin binding assay using BT474 cells" is available. Fab' fragment was separately secreted from E. coli and The BT474 cells are cultured and seeded in dishes as subjected to directed chemical coupling in vitro to form the discussed in the preceding paragraph. The medium is then bispecific antibody. The bispecific antibody thus formed was 30 removed and replaced with fresh medium alone or medium able to bind to cells overexpressing the ErbB2 receptor and containing 10,ug/ml of the MAb. Following a three day normal human T cells, as well as trigger the lytic activity of incubation period, monolayers are washed with PBS and human cytotoxic lymphocytes against human breast tumor detached by trypsinization. Cells are then centrifuged, resus 2 targets. pended in Ca + binding buffer and aliquoted into tubes as Various techniques for making and isolating bispecific 35 discussed above for the cell death assay. Tubes then receive antibody fragments directly from recombinant cell culture labeled annexin (e.g. annexin V-FTIC) (1 ,ug/ml). Samples have also been described. For example, bispecific antibodies may be analyzed using a FACSCAN™ flow cytometer and have been produced using leucine zippers. Kostelny et al., J. FACSCONVERT™ CellQuest software (Becton Immunol., 148(5):1547-1553 (1992). The leucine zipper Dickinson). Those antibodies which induce statistically sig peptides from the Fos and Jun proteins were linked to the 40 nificant levels of annexin binding relative to control are Fab' portions of two different antibodies by gene fusion. The selected as apoptosis-inducing antibodies. antibody homodimers were reduced at the hinge region to In addition to the annexin binding assay, a "DNA staining form monomers and then re-oxidized to form the antibody assay using BT474 cells" is available. In order to perform heterodimers. This method can also be utilized for the this assay, BT474 cells which have been treated with the production of antibody homodimers. The "diabody" tech 45 antibody of interest as described in the preceding two nology described by Hollinger et al., Proc. Natl. Acad. Sci. paragraphs are incubated with 9,ug/ml HOECHST 33342™ USA, 90:6444-6448 (1993) has provided an alternative for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flow mechanism for making bispecific antibody fragments. The cytometer (Coulter Corporation) using MOD FIT LT™ soft fragments comprise a heavy-chain variable domain (VH) ware (Verity Software House). Antibodies which induce a connected to a light-chain variable domain (VL) by a linker 50 change in the percentage of apoptotic cells which is 2 fold which is too short to allow pairing between the two domains or greater (and preferably 3 fold or greater) than untreated on the same chain. Accordingly, the VHand VL domains of cells (up to 100% apoptotic cells) may be selected as one fragment are forced to pair with the complementary VL pro-apoptotic antibodies using this assay. and VH domains of another fragment, thereby forming two To screen for antibodies which bind to an epitope on antigen-binding sites. Another strategy for making bispecific 55 ErbB2 bound by an antibody of interest, a routine cross antibody fragments by the use of single-chain Fv (sFv) blocking assay such as that described in Antibodies, A dimers has also been reported. See Gruber et al., J. Laboratory Manual, Cold Spring Harbor Laboratory, Ed Immunol., 152:5368 (1994). Harlow and David Lane (1988), can be performed. Antibodies with more than two valencies are contem Alternatively, epitope mapping can be performed by meth- plated. For example, trispecific antibodies can be prepared. 60 ods known in the art. Tutt et al., J. Immunol. 147: 60 (1991). To identify anti-ErbB2 antibodies which inhibit growth of (vi) Screening for Antibodies With the Desired Properties SKBR3 cells in cell culture by 50-100%, the SKBR3 assay Techniques for generating antibodies have been described described in WO 89/06692 can be performed. According to above. Those antibodies having the characteristics described this assay, SKBR3 cells are grown in a 1:1 mixture of F12 herein are selected. 65 and DMEM medium supplemented with 10% fetal bovine To select for antibodies which induce cell death, loss of serum, glutamine and penicillinstreptomycin. The SKBR3 membrane integrity as indicated by, e.g., PI, trypan blue or cells are plated at 20,000 cells in a 35 mm cell culture dish
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US 6,627,196 Bl 27 28 (2 mls/35 mm dish) 2.5,ug/ml of the anti-ErbB2 antibody is conjugate from the circulation using a clearing agent and added per dish. After six days, the number of cells, com then administration of a "ligand" (e.g. avidin) which 1s pared to untreated cells are counted using an electronic conjugated to a cytotoxic agent (e.g. a radionucleotide). COULTER TM cell counter. Those antibodies which inhibit (ix) Immunoliposomes growth of the SKBR3 cells by 50-100% are selected for 5 The anti-ErbB2 antibodies disclosed herein may also be combination with the apoptotic antibodies as desired. formulated as inununoliposomes. Liposomes containing the (vii) Effector Function Engineering antibody are prepared by methods known in the art, such as It may be desirable to modify the antibody of the inven described in Epstein et al., Proc. Natl. Acad. Sci. USA, tion with respect to effector function, so as to enhance the 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, effectiveness of the antibody in treating cancer, for example. 10 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544, For example, cysteine residue(s) may be introduced in the Fe 545. Liposomes with enhanced circulation time are dis- region, thereby allowing interchain disulfide bond formation closed in U.S. Pat. No. 5,013,556. in this region. The homodimeric antibody thus generated Particularly useful liposomes can be generated by the may have improved internalization capability and/or reverse phase evaporation method with a lipid composition increased complement-mediated cell killing and antibody- 15 comprising phosphatidylcholine, cholesterol and PEG dependent cellular cytotoxicity (ADCC). See Caron et al., J. derivatized phosphatidylethanolamine (PEG-PE). S Lipo- Exp Med. 176:1191-1195 (1992) and Shapes, B. J. Immu somes are extruded through filters of defined pore size to nol. 148:2918-2922 (1992). Homodimeric antibodies with yield liposomes with the desired diameter. Fab' fragments of enhanced anti-tumor activity may also be prepared using the antibody of the present invention can be conjugated to heterobifunctional cross-linkers as described in Wolff et al. 20 the liposomes as described in Martin et al., J. Bioi. Chern. Cancer Research 53:2560-2565 (1993). Alternatively, an 257: 286-288 (1982) via a disulfide interchange reaction. A antibody can be engineered which has dual Fe regions and chemotherapeutic agent is optionally contained within the may thereby have enhanced complement lysis and ADCC liposome. See Gabizon et al., J. National Cancer Inst. capabilities. See Stevenson et al. Anti-Cancer Drug Design 81(19)1484 (1989). 3:219-230 (1989). 25 (x) Antibody Dependent Enzyme Mediated Prodrug (viii) Immunoconjugates Therapy (ADEPT) The invention also pertains to immunoconjugates com The antibodies of the present invention may also be used prising the antibody described herein conjugated to a cyto in ADEPT by conjugating the antibody to a prodrug toxic agent such as a chemotherapeutic agent, toxin (e.g. an activating enzyme which converts a prodrug (e.g. a peptidyl enzymatically active toxin of bacterial, fungal, plant or 30 chemotherapeutic agent, see WO 81/01145) to an active animal origin, or fragments thereof), or a radioactive isotope anti-cancer drug. See, for example, WO 88/07378 and U.S. (i.e., a radioconjugate ). Pat. No. 4,975,278. Chemotherapeutic agents useful in the generation of such The enzyme component of the immunoconjugate useful immunoconjugates have been described above. Enzymati for ADEPT includes any enzyme capable of acting on a cally active toxins and fragments thereof which can be used 35 prodrug in such a way so as to covert it into its more active, include diphtheria A chain, nonbinding active fragments of cytotoxic form. diphtheria toxin, exotoxin A chain (from Pseudomonas Enzymes that are useful in the method of this invention aeruginosa), ricin A chain, abrinAchain, modeccinAchain, include, but are not limited to, alkaline phosphatase useful alpha-sarcin, Aleurites fordii proteins, dianthin proteins, for converting phosphate-containing prodrugs into free Phytolaca americana proteins (PAPI, PAPII, and PAP-S), 40 drugs; arylsulfatase useful for converting sulfate-containing momordica charantia inhibitor, curcin, crotin, sapaonaria prodrugs into free drugs; cytosine deaminase useful for officinalis inhibitor, gelonin, mitogellin, restrictocin, converting non-toxic 5-fiuorocytosine into the anti-cancer phenomycin, enomycin and the tricothecenes. A variety of drug, 5-fiuorouracil; proteases, such as serratia protease, radionuclides are available for the production of radiocon thermolysin, subtilisin, carboxypeptidases and cathepsins 212 131 jugated anti-ErbB2 antibodies. Examples include Bi, I, 45 (such as cathepsins B and L), that are useful for converting 131In, goy and 1s6Re. peptide-containing prodrugs into free drugs; Conjugates of the antibody and cytotoxic agent are made D-alanylcarboxypeptidases, useful for converting prodrugs using a variety of bifunctional protein coupling agents such that contain D-amino acid substituents; carbohydrate as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), cleaving enzymes such as ~-galactosidase and neuramini iminothiolane (IT), bifunctional derivatives of imidoesters 50 dase useful for converting glycosylated prodrugs into free (such as dimethyl adipimidate HCL), active esters (such as drugs; ~-lactamase useful for converting drugs derivatized disuccinimidyl suberate), aldehydes (such as with ~-lactams into free drugs; and penicillin amidases, such glutareldehyde ), bis-azido compounds (such as bis as penicillin V amidase or penicillin G amidase, useful for (p-azidobenzoyl) hexanediamine), bis-diazonium deriva converting drugs derivatized at their amine nitrogens with tives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine ), 55 phenoxyacetyl or phenylacetyl groups, respectively, into diisocyanates (such as tolyene 2,6-diisocyanate), and his free drugs. Alternatively, antibodies with enzymatic activity, active fluorine compounds (such as 1,5-difiuoro-2,4- also known in the art as "abzymes", can be used to convert dinitrobenzene). For example, a ricin immunotoxin can be the prodrugs of the invention into free active drugs (see, e.g., prepared as described in Vitetta et al. Science 238: 1098 Massey, Nature 328: 457458 [1987]). Antibody-abzyme (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3- 60 conjugates can be prepared as described herein for delivery methyldiethylene triaminepentaacetic acid (MX-DTPA) is of the abzyme to a tumor cell population. an exemplary chelating agent for conjugation of radio nucle The enzymes of this invention can be covalently bound to otide to the antibody. See WO 94/11026. the anti-ErbB2 antibodies by techniques well known in the In another embodiment, the antibody may be conjugated art such as the use of the heterobifunctional crosslinking to a "receptor" (such streptavidin) for utilization in tumor 65 reagents discussed above. Alternatively, fusion proteins pretargeting wherein the antibody-receptor conjugate is comprising at least the antigen binding region of an antibody administered to the patient, followed by removal of unbound of the invention linked to at least a functionally active
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US 6,627,196 Bl 29 30 portion of an enzyme of the invention can be constructed centrifugation or ultrafiltration. Carter et al., Bio/Technology using recombinant DNA techniques well known in the art 10:163-167 (1992) describe a procedure for isolating anti (see, e.g., Neuberger et al., Nature, 312: 604-608 [1984]). bodies which are secreted to the periplasmic space of E. coli. (xi) Antibody-salvage Receptor Binding Epitope Fusions Briefly, cell paste is thawed in the presence of sodium In certain embodiments of the invention, it may be 5 acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride desirable to use an antibody fragment, rather than an intact (PMSF) over about 30 min. Cell debris can be removed by antibody, to increase tumor penetration, for example. In this centrifugation. Where the antibody is secreted into the case, it may be desirable to modify the antibody fragment in medium, supernatants from such expression systems are order to increase its serum half life. This may be achieved, preferably first concentrated using a commercially available for example, by incorporation of a salvage receptor binding 10 protein concentration filter, for example, an Amicon or epitope into the antibody fragment (e.g. by mutation of the Millipore Pellicon ultrafiltration unit. A protease inhibitor appropriate region in the antibody fragment or by incorpo such as PMSF may be included in any of the foregoing steps rating the epitope into a peptide tag that is then fused to the to inhibit proteolysis and antibiotics may be included to antibody fragment at either end or in the middle, e.g., by prevent the growth of adventitious contaminants. DNA or peptide synthesis). 15 The antibody composition prepared from the cells can be A systematic method for preparing such an antibody purified using, for example, hydroxylapatite variant having an increased in vivo half-life comprises chromatography, gel electrophoresis, dialysis, and affinity several steps. The first involves identifying the sequence and chromatography, with affinity chromatography being the conformation of a salvage receptor binding epitope of an Fe preferred purification technique. The suitability of protein A region of an IgG molecule. Once this epitope is identified, 20 as an affinity ligand depends on the species and isotype of the sequence of the antibody of interest is modified to any immunoglobulin Fe domain that is present in the anti include the sequence and conformation of the identified body. Protein A can be used to purify antibodies that are binding epitope. After the sequence is mutated, the antibody based on human y1, y2, or y4 heavy chains (Lindmark et al., variant is tested to see if it has a longer in vivo half-life than J. Immunol. Meth. 62:1-13 [1983]). Protein G is recom- that of the original antibody. If the antibody variant does not 25 mended for all mouse isotypes and for human y3 (Gusset al., have a longer in vivo half-life upon testing, its sequence is EMBO J. 5:15671575 [1986]). The matrix to which the further altered to include the sequence and conformation of affinity ligand is attached is most often agarose, but other the identified binding epitope. The altered antibody is tested matrices are available. Mechanically stable matrices such as for longer in vivo half-life, and this process is continued controlled pore glass or poly(styrenedivinyl)benzene allow until a molecule is obtained that exhibits a longer in vivo 30 for faster flow rates and shorter processing times than can be half-life. achieved with agarose. Where the antibody comprises a CH3 The salvage receptor binding epitope being thus incorpo domain, the Bakerbond ABX™ resin (J. T. Baker, rated into the antibody of interest is any suitable such Phillipsburg, N.J.) is useful for purification. Other tech epitope as defined above, and its nature will depend, e.g., on niques for protein purification such as fractionation on an the type of antibody being modified. The transfer is made 35 ion-exchange column, ethanol precipitation, Reverse Phase such that the antibody of interest still possesses the biologi HPLC, chromatography on silica, chromatography on hep cal activities described herein. arin SEPHAROSE™ chromatography on an anion or cation The epitope preferably constitutes a region wherein any exchange resin (such as a polyaspartic acid column), one or more amino acid residues from one or two loops of chromatofocusing, SDS-PAGE, and ammonium sulfate pre- a Fe domain are transferred to an analogous position of the 40 cipitation are also available depending on the antibody to be antibody fragment. Even more preferably, three or more recovered. residues from one or two loops of the Fe domain are Following any preliminary purification step(s), the mix transferred. Still more preferred, the epitope is taken from ture comprising the antibody of interest and contaminants the CH2 domain of the Fe region (e.g., of an IgG) and may be subjected to low pH hydrophobic interaction chro- transferred to the CH1, CH3, or VH, region, or more than 45 matography using an elution buffer at a pH between about one such region, of the antibody. Alternatively, the epitope 2.5-4.5, preferably performed at low salt concentrations is taken from the CH2 domain of the Fe region and trans (e.g. from about 0-0.25M salt). ferred to the CL region or VL region, or both, of the antibody III. Determination of anti-ErbB2 Antibody Concentration in fragment. Serum In one most preferred embodiment, the salvage receptor 50 The following non-limiting assay is useful for determin- binding epitope comprises the sequence (5' to 3'): PKNS ing the presence of and to quantitate the amount of specific SMISNTP (SEQ ID N0:3), and optionally further comprises rhuMAb HER2 (humanized anti-p185HER2 monoclonal a sequence selected from the group consisting of HQSLGTQ antibody, including HERCEPTIN® anti-ErbB2 antibody) in (SEQ ID N0:4), HQNLSDGK (SEQ ID N0:5), HQNIS a body fluid of a mammal including, but not limited to, DGK (SEQ ID N0:6), or VISSHLGQ (SEQ ID N0:7), 55 serum, amniotic fluid, milk, umbilical cord serum, ocular particularly where the antibody fragment is a Fab or F(ab')2 . aqueous and vitreous liquids, and ocular vitreous gel. In another most preferred embodiment, the salvage receptor Plate Binding Activity Assay for rhuMAb HER2 binding epitope is a polypeptide containing the sequence(s) (Humanized Anti-p185HER2 Monoclonal Antibody (5' to 3'): HQNLSDGK (SEQ ID N0:5), HQNISDGK (SEQ The method of assaying rhuMAb HER2 described herein ID N0:6), or VISSHLGQ (SEQ ID N0:7) and the sequence: 60 is meant as an example of such a method and is not meant PKNSSMISNTP (SEQ ID N0:3). to be limiting. A standardized preparation of rhuMAb HER2 (xii) Purification of anti-ErbB2 Antibody (Genentech, Inc., South San Francisco, Calif.), controls, and When using recombinant techniques, the antibody can be serum samples were diluted with Assay Diluent (PBS/0.5% produced intracellularly, in the periplasmic space, or directly BSN0.05% Polysorbate 20/0.01% Thimerosal). The dilu- secreted into the medium. If the antibody is produced 65 tions of standardized rhuMAb HER2 were prepared to span intracellularly, as a first step, the particulate debris, either a range of concentrations useful for a standard curve. The host cells or lysed fragments, is removed, for example, by samples were diluted to fall within the standard curve.
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US 6,627,196 Bl 31 32 An aliquot of Coat Antigen in Coating buffer ties that do not adversely affect each other. For example, it (recombinant p185HER2 (Genentech, Inc.) in 0.05 M sodium may be desirable to further provide antibodies which bind to carbonate buffer) was added to each well of a microtiter EGFR, ErbB2 (e.g. an antibody which binds a different plate and incubated at 2-8° C. for 12-72 hours. The coatin epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial solution was removed and each well was washed six times 5 growth factor (VEGF) in the one formulation. Alternatively, with water, then blotted to remove excess water. or in addition, the composition may comprise a cytotoxic An aliquot of Assay Diluent was added to each well and agent, cytokine or growth inhibitory agent. Such molecules incubated for 1-2 hours at ambient temperature with agita are suitably present in combination in amounts that are tion. The wells were washed as in the previous step. effective for the purpose intended. Aliquots of diluted standard, control and sample solutions 10 The active ingredients may also be entrapped in micro- were added to the wells and incubated at ambient tempera capsules prepared, for example, by coacervation techniques ture for 1 hour with agitation to allow binding of the or by interfacial polymerization, for example, hydroxym antibody to the coating antigen. The wells are washed again ethylcellulose or gelatin-microcapsules and poly with water as in previous steps. (methylmethacylate) microcapsules, respectively, in colloi- Horse radish peroxidase-conjugate (HRP-conjugate, Goat 15 dal drug delivery systems (for example, liposomes, albumin anti-human IgG Fe conjugated to horseradish peroxidase; microspheres, microemulsions, nano-particles and Organon Teknika catalog #55253 or equivalent) was diluted nanocapsules) or in macroemulsions. Such techniques are with Assay Diluent to yield an appropriate optical density disclosed in Remington's Pharmaceutical Sciences 16th range between the highest and lowest standards. An aliquot edition, Osol, A Ed. (1980). of the HRP-conjugate solution was added to each well and 20 The formulations to be used for in vivo administration incubated at ambient temperature for 1 hour with agitation. must be sterile. This is readily accomplished by filtration The wells were washed with water as in previous steps. through sterile filtration membranes. An aliquot of Substrate Solution (a-phenylenediamine Sustained-release preparations may be prepared. Suitable (OPD) 5 mg tablet (Sigma P6912 or equivalent) in 12.5 ml examples of sustained-release preparations include semiper 4 mM H2 0 2 in PBS) was added to each well and incubated 25 meable matrices of solid hydrophobic polymers containing for a sufficient period of time (approximately 8-10 minutes) the antibody, which matrices are in the form of shaped in the dark at ambient temperature to allow color develop articles, e.g. films, or microcapsules. Examples of sustained ment. The reaction was stopped with an aliquot of 4.5 N release matrices include polyesters, hydrogels (for example, sulfuric acid. Optical density was read at 490-492 nm for poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), detection absorbance and 405 nm for reference absorbance. 30 polylactides (U.S. Pat. No. 3,773,919), copolymers of The standard curve data are plotted and the results for the L-glutamic acid and y ethyl-L-glutamate, non-degradable controls and samples are determined from the standard ethylene-vinyl acetate, degradable lactic acid-glycolic acid curve. copolymers such as the LUPRON DEPOT™ (injectable IV. Pharmaceutical Formulations microspheres composed of lactic acid-glycolic acid copoly- 35 mer and leuprolide acetate), and poly-D-( -)-3- Therapeutic formulations of the antibodies used in accor hydroxybutyric acid. While polymers such as ethylene-vinyl dance with the present invention are prepared for storage by acetate and lactic acid-glycolic acid enable release of mol mixing an antibody having the desired degree of purity with ecules for over 100 days, certain hydrogels release proteins optional pharmaceutically acceptable carriers, excipients or for shorter time periods. When encapsulated antibodies stabilizers (Remington's Pharmaceutical Sciences 16th 40 remain in the body for a long time, they may denature or edition, Osol, A Ed. [1980]), in the form of lyophilized aggregate as a result of exposure to moisture at 37° C., formulations or aqueous solutions. Acceptable carriers, resulting in a loss of biological activity and possible changes excipients, or stabilizers are nontoxic to recipients at the in immunogenicity. Rational strategies can be devised for dosages and concentrations employed, and include buffers stabilization depending on the mechanism involved. For such as phosphate, citrate, and other organic acids; antioxi 45 example, if the aggregation mechanism is discovered to be dants including ascorbic acid and methionine; preservatives intermolecular S-S bond formation through thio-disulfide (such as octadecyldimethylbenzyl ammonium chloride; hex interchange, stabilization may be achieved by modifying amethonium chloride; benzalkonium chloride, benzetho sulhydryl residues, lyophilizing from acidic solutions, con nium chloride; phenol, butyl or benzyl alcohol; alkyl para trolling moisture content, using appropriate additives, and hens such as methyl or propylparaben; catechol; resorcinol; 50 developing specific polymer matrix compositions. cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, V. Treatment With the Anti-ErbB2 Antibodies such as serum albumin, gelatin, or immunoglobulins; hydro It is contemplated that, according to the present invention, philic polymers such as polyvinylpyrrolidone; amino acids the anti-ErbB2 antibodies may be used to treat various such as glycine, glutamine, asparagine, histidine, arginine, 55 conditions characterized by overexpression and/or activa or lysine; monosaccharides, disaccharides, and other carbo tion of the ErbB2 receptor. Exemplary conditions or disor- hydrates including glucose, mannose, or dextrins; chelating ders include benign or malignant tumors (e.g. renal, liver, agents such as EDTA; sugars such as sucrose, mannitol, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, trehalose or sorbitol; salt-forming counter-ions such as pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarco sodium; met al complexes (e.g. Zn-protein complexes); 60 mas; glioblastomas; and various head and neck tumors); and/or non-ionic surfactants such as TWEEN™, PLURON leukemias and lymphoid malignancies; other disorders such ICS™ or polyethylene glycol (PEG). Preferred lyophilized as neuronal, glial, astrocytal, hypothalamic and other anti-ErbB2 antibody formulations are described in WO glandular, macrophagal, epithelial, stromal and blastocoelic 97/04801, expressly incorporated herein be reference. disorders; and inflammatory, angiogenic and immunologic The formulation herein may also contain more than one 65 disorders. active compound as necessary for the particular indication The antibodies of the invention are administered to a being treated, preferably those with complementary activi- human patient, in accord with known methods, such as
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US 6,627,196 Bl 33 34 intravenous administration as a bolus or by continuous whether, for example, by one or more separate infusion over a period of time, by intramuscular, administrations, or by continuous infusion. A typical daily intraperitoneal, intracerobrospinal, subcutaneous, intra dosage might range from about 1 ,ug!kg to 100 mg/kg or articular, intrasynovial, intrathecal, oral, topical, or inhala more, depending on the factors mentioned above. For tion routes. Intravenous or subcutaneous administration of 5 repeated administrations over several days or longer, the antibody is preferred. depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The The treatment of the present invention involves the progress of this therapy is easily monitored by conventional administration of an anti-ErbB2 antibody to an animal or techniques and assays. human patient, followed at intervals by subsequent doses of equal or smaller doses such that a target serum concentration 10 According to the invention, dosage regimens may include is achieved and maintained during treatment. Preferably, an initial dose of anti-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 maintenance doses are delivered by bolus delivery, prefer mg/kg delivered by intravenous or subcutaneous infusion, ably by subcutaneous bolus administration, making treat followed by subsequent weekly maintenance doses of 2 ment convenient and cost-effective for the patient and health mg/kg by intravenous infusion, intravenous bolus injection, care professionals. subcutaneous infusion, or subcutaneous bolus injection. 15 Where the antibody is well-tolerated by the patient, the time Where combined administration of a chemotherapeutic of infusion may be reduced. agent (other than an an tracy cline) is desired, the combined administration includes coadministration, using separate for Alternatively, the invention includes an initial dose of 12 mulations or a single pharmaceutical formulation, and con mg/kg anti-ErbB2 antibody, followed by subsequent main tenance doses of 6 mg/kg once per 3 weeks. secutive administration in either order, wherein preferably 20 there is a time period while both (or all) active agents Another dosage regimen involves an initial dose of 8 simultaneously exert their biological activities. Preparation mg/kg anti-ErbB2 antibody, followed by 6 mg/kg once per and dosing schedules for such chemotherapeutic agents may 3 weeks. be used according to manufacturers' instructions or as Still another dosage regimen involves an initial dose of 8 determined empirically by the skilled practitioner. Prepara- mg/kg anti-ErbB2 antibody, followed by subsequent main tion and dosing schedules for such chemotherapy are also 25 tenance doses of 8 mg/kg once per week or 8 mg/kg once described in Chemotherapy Service Ed., M. C. Perry, Wil every 2 to 3 weeks. liams & Wilkins, Baltimore, Md. (1992). The chemothera As an alternative regimen, initial doses of 4 mg!kg peutic agent may precede, or follow administration of the anti-ErbB2 antibody may be administered on each of days 1, antibody or may be given simultaneously therewith. The 2 and 3, followed by subsequent maintenance doses of 6 antibody may be combined with an anti-estrogen compound 30 mg/kg once per 3 weeks. such as tamoxifen or an anti-progesterone such as onapris An additional regimen involves an initial dose of 4 mg!kg tone (see, EP 616 812) in dosages known for such mol anti-ErbB2 antibody, followed by subsequent maintenance ecules. doses of 2 mg/kg twice per week, wherein the maintenance It may be desirable to also administer antibodies against doses are separated by 3 days. other tumor associated antigens, such as antibodies which 35 Alternatively, the invention may include a cycle of dosing bind to the EGFR, ErbB3, ErbB4, or vascular endothelial in which delivery of anti-ErbB2 antibody is 2-3 times per growth factor (VEGF). Alternatively, or additionally, two or week for 3 weeks. The 3 week cycle is preferably repeated more anti-ErbB2 antibodies may be co-administered to the as necessary to achieve suppression of disease symptoms. patient. Sometimes, it may be beneficial to also administer The invention further includes a cyclic dosage regimen in one or more cytokines to the patient. The ErbB2 antibody 40 which delivery of anti-ErbB2 antibody is daily for 5 days. may be co-administered with a growth inhibitory agent. For According to the invention, the cycle is preferably repeated example, the growth inhibitory agent may be administered as necessary to achieve suppression of disease symptoms. first, followed by the ErbB2 antibody. However, simulta Further information about suitable dosages is provided in the neous administration, or administration of the ErbB2 anti Examples below. body first is also contemplated. Suitable dosages for the 45 growth inhibitory agent are those presently used and may be VI. Articles of Manufacture lowered due to the combined action (synergy) of the growth In another embodiment of the invention, an article of inhibitory agent and anti-ErbB2 antibody. manufacture containing materials useful for the treatment of In addition to the above therapeutic regimens, the patient the disorders described above is provided. The article of may be subjected to surgical removal of cancer cells and/ 50 manufacture comprises a container, a label and a package or-radiation therapy. insert. Suitable containers include, for example, bottles, For the prevention or treatment of disease, the appropriate vials, syringes, etc. The containers may be formed from a dosage of anti-ErbB2 antibody will depend on the type of variety of materials such as glass or plastic. The container disease to be treated, as defined above, the severity and holds a composition which is effective for treating the course of the disease, whether the antibody is administered 55 condition and may have a sterile access port (for example, for preventive or therapeutic purposes, previous therapy, the the container may be an intravenous solution bag or a vial patient's clinical history and response to the antibody, and having a stopper pierceable by a hypodermic injection the discretion of the attending physician. The antibody is needle). At least one active agent in the composition is an suitably administered to the patient at one time or over a anti-ErbB2 antibody. The label on, or associated with, the series of treatments. Where the treatment involves a series of container indicates that the composition is used for treating treatments, the initial dose or initial doses are followed at 60 the condition of choice. The article of manufacture may daily or weekly intervals by maintenance doses. Each main further comprise a second container comprising a tenance dose provides the same or a smaller amount of pharmaceutically-acceptable buffer, such as phosphate antibody compared to the amount of antibody administered buffered saline, Ringer's solution and dextrose solution. It in the initial dose or doses. may further include other materials desirable from a com Depending on the type and severity of the disease, about 65 mercial and user standpoint, including other buffers, 1 ,ug!kg to 15 mg!kg (e.g. 0.1-20 mg!kg) of antibody is an diluents, filters, needles, and syringes. In addition, the article initial candidate dosage for administration to the patient, of manufacture may comprise a package inserts with instruc-
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US 6,627,196 Bl 35 36 tions for use, including, e.g., a warning that the composition Eligibility Criteria is not to be used in combination with anthacycline-type Patients had to fulfill all of the following criteria to be chemotherapeutic agent, e.g. doxorubicin or epirubicin. eligible for study admission: Deposit of Materials Metastatic breast cancer The following hybridoma cell lines have been deposited 5 Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ with the American Type Culture Collection, 12301 Parklawn as determined by immunohistochemistry or fluores Drive, Rockville, Md., USA (ATCC): cence in situ hybridization (FISH). [Tumor expression of ErbB2 can be determined by immunohistochemical analysis, as previously described (Slamon et al., [ 1987] 10 and [1989], supra), of a set of thin sections prepared Antibody Designation ATCC No. Deposit Date from the patient's paraffim-archived tumor blocks. The 7C2 ATCC HB-12215 Oct. 17, 1996 primary detecting antibody used is murine 4D5 MAb, 7F3 ATCC HB-12216 Oct. 17, 1996 which has the same CDRs as the humanized antibody 4D5 ATCC CRL 10463 May 24, 1990 used for the treatment. Tumors are considered to over 2C4 ATCC HB-12697 Apr. 8, 1999 15 express ErbB2 if at least 25% of tumor cells exhibit 2 p HER ]. characteristic membrane staining for 185 Further details of the invention are illustrated by the Bidimensionally measurable disease (including lytic bone following non-limiting Examples. lesions) by radiographic means, physical examination, or photographs EXAMPLES 20 Measurable disease was defined as any mass reproducibly Example 1 measurable in two perpendicular diameters by physical examination, X-ray (plain films), computerized tomography Preparation and Efficacy of HERCEPTIN® Anti (CT), magnetic resonance imaging (MRI), ultrasound, or ErbB2 Antibody photographs. Materials and Methods 25 Osteoblastic metastases, pleural effusions, or ascites were not considered to be measurable. Measurable lesions must Anti-ErbB2 monoclonal antibody The anti-ErbB2 IgG K 1 be at least 1 em in greatest dimension. Enumeration of murine monoclonal antibody 4D5, specific for the extracel evaluable sites of metastatic disease and number of lesions lular domain of ErbB2, was produced as described in Fendly in an evaluable site (e.g. lung) had to be recorded on the et al., Cancer Research 50: 1550--1558 (1990) and W089/ appropriate Case Report Form (CRF). If a large number of 06692. Briefly, NIH 3T3/HER2-3 cells (expressing 400 30 pulmonary or hepatic lesions were present, the six largest approximately 1x105 ErbB2 molecules/cell) produced as lesions per site were followed. described in Hudziak et al., Proc. Natl. Acad. Sci. (USA) 84:7159 (1987) were harvested with phosphate buffered The ability to understand and willingness to sign a written saline (PBS) containing 25 mM EDTA and used to immu informed consent form Women ~ 18 years nize BALB/c mice. The mice were given injections i.p. of 35 107 cells in 0.5 ml PBS on weeks, 0, 2, 5 and 7. The mice Suitable candidates for receiving concomitant cytotoxic with antisera that immunoprecipitated 32P-labeled ErbB2 chemotherapy as evidenced by screening laboratory were given i.p. injections of a wheat germ agglutinin assessments of hematologic, renal, hepatic, and meta Sepharose (WGA) purified ErbB2 membrane extract on bolic functions. weeks 9 and 13. This was followed by an i.v. injection of0.1 40 Exclusion Criteria ml of the ErbB2 preparation and the splenocytes were fused Patients with any of the following were excluded from with mouse myeloma line X63-Ag8.653. Hybridoma super study entry: natants were screened for ErbB2-binding by ELISA and Prior cytotoxic chemotherapy for metastatic breast cancer radioimmunoprecipitation. MOPC-21 (IgG1), (Cappell, Patients may have received prior hormonal therapy (e.g. Durham, N.C.), was used as an isotype-matched control. tamoxifen) for metastatic disease or cytotoxic therapy The treatment was performed with a humanized version 45 of the murine 4D5 antibody (HERCEPTIN® anti-ErbB2 in the adjuvant setting. antibody). The humanized antibody was engineered by Concomitant malignancy that has not been curatively inserting the complementarity determining regions of the treated murine 4D5 antibody into the framework of a consensus A performance status of <60% on the Karnofsky scale
human immunoglobulin IgG1 (IgG1) (Carter et al., Proc. 50 Pregnant or nursing women; women of childbearing Natl. Acad. Sci. USA 89:4285-4289 [1992]). The resulting potential, unless using effective contraception as deter humanized anti-ErbB2 monoclonal antibody has high affin- mined by the investigator 2 ity for p185HER (Dillohiation constant [Ka]=0.1 nmol/L), Bilateral breast cancer (either both primary tumors must markedly inhibits, in vitro and in human xenografts, the have 2+ to 3+ HER2 overexpression, or the metastatic growth of breast cancer cells that contain high levels of 2 55 site must have 2+ to 3+ HER2 overexpression) p 185HER , induces antibody-dependent cellular cytotoxicity (ADCC), and has been found clinically active, as a single Use of investigational or unlicensed agents within 30 days agent, in patients with ErbB2-overexpressing metastatic prior to study entry breast cancers that had received extensive prior therapy. Clinically unstable or untreated metastases to the brain HERCEPTIN® anti-ErbB2 antibody is produced by a (e.g. requiring radiation therapy) genetically engineered Chinese Hamster Ovary (CHO) cell 60 Based upon the foregoing criteria, 469 patients were line, grown in large scale, that secretes the antibody into the chosen, and enrolled in the study. Half the patients (stratified culture medium. The antibody is purified from the CHO by chemotherapy) were randomized to additionally receive culture media using standard chromatographic and filtration the HERCEPTIN® anti-ErbB2 antibody (see below). methods. Each lot of antibody used in this study was assayed Administration and Dosage to verify identity, purity, and potency, as well as to meet 65 Anti-ErbB2 Antibody Food and Drug Administration requirements for sterility and On day 0, a 4 mg/kg dose of humanized anti-ErbB2 safety. antibody (HERCEPTIN®, H) was administered
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US 6,627,196 Bl 37 38 intravenously, over a 90-minute period. Beginning on day 7, rates (RR) showed a significant augmentation of the che patients received weekly administration of 2 mg/kg antibody motherapeutic effect by HERCEPTIN® anti-ErbB2 (i.v.) over a 90-minute period. antibody, without increase in overall severe adverse events Chemotherapy (AE): The patients received one of two chemotherapy regimens 5 for a minimum of six cycles, provided their disease was not TABLE 1 progressing: a) cyclophosphamide and doxorubicin or epi rubicin (AC), if patients have not received anthracycline HERCEPTIN ® Anti-ErbB2 Antibody Efficacy therapy in the adjuvant setting, or b) paclitaxel (T, TAXOL®), if patients have received any anthracycline Enrolled TIP (months) RR(%) AE(%) therapy in the adjuvant setting. The initial dose of the 10 HERCEPTIN® anti-ErbB2 antibody preceded the first cycle CRx 234 5.5 36.2 66 CRx + 14 235 8.6* 62.00** 69 of either chemotherapy regimen by 24 hours. Subsequent AC 145 6.5 42.1 71 doses of the antibody were given immediately before che AC+H 146 9.0 64.9 68 motherapy administration, if the initial dose of the antibody T 89 4.2 25.0 59 was well tolerated. If the first dose of the antibody was not T+H 89 7.1 57.3 70 well tolerated, subsequent infusions continued to precede 15 chemotherapy administration by 24 hours. Patients were *p < 0.001 by log-rank test; ** p < 0.01 by X2 test; CRx: chemotherapy; permitted to continue receiving chemotherapy beyond six AC: anthracycline/cyclophosphamide treatment; H: HERCEPTIN ® anti cycles if, in the opinion of the treating physician, they were ErbB2 antibody; T: TAXOL ® continuing to receive treatment benefit. A syndrome of myocardial dysfunction similar to that 2 Cyclophosphamide ( 600 mg/m ) was given either by iv 20 observed with anthracyclines was reported more commonly push over a minimum period of 3 minutes or by infusion with a combined treatment of AC+H (18% Grade %) than over a maximum period of 2 hours. with AC alone (3%), T (0%), or T+H (2%). 2 2 Doxorubicin (60 mg/m ) or epirubicin (75 mg/m ) were These data indicate that the combination of anti-ErbB2 given either by slow iv push over a minimum period of 3-5 antibody treatment with chemotherapy markedly increases minutes or by infusion over a maximum period of 2 hours, 25 the clinical benefit, as assessed by response rates and the according to institutional protocol. evaluation of disease progression. However, due to the Paciltaxel (TAXOL®) was given at a dose of 175 mg/m2 increased cardiac side-effects of doxorubicin or epirubicin, over 3 hours by intravenous administration. All patients the combined use of anthracyclines with anti-ErbB2 anti receiving paclitaxel were premedicated with dexamethasone body therapy is contraindicated. The results, taking into (or its equivalent) 20 mgx2, administered orally 12 and 6 30 account risk and benefit, favor treatment with HERCEP hours prior to paclitaxel; diphenhydramine (or its TIN® anti-ErbB2 antibody and paclitaxel (TAXOL®) equivalent) 50 mg, iv, administered 30 minutes prior to where a combined treatment regimen is desired. paclitaxel, and dimetidine (or another H2 blocker) 300 mg, iv, administered 30 minutes prior to paclitaxel. Example 2 Response Criteria Pharmacokinetic and Pharmacodynamic Properties Progressive Disease 35 of Anti-ErbB2 Antibody (HERCEPTIN®) Objective evidence of an increase of 25% or more in any measurable lesion. Progressive disease also includes those HERCEPTIN® anti-ErbB2 antibody was administered by instances when new lesions have appeared. For bone lesions, intravenous, infusion to human patients selected according progression is defined as a 25% increase in objective mea to the criteria provided in Example 1. An initial dose of 4 surement by plain film, CT, MRI; symptomatic new lesions 40 mg/kg HERCEPTIN® anti-ErbB2 antibody was delivered not due to fracture; or requirement for palliative radio by intravenous infusion, followed by subsequent i.v. infu- therapy. sions of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody Complete Response weekly for several weeks. Two hundred thirteen patients Disappearance of all radiographically and/or visually began this treatment regimen and serum drug concentration apparent tumor for a minimum of 4 weeks. Skin and chest 45 was obtained beyond 8 weeks for fewer than 90 patients as wall complete responses had to be confirmed by biopsy. selective discontinuation of patients with rapidly progress- Partial Response ing disease occurred. Of the 213 patients who began A reduction of at least 50% in the sum of the products of treatment, serum trough concentration data were available the perpendicular diameters of all measurable lesions for a for 80 patients at Week 12, for 77 patients at Week 16, for 44 patients at Week 20, for 51 patients at Week 24, for 25 minimum period of 4 weeks. No new lesions may have 50 appeared, nor may any lesions have progressed in size. patients at Week 28, for 23 patients at Week 32, and for 37 Minor Response patients at Week 36. A reduction of 25% to 49% in the sum of the products of HERCEPTIN® anti-ErbB2Antibody Trough Serum Con the perpendicular diameters of all measurable lesions. No centrations for Weeks 0-36 new lesions may have appeared, nor may any lesions have The HERCEPTIN® anti-ErbB2 antibody trough serum 55 progressed in size. concentrations (ug/ml, mean±SE) from Week 2 through Stable Disease Week 36 are plotted in FIG. 3 (dark circles). The number of No change of greater than 25% in the size of measurable patients was fairly constant because data from patients lesions. No lesions may have appeared. discontinued from the program due to rapidly progressing Time to disease progression (TTP) was calculated from disease were excluded from this analysis. Trough serum the beginning of therapy to progression. Confidence limits 60 concentrations tended to increase through Week 12 and for response rates were calculated using the exact method tended to plateau after that time. for a single proportion. (Fleiss, 1 L, Statistical Methods for HERCEPTIN® anti-ErbB2 Antibody Trough and Peak Rates and Proportions (ed.2), New York, N.Y., Wiley, 1981, Serum Concentrations for Weeks 1-8 pp 13-17). Some HERCEPTIN® anti-ErbB2 antibody serum con- Results 65 centration data were available for 212 of the original 213 At a median follow-up of 10.5 months, assessments of patients. Trough and peak serum concentration data reflect time to disease progression (TTP in months) and response ing the first HERCEPTIN® anti-ErbB2 antibody infusion
Appx39 HOSPIRA EX. 1001 Page 28 Case: 19-2156 Document: 19 Page: 111 Filed: 07/26/2019
US 6,627,196 Bl 39 40 were available for 195 of the 212 patients. For the seventh serum concentrations (n=12, mean 34.1 ,ug/ml, SD=12.0) infusion, trough serum concentration data were available for compared with patients with visceral disease (n=183, mean= 137/212 patients and peak serum concentration data were 24.4 ,ug!ml, SD=12.6). This difference in the average trough available for 114/212 patients. Table 2 presents a summary serum concentrations for Weeks 7 and 8 was significant. of statistics from trough and peak serum concentrations for 5 These data indicate that the rise in trough serum concentra the first 8 weeks of treatment. Peak samples were drawn tions between Weeks 2 and 7/8 occurs for human patients shortly after the end of HERCEPTIN® anti-ErbB2 antibody with various disease profiles. administration; trough samples were drawn prior to the In a subsequent, similarly designed study, human breast subsequent dose (i.e., 1 week later). Serum concentrations of cancer patients were treated with a loading dose of 8 mg!kg HERCEPTIN® anti-ErbB2 antibody were determined as followed by maintenance doses of 4 mg!kg weekly. The disclosed herein. 10 results of this preliminary human study indicated that an 8mg!kg load:4 mg/kg weekly maintenance regimen was TABLE 2 efficacious in reducing tumor volume in the patients. HERCEPTIN ® Anti-ErbB2 Antibody Trough and Peak Serum The data disclosed in this Example indicate that front Concentrations for the First 8 Weeks of Treatment (gg!ml) loading of antibody, such that a target serum concentration 15 is reached more quickly, may be associated with improved Dose outcomes. Number n Mean so Minimum Maximum Example 3 Peak 195 100.3 35.2 30.7 274.6 Trough 195 25.0 12.7 0.16 60.7 I.V. Bolus Delivery and Subcutaneous Infusion of Peak 2 190 74.3 31.3 20.8 307.9 20 Trough 167 30.4 16.0 0.2 74.4 HERCEPTIN® Anti-ErbB2 Antibody Effectively Peak 3 167 75.3 26.8 16.1 194.8 Decrease Tumor Volume in the Mouse Trough 179 33.7 17.9 0.2 98.2 Peak 4 175 80.2 26.9 22.2 167 The efficacy of infusion or bolus delivery of humanized Trough 132 38.6 20.1 0.2 89.4 anti-ErbB2 antibody (HERCEPTIN®, see Example 1 for Peak 5 128 85.9 29.2 27.8 185.8 25 preparation), either by intravenous injection or subcutaneous Trough 141 42.1 24.8 0.2 148.7 injection, was examined. The purpose of the study was to Peak 137 87.2 32.2 28.9 218.1 Trough 115 43.2 24.0 0.2 109.9 ask whether subcutaneous delivery was feasible and whether Peak 7 114 89.7 32.5 16.3 187.8 the convenient subcutaneous bolus delivery was useful in Trough 137 48.8 24.9 0.2 105.2 treating metastatic breast cancer in animals inoculated with Peak 8 133 95.6 35.9 11.4 295.6 30 a cell line that overexpresses the HER2 gene. The results, detailed below, show that i.v. and s.c. infusion and bolus The data in Table 2 suggest that there was an increase in delivery are feasible treatment methodologies. trough serum concentration over time. Of the many patients A study in a nude mouse xenograft model, which incor studied, there were 18 patients for whom the trough con porates a human breast cancer cell line that naturally over- centrations did not exceed 20 ,ug!ml from Week 2 through 35 expresses the HER2 gene (BT-474MI, derived from BT-474 Week 8. A HERCEPTIN® anti-ErbB2 antibody trough cells, ATCC Accession number HTB-20), comparing tumor serum concentration of 20 ,ug/ml was nominally targeted for volume as a function of i.v. bolus versus s.c. infusion was these studies based on prior pharmacologic studies in ani performed as follows. In the first study athymic nude nu nu mals and exploratory analyses in clinical trials. 7-9 week old female mice were obtained from Taconic Inc Patient response status was evaluated relative to serum (Germantown, N.Y.). To initiate tumor development, each concentration of HERCEPTIN® anti-ErbB2 antibody. For 40 mouse was inoculated subcutaneously with 3x106 BT474M this purpose, mean serum concentration (an average of 1 cells suspended in Matrigel TM. When tumor nodules 3 troughs and peaks) was calculated for various times and reached a volume of approximately 100 mm , animals were patient response status (where the patient response status randomized to 4 treatment groups. The groups were treated was determined by an independent Response Evaluation according to Table 3. Committee). The increase in serum concentration between 45 Weeks 2 and 8 appeared to be greater in responders than in TABLE 3 nonresponders, suggesting that there is a relationship between response status and HERCEPTIN® anti-ErbB2 Animal Groups and Doses for Comparison of I. V Bolus and antibody serum concentration. A statistical analysis (analysis S.C. Infusion of variance) of trough serum concentration values at Week 50 Target 2 and an average of Weeks 7 and 8 in relation to response Group, Serum Loading status indicated a highly significant relationship between Dose, Cone. Route of Dose Maintenance response status and average trough of Weeks 7 and 8 Antibody .ug/ml Administration (mg!kg) Dose (p Appx40 HOSPIRA EX. 1001 Page 29 Case: 19-2156 Document: 19 Page: 112 Filed: 07/26/2019 US 6,627,196 Bl 41 42 dosing to promote growth of grafted tumor cells. The This study was set up as disclosed herein in Example 3 for animals were inoculated with the BT474M 1 cells 8 days the comparison of intravenous bolus and subcutaneous infu before the beginning of treatment and tumors were allowed sian delivery. A sustained release estrogen implant was to grow. The animals were then treated with nonrelevant inserted subcutaneously one day before tumor cell innocu- antibody E25 (non-specific for HER2 receptor, but a mem 5 lation as described in Example 3. Six days after tumor cell ber of the monoclonal IgG class) or test antibody HERCEP innoculation, the initial tumor measurement was performed. TIN® anti-ErbB2 anitbody as indicated in Table 3. The dosage levels were selected to achieve target serum concen Seven days after tumor cell innoculation, the first dose of trations of HERCEPTIN®, either 1 ,ug/ml or 20 ,ug/ml, by control antibody or HERCEPTIN® anti-ErbB2 antibody subcutaneous pump infusion or by i.v. bolus delivery. The was delivered. The animal groups, type of delivery, loading 10 study groups were treated until day 35. The serum concen dose and maintenance doses are provided in Table 4. Ani tration of HERCEPTIN® anti-ErbB2 antibody was mea mals were dosed once weekly for 4 weeks. sured weekly (Oust prior to dosing for Group 4) using 3 mice/group/time point. The anti-ErbB2 antibody concentra TABLE 5 tion was determined according to the method disclosed herein involving standard techniques. Tumor volumes were 15 Animal Groups and Doses for Comparison of I. V Bolus and measured two days before dosing began and twice per week S.C Bolus Delivery from day 6 to day 35 in the study for which data is tabulated Loading Maintenance below. Tumors were measured in three dimensions and Route of Dose Dose 3 volumes were expressed in mm . Efficacy was determined Group Administration (mg!kg) (mg!kg!week) n by a statistical comparison (ANOVA) of tumor volumes of 20 test animals relative to untreated control animals. 1-Control IV 8 4 10 rbuMAb E25 As shown in Table 4, below, treatment of the BT474M 1 2-rhuMAb HER2 IV 2 10 tumor-bearing mice with HERCEPTIN® anti-ErbB2 anti 3-rhuMAb HER2 IV 4 2 10 body by the indicated dosage methods significantly inhibited 4-rhuMAb HER2 IV 8 4 10 5-rhuMAb HER2 sc 4 2 10 the growth of the tumors. All HERCEPTIN® -treated groups 25 showed similar inhibition of tumor growth relative to the IV = intraveneous; SC = subcutaneous; n = number of animals per group. control group. No dose-response was observed. TABLE 4 The mice were treated according to the information in 30 Comparison of S.C. Infusion and I.V Bolus Delivery Table 4 and using the techniques disclosed in Example 3. The serum concentration of HERCEPTIN® anti-ErbB2 Tumor Volume Tumor Volume HERCEPT® (area under antibody was measured weekly before each weekly i.v. 3 (mm ), curve) Serum Cone. maintenance dose according to the procedure described Day 35, Day 6-Day 35 (ugiml), Day 27, herein and using standard techniques. The control E25 Treatment Group (n ~ 14) (n ~ 13) (n ~ 3) 35 antibody serum concentration was determined according to control s.c. infusion 764 ± 700 5650 ± 4700 4.16 ± 1.94 standard immunoassay techniques. Table 6 shows the s.c. infusion 80.6 ± 158 1610 ± 1250 2.11 ± 1.74 (low dose) increase in HERCEPTIN® anti-ErbB2 antibody serum con s.c. infusion 31 ± 75.6 1440 ± 1140 22.1 ± 5.43 centrations with time. (high dose) 40 i.v. bolus dose* 49.7 ± 95.7 2150 ± 1480 21.7 ± 17.1 ** TABLE 6 s.c. = subcutaneous delivery; i.v. = intravenous delivery. *4.0 mg/k:g Loading Dose and 2.0 mg/k:g!week Maintenance Dose. IV versus SC Bolus Delivery: **at predose (trough serum concentration immediately prior to a mainte Serum HERCEPTIN ® Anti-ErbB2 Antibody Concentration nance dose) Serum Concentration, yg/ml 45 The results tabulated above indicate that maintenance of Day 0 Day 7 Day 14 Day 21 Treatment Group Mean Mean Mean Mean a serum concentration of approximately 2 ,ug/ml was as (delivery, MD) (SO) (SO) (SO) (SO) effective as a concentration of 20 ,ug!ml in this study. The results indicated that dosing by subcutaneous infusion was 1-Control rhu MAb E25 0 25.9 34.6 38.5 as effective as intravenous bolus dosing and achieved similar 50 (IV, 4mg!kg) (0) (8.29) (11.2) (14.4) 2-rhu MAb HER2 0 4.96 8.55 8.05 trough serum concentrations. The results also indicate that (IV, 1 mg/k:g) (0) (3.79) (5.83) (4.67) the dose levels studied are at the top of the dose-response 3-rhu MAb HER2 0 13.4 18.9 22.6 curve in this model and that subcutaneous dosing is effective (IV, 2 mg/k:g) (0) (9.24) (12.0) (9.21) in treating breast cancer tumors. Thus, subcutaneous admin 4-rhu MAb HER2 0 29.6 37.7 46.2 istration of maintenance doses is feasible as part of a 55 (IV, 4 mg/k:g) (0) (13.5) (14.4) (13.8) HERCEPTIN® anti-ErbB2 antibody treatment regimen. 5-rhu MAb HER2 0 12.5 16.9 17.6 (SC, 2 mg/k:g) (0) (7.33) (10.2) (10.7) Example 4 n ~ 10 for time points Days 0, 7 and 14. N ~ 9 for Day 21. I.V. Bolus and Subcutaneous Bolus Deliveries of 60 HERCEPTIN® Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse Table 7 shows the relative efficacy of intravenous bolus Subcutaneous bolus delivery is convenient and cost delivery and subcutaneous bolus delivery for Groups 1-5 effective for the patient and health care professionals. The having achieved the serum antibody concentrations pre- results of the study disclosed in this example indicate that 65 sented in Table 6. For this study, efficacy was measured as subcutaneous bolus delivery was as effective as intravenous a decrease in tumor volume. Tumor volume was measured bolus delivery in reducing breast cell tumor size in a mouse. twice weekly. Appx41 HOSPIRA EX. 1001 Page 30 Case: 19-2156 Document: 19 Page: 113 Filed: 07/26/2019 US 6,627,196 Bl 43 44 week or less, including 1 day or less. The number of TABLE 7 maintenance doses may be one or more doses sufficient to achieve suppression of disease symptoms, such as a Efficacy of HERCEPTIN ® Anti-ErbB2 Antibody Measured as a decrease in tumor volume. The maintenance doses are equal Change in Tumor Volume Comparing Intravenous Bolus and Subcutaneous Bolus Delivery, Mean (SO) 5 to or smaller than the initial dose or doses, consistent with an object of the invention of administering HERCEPTIN® Tumor anti-ErbB2 antibody by regimens providing greater front Tumor Tumor Tumor Day 6-Day 31 * Growth loading. The specific drug delivery regimens disclosed Treatment Vol. Vol. Vol. Area Under Rate on Group Day 6, Day 28 Day 31, Curve Tumor Log herein are representative of the invention and are not meant (Delivery, MD) mm3 mm3 mm3 Vol., mm3 (TM + 1) 10 to be limiting. 1-IV Control 321 1530 1630 13600 0.0660 In one trial, an initial dose of 6 mg/kg, 8 mg!kg, or 12 (190) (1040) (1170) (7230) (0.0200) mg/kg of HERCEPTIN® anti-ErbB2 antibody is delivered 2-IV Herceptin 297 175 151 4690 -0.0505 to human patients by intravenous or subcutaneous injection. 1 mg/k:g (130) (215) (188) (1400) (0.142) Initial doses (loading doses) are delivered by intravenous 3-IV Herceptin 269 75.7 73.6 3510 -0.0608 15 infusion or bolus injection or preferably subcutaneous bolus 2 mg/k:g (129) (92.4) (84.5) (1220) (0.110) 4-IV Herceptin 272 25.3 25.8 2880 -0.0810 injection. Preferably a target trough serum concentration of 4 mg/k:g (117) (75.9) (72.9) (1230) (0.0859) HERCEPTIN® anti-ErbB2 antibody of approximately 5-SC Herceptin 268 76.2 90.4 3230 -0.0304 10--20 ,ug/ml is achieved.(averaged for all patients in the 2 mg/k:g (117) (98.8) (105) (1440) (0.104) treatment group) and maintained by subsequent doses of N ~ 10 for each data point. 20 anti-ErbB2 antibody that are equal to or smaller than the TM = tumor measurement. initial dose. In one method, a target trough serum concen IV = intravenous. tration is achieved and maintained by once-per-week deliv SC = subcutaneous. eries of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody by MD = maintenance dose. 3 Tumor Vol. = tumor volume, mm . intravenous or subcutaneous injection for at least eight *Day 17 excluded due to measurement error. 25 weeks. Alternatively, for this or any dosage regimen dis Tumor growth rate calculated on Day 21-Day 31 Log(TM + 1). Area closed herein, subcutaneous continuous infusion by subcu under the curve is the area beneath a plot of tumor volume versus time. taneous pump is used to delivery subsequent maintenance FIGS. 4A and 4B are graphical plots of changes in tumor doses. volume over time, some of which data is found in Table 7. In another method, an initial (front loading) dose of 8 FIG. 4A is a linear plot of tumor volume versus time. FIG. 30 mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by 4B is a semilogarithmic plot of the same data, allowing the intravenous injection (infusion or bolus injection) or by test points be viewed more clearly. The data in Table 7 and subcutaneous bolus injection. This is followed by intrave FIGS. 4A and 4B indicate that, although a dose-related nous bolus injections, intravenous infusion, subcutaneous response was not observed between HERCEPTIN-treated infusion, or subcutaneous bolus injection of 6 mg/kg at groups, dosing by subcutaneous bolus was as effective as 35 3-week intervals to maintain a trough serum concentration intravenous bolus dosing and achieved similar trough serum of approximately 10--20 ,ug/ml, averaged for an entire treat concentrations. ment group. In another method, an initial (front loading) dose of 12 Example 5 mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by 40 intravenous injection (infusion or bolus injection) or by Regimens for Intravenous and Subcutaneous subcutaneous bolus injection. This is followed by intrave Delivery of Anti-ErbB2 Antibody nous bolus injections, intravenous infusion, subcutaneous According to the invention, methods of anti-ErbB2 anti infusion, or subcutaneous bolus injection of 6 mg/kg at 3-week intervals to maintain a trough serum concentration body (e.g., HERCEPTIN®) delivery comprise greater front 45 loading of the drug to achieve a target serum concentration of approximately 10-20 ,ug/ml. in approximately 4 weeks or less, preferably 3 weeks or less, In yet another method, an initial (front loading) dose of 8 more preferably 2 weeks or less, and most preferably 1 week mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by or less, including one day or less. According to the intravenous infusion or bolus injection, or preferably by invention, this initial dosing is followed by dosing that 50 subcutaneous bolus injection or infusion. maintains the target serum concentration by subsequent This is followed by administration of 8 mg/kg per week doses of equal or smaller amount. An advantage of the or 8 mg/kg per 2-3 weeks to maintain a trough serum methods of the invention is that the maintenance dosing may concentration of HERCEPTIN® anti-ErbB2 antibody of be less frequent and/or delivered by subcutaneous injection, approximately 10--20 ,ug/ml. Maintenance doses are deliv- making the treatment regimens of the invention convenient ss ered by intravenous infusion or bolus injection, or preferably and cost-effective for the patient and medical professionals by subcutaneous infusion or bolus injection. administering the antibody. In addition, a subcutaneous In another method, the front loading initial dose is a series maintenance dose regimen may be interrupted by intrave of intravenous or subcutaneous injections, for example, one nous dosing (such as infusion) when the patient's chemo on each of days 1, 2, and 3 of at least 1 mg/kg for each therapy requires delivery of other drugs by intravenous 60 injection (where the amount of anti-ErbB2 antibody deliv injection. ered by the sum of initial injections is more than 4 mg/kg), To test the following dosage regimens, human subjects are followed by maintenance doses of 6 mg/kg once each 3 selected according to the criteria disclosed in Example 1, week interval to maintain a target trough serum concentra above. The number of initial doses is one or more doses tion (for example, approximately 10--20 ,ug/ml) of HER- sufficient to achieve an efficacious target serum concentra 65 CEPTIN® anti-ErbB2 antibody. The maintenance doses are tion in approximately 4 weeks or less, preferably 3 weeks or delivered by intravenous infusion or bolus injection or by less, more preferably 2 weeks or less, and most preferably 1 subcutaneous infusion or subcutaneous bolus injection. Appx42 HOSPIRA EX. 1001 Page 31 Case: 19-2156 Document: 19 Page: 114 Filed: 07/26/2019 US 6,627,196 Bl 45 46 In yet another method, the front loading is by intravenous week. According to another embodiment of the invention, infusion of at least 1 mg!kg, preferably 4 mg/kg on each of the taxoid is docetaxel and is administered at a dose of five consecutive days, followed by repeats of this cycle a 30--70 mg/m2 /week. sufficient number of times to achieve suppression of disease symptoms. Following the initial dose or doses, subsequent 5 Example 6 doses may be delivered by subcutaneous infusion or bolus HERCEPTIN® Administered Intravenously Every injection if tolerated by the patient. Such subcutaneous Three Weeks in Combination with Paclitaxel delivery is convenient and cost-effective for the patient and administering health care professionals. Currently, the recommended dose of HERCEPTIN® is 2 In still another method, HERCEPTIN® anti-ErbB2 anti- 10 mg/kg once weekly. Patients will be administered HERCEP TIN® every three weeks instead of weekly, along with body is delivered initially as at least 2 intravenous infusions 2 per week for three weeks, followed by repeats of this cycle paclitaxel (175 mg/m every three weeks). Simulation of the to maintain an efficacious trough serum concentration of proposed treatment regimen suggests that the trough serum HERCEPTIN® 0 anti-ErbB2 antibody. The dose is at least concentrations will be 17 mcg/ml, in the range (10-20mcg/ 4 mg/kg of anti-ErbB2 antibody, preferably at least 5 mg/kg. 15 ml) of the targeted trough serum concentrations from pre The maintenance drug deliveries may be intravenous or vious HERCEPTIN® IV clinical trials. After the first 12 subcutaneous. patients the PK parameters will be assessed, if exposure is felt inadequate, then the dose will be increased to 8 mg!kg Where the animal or patient tolerates the antibody during every three weeks for the remaining 12 patients. and after an initial dose, delivery of subsequent doses may Inclusion Criteria be subcutaneous, thereby providing greater convenience and 20 cost-effectiveness for the patient and health care profession 1) Females~18 years of age als. 2) Histologically confirmed ErbB2 over-expressing meta static breast cancer In animal studies, an initial dose of more than 4 mg/kg, preferably more than 5 mg/kg delivered by intravenous or 3) Patients who have been newly diagnosed with meta 25 subcutaneous injection, is followed by subcutaneous bolus static disease injections of 2 mg/kg twice per week (separated by 3 days) 4) Have a Karnofsky performance status of~70% to maintain a trough serum concentration of approximately 5) Give written informed consent prior to any study 10-20 ,ug!ml. In addition, where the animal or patient is specific screening procedures with the understanding known to tolerate the antibody, an initial dose of HERCEP- that the patient has the right to withdraw from the study 30 TIN® anti-ErbB2 antibody is optionally and preferably at any time, without prejudice. deliverable by subcutaneous bolus injection followed by Exclusion Criteria subcutaneous maintenance injections. 1) Pregnant or lactating women While target serum concentrations are disclosed herein for 2) Women of childbearing potential unless (1) surgically the purpose of comparing animal studies and human trials, 35 sterile or (2) using adequate measures of contraception target serum concentrations in clinical uses may differ. The such as oral contraceptive, intra-uterine device or bar disclosure provided herein guides the user in selecting a rier method of contraception in conjunction with sper front loading drug delivery regimen that provides an effi micidal jelly. cacious target trough serum concentration. 3) Clinical or radiologic evidence of CNS metastases. The methods of the invention disclosed herein optionally 40 4) History of any significant cardiac disease include the delivery of HERCEPTIN® anti-ErbB2 antibody 5) LVEF~50% in combination with a chemotherapeutic agent (other than an 6) No prior taxane therapy in any treatment setting. anthrocycline derivative) to achieve suppression of disease symptoms. The chemotherapeutic agent may be delivered 7) Any of the following abnormal baseline hematologic with HERCEPTIN® anti-ErbB2 antibody or separately and 45 values: Hb less than 9 g/dl according to a different dosing schedule. For example, 9 subcutaneous delivery of HERCEPTIN® anti-ErbB2 anti WBC less than 3.0x10 /l body with TAXOL® is included in the invention. In Granulocytes less than 1.5 x 109 !1 addition, intravenous or subcutaneous injection of 8 mg!kg Platelets less than 100x109/l HERCEPTIN® anti-ErbB2 antibody, followed by intrave- 50 8) Any of the following abnormal baseline liver function nous or subcutaneous injection of 6 mg/kg HERCEPTIN® tests: anti-ErbB2 antibody every 3 weeks is administered in com Serum bilirubin greater than 1.5x ULN (upper normal bination with a chemotherapeutic agent, such as a taxoid limit) (e.g. paclitaxel175mg/m2 every 3 weeks) or an anthracy ALT and/or AST greater than 2.5x ULN (greater than 2 cline derivative (e.g. doxorubicin 60 mg/m or epirubicin 75 55 4.0x ULN if liver or bone metastasis) mg/m2 every 3 weeks). Optionally, where an anthracycline Alkaline phosphatase greater than 2.5x ULN (greater derivative is administered, a cardioprotectant (e.g. 600 than 4.0x ULN if liver or bone metastasis) mg/m2 cyclophosphamide every 3 weeks) is also adminis 9) The following abnormal baseline renal function tests: tered. In another combination therapy, anti-ErbB2 antibody serum creatinine greater than 1.5x ULN is administered in a loading dose of more than 4 mg/kg, 60 10) History of other serious medical conditions that would preferably more than 5 mg/kg, and more preferably at least preclude patient participation in an investigational 8 mg!kg. The loading dose is followed by maintenance doses study. of at least 2 mg!kg weekly, preferably 6 mg!kg every 3 HERCEPTIN® Loading dose and schedule: 8 mg!kg for weeks. The combination therapy includes administration of first dose. Maintenance dose and schedule: 6 mg/kg every 3 a taxoid during treatment with anti-ErbB2 antibody. Accord- 65 weeks. ing to one embodiment of the invention, the taxoid is Paclitaxel-175 mg/m2 IV every 3 weeksx6 cycles as a 2 paclitaxel and is administered at a dose of 70-100 mg/m / 3-hour infusion. NOTE: On the first cycle of treatment, Appx43 HOSPIRA EX. 1001 Page 32 Case: 19-2156 Document: 19 Page: 115 Filed: 07/26/2019 US 6,627,196 Bl 47 48 paclitaxel will be dosed 8 hours prior to HERCEPTIN® to It is believed that the above treatment regimen will be determine the PK of paclitaxel alone. HERCEPTIN® will be effective in treating metastatic breast cancer, despite the administered 8 hours post-paclitaxel for the 1st cycle only. In infrequency with which HERCEPTIN® is administered to the patient. subsequent treatment cycles, HERCEPTIN® will be admin 5 While the particular aspects and embodiments of the istered prior to paclitaxel. invention as herein shown and disclosed in detail is fully The total duration of this study is 18 weeks. Study capable of obtaining the objects and providing the advan subjects will receive up to 6 total HERCEPTIN® doses. tages herein before stated, it is to be understood that it is After the last subject has received the last cycle of paclitaxel, merely illustrative of some of the presently preferred embodiments of the invention and that no limitations are data collection for safety and pharmacokinetic analysis will 10 intended to the details of methods and articles of manufac stop, and the study will close to protocol specified treatment. ture shown other than as described in the appended claims. Study subjects may continue to receive the HERCEPTIN® The disclosures of all citations in the specification are +/- paclitaxel at the discretion of the investigator. expressly incorporated herein by reference. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO <211> LENGTH: 166 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro Glu 1 5 10 15 Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val 20 25 30 Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu 50 55 60 Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg 65 70 75 Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala 80 85 90 Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr 95 100 105 Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu 110 115 120 Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 125 130 135 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys 140 145 150 Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg 155 160 165 Ala <210> SEQ ID NO 2 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro 5 10 15 Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln 20 25 30 Appx44 HOSPIRA EX. 1001 Page 33 Case: 19-2156 Document: 19 Page: 116 Filed: 07/26/2019 US 6,627,196 Bl 49 50 -continued Gly Cys <210> SEQ ID NO 3 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: salvage receptor binding epitope <400> SEQUENCE: Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro 5 10 <210> SEQ ID NO <2 11 > LENGTH : 7 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: salvage receptor binding epitope <400> SEQUENCE: His Gln Ser Leu Gly Thr Gln 5 <210> SEQ ID NO 5 <2 11 > LENGTH : 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: salvage receptor binding epitope <400> SEQUENCE: 5 His Gln Asn Leu Ser Asp Gly Lys 5 <210> SEQ ID NO <2 11 > LENGTH : 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: salvage receptor binding epitope <400> SEQUENCE: His Gln Asn Ile Ser Asp Gly Lys 5 <210> SEQ ID NO <2 11 > LENGTH : 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: salvage receptor binding epitope <400> SEQUENCE: Val Ile Ser Ser His Leu Gly Gln 1 5 <210> SEQ ID NO 8 <211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val 1 5 10 15 Appx45 HOSPIRA EX. 1001 Page 34 Case: 19-2156 Document: 19 Page: 117 Filed: 07/26/2019 US 6,627,196 Bl 51 52 -continued Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln 20 25 30 Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val 35 40 45 Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 50 55 <210> SEQ ID NO 9 <211> LENGTH: 65 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr 5 10 15 Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr 20 25 30 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys 35 40 45 Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu 50 55 60 Gly Ala Cys Gln Pro 65 <210> SEQ ID NO 10 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Mus Musculus <400> SEQUENCE: 10 Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys <210> SEQ ID NO 11 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 11 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu 35 40 45 Appx46 HOSPIRA EX. 1001 Page 35 Case: 19-2156 Document: 19 Page: 118 Filed: 07/26/2019 US 6,627,196 Bl 53 54 -continued Glu Trp Ile Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn Gln Arg Phe Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser 65 70 75 Ser Arg Ile Val Tyr Met Glu Leu Arg Ser Leu Thr Phe Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115 <210> SEQ ID NO 12 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: humanized VL sequence <400> SEQUENCE: 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys <210> SEQ ID NO 13 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: humanized VH sequence <400> SEQUENCE: 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Appx47 HOSPIRA EX. 1001 Page 36 Case: 19-2156 Document: 19 Page: 119 Filed: 07/26/2019 US 6,627,196 Bl 55 56 -continued Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 <210> SEQ ID NO 14 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: VL consensus sequence <400> SEQUENCE: 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys <210> SEQ ID NO 15 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: VH consensus sequence <400> SEQUENCE: 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu 95 100 105 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 What is claimed is: administering to the patient an initial dose of at least 1. A method for the treatment of a human patient diag approximately 5 mg/kg of the anti-ErbB2 antibody; and nosed with cancer characterized by overexpression of ErbB2 receptor, comprising administering an effective amount of 65 administering to the patient a plurality of subsequent an anti-ErbB2 antibody to the human patient, the method doses of the antibody in an amount that is approxi comprising: mately the same or less than the initial dose, wherein Appx48 HOSPIRA EX. 1001 Page 37 Case: 19-2156 Document: 19 Page: 120 Filed: 07/26/2019 US 6,627,196 Bl 57 58 the subsequent doses are separated in time from each the first subsequent and additional subsequent doses are other by at least two weeks. separated in time by at least 3 weeks. 2. The method of claim 1, wherein the initial dose is at 17. The method of claim 1, wherein said cancer is selected least approximately 6 mg/kg. from the group consisting of breast cancer, leukemia, squa- 3. The method of claim 2, wherein the initial dose is at 5 mous cell cancer, small-celllung cancer, non-small cell lung least approximately 8 mg/kg. cancer, gastrointestinal cancer, pancreatic cancer, 4. The method of claim 3, wherein the initial dose is at glioblastoma, cervical cancer, ovarian cancer, liver cancer, least approximately 12 mg/kg. bladder cancer, hepatoma, colon cancer, colorectal cancer, 5. The method of claim 1, wherein the subsequent doses endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid are separated in time from each other by at least three weeks. 10 cancer, hepatic carcinoma and various types of head and 6. The method of claim 1, wherein the initial dose is neck cancer. administered by intravenous injection, and wherein at least 18. The method of claim 17, wherein said cancer is breast one subsequent dose is administered by subcutaneous injec cancer. tion. 19. The method of claim 18, wherein said cancer is 7. The method of claim 1, wherein the initial dose is 15 metastatic breast carcinoma. administered by intravenous injection, wherein at least two 20. The method of claim 1, wherein said antibody binds subsequent doses are administered, and wherein each sub to the extracellular domain of the ErbB2 receptor. sequent dose is administered by a method selected from the 21. The method of claim 20, wherein said antibody binds group consisting of intravenous injection and subcutaneous to epitope 4D5 within the ErbB2 extracellular domain injection. 20 sequence. 8. The method of claim 1, wherein the initial dose and at 22. The method of claim 21, wherein said antibody is a least one subsequent dose are administered by subcutaneous humanized 4D5 anti-ErbB2 antibody. injection. 23. The method of claim 1, wherein efficacy is measured 9. The method of claim 1, wherein the initial dose is by determining the time to disease progression or the selected from the group consisting of approximately 6 25 response rate. mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of 24. A method for the treatment of cancer in a human subsequent doses are at least approximately 2 mg/kg. patient comprising administering to the patient a first dose of 10. The method of claim 9, wherein the plurality of an anti-ErbB2 antibody followed by two or more subsequent subsequent doses are separated in time from each other by doses of the antibody, wherein the subsequent doses are at least three weeks. 30 separated in time from each other by at least two weeks. 11. The method of claim 10, wherein the initial dose is 25. The method of claim 24, wherein the first dose and a approximately 8 mg/kg, and wherein at least one subsequent first subsequent dose are separated from each other in time dose is approximately 6 mg/kg. by at least about three weeks. 12. The method of claim 10, wherein the initial dose is 26. The method of claim 24, wherein the first dose and approximately 12 mg/kg, and wherein at least one subse- 35 subsequent doses are each from about 2 mg/kg to about 16 quent dose is approximately 6 mg/kg. mg/kg. 13. The method of claim 9, wherein the initial dose is 27. The method of claim 26, wherein the first dose and approximately 8 mg/kg, and wherein at least one subsequent subsequent doses are each from about 4 mg/kg to about 12 dose is approximately 8 mg/kg. mg/kg. 14. The method of claim 9, wherein the initial dose is 40 28. The method of claim 27, wherein the first dose and approximately 8 mg/kg, wherein at least one subsequent subsequent doses are each from about 6 mg/kg to about 12 dose is 8 mg/kg, and wherein administration of the initial mg/kg. dose and subsequent doses are separated in time by at least 29. The method of claim 24, wherein from about two to 2 weeks. about ten subsequent doses of the antibody are administered 15. The method of claim 14, wherein the initial dose and 45 to the patient. subsequent doses are separated in time by at least 3 weeks. 30. The method of claim 24, wherein the subsequent doses 16. A method for the treatment of a human patient are separated in time from each other by at least about three diagnosed with cancer characterized by overexpression of weeks. ErbB2 receptor, comprising administering an effective 31. The method of claim 24, wherein the two or more amount of an anti-ErbB2 antibody to the human patient, the 50 subsequent doses are each from about 2 mg/kg to about 16 method comprising: mg/kg. administering to the patient an initial dose of the antibody, 32. The method of claim 24, wherein the two or more wherein the initial dose is a plurality of doses, wherein subsequent doses are each from about 4 mg/kg to about 12 each of the plurality of initial doses is at least appro xi mg/kg. mately 1 mg/kg and is administered on at least 3 55 33. The method of claim 24, wherein the two or more consecutive days, and administering to the patient at subsequent doses are each from about 6 mg/kg to about 12 least 1 subsequent dose of the antibody, wherein at least mg/kg. one subsequent dose is at least approximately 6 mg/kg, and wherein administration of the last initial dose and * * * * * Appx49 HOSPIRA EX. 1001 Page 38 Case: 19-2156 Document: 19 Page: 121 Filed: 07/26/2019 111111 1111111111111111111111111111111111111111111111111111111111111 US007371379B2 c12) United States Patent (10) Patent No.: US 7,371,379 B2 Baughman et al. (45) Date of Patent: May 13,2008 (54) DOSAGES FOR TREATMENT WITH 5,726,023 A 3/1998 Cheever et a!. ANTI-ERBB2 ANTIBODIES 5,728,687 A 3/1998 Bissery 5,747,261 A 5/1998 King et al. (75) Inventors: Sharon A. Baughman, Ventura, CA 5,770,195 A 6/1998 Hudziak et a!. (US); Steven Shak, Burlingame, CA 5,772,997 A 6/1998 Hudziak et a!. 5,776,427 A 7/1998 Thorpe eta!. (US) 5,783,186 A 7/1998 Arakawa et a!. 5,801,005 A 9/1998 Cheever et a!. (73) Assignee: Genentech, Inc., South San Francisco, 5,821,337 A 10/1998 Carteret a!. CA (US) 5,824,311 A 10/1998 Greene eta!. 5,834,229 A 1111998 Vandlen et a!. ( *) Notice: Subject to any disclaimer, the term of this 5,837,243 A 1111998 Deo et al. patent is extended or adjusted under 35 5,837,523 A 1111998 Greene eta!. U.S.C. 154(b) by 540 days. 5,840,525 A 1111998 Vandlen et a!. 5,846,538 A 12/1998 Cheever et a!. (21) Appl. No.: 10/600,152 5,856,110 A 111999 Vandlen et a!. 5,859,206 A 111999 Vandlen et a!. (22) Filed: Jun. 20, 2003 5,869,445 A 2/1999 Cheever et a!. 5,876,712 A 3/1999 Cheever et a!. (65) Prior Publication Data 5,877,305 A 3/1999 Huston eta!. 5,908,835 A 6/1999 Bissery US 2004/0037824 Al Feb. 26, 2004 5,910,486 A 6/1999 Curiel eta!. 5,922,845 A 7/1999 Deo et al. Related U.S. Application Data 5,939,531 A 8/1999 Wels eta!. 5,968,511 A 10/1999 Akita eta!. (62) Division of application No. 09/648,067, filed on Aug. 5,977,322 A 1111999 Marks eta!. 25, 2000, now Pat. No. 6,627,196. 5,985,553 A 1111999 King et al. 6,015,567 A 1/2000 Hudziak et a!. (60) Provisional application No. 60/213,822, filed on Jun. 6,028,059 A 212000 Curiel eta!. 23, 2000, provisional application No. 60/151,018, filed on Aug. 27, 1999. (Continued) (51) Int. Cl. FOREIGN PATENT DOCUMENTS A61K 391395 (2006.01) EP 0003089 A1 7/1979 (52) U.S. Cl...... 424/138.1; 424/130.1; EP 0599274 A1 6/1994 424/133.1; 424/141.1; 424/142.1; 424/143.1; EP 616812 A1 9/1994 424/155.1; 424/156.1; 424/174.1 EP 711565 8/1998 (58) Field of Classification Search ...... 424/130.1, JP 3-240498 10/1991 424/133.1, 138.1, 141.1, 142.1, 143.1, 155.1, JP 5-117165 5/1993 424/156.1, 174.1 JP 5-170667 7/1993 JP 5-213775 8/1993 See application file for complete search history. JP 5-317084 12/1993 (56) References Cited JP 95006982 B2 111995 U.S. PATENT DOCUMENTS (Continued) 4,676,980 A 6/1987 Segal eta!. 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Harris et a!., "A population pharmacokinetic (PK) model for Pegram et a!., "Inhibitory effects of combinaitons of HER-2/neu trastuzumab (Herception) and implications for clinical dosing" Proc antibody and chemotherapeutic agents used for treatment of human Am Soc Clin Oneal (Abstact #488) 21:1231 (2002). breast cancers" Oncogene 18:2241-2251 ( 1999). Harwerth et a!., "Monoclonal Antibodies Against the Extracellular Pegram et al., "Phase II study of receptor-enhanced chemosensitvity Domain of the rebB-2 Receptor Function as Partial Ligand using recombinant humanized anti-p185HER2/neu monoclonal anti Agonists" Journal of Biological Chemistry 267921):15160-15167 body plus cisplatin in patients with HER2/neu-overexpressing (Jul. 25, 1992). metastatic breast cancer refractrory to chemotherapy treatment" Hudziak et al., "Increased Expresssion of the Putative Growth Journal ofClin Onco/16(8):2659-2671 (Aug. 1998). Factor Receptor p185HER2 Causes Transformation and Pietras et a!., "Antibody to HER-2/neu Receptor Blocks DNA Tumorigenesis of NIH 3T3 Cells." Proc. Nat!. Acad. Sci. USA Repair After Cisplatin in Human Breast and Ovarian Cancer Cells" 84(20):7159-7163 (Oct. Oncogene 9:182901839 (1994). Hudziak eta!., "p185HER2 Monoclonal Antibody Has Antiprolifera Presta et al., "Humanizaiton of an Antibody Directed Against IgE" tive Effects In Virto and Sensitizes Human Breast Tumor Cells to J Immunol. 151(5):2623-2632 (Sep. 1, 1993). Tumor Necrosis Factor" Molecular & Cellular Biology 9(3):1165- Raefsky eta!., "Phase II Trial of Docetaxel and Herceptin as First 1172 (Mar. 1989). or Second-Line Chemotherapy for Women with Metastatic Breast Hynes and Stern., "The Biology of erbB-2/neu/HER-2 and Its Role Cancer Whose Tumors Overexpress HER2" Proceedings ofASCO in Cancer." Biochimica et Biophysica Acta 1198 (2-2):165-184 (Abstract n523) 18: 137a (1999). (Dec. 30, 1994). Ravdin and Chamness, "The c-erbB-2 proto-oncogene as a prog ligen et a!., "Characterization of anti-HER/2 antibodies which nostic and predictive marker in breast cancer: a paradigm for the inhibit the growth of breast tumor cells in vitro" Proceedings ofthe developement of other macromolecular marker-a review" Gene American Association for Cancer Research (abstract #3209) 37:470 159( 1): 19-27 (Jun. 14, 1995). (Mar 1996). Renz, M.E. eta!., "Structural requirements for adhesion of soluble Jones eta!., "Replacing the Complementarity-Determining Regions recombinant murine vascular cell adhesion molecule-1 to a4f:l1" in a Human Antibody with Those From a Mouse." Nature. 321- Journal of Cell Biology 125(6):1395-1406 (Jun. 1994). 522-525 (May 29, 1986). Riechmann et a!., "Reshaping Human Antibodies for Therapy" Kasprzyk et al., "Therapy of an Animal Model of Human Gastric Nature 332:323-327 (Mar. 24, 1988). Cancer Using a Combination of Anti-erbB-2 Monoclonal Antibod Rodeck et a!., "Interactions between growth factor receptors and ies" Cancer Research 52(10):2771-2776 (May 15, 1992). corresponding monoclonal antibodies in human tumors" J Cellular Kotts et a!., "Differential Growth Inhibition of Human Carcinoma Biochem. 35(4):315-320 (1987). Cells Exposed to Monoclonal Antibodies Directed against the Sarup et a!., "Characterization of an Anti-P185HER2 Monoclonal Extracellular Domain of the HER2/ERBB2 protooncogene" In Vitro Antibody that Stimulates Receptor Function and Inhibits Tumor (Abstract #176) 26(3):59A (1990). Cell Growth" Growth Regulation 1:72-82 (1991). Kumar eta!., "Regulation of Phosphorylation of the c-erbB-2/HER2 Schlom, J., "Monoclonal Antibodies: They're More and Less Than Gene Product by a Monoclonal Antibody and Serum Growth You Think" Molecular Foundations of Oncology, Broder, S. ed., Factor(s) in Human Mammary Carcinoma Cells" Molecular & Baltimore, MD:Williams & Wilkins, Chapter Cellular Biology 11(2):979-986 (Feb. 1991). Scott eta!., "p185HER2 Signal Transduction in Breast Cancer Cells" Lewis eta!., "Differential Responses of Human Tumor Cell Lines to Journal of Biological Chemistry 266(22): 14300-14305 (Aug. 5, Anti-p185HER2 Monoclonal Antibodies." Cancer Immunol. 1991). Immunother. 37:255-263 (1993). Seifert et a!., "Dextrazoxane in the prevention of doxorubicin Lewis et a!., "Growth Regulation of Human Breast and Ovarian induced cardiotoxicity" Annals of Pharmacotherapy 28(9):1063- Tumor Cells by Heregulin: Evidence for the Requirement of ErbB2 1072 (Sep. 1994). as a Critical Component in Mediating Heregulin Responsiveness" Semba eta!., "A v-erbB-relatd protooncogene, c-erbB-2, is distinct Cancer Research 56:1457-1465 (Mar. 15, 1996). from the c-erbB-1/epidermal growth factor-receptor gene and is Leyland-Jones eta!., "Pharmacokinetics of Herceptin adminstered amplified in a human salivary gland adenocarcinoma" Proc. Nat!. with paclitaxel every three weeks" Breast Cancer Res Treat Acad. Sci. USA 82:6497-6501 (1985). (abstract only) 64:124 (2000). Shawver et a!., "Ligand-Like Effects Induced by Anti-c-erbB-2 Maier et a!., "Requirements for the Internalization of a Murine Antibodies Do Not Correlate with and Are Not Required for Growth Monoclonal Antibody Directed against the HER-2/neu Gene Prod Inhibition of Human Carcinoma Cells" Cancer Research uct c-erb-2" Cancer Research 51(19):5361-5369 (Oct. 1, 1991). 54(5):1367-1373 (Mar. 1, 1994). Masui eta!., "Growth Inhibition of Human Tumor Cells in Athymic Shepard eta!., "Monoclonal Antibody Therapy of Human Cancer: Mice by Anti-Epidermal Growth Factor Receptor Monoclonal Anti Taking the HER2 Protooncogens to the Clinic" J Clin. Immunol. bodies" Cancer Research 44(3): 1002-1007 (Mar. 1984). 11(3):117-127 (1991). Maskuo eta!., "A murine Monoclonal Antibody That Recognizes an Sims et al., "A Humanized CD18 Antibody Can Block Function Extracellualr Domain of the Human c-erbB-2 Protooncogene Prod Without Cell Destruction" The Journal of Immunology uct" Jpn J Cancer Res. 80:10-14 (Jan. 1989). 151(4):2296-2308 (Aug. 1993). Appx52 HOSPIRA EX. 1001 Page 3 Case: 19-2156 Document: 19 Page: 124 Filed: 07/26/2019 US 7,371,379 B2 Page 4 Singal and Iliskovic, "Doxorubicin-Induced cardiomyopathy" New Verma et al., "Efficacy and safety of three-weekly herceptin with England J of Medicine 339(13):900-905 (Sep. 24, 1998). paclitaxel in women with her2-positive metastatic breast cancer: Singal et al., "Combination therapy with probucol prevents preliminary results of a phase II trial II trial" European Journal of adriamycin-induced cardiomyopathy" Journal ofMolecular & Cel Cancer (abstract only) 37:S146 (2001). lular Cardiology 27(4):1055-1063 (Apr. 1995). Vitetta and Uhr, "Monoclonal Antibodies as Agonists: An Expanded Slamon et al., "Human Breast Cancer: Correlation of Relapse and Role for Their Use in Cancer Therapy" Cancer Research Survival with Amplification of the HER-2/neu Oncogene" Science 54(20):5301-5309 (Oct. 15, 1994). 235:177-182 (Jan. 9, 1987). Vogel et a!., "First-Line Herceptin Monotherapy in Metastatic Slamon et al., "Studies of the HER-2/ney Proto-Oncogene in Breast Cancer" Oncology 61(Suppl. 2):37-42 (2001). Human Breast and Ovarian Cancer" Science 244:707-712 (May 12, Washington et a!., "A population pharmacokinetic (PK) model for 2989). trastuzumab (T) following weekly dosing" Clin Pharmacal Ther Slamon eta!., "Use of chemotherapy plus a monoclonal antibody (abstract only) 71:P12 (2002). against HER2 for metastatic breast cancer that overexpresses Watanabe et a!., "Pharmacokinetically guided dose escalation study HER2" New England J of Medicine 344(11):783-792 (Mar. 15, of anit-HER2 monoclonal antibody in patients with HER2/NEU 2001). overexpressing metastatic breast cancer" Proceedings ofthe Ameri Sliwkowaki et al., "A humanized monoclonal antibody for the can Society of Clinical Oncology (Abstract 702 presented at the treatment of HER2 overexpression breast cancer" Proceedings of Annual ASCO meeting held May 15-18, 1998) 17: 182a (1998). the American Association for Cancer Research (abstract only) Wolff et a!., "Monoclonal antibody homodimers: enhanced antitu 37:625-626 (Mar. 1996). mor activity in nude mice" Cancer Research 53(11):2560-2565 Sliwkowski et al., "Coexpression of erbB2 and erbB3 Proteins (1993). Reconstitutes a High Affinity Receptor for Heregulin" Journal of Biological Chemistry 269(20):14661-14665 (May 20, 1994). Xu et a!., "Antibody-Induced Growth Inhibition is Mediated Stancovski et a!., "Mechanistic Aspects of the Opposing Effects of Through Immunochemically and Functionally Distinct Epitopes on Monocolonal Antibodies to the ERBB2 Receptor on Tumor the Extracellular Domain of the c-erb-2 (HER-2/neu) Gene Product Growth" Proc. Nat!. Acad. Sci. USA 88(19):8691-8695 (Oct. 1, p185" International Journal of Cancer 53(3):401-408 (Feb. 1, 1991). 1993). Stevenson et a!., "A Chimeric Antibody With Dual Fe Regions Yamamoto et a!., "Similarity of protein encoded by the human (bisFabFc) Prepared by Manipulations at the IGG Hinge." Anti c-erb-B-2 gene to epidermal growth factor receptor" (GenBank Cancer Drug Design. 3(4):219-230 (1989). accession No. X03363) (Mar. 30, 1995). Suresh et a!., "Bispecific Monoclonal Antibodies from Hybrid Yamamoto et a!., "Similarity of protein encoded by the human Hybridomas" Methods in Enzymology 121:210-228 (1986). c-erb-B-2 gene to epidermal growth factor receptor" Nature Tagliabue et a!., "Selection of Monoclonal Antibodies Which 319:230-234 (1986). Induce Internalization and Phosphorylation of p185hER2 and Growth Zhang et a!., "Shared antigenic epitopes and pathobiological func Inhibition of Cells With HER2/NEU Gene Amplification" Interna tions of anit-pl85her2/neu monoclonal antibodies" Experimental and tional Journal of Cancer 47(6):933-937 (Apr. 1, 1991). Molecular Pathology 67:15-25 (1999). Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity" Science 239:1534-1536 (Mar. 25, 1988). * cited by examiner Appx53 HOSPIRA EX. 1001 Page 4 Case: 19-2156 Document: 19 Page: 125 Filed: 07/26/2019 U.S. Patent May 13,2008 Sheet 1 of 5 US 7,371,379 B2 3H4 aa 541-599 405 aa 529-625 7C2 aa 22-53 7F3 aa 22-53 100 200 300 400 500 600 I I I I I I 2 3 I' ' ~ -5 7C2 7F3 22 248 645 3H4 541 599 405 I 561 625 100 200 300 400 500 600 I I .I . . I 3B4 epitope {SEQ ID N0:8) 58 residues VEECRVLQGLPREYVNARHCLPCBPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVAR I I 541 599 4D5 epitope (SEQ ID N0:9) 64 residues LPCBPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQP I I 561 625 FIG._1 1 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPA 38 SPETBLDMLRBLYQGCQVVQGNLELTYLPTNASLSFL 75 QDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDN 112 YALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEI 149 LKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLID 186 TNRSRA FIG._2 Appx54 HOSPIRA EX. 1001 Page 5 e• 00 Case: 19-2156Document:19Page:126Filed:07/26/2019 • ~ ...,__ Mean Trough Serum Concentration (~J.g/ml) ~ ····0····n ~ 80 =~ 200 70 ~ ~ .... (.H ~ 60 N 0 150 0 QO Appx55 Mean Trough 50 Concentration M rFJ (~J.g/ml) ('D= 40 ('D ..... 100 N 0 ..... 30 Ul 20 50 .... a ···i::J······r •. .. _•..•• O.I. ~ 1.0 10 •• .. •• t ...... •••• •• d 0··· ···IT rJl -....l FIG._3 I I I I I I I I I I I I I I I l I I I I I I I I I I I I I I I I I I I I I I I I I 0 w 0 I -....l 0 5 10 15 20 25 30 35 40 w""""' -....l Week \C =N HOSPIRA EX. 1001 Page 6 e 3000~------~------.------.------~------.------~~----~ • 00 Case: 19-2156Document:19Page:127Filed:07/26/2019 • ~ ~ - -o- • E25_ 4 mg_kg_IV ~ ~ 2500~ I --o- IV _1_mg_kg_anti-HER2 =~ ···6··· IV _2_mg_kg_anti-HER2 ···<>···IV_ 4 mg_kg_anti-HER2 SC_2_mg_kg_anti-HER2 2000 _j I .._ ~ ~ .... (.H ~ N ____...... 0 0 QO Appx56 1500 # # # Cell Mean for # TUMOR VOL , # (mm3) , # rFJ # ('D= , ('D 1000 , ..... , (.H 0 # ..... # # Ul # # # 500 # # # I ------~,'1 d rJl w-...l -...l FIG._4A -5oo..J_------r----r------,-----.---.------.---- w""""' -...l DAY6 DAY 10 DAY21 DAY24 DAY28 DAY31 \C =N HOSPIRA EX. 1001 Page 7 Case: 19-2156 Document: 19 Page: 128 Filed: 07/26/2019 Page 8 ~ ~ """"' """"' ~ ~ = .j;o. .j;o. ~ ~ ...... ~ ~ ...... (.H (.H -....l -....l = = 0 0 N N -....l -....l rFJ rFJ N N 00 00 ('D ('D rJl rJl ~ ~ ('D ('D • • ~ ~ \C \C • • Ul Ul d d QO QO 0 0 -....l -....l 0 0 e e = = ~ ~ w w ~ ~ w w HOSPIRA EX. 1001 DAY31 DAY31 ----<> ----<> ------ ···············~ ···············~ .o------o .o------o DAY28 DAY28 ·····o----- ,' ,' ··. ··. ,, ,, .. .. .. .. . . ,, ,, ,, ,, ...... p p # # # # DAY24 DAY24 # # # # # # # # # # # # # # # # ------····---0 ------····---0 '"0# '"0# ER2 ER2 DAY21 DAY21 ...... .. .. .. .. 10 10 mg_kg_IV mg_kg_IV DAY DAY mg_kg_anti-HER2 mg_kg_anti-HER2 4 4 _2_mg_kg_anti-H _2_mg_kg_anti-H _2_mg_kg_anti-HER2 _2_mg_kg_anti-HER2 _1_mg_kg_anti-HER2 _1_mg_kg_anti-HER2 IV_4 IV_4 IV IV E25_ E25_ IV IV SC SC DAY6 DAY6 ···~··· ···~··· ---0--- -o- --o-- 10~------.------.------.------.------~------.------~ 10~------.------.------.------.------~------.------~ 100 100 1000 1000 10000~------~------r------.------.------.------,------~ 10000~------~------r------.------.------.------,------~ for for VOL VOL Mean Mean (mm3) (mm3) Cell Cell TUMOR TUMOR FIG._48 FIG._48 Appx57 Case: 19-2156 Document: 19 Page: 129 Filed: 07/26/2019 U.S. Patent May 13,2008 Sheet 5 of 5 US 7,371,379 B2 VARIABLE LIGHT 1 10 20 30 40 2C4 DTVMTQSHKIMSTSVGDRVSITC [KASQDVSIGVA----] WYQQRP ** **** * * * 574 DIQMTQSPSSLSASVGDRVTITC [KASQDVSIGVA----] WYQQKP * * **** hum ki DIQMTQSPSSLSASVGDRVTITC [RASQSVSTSSYSYMH] WYQQKP 50 60 70 80 2C4 GQSPKLLIY [SASYRYT] GVPDRFTGSGSGTDFTFTISSVQA ** * * * * * 574 GKAPKLLIY [SASYRYT] GVPSRFSGSGSGTDFTLTISSLQP * **** hum ki GKAPKLLIY [AASSLES] GVPSRFSGSGSGTDFTLTISSLQP 90 100 2C4 EDLAVYYC [QQYYIYPYT] FGGGTKLEIK (SEQ ID N0:10) * * * * 574 EDFATYYC [QQYYIYPYT] FGQGTKVEIK (SEQ ID N0:12) *** hum ki EDFATYYC [QQYNSLPYT] FGQGTKVEIK (SEQ ID N0:14) FIG._5A VARIABLE HEAVY 1 10 20 30 40 2C4 EVQLQQSGPELVKPGTSVKISCKAS [GFTFTDYTMD] WVKQS ** ** * * *** ** * 574 EVQLVESGGGLVQPGGSLRLSCAAS [GFTFTDYTMD] WVRQA ** * * humiii EVQLVESGGGSVQPGGSLRLSCAAS [GFTFSSYAMS] WVRQA 50 60 70 80 2C4 HGKSLEWIG [DVNPNSGGSIYNQRFKG] KASLTVDRSSRIVYM * * ** *** * **** * 574 PGKGLEWVA [DVNPNSGGSIYNQRFKG] RFTLSVDRSKNTLYL * ****** ** **** *· * * humiii PGKGLEWVS [VISGDGGSTYYADSVKG] RFTISRDDSKNTLYL 90 100 110 2C4 ELRSLTFEDTAVYYCAR [NLGPSFYFDY] WGQGTTLVTSS (SEQ ID N0:11) *** ** * 574 QMNSLRAEDTAVYYCAR [NLGPSFYFDY] WGQGTLVTVSS (SEQ ID N0:13) ** *** humiii QMNSLRAEDTAVYYCAR [GRGGGS--DY] WGQGTLVTVSS (SEQ ID N0:15) FIG._5B Appx58 HOSPIRA EX. 1001 Page 9 Case: 19-2156 Document: 19 Page: 130 Filed: 07/26/2019 US 7,371,379 B2 1 2 DOSAGES FOR TREATMENT WITH these cells. In Drebin et a!. PNAS (USA) 83:9129-9133 ANTI-ERBB2 ANTIBODIES (1986), the 7.16.4 antibody was shown to inhibit the tum origenic growth of neu-transformed NIH-3T3 cells as well RELATED APPLICATIONS as rat neuroblastoma cells (from which the neu oncogene 5 was initially isolated) implanted into nude mice. Drebin et This application is divisional of U.S. Ser. No. 09/648,067 a!. in Oncogene 2:387-394 (1988) discuss the production of filed Aug. 25, 2000 (now U.S. Pat. No. 6,627,196), which a panel of antibodies against the rat neu gene product. AI of claims priority under 35 USC 119(e) to provisional appli the antibodies were found to exert a cytostatic effect on the cation Nos. 60/151,018, filed Aug. 27, 1999 and 60/213,822, growth of neu-transformed cells suspended in soft agar. filed Jun. 23, 2000, the contents of which are incorporated 10 Antibodies of the IgM, IgG2a and IgG2b isotypes were able heroin by reference. to mediate significant in vitro lysis ofneu-transformed cells FIELD OF THE INVENTION in the presence of complement, whereas none of the anti bodies were able to mediate high levels of antibody-depen- The present invention concerns the treatment of disorders 15 dent cellular cytotoxicity (ADCC) of the neu-transformed characterized by the overexpression of ErbB2 or disorders cells. Drebin eta!. Oncogene 2:273-277 (1988) report that expressing epidermal growth factor receptor (EGFR), com mixtures of antibodies reactive with two distinct regions on prising administering to a human or animal presenting the the pl85 molecule result in synergistic anti-tumor effects on disorders a therapeutically effective amount of an antibody neu-transformed NIH-3T3 cells implanted into nude mice. that binds ErbB2. More specifically, the invention concerns 20 Biological effects of anti-neu antibodies are reviewed in the treatment of human patients susceptible to or diagnosed Myers et a!., Meth. Enzyrn. 198:277-290 (1991). See also with cancer overexpressing ErbB2 or expressing EGFR, W094/22478 published Oct. 13, 1994. Hudziak eta!., Mol. where the treatment is with an anti-ErbB2 antibody admin Cell. Biol. 9(3): 1165-1172 (1989) describe the generation of istered by front loading the dose of antibody during treat a panel of anti-ErbB2 antibodies which were characterized ment by intravenous and/or subcutaneous administration. 25 using the human breast tumor cell line SKBR3. Relative cell The invention optionally includes treatment of cancer in a proliferation of the SKBR3 cells following exposure to the human patient with a combination of an anti -ErbB2 antibody antibodies was determined by crystal violet staining of the and a chemotherapeutic agent, such as, but not limited to, a monolayers after 72 hours. Using this assay, maximum taxoid. The taxoid may be, but is not limited to paclitaxel or inhibition was obtained with the antibody called 4D5 which docetaxel. The invention further includes treatment of can 30 inhibited cellular proliferation by 56%. Other antibodies in cer in a human patient with a combination of anti-ErbB2 the panel, including 7C2 and 7F3, reduced cellular prolif antibody and a chemotherapeutic agent, such as, but not limited to, an anthracycline derivative. Optionally, treatment eration to a lesser extent in this assay. Hudziak et a!. with a combination of anti-ErbB2 and an anthracycline conclude that the effect of the 4D5 antibody on SKBR3 cells was cytostatic rather than cytotoxic, since SKBR3 cells derivative includes treatment with an effective amount of a 35 cardioprotectant. The present invention further concerns resumed growth at a nearly normal rate following removal infrequent dosing of anti-ErbB2 antibodies. of the antibody from the medium. The antibody 4D5 was 6 2 further found to sensitize p 185er B -overexpressing breast BACKGROUND OF THE INVENTION tumor cell lines to the cytotoxic effects of TNF -a. See also W089/06692 published Jul. 27, 1989. The anti-ErbB2 anti 40 Proto-oncogenes that encode growth factors and growth bodies discussed in Hudziak et a!. are further characterized factor receptors have been identified to play important roles in Fendly et a!. Cancer Research 50:1550-1558 (1990); in the pathogenesis of various human malignancies, includ Kotts eta!. In Vitro 26(3):59A (1990); Sarup eta!. Growth ing breast cancer. It has been found that the human ErbB2 Regulation 1:72-82 (1991); Shepard eta!. J. Clin. Irnrnunol. 11(3):117-127 (1991); Kumar eta!. Mol. Cell. Biol. 11(2): gene (erbB2, also known as her2, or c-erbB-2), which 45 encodes a 185-kd transmembrane glycoprotein receptor 979-986 (1991); Lewis eta!. Cancer Irnrnunol. Irnrnunother. 2 (p185HER ) related to the epidermal growth factor receptor 37:255-263 (1993); Pietras et a!. Oncogene 9:1829-1838 (EGFR), is overexpressed in about 25% to 30% of human (1994); Vitetta eta!. Cancer Research 54:5301-5309 (1994); breast cancer (Slamon eta!., Science 235:177-182 [1987]; Sliwkowski et a!. J. Biol. Chern. 269(20):14661-14665 (1994); Scott eta!. J. Biol. Chern. 266:14300-5 (1991); and Slamon eta!., Science 244:707-712 [1989]). 50 Several lines of evidence support a direct role for ErbB2 D'souza eta!. Proc. Nat!. Acad. Sci. 91:7202-7206 (1994). in the pathogenesis and clinical aggressiveness of ErbB2- Tagliabue eta!. Int. J. Cancer 47:933-937 (1991) describe overexpressing tumors. The introduction of ErbB2 into two antibodies which were selected for their reactivity on non-neoplastic cells has been shown to cause their malignant the lung adenocarcinoma cell line (Calu-3) which overex transformation (Hudziak et a!., Proc. Nat!. Acad. Sci. USA 55 presses ErbB2. One of the antibodies, called MGR3, was 84:7159-7163 [1987]; DiFiore et a!., Science 237:78-182 found to internalize, induce phosphorylation of ErbB2, and [1987]). Transgenic mice that express HER2 were found to inhibit tumor cell growth in vitro. develop mammary tumors (Guy eta!., Proc. Nat!. Acad. Sci. McKenzie eta!. Oncogene 4:543-548 (1989) generated a USA 89:10578-10582 [1992]). panel of anti-ErbB2 antibodies with varying epitope speci- Antibodies directed against human erbB2 protein prod 60 ficities, including the antibody designated TAl. This TAl ucts and proteins encoded by the rat equivalent of the erbB2 antibody was found to induce accelerated endocytosis of gene (neu) have been described. Drebin eta!., Cell 41:695- ErbB2 (see Maier eta!. Cancer Res. 51:5361-5369 [1991]). 706 (1985) refer to an IgG2a monoclonal antibody which is Bacus et a!. Molecular Carcinogenesis 3:350-362 (1990) directed against the rat neu gene product. This antibody reported that the TAl antibody induced maturation of the called 7.16.4 causes down-modulation of cell surface pl85 65 breast cancer cell lines AU-565 (which overexpresses the expression on Bl04-l-l cells (NIH-3T3 cells transfected erbB2 gene) and MCF-7 (which does not). Inhibition of with the neu proto-oncogene) a inhibits colony formation of growth and acquisition of a mature phenotype in these cells Appx59 HOSPIRA EX. 1001 Page 10 Case: 19-2156 Document: 19 Page: 131 Filed: 07/26/2019 US 7,371,379 B2 3 4 was found to be associated with reduced levels of ErbB2 mended weekly maintenance dose is 2 mg/kg and can be receptor at the cell surface and transient increased levels in administered as a 30-minute infusion if the initial loading the cytoplasm. dose is well tolerated. Stancovski eta!. PNAS (USA) 88:8691-8695 (1991) gen ErbB2 overexpression is commonly regarded as a predic- erated a panel of anti-ErbB2 antibodies, injected them i.p. 5 tor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et a!., into nude mice and evaluated their effect on tumor growth of [1987] and [1989], supra; Ravdin and Chanmess, Gene murine fibroblasts transformed by overexpression of the 159:19-27 [1995]; and Hynes and Stem, Biochim Biophys erbB2 gene. Various levels of tumor inhibition were detected Acta 1198:165-184 [1994]), and has been linked to sensi- for four of the antibodies, but one of the antibodies (N28) 10 tivity and/or resistance to hormone therapy and chemothera consistently stimulated tumor growth. Monoclonal antibody peutic regimens, including CMF (cyclophosphamide, meth N28 induced significant phosphorylation of the ErbB2 otrexate, and fluoruracil) and anthracyclines (Baselga eta!., receptor, whereas the other four antibodies generally dis Oncology 11(3 Supp11):43-48 [1997]). However, despite played low or no phosphorylation-inducing activity. The the association of ErbB2 overexpression with poor progno- effect of the anti-ErbB2 antibodies on proliferation of 15 sis, the odds ofHER2-positive patients responding clinically SKBR3 cells was also assessed. In this SKBR3 cell prolif to treatment with taxanes were greater than three times those eration assay, two of the antibodies (N12 and N29) caused ofHER2-negative patients (Ibid). rhuMab HER2 was shown a reduction in cell proliferation relative to control. The to enhance the activity of paclitaxel (TAXOL®) and doxo ability of the various antibodies to induce cell lysis in vitro rubicin against breast cancer xenografts in nude mice via complement-dependent cytotoxicity (CDC) and anti 20 injected with BT-474 human breast adenocarcinoma cells, body-mediated cell-dependent cytotoxicity (ADCC) was which express high levels of HER2 (Baselga et a!., Breast assessed, with the authors of this paper concluding that the Cancer, Proceedings ofASCO, Vol. 13, Abstract 53 [1994]). inhibitory function of the antibodies was not attributed significantly to CDC or ADCC. SUMMARY OF THE INVENTION 25 Bacus eta!. Cancer Research 52:2580-2589 (1992) fur- The present invention concerns the discovery that an early ther characterized the antibodies described in Bacus et a!. attainment of an efficacious target trough serum concentra (1990) and Stancovski et a!. of the preceding paragraphs. tion by providing an initial dose or doses of anti-ErbB2 Extending the i.p. studies of Stancovski et a!., the effect of antibodies followed by subsequent doses of equal or smaller the antibodies after i.v. injection into nude mice harboring 30 amounts of antibody (greater front loading) is more effica mouse fibroblasts overexpressing human ErbB2 was cious than conventional treatments. The efficacious target assessed. As observed in their earlier work, N28 accelerated trough serum concentration is reached in 4 weeks or less, tumor growth, whereas N12 and N29 significantly inhibited preferably 3 weeks or less, more preferably 2 weeks or less, growth of the ErbB2-expressing cells. Partial tumor inhibi and most preferably 1 week or less, including 1 day or less. tion was also observed with the N24 antibody. Bacus et a!. 35 The target serum concentration is thereafter maintained by also tested the ability of the antibodies to promote a mature the administration of maintenance doses of equal or smaller phenotype in the human breast cancer cell lines AU-565 and amounts for the remainder of the treatment regimen or until MDA-MB453 (which overexpress ErbB2) as well as MCF -7 suppression of disease symptoms is achieved. (containing low levels of the receptor). Bacus eta!. saw a The invention further concerns a method for the treatment correlation between tumor inhibition in vivo and cellular 40 of a human patient susceptible to or diagnosed with a differentiation; the tumor-stimulatory antibody N28 had no disorder characterized by overexpression of ErbB2 receptor effect on differentiation, and the tumor inhibitory action of comprising administering a therapeutically effective amount the N12, N29 and N24 antibodies correlated with the extent of an anti-ErbB2 antibody subcutaneously. Preferably, the of differentiation they induced. initial dose (or doses) as well as the subsequent maintenance Xu et a!. Int. J. Cancer 53:401-408 (1993) evaluated a 45 dose or doses are administered subcutaneously. Optionally, panel of anti-ErbB2 antibodies for their epitope binding where the patient's tolerance to the anti-ErbB2 antibody is specificities, as well as their ability to inhibit anchorage unknown, the initial dose is administered by intravenous independent and anchorage-dependent growth of SKBR3 infusion, followed by subcutaneous administration of the cells (by individual antibodies and in combinations), modu maintenance doses if the patient's tolerance for the antibody late cell-surface ErbB2, and inhibit ligand stimulated 50 is acceptable. anchorage-independent growth. See also W094/00136 pub According to the invention, the method of treatment lished Jan. 6, 1994 and Kasprzyk et a!. Cancer Research involves administration of an initial dose of anti-ErbB2 52:2771-2776 (1992) concerning anti-ErbB2 antibody com antibody of more than approximately 4 mg/kg, preferably binations. Other anti-ErbB2 antibodies are discussed in more than approximately 5 mg/kg. The maximum initial Hancock eta!. Cancer Res. 51:4575-4580 (1991); Shawver 55 dose or a subsequent dose does not exceed 50 mg/kg, et a!. Cancer Res. 54:1367-1373 (1994); Arteaga et a!. preferably does not exceed 40 mg/kg, and more preferably Cancer Res. 54:3758-3765 (1994); and Harwerth et a!. J. does not exceed 30 mg/kg. Administration is by intravenous Biol. Chern. 267:15160-15167 (1992). or subcutaneous administration, preferably intravenous infu A recombinant humanized anti-ErbB2 monoclonal anti sion or bolus injection, or more preferably subcutaneous body (a humanized version of the murine anti-ErbB2 anti 60 bolus injection. The initial dose may be one or more body 4D5, referred to as rhuMAb HER2, HERCEPTIN®, or administrations of drug sufficient to reach the target trough HERCEPTIN® anti-ErbB2 antibody) has been clinically serum concentration in 4 weeks or less, preferably 3 weeks active in patients with ErbB2-overexpressing metastatic or less, more preferably 2 weeks or less, and most preferably breast cancers that had received extensive prior anti-cancer 1 week or less, including one day or less. therapy (Baselga eta!., J. Clin. Oneal. 14:737-744 [1996]). 65 According to the invention, the initial dose or doses is/are The recommended initial loading dose for HERCEPTIN® is followed by subsequent doses of equal or smaller amounts 4 mg/kg administered as a 90-minute infusion. The recom- of antibody at intervals sufficiently close to maintain the Appx60 HOSPIRA EX. 1001 Page 11 Case: 19-2156 Document: 19 Page: 132 Filed: 07/26/2019 US 7,371,379 B2 5 6 trough serum concentration of antibody at or above an trointestinal cancer, pancreatic cancer, glioblastoma, cervi efficacious target level. Preferably, an initial dose or subse cal cancer, ovarian cancer, liver cancer, bladder cancer, quent dose does not exceed 50 mg/kg, and each subsequent hepatoma, colon cancer, colorectal cancer, endometrial car dose is at least 0.01 mg/kg. Preferably the amount of drug cinoma, salivary gland carcinoma, kidney cancer, liver can administered is sufficient to maintain the target trough serum cer, prostate cancer, vulval cancer, thyroid cancer, hepatic concentration such that the interval between administration carcinoma and various types of head and neck cancer. The cycles is at least one week. Preferably the trough serum method of the invention may further comprise administra concentration does not exceed 2500 f.tg/ml and does not fall tion of a chemotherapeutic agent other than an anthracy below 0.01 flg/ml during treatment. The front loading drug cline, e.g. doxorubicin or epirubicin. The chemotherapeutic treatment method of the invention has the advantage of 10 agent preferably is a taxoid, such as TAXOL® (paclitaxel) increased efficacy by reaching a target serum drug concen or a TAXOL® derivative. tration early in treatment. The subcutaneous delivery of Preferred anti-ErbB2 antibodies bind the extracellular maintenance doses according to the invention has the advan domain of the ErbB2 receptor, and preferably bind to the tage of being convenient for the patient and health care epitope 4D5 or 3H4 within the ErbB2 extracellular domain professionals, reducing time and costs for drug treatment. 15 sequence. More preferably, the antibody is the antibody Preferably, the initial dose (or the last dose within an initial 4D5, most preferably in a humanized form. Other preferred dose series) is separated in time from the first subsequent ErbB2-binding antibodies include, but are not limited to, dose by 4 weeks or less, preferably 3 weeks or less, more antibodies 7C2, 7F3, and 2C4, preferably in a humanized preferably 3 weeks or less, most preferably 1 week or less. form. In an embodiment of the invention, the initial dose of 20 The method of the present invention is particularly suit anti-ErbB2 is 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by able for the treatment of breast or ovarian cancer, charac intravenous or subcutaneous administration, such as intra terized by the overexpression of the ErbB2 receptor. venous infusion or subcutaneous bolus injection. The sub The present application also provides a method of therapy sequent maintenance doses are 2 mg/kg delivered once per involving infrequent dosing of an anti-ErbB2 antibody. In week by intravenous infusion, intravenous bolus injection, 25 particular, the invention provides a method for the treatment subcutaneous infusion, or subcutaneous bolus injection. The of cancer (e.g. cancer characterized by overexpression of the choice of delivery method for the initial and maintenance ErbB2 receptor) in a human patient comprising administer doses is made according to the ability of the animal or ing to the patient a first dose of an anti-ErbB2 antibody human patient to tolerate introduction of the antibody into followed by at least one subsequent dose of the antibody, the body. Where the antibody is well-tolerated, the time of 30 wherein the first dose and subsequent dose are separated infusion may be reduced. The choice of delivery method as from each other in time by at least about two weeks (e.g. disclosed for this embodiment applies to all drug delivery from about two weeks to about two months), and optionally regimens contemplated according to the invention. at least about three weeks (e.g. from about three weeks to In another embodiment, the invention includes an initial about six weeks). For instance, the antibody may be admin dose of 12 mg/kg anti-ErbB2 antibody, followed by subse- 35 istered about every three weeks, about two to about 20 times, quent maintenance doses of 6 mg/kg once per 3 weeks. e.g. about six times. The first dose and subsequent dose may In still another embodiment, the invention includes an each be from about 2 mg/kg to about 16 mg/kg; e.g. from initial dose of 8 mg/kg anti-ErbB2 antibody, followed by 6 about 4 mg/kg to about 12 mg/kg; and optionally from about mg/kg once per 3 weeks. 6 mg/kg to about 12 mg/kg. Generally, two or more subse In yet another embodiment, the invention includes an 40 quent doses (e.g. from about two to about ten subsequent initial dose of 8 mg/kg anti-ErbB2 antibody, followed by doses) of the antibody are administered to the patient, and subsequent maintenance doses of 8 mg/kg once per week or those subsequent doses are preferably separated from each 8 mg/kg once every 2 to 3 weeks. other in time by at least about two weeks (e.g. from about In another embodiment, the invention includes initial two weeks to about two months), and optionally at least doses of at least 1 mg/kg, preferably 4 mg/kg, anti-ErbB2 45 about three weeks (e.g. from about three weeks to about six antibody on each of days 1, 2 and 3, followed by subsequent weeks). The two or more subsequent doses may each be maintenance doses of 6 mg/kg once per 3 weeks. from about 2 mg/kg to about 16 mg/kg; or from about 4 In another embodiment, the invention includes an initial mg/kg to about 12 mg/kg; or from about 6 mg/kg to about dose of 4 mg/kg anti-ErbB2 antibody, followed by subse 12 mg/kg. The invention additionally provides an article of quent maintenance doses of 2 mg/kg twice per week, 50 manufacture, comprising a container, a composition within wherein the maintenance doses are separated by 3 days. the container comprising an anti-ErbB2 antibody, and a In still another embodiment, the invention includes a package insert containing instructions to administer the cycle of dosing in which delivery of anti-ErbB2 antibody is antibody according to such methods. 2-3 times per week for 3 weeks. In one embodiment of the The presently described dosing protocols may be applied invention, each dose is approximately 25 mg/kg or less for 55 to other anti-ErbB antibodies such as anti-epidermal growth a human patient, preferably approximately 10 mg/kg or less. factor receptor (EGFR), anti-ErbB3 and anti-ErbB4 anti This 3 week cycle is preferably repeated as necessary to bodies. Thus, the invention provides a method for the achieve suppression of disease symptoms. treatment of cancer in a human patient, comprising admin In another embodiment, the invention includes a cycle of istering an effective amount of an anti-ErbB antibody to the dosing in which delivery of anti-ErbB2 antibody is daily for 60 human patient, the method comprising administering to the 5 days. According to the invention, the cycle is preferably patient an initial dose of at least approximately 5 mg/kg of repeated as necessary to achieve suppression of disease the anti-ErbB antibody; and administering to the patient a symptoms. plurality of subsequent doses of the antibody in an amount The disorder preferably is a benign or malignant tumor that is approximately the same or less than the initial dose. characterized by the overexpression of the ErbB2 receptor, 65 Alternatively, or additionally, the invention pertains to a e.g. a cancer, such as, breast cancer, squamous cell cancer, method for the treatment of cancer in a human patient small-cell lung cancer, non-small cell lung cancer, gas- comprising administering to the patient a first dose of an Appx61 HOSPIRA EX. 1001 Page 12 Case: 19-2156 Document: 19 Page: 133 Filed: 07/26/2019 US 7,371,379 B2 7 8 anti-ErbB antibody followed by at least one subsequent dose body) or an antibody fragment (e.g., a Fab, F(ab')2 , diabody, of the antibody, wherein the first dose and subsequent dose and the like). The variable light chain and variable heavy are separated from each other in time by at least about two chain regions of humanized anti-ErbB2 antibody 2C4 are weeks. The invention additionally provides an article of shown in FIGS. SA and SB. manufacture, comprising a container, a composition within 5 the container comprising an anti-ErbB antibody, and a BRIEF DESCRIPTION OF THE DRAWINGS package insert containing instructions to administer the antibody according to such methods. FIG. 1 shows epitope-mapping of the extracellular In another aspect, the invention concerns an article of domain ofErbB2 as determined by truncation mutant analy manufacture, comprising a container, a composition within 10 sis and site-directed mutagenesis (Nakamura et a!. J. of the container comprising an anti-ErbB2 antibody, optionally Virology 67(10):6179-6191 [October 1993]; Renz eta!. J. a label on or associated with the container that indicates that Cell Biol. 125(6):1395-1406 [June 1994]). The anti-prolif the composition can be used for treating a condition char erative Mabs 4D5 and 3H4 bind adjacent to the transmem acterized by overexpression of ErbB2 receptor, and a pack brane domain. The various ErbB2-ECD truncations or point age insert containing instructions to avoid the use of anthra 15 mutations were prepared from eDNA using polymerase cycline-type chemotherapeutics in combination with the chain reaction technology. The ErbB2 mutants were composition. According to the invention, the package insert expressed as gD fusion proteins in a mammalian expression further includes instructions to administer the anti-ErbB2 plasmid. This expression plasmid uses the cytomegalovirus antibody at an initial dose of 5 mg/kg followed by the same promoter/enhancer with SV40 termination and polyadeny- or smaller subsequent dose or doses. In another embodiment 20 lation signals located downstream of the inserted eDNA. of the invention, the package insert further includes instruc Plasmid DNA was transfected into 293S cells. One day tions to administer the anti-ErbB2 antibody subcutaneously following transfection, the cells were metabolically labeled for at least one of the doses, preferably for all of the overnight in methionine and cysteine-free, low glucose subsequent doses following the initial dose, most preferably DMEM containing 1% dialyzed fetal bovine serum and 25 for all doses. 25 f.l.Ci each of 35S methionine and 35S cysteine. Supernatants In a further aspect, the invention provides a method of were harvested either the ErbB2 MAbs or control antibodies treating ErbB2 expressing cancer in a human patient com were added to the supernatant and incubated 24 hours at 4 o prising administering to the patient effective amounts of an C. The complexes were precipitated, applied to a 10-20% anti-ErbB2 antibody and a chemotherapeutic agent. In one Tricine SDS gradient gel and electrophoresed at 100 V. The embodiment of the invention, the chemotherapeutic agent is 30 gel was electroblotted onto a membrane and analyzed by a taxoid including, but not limited to, paclitaxel and doc autoradiography. SEQ ID NOs:S and 9 depict the 3H4 and etaxel. In another embodiment, the chemotherapeutic agent 4D5 epitopes, respectively. is an anthracyline derivative including, but not limited to, FIG. 2 depicts with underlining the amino acid sequence doxorubicin or epirubicin. In still another embodiment of the of Domain 1 of ErbB2 (SEQ ID N0:1). Bold amino acids invention, treatment with an anti-ErbB2 antibody and an 35 indicate the location of the epitope recognized by MAbs 7C2 anthracycline derivative further includes administration of a and 7F3 as determined by deletion mapping, i.e. the "7C2/ cardioprotectant to the patient. In still another embodiment, 7F3 epitope" (SEQ ID N0:2). an anthracycline derivative is not administered to the patient FIG. 3 is a graph of anti-ErbB2 antibody (HERCEP with the anti-ErbB2 antibody. One or more additional che TIN®) trough serum concentration (f.lg/ml, mean±SE, dark motherapeutic agents may also be administered to the 40 circles) by week from week 2 through week 36 for ErbB2 patient. The cancer is preferably characterized by overex overexpressing patients treated with HERCEPTIN® anti- pression of ErbB2. ErbB2 antibody at 4 mg/kg initial dose, followed by 2 mg/kg The invention further provides an article of manufacture weekly. The number of patients at each time point is comprising a container, a composition within the container represented by "n" (white squares). comprising an anti-ErbB2 antibody and a package insert 45 FIG. 4A is a linear plot of tumor volume changes over instructing the user of the composition to administer the time in mice treated with HERCEPTIN® anti-ErbB2 anti anti-ErbB2 antibody composition and a chemotherapeutic body. FIG. 4B is a semi-logarithmic plot of the same data as agent to a patient. In another embodiment, the chemothera in FIG. 4A such that the variation in tumor volume for the peutic agent is other than an anthracycline, and is preferably treated animals is observed more readily. a taxoid, such as TAXOL®. In still another embodiment, the 50 FIGS. SA and SB depict alignments of the amino acid chemotherapeutic agent is an anthracycline, including but sequences of the variable light (VL) (FIG. SA) and variable not limited to, doxorubicin or epirubicin. In yet another heavy (VH) (FIG. SB) domains of murine monoclonal anti embodiment, the chemotherapeutic agent is an anthracycline body 2C4 (SEQ ID Nos. 10 and 11, respectively); VL and VH and the package insert further instructs the user to administer domains of humanized Fab version 574 (SEQ ID Nos. 12 a cardioprotectant. 55 and 13, respectively), and human VL and VH consensus The methods and compositions of the invention comprise frameworks (hum K1, light kappa subgroup I; humiii, heavy an anti-ErbB2 antibody and include a humanized anti-ErbB2 subgroup III) (SEQ ID Nos. 14 and 15, respectively). antibody. Thus, the invention further pertains to a compo Asterisks identifY differences between humanized Fab ver- sition comprising an antibody that binds ErbB2 and the use sion 574 and murine monoclonal antibody 2C4 or between of the antibody for treating ErbB2 expressing cancer, e.g., 60 humanized Fab version 574 and the human framework. ErbB2 overexpressing cancer, in a human. The invention Complementarity Determining Regions (CDRs) are in also pertains to the use of the antibody for treating EGFR brackets. Humanized Fab version 574, with the changes expressing cancer. Preferably the antibody is a monoclonal ArgH71 Val, AspH73Arg and IleH69Leu, appears to have antibody 4D5, e.g., humanized 4D5 (and preferably binding restored to that of the original chimeric 2C4 Fab huMAb4D5-8 (HERCEPTIN® anti-ErbB2 antibody); or 65 fragment. Additional FR and/or CDR residues, such as L2, monoclonal antibody 2C4, e.g., humanized 2C4. The anti L54, L55, L56, H35 and/or H48, may be modified (e.g. body may be an intact antibody (e.g., an intact IgG1 anti- substituted as follows-IleL2Thr; ArgL54Leu; TyrL55Glu; Appx62 HOSPIRA EX. 1001 Page 13 Case: 19-2156 Document: 19 Page: 134 Filed: 07/26/2019 US 7,371,379 B2 9 10 ThrL56Ser; AspH35Ser; and Va!H48Ile) in order to further be performed. Alternatively, epitope mapping can be per refine or enhance binding of the humanized antibody. Alter formed (see FIG. 1) to assess whether the antibody binds to natively, or additionally, the humanized antibody may be the 4D5 epitope of ErbB2 (i.e. any one or more residues in affinity matured in order to further improve or refine its the region from about residue 529, e.g. about residue 561 to affinity and/or other biological activities. 5 about residue 625, inclusive). The "epitope 3H4" is the region in the extracellular DETAILED DESCRIPTION OF THE domain of ErbB2 to which the antibody 3H4 binds. This PREFERRED EMBODIMENTS epitope is shown in FIG. 1, and includes residues from about 541 to about 599, inclusive, in the amino acid sequence of 10 ErbB2 extracellular domain. I. Definitions The "epitope 7C2/7F3" is the region at theN-terminus of An "ErbB receptor" is a receptor protein tyrosine kinase the extracellular domain of ErbB2 to which the 7C2 and/or which belongs to the ErbB receptor family and includes 7F3 antibodies (each deposited with the ATCC, see below) EGFR, HER2, ErbB3 and ErbB4 receptors as well as bind. To screen for antibodies which bind to the 7C2/7F3 TEGFR (U.S. Pat. No. 5,708,156) and other members of this 15 epitope, a routine cross-blocking assay such as that family to be identified in the future. The ErbB receptor will described in Antibodies, A Laboratory Manual, Cold Spring generally comprise an extracellular domain, which may bind Harbor Laboratory, E d Harlow and David Lane (1988), can an ErbB ligand; a lipophilic transmembrane domain; a be performed. Alternatively, epitope mapping can be per conserved intracellular tyrosine kinase domain; and a car formed to establish whether the antibody binds to the boxyl-terminal signaling domain harboring several tyrosine 20 7C2/7F3 epitope on ErbB2 (i.e. any one or more of residues residues which can be phosphorylated. The ErbB receptor in the region from about residue 22 to about residue 53 of may be a native sequence ErbB receptor or an amino acid ErbB2; SEQ ID NO: 2). sequence variant thereof. Preferably the ErbB receptor is The term "induces cell death" or "capable of inducing cell native sequence human ErbB receptor. death" refers to the ability of the antibody to make a viable The terms "ErbB1", "epidermal growth factor receptor" 25 cell become nonviable. The "cell" here is one which and "EGFR" are used interchangeably herein and refer to expresses the ErbB2 receptor, especially where the cell native sequence EGFR as disclosed, for example, in Car overexpresses the ErbB2 receptor. A cell which "overex penter eta!. Ann. Rev. Biochem. 56:881-914 (1987), includ presses" ErbB2 has significantly higher than normal ErbB2 ing variants thereof (e.g. a deletion mutant EGFR as in levels compared to a noncancerous cell of the same tissue Humphrey eta!. PNAS (USA) 87:4207-4211 (1990)). erbB1 30 type. Preferably, the cell is a cancer cell, e.g. a breast, refers to the gene encoding the EGFR protein product. ovarian, stomach, endometrial, salivary gland, lung, kidney, Examples of antibodies which bind to EGFR include MAb colon, thyroid, pancreatic or bladder cell. In vitro, the cell 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL may be a SKBR3, BT474, Calu 3, MDA-MB-453, MDA HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC MB-361 or SKOV3 cell. Cell death in vitro may be deter- CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn eta!.) 35 mined in the absence of complement and immune effector and variants thereof, such as chimerized 225 (C225) and cells to distinguish cell death induced by antibody dependent reshaped human 225 (H225) (see, WO 96/40210, Imclone cellular cytotoxicity (ADCC) or complement dependent Systems Inc.). cytotoxicity (CDC). Thus, the assay for cell death may be "ErbB3" and "HER3" refer to the receptor polypeptide as performed using heat inactivated serum (i.e. in the absence disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 40 of complement) and in the absence of immune effector cells. 5,480,968 as well as Kraus eta!. PNAS (USA) 86:9193-9197 To determine whether the antibody is able to induce cell (1989), including variants thereof Examples of antibodies death, loss of membrane integrity as evaluated by uptake of which bind HER3 are described in U.S. Pat. No. 5,968,511 propidium iodide (PI), trypan blue (see Moore et a!. Cyto (Akita and Sliwkowski), e.g. the 8B8 antibody (ATCC HB technology 17:1-11 [1995]) or 7AAD can be assessed rela- 12070) or a humanized variant thereof 45 tive to untreated cells. Preferred cell death-inducing anti The terms "ErbB4" and "HER4" herein refer to the bodies are those which induce PI uptake in the "PI uptake receptor polypeptide as disclosed, for example, in EP Pat assay in BT474 cells". Appln No 599,274; Plowman et a!., Proc. Nat!. Acad. Sci. The phrase "induces apoptosis" or "capable of inducing USA, 90:1746-1750 (1993); and Plowman eta!., Nature, apoptosis" refers to the ability of the antibody to induce 366:473-475 (1993), including variants thereof such as the 50 programmed cell death as determined by binding of annexin HER4 isoforms disclosed in WO 99/19488. V, fragmentation of DNA, cell shrinkage, dilation of endo The terms "HER2", "ErbB2" "c-Erb-B2" are used inter plasmic reticulum, cell fragmentation, and/or formation of changeably. Unless indicated otherwise, the terms "ErbB2" membrane vesicles (called apoptotic bodies). The cell is one "c-Erb-B2" and "HER2" when used herein refer to the which overexpresses the ErbB2 receptor. Preferably the human protein, and "erbB2," "c-erb-B2," and "her2" refer to 55 "cell" is a tumor cell, e.g. a breast, ovarian, stomach, human gene. The human erbB2 gene and ErbB2 protein are, endometrial, salivary gland, lung, kidney, colon, thyroid, for example, described in Semba et a!., PNAS (USA) pancreatic or bladder cell. In vitro, the cell may be a SKBR3, 82:6497-6501 (1985) and Yamamoto eta!. Nature 319:230- BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or 234 (1986) (Genebank accession number X03363). ErbB2 SKOV3 cell. Various methods are available for evaluating comprises four domains (Domains 1-4). 60 the cellular events associated with apoptosis. For example, The "epitope 4D5" is the region in the extracellular phosphatidyl serine (PS) translocation can be measured by domain of ErbB2 to which the antibody 4D5 (ATCC CRL aunexin binding; DNA fragmentation can be evaluated 10463) binds. This epitope is close to the transmembrane through DNA laddering as disclosed in the example herein; region of ErbB2. To screen for antibodies which bind to the and nuclear/chromatin condensation along with DNA frag- 4D5 epitope, a routine cross-blocking assay such as that 65 mentation can be evaluated by any increase in hypodiploid described in Antibodies, A Laboratory Manual, Cold Spring cells. Preferably, the antibody which induces apoptosis is Harbor Laboratory, Ed Harlow and David Lane (1988), can one which results in about 2 to 50 fold, preferably about 5 Appx63 HOSPIRA EX. 1001 Page 14 Case: 19-2156 Document: 19 Page: 135 Filed: 07/26/2019 US 7,371,379 B2 11 12 to 50 fold, and most preferably about I 0 to 50 fold, induction residues are believed to form an interface between the light of annexin binding relative to untreated cell in an "annexin and heavy-chain variable domains. binding assay using BT474 cells" (see below). The term "variable" refers to the fact that certain portions Sometimes the pro-apoptotic antibody will be one which of the variable domains differ extensively in sequence blocks HRG binding/activation of the ErbB2/ErbB3 com- 5 among antibodies and are used in the binding and specificity plex (e.g. 7F3 antibody). In other situations, the antibody is of each particular antibody for its particular antigen. How- one which does not significantly block activation of the ever, the variability is not evenly distributed throughout the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2). Further, variable domains of antibodies. It is concentrated in three the antibody may be one like 7C2 which, while inducing segments called complementarity determining regions apoptosis, does not induce a large reduction in the percent of 10 (CDRs) or hypervariable regions both in the light-chain and cells inS phase (e.g. one which only induces about 0-10% the heavy-chain variable domains. The more highly con- reduction in the percent of these cells relative to control). served portions of variable domains are called the frame The antibody of interest may be one like 7C2 which binds work region (FR). The variable domains of native heavy and specifically to human ErbB2 and does not significantly light chains each comprise four FR regions, largely adopting cross-react with other proteins such as those encoded by the 15 a ~-sheet configuration, connected by three CDRs, which erbBI, erbB3 and/or erbB4 genes. Sometimes, the antibody form loops connecting, and in some cases forming part of, may not significantly cross-react with the rat neu protein, the ~-sheet structure. The CDRs in each chain are held e.g., as described in Schecter eta!. Nature 312:513 (1984) together in close proximity by the FRs and, with the CDRs and Drebin et a!., Nature 312:545-548 (1984). In such from the other chain, contribute to the formation of the embodiments, the extent of binding of the antibody to these 20 antigen-binding site of antibodies (see Kabat et a!., NIH proteins (e.g., cell surface binding to endogenous receptor) Pub!. No. 91-3242, Vol. I, pages 647-669 [1991]). The will be less than about 10% as determined by fluorescence constant domains are not involved directly in binding an activated cell sorting (FACS) analysis or radioimmunopre antibody to an antigen, but exhibit various effector func cipitation (RIA). tions, such as participation of the antibody in antibody "Heregulin" (HRG) when used herein refers to a polypep- 25 dependent cellular cytotoxicity. tide which activates the ErbB2-ErbB3 and ErbB2-ErbB4 Papain digestion of antibodies produces two identical protein complexes (i.e. induces phosphorylation of tyrosine antigen-binding fragments, called "Fab" fragments, each residues in the complex upon binding thereto). Various with a single antigen-binding site, and a residual "Fe" heregulin polypeptides encompassed by this term are dis fragment, whose name reflects its ability to crystallize closed in Holmes eta!., Science, 256:1205-1210 (1992); WO 30 readily. Pepsin treatment yields an F(ab')2 fragment that has 92/20798; Wen et a!., Mol. Cell. Biol., 14(3):1909-1919 two antigen-combining sites and is still capable of cross (1994); and Marchionni eta!., Nature, 362:312-318 (1993), linking antigen. for example. The term includes biologically active frag "Fv" is the minimum antibody fragment which contains a ments and/or variants of a naturally occurring HRG complete antigen-recognition and -binding site. This region polypeptide, such as an EGF-like domain fragment thereof 35 consists of a dimer of one heavy- and one light-chain (e.g. HRG~I 177 _ 244). variable domain in tight, non-covalent association. It is in The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 this configuration that the three CDRs of each variable protein complex" are noncovalently associated oligomers of domain interact to define an antigen-binding site on the the ErbB2 receptor and the ErbB3 receptor or ErbB4 recep surface of the VErVL dimer. Collectively, the six CDRs tor, respectively. The complexes form when a cell express 40 confer antigen-binding specificity to the antibody. However, ing both of these receptors is exposed to HRG and can be even a single variable domain (or half of an Fv comprising isolated by immunoprecipitation and analyzed by SDS only three CDRs specific for an antigen) has the ability to PAGE as described in Sliwkowski et a!., J. Biol. Chern., recognize and bind antigen, although at a lower affinity than 269(20):14661-14665 (1994). the entire binding site. "Antibodies" (Abs) and "immunoglobulins" (Igs) are 45 The Fab fragment also contains the constant domain of glycoproteins having the same structural characteristics. the light chain and the first constant domain (CHI) of the While antibodies exhibit binding specificity to a specific heavy chain. Fab' fragments differ from Fab fragments by antigen, immunoglobulins include both antibodies and other the addition of a few residues at the carboxy terminus of the antibody-like molecules which lack antigen specificity. heavy chain CHI domain including one or more cysteines Polypeptides of the latter kind are, for example, produced at 50 from the antibody hinge region. Fab'-SH is the designation low levels by the lymph system and at increased levels by herein for Fab' in which the cysteine residue(s) of the myelomas. constant domains bear a free thiol group. F(ab')2 antibody "Native antibodies" and "native immunoglobulins" are fragments originally were produced as pairs of Fab' frag usually heterotetrameric glycoproteins of about 150,000 ments which have hinge cysteines between them. Other daltons, composed of two identical light (L) chains and two 55 chemical couplings of antibody fragments are also known. identical heavy (H) chains. Each light chain is linked to a The "light chains" of antibodies (immunoglobulins) from heavy chain by one covalent disulfide bond, while the any vertebrate species can be assigned to one of two clearly number of disulfide linkages varies among the heavy chains distinct types, called kappa (K) and lambda (A.), based on the of different immunoglobulin isotypes. Each heavy and light amino acid sequences of their constant domains. chain also has regularly spaced intrachain disulfide bridges. 60 Depending on the amino acid sequence of the constant Each heavy chain has at one end a variable domain (VH) domain of their heavy chains, immunoglobulins can be followed by a number of constant domains. Each light chain assigned to different classes. There are five major classes of has a variable domain at one end (VL) and a constant domain immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several at its other end; the constant domain of the light chain is of these maybe further divided into subclasses (isotypes), aligned with the first constant domain of the heavy chain, 65 e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy and the light-chain variable domain is aligned with the chain constant domains that correspond to the different variable domain of the heavy chain. Particular amino acid classes of immunoglobulins are called a, ll, E, y, and fl, Appx64 HOSPIRA EX. 1001 Page 15 Case: 19-2156 Document: 19 Page: 136 Filed: 07/26/2019 US 7,371,379 B2 13 14 respectively. The subunit structures and three-dimensional human immunoglobulin are replaced by corresponding non configurations of different classes of immunoglobulins are human residues. Furthermore, humanized antibodies may well known. comprise residues which are found neither in the recipient The term "antibody" is used in the broadest sense and antibody nor in the imported CDR or framework sequences. specifically covers intact monoclonal antibodies, polyclonal These modifications are made to further refine and maximize antibodies, multispecific antibodies (e.g. bispecific antibod antibody performance. In general, the humanized antibody ies) formed from at least two intact antibodies, and antibody will comprise substantially all of at least one, and typically fragments so long as they exhibit the desired biological two, variable domains, in which all or substantially all of the activity. CDRs correspond to those of a non-human immunoglobulin "Antibody fragments" comprise a portion of an intact 10 and all or substantially all of the FRs are those of a human antibody, preferably the antigen binding or variable region immunoglobulin sequence. The humanized antibody opti- of the intact antibody. Examples of antibody fragments mally also will comprise at least a portion of an immuno include Fab, Fab', F(ab')2 , and Fv fragments; diabodies; globulin constant region (Fe), typically that of a human linear antibodies (Zapata et a!., Protein Eng. 8(10):1057- immunoglobulin. For further details, see Jones eta!., Nature, 1062 [1995]); single-chain antibody molecules; and multi- 15 321:522-525 (1986); Reichmann eta!., Nature, 332:323-329 specific antibodies formed from antibody fragments. (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 The term "monoclonal antibody" as used herein refers to (1992). The humanized antibody includes a PRIMA an antibody obtained from a population of substantially TIZED™ antibody wherein the antigen-binding region of homogeneous antibodies, i.e., the individual antibodies the antibody is derived from an antibody produced by comprising the population are identical except for possible 20 immunizing macaque monkeys with the antigen of interest. naturally occurring mutations that may be present in minor "Single-chain Fv" or "sFv" antibody fragments comprise amounts. Monoclonal antibodies are highly specific, being the V Hand V L domains of antibody, wherein these domains directed against a single antigenic site. Furthermore, in are present in a single polypeptide chain. Preferably, the Fv contrast to conventional (polyclonal) antibody preparations polypeptide further comprises a polypeptide linker between which typically include different antibodies directed against 25 the V H and V L domains which enables the sFv to form the different determinants ( epitopes ), each monoclonal antibody desired structure for antigen binding. For a review of sFv see is directed against a single determinant on the antigen. In Pluckthun in The Pharmacology ofMonoclonal Antibodies, addition to their specificity, the monoclonal antibodies are vol. 113, Rosenburg and Moore eds., Springer-Verlag, New advantageous in that they are synthesized by the hybridoma York, pp. 269-315 (1994). culture, uncontaminated by other immunoglobulins. The 30 The term "diabodies" refers to small antibody fragments modifier "monoclonal" indicates the character of the anti- with two antigen-binding sites, which fragments comprise a body as being obtained from a substantially homogeneous heavy-chain variable domain (VH) connected to a light population of antibodies, and is not to be construed as chain variable domain (VL) in the same polypeptide chain requiring production of the antibody by any particular (VH- V L). By using a linker that is too short to allow pairing method. For example, the monoclonal antibodies to be used 35 between the two domains on the same chain, the domains are in accordance with the present invention may be made by the forced to pair with the complementary domains of another hybridoma method first described by Kohler et a!., Nature, chain and create two antigen-binding sites. Diabodies are 256:495 (1975), or may be made by recombinant DNA described more fully in, for example, EP 404,097; WO methods (see, e.g., U.S. Pat. No. 4,816,567). The "mono 93/11161; and Hollinger eta!., Proc. Nat!. Acad. Sci. USA, clonal antibodies" may also be isolated from phage antibody 40 90:6444-6448 (1993). libraries using the techniques described in Clackson et a!., Nature, 352:624-628 (1991) and Marks eta!., J. Mol. Biol., An "isolated" antibody is one which has been identified 222:581-597 (1991), for example. and separated and/or recovered from a component of its The monoclonal antibodies herein specifically include natural environment. Contaminant components of its natural environment are materials which would interfere with diag "chimeric" antibodies (immunoglobulins) in which a portion 45 of the heavy and/or light chain is identical with or homolo nostic or therapeutic uses for the antibody, and may include gous to corresponding sequences in antibodies derived from enzymes, hormones, and other proteinaceous or nonpro- a particular species or belonging to a particular antibody teinaceous solutes. In preferred embodiments, the antibody class or subclass, while the remainder of the chain(s) is will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most identical with or homologous to corresponding sequences in 50 antibodies derived from another species or belonging to preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biologi- internal amino acid sequence by use of a spinning cup cal activity (U.S. Pat. No. 4,816,567; Morrison eta!., Proc. sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue Nat!. Acad. Sci. USA, 81:6851-6855 [1984]). 55 "Humanized" forms of non-human (e.g., murine) anti or, preferably, silver stain. Isolated antibody includes the bodies are chimeric immunoglobulins, immunoglobulin antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which present. Ordinarily, however, isolated antibody will be pre- pared by at least one purification step. contain minimal sequence derived from non-human immu- 60 noglobulin. For the most part, humanized antibodies are As used herein, the term "salvage receptor binding human immunoglobulins (recipient antibody) in which resi epitope" refers to an epitope of the Fe region of an IgG dues from a complementarity determining region (CDR) of molecule (e.g., IgGu IgG2 , IgG3 , or IgG4 ) that is responsible the recipient are replaced by residues from a CDR of a for increasing the in vivo serum half-life of the IgG mol non-human species (donor antibody) such as mouse, rat or 65 ecule. rabbit having the desired specificity, affinity, and capacity. In "Treatment" refers to both therapeutic treatment and some instances, framework region (FR) residues of the prophylactic or preventative measures. Those in need of Appx65 HOSPIRA EX. 1001 Page 16 Case: 19-2156 Document: 19 Page: 137 Filed: 07/26/2019 US 7,371,379 B2 15 16 treatment include those already with the disorder as well as carminomycin, carzinophilin, chromomycins, dactinomy those in which the disorder is to be prevented. cin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, "Mammal" for purposes of treatment refers to any animal doxorubicin, epirubicin, esorubicin, idarubicin, marcello classified as a mammal, including humans, domestic and mycin, mitomycins, mycophenolic acid, nogalamycin, farm animals, and zoo, sports, or pet animals, such as dogs, olivomycins, peplomycin, potfiromycin, puromycin, quela horses, cats, cows, etc. Preferably, the mammal is human. mycin, rodorubicin, streptonigrin, streptozocin, tubercidin, A "disorder" is any condition that would benefit from ubenimex, zinostatin, zorubicin; anti-metabolites such as treatment with the anti-ErbB2 antibody. This includes methotrexate and 5-fluorouracil (5-FU); folic acid analogues chronic and acute disorders or diseases including those such as denopterin, methotrexate, pteropterin, trimetrexate; pathological conditions which predispose the mammal to the 10 purine analogs such as fludarabine, 6-mercaptopurine, thia disorder in question. Non-limiting examples of disorders to miprine, thioguanine; pyrimidine analogs such as ancitab be treated herein include benign and malignant tumors; ine, azacitidine, 6-azauridine, carmofur, cytarabine, dideox leukemias and lymphoid malignancies; neuronal, glial, yuridine, doxifluridine, enocitabine, floxuridine, 5-FU; astrocytal, hypothalamic and other glandular, macrophagal, androgens such as calusterone, dromostanolone propionate, epithelial, stromal and blastocoelic disorders; and inflam 15 epitiostanol, mepitiostane, testolactone; anti-adrenals such matory, angiogenic and immunologic disorders. as amino glutethimide, mitotane, trilostane; folic acid replen The term "therapeutically effective amount" is used to isher such as frolinic acid; aceglatone; aldophosphamide refer to an amount having antiproliferative effect. Preferably, glycoside; aminolevulinic acid; amsacrine; bestrabucil; the therapeutically effective amount has apoptotic activity, bisantrene; edatraxate; defofamine; demecolcine; diazi- or is capable of inducing cell death, and preferably death of 20 quone; elfomithine; elliptinium acetate; etoglucid; gallium benign or malignant tumor cells, in particular cancer cells. nitrate; hydroxyurea; lentinan; lonidaniine; mitoguazone; Efficacy can be measured in conventional ways, depending mitoxantrone; mopidamol; nitracrine; pentostatin; phe on the condition to be treated. For cancer therapy, efficacy namet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; can, for example, be measured by assessing the time to procarbazine; PSK®; razoxane; sizofiran; spirogermanium; disease progression (TTP), or determining the response rates 25 tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; (RR) (see Example 1, below). Therapeutically effective urethan; vindesine; dacarbazine; maunomustine; mitobroni amount also refers to a target serum concentration, such as tol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara a trough serum concentration, that has been shown to be C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel effective in suppressing disease symptoms when maintained (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, for a period of time. 30 N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, The terms "cancer" and "cancerous" refer to or describe Antony, France); chlorambucil; gemcitabine; 6-thioguanine; the physiological condition in mammals that is typically mercaptopurine; methotrexate; platinum analogs such as characterized by unregulated cell growth. Examples of can cisplatin and carboplatin; vinblastine; platinum; etoposide cer include, but are not limited to, carcinoma, lymphoma, (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincris- blastoma, sarcoma, and leukemia. More particular examples 35 tine; vinorelbine; navelbine; novantrone; teniposide; dauno of such cancers include squamous cell cancer, small-cell mycin; aminopterin; xeloda; ibandronate; CPT-11; to poi lung cancer, non-small cell lung cancer, gastrointestinal somerase inhibitor RFS 2000; difluoromethylomithine cancer, pancreatic cancer, glioblastoma, cervical cancer, (DMFO); retinoic acid; esperamicins; capecitabine; and ovarian cancer, liver cancer, bladder cancer, hepatoma, pharmaceutically acceptable salts, acids or derivatives of breast cancer, colon cancer, colorectal cancer, endometrial 40 any of the above. Also included in this definition are carcinoma, salivary gland carcinoma, kidney cancer, pros anti-hormonal agents that act to regulate or inhibit hormone tate cancer, vulval cancer, thyroid cancer, hepatic carcinoma action on tumors such as anti-estrogens including for and various types of head and neck cancer. example tamoxifen, raloxifene, aromatase inhibiting 4(5) The term "cytotoxic agent" as used herein refers to a imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, substance that inhibits or prevents the function of cells 45 LY117018, onapristone, and toremifene (Fareston); and anti and/or causes destruction of cells. The term is intended to androgens such as flutamide, nilutamide, bicalutamide, leu 131 125 90 186 include radioactive isotopes (e.g. 1 , 1 , Y and Re ), prolide, and goserelin; and pharmaceutically acceptable chemotherapeutic agents, and toxins such as enzymatically salts, acids or derivatives of any of the above. active toxins of bacterial, fungal, plant or animal origin, or A "growth inhibitory agent" when used herein refers to a fragments thereof 50 compound or composition which inhibits growth of a cell, A "chemotherapeutic agent" is a chemical compound especially an ErbB2-overexpressing cancer cell either in useful in the treatment of cancer. Examples of chemothera vitro or in vivo. Thus, the growth inhibitory agent is one peutic agents include alkylating agents such as thiotepa and which significantly reduces the percentage of ErbB2 over cyclosphosphamide (CYTOXAN™); alkyl sulfonates such expressing cells in S phase. Examples of growth inhibitory as busulfan, improsulfan and piposulfan; aziridines such as 55 agents include agents that block cell cycle progression (at a benzodopa, carboquone, meturedopa, and uredopa; ethylen place other than S phase), such as agents that induce G 1 imines and methylamelamines including altretamine, trieth arrest and M-phase arrest. Classical M-phase blockers ylenemelamine, trietylenephosphoramide, triethylenethio include the vincas (vincristine and vinblastine), TAXOL®, phosphaoramide and trimethylolomelamine; nitrogen and topo II inhibitors such as doxorubicin, epirubicin, mustards such as chlorambucil, chlornaphazine, cholophos 60 daunorubicin, etoposide, and bleomycin. Those agents that phamide, estramustine, ifosfamide, mechlorethamine, arrest G1 also spill over into S-phase arrest, for example, mechlorethamine oxide hydrochloride, melphalan, novem DNA alkylating agents such as tamoxifen, prednisone, dac bichin, phenesterine, prednimustine, trofosfamide, uracil arbazine, mechlorethamine, cisplatin, methotrexate, 5-fluo mustard; nitrosureas such as carmustine, chlorozotocin, rouracil, and ara-C. Further information can be found in The fotemustine, lomustine, nimustine, ranimustine; antibiotics 65 Molecular Basis of Cancer, Mendelsohn and Israel, eds., such as aclacinomysins, actinomycin, authramycin, azaser Chapter 1, entitled "Cell cycle regulation, oncogenes, and ine, bleomycins, cactinomycin, calicheamicin, carabicin, antineoplastic drugs" by Murakami et a!. (W B Saunders: Appx66 HOSPIRA EX. 1001 Page 17 Case: 19-2156 Document: 19 Page: 138 Filed: 07/26/2019 US 7,371,379 B2 17 18 Philadelphia, 1995), especially p. 13. The 4D5 antibody (and phase can comprise the well of an assay plate; in others it is functional equivalents thereof) can also be employed for this a purification colunm (e.g.,an affinity chromatography col purpose. unm). This term also includes a discontinuous solid phase of "Doxorubicin" is an athracycline antibiotic. The full discrete particles, such as those described in U.S. Pat. No. chemical name of doxorubicin is (8S-cis)-10-[(3amino-2,3, 4,275,149. 6-trideoxy-a-L-lyxo-hexopyranosyl)oxy ]-7,8,9, 10-tetrahy A "liposome" is a small vesicle composed of various dro-6,8, 11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5, 12- types of lipids, phospholipids and/or surfactant which is naphthacenedione. useful for delivery of a drug (such as the anti-ErbB2 The term "cytokine" is a generic term for proteins antibodies disclosed herein and, optionally, a chemothera released by one cell population which act on another cell as 10 peutic agent) to a mammal. The components of the liposome intercellular mediators. Examples of such cytokines are are commonly arranged in a bilayer formation, similar to the lymphokines, monokines, and traditional polypeptide hor lipid arrangement of biological membranes. mones. Included among the cytokines are growth hormone The term "package insert" is used to refer to instructions such as human growth hormone, N-methionyl human customarily included in commercial packages of therapeutic growth hormone, and bovine growth hormone; parathyroid 15 products, that contain information about the indications, hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; usage, dosage, administration, contraindications and/or glycoprotein hormones such as follicle stimulating hormone warnings concerning the use of such therapeutic products. (FSH), thyroid stimulating hormone (TSH), and luteinizing The term "serum concentration," "serum drug concentra hormone (LH); hepatic growth factor; fibroblast growth tion," or "serum HERCEPTIN® anti-ErbB2 antibody con factor; prolactin; placental lactogen; tumor necrosis factor-a 20 centration" refers to the concentration of a drug, such as and -~; mullerian-inhibiting substance; mouse gonadotro HERCEPTIN® anti-ErbB2 antibody, in the blood serum of pin-associated peptide; inhibin; activin; vascular endothelial an animal or human patient being treated with the drug. growth factor; integrin; thrombopoietin (TPO); nerve Serum concentration of HERCEPTIN® anti-ErbB2 anti growth factors such as NGF-~; platelet-growth factor; trans body, for example, is preferably determined by immunoas forming growth factors (TGFs) such as TGF-a and TGF-~; 25 say. Preferably, the immunoassay is an ELISA according to insulin-like growth factor-! and -II; erythropoietin (EPO); the procedure disclosed herein. osteoinductive factors; interferons such as interferon-a, -~, The term "peak serum concentration" refers to the maxi and-y; colony stimulating factors (CSFs) such as macroph mal serum drug concentration shortly after delivery of the age-CSF (M-CSF); granulocyte-macrophage-CSF (GM drug into the animal or human patient, after the drug has CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) 30 been distributed throughout the blood system, but before such as IL-l, IL-l a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, significant tissue distribution, metabolism or excretion of IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-a drug by the body has occurred. or TNF-~; and other polypeptide factors including LIF and The term "trough serum concentration" refers to the kit ligand (KL). As used herein, the term cytokine includes serum drug concentration at a time after delivery of a proteins from natural sources or from recombinant cell 35 previous dose and immediately prior to delivery of the next culture and biologically active equivalents of the native subsequent dose of drug in a series of doses. Generally, the sequence cytokines. trough serum concentration is a minimum sustained effica The term "prodrug" as used in this application refers to a cious drug concentration in the series of drug administra precursor or derivative form of a pharmaceutically active tions. Also, the trough serum concentration is frequently substance that is less cytotoxic to tumor cells compared to 40 targeted as a minimum serum concentration for efficacy the parent drug and is capable of being enzymatically because it represents the serum concentration at which activated or converted into the more active parent form. See, another dose of drug is to be administered as part of the e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Bio treatment regimen. If the delivery of drug is by intravenous chemical Society Transactions, 14, pp. 375-382, 615th administration, the trough serum concentration is most pref Meeting Belfast (1986) and Stella et a!., "Prodrugs: A 45 erably attained within 1 day of a front loading initial drug Chemical Approach to Targeted Drug Delivery," Directed delivery. If the delivery of drug is by subcutaneous admin Drug Delivery, Borchardt eta!., (ed.), pp. 247-267, Humana istration, the peak serum concentration is preferably attained Press (1985). The prodrugs of this invention include, but are in 3 days or less. According to the invention, the trough not limited to, phosphate-containing prodrugs, thiophos serum concentration is preferably attained in 4 weeks or less, phate-containing prodrugs, sulfate-containing prodrugs, 50 preferably 3 weeks or less, more preferably 2 weeks or less, peptide-containing prodrugs, D-amino acid-modified pro most preferably in 1 week or less, including 1 day or less drugs, glycosylated prodrugs, ~-lactam-containing pro using any of the drug delivery methods disclosed herein. drugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-contain The term "intravenous infusion" refers to introduction of ing prodrugs, 5-fluorocytosine and other 5-fluorouridine 55 a drug into the vein of an animal or human patient over a prodrugs which can be converted into the more active period of time greater than approximately 5 minutes, pref cytotoxic free drug. Examples of cytotoxic drugs that can be erably between approximately 30 to 90 minutes, although, derivatized into a prodrug form for use in this invention according to the invention, intravenous infusion is alterna include, but are not limited to, those chemotherapeutic tively administered for 10 hours or less. agents described above. 60 The term "intravenous bolus" or "intravenous push" By "solid phase" is meant a non-aqueous matrix to which refers to drug administration into a vein of an animal or the antibodies used in accordance with the present invention human such that the body receives the drug in approximately can adhere. Examples of solid phases encompassed herein 15 minutes or less, preferably 5 minutes or less. include those formed partially or entirely of glass (e.g., The term "subcutaneous administration" refers to intro controlled pore glass), polysaccharides (e.g., agarose), poly- 65 duction of a drug under the skin of an animal or human acrylamides, polystyrene, polyvinyl alcohol and silicones. In patient, preferable within a pocket between the skin and certain embodiments, depending on the context, the solid underlying tissue, by relatively slow, sustained delivery Appx67 HOSPIRA EX. 1001 Page 18 Case: 19-2156 Document: 19 Page: 139 Filed: 07/26/2019 US 7,371,379 B2 19 20 from a drug receptacle. The pocket may be created by duction of antibodies may be, e.g., a soluble form of the pinching or drawing the skin up and away from underlying extracellular domain ofErbB2 or a portion thereof, contain tissue. ing the desired epitope. Alternatively, cells expressing The term "subcutaneous infusion" refers to introduction ErbB2 at their cell surface (e.g. NIH-3T3 cells transformed of a drug under the skin of an animal or human patient, to overexpress ErbB2; or a carcinoma cell line such as preferably within a pocket between the skin and underlying SKBR3 cells, see Stancovski eta!., PNAS (USA) 88:8691- tissue, by relatively slow, sustained delivery from a drug 8695 [1991]) can be used to generate antibodies. Other receptacle for a period of time including, but not limited to, forms of ErbB2 useful for generating antibodies will be 30 minutes or less, or 90 minutes or less. Optionally, the apparent to those skilled in the art. infusion may be made by subcutaneous implantation of a 10 (i) Polyclonal Antibodies drug delivery pump implanted under the skin of the animal Polyclonal antibodies are preferably raised in animals by or human patient, wherein the pump delivers a predeter multiple subcutaneous (sc) or intraperitoneal (ip) injections mined amount of drug for a predetermined period of time, of the relevant antigen and an adjuvant. It may be useful to such as 30 minutes, 90 minutes, or a time period spanning conjugate the relevant antigen to a protein that is immuno- the length of the treatment regimen. 15 genic in the species to be immunized, e.g., keyhole limpet The term "subcutaneous bolus" refers to drug adminis hemocyanin, serum albumin, bovine thyroglobulin, or soy tration beneath the skin of an animal or human patient, bean trypsin inhibitor using a bifunctional or derivatizing where bolus drug delivery is preferably less than approxi agent, for example, maleimidobenzoyl sulfosuccinimide mately 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds. Administration is ester (conjugation through cysteine residues), N-hydrox- 20 ysuccinimide (through lysine residues), glutaraldehyde, suc preferably within a pocket between the skin and underlying 1 1 tissue, where the pocket is created, for example, by pinching cinic anhydride, SOC!', or R N=C=NR, where Rand R or drawing the skin up and away from underlying tissue. are different alkyl groups. The term "front loading" when referring to drug admin Animals are immunized against the antigen, immuno istration is meant to describe an initially higher dose fol genic conjugates, or derivatives by combining, e.g., 100 flg lowed by the same or lower doses at intervals. The initial 25 or 5 flg of the protein or conjugate (for rabbits or mice, higher dose or doses are meant to more rapidly increase the respectively) with 3 volumes of Freund's complete adjuvant animal or human patient's serum drug concentration to an and injecting the solution intradermally at multiple sites. efficacious target serum concentration. According to the One month later the animals are boosted with 1/s to 1/Jo the present invention, front loading is achieved by an initial dose original amount of peptide or conjugate in Freund's com or doses delivered over three weeks or less that causes the 30 plete adjuvant by subcutaneous injection at multiple sites. animal's or patient's serum concentration to reach a target Seven to 14 days later the animals are bled and the serum is serum trough concentration. Preferably, the initial front assayed for antibody titer. Animals are boosted until the titer loading dose or series of doses is administered in two weeks plateaus. Preferably, the animal is boosted with the conju or less, more preferably in 1 week or less, including 1 day gate of the same antigen, but conjugated to a different or less. Most preferably, where the initial dose is a single 35 protein and/or through a different cross-linking reagent. dose and is not followed by a subsequent maintenance dose Conjugates also can be made in recombinant cell culture as for at least 1 week, the initial dose is administered in 1 day protein fusions. Also, aggregating agents such as alum are or less. Where the initial dose is a series of doses, each dose suitably used to enhance the immune response. is separated by at least 3 hours, but not more than 3 weeks (ii) Monoclonal Antibodies or less, preferably 2 weeks or less, more preferably 1 week or less, most preferably 1 day or less. To avoid adverse 40 Monoclonal antibodies are obtained from a population of immune reaction to an antibody drug such as an anti-ErbB2 substantially homogeneous antibodies, i.e., the individual antibody (e.g., HERCEPTIN® anti-ErbB2 antibody) in an antibodies comprising the population are identical except for animal or patient who has not previously been treated with possible naturally occurring mutations that may be present the antibody, it may be preferable to deliver initial doses of in minor amounts. Thus, the modifier "monoclonal" indi- the antibody by intravenous infusion. The present invention 45 cates the character of the antibody as not being a mixture of includes front loading drug delivery of initial and mainte discrete antibodies. nance doses by infusion or bolus administration, intrave For example, the monoclonal antibodies may be made nously or subcutaneously. using the hybridoma method first described by Kohler eta!., Published information related to anti-ErbB2 antibodies Nature, 256:495 (1975), or may be made by recombinant includes the following issued patents and published appli- 50 DNA methods (U.S. Pat. No. 4,816,567). cations: PCT/US89/0005 1, published Jan. 5, 1989; PCT/ In the hybridoma method, a mouse or other appropriate US90/02697, published May 18, 1990; EU 0474727 issued host animal, such as a hamster, is immunized as hereinabove Jul. 23, 1997; DE 69031120.6, issued Jul. 23, 1997; PCT/ described to elicit lymphocytes that produce or are capable US97/18385, published Oct. 9, 1997; SA 97/9185, issued of producing antibodies that will specifically bind to the Oct. 14, 1997; U.S. Pat. No. 5,677,171, issued Oct. 14, 1997; 55 protein used for immunization. Alternatively, lymphocytes U.S. Pat. No. 5,720,937, issued Feb. 24, 1998; U.S. Pat. No. may be immunized in vitro. Lymphocytes then are fused 5,720,954, issued Feb. 24, 1998; U.S. Pat. No. 5,725,856, with myeloma cells using a suitable fusing agent, such as issued Mar. 10, 1998; U.S. Pat. No. 5,770,195, issued Jun. polyethylene glycol, to form a hybridoma cell (Goding, 23, 1998; U.S. Pat. No. 5,772,997, issued Jun. 30, 1998; Monoclonal Antibodies: Principles and Practice, pp. 59-103 PCTIUS98/2626, published Dec. 10, 1998; and PCT/US99/ 60 [Academic Press, 1986]). 06673, published Mar. 26, 1999, each of which patents and publications is herein incorporated by reference in its The hybridoma cells thus prepared are seeded and grown entirety. in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the II. Production of anti-ErbB2 Antibodies unfused, parental myeloma cells. For example, if the paren- A description follows as to exemplary techniques for the 65 tal myeloma cells lack the enzyme hypoxanthine guanine production of the antibodies used in accordance with the phosphoribosyl transferase (HGPRT or HPRT), the culture present invention. The ErbB2 antigen to be used for pro- medium for the hybridomas typically will include hypox- Appx68 HOSPIRA EX. 1001 Page 19 Case: 19-2156 Document: 19 Page: 140 Filed: 07/26/2019 US 7,371,379 B2 21 22 anthine, aminopterin, and thymidine (HAT medium), which infection and in vivo recombination as a strategy for con substances prevent the growth of HGPRT-deficient cells. structing very large phage libraries (Waterhouse et a!., Nuc. Preferred myeloma cells are those that fuse efficiently, Acids. Res., 21:2265-2266 [1993]). Thus, these techniques support stable high-level production of antibody by the are viable alternatives to traditional monoclonal antibody selected antibody-producing cells, and are sensitive to a 5 hybridoma techniques for isolation of monoclonal antibod medium such as HAT medium. Among these, preferred Ies. myeloma cell lines are murine myeloma lines, such as those The DNA also may be modified, for example, by substi derived from MOPC-21 and MPC-11 mouse tumors avail tuting the coding sequence for human heavy- and light-chain able from the Salk Institute Cell Distribution Center, San constant domains in place of the homologous murine Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells avail 10 sequences (U.S. Pat. No. 4,816,567; Morrison, eta!., Proc. able from the American Type Culture Collection, Rockville, Nat! Acad. Sci. USA, 81:6851 [1984]), or by covalently Md. USA. Human myeloma and mouse-human heteromy joining to the immunoglobulin coding sequence all or part of eloma cell lines also have been described for the production the coding sequence for a non-immunoglobulin polypeptide. of human monoclonal antibodies (Kozbor, J. Immunol., Typically such non-immunoglobulin polypeptides are 133:3001 (1984); Brodeur eta!., Monoclonal Antibody Pro 15 substituted for the constant domains of an antibody, or they duction Techniques and Applications, pp. 51-63 [Marcel are substituted for the variable domains of one antigen Dekker, Inc., New York, 1987]). combining site of an antibody to create a chimeric bivalent Culture medium in which hybridoma cells are growing is antibody comprising one antigen-combining site having assayed for production of monoclonal antibodies directed specificity for an antigen and another antigen-combining site against the antigen. Preferably, the binding specificity of 20 having specificity for a different antigen. monoclonal antibodies produced by hybridoma cells is (iii) Humanized and Human Antibodies determined by immunoprecipitation or by an in vitro binding Methods for humanizing non-human antibodies are well assay, such as radioimmunoassay (RIA) or enzyme-linked known in the art. Preferably, a humanized antibody has one immunoabsorbent assay (ELISA). or more amino acid residues introduced into it from a source The binding affinity of the monoclonal antibody can, for 25 which is non-human. These non-human amino acid residues example, be determined by the Scatchard analysis of Mun are often referred to as "import" residues, which are typi son eta!., Anal. Biochem., 107:220 (1980). cally taken from an "import" variable domain. Humaniza After hybridoma cells are identified that produce antibod- tion can be essentially performed following the method of ies of the desired specificity, affinity, and/or activity, the Winter and co-workers (Jones et a!., Nature, 321:522-525 clones may be subcloned by limiting dilution procedures and 30 (1986); Riechmann et a!., Nature, 332:323-327 (1988); grown by standard methods (Goding, Monoclonal Antibod Verhoeyen eta!. Science, 239:1534-1536 [1988]), by sub- ies: Principles and Practice, pp. 59-103 [Academic Press, stituting rodent CDRs or CDR sequences for the correspond 1986]). Suitable culture media for this purpose include, for ing sequences of a human antibody. Accordingly, such example, D-MEM or RPMI-1640 medium. In addition, the "humanized" antibodies are chimeric antibodies (U.S. Pat. hybridoma cells may be grown in vivo as ascites tumors in 35 No. 4,816,567) wherein substantially less than an intact an animal. human variable domain has been substituted by the corre- The monoclonal antibodies secreted by the subclones are sponding sequence from a non-human species. In practice, suitably separated from the culture medium, ascites fluid, or humanized antibodies are typically human antibodies in serum by conventional immunoglobulin purification proce which some CDR residues and possibly some FR residues dures such as, for example, protein A-Sepharose, hydroxy- 40 are substituted by residues from analogous sites in rodent !apatite chromatography, gel electrophoresis, dialysis, or antibodies. affinity chromatography. The choice of human variable domains, both light and DNA encoding the monoclonal antibodies is readily iso heavy, to be used in making the humanized antibodies is lated and sequenced using conventional procedures (e.g., by very important to reduce antigenicity. According to the using oligonucleotide probes that are capable of binding 45 so-called "best-fit" method, the sequence of the variable specifically to genes encoding the heavy and light chains of domain of a rodent antibody is screened against the entire murine antibodies). The hybridoma cells serve as a preferred library of known human variable-domain sequences. The source of such DNA. Once isolated, the DNA may be placed human sequence which is closest to that of the rodent is then into expression vectors, which are then transfected into host accepted as the human framework region (FR) for the cells such as E. coli cells, simian COS cells, Chinese 50 humanized antibody (Sims et a!., J. Immunol., 151:2296 Hamster Ovary (CHO) cells, or myeloma cells that do not (1993); Chothia et a!., J. Mol. Biol., 196:901 [1987]). otherwise produce immunoglobulin protein, to obtain the Another method uses a particular framework region derived synthesis of monoclonal antibodies in the recombinant host from the consensus sequence of all human antibodies of a cells. Review articles on recombinant expression in bacteria particular subgroup of light or heavy chains. The same of DNA encoding the antibody include Skerra et a!., Curr. 55 framework may be used for several different humanized Opinion in Immunol., 5:256-262 (1993) and Pluckthun, antibodies (Carteret a!., Proc. Nat!. A cad. Sci. USA, 89:4285 Immunol. Revs., 130:151-188 (1992). (1992); Presta eta!., J. Immnol., 151:2623 [1993]). In a further embodiment, antibodies or antibody frag It is further important that antibodies be humanized with ments can be isolated from antibody phage libraries gener retention of high affinity for the antigen and other favorable ated using the techniques described in McCafferty et a!., 60 biological properties. To achieve this goal, according to a Nature, 348:552-554 (1990). Clackson eta!., Nature, 352: preferred method, humanized antibodies are prepared by a 624-628 (1991) and Marks eta!., J. Mol. Biol, 222:581-597 process of analysis of the parental sequences and various (1991) describe the isolation of murine and human antibod conceptual humanized products using three-dimensional ies, respectively, using phage libraries. Subsequent publica models of the parental and humanized sequences. Three tions describe the production of high affinity (nM range) 65 dimensional immunoglobulin models are commonly avail human antibodies by chain shuffling (Marks et a!., Bioi able and are familiar to those skilled in the art. Computer Technology, 10:779-783 [1992]), as well as combinatorial programs are available which illustrate and display probable Appx69 HOSPIRA EX. 1001 Page 20 Case: 19-2156 Document: 19 Page: 141 Filed: 07/26/2019 US 7,371,379 B2 23 24 three-dimensional conformational structures of selected can hapten). Bispecific antibodies can be prepared as full length didate immunoglobulin sequences. Inspection of these dis antibodies or antibody fragments (e.g. F(ab')2 bispecific plays permits analysis of the likely role of the residues in the antibodies). functioning of the candidate immunoglobulin sequence, i.e., Methods for making bispecific antibodies are known in the analysis of residues that influence the ability of the 5 the art. Traditional production of full length bispecific anti candidate immunoglobulin to bind its antigen. In this way, bodies is based on the coexpression of two immunoglobulin FR residues can be selected and combined from the recipient heavy chain-light chain pairs, where the two chains have and import sequences so that the desired antibody charac different specificities (Millstein eta!., Nature, 305:537-539 teristic, such as increased affinity for the target antigen( s), is [1983]). Because of the random assortment of immunoglo achieved. In general, the CDR residues are directly and most 10 bulin heavy and light chains, these hybridomas (quadromas) substantially involved in influencing antigen binding. produce a potential mixture of 10 different antibody mol Alternatively, it is now possible to produce transgenic ecules, of which only one has the correct bispecific structure. animals (e.g., mice) that are capable, upon immunization, of Purification of the correct molecule, which is usually done producing a full repertoire of human antibodies in the by affinity chromatography steps, is rather cumbersome, and absence of endogenous immunoglobulin production. For 15 the product yields are low. Similar procedures are disclosed example, it has been described that the homozygous deletion in WO 93/08829, and in Traunecker et a!., EMBO J., of the antibody heavy-chain joining region (JH) gene in 10:3655-3659 (1991 ). chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of According to a different approach, antibody variable domains with the desired binding specificities (antibody the human germ-line immunoglobulin gene array in such 20 germ-line mutant mice will result in the production of antigen combining sites) are fused to immunoglobulin con human antibodies upon antigen challenge. See, e.g., Jakobo stant domain sequences. The fusion preferably is with an vits et a!., Proc. Nat!. Acad. Sci. USA, 90:2551 (1993); immunoglobulin heavy chain constant domain, comprising Jakobovits eta!., Nature, 362:255-258 (1993); Bruggermann at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region eta!. Year in Immuno., 7:33 (1993). Human antibodies can 25 also be derived from phage-display libraries (Hoogenboom (CHI) containing the site necessary for light chain binding, eta!., J. Mol. Biol., 227:381 (1991); Marks eta!., J. Mol. present in at least one of the fusions. DNAs encoding the Biol., 222:581-597 [1991]). immunoglobulin heavy chain fusions and, if desired, the (iv) Antibody Fragments immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable Various techniques have been developed for the produc- 30 tion of antibody fragments. Traditionally, these fragments host organism. This provides for great flexibility in adjusting were derived via proteolytic digestion of intact antibodies the mutual proportions of the three polypeptide fragments in (see, e.g., Morimoto et a!., Journal of Biochemical and embodiments when unequal ratios of the three polypeptide Biophysical Methods 24:107-117 (1992) and Brennan et a!., chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for Science, 229:81 [1985]). However, these fragments can now 35 be produced directly by recombinant host cells. For two or all three polypeptide chains in one expression vector example, the antibody fragments can be isolated from the when the expression of at least two polypeptide chains in antibody phage libraries discussed above. Alternatively, equal ratios results in high yields or when the ratios are of Fab'-SH fragments can be directly recovered from E. coli no particular significance. In a preferred embodiment of this approach, the bispecific and chemically coupled to form F(ab')2 fragments (Carteret 40 a!., Bio/Technology 10: 163-167 [1992]). According to antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the immunoglobulin heavy chain-light chain pair (providing a production of antibody fragments will be apparent to the second binding specificity) in the other arm. It was found skilled practitioner. In other embodiments, the antibody of 45 that this asymmetric structure facilitates the separation of the choice is a single chain Fv fragment (scFv). See WO desired bispecific compound from unwanted immunoglobu- 93/16185. lin chain combinations, as the presence of an immunoglo (v) Bispecific Antibodies bulin light chain in only one half of the bispecific molecule Bispecific antibodies are antibodies that have binding provides for a facile way of separation. This approach is specificities for at least two different epitopes. Exemplary 50 disclosed in WO 94/04690. For further details of generating bispecific antibodies may bind to two different epitopes of bispecific antibodies see, for example, Suresh eta!., Methods the ErbB2 protein. For example, one arm may bind an in Enzymology, 121:210 (1986). epitope in Domain 1 ofErbB2 such as the 7C2/7F3 epitope, According to another approach described in W096/ the other may bind a different ErbB2 epitope, e.g. the 4D5 27011, the interface between a pair of antibody molecules epitope. Other such antibodies may combine an ErbB2 55 can be engineered to maximize the percentage of het binding site with binding site(s) for EGFR, ErbB3 and/or erodimers which are recovered from recombinant cell cul- ErbB4. Alternatively, an anti-ErbB2 arm may be combined ture. The preferred interface comprises at least a part of the with an arm which binds to a triggering molecule on a CH3 domain of an antibody constant domain. In this method, leukocyte such as a T-cell receptor molecule (e.g. CD2 or one or more small amino acid side chains from the interface CD3), or Fe receptors for IgG (FcyR), such as FcyRI 60 of the first antibody molecule are replaced with larger side (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus chains (e.g. tyrosine or tryptophan). Compensatory "cavi cellular defense mechanisms to the ErbB2-expressing cell. ties" of identical or similar size to the large side chain(s) are Bispecific antibodies may also be used to localize cytotoxic created on the interface of the second antibody molecule by agents to cells which express ErbB2. These antibodies replacing large amino acid side chains with smaller ones possess an ErbB2-binding arm and an arm which binds the 65 (e.g. alanine or threonine). This provides a mechanism for cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alka increasing the yield of the heterodimer over other unwanted loid, ricin A chain, methotrexate or radioactive isotope end-products such as homodimers. Appx70 HOSPIRA EX. 1001 Page 21 Case: 19-2156 Document: 19 Page: 142 Filed: 07/26/2019 US 7,371,379 B2 25 26 Bispecific antibodies include cross-linked or "heterocon (vi) Screening for Antibodies with the Desired Properties jugate" antibodies. For example, one of the antibodies in the Techniques for generating antibodies have been described heteroconjugate can be coupled to avidin, the other to biotin. above. Those antibodies having the characteristics described Such antibodies have, for example, been proposed to target herein are selected. immune system cells to unwanted cells (U.S. Pat. No. 5 To select for antibodies which induce cell death, loss of 4,676,980), and for treatment of HIV infection (WO membrane integrity as indicated by, e.g., PI, trypan blue or 91/00360, WO 92/200373, and EP 03089). Heteroconjugate 7 AAD uptake is assessed relative to control. The preferred antibodies may be made using any convenient cross-linking assay is the "PI uptake assay using BT474 cells". According methods. Suitable cross-linking agents are well known in the to this assay, BT474 cells (which can be obtained from the art, and are disclosed in U.S. Pat. No. 4,676,980, along with 10 American Type Culture Collection [Rockville, Md.]) are a number of cross-linking techniques. cultured in Dulbecco's Modified Eagle Medium (D-MEM): Techniques for generating bispecific antibodies from anti Ham's F-12 (50:50) supplemented with 10% heat-inacti body fragments have also been described in the literature. vated FBS (Hyclone) and 2 mM L-glutamine. (Thus, the assay is performed in the absence of complement and For example, bispecific antibodies can be prepared using 15 immune effector cells). The BT474 cells are seeded at a chemical linkage. Brennan et a!., Science, 229: 81 (1985) density of3x106 per dish in 100x20 mm dishes and allowed describe a procedure wherein intact antibodies are pro to attach overnight. The medium is then removed and teolytically cleaved to generate F(ab')2 fragments. These replaced with fresh medium alone or medium containing 10 fragments are reduced in the presence of the dithiol com f.tg/ml of the appropriate MAb. The cells are incubated for a plexing agent sodium arsenite to stabilize vicinal dithiols 20 3 day time period. Following each treatment, mono layers are and prevent intermolecular disulfide formation. The Fab' washed with PBS and detached by trypsinization. Cells are fragments generated are then converted to thionitrobenzoate then centrifuged at 1200 rpm for 5 minutes at 4° C., the 2 (TNB) derivatives. One of the Fab'-TNB derivatives is then pellet resuspended in 3 ml ice cold Ca + binding buffer (10 reconverted to the Fab'-thiol by reduction with mercaptoet mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaC12 ) and hylamine and is mixed with an equimolar amount of the 25 aliquoted into 35 mm strainer-capped 12x75 tubes (1 ml per other Fab'-TNB derivative to form the bispecific antibody. tube, 3 tubes per treatment group) for removal of cell The bispecific antibodies produced can be used as agents for clumps. Tubes then receive PI (10 flg/ml). Samples may be the selective immobilization of enzymes. analyzed using a FACSCAWM flow cytometer and FAC Recent progress has facilitated the direct recovery of SCONVERT™ CellQuest software (Becton Dickinson). 30 Fab'-SH fragments from E. coli, which can be chemically Those antibodies which induce statistically significant levels coupled to form bispecific antibodies. Shalaby eta!., J. Exp. of cell death as determined by PI uptake are selected. Me d., 175: 217-225 (1992) describe the production of a fully In order to select for antibodies which induce apoptosis, humanized bispecific antibody F(ab')2 molecule. Each Fab' an "annexin binding assay using BT474 cells" is available. fragment was separately secreted from E. coli and subjected The BT474 cells are cultured and seeded in dishes as 35 to directed chemical coupling in vitro to form the bispecific discussed in the preceding paragraph. The medium is then antibody. The bispecific antibody thus formed was able to removed and replaced with fresh medium alone or medium bind to cells overexpressing the ErbB2 receptor and normal containing 10 f.tg/ml of the MAb. Following a three day human T cells, as well as trigger the lytic activity of human incubation period, monolayers are washed with PBS and cytotoxic lymphocytes against human breast tumor targets. detached by trypsinization. Cells are then centrifuged, resus 40 2 Various techniques for making and isolating bispecific pended in Ca + binding buffer and aliquoted into tubes as antibody fragments directly from recombinant cell culture discussed above for the cell death assay. Tubes then receive have also been described. For example, bispecific antibodies labeled annexin (e.g. armexin V-FTIC) (1 flg/ml). Samples have been produced using leucine zippers. Kostelny eta!., J. may be analyzed using a FACSCAWM flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickin Immunol., 148(5):1547-1553 (1992). The leucine zipper 45 peptides from the Fos and Jun proteins were linked to the son). Those antibodies which induce statistically significant Fab' portions of two different antibodies by gene fusion. The levels of annexin binding relative to control are selected as antibody homodimers were reduced at the hinge region to apoptosis-inducing antibodies. form monomers and then re-oxidized to form the antibody In addition to the armexin binding assay, a "DNA staining heterodimers. This method can also be utilized for the 50 assay using BT474 cells" is available. In order to perform production of antibody homodimers. The "diabody" tech this assay, BT474 cells which have been treated with the nology described by Hollinger et a!., Proc. Nat!. Acad. Sci. antibody of interest as described in the preceding two USA, 90:6444-6448 (1993) has provided an alternative paragraphs are incubated with 9 flg/ml HOECHST 33342™ mechanism for making bispecific antibody fragments. The for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flow fragments comprise a heavy-chain variable domain (VH) 55 cytometer (Coulter Corporation) using MOD FIT LT™ soft connected to a light-chain variable domain (VL) by a linker ware (Verity Software House). Antibodies which induce a which is too short to allow pairing between the two domains change in the percentage of apoptotic cells which is 2 fold on the same chain. Accordingly, the V H and V L domains of or greater (and preferably 3 fold or greater) than untreated one fragment are forced to pair with the complementary V L cells (up to 100% apoptotic cells) may be selected as and V H domains of another fragment, thereby forming two 60 pro-apoptotic antibodies using this assay. antigen-binding sites. Another strategy for making bispecific To screen for antibodies which bind to an epitope on antibody fragments by the use of single-chain Fv (sFv) ErbB2 bound by an antibody of interest, a routine cross dimers has also been reported. See Gruber et a!., J. Immu blocking assay such as that described in Antibodies, A nol., 152:5368 (1994). Laboratory Manual, Cold Spring Harbor Laboratory, Ed Antibodies with more than two valencies are contem- 65 Harlow and David Lane (1988), can be performed. Alter plated. For example, trispecific antibodies can be prepared. natively, epitope mapping can be performed by methods Tutt eta!., J. Immunol. 147: 60 (1991). known in the art. Appx71 HOSPIRA EX. 1001 Page 22 Case: 19-2156 Document: 19 Page: 143 Filed: 07/26/2019 US 7,371,379 B2 27 28 To identify anti-ErbB2 antibodies which inhibit growth of taacetic acid (MX-DTPA) is an exemplary chelating agent SKBR3 cells in cell culture by 50-100%, the SKBR3 assay for conjugation of radionucleotide to the antibody. See WO described in WO 89/06692 can be performed. According to 94/11026. this assay, SKBR3 cells are grown in a 1:1 mixture ofF 12 In another embodiment, the antibody may be conjugated and DMEM medium supplemented with 10% fetal bovine 5 to a "receptor" (such streptavidin) for utilization in tumor serum, glutamine and penicillinstreptomycin. The SKBR3 pretargeting wherein the antibody-receptor conjugate is cells are plated at 20,000 cells in a 35 mm cell culture dish administered to the patient, followed by removal of unbound (2 mls/35 mm dish). 2.5 flg/ml of the anti-ErbB2 antibody is conjugate from the circulation using a clearing agent and added per dish. After six days, the number of cells, com then administration of a "ligand" (e.g. avidin) which 1s pared to untreated cells are counted using an electronic 10 COULTER™ cell counter. Those antibodies which inhibit conjugated to a cytotoxic agent (e.g. a radionucleotide). growth of the SKBR3 cells by 50-100% are selected for (ix) Immunoliposomes combination with the apoptotic antibodies as desired. The anti-ErbB2 antibodies disclosed herein may also be (vii) Effector Function Engineering formulated as immunoliposomes. Liposomes containing the It may be desirable to modifY the antibody of the inven- 15 antibody are prepared by methods known in the art, such as tion with respect to effector function, so as to enhance the described in Epstein et a!., Proc. Nat!. Acad. Sci. USA, effectiveness of the antibody in treating cancer, for example. 82:3688 (1985); Hwang eta!., Proc. Nat! Acad. Sci. USA, For example, cysteine residue(s) may be introduced in the Fe 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544, region, thereby allowing interchain disulfide bond formation 545. Liposomes with enhanced circulation time are dis in this region. The homodimeric antibody thus generated 20 closed in U.S. Pat. No. 5,013,556. may have improved internalization capability and/or Particularly useful liposomes can be generated by the increased complement-mediated cell killing and antibody reverse phase evaporation method with a lipid composition dependent cellular cytotoxicity (ADCC). See Caron et a!., J. comprising phosphatidylcholine, cholesterol and PEG-de Exp Me d. 176:1191-1195 (1992) and Shapes, B. J. Immunol. rivatized phosphatidylethanolamine (PEG-PE). Liposomes 148:2918-2922 (1992). Homodimeric antibodies with 25 are extruded through filters of defined pore size to yield enhanced anti-tumor activity may also be prepared using liposomes with the desired diameter. Fab' fragments of the heterobifunctional cross-linkers as described in Wolff eta!. antibody of the present invention can be conjugated to the Cancer Research 53:2560-2565 (1993). Alternatively, an liposomes as described in Martinet a!., J. Biol. Chern. 257: antibody can be engineered which has dual Fe regions and 286-288 (1982) via a disulfide interchange reaction. A may thereby have enhanced complement lysis and ADCC 30 chemotherapeutic agent is optionally contained within the capabilities. See Stevenson et a!. Anti-Cancer Drug Design liposome. See Gabizon et a!., J. National Cancer Inst. 3:219-230 (1989). 81(19)1484 (1989). (viii) Immunoconjugates (x) Antibody Dependent Enzyme Mediated Prodrug The invention also pertains to immunoconjugates com Therapy (ADEPT) prising the antibody described herein conjugated to a cyto 35 toxic agent such as a chemotherapeutic agent, toxin (e.g. an The antibodies of the present invention may also be used enzymatically active toxin of bacterial, fungal, plant or in ADEPT by conjugating the antibody to a prodrug-acti animal origin, or fragments thereof), or a radioactive isotope vating enzyme which converts a prodrug (e.g. a peptidyl (i.e., a radioconjugate ). chemotherapeutic agent, see WO 81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Chemotherapeutic agents useful in the generation of such 40 immunoconjugates have been described above. Enzymati Pat. No. 4,975,278. cally active toxins and fragments thereof which can be used The enzyme component of the immunoconjugate useful include diphtheria A chain, nonbinding active fragments of for ADEPT includes any enzyme capable of acting on a diphtheria toxin, exotoxin A chain (from Pseudomonas prodrug in such a way so as to covert it into its more active, aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, 45 cytotoxic form. alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Enzymes that are useful in the method of this invention Phytolaca americana proteins (PAPI, PAPII, and PAP-S), include, but are not limited to, alkaline phosphatase useful momordica charantia inhibitor, curcin, crotin, sapaonaria for converting phosphate-containing prodrugs into free officinalis inhibitor, gelonin, mitogellin, restrictocin, pheno drugs; arylsulfatase useful for converting sulfate-containing mycin, enomycin and the tricothecenes. A variety of radio- 50 prodrugs into free drugs; cytosine deaminase useful for nuclides are available for the production of radioconjugated converting non-toxic 5-fluorocytosine into the anti-cancer anti-ErbB2 antibodies. Examples include 212Bi, 1311, 131 In, drug, 5-fluorouracil; proteases, such as serratia protease, 9oy and 186Re. thermolysin, subtilisin, carboxypeptidases and cathepsins Conjugates of the antibody and cytotoxic agent are made (such as cathepsins B and L), that are useful for converting using a variety of bifunctional protein coupling agents such 55 peptide-containing prodrugs into free drugs; D-alanylcar as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), boxypeptidases, useful for converting prodrugs that contain iminothiolane (IT), bifunctional derivatives of imidoesters D-amino acid substituents; carbohydrate-cleaving enzymes (such as dimethyl adipimidate HCL), active esters (such as such as ~-galactosidase and neuraminidase useful for con disuccinimidyl suberate), aldehydes (such as glutarelde verting glycosylated prodrugs into free drugs; ~-lactamase hyde), bis-azido compounds (such as his (p-azidobenzoyl) 60 useful for converting drugs derivatized with ~-lactams into hexanediamine), bis-diazonium derivatives (such as bis-(p free drugs; and penicillin amidases, such as penicillin V diazoniumbenzoy I)-ethylenediamine), diisocyanates (such amidase or penicillin G amidase, useful for converting drugs as tolyene 2,6-diisocyanate), and his-active fluorine com derivatized at their amine nitrogens with phenoxyacetyl or pounds (such as 1,5-difluoro-2,4-dinitrobenzene). For phenylacetyl groups, respectively, into free drugs. Alterna example, a ricin immunotoxin can be prepared as described 65 tively, antibodies with enzymatic activity, also known in the in Vitetta eta!. Science 238: 1098 (1987). Carbon-14-labeled art as "abzymes", can be used to convert the prodrugs of the 1-isothiocyanato benzy1-3- methyldiethylene triaminepen- invention into free active drugs (see, e.g., Massey, Nature Appx72 HOSPIRA EX. 1001 Page 23 Case: 19-2156 Document: 19 Page: 144 Filed: 07/26/2019 US 7,371,379 B2 29 30 328: 457-458 [1987]). Antibody-abzyme conjugates can be (5' to 3'): HQNLSDGK (SEQ ID N0:5), HQNISDGK (SEQ prepared as described herein for delivery of the abzyme to ID N0:6), or VISSHLGQ (SEQ ID N0:7) and the sequence: a tumor cell population. PKNSSMISNTP (SEQ ID N0:3). The enzymes of this invention can be covalently bound to (xii) Purification of anti-ErbB2 Antibody the anti-ErbB2 antibodies by techniques well known in the 5 art such as the use of the heterobifunctional crosslinking When using recombinant techniques, the antibody can be reagents discussed above. Alternatively, fusion proteins produced intracellularly, in the periplasmic space, or directly comprising at least the antigen binding region of an antibody secreted into the medium. If the antibody is produced of the invention linked to at least a functionally active intracellularly, as a first step, the particulate debris, either portion of an enzyme of the invention can be constructed 10 host cells or lysed fragments, is removed, for example, by using recombinant DNA techniques well known in the art centrifugation or ultrafiltration. Carteret a!., Bio/Technology (see, e.g., Neuberger eta!., Nature, 312: 604-608 [1984]). 10:163-167 (1992) describe a procedure for isolating anti (xi) Antibody-Salvage Receptor Binding Epitope Fusions bodies which are secreted to the periplasmic space of E. coli. In certain embodiments of the invention, it may be Briefly, cell paste is thawed in the presence of sodium desirable to use an antibody fragment, rather than an intact 15 acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride antibody, to increase tumor penetration, for example. In this (PMSF) over about 30 min. Cell debris can be removed by case, it may be desirable to modifY the antibody fragment in centrifugation. Where the antibody is secreted into the order to increase its serum half life. This may be achieved, medium, supernatants from such expression systems are for example, by incorporation of a salvage receptor binding preferably first concentrated using a commercially available epitope into the antibody fragment (e.g. by mutation of the 20 protein concentration filter, for example, an Amicon or appropriate region in the antibody fragment or by incorpo Millipore Pellicon ultrafiltration unit. A protease inhibitor rating the epitope into a peptide tag that is then fused to the such as PMSF may be included in any of the foregoing steps antibody fragment at either end or in the middle, e.g. by to inhibit proteolysis and antibiotics may be included to DNA or peptide synthesis). prevent the growth of adventitious contaminants. A systematic method for preparing such an antibody 25 variant having an increased in vivo half-life comprises The antibody composition prepared from the cells can be several steps. The first involves identifying the sequence and purified using, for example, hydroxylapatite chromatogra conformation of a salvage receptor binding epitope of an Fe phy, gel electrophoresis, dialysis, and affinity chromatogra region of an IgG molecule. Once this epitope is identified, phy, with affinity chromatography being the preferred puri the sequence of the antibody of interest is modified to 30 fication technique. The suitability of protein A as an affinity include the sequence and conformation of the identified ligand depends on the species and isotype of any immuno- binding epitope. After the sequence is mutated, the antibody globulin Fe domain that is present in the antibody. Protein A variant is tested to see if it has a longer in vivo half-life than can be used to purifY antibodies that are based on human yl, that of the original antibody. If the antibody variant does not y2, or y4 heavy chains (Lindmark et a!., J. Immunol. Meth. have a longer in vivo half-life upon testing, its sequence is 35 62:1-13 [1983]). G is recommended for all mouse isotypes further altered to include the sequence and conformation of and for human y3 (Guss et a!., EMBO J. 5:15671575 the identified binding epitope. The altered antibody is tested [1986]). The matrix to which the affinity ligand is attached for longer in vivo half-life, and this process is continued is most often agarose, but other matrices are available. until a molecule is obtained that exhibits a longer in vivo Mechanically stable matrices such as controlled pore glass half-life. 40 or poly(styrenedivinyl)benzene allow for faster flow rates The salvage receptor binding epitope being thus incorpo and shorter processing times than can be achieved with rated into the antibody of interest is any suitable such agarose. Where the antibody comprises a CH3 domain, the epitope as defined above, and its nature will depend, e.g., on Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is the type of antibody being modified. The transfer is made useful for purification. Other techniques for protein purifi- such that the antibody of interest still possesses the biologi 45 cation such as fractionation on an ion-exchange column, cal activities described herein. ethanol precipitation, Reverse Phase HPLC, chromatogra The epitope preferably constitutes a region wherein any phy on silica, chromatography on heparin SEPHAROSE™ one or more amino acid residues from one or two loops of chromatography on an anion or cation exchange resin (such a Fe domain are transferred to an analogous position of the as a polyaspartic acid column), chromatofocusing, SDS- antibody fragment. Even more preferably, three or more 50 PAGE, and ammonium sulfate precipitation are also avail residues from one or two loops of the Fe domain are able depending on the antibody to be recovered. transferred. Still more preferred, the epitope is taken from Following any preliminary purification step(s), the mix the CH2 domain of the Fe region (e.g., of an IgG) and ture comprising the antibody of interest and contaminants transferred to the CHI, CH3, or VH region, or more than one may be subjected to low pH hydrophobic interaction chro such region, of the antibody. Alternatively, the epitope is 55 matography using an elution buffer at a pH between about taken from the CH2 domain of the Fe region and transferred 2.5-4.5, preferably performed at low salt concentrations (e.g. to the CL region or VL region, or both, of the antibody from about 0-0.25M salt). fragment. In one most preferred embodiment, the salvage receptor III. Determination of anti-ErbB2 Antibody Concentration in binding epitope comprises the sequence (5' to 3'): PKNS 60 Serum SMISNTP (SEQ ID N0:3 ), and optionally further comprises The following non-limiting assay is useful for determin a sequence selected from the group consisting ofHQSLGTQ ing the presence of and to quantitate the amount of specific (SEQ ID N0:4), HQNLSDGK (SEQ ID N0:5), HQNIS rhuMAb HER2 (humanized anti -p 185HER 2 monoclonal anti DGK (SEQ ID N0:6), or VISSHLGQ (SEQ ID N0:7), body, including HERCEPTIN® anti-ErbB2 antibody) in a particularly where the antibody fragment is a Fab or F(ab')2 . 65 body fluid of a mammal including, but not limited to, serum, In another most preferred embodiment, the salvage receptor amniotic fluid, milk, umbilical cord serum, ocular aqueous binding epitope is a polypeptide containing the sequence(s) and vitreous liquids, and ocular vitreous gel. Appx73 HOSPIRA EX. 1001 Page 24 Case: 19-2156 Document: 19 Page: 145 Filed: 07/26/2019 US 7,371,379 B2 31 32 Plate Binding Activity Assay for rhuMAb HER2 (Human hydrates including glucose, mannose, or dextrins; chelating ized Anti-p185HER2 Monoclonal Antibody agents such as EDTA; sugars such as sucrose, mannitol, The method of assaying rhuMAb HER2 described herein trehalose or sorbitol; salt-forming counter-ions such as is meant as an example of such a method and is not meant sodium; metal complexes (e.g. Zn-protein complexes); and/ to be limiting. A standardized preparation of rhuMAb HER2 or non-ionic surfactants such as TWEEN™, PLURON (Genentech, Inc., South San Francisco, Calif.), controls, and ICS™ or polyethylene glycol (PEG). Preferred lyophilized serum samples were diluted with Assay Diluent (PBS/0.5% anti-ErbB2 antibody formulations are described in WO BSA/0.05% Polysorbate 20/0.01% Thimerosal). The dilu 97/04801, expressly incorporated herein be reference. tions of standardized rhuMAb HER2 were prepared to span The formulation herein may also contain more than one a range of concentrations useful for a standard curve. The 10 active compound as necessary for the particular indication samples were diluted to fall within the standard curve. being treated, preferably those with complementary activi An aliquot of Coat Antigen in Coating buffer (recombi ties that do not adversely affect each other. For example, it nant p185HER2 (Genentech, Inc.) in 0.05 M sodium carbon- may be desirable to further provide antibodies which bind to ate buffer) was added to each well of a microtiter plate and EGFR, ErbB2 (e.g. an antibody which binds a different incubated at 2-8° C. for 12-72 hours. The coating solution 15 epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial was removed and each well was washed six times with growth factor (VEGF) in the one formulation. Alternatively, water, then blotted to remove excess water. or in addition, the composition may comprise a cytotoxic An aliquot of Assay Diluent was added to each well and agent, cytokine or growth inhibitory agent. Such molecules incubated for 1-2 hours at ambient temperature with agita are suitably present in combination in amounts that are tion. The wells were washed as in the previous step. 20 effective for the purpose intended. Aliquots of diluted standard, control and sample solutions The active ingredients may also be entrapped in micro were added to the wells and incubated at ambient tempera capsules prepared, for example, by coacervation techniques ture for 1 hour with agitation to allow binding of the or by interfacial polymerization, for example, hydroxym antibody to the coating antigen. The wells are washed again ethylcellulose or gelatin-microcapsules and poly-(methyl- with water as in previous steps. 25 methacylate) microcapsules, respectively, in colloidal drug Horse radish peroxidase-conjugate (HRP-conjugate, Goat delivery systems (for example, liposomes, albumin micro anti-human IgG Fe conjugated to horseradish peroxidase; spheres, microemulsions, nano-particles and nanocapsules) Organon Teknika catalog #55253 or equivalent) was diluted or in macroemulsions. Such techniques are disclosed in with Assay Diluent to yield an appropriate optical density Remington's Pharmaceutical Sciences 16 th edition, Osol, range between the highest and lowest standards. An aliquot 30 A. Ed. (1980). of the HRP-conjugate solution was added to each well and The formulations to be used for in vivo administration incubated at ambient temperature for I hour with agitation. must be sterile. This is readily accomplished by filtration The wells were washed with water as in previous steps. through sterile filtration membranes. An aliquot of Substrate Solution (a-phenylenediamine Sustained-release preparations may be prepared. Suitable (OPD) 5 mg tablet (Sigma P6912 or equivalent) in 12.5 ml 35 examples of sustained-release preparations include semiper 4 mM H2 0 2 in PBS) was added to each well and incubated meable matrices of solid hydrophobic polymers containing for a sufficient period of time (approximately 8-10 minutes) the antibody, which matrices are in the form of shaped in the dark at ambient temperature to allow color develop articles, e.g. films, or microcapsules. Examples of sustained ment. The reaction was stopped with an aliquot of 4.5 N release matrices include polyesters, hydrogels (for example, sulfuric acid. Optical density was read at 490-492 urn for 40 poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), detection absorbance and 405 nm for reference absorbance. polylactides (U.S. Pat. No. 3,773,919), copolymers of The standard curve data are plotted and the results for the L-glutamic acid and y ethyl-L-glutamate, non-degradable controls and samples are determined from the standard ethylene-vinyl acetate, degradable lactic acid-glycolic acid curve. copolymers such as the LUPRON DEPOT™ (injectable 45 microspheres composed of lactic acid-glycolic acid copoly IV. Pharmaceutical Formulations mer and leuprolide acetate), and poly-D-(-)-3-hydroxybu Therapeutic formulations of the antibodies used in accor tyric acid. While polymers such as ethylene-vinyl acetate dance with the present invention are prepared for storage by and lactic acid-glycolic acid enable release of molecules for mixing an antibody having the desired degree of purity with over 100 days, certain hydro gels release proteins for shorter optional pharmaceutically acceptable carriers, excipients or 50 time periods. When encapsulated antibodies remain in the stabilizers (Remington's Pharmaceutical Sciences 16 th body for a long time, they may denature or aggregate as a edition, Osol, A. Ed. [1980]), in the form of lyophilized result of exposure to moisture at 37° C., resulting in a loss formulations or aqueous solutions. Acceptable carriers, of biological activity and possible changes in immunoge excipients, or stabilizers are nontoxic to recipients at the nicity. Rational strategies can be devised for stabilization dosages and concentrations employed, and include buffers 55 depending on the mechanism involved. For example, if the such as phosphate, citrate, and other organic acids; antioxi aggregation mechanism is discovered to be intermolecular dants including ascorbic acid and methionine; preservatives S-S bond formation through thio-disulfide interchange, (such as octadecyldimethylbenzyl ammonium chloride; hex stabilization may be achieved by modifying sulfhydryl resi amethonium chloride; benzalkonium chloride, benzetho dues, lyophilizing from acidic solutions, controlling mois- nium chloride; phenol, butyl or benzyl alcohol; alkyl para 60 ture content, using appropriate additives, and developing hens such as methyl or propylparaben; catechol; resorcinol; specific polymer matrix compositions. cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, V. Treatment with the Anti-ErbB2 Antibodies such as serum albumin, gelatin, or immunoglobulins; hydro It is contemplated that, according to the present invention, philic polymers such as polyvinylpyrrolidone; amino acids 65 the anti-ErbB2 antibodies may be used to treat various such as glycine, glutamine, asparagine, histidine, arginine, conditions characterized by overexpression and/or activa or lysine; monosaccharides, disaccharides, and other carbo- tion of the ErbB2 receptor. Exemplary conditions or disor- Appx74 HOSPIRA EX. 1001 Page 25 Case: 19-2156 Document: 19 Page: 146 Filed: 07/26/2019 US 7,371,379 B2 33 34 ders include benign or malignant tumors (e.g. renal, liver, for preventive or therapeutic purposes, previous therapy, the kidney, bladder, breast, gastric, ovarian, colorectal, prostate, patient's clinical history and response to the antibody, and pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarco the discretion of the attending physician. The antibody is mas; glioblastomas; and various head and neck tumors); suitably administered to the patient at one time or over a leukemias and lymphoid malignancies; other disorders such series of treatments. Where the treatment involves a series of as neuronal, glial, astrocytal, hypothalamic and other glan treatments, the initial dose or initial doses are followed at dular, macrophagal, epithelial, stromal and blastocoelic dis daily or weekly intervals by maintenance doses. Each main orders; and inflammatory, angiogenic and immunologic dis tenance dose provides the same or a smaller amount of orders. antibody compared to the amount of antibody administered The antibodies of the invention are administered to a 10 in the initial dose or doses. human patient, in accord with known methods, such as Depending on the type and severity of the disease, about intravenous administration as a bolus or by continuous 1 flg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an infusion over a period of time, by intramuscular, intraperi initial candidate dosage for administration to the patient, toneal, intracerobrospinal, subcutaneous, intra-articular, whether, for example, by one or more separate administra- intrasynovial, intrathecal, oral, topical, or inhalation routes. 15 tions, or by continuous infusion. A typical daily dosage Intravenous or subcutaneous administration of the antibody might range from about 1 flg/kg to 100 mg/kg or more, is preferred. depending on the factors mentioned above. For repeated The treatment of the present invention involves the administrations over several days or longer, depending on administration of an anti-ErbB2 antibody to an animal or the condition, the treatment is sustained until a desired human patient, followed at intervals by subsequent doses of 20 suppression of disease symptoms occurs. The progress of equal or smaller doses such that a target serum concentration this therapy is easily monitored by conventional techniques is achieved and maintained during treatment. Preferably, and assays. maintenance doses are delivered by bolus delivery, prefer According to the invention, dosage regimens may include ably by subcutaneous bolus administration, making treat an initial dose of anti-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 ment convenient and cost-effective for the patient and health 25 mg/kg delivered by intravenous or subcutaneous infusion, care professionals. followed by subsequent weekly maintenance doses of 2 Where combined administration of a chemotherapeutic mg/kg by intravenous infusion, intravenous bolus injection, agent (other than an antracycline) is desired, the combined subcutaneous infusion, or subcutaneous bolus injection. administration includes coadministration, using separate for Where the antibody is well-tolerated by the patient, the time mulations or a single pharmaceutical formulation, and con 30 of infusion may be reduced. secutive administration in either order, wherein preferably Alternatively, the invention includes an initial dose of 12 there is a time period while both (or all) active agents mg/kg anti-ErbB2 antibody, followed by subsequent main simultaneously exert their biological activities. Preparation tenance doses of 6 mg/kg once per 3 weeks. and dosing schedules for such chemotherapeutic agents may Another dosage regimen involves an initial dose of 8 be used according to manufacturers' instructions or as 35 mg/kg anti-ErbB2 antibody, followed by 6 mg/kg once per determined empirically by the skilled practitioner. Prepara 3 weeks. tion and dosing schedules for such chemotherapy are also Still another dosage regimen involves an initial dose of 8 described in Chemotherapy Service Ed., M. C. Perry, Wil mg/kg anti-ErbB2 antibody, followed by subsequent main liams & Wilkins, Baltimore, Md. (1992). The chemothera tenance doses of 8 mg/kg once per week or 8 mg/kg once peutic agent may precede, or follow administration of the 40 every 2 to 3 weeks. antibody or may be given simultaneously therewith. The As an alternative regimen, initial doses of 4 mg/kg antibody may be combined with an anti-estrogen compound anti-ErbB2 antibody may be administered on each of days 1, such as tamoxifen or an anti-progesterone such as onapris 2 and 3, followed by subsequent maintenance doses of 6 tone (see, EP 616 812) in dosages known for such mol mg/kg once per 3 weeks. ecules. 45 An additional regimen involves an initial dose of 4 mg/kg It maybe desirable to also administer antibodies against anti-ErbB2 antibody, followed by subsequent maintenance other tumor associated antigens, such as antibodies which doses of 2 mg/kg twice per week, wherein the maintenance bind to the EGFR, ErbB3, ErbB4, or vascular endothelial doses are separated by 3 days. growth factor (VEGF). Alternatively, or additionally, two or Alternatively, the invention may include a cycle of dosing more anti-ErbB2 antibodies may be co-administered to the 50 in which delivery of anti-ErbB2 antibody is 2-3 times per patient. Sometimes, it may be beneficial to also administer week for 3 weeks. The 3 week cycle is preferably repeated one or more cytokines to the patient. The ErbB2 antibody as necessary to achieve suppression of disease symptoms. may be co-administered with a growth inhibitory agent. For The invention further includes a cyclic dosage regimen in example, the growth inhibitory agent may be administered which delivery of anti-ErbB2 antibody is daily for 5 days. first, followed by the ErbB2 antibody. However, simulta- 55 According to the invention, the cycle is preferably repeated neous administration, or administration of the ErbB2 anti as necessary to achieve suppression of disease symptoms. body first is also contemplated. Suitable dosages for the Further information about suitable dosages is provided in the growth inhibitory agent are those presently used and may be Examples below. lowered due to the combined action (synergy) of the growth inhibitory agent and anti-ErbB2 antibody. 60 VI. Articles of Manufacture In addition to the above therapeutic regimens, the patient In another embodiment of the invention, an article of may be subjected to surgical removal of cancer cells and/or manufacture containing materials useful for the treatment of radiation therapy. the disorders described above is provided. The article of For the prevention or treatment of disease, the appropriate manufacture comprises a container, a label and a package dosage of anti-ErbB2 antibody will depend on the type of 65 insert. Suitable containers include, for example, bottles, disease to be treated, as defined above, the severity and vials, syringes, etc. The containers may be formed from a course of the disease, whether the antibody is administered variety of materials such as glass or plastic. The container Appx75 HOSPIRA EX. 1001 Page 26 Case: 19-2156 Document: 19 Page: 147 Filed: 07/26/2019 US 7,371,379 B2 35 36 holds a composition which is effective for treating the human immunoglobulin IgG1 (IgG1) (Carter et a!., Proc. condition and may have a sterile access port (for example, Nat!. Acad. Sci. USA 89:4285-4289 [1992]). The resulting the container may be an intravenous solution bag or a vial humanized anti-ErbB2 monoclonal antibody has high affin- having a stopper pierceable by a hypodermic injection ity for p185HER 2 (Dillohiation constant [Kd]=0.1 nmol/L), needle). At least one active agent in the composition is an 5 markedly inhibits, in vitro and in human xenografts, the anti-ErbB2 antibody. The label on, or associated with, the growth of breast cancer cells that contain high levels of 2 container indicates that the composition is used for treating p185HER , induces antibody-dependent cellular cytotoxicity the condition of choice. The article of manufacture may (ADCC), and has been found clinically active, as a single further comprise a second container comprising a pharma agent, in patients with ErbB2-overexpressing metastatic ceutically-acceptable buffer, such as phosphate-buffered 10 breast cancers that had received extensive prior therapy. saline, Ringer's solution and dextrose solution. It may HERCEPTIN® anti-ErbB2 antibody is produced by a further include other materials desirable from a commercial genetically engineered Chinese Hamster Ovary (CHO) cell and user standpoint, including other buffers, diluents, filters, line, grown in large scale, that secretes the antibody into the needles, and syringes. In addition, the article of manufacture culture medium. The antibody is purified from the CHO may comprise a package inserts with instructions for use, 15 culture media using standard chromatographic and filtration including, e.g., a warning that the composition is not to be methods. Each lot of antibody used in this study was assayed used in combination with anthacycline-type chemotherapeu- to verifY identity, purity, and potency, as well as to meet tic agent, e.g. doxorubicin or epirubicin. Food and Drug Administration requirements for sterility and safety. Deposit of Materials 20 Eligibility Criteria Patients had to fulfill all of the follow The following hybridoma cell lines have been deposited ing criteria to be eligible for study admission: with the American Type Culture Collection, 12301 Parklawn Metastatic breast cancer Drive, Rockville, Md., USA (ATCC): Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ as determined by immunohistochemistry or fluores 25 cence in situ hybridization (FISH). [Tumor expression Antibody Designation ATCC No. Deposit Date of ErbB2 can be determined by immunohistochemical analysis, as previously described (Slamon et a!., [ 1987] 7C2 ATCC HB-12215 Oct. 17, 1996 and [1989], supra), of a set of thin sections prepared 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRT 10463 May 24, 1990 from the patient's paraffin-archived tumor blocks. The 2C4 ATCC HB-12697 Apr. 8, 1999 30 primary detecting antibody used is murine 4D5 MAb, which has the same CDRs as the humanized antibody used for the treatment. Tumors are considered to over Further details of the invention are illustrated by the express ErbB2 if at least 25% of tumor cells exhibit following non-limiting Examples. 2 characteristic membrane staining for p 185HER ]. 35 Bidimensionally measurable disease (including lytic bone EXAMPLES lesions) by radiographic means, physical examination, or photographs Example 1 Measurable disease was defined as any mass reproducibly measurable in two perpendicular diameters by physical Preparation and Efficacy of HERCEPTIN® 40 examination, X-ray (plain films), computerized tomography Anti-ErbB2 Antibody Materials and Methods (CT), magnetic resonance imaging (MRI), ultrasound, or photographs. Anti-ErbB2 monoclonal antibody The anti-ErbB2 IgG1K Osteoblastic metastases, pleural effusions, or ascites were murine monoclonal antibody 4D5, specific for the extracel not considered to be measurable. Measurable lesions must lular domain ofErbB2, was produced as described in Fendly 45 be at least 1 em in greatest dimension. Enumeration of et a!., Cancer Research 5o: 1550-1558 (1990) and W089/ evaluable sites of metastatic disease and number of lesions 06692. Briefly, NIH 3T3/HER2-3400 cells (expressing 5 in an evaluable site (e.g. lung) had to be recorded on the approximately 1x10 ErbB2 molecules/cell) produced as appropriate Case Report Form (CRF). If a large number of described in Hudziak et a!., Proc. Nat!. Acad. Sci. (USA) pulmonary or hepatic lesions were present, the six largest 84:7159 (1987) were harvested with phosphate buffered 50 lesions per site were followed. saline (PBS) containing 25 mM EDTA and used to immu The ability to understand and willingness to sign a written ni:z;e BALB/c mice. The mice were given injections i.p. of informed consent form 10 cells in 0.5 ml PBS on weeks, 0, 2, 5 and 7. The mice Women>18 years with antisera that immunoprecipitated 32P-labeled ErbB2 Suitable candidates for receiving concomitant cytotoxic were given i.p. injections of a wheat germ agglutinin- 55 Sepharose (WGA) purified ErbB2 membrane extract on chemotherapy as evidenced by screening laboratory weeks 9 and 13. This was followed by an i.v. injection of0.1 assessments of hematologic, renal, hepatic, and meta ml of the ErbB2 preparation and the splenocytes were fused bolic functions. with mouse myeloma line X63-Ag8.653. Hybridoma super Exclusion Criteria Patients with any of the following were natants were screened for ErbB2-binding by ELISA and 60 excluded from study entry: radioimmunoprecipitation. MOPC-21 (IgG1), (Cappell, Prior cytotoxic chemotherapy for metastatic breast cancer Durham, N.C.), was used as an isotype-matched control. Patients may have received prior hormonal therapy (e.g. The treatment was performed with a humanized version tamoxifen) for metastatic disease or cytotoxic therapy of the murine 4D5 antibody (HERCEPTIN® anti-ErbB2 in the adjuvant setting. antibody). The humanized antibody was engineered by 65 Concomitant malignancy that has not been curatively inserting the complementarity determining regions of the treated murine 4D5 antibody into the framework of a consensus A performance status of <60% on the Kamofsky scale Appx76 HOSPIRA EX. 1001 Page 27 Case: 19-2156 Document: 19 Page: 148 Filed: 07/26/2019 US 7,371,379 B2 37 38 Pregnant or nursing women; women of childbearing Partial Response A reduction of at least 50% in the sum potential, unless using effective contraception as deter of the products of the perpendicular diameters of all mea mined by the investigator surable lesions for a minimum period of 4 weeks. No new Bilateral breast cancer (either both primary tumors must lesions may have appeared, nor may any lesions have have 2+ to 3+ HER2 overexpression, or the metastatic progressed in size. site must have 2+ to 3+ HER2 overexpression) Minor Response A reduction of 25% to 49% in the sum of Use of investigational or unlicensed agents within 30 days the products of the perpendicular diameters of all measur prior to study entry able lesions. No new lesions may have appeared, nor may Clinically unstable or untreated metastases to the brain any lesions have progressed in size. (e.g. requiring radiation therapy) 10 Stable Disease No change of greater than 25% in the size Based upon the foregoing criteria, 469 patients were of measurable lesions. No lesions may have appeared. chosen, and enrolled in the study. Half the patients (stratified Time to disease progression (TTP) was calculated from by chemotherapy) were randomized to additionally receive the beginning of therapy to progression. Confidence limits the HERCEPTIN® anti-ErbB2 antibody (see below). for response rates were calculated using the exact method Administration and Dosage 15 for a single proportion. (Fleiss, J L, Statistical Methods for Anti-ErbB2 Antibody Rates and Proportions (ed. 2), New York, N.Y., Wiley, 1981, On day 0, a 4 mg/kg dose of humanized anti-ErbB2 pp 13-17). antibody (HERCEPTIN®, H) was administered intrave Results nously, over a 90-minute period. Beginning on day 7, 20 At a median follow-up of 10.5 months, assessments of patients received weekly administration of2 mg/kg antibody time to disease progression (TTP in months) and response (i.v.) over a 90-minute period. rates (RR) showed a significant augmentation of the che Chemotherapy motherapeutic effect by HERCEPTIN® anti-ErbB2 anti The patients received one of two chemotherapy regimens body, without increase in overall severe adverse events for a minimum of six cycles, provided their disease was not (AE): progressing: a) cyclophosphamide and doxorubicin or epi- 25 rubicin (AC), if patients have not received anthracycline TABLE 1 therapy in the adjuvant setting, or b) paclitaxel (T, TAXOL®), if patients have received any anthracycline HERCEPTIN ® Anti-ErbB2 Antibody Efficacy therapy in the adjuvant setting. The initial dose of the HERCEPTIN® anti-ErbB2 antibody preceded the first cycle 30 Enrolled TTP(months) RR(%) AE(%) of either chemotherapy regimen by 24 hours. Subsequent CRx 234 5.5 36.2 66 doses of the antibody were given immediately before che CRx + H 235 8.6* 62.00** 69 motherapy administration, if the initial dose of the antibody AC 145 6.5 42.1 71 AC + H 146 9.0 64.9 68 was well tolerated. If the first dose of the antibody was not 35 T 89 4.2 25.0 59 well tolerated, subsequent infusions continued to precede T+H 89 7.1 57.3 70 chemotherapy administration by 24 hours. Patients were permitted to continue receiving chemotherapy beyond six *p < 0.001 by log-rank test; **p < 0.01 by X2 test; cycles if, in the opinion of the treating physician, they were CRx: chemotherapy; continuing to receive treatment benefit. AC: anthracycline/cyclophosphamide treatment; 2 40 Cyclophosphamide (600 mg/m ) was given either by iv H: HERCEPTIN ® anti-ErbB2 antibody; push over a minimum period of 3 minutes or by infusion T: TAXOL® over a maximum period of 2 hours. A syndrome of myocardial dysfunction similar to that 2 2 Doxorubicin (60 mg/m ) or epirubicin (75 mg/m ) were observed with anthracyclines was reported more commonly given either by slow iv push over a minimum period of 3-5 45 with a combined treatment of AC+H (18% Grade%) than minutes or by infusion over a maximum period of 2 hours, with AC alone (3%), T (0%), or T+H (2%). according to institutional protocol. These data indicate that the combination of anti-ErbB2 2 Paciltaxel (TAXOL®) was given at a dose of 175 mg/m antibody treatment with chemotherapy markedly increases over 3 hours by intravenous administration. All patients the clinical benefit, as assessed by response rates and the receiving paclitaxel were premedicated with dexamethasone 50 evaluation of disease progression. However, due to the (or its equivalent) 20 mgx2, administered orally 12 and 6 increased cardiac side-effects of doxorubicin or epirubicin, hours prior to paclitaxel; diphenhydramine (or its equiva the combined use of anthracyclines with anti-ErbB2 anti lent) 50 mg, iv, administered 30 minutes prior to paclitaxel, body therapy is contraindicated. The results, taking into and dimetidine (or another H2 blocker) 300 mg, iv, admin account risk and benefit, favor treatment with HERCEP- istered 30 minutes prior to paclitaxel. 55 TIN® anti-ErbB2 antibody and paclitaxel (TAXOL®) Response Criteria where a combined treatment regimen is desired. Progressive Disease Objective evidence of an increase of 25% or more in any measurable lesion. Progressive disease Example 2 also includes those instances when new lesions have appeared. For bone lesions, progression is defined as a 25% 60 Pharmacokinetic and Pharmacodynamic Properties increase in objective measurement by plain film, CT, MRI; of Anti-ErbB2 Antibody (HERCEPTIN®) symptomatic new lesions not due to fracture; or requirement for palliative radiotherapy. HERCEPTIN® anti-ErbB2 antibody was administered by Complete Response Disappearance of all radiographically intravenous infusion to human patients selected according to and/or visually apparent tumor for a minimum of 4 weeks. 65 the criteria provided in Example 1. An initial dose of 4 Skin and chest wall complete responses had to be confirmed mg/kg HERCEPTIN® anti-ErbB2 antibody was delivered by biopsy. by intravenous infusion, followed by subsequent i.v. infu- Appx77 HOSPIRA EX. 1001 Page 28 Case: 19-2156 Document: 19 Page: 149 Filed: 07/26/2019 US 7,371,379 B2 39 40 sions of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody Patient response status was evaluated relative to serum weekly for several weeks. Two hundred thirteen patients concentration of HERCEPTIN® anti-ErbB2 antibody. For began this treatment regimen and serum drug concentration this purpose, mean serum concentration (an average of was obtained beyond 8 weeks for fewer than 90 patients as troughs and peaks) was calculated for various times and selective discontinuation of patients with rapidly progress patient response status (where the patient response status was determined by an independent Response Evaluation ing disease occurred. Of the 213 patients who began treat Committee). The increase in serum concentration between ment, serum trough concentration data were available for 80 Weeks 2 and 8 appeared to be greater in responders than in patients at Week 12, for 77 patients at Week 16, for 44 nonresponders, suggesting that there is a relationship patients at Week 20, for 51 patients at Week 24, for 25 between response status and HERCEPTIN® anti-ErbB2 10 patients at Week 28, for 23 patients at Week 32, and for 37 antibody serum concentration. A statistical analysis (analysis patients at Week 36. of variance) of trough serum concentration values at Week HERCEPTIN® Anti-ErbB2 Antibody Trough Serum 2 and an average of Weeks 7 and 8 in relation to response Concentrations for Weeks 0-36 status indicated a highly significant relationship between response status and average trough of Weeks 7 and 8 The HERCEPTIN® anti-ErbB2 antibody trough serum 15 (p Peak 195 100.3 35.2 30.7 274.6 Example 3 Trough 195 25.0 12.7 0.16 60.7 Peak 2 190 74.3 31.3 20.8 307.9 Trough 167 30.4 16.0 0.2 74.4 I.V. Bolus Delivery and Subcutaneous Infusion of Peak 167 75.3 26.8 16.1 194.8 50 HERCEPTIN® Anti-ErbB2 Antibody Effectively Trough 179 33.7 17.9 0.2 98.2 Decrease Tumor Volume in the Mouse Peak 4 175 80.2 26.9 22.2 167 Trough 132 38.6 20.1 0.2 89.4 Peak 128 85.9 29.2 27.8 185.8 The efficacy of infusion or bolus delivery of humanized Trough 141 42.1 24.8 0.2 148.7 anti-ErbB2 antibody (HERCEPTIN®, see Example 1 for Peak 137 87.2 32.2 28.9 218.1 preparation), either by intravenous injection or subcutaneous 55 Trough 115 43.2 24.0 0.2 109.9 injection, was examined. The purpose of the study was to Peak 7 114 89.7 32.5 16.3 187.8 ask whether subcutaneous delivery was feasible and whether Trough 137 48.8 24.9 0.2 105.2 Peak 133 95.6 35.9 11.4 295.6 the convenient subcutaneous bolus delivery was useful in treating metastatic breast cancer in animals inoculated with a cell line that overexpresses the HER2 gene. The results, The data in Table 2 suggest that there was an increase in 60 detailed below, show that i.v. and s.c. infusion and bolus trough serum concentration over time. Of the many patients delivery are feasible treatment methodologies. studied, there were 18 patients for whom the trough con A study in a nude mouse xenograft model, which incor centrations did not exceed 20 f.tg/ml from Week 2 through porates a human breast cancer cell line that naturally over Week 8. A HERCEPTIN® anti-ErbB2 antibody trough expresses the HER2 gene (BT-474Ml, derived from BT-474 serum concentration of 20 flg/ml was nominally targeted for 65 cells, ATCC Accession number HTB-20), comparing tumor these studies based on prior pharmacologic studies in ani volume as a function of i.v. bolus versus s.c. infusion was mals and exploratory analyses in clinical trials. performed as follows. In the first study athymic nude nu nu Appx78 HOSPIRA EX. 1001 Page 29 Case: 19-2156 Document: 19 Page: 150 Filed: 07/26/2019 US 7,371,379 B2 41 42 7-9 week old female mice were obtained from Taconic Inc (Germantown, N.Y.). To initiate tumor development, each TABLE 4-continued mouse was inoculated subcutaneously with 3x106 BT474M1 cells suspended in Matrigel™. When tumor nod Comparison of S.C. Infusion and I.V Bolus Delivery 3 ules reached a volume of approximately 100 mm , animals Tumor Volume HERCEPTIN® were randomized to 4 treatment groups. The groups were Twnor Volume (area under curve) Serum Cone. 3 treated according to Table 3. (mm ), Day 35, Day 6-Day 35 (f!g/ml), Day 27, Treatment Group (n ~ 14) (n ~ 13) (n ~ 3) TABLE 3 s.c. infusion 80.6 ± 158 1610 ± 1250 2.11 ± 1.74 10 (low dose) Animal Groups and Doses for Comparison of I.V Bolus and S.C. s.c. infusion 31 ± 75.6 1440 ± 1140 22.1 ± 5.43 Infusion (high dose) i.v. bolus dose* 49.7 ± 95.7 2150 ± 1480 21.7 ± 17.1 ** Group, Target Loading Dose, Serum Cone. Route of Dose Maintenance s.c. = subcutaneous delivery; i.v. = intravenous delivery. Antibody f!g/ml Administration (mg/kg) Dose 15 *4.0 mg/kg Loading Dose and 2.0 mg/kg/week Maintenance Dose. **at predose (trough serum concentration immediately prior to a mainte 1 -Control, 20 NLD and 2.20 0.250 mg/ml nance dose) rhuMAb E25 SC infusion (infusate) 2 - Low Dose SC NLD and 0.313 0.050 mg/ml The results tabulated above indicate that maintenance of rhuMAb HER2 SC infusion (infusate) 3 - High Dose SC 20 NLD and 6.25 1.00 mg/ml a serum concentration of approximately 2 flg/ml was as rhuMAb HER2 SC infusion (infusate) 20 effective as a concentration of 20 flg/ml in this study. The 4- N Multi-Dose 20 NLD and MD 4.00 2 mg/kg/ results indicated that dosing by subcutaneous infusion was rhuMAb HER2 (trough) week as effective as intravenous bolus dosing and achieved similar (IV bolus) trough serum concentrations. The results also indicate that Serum Cone. = concentration in serum. LD = loading dose. MD = mainte the dose levels studied are at the top of the dose-response nance dose. 25 curve in this model and that subcutaneous dosing is effective Infusate concentration was calculated to achieve targeted serum concentra in treating breast cancer tumors. Thus, subcutaneous admin tion using Alzet ®osmotic mini pumps (Alza Corp., Palo Alto, CA). istration of maintenance doses is feasible as part of a Animals were exposed to estrogen by subcutaneous sus HERCEPTIN® anti-ErbB2 antibody treatment regimen. tained release estrogen pellet 9 days before the start of dosing to promote growth of grafted tumor cells. The 30 Example 4 animals were inoculated with the BT474M 1 cells 8 days before the beginning of treatment and tumors were allowed I.V. Bolus and Subcutaneous Bolus Deliveries of to grow. The animals were then treated with nonrelevant HERCEPTIN® Anti-ErbB2 Antibody Effectively antibody E25 (non-specific for HER2 receptor, but a mem Decrease Tumor Volume in the Mouse ber of the monoclonal IgG class) or test antibody HERCEP- 35 TIN® anti-ErbB2 anitbody as indicated in Table 3. The Subcutaneous bolus delivery is convenient and cost dosage levels were selected to achieve target serum concen effective for the patient and health care professionals. The trations of HERCEPTIN®, either 1 flg/ml or 20 flg/ml, by results of the study disclosed in this example indicate that subcutaneous pump infusion or by i.v. bolus delivery. The subcutaneous bolus delivery was as effective as intravenous study groups were treated until day 35. The serum concen- 40 bolus delivery in reducing breast cell tumor size in a mouse. tration of HERCEPTIN® anti-ErbB2 antibody was mea This study was set up as disclosed herein in Example 3 for sured weekly (just prior to dosing for Group 4) using 3 mice/group/time point. The anti-ErbB2 antibody concentra the comparison of intravenous bolus and subcutaneous infu tion was determined according to the method disclosed sion delivery. A sustained release estrogen implant was herein involving standard techniques. Tumor volumes were inserted subcutaneously one day before tumor cell innocu measured two days before dosing began and twice per week 45 lation as described in Example 3. Six days after tumor cell from day 6 to day 35 in the study for which data is tabulated innoculation, the initial tumor measurement was performed. below. Tumors were measured in three dimensions and Seven days after tumor cell innoculation, the first dose of 3 volumes were expressed in mm . Efficacy was determined control antibody or HERCEPTIN® anti-ErbB2 antibody by a statistical comparison (ANOVA) of tumor volumes of was delivered. The animal groups, type of delivery, loading test animals relative to untreated control animals. 50 dose and maintenance doses are provided in Table 4. Ani As shown in Table 4, below, treatment of the BT474M 1 mals were dosed once weekly for 4 weeks. tumor-bearing mice with HERCEPTIN® anti-ErbB2 anti body by the indicated dosage methods significantly inhibited TABLE 5 the growth of the tumors. All HERCEPTIN®-treated groups showed similar inhibition of tumor growth relative to the 55 Animal Groups and Doses for Comparison of I.V Bolus and S.C. control group. No dose-response was observed. Bolus Delive Maintenance TABLE 4 Route of Ad- Loading Dose Dose Group ministration (mg/kg) (mg/kg/week) n Comparison of S.C. Infusion and I.V Bolus Delivery 60 1 -Control IV 4 10 Tumor Volume HERCEPTIN ® rhuMAb E25 Tumor Volume (area under curve) Serum Cone. 2 - rhuMAb HER2 IV 2 10 3 (mm ), Day 35, Day 6-Day 35 (f!g/ml), Day 27, 3 - rhuMAb HER2 IV 4 2 10 Treatment Group (n ~ 14) (n ~ 13) (n ~ 3) 4 - rhuMAb HER2 IV 4 10 5 - rhuMAb HER2 sc 4 2 10 control s.c. 764 ± 700 5650 ± 4700 4.16 ± 1.94 65 infusion IV = intraveneous; SC = subcutaneous; n = nwnber of animals per group. Appx79 HOSPIRA EX. 1001 Page 30 Case: 19-2156 Document: 19 Page: 151 Filed: 07/26/2019 US 7,371,379 B2 43 44 The mice were treated according to the information in groups, dosing by subcutaneous bolus was as effective as Table 4 and using the techniques disclosed in Example 3. intravenous bolus dosing and achieved similar trough serum The serum concentration of HERCEPTIN® anti-ErbB2 concentrations. antibody was measured weekly before each weekly i.v. maintenance dose according to the procedure described Example 5 herein and using standard techniques. The control E25 antibody serum concentration was determined according to Regimens for Intravenous and Subcutaneous standard immunoassay techniques. Table 6 shows the Delivery of Anti-ErbB2 Antibody increase in HERCEPTIN® anti-ErbB2 antibody serum con centrations with time. 10 According to the invention, methods of anti-ErbB2 anti- body (e.g., HERCEPTIN®) delivery comprise greater front TABLE 6 loading of the drug to achieve a target serum concentration in approximately 4 weeks or less, preferably 3 weeks or less, N versus SC Bolus Delivery: Serum HERCEPTIN ® Anti-ErbB2 more preferably 2 weeks or less, and most preferably 1 week Antibody Concentration 15 or less, including one day or less. According to the inven Serum Concentration, ug/ml tion, this initial dosing is followed by dosing that maintains the target serum concentration by subsequent doses of equal Day 0 Day 7 Day 14 Day 21 or smaller amount. An advantage of the methods of the Treatment Group Mean Mean Mean Mean (delivery, MD) (SD) (SD) (SD) (SD) invention is that the maintenance dosing may be less fre- 20 quent and/or delivered by subcutaneous injection, making 1 - Control rhu MAb E25 0 25.9 34.6 38.5 the treatment regimens of the invention convenient and (N, 4 mg/kg) (0) (8.29) (11.2) (14.4) 2 - rhu MAb HER2 0 4.96 8.55 8.05 cost-effective for the patient and medical professionals (N, 1 mg/kg) (0) (3.79) (5.83) (4.67) administering the antibody. In addition, a subcutaneous 3 - rhu MAb HER2 0 13.4 18.9 22.6 maintenance dose regimen may be interrupted by intrave- (N, 2 mg/kg) (0) (9.24) (12.0) (9.21) 25 nous dosing (such as infusion) when the patient's chemo 4 - rhu MAb HER2 0 29.6 37.7 46.2 (N, 4 mg/kg) (0) (13.5) (14.4) (13.8) therapy requires delivery of other drugs by intravenous 5 - rhu MAb HER2 0 12.5 16.9 17.6 injection. (SC, 2 mg/kg) (0) (7.33) (10.2) (10.7) To test the following dosage regimens, human subjects are selected according to the criteria disclosed in Example 1, n ~ 10 for time points Days 0, 7 and 14. N ~ 9 for Day 21. 30 above. The number of initial doses is one or more doses Table 7 shows the relative efficacy of intravenous bolus sufficient to achieve an efficacious target serum concentra delivery and subcutaneous bolus delivery for Groups 1-5 tion in approximately 4 weeks or less, preferably 3 weeks or having achieved the serum antibody concentrations pre less, more preferably 2 weeks or less, and most preferably 1 sented in Table 6. For this study, efficacy was measured as week or less, including 1 day or less. The number of a decrease in tumor volume. Tumor volume was measured 35 maintenance doses may be one or more doses sufficient to twice weekly. achieve suppression of disease symptoms, such as a TABLE 7 Efficacy of HERCEPTIN ® Anti-ErbB2 Antibody Measured as a Change in Twnor Volume Comparing Intravenous Bolus and Subcutaneous Bolus Delive!JS Mean (SD) Treatment Twnor Tumor Vol. Day 6-Day 31 * Tumor Growth Group Tumor Vol. Vol. Day Day 31, Area Under Curve Rate (Delivery, MD) Day 6, mm3 28, mm3 mm3 Tumor Vol., mm3 on Log (TM + 1) 1-N Control 321 1530 1630 13600 0.0660 (190) (1040) (1170) (7230) (0.0200) 2-N Herceptin 297 175 151 4690 -0.0505 1 mg/kg (130) (215) (188) (1400) (0.142) 3-N Herceptin 269 75.7 73.6 3510 -0.0608 2 mg/kg (129) (92.4) (84.5) (1220) (0.110) 4-N Herceptin 272 25.3 25.8 2880 -0.0810 4 mg/kg (117) (75.9) (72.9) (1230) (0.0859) 5-SC Herceptin 268 76.2 90.4 3230 -0.0304 2 mg/kg (117) (98.8) (105) (1440) (0.104) N = 10 for each data point. TM =tumor measurement. N =intravenous. SC = subcutaneous. MD 3 =maintenance dose. Tumor Vol. =tumor volwne, mm . *Day 17 excluded due to measurement error. Tumor growth rate calculated on Day 21-Day 31 Log (TM + 1 ). Area under the curve is the area beneath a plot of tumor volume versus time. 60 FIGS. 4A and 4B are graphical plots of changes in tumor decrease in tumor volume. The maintenance doses are equal volume over time, some of which data is found in Table 7. to or smaller than the initial dose or doses, consistent with FIG. 4A is a linear plot of tumor volume versus time. FIG. an object of the invention of administering HERCEPTIN® 4B is a semilogarithmic plot of the same data, allowing the anti-ErbB2 antibody by regimens providing greater front test points be viewed more clearly. The data in Table 7 and 65 loading. The specific drug delivery regimens disclosed FIGS. 4A and 4B indicate that, although a dose-related herein are representative of the invention and are not meant response was not observed between HERCEPTIN-treated to be limiting. Appx80 HOSPIRA EX. 1001 Page 31 Case: 19-2156 Document: 19 Page: 152 Filed: 07/26/2019 US 7,371,379 B2 45 46 In one trial, an initial dose of 6 mg/kg, 8 mg/kg, or 12 per week for three weeks, followed by repeats of this cycle mg/kg of HERCEPTIN® anti-ErbB2 antibody is delivered to maintain an efficacious trough serum concentration of to human patients by intravenous or subcutaneous injection. HERCEPTIN® anti-ErbB2 antibody. The dose is at least 4 Initial doses (loading doses) are delivered by intravenous mg/kg of anti-ErbB2 antibody, preferably at least 5 mg/kg. infusion or bolus injection or preferably subcutaneous bolus The maintenance drug deliveries may be intravenous or injection. Preferably a target trough serum concentration of subcutaneous. HERCEPTIN® anti-ErbB2 antibody of approximately Where the animal or patient tolerates the antibody during 10-20 flg/ml is achieved (averaged for all patients in the and after an initial dose, delivery of subsequent doses may treatment group) and maintained by subsequent doses of be subcutaneous, thereby providing greater convenience and anti-ErbB2 antibody that are equal to or smaller than the 10 cost-effectiveness for the patient and health care profession initial dose. In one method, a target trough serum concen als. tration is achieved and maintained by once-per-week deliv In animal studies, an initial dose of more than 4 mg/kg, eries of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody by preferably more than 5 mg/kg delivered by intravenous or intravenous or subcutaneous injection for at least eight subcutaneous injection, is followed by subcutaneous bolus weeks. Alternatively, for this or any dosage regimen dis- 15 injections of 2 mg/kg twice per week (separated by 3 days) closed herein, subcutaneous continuous infusion by subcu to maintain a trough serum concentration of approximately taneous pump is used to delivery subsequent maintenance 10-20 flg/ml. In addition, where the animal or patient is doses. known to tolerate the antibody, an initial dose of HERCEP In another method, an initial (front loading) dose of 8 TIN® anti-ErbB2 antibody is optionally and preferably mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by 20 deliverable by subcutaneous bolus injection followed by intravenous injection (infusion or bolus injection) or by subcutaneous maintenance injections. subcutaneous bolus injection. This is followed by intrave While target serum concentrations are disclosed herein for nous bolus injections, intravenous infusion, subcutaneous the purpose of comparing animal studies and human trials, infusion, or subcutaneous bolus injection of 6 mg/kg at target serum concentrations in clinical uses may differ. The 3-week intervals to maintain a trough serum concentration 25 disclosure provided herein guides the user in selecting a of approximately 10-20 f.tg/ml, averaged for an entire treat front loading drug delivery regimen that provides an effi- ment group. cacious target trough serum concentration. In another method, an initial (front loading) dose of 12 The methods of the invention disclosed herein optionally mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by include the delivery ofHERCEPTIN® anti-ErbB2 antibody intravenous injection (infusion or bolus injection) or by 30 in combination with a chemotherapeutic agent (other than an subcutaneous bolus injection. This is followed by intrave anthrocycline derivative) to achieve suppression of disease nous bolus injections, intravenous infusion, subcutaneous symptoms. The chemotherapeutic agent may be delivered infusion, or subcutaneous bolus injection of 6 mg/kg at with HERCEPTIN® anti-ErbB2 antibody or separately and 3-week intervals to maintain a trough serum concentration according to a different dosing schedule. For example, of approximately 10-20 f.tg/ml. 35 subcutaneous delivery of HERCEPTIN® anti-ErbB2 anti In yet another method, an initial (front loading) dose of 8 body with TAXOL® is included in the invention. In addi mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by tion, intravenous or subcutaneous injection of 8 mg/kg intravenous infusion or bolus injection, or preferably by HERCEPTIN® anti-ErbB2 antibody, followed by intrave- subcutaneous bolus injection or infusion. This is followed by nous or subcutaneous injection of 6 mg/kg HERCEPTIN® administration of 8 mg/kg per week or 8 mg/kg per 2-3 40 anti-ErbB2 antibody every 3 weeks is administered in com weeks to maintain a trough serum concentration of HER bination with a chemotherapeutic agent, such as a taxoid CEPTIN® anti-ErbB2 antibody of approximately 10-20 (e.g. paclitaxel 175 mg/m2 every 3 weeks) or an anthracy flg/ml. Maintenance doses are delivered by intravenous cline derivative (e.g. doxorubicin 60 mg/m2 or epirubicin 75 infusion or bolus injection, or preferably by subcutaneous mg/m2 every 3 weeks). Optionally, where an anthracycline infusion or bolus injection. 45 derivative is administered, a cardioprotectant (e.g. 600 In another method, the front loading initial dose is a series mg/m2 cyclophosphamide every 3 weeks) is also adminis of intravenous or subcutaneous injections, for example, one tered. In another combination therapy, anti-ErbB2 antibody on each of days 1, 2, and 3 of at least 1 mg/kg for each is administered in a loading dose of more than 4 mg/kg, injection (where the amount of anti-ErbB2 antibody deliv preferably more than 5 mg/kg, and more preferably at least ered by the sum of initial injections is more than 4 mg/kg), 50 8 mg/kg. The loading dose is followed by maintenance doses followed by maintenance doses of 6 mg/kg once each 3 of at least 2 mg/kg weekly, preferably 6 mg/kg every 3 week interval to maintain a target trough serum concentra weeks. The combination therapy includes administration of tion (for example, approximately 10-20 f.tg/ml) ofHERCEP a taxoid during treatment with anti-ErbB2 antibody. Accord TIN® anti-ErbB2 antibody. The maintenance doses are ing to one embodiment of the invention, the taxoid is 2 delivered by intravenous infusion or bolus injection or by 55 paclitaxel and is administered at a dose of 70-100 mg/m / subcutaneous infusion or subcutaneous bolus injection. week. According to another embodiment of the invention, In yet another method, the front loading is by intravenous the taxoid is docetaxel and is administered at a dose of 30-70 infusion of at least 1 mg/kg, preferably 4 mg/kg on each of mg/m2 /week. five consecutive days, followed by repeats of this cycle a sufficient number of times to achieve suppression of disease 60 Example 6 symptoms. Following the initial dose or doses, subsequent doses may be delivered by subcutaneous infusion or bolus HERCEPTIN® Administered Intravenously Every injection if tolerated by the patient. Such subcutaneous Three Weeks in Combination with Paclitaxel delivery is convenient and cost-effective for the patient and administering health care professionals. 65 Currently, the recommended dose of HERCEPTIN® is 2 In still another method, HERCEPTIN® anti-ErbB2 anti mg/kg once weekly. Patients will be administered HERCEP body is delivered initially as at least 2 intravenous infusions TIN® every three weeks instead of weekly, along with Appx81 HOSPIRA EX. 1001 Page 32 Case: 19-2156 Document: 19 Page: 153 Filed: 07/26/2019 US 7,371,379 B2 47 48 paclitaxel (17 5 mg/m2 every three weeks). Simulation of the Alkaline phosphatase greater than 2.5xULN (greater than proposed treatment regimen suggests that the trough serum 4.0xULN if liver or bone metastasis) concentrations will be 17 mcg/ml, in the range (1 0-20 9) The following abnormal baseline renal function tests: mcg/ml) of the targeted trough serum concentrations from serum creatinine greater than 1.5xULN previous HERCEPTIN® IV clinical trials. After the first 12 patients the PK parameters will be assessed, if exposure is 10) History of other serious medical conditions that would felt inadequate, then the dose will be increased to 8 mg/kg preclude patient participation in an investigational study. every three weeks for the remaining 12 patients. HERCEPTIN® Loading dose and schedule: 8 mg/kg for Inclusion Criteria first dose. Maintenance dose and schedule: 6 mg/kg every 3 1) Females24 18 years of age 10 weeks. 2) Histologically confirmed ErbB2 over-expressing meta Paclitaxel-17 5 mg/m2 IV every 3 weeksx6 cycles as a static breast cancer 3-hour infusion. 3) Patients who have been newly diagnosed with metastatic disease NOTE: On the first cycle of treatment, paclitaxel will be 4) Have a Kamofsky performance status of 24 70% 15 dosed 8 hours prior to HERCEPTIN® to determine the PK 5) Give written informed consent prior to any study specific of paclitaxel alone. HERCEPTIN® will be administered 8 screening procedures with the understanding that the hours post-paclitaxel for the 1st cycle only. In subsequent patient has the right to withdraw from the study at any treatment cycles, HERCEPTIN® will be administered prior time, without prejudice. to paclitaxel. Exclusion Criteria 20 1) Pregnant or lactating women The total duration of this study is 18 weeks. Study 2) Women of childbearing potential unless (1) surgically subjects will receive up to 6 total HERCEPTIN® doses. sterile or (2) using adequate measures of contraception After the last subject has received the last cycle of paclitaxel, such as oral contraceptive, intra-uterine device or barrier data collection for safety and pharmacokinetic analysis will method of contraception in conjunction with spermicidal 25 stop, and the study will close to protocol specified treatment. jelly. Study subjects may continue to receive the HERCEPTIN® 3) Clinical or radiologic evidence of CNS metastases. +1- paclitaxel at the discretion of the investigator. 4) History of any significant cardiac disease It is believed that the above treatment regimen will be 5) LVEF~50% effective in treating metastatic breast cancer, despite the 6) No prior taxane therapy in any treatment setting. 30 infrequency with which HERCEPTIN® is administered to 7) Any of the following abnormal baseline hematologic the patient. values: Hb less than 9 g/dl While the particular aspects and embodiments of the WBC less than 3.0x109 /l invention as herein shown and disclosed in detail is fully 9 Granulocytes less than 1.5x10 /l 35 capable of obtaining the objects and providing the advan Platelets less than 1OOx 109 /1 tages herein before stated, it is to be understood that it is 8) Any of the following abnormal baseline liver function merely illustrative of some of the presently preferred tests: embodiments of the invention and that no limitations are Serum bilirubin greater than 1.5xULN (upper normal intended to the details of methods and articles of manufac- limit) 40 ture shown other than as described in the appended claims. ALT and/or AST greater than 2.5xULN (greater than The disclosures of all citations in the specification are 4.0xULN if liver or bone metastasis) expressly incorporated herein by reference. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS, 15 <210> SEQ ID NO 1 <211> LENGTH, 166 <212 > TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 1 Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro Glu 1 5 10 15 Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val 20 25 30 Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu 50 55 60 Appx82 HOSPIRA EX. 1001 Page 33 Case: 19-2156 Document: 19 Page: 154 Filed: 07/26/2019 US 7,371,379 B2 49 50 -continued Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg 65 70 75 Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala 80 85 90 Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr 95 100 105 Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu 110 115 120 Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 125 130 135 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys 140 145 150 Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg 155 160 165 Ala <210> SEQ ID NO 2 <211> LENGTH, 32 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 2 Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro 1 5 10 15 Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln 20 25 30 Gly Cys <210> SEQ ID NO 3 <211> LENGTH, 11 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, salvage receptor binding epitope <400> SEQUENCE, 3 Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro 1 5 10 <210> SEQ ID NO 4 <211> LENGTH, 7 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, salvage receptor binding epitope <400> SEQUENCE, 4 His Gln Ser Leu Gly Thr Gln 1 5 <210> SEQ ID NO 5 <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, salvage receptor binding epitope <400> SEQUENCE, 5 His Gln Asn Leu Ser Asp Gly Lys 1 5 Appx83 HOSPIRA EX. 1001 Page 34 Case: 19-2156 Document: 19 Page: 155 Filed: 07/26/2019 US 7,371,379 B2 51 52 -continued <210> SEQ ID NO <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, salvage receptor binding epitope <400> SEQUENCE, His Gln Asn Ile Ser Asp Gly Lys 1 5 <210> SEQ ID NO 7 <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, salvage receptor binding epitope <400> SEQUENCE, 7 Val Ile Ser Ser His Leu Gly Gln 1 5 <210> SEQ ID NO 8 <211> LENGTH, 59 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 8 Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val 1 5 10 15 Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln 20 25 30 Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val 35 40 45 Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 50 55 <210> SEQ ID NO 9 <211> LENGTH, 65 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 9 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr 1 5 10 15 Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr 20 25 30 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys 35 40 45 Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu 50 55 60 Gly Ala Cys Gln Pro 65 <210> SEQ ID NO 10 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Mus Musculus Appx84 HOSPIRA EX. 1001 Page 35 Case: 19-2156 Document: 19 Page: 156 Filed: 07/26/2019 US 7,371,379 B2 53 54 -continued <400> SEQUENCE, 10 Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys <210> SEQ ID NO 11 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Mus musculus <400> SEQUENCE, 11 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu 35 40 45 Glu Trp Ile Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn Gln Arg Phe Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser 65 70 75 Ser Arg Ile Val Tyr Met Glu Leu Arg Ser Leu Thr Phe Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115 <210> SEQ ID NO 12 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, humanized VL sequence <400> SEQUENCE, 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser 50 55 60 Appx85 HOSPIRA EX. 1001 Page 36 Case: 19-2156 Document: 19 Page: 157 Filed: 07/26/2019 US 7,371,379 B2 55 56 -continued Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys <210> SEQ ID NO 13 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, humanized VH sequence <400> SEQUENCE, 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 6 0 Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 <210> SEQ ID NO 14 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, VL consensus sequence <400> SEQUENCE, 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 50 55 6 0 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys Appx86 HOSPIRA EX. 1001 Page 37 Case: 19-2156 Document: 19 Page: 158 Filed: 07/26/2019 US 7,371,379 B2 57 58 -continued <210> SEQ ID NO 15 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Artificial sequence <220> FEATURE, <223> OTHER INFORMATION, VH consensus sequence <400> SEQUENCE, 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu 95 100 105 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 The invention claimed is: 9. The method of claim 1, wherein the initial dose is 1. A method for the treatment of a human patient diag selected from the group consisting of approximately 6 nosed with cancer characterized by overexpression of ErbB2 mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of receptor, comprising administering an effective amount of 35 subsequent doses are at least approximately 2 mg/kg. an anti-ErbB2 antibody to the human patient, the method 10. The method of claim 9, wherein the plurality of comprising: subsequent doses are separated in time from each other by administering to the patient an initial dose of at least at least three weeks. approximately 5 mg/kg of the anti-ErbB2 antibody; and 11. The method of claim 10, wherein the initial dose is administering to the patient a plurality of subsequent 40 approximately 8 mg/kg, and wherein at least one subsequent doses of the antibody in an amount that is approxi dose is approximately 6 mg/kg. mately the same or less than the initial dose, wherein 12. The method of claim 10, wherein the initial dose is the subsequent doses are separated in time from each approximately 12 mg/kg, and wherein at least one subse other by at least two weeks; and quent dose is approximately 6 mg/kg. further comprising administering an effective amount of a 45 chemotherapeutic agent to the patient. 13. The method of claim 9, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent 2. The method of claim 1, wherein the initial dose is at least approximately 6 mg/kg. dose is approximately 8 mg/kg. 3. The method of claim 2, wherein the initial dose is at 14. The method of claim 9, wherein the initial dose is approximately 8 mg/kg, wherein at least one subsequent least approximately 8 mg/kg. 50 4. The method of claim 3, wherein the initial dose is at dose is 8 mg/kg, and wherein administration of the initial least approximately 12 mg/kg. dose and subsequent doses are separated in time by at least 5. The method of claim 1, wherein the subsequent doses 2 weeks. are separated in time from each other by at least three weeks. 15. The method of claim 14, wherein the initial dose and 6. The method of claim 1, wherein the initial dose is 55 subsequent doses are separated in time by at least 3 weeks. administered by intravenous injection, and wherein at least 16. The method of claim 1, wherein said cancer is selected one subsequent dose is administered by subcutaneous injec from the group consisting of breast cancer, leukemia, squa tion. mous cell cancer, small-celllung cancer, non-small cell lung 7. The method of claim 1, wherein the initial dose is cancer, gastrointestinal cancer, pancreatic cancer, glioblas administered by intravenous injection, wherein at least two 60 toma, cervical cancer, ovarian cancer, liver cancer, bladder subsequent doses are administered, and wherein each sub cancer, hepatoma, colon cancer, colorectal cancer, endome- sequent dose is administered by a method selected from the trial carcinoma, salivary gland carcinoma, kidney cancer, group consisting of intravenous injection and subcutaneous liver cancer, prostate cancer, vulval cancer, thyroid cancer, injection. hepatic carcinoma and various types of head and neck 8. The method of claim 1, wherein the initial dose and at 65 cancer. least one subsequent dose are administered by subcutaneous 17. The method of claim 16, wherein said cancer is breast injection. cancer. Appx87 HOSPIRA EX. 1001 Page 38 Case: 19-2156 Document: 19 Page: 159 Filed: 07/26/2019 US 7,371,379 B2 59 60 18. The method of claim 17, wherein said cancer is time by at least 3 weeks, and further comprising adminis metastatic breast carcinoma. tering an effective amount of a chemotherapeutic agent to 19. The method of claim 1, wherein said antibody binds the patient. to the extracellular domain of the ErbB2 receptor. 30. A method for the treatment of cancer in a human 20. The method of claim 19, wherein said antibody binds patient comprising administering to the patient a first dose of to epitope 4D5 within the ErbB2 extracellular domain an anti-ErbB2 antibody followed by two or more subsequent sequence. doses of the antibody, wherein the subsequent doses are 21. The method of claim 20, wherein said antibody is a separated from each other in time by at least about two humanized 4D5 anti-ErbB2 antibody. weeks, and further comprising administering an effective 22. The method of claim 1, wherein the chemotherapeutic 10 amount of a chemotherapeutic agent to the patient. agent is a taxoid. 31. The method of claim 30, wherein the first dose and a 23. The method of claim 22, wherein said taxoid is first subsequent dose are separated from each other in time paclitaxel or docetaxel. by at least about three weeks. 24. The method of claim 1, wherein the effective amount 32. The method of claim 30, wherein the first dose and of the anti-ErbB2 antibody and the effective amount of the 15 subsequent doses are each from about 2 mg/kg to about 16 chemotherapeutic agent as a combination is lower than the mg/kg. sum of the effective amounts of said anti-ErbB2 antibody 33. The method of claim 32, wherein the first dose and and said chemotherapeutic agent, when administered indi subsequent doses are each from about 4 mg/kg to about 12 vidually, as single agents. mg/kg. 25. The method of claim 1, wherein the chemotherapeutic 20 34. The method of claim 33, wherein the first dose and agent is an anthracycline. subsequent doses are each from about 6 mg/kg to about 12 26. The method of claim 25, wherein the anthracycline is mg/kg. doxorubicin or epirubicin. 35. The method of claim 30, wherein from about two to 27. The method of claim 25, wherein the method further about ten subsequent doses of the antibody are administered comprises administration of a cardioprotectant. 25 to the patient. 28. The method of claim 1, wherein efficacy is measured 36. The method of claim 30, wherein the two or more by determining the time to disease progression or the subsequent doses are separated from each other in time by response rate. at least about three weeks. 29. A method for the treatment of a human patient 37. The method of claim 30, wherein the two or more diagnosed with cancer characterized by overexpression of 30 subsequent doses are each from about 2 mg/kg to about 16 ErbB2 receptor, comprising administering an effective mg/kg. amount of an anti-ErbB2 antibody to the human patient, the 38. The method of claim 30, wherein the two or more method comprising: administering to the patient an initial subsequent doses are each from about 4 mg/kg to about 12 dose of the antibody, wherein the initial dose is a plurality mg/kg. of doses, wherein each of the plurality of initial doses is at 35 39. The method of claim 30, wherein the two or more least approximately 1 mg/kg and is administered on at least subsequent doses are each from about 6 mg/kg to about 12 3 consecutive days, and administering to the patient at least mg/kg. one subsequent dose of the antibody, wherein at least one 40. The method of claim 30, wherein the chemotherapeu subsequent dose is at least approximately 6 mg/kg, and tic agent is a taxoid. wherein administration of the last initial dose and the first 40 subsequent and additional subsequent doses are separated in * * * * * Appx88 HOSPIRA EX. 1001 Page 39 Case: 19-2156 Document: 19 Page: 160 Filed: 07/26/2019 IIIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111 US010160811B2 c12) United States Patent (IO) Patent No.: US 10,160,811 B2 Baughman et al. (45) Date of Patent: *Dec. 25, 2018 (54) TREATMENT WITH ANTI-ERBB2 (56) References Cited ANTIBODIES U.S. PATENT DOCUMENTS (71) Applicant: GENENTECH, INC., South San 4,676,980 A 6/1987 Segal et al. Francisco, CA (US) 4,753,894 A 6/1988 Frankel et al. 4,816,567 A 3/1989 Cabilly et al. (72) Inventors: Sharon A. Baughman, Ventura, CA 4,935,341 A 6/1990 Bargmann et al. (US); Steven Shak, Burlingame, CA 4,943,533 A 7/1990 Mendelsohn et al. 4,968,603 A 11/1990 Slamon et al. 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Genentech trial in the High Court of the Kong Special Genentech's Brief of Final Allegations filed with Federal Court of Administrative Region, Court of First Instance, Case No. 1873 Fiscal and Administrative Justice regarding Mexican Patent No. regarding HK Patent No. 1048260, Order for Advertisement, Date: 259512 dated Jun. 24, 2015 (in Spanish with English translation) Sep. 17, 2014 (3 pages). (28 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Invalidation Action filed by Celltrion against Polish Patent No. Administrative Region, Court of First Instance, Case No. 1873 202369. regarding HK Patent No. 1048260, Sealed Order for Adjournment, Invalidation Action filed by Celltrion against Polish Patent No. Date: Sep. 17, 2014 (3 pages). 202369 dated Aug. 23, 2011 (21 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Genentech's Motion for Suspension of Invalidation Action filed by Administrative Region, Court of First Instance, Case No. 1873 Celltrion against Polish Patent No. 202369 dated Jan. 20, 2012 (3 regarding HK Patent No. 1048260, Sealed Order regarding Claim pages). Amendment, Date: Sep. 17, 2014 (3 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion's Reply to Genentech's Motion for Suspension of Invali Administrative Region, Court of First Instance, Case No. 1873 dation Action filed by Celltrion against Polish Patent No. 202369 regarding HK Patent No. 1048260, Note from Master Ho regarding dated May 11, 2012 (in Polish with English translation) (7 pages). Expert Directions, Date: Oct. 7, 2014 ( 1 page). Genentech's Supplemental Reply regarding invalidation against Celltrion v. Genentech trial in the High Court of the Kong Special Polish Patent No. 202369 dated Oct. 2, 2012 (21 pages). 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Genentech trial in the High Court of the Kong Special Celltrion's Pleading submitted in invalidation against Polish Patent Administrative Region, Court of First Instance, Case No. 1873 No. 202369 dated Jul. 24, 2013 (11 pages). regarding HK Patent No. 1048260, Order for Cost Summary Attachment to Celltrion's pleading, 2nd Expert Report of Dr. Martin Assessment, Date: Jan. 23, 2015 ( 1 page). J. Glennie dated Jan. 23, 2012 in invalidation against Polish Patent Celltrion v. Genentech trial in the High Court of the Kong Special No. 202369 (12 pages). Administrative Region, Court of First Instance, Case No. 1873 Decision of Polish Patent Office in invalidation against Polish regarding HK Patent No. 1048260, Sealed Order for Claim Amend Patent No. 202369 dated Oct. 23, 2013 (26 pages). ment, Date: Jan. 26, 2015 (3 pages). Documents Submitted by Celltrion to EMEA, Submitted as evi Celltrion v. Genentech trial in the High Court of the Kong Special dence in invalidation against Polish Patent No. 202369, Date: May Administrative Region, Court of First Instance, Case No. 1873 24, 2013 (13 pages). regarding HK Patent No. 1048260, Consent Summons for Exten EMEA Scientific Advice for Trastuzumab, Submitted as evidence in sion of Time to File List of Documents, Date: Feb. 12, 2015 (3 invalidation against Polish Patent No. 202369, Date: Dec. 18, 2008 pages). (29 pages). Celltrion v. Genentech trial in the High Court of the Kong Special 151 Central-Eastern European Congress on Biosimilars, "Biosimilar, Administrative Region, Court of First Instance, Case No. 1873 a New Treatment Option for Breast Cancer", Submitted as evidence regarding HK Patent No. 1048260, Consent Summons for Section in invalidation against Polish Patent No. 202369, Date: Oct. 13, 102 Timings for Fact Evidence, Date: Mar. 9, 2015 (3 pages). 2011 (29 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion's motion for invalidation of the Decision of Patent Office Administrative Region, Court of First Instance, Case No. 1873 for changing the patent in invalidation against Polish Patent No. regarding HK Patent No. 1048260, Consent Summons for Discov 202369 dated Nov. 3, 2013 (29 pages). ery, Date: Mar. 13, 2015 (2 pages). Celltrion's Motion to Suspend Court Proceedings in invalidation Celltrion v. Genentech trial in the High Court of the Kong Special against Polish Patent No. 202369 dated Nov. 3, 2013 (in Polish)(l5 Administrative Region, Court of First Instance, Case No. 1873 pages). regarding HK Patent No. 1048260, Letter from the Court with Celltrion's Further Pleading submitted in invalidation against Polish Queries Regarding Consent Summons, Date: Mar. 27, 2015 (1 Patent No. 202369 dated Nov. 3, 2013 (in Polish with English page). translation) (25 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Genentech's Answer to the Application submitted in invalidation Administrative Region, Court of First Instance, Case No. 1873 against Polish Patent No. 202369 dated Apr. 8, 2015 (10 pages). regarding HK Patent No. 1048260, Order of Master Ho regarding Judgement by the Provincial Administrative Court of Warsaw in Case Management Conference, Date: Jul. 23, 2015 (3 pages). invalidation against Polish Patent No. 202369 dated Apr. 22, 2015 Celltrion v. Genentech trial in the High Court of the Kong Special (in Polish with English translation) (78 pages). Administrative Region, Court of First Instance, Case No. 1873 Genentech's Cassation Appeal submitted in invalidation against regarding HK Patent No. 1048260, Particulars of Objections, Date: Polish Patent No. 202369 dated Aug. 3, 2015 (in Polish with English Oct. 2, 2013 (7 pages). translation) (12 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion v. Genentech trial in the High Court of the Kong Special Administrative Region, Court of First Instance, Case No. 1873 Administrative Region, Court of First Instance, Case No. 1873 regarding HK Patent No. 1048260, Writ of Summons, Date: Oct. 2, regarding HK Patent No. 1048260. 2013 (5 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion v. Genentech trial in the High Court of the Kong Special Administrative Region, Court of First Instance, Case No. 1873 Administrative Region, Court of First Instance, Case No. 1873 regarding HK Patent No. 1048260, Order regarding Filing Defence, regarding HK Patent No. 1048260, Acknowledgement of Service of Date: Feb. 18, 2013 (1 page). Writ of Summons, Date: Oct. 16, 2013 (1 page). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion v. Genentech trial in the High Court of the Kong Special Administrative Region, Court of First Instance, Case No. 1873 Administrative Region, Court of First Instance, Case No. 1873 regarding HK Patent No. 1048260, Sealed Order for Directions, regarding HK Patent No. 1048260, Defence, Date: Jan. 22, 2014 (2 Date: Jul. 14, 2013 (6 pages). pages). Appx96 GNE-HER_002953474 Case: 19-2156 Document: 19 Page: 168 Filed: 07/26/2019 US 10,160,811 B2 Page 9 (56) References Cited Celltrion v. Genentech trial in the High Court of the Kong Special Administrative Region, Court of First Instance, Case No. 1873 OTHER PUBLICATIONS regarding HK Patent No. 1048260, Third Affidavit of Anthony Clinton Dudley Evans, Date: Oct. 3, 2014 (11 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Celltrion v. Genentech trial in the High Court of the Kong Special Administrative Region, Court of First Instance, Case No. 1873 Administrative Region, Court of First Instance, Case No. 1873 regarding HK Patent No. 1048260, Timetabling Questionnaire of regarding HK Patent No. 1048260, Witness Statement and Affir the Plaintiff, Date; Mar. 28, 2014 (9 pages). mation of David Kim, Date: Mar. 20, 2015 (32 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Invalidation Action by Celltrion against South African Patent No. Administrative Region, Court of First Instance, Case No. 1873 2002/1229. regarding HK Patent No. 1048260, Further and Better Particulars of Invalidation Action filed by Celltrion against South African Patent Objections, Date: Jul. 4, 2014 ( 11 pages). No. 2002/1229 dated Oct. 28, 2011 (9 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Genentech's Request for Extension of Time in Invalidation Action Administrative Region, Court of First Instance, Case No. 1873 against South African Patent No. 2002/1229 dated Jan. 3, 2012 (2 regarding HK Patent No. 1048260, Summons for Further and Better pages). Particulars of Objections, Date: Jul. 23, 2014 (5 pages). Grounds for Genentech's Request for Extension of Time Celltrion v. Genentech trial in the High Court of the Kong Special Founding affidavit and motion to amend in Invalidation Action Administrative Region, Court of First Instance, Case No. 1873 against South African Patent No. 2002/1229 dated Jan. 3, 2012 (2 regarding HK Patent No. 1048260, Mediation Certificate of the pages). Defendant, Date: Jul. 28, 2014 (4 pages). Notice of Intention to Amend in Terms of Rule 28 in Invalidation Celltrion v. Genentech trial in the High Court of the Kong Special Action Against South African Patent No. 2002/1229 dated Jun. 11, Administrative Region, Court of First Instance, Case No. 1873 2012 (9 pages). regarding HK Patent No. 1048260, Timetabling Questionnaire of Founding Affidavit of Diane Marschang in Invalidation Action the Defendant, Date; Jul. 28, 2014 (9 pages). Against South African Patent No. 2002/1229 dated Oct. 4, 2012 (23 Celltrion v. Genentech trial in the High Court of the Kong Special pages). Administrative Region, Court of First Instance, Case No. 1873 Order to Amend Patent in Invalidation Action Against South African regarding HK Patent No. 1048260, Agreed English Translation of Patent No. 2002/1229 dated Mar. 12, 2013 (2 pages). Hong Kong Standard Patent No. 1048260, Date: Aug. 15, 2014 (67 Celltrion's Amended Motion for Revocation in Invalidation Actin pages). Against South African Patent No. 2002/1229 dated Jul. 6, 2012 (18 Celltrion v. Genentech trial in the High Court of the Kong Special pages). Administrative Region, Court of First Instance, Case No. 1873 Kostowski, W., Excerpt from "Pharmacology, Basics of Pharma regarding HK Patent No. 1048260, Plaintiff's Notice to Admit, cology, Coursebook for Students of Medicine and Physicians", Date: Sep. 26, 2014 (7 pages). Warsaw, First Edition, 1998, pp. 89-91. Celltrion v. Genentech trial in the High Court of the Kong Special Founding Affidavit of Martin John Glennie in Invalidation Action Administrative Region, Court of First Instance, Case No. 1873 Against South African Patent No. 2002/1229 dated Aug. 23, 2013 regarding HK Patent No. 1048260, Defendant's Admissions to (82 pages). Facts, Date: Oct. 23, 2014 (7 pages). Supporting Affidavit ofKyungha Shin in Invalidation Action Against Celltrion v. Genentech trial in the High Court of the Kong Special South African Patent No. 2002/1229 dated Aug. 20, 2013 (2 pages). 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Administrative Region, Court of First Instance, Case No. 1873 Replying Affidavit of Robert Howard Earhart in Invalidation Action regarding HK Patent No. 1048260, Plaintiff's Listing Question Against South African Patent No. 2002/1229 dated Sep. 30, 2014 naire, Date: Jul. 20, 2015 (7 pages). (129 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Replying Affidavit of Robert Charles Frederick Leonard in Invali Administrative Region, Court of First Instance, Case No. 1873 dation Action Against South African Patent No. 2002/1229 dated regarding HK Patent No. 1048260, Defendant's Listing Question Sep. 30, 2014 (59 pages). naire, Date: Jul. 21, 2015 (7 pages). Invalidation Action Against Chinese Patent No. CN 00814890.3. Celltrion v. Genentech trial in the High Court of the Kong Special Koizumi, et. al., "Multicompartmental Analysis of the Kinetics of Administrative Region, Court of First Instance, Case No. 1873 Radioiodinated Monoclonal Antibody in Patients with Cancer", J. regarding HK Patent No. 1048260, Affidavit of Anthony Clinton Nucl. Med., 1986, vol. 27, pp. 1243-1254. Dudley Evans, Date: Jul. 24, 2014 (7 pages). Eger, et. al., "Kinetic Model for the Biodistribution of an 111 In Celltrion v. Genentech trial in the High Court of the Kong Special labeled Monoclonal Antibody in Humans", Cancer Res., 1987, vol. Administrative Region, Court of First Instance, Case No. 1873 47, pp. 3328-3336. regarding HK Patent No. 1048260, Second Affidavit of Anthony Genentech's Appeal Brief submitted in Invalidation of Chinese Clinton Dudley Evans, Date: Aug. 29, 2014 (6 pages). Decision No. 19128 dated Nov. 16, 2012 (Chinese with English Celltrion v. Genentech trial in the High Court of the Kong Special translation (16 pages). Administrative Region, Court of First Instance, Case No. 1873 Administrative Judgement in Request for invalidation of Chinese regarding HK Patent No. 1048260, Affidavit of Wong Tak Kay Patent No. CN 00814890.3 dated Aug. 23, 2010 (English transla Alison, Date: Sep. 17, 2014 (7 pages). tion) (12 pages). Celltrion v. Genentech trial in the High Court of the Kong Special Request for Invalidation filed against Chinese Patent No. ZL00814590.3 Administrative Region, Court of First Instance, Case No. 1873 dated Dec. 30, 2011 (11 pages). regarding HK Patent No. 1048260, Exhibit WTKA-5, Proposed Genentech's Response to Request for invalidation of Chinese Patent Claim Amendments to Claims of Hong Kong Standard Patent No. No. CN 00814890.3 dated Feb. 26, 2012 (9 pages). HK 1048260, in Affidavit of Wong Tak Kay Alison, Date: Sep. 17, Genentech's Supplemental Response to Request for invalidation of 2014 (5 pages). Chinese Patent No. CN 00814890.3 dated May 31, 2012 (11 pages). Appx97 GNE-HER_002953475 Case: 19-2156 Document: 19 Page: 169 Filed: 07/26/2019 US 10,160,811 B2 Page 10 (56) References Cited Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP OTHER PUBLICATIONS (UK) 1 210 ll5. Skeleton Argument of the Claimant for Case Management Conference, Date: Nov. 22, 2012 (5 pages). Decision of Examination of the Request for Invalidation No. 19128 Hospira v. Genentech trial in the High Court of Justice Chancery relating to Chinese Patent No. 00814590.3, Date: Aug. 8, 2012 Division 2 Patents Court, Case No. HC12C03487 regarding EP (English translation) (20 pages). (UK) 1 210 ll5. Supplementary Skeleton Argument of the Claim Administrative Judgement by Beijing First Intermediate People's ant, Date: Nov. 22, 2012 (1 page). Court of the PRC in Request for invalidation of Chinese Patent No. Hospira v. Genentech trial in the High Court of Justice Chancery CN 000814890.3, Date: Dec. 19, 2013 (English translation) (20 Division 2 Patents Court, Case No. HC12C03487 regarding EP pages). (UK) 1 210 ll5. Transcript of the Case Management Conference, Administrative Judgement by Beijing High Court of the PRC in Date: Nov. 22, 2012 (21 pages). Request for invalidation of Chinese Patent No. CN 000814890.3, Hospira v. Genentech trial in the High Court of Justice Chancery Date: Dec. 19, 2013 (English translation) (21 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Petition to Supreme People's Court for Retrial in Request for (UK) 1 210 ll5. First Witness Statement of Nigel Martin Stoate, invalidation of Chinese Patent No. CN 000814890.3, Date: Oct. 30, Date: Dec. 3, 2012 (4 pages). 2015 (Chinese with English translation) (16 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Order for Directions following Case Management (UK) 1 210 ll5. Grounds of Invalidity dated Sep. 13, 2012 (7 Conference, Date: Nov. 22, 2012 (5 pages). pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Consent Order demanding further information, (UK) 1 210 ll5. Amended Particulars of Claim, Date: Sep. 13, 2012 Date: Dec. 12, 2012 (3 pages). (3 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Notice to Admit Facts and Request for Further (UK) 1 210 ll5. Re-amended Grounds of Invalidity, Date: Sep. 13, Information from the Defendant, Date: Feb. 15, 2013 (7 pages). 2012 (8 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Defendant's Response to the Claimant's Notice to Division 2 Patents Court, Case No. HC12C03487 regarding EP Admit Facts and Part 18 Request for Further Information, Date: (UK) 1 210 ll5. Re-Amended Defence, Date: Oct. 29, 2012 (3 Mar. 1, 2013 (3 pages). pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Supplemental Claimant's Notice to Admit Facts (UK) 1 210 ll5. Confidential Witness Statement of Michael John and Part 18 Request for Further Information from the Defendant, Gilbert, Date: Nov. 21, 2012 (7 pages). Date: Jul. 4, 2013 (3 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit MJG-1, correspondence between Taylor (UK) 1 210 ll5. Defendant's Response to the Claimant's Supple Wessing and Mewburn Ellis in Confidential Witness Statement of mental Notice to Admit Facts and Part 18 Request for Further Michael John Gilbert, Date: Nov. 21, 2012 (7 pages). Information, Date: Jul. 15, 2013 (3 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit MJG-2, Hospira Press Release, "Hospira, (UK) 1 210 ll5. Witness Statement of Charles Anthony Nettleton Celltrion, Enter Business Cooperation Agreement to Develop and Balme, Date: Jul. 18, 2013 (7 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Market Biogeneric Drugs", 2009, in Confidential Witness Statement Division 2 Patents Court, Case No. HC12C03487 regarding EP of Michael John Gilbert, Date: Nov. 21, 2012 (2 pages). (UK) 1 210 ll5. Exhibit CAB-1, inter partes correspondence Hospira v. Genentech trial in the High Court of Justice Chancery regarding confidentiality of material, in Witness Statement of Charles Division 2 Patents Court, Case No. HC12C03487 regarding EP Anthony Nettleton Balme, Date: Jul. 18, 2013 (9 pages). (UK) 1 210 ll5. Exhibit MJG-3, Hospira website, "About Hospira", Hospira v. Genentech trial in the High Court of Justice Chancery in Confidential Witness Statement of Michael John Gilbert, Date: Division 2 Patents Court, Case No. HC12C03487 regarding EP Nov. 21, 2012 (2 pages). (UK) 1 210 ll5. Exhibit CAB-2, Affidavit by HyukJae Lee dated Hospira v. Genentech trial in the High Court of Justice Chancery Dec. 5, 2012, in Witness Statement of Charles Anthony Nettleton Division 2 Patents Court, Case No. HC12C03487 regarding EP Balme, Date: Jul. 18, 2013 (6 pages). (UK) 1 210 ll5. Exhibit MJG-4, United States Securities and Hospira v. Genentech trial in the High Court of Justice Chancery Exchange Commission, Form 10-Q Quarterly report for Hospira, Division 2 Patents Court, Case No. HC12C03487 regarding EP Inc., dated Aug. 1, 2012 in Confidential Witness Statement of (UK) 1 210 ll5. Claimant's Pleading of Matters Falling within the Michael John Gilbert, Date: Nov. 21, 2012 (69 pages). Common General Knowledge, Date: Sep. 27, 2013 (7 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit MJG-5, Celltrion Outlook Presentation, (UK) 1 210 ll5. Defendant's Reply to the Claimant's Pleading of Matters Falling within the Common General Knowledge, Date: Oct. 2nd Quarter 2012, in Confidential Witness Statement of Michael 29, 2013 (17 pages). John Gilbert, Date: Nov. 21, 2012 (15 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Witness Statement of Simon Charles Cohen, Date: (UK) 1 210 ll5. Exhibit MJG-6, Celltrion Outlook Presentation, Nov. 18, 2013 (5 pages). 3rd Quarter 2012, in Confidential Witness Statement of Michael Hospira v. Genentech trial in the High Court of Justice Chancery John Gilbert, Date: Nov. 21, 2012 (16 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit SCC-1, Correspondence between Taylor Division 2 Patents Court, Case No. HC12C03487 regarding EP Wessing and Marks & Clerk regarding slide presentation of Dr. (UK) 1 210 ll5. Skeleton Argument of the Defendant for Case Leyland-Jones, in Witness Statement of Simon Charles Cohen, Management Conference, Date: Nov. 22, 2012 (4 pages). Date: Nov. 18, 2013 (9 pages). Appx98 GNE-HER_002953476 Case: 19-2156 Document: 19 Page: 170 Filed: 07/26/2019 US 10,160,811 B2 Page 11 (56) References Cited Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP OTHER PUBLICATIONS (UK) 1 210 ll5. Exhibit, AVB-4, Schedule of Sources for Figures, in Expert Report of Professor Alan Vincent Boddy, Date: Dec. 18, Hospira v. Genentech trial in the High Court of Justice Chancery 2013 (3 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Second Witness Statement of Michael John Division 2 Patents Court, Case No. HC12C03487 regarding EP Gilbert, Date: Nov. 22, 2013 (4 pages). (UK) 1 210 ll5. Exhibit, AVB-5, WinNoLin output file regarding Hospira v. Genentech trial in the High Court of Justice Chancery one-compartment analysis, in Expert Report of Professor Alan Division 2 Patents Court, Case No. HC12C03487 regarding EP Vincent Boddy, Date: Dec. 18, 2013 (13 pages). (UK) 1 210 ll5. Exhibit MJG-7, Correspondence between Taylor Hospira v. Genentech trial in the High Court of Justice Chancery Wessing and Marks & Clerk regarding slide presentation of Dr. Division 2 Patents Court, Case No. HC12C03487 regarding EP Leyland-Jones in Second Witness Statement of Michael John Gil (UK) 1 210 ll5. Exhibit, AVB-6, WinNoLin output file regarding bert, Date: Nov. 22, 2013 (6 pages). analysis incorporating data from figure 3, in Expert Report of Hospira v. Genentech trial in the High Court of Justice Chancery Professor Alan Vincent Boddy, Date: Dec. 18, 2013 (13 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Claimant's Skeleton Argument for Letter of Division 2 Patents Court, Case No. HC12C03487 regarding EP Request Application for Examination of Brian Leyland-Jones, Date: (UK) 1 210 ll5. Exhibit, AVB-7, WinNoLin output file regarding Nov. 26, 2013 (7 pages). two-compartment analysis of data from Table 2, in Expert Report of Hospira v. Genentech trial in the High Court of Justice Chancery Professor Alan Vincent Boddy, Date: Dec. 18, 2013 (13 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Defendant's Skeleton Argument for Letter of Hospira v. Genentech trial in the High Court of Justice Chancery Request Application for Examination of Brian Leyland-Jones, Date: Division 2 Patents Court, Case No. HC12C03487 regarding EP Nov. 26, 2013 (6 pages). (UK) 1 210 ll5. Exhibit, AVB-8, WinNoLin output file regarding Hospira v. Genentech trial in the High Court of Justice Chancery two-compartment analysis of data from Table 2 and Table 3, in Division 2 Patents Court, Case No. HC12C03487 regarding EP Expert Report of Professor Alan Vincent Boddy, Date: Dec. 18, (UK) 1 210 ll5. Transcript of Hearing for Letter of Request 2013 (13 pages). Application for Examination of Brian Leyland-Jones, Date: Nov. Hospira v. Genentech trial in the High Court of Justice Chancery 26, 2013 (8 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit, AVB-9, Output of data underlying Figure Division 2 Patents Court, Case No. HC12C03487 regarding EP 26, in Expert Report of Professor Alan Vincent Boddy, Date: Dec. (UK) 1 210 ll5. Order Directing Issuance of Letter of Request 18, 2013 (100 pages). Application for Examination of Brian Leyland-Jones, Date: Nov. Hospira v. Genentech trial in the High Court of Justice Chancery 28, 2013 (3 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Expert Report of Professor Robert Charles Fred Division 2 Patents Court, Case No. HC12C03487 regarding EP erick Leonard, Date: Dec. 18, 2013 (32 pages). (UK) 1 210 ll5. Expert Report of Professor Peter Barrett-Lee, Date: Hospira v. Genentech trial in the High Court of Justice Chancery Dec. 18, 2013 (43 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit RFCL-1, Curriculum Vitae of Robert Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit PBL-1, CV of Professor Robert Barrett Charles Frederick Leonard dated Oct. 2012, in Expert Report of Lee, in Expert Report of Professor Peter Barrett-Lee, Date: Dec. 18, Professor Robert Charles Frederick Leonard, Date: Dec. 18, 2013 2013 (8 pages). (42 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit PBL-2, List of Publications Authored or (UK) 1 210 ll5. Exhibit RFCL-2, Devita, et. al., "Therapy of Co-Authored by Professor Robert Barrett-Lee, in Expert Report of Locally Advanced and Inflammatory Breast Cancer", Cancer: Prin Professor Peter Barrett-Lee, Date: Dec. 18, 2013 (8 pages). ciples & Practices of Oncology, Caputo, Grace R., Lippincott Hospira v. Genentech trial in the High Court of Justice Chancery Raven Publishers, 5th Edition, 1997, p. 1599, in Expert Report of Division 2 Patents Court, Case No. HC12C03487 regarding EP Professor Robert Charles Frederick Leonard, Date: Dec. 18, 2013 (3 (UK) 1 210 ll5. Exhibit PBL-3, Publication of European Patent pages). Specification, Patent No. EP 1210 ll5 Bl, Published Aug. 5, 2009, Hospira v. Genentech trial in the High Court of Justice Chancery in Expert Report of Professor Peter Barrett-Lee, Date: Dec. 18, 2013 Division 2 Patents Court, Case No. HC12C03487 regarding EP (51 pages). (UK) 1 210 ll5. Exhibit RFCL-3, Taylor, et. al., "The therapeutic Hospira v. Genentech trial in the High Court of Justice Chancery potential of novel aromatase inhibitors in breast cancer", Exp. Opin. Division 2 Patents Court, Case No. HC12C03487 regarding EP Invest. Drugs, Mar. 1999, vol. 8, No. 3, pp. 269-279, in Expert (UK) 1 210 ll5. Expert Report of Alan Vincent Boddy, Date: Dec. Professor Robert Charles Frederick Leonard, Date: Dec. 18, 2013 18, 2013 (57 pages). (ll pages). Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit, AVB-1, Curriculum Vitae of Professor Division 2 Patents Court, Case No. HC12C03487 regarding EP Alan Vincent Boddy, in Expert Report of Professor Alan Vincent (UK) 1 210 ll5. Exhibit RFCL-4, Blainey, R. W., "The Role of Boddy, Date: Dec. 18, 2013 (4 pages). Selective Non-Steroidal Aromatase Inhibitors in Future Treatment Hospira v. Genentech trial in the High Court of Justice Chancery Strategies", Oncology, 1997, vol. 54, Suppl. 2, pp. 27-31, in Expert Division 2 Patents Court, Case No. HC12C03487 regarding EP Report of Professor Robert Charles Frederick Leonard, Date: Dec. (UK) 1 210 ll5. Exhibit, AVB-2, List of Publications Authored and 18, 2013 (5 pages). Co-Authored by Professor Alan Vincent Boddy, in Expert Report of Hospira v. Genentech trial in the High Court of Justice Chancery Professor Alan Vincent Boddy, Date: Dec. 18, 2013 (13 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit RFCL-5, Dixon, et. al., "Lessons from the (UK) 1 210 ll5. Exhibit, AVB-3, List of patents and patent use of aromatase inhibitors in the neoadjuvant setting", Endocrine applications in which Alan Boddy is a named inventor, in Expert Related Cancer, 1999, vol. 6, pp. 227-230 in Expert Report of Report of Professor Alan Vincent Boddy, Date: Dec. 18, 2013 (3 Professor Robert Charles Frederick Leonard, Date: Dec. 18, 2013 (5 pages). pages). Appx99 GNE-HER_002953477 Case: 19-2156 Document: 19 Page: 171 Filed: 07/26/2019 US 10,160,811 B2 Page 12 (56) References Cited Exhibit 2 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. Undated slide deck titled "Pharmacological/Screening Insights into OTHER PUBLICATIONS the Future of Herceptin." (34 pages). Exhibit 3 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. Hospira v. Genentech trial in the High Court of Justice Chancery Undated slide deck titled "Dose-Scheduling: Herceptin®." (42 Division 2 Patents Court, Case No. HC12C03487 regarding EP pages). (UK) 1210 115. Exhibit RFCL-6, ATAC Trialists' Group, "Anastrozole Exhibit 4 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. alone or in combination with tamoxifen versus tamoxifen alone for Meta data on electronic files for Exhibits 2 and 3. (2 pages). adjuvant treatment of postmenopausal women with early breast Exhibit 5 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. cancer: first results of the ATAC randomized trial", Lancet, 2002, "HER2 State-of-the Art." (26 pages). vol. 159, pp. 2131-2139, in Expert Report of Professor Robert Charles Frederick Leonard, Date: Dec. 18, 2013 (9 pages). Exhibit 6 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. Hospira v. Genentech trial in the High Court of Justice Chancery HER2 State-of-the-Art Conference Report Nov. 21-23, 1999, Work Division 2 Patents Court, Case No. HC12C03487 regarding EP shop Proceedings, from the Annals of Oncology, vol. 12, 6 supple (UK) 1 210 115. Exhibit RFCL-9, Slamon, et. al., "Addition of ment 1, 2001. (10 pages). Herceptin (Humanized Anti-Her2 Antibody) for HER2 Overexpress Exhibit 7 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. ing Metastatic Breast Cancer (HER2+/MBC) Markedly Increases Leyland-Jones, "Dose Scheduling-Herceptin®," Oncology, 2001; Anticancer Activity: A Randomised, Multinational Controlled Phase 61 (suppl 2):31-36. III Trial", Proceedings of ASCO, 1998, vol. 17, Abstract #377, p. Exhibit 8 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. 98A, in Expert Report of Professor Robert Charles Frederick "Pharmacokinetics: Safety and efficacy of trastuzurnab adminis Leonard, Date: Dec. 18, 2013 (5 pages). tered every three weeks in combination with paclitaxel" Journal of Hospira v. Genentech trial in the High Court of Justice Chancery Clinical Oncology, 2003, vol. 21, 3965-3971. Division 2 Patents Court, Case No. HC12C03487 regarding EP Exhibit 9 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. (UK) 1210 115. Expert Report of Dr. Robert Howard Earhart, Date: Face page of a publication titled "Optimising the Role of Herceptin Dec. 18, 2013 (39 pages). in Breast Cancer." (1 page). Hospira v. Genentech trial in the High Court of Justice Chancery Undated slide deck titled: "Pharmacological/Screening Insights into Division 2 Patents Court, Case No. HC12C03487 regarding EP the Future of Herceptin." (28 pages). (UK) 1 210 115. Exhibit RHE-1, Curriculum Vitae of Robert Undated slide deck titled: "Optimising the role of Herceptin® in Howard Earhart, in Expert Report of Dr. Robert Howard Earhart, breast cancer." (87 pages). Date: Dec. 18, 2013 (16 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 115. Second Witness Statement of Simon Charles (UK) 1 210 115. Exhibit RHE-2, Baselga, et. al., "Phase II Study of Cohen, Date: Jan. 27, 2014 (12 pages). Weekly Intravenous Recombinant HumanizedAnti-pl85HER2 Mono Hospira v. Genentech trial in the High Court of Justice Chancery clonal Antibody in Patients with HER2/neu-Overexpressing Meta Division 2 Patents Court, Case No. HC12C03487 regarding EP static Breast Cancer", Journal of Clinical Oncology, Mar. 1996, vol. (UK) 1 210 115. Exhibit SCC-2, inter partes correspondence with 14, No. 3, pp. 737-744, in Expert Report of Dr. Robert Howard regard to independently valid claims, in Second Witness Statement Earhart, Date: Dec. 18, 2013 (8 pages). of Simon Charles Cohen, Date: Jan. 27, 2014 (3 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 115. Exhibit RHE-3, Pegram, et. al., "Phase II Study of (UK) 1 210 115. Exhibit SCC-3, inter partes correspondence with Receptor-Enhanced Chemosensitivity Using Recombinant Human regard to the declaration of non-infringement, in Second Witness ized Anti-p 135HERllneu Monoclonal Antibody Plus Cisplatin in Patients Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (29 pages). with HER2/neu-Overexpressing Metastatic Breast Cancer Refrac Hospira v. Genentech trial in the High Court of Justice Chancery tory to Chemotherapy Treatment", Journal of Clinical Oncology, Division 2 Patents Court, Case No. HC12C03487 regarding EP Aug. 1998. vol. 16, No. 8, pp. 2659-2671, in Expert Report of Dr. (UK) 1 210 115. Exhibit SCC-4, Application Notice, request that Robert Howard Earhart, Date: Dec. 18, 2013 (8 pages). Hospira provide further information dated Nov. 23, 2012, in Second Hospira v. Genentech trial in the High Court of Justice Chancery Witness Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (6 Division 2 Patents Court, Case No. HC12C03487 regarding EP pages). (UK) 1 210 115. Order dispensing with expert meetings and joint Hospira v. Genentech trial in the High Court of Justice Chancery reports, Date: Jan. 16, 2014 (2 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 115. Exhibit SCC-5, Consent Order requesting provi Division 2 Patents Court, Case No. HC12C03487 regarding EP sion of a confidential statement of case dated Dec. 12, 2012, in (UK) 1 210 115. Second Expert Report of Professor Peter Barrett Second Witness Statement of Simon Charles Cohen, Date: Jan. 27, Lee, Date: Jan. 24, 2014 (4 pages). 2014 (4 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 115. Second Expert Report of Alan Vincent Boddy, (UK) 1 210 115. Exhibit SCC-6, Confidential Statement of Case Date: Jan. 24, 2014 (24 pages). Served Pursuant to Court Order dated Dec. 12, 2012, in Second Hospira v. Genentech trial in the High Court of Justice Chancery Witness Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (8 Division 2 Patents Court, Case No. HC12C03487 regarding EP pages). (UK) 1210115. Second Expert Report of Professor Robert Charles Hospira v. Genentech trial in the High Court of Justice Chancery Frederick Leonard, Date: Jan. 24, 2014 (3 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 115. Exhibit SCC-7, Expert Report of Professor Division 2 Patents Court, Case No. HC12C03487 regarding EP Zhaohui Sunny Zhou dated Dec. 18, 2013, in Second Witness (UK) 1 210 115. Second Expert Report of Dr. Robert Howard Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (5 pages). Earhart, Date: Jan. 24, 2014 (9 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Transcript of Deposition of Brian Leyland-Jones dated Jan. 27, 2014 Division 2 Patents Court, Case No. HC12C03487 regarding EP (92 pages). (UK) 1 210 115. Exhibit SCC-8, Expert Report of Dr. Uwe Exhibit 1 from Jan. 27, 2014 Deposition of Brian Leyland-Jones. Gottschalk dated Dec. 18, 2013, in Second Witness Statement of Subpoena for Dr. Leyland-Jones, (10 pages). Simon Charles Cohen, Date: Jan. 27, 2014 (4 pages). Appx100 GNE-HER_002953478 Case: 19-2156 Document: 19 Page: 172 Filed: 07/26/2019 US 10,160,811 B2 Page 13 (56) References Cited Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP OTHER PUBLICATIONS (UK) 1 210 ll5. Third Expert Report of Professor Robert Charles Frederick Leonard, Date: Feb. 10, 2014 (5 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit SCC-9, Second Expert Report of Dr. Uwe (UK) 1 210 ll5. Third Expert Report of Dr. Robert Howard Earhart, Gottschalk dated Dec. 18, 2013, in Second Witness Statement of Date: Feb. 10, 2014 (18 pages). Simon Charles Cohen, Date: Jan. 27, 2014 (4 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit RHE-4, WinNoLin analysis using Model (UK) 1 210 ll5. Exhibit SCC-10, inter partes correspondence with 8, in Third Expert Report of Dr. Robert Howard Earhart, Date: Feb. regard to directions for the EP '455 amendment, in Second Witness 10, 2014 (12 pages). Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (12 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit RHE-5, WinNoLin analysis using Model (UK) 1 210 ll5. Exhibit SCC-ll, Comptroller's objections to claim 1, in Third Expert Report of Dr. Robert Howard Earhart, Date: Feb. set 2 dated Nov. 26, 2013, in Second Witness Statement of Simon 10, 2014 (12 pages). Charles Cohen, Date: Jan. 27, 2014 (4 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit RHE-6, Except from Biological Licensing (UK) 1 210 ll5. Exhibit SCC-12, Draft Trial Timetable, in Second Application submitted to the FDA, May 1, 1998, p. 14, in Third Witness Statement of Simon Charles Cohen, Date: Jan. 27, 2014 (2 Expert Report of Dr. Robert Howard Earhart, Date: Feb. 10, 2014 pages). (1 page). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit SCC-13, inter partes correspondence with (UK) 1 210 ll5. Exhibit RHE-8, Simulation of alternative dosing regard to costs for EP '632, in Second Witness Statement of Simon regimens, May 21, 1998, in Third Expert Report of Dr. Robert Charles Cohen, Date: Jan. 27, 2014 (4 pages). Howard Earhart, Date: Feb. 10, 2014 (10 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit SCC-14, inter partes correspondence with (UK) 1 210 ll5. Exhibit RHE-9, Simulation of alternative dosing regard to disclosure of documents, in Second Witness Statement of regimens, May 22, 1998, in Third Expert Report of Dr. Robert Simon Charles Cohen, Date: Jan. 27, 2014 (16 pages). Howard Earhart, Date: Feb. 10, 2014 (10 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Hospira's Skeleton Argument for the Pre-Trial (UK) 1 210 ll5. Exhibit RHE-10, Excerpt ofletter sent by Genentech Review, Date: Jan. 29, 2014 (15 pages). to the FDA, Feb. 20, 2001, in Third Expert Report of Dr. Robert Hospira v. Genentech trial in the High Court of Justice Chancery Howard Earhart, Date: Feb. 10, 2014 (9 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Genentech's Skeleton Argument for the Pre-Trial Division 2 Patents Court, Case No. HC12C03487 regarding EP Review, Date: Jan. 29, 2014 (8 pages). (UK) 1 210 ll5, Claimant's Response to Defendant's Statement of Hospira v. Genentech trial in the High Court of Justice Chancery Case on Independent Validity, Date: Feb. 17, 2014 (3 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Claimant's Civil Evidence Act Notice, Date: Jan. Division 2 Patents Court, Case No. HC12C03487 regarding EP 2014 (3 pages). (UK) 1 210 ll5. Genentech's Skeleton Argument, Date: Feb. 27, Hospira v. Genentech trial in the High Court of Justice Chancery 2014 (37 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Approved Judgement-Pre-trial review, Date: Jan. Division 2 Patents Court, Case No. HC12C03487 regarding EP 2014 (6 pages). (UK) 1 210 ll5. Hospira's Skeleton Argument, Date: Feb. 28, 2014 (61 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Order from Pre-trial review, Date: Jan. 30, 2014 (6 (UK) 1 210 ll5. Genentech's Supplemental Skeleton Argument, pages). Date: Mar. 4, 2014 (4 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Transcript of the Pre-trial Review, Date: Jan. 30, (UK) 1 210 ll5. Transcript of UK Patents Court Trial (Days 1-9), 2014 (63 pages). Date: Mar. 6-19, 2014 (487 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Defendant's Statement of Case on Independent (UK) 1 210 ll5. Hospira's Written Closing, Date: Mar. 17, 2014 (60 Validity, Date: Feb. 10, 2014 (4 pages). pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Third Expert Report of Professor Peter Barrett-Lee, (UK) 1 210 ll5. Genentech's Closing Submissions, Date: Mar. 17, Date: Feb. 10, 2014 (3 pages). 2014 (86 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Third Expert Report of Alan Vincent Boddy, Date: (UK) 1 210 ll5. Consent Order for suspending time to appeal, Date: Feb. 10, 2014 (3 pages). Apr. 10, 2014 (2 pages). Appx101 GNE-HER_002953479 Case: 19-2156 Document: 19 Page: 173 Filed: 07/26/2019 US 10,160,811 B2 Page 14 (56) References Cited Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP OTHER PUBLICATIONS (UK) 1 210 ll5. Transcript of Form of Order Hearing, Date: May 16, 2014 (26 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Approved Judgement, Date: Apr. 10, 2014 (47 (UK) 1 210 ll5. Genentech's Skeleton Argument for Petition to pages). Appeal, Date: Jun. 6, 2014 (17 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Third Witness Statement of Simon Charles Cohen, (UK) 1 210 ll5. Notice of Appeal and Grounds of Appeal, Date: Date: May 2, 2014 (9 pages). Jun. 6, 2014 (12 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Fourth Witness Statement of Simon Charles Division 2 Patents Court, Case No. HC12C03487 regarding EP Cohen, Date: May 13, 2014 (5 pages). (UK) 1 210 ll5. Defendant's list of documents: standard disclosure, Hospira v. Genentech trial in the High Court of Justice Chancery Date: Jun. 13, 2014 (12 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit SCC-15, Letter from Marks & Clerk Division 2 Patents Court, Case No. HC12C03487 regarding EP regarding draft order and supporting evidence dated Apr. 26, 2014, (UK) 1 210 ll5. Defendant's supplemental list of documents: in Fourth Witness Statement of Simon Charles Cohen, Date: May standard disclosure, Date: Jun. 13, 2014 (12 pages). 13, 2014 (2 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Claimant's list of documents: standard disclosure, (UK) 1 210 ll5. Exhibit SCC-16, Letter from Marks & Clerk Date: Jun. 14, 2014 (3 pages). requesting further information and clarification regarding statement Hospira v. Genentech trial in the High Court of Justice Chancery of costs dated May 9, 2014, in Fourth Witness Statement of Simon Division 2 Patents Court, Case No. HC12C03487 regarding EP Charles Cohen, Date: May 13, 2014 (3 pages). (UK) 1 210 ll5. Fifth Witness Statement of Simon Charles Cohen, Hospira v. Genentech trial in the High Court of Justice Chancery Date: Jul. 9, 2014 (8 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit SCC-17, Breakdown of costs incurred by Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira, in Fourth Witness Statement of Simon Charles Cohen, (UK) 1 210 ll5. Exhibit SCC-18, Decision of Opposition Division Date: May 13, 2014 (2 pages). Hospira v. Genentech trial in the High Court of Justice Chancery regarding' ll5 patent dated May 4, 2012, in Fifth Witness Statement Division 2 Patents Court, Case No. HC12C03487 regarding EP of Simon Charles Cohen, Date: Jul. 9, 2014 (16 pages). (UK) 1 210 ll 5. Third Witness Statement of Michael John Gilbert, Hospira v. Genentech trial in the High Court of Justice Chancery Date: May 13, 2014 (10 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit SCC-19, Proprietor's Grounds of Appeal Division 2 Patents Court, Case No. HC12C03487 regarding EP regarding 'll5 patent dated Sep. 13, 2012, in Fifth Witness State (UK) 1 210 ll5. Exhibit MJC-8, Letter from Marks & Clerk ment of Simon Charles Cohen, Date: Jul. 9, 2014 (41 pages). requesting a limited breakdown of global costs dated May 9, 2014, Hospira v. Genentech trial in the High Court of Justice Chancery in Third Witness Statement of Michael John Gilbert, Date: May 13, Division 2 Patents Court, Case No. HC12C03487 regarding EP 2014 (3 pages). (UK) 1 210 ll5. Order granting permission to appeal, Date: Jul. 21, Hospira v. Genentech trial in the High Court of Justice Chancery 2014 (2 pages). Division 2 Patents Court, Case No. HC12C03487 regarding EP Hospira v. Genentech trial in the High Court of Justice Chancery (UK) 1 210 ll5. Exhibit MJC-9, inter partes correspondence Division 2 Patents Court, Case No. HC12C03487 regarding EP regarding declaration about construction, in Third Witness State (UK) 1 210 ll5. Genentech's Skeleton Argument for hearing on Jul. ment of Michael John Gilbert, Date: May 13, 2014 (53 pages). 22, 2014, Date: Jul. 21, 2014 (6 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Genentech's Skeleton Argument on the Final (UK) 1 210 ll5. Hospira's Confidential Skeleton Argument, Date: Order, Date: May 14, 2014 (9 pages). Jul. 21, 2014 (ll pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Fourth Witness Statement of Michael John Gilbert, (UK) 1 210 ll5. Transcript of Confidentiality Hearing, Date: Jul. Date: May 15, 2014 (6 pages). 22, 2014 (23 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Exhibit MJC-10, Roche Media Release, "Roche (UK) 1 210 ll5. Order of Confidentiality, Date: Jul. 22, 2014 (3 delivers strong 2013 results" dated Jan. 30, 2014, in Fourth Witness pages). Statement of Michael John Gilbert, Date: May 15, 2014 (3 pages). Hospira v. Genentech trial in the High Court of Justice Chancery Hospira v. Genentech trial in the High Court of Justice Chancery Division 2 Patents Court, Case No. HC12C03487 regarding EP Division 2 Patents Court, Case No. HC12C03487 regarding EP (UK) 1 210 ll5. Respondent's Notice for Upholding Revocation (UK) 1 210 ll5. Hospira's Skeleton Argument for the form-of-order Order and Grounds for Upholding Revocation Order, Date: Aug. 6, hearing, Date: May 16, 2014 (15 pages). 2014 (ll pages). Hospira v. 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No. 7,371,379, PTAB Rowland and Tozer (1995), "Chapter 22: Dose and Time Depen IPR2017-00805 by Hospira, Inc. dencies," in Clinical Pharmacokinetics, 3rd ed. Williams & Wilkins, Petition for Inter Partes Review of U.S. Pat. No. 7,371,379, under pp. 394-423. 35 U.S.C. § 311 and 37 C.F.R. § 42.100 by Hospira, Inc., dated Jan. Sarfaraz Niazi, "Chapter 7: Pharmacokinetic Principles", in Text 30, 2017 (77 pages). book of Biopharrnaceutics and Clinical Pharmacokinetics, 141-203 Declaration of Dr. Allan Lipton filed in Inter Partes Review No. (1979). IPR2017-00805 re: U.S. Pat. No. 7,371,379, dated Jan. 30, 2017 (169 pages). * cited by examiner Appx105 GNE-HER_002953483 Case: 19-2156 Document: 19 Page: 177 Filed: 07/26/2019 U.S. Patent Dec. 25, 2018 Sheet 1 of 5 US 10,160,811 B2 3H4 aa 541-599 405 aa 529-625 7C2 aa 22-53 7F3 aa 22-53 300 400 500 600 I 100I 200I I I I I 2 53 ~ - 7C2 7F3 5 5 22 248 645 3H4 . 541 599 . 405 . 561 625 100 200 300 400 500 600 . . . . I . . . . I I 3H4 epitope {SEQ ID N0:8} 58 residues VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVAR I I 541 599 4D5 epitope {SEQ ID N0:9} 64 residues LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQP I I 561 625 FIG._ 1 1 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPA 38 SPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFL 75 QDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDN 112 YALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEI 149 LKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLID 186 TNRSRA FIG._2 Appx106 GNE-HER_002953484 Case: 19-2156 Document: 19 Page: 178 Filed: 07/26/2019 U.S. Patent Dec. 25, 2018 Sheet 2 of 5 US 10,160,811 B2 0 0 0 0 U') 0 0 C\J ,- ,- I.!) 0 0 ~ D . LO .. (V) .... -E b 0) . -:::i. . ..._.. 0 . (V) C . 0 :;::::; 0 ro...... C • -Q) .. LO (.) (\J C 0 Q () ... E . ::, ':. ~ .... 0 Q) Q) . (\J Q) (/) .. $ .c .. 0) .. ::,e • I- 0 LO . T""" C . ro . Q) . ::E C 0. . 0 f9 . T""" LO 0 0 0 0 0 0 0 0 0 0 co I'-- (0 LO ~ (") C\J ,- .C C CW) Ol.Q ::, cii - I E 1-c-e~ • C Q) O> (!) ro oc- :::i. Q) 0 ~() ii: Appx107 GNE-HER_002953485 Case: 19-2156 Document: 19 Page: 179 Filed: 07/26/2019 ~ ~ ~ 00 • • e ~ ~ N c ~ = N ('D .... QO 0 rJJ ..... = .... ~ ('D ('D Ul 0 d """' """' N O', r.,;_ """' """' 00 = '"Ul '"= '"= I DAY31 -- ...... v------\2 , , DAY28 , , , , , , , , , ...... , ¥ DAY24 ______7 , DAY21 10 mg_kg_lV DAY mg_kg_anti-HER2 4 _mg_kg_anti-HER2 1 4 _2_mg_kg_anti-HER2 E25_ IV_ IV IV_ SC_2_mg_kg_anti-HER2 • iJ,.. DAY6 -o- -0- • .... ···<>··· -e- .J._--,-----,---,----,----,--r--- O I 500 -soo 1500 2500 1000 3000~~~-.-~~----r~~---,r---~~,--~~-.-~~---.--~~~ 2000 for VOL Mean (mm3) Cell TUMOR FIG._4A Appx108 GNE-HER_002953486 Case: 19-2156 Document: 19 Page: 180 Filed: 07/26/2019 ~ ~ ~ 00 • • e ~ ~ N c ~ = N ('D .... QO 0 rJJ .i;...... = .... ('D ('D Ul 0 d """' """' N O', r.,;_ """' """' 00 = '"Ul '"= '"= ,0 • DAY31 • • • • • • O ...... ,,,O• DAY28 ,, ·····0, , .. .. . " ,, .... 11, ,, •• .. p' o , , DAY24 , , , , ...... , , , , •••••• DAY21 ER2 ···o' .. .. 10 mg_kg_lV DAY mg_kg_anti-HER2 4 _mg_kg_anti-H 1 4 _2_mg_kg_anti-HER2 E25_ IV_ IV IV_ SC_2_mg_kg_anti-HER2 DAY6 •O•. • -0- ···il··· ---- ···O··· 10...J__----..-----..---,-----.---.-----' 100 1000 10000~~~~-r---~~---.~~~--.~~~-,-~~~,--~~---.~~~ for VOL 48 Mean (mm3) Cell TUMOR FIG._ Appx109 GNE-HER_002953487 Case: 19-2156 Document: 19 Page: 181 Filed: 07/26/2019 U.S. Patent Dec. 25, 2018 Sheet 5 of 5 US 10,160,811 B2 VARIABLE LIGHT 1 10 20 30 40 2C4 DTVMTQSHKIMSTSVGDRVSITC [KASQDVSIGVA----1 WYQQRP ** **** * * * 574 DIQMTQSPSSLSASVGDRVTITC [KASQDVSIGVA----] WYQQKP * * **** hum kI DIQMTQSPSSLSASVGDRVTITC [RASQSVSTSSYSYMHJ WYQQKP 50 60 70 80 2C4 GQSPKLLIY {SASYRYT] GVPDRFTGSGSGTDFTFTISSVQA ** * * * * * 574 GKAPKLLIY [SASYRYT] GVPSRFSGSGSGTDFTLTISSLQP * **** hum kI GKAPKLLIY [AASSLES] GVPSRFSGSGSGTDFTLTISSLQP 90 100 2C4 EDLAVYYC [QQYYIYPYT] FGGGTKLEIK (SEQ ID N0:10) * * * * 574 EDFATYYC [QQYYIYPYT] FGQGTKVEIK (SEQ ID N0:12) *** hum kI EDFATYYC [QQYNSLPYT] FGQGTKVEIK (SEQ ID N0:14) FIG._5A VARIABLE HEAVY 1 10 20 30 40 2C4 EVQLQQSGPELVKPGTSVKISCKAS [GFTFTDYTMD] WVKQS ** ** * * *** ** * 574 EVQLVESGGGLVQPGGSLRLSCAAS [GFTFTDYTMD] WVRQA ** * * humIII EVQLVESGGGSVQPGGSLRLSCAAS [GFTFSSYAMS] WVRQA 50 60 70 80 2C4 HGKSLEWIG [DVNPNSGGSIYNQRFKG] KASLTVDRSSRIVYM * * ** *** * **** * 574 PGKGLEWVA [DVNPNSGGSIYNQRFKG] RFTLSVDRSKNTLYL * ****** ** **** * * * humIII PGKGLEWVS [VISGDGGSTYYADSVKG] RFTISRDDSKNTLYL 90 100 110 2C4 ELRSLTFEDTAVYYCAR [NLGPSFYFDY] WGQGTTLVTSS (SEQ ID NO: 11) *** ** * 574 QMNSLRAEDTAVYYCAR [NLGPSFYFDYJ WGQGTLVTVSS {SEQ ID NO: 13) ** *** humIII QMNSLRAEDTAVYYCAR [GRGGGS--DY] WGQGTLVTVSS (SEQ ID NO: 15) FIG._58 Appx110 GNE-HER_002953488 Case: 19-2156 Document: 19 Page: 182 Filed: 07/26/2019 US 10,160,811 B2 1 2 TREATMENT WITH ANTI-ERBB2 with the neu proto-oncogene) and inhibits colony formation ANTIBODIES of these cells. In Drebin et al. PNAS (USA) 83:9129-9133 (1986), the 7.16.4 antibody was shown to inhibit the tum RELATED APPLICATIONS origenic growth of neu-transformed NIH-3T3 cells as well 5 as rat neuroblastoma cells (from which the neu oncogene This application is divisional of U.S. Ser. No. 10/600,152 was initially isolated) implanted into nude mice. Drebin et filed Jun. 20, 2003, which is a divisional of U.S. Ser. No. al. in Oncogene 2:387-394 (1988) discuss the production of 09/648,067 filed Aug. 25, 2000 (now U.S. Pat. No. 6,627, a panel of antibodies against the rat neu gene product. All of 196), which claims priority under 35 USC 119(e) to provi the antibodies were found to exert a cytostatic effect on the sional application Nos. 60/151,018, filed Aug. 27, 1999 and 10 growth of neu-transformed cells suspended in soft agar. 60/213,822, filed Jun. 23, 2000, the contents of which are Antibodies of the IgM, IgG2a and IgG2b isotypes were able incorporated herein by reference. to mediate significant in vitro lysis ofneu-transformed cells in the presence of complement, whereas none of the anti- FIELD OF THE INVENTION 15 bodies were able to mediate high levels of antibody-depen The present invention concerns the treatment of disorders dent cellular cytotoxicity (ADCC) of the neu-transformed characterized by the overexpression of ErbB2 or disorders cells. Drebin et al. Oncogene 2:273-277 (1988) report that expressing epidermal growth factor receptor (EGFR), com mixtures of antibodies reactive with two distinct regions on prising administering to a human or animal presenting the the p185 molecule result in synergistic anti-tumor effects on disorders a therapeutically effective amount of an antibody 20 neu-transformed NIH-3T3 cells implanted into nude mice. that binds ErbB2. More specifically, the invention concerns Biological effects of anti-neu antibodies are reviewed in the treatment of human patients susceptible to or diagnosed Myers et al., Meth. Enzym. 198:277-290 (1991). See also with cancer overexpressing ErbB2 or expressing EGFR, W094/22478 published Oct. 13, 1994. Hudziak et al., Mal. where the treatment is with an anti-ErbB2 antibody admin Cell. Biol. 9(3): 1165-1172 (1989) describe the generation of istered by front loading the dose of antibody during treat 25 a panel of anti-ErbB2 antibodies which were characterized ment by intravenous and/or subcutaneous administration. using the human breast tumor cell line SKBR3. Relative cell The invention optionally includes treatment of cancer in a proliferation of the SKBR3 cells following exposure to the human patient with a combination of an anti-ErbB2 antibody antibodies was determined by crystal violet staining of the and a chemotherapeutic agent, such as, but not limited to, a monolayers after 72 hours. Using this assay, maximum taxoid. The taxoid may be, but is not limited to paclitaxel or 30 inhibition was obtained with the antibody called 4D5 which docetaxel. The invention further includes treatment of can inhibited cellular proliferation by 56%. Other antibodies in cer in a human patient with a combination of anti-ErbB2 the panel, including 7C2 and 7F3, reduced cellular prolif antibody and a chemotherapeutic agent, such as, but not eration to a lesser extent in this assay. Hudziak et al. limited to, an anthracycline derivative. Optionally, treatment conclude that the effect of the 4D5 antibody on SKBR3 cells with a combination of anti-ErbB2 and an anthracycline 35 was cytostatic rather than cytotoxic, since SKBR3 cells derivative includes treatment with an effective amount of a resumed growth at a nearly normal rate following removal cardioprotectant. The present invention further concerns of the antibody from the medium. The antibody 4D5 was infrequent dosing of anti-ErbB2 antibodies. further found to sensitize pl 85erbB2 -overexpressing breast tumor cell lines to the cytotoxic effects of TNF -a. See also BACKGROUND OF THE INVENTION 40 W089/06692 published Jul. 27, 1989. The anti-ErbB2 anti bodies discussed in Hudziak et al. are further characterized Proto-oncogenes that encode growth factors and growth in Fendly et al. Cancer Research 50:1550-1558 (1990); factor receptors have been identified to play important roles Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth in the pathogenesis of various human malignancies, includ Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. ing breast cancer. It has been found that the human ErbB2 45 11(3):117-127 (1991); Kumar et al. Mal. Cell. Biol. 11(2): gene (erbB2, also known as her2, or c-erbB-2), which 979-986 (1991); Lewis et al. Cancer Immunol. Immunother. encodes a 185-kd transmembrane glycoprotein receptor 37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 2 (pl85HER ) related to the epidermal growth factor receptor (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); (EGFR), is overexpressed in about 25% to 30% of human Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 breast cancer (Slamon et al., Science 235:177-182 [1987]; 50 (1994); Scott et al. J. Biol. Chem. 266:14300-5 (1991); and Slamon et al., Science 244:707-712 [1989]). D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994). Several lines of evidence support a direct role for ErbB2 Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe in the pathogenesis and clinical aggressiveness of ErbB2- two antibodies which were selected for their reactivity on overexpressing tumors. The introduction of ErbB2 into the lung adenocarcinoma cell line (Calu-3) which overex- non-neoplastic cells has been shown to cause their malignant 55 presses ErbB2. One of the antibodies, called MGR3, was transformation (Hudziak et al., Proc. Natl. Acad. Sci. USA found to internalize, induce phosphorylation of ErbB2, and 84:7159-7163 [1987]; Difiore et al., Science 237:78-182 inhibit tumor cell growth in vitro. [1987]). Transgenic mice that express HER2 were found to McKenzie et al. Oncogene 4:543-548 (1989) generated a develop mammary tumors (Guy et al., Proc. Natl. Acad. Sci. panel of anti-ErbB2 antibodies with varying epitope speci- USA 89:10578-10582 [1992]). 60 ficities, including the antibody designated TAI. This TAI Antibodies directed against human erbB2 protein prod antibody was found to induce accelerated endocytosis of ucts and proteins encoded by the rat equivalent of the erbB2 ErbB2 (see Maier et al. Cancer Res. 51:5361-5369 [1991]). gene (neu) have been described. Drebin et al., Cell 41:695- Bacus et al. Molecular Carcinogenesis 3:350-362 (1990) 706 (1985) refer to an IgG2a monoclonal antibody which is reported that the TAI antibody induced maturation of the directed against the rat neu gene product. This antibody 65 breast cancer cell lines AU-565 (which overexpresses the called 7.16.4 causes down-modulation of cell surface p185 erbB2 gene) and MCF-7 (which does not). Inhibition of expression on B104-1-1 cells (NIH-3T3 cells transfected growth and acquisition of a mature phenotype in these cells Appx111 GNE-HER_002953489 Case: 19-2156 Document: 19 Page: 183 Filed: 07/26/2019 US 10,160,811 B2 3 4 was found to be associated with reduced levels of ErbB2 mended weekly maintenance dose is 2 mg/kg and can be receptor at the cell surface and transient increased levels in administered as a 30-minute infusion if the initial loading the cytoplasm. dose is well tolerated. Stancovski et al. PNAS (USA) 88:8691-8695 (1991) gen ErbB2 overexpression is commonly regarded as a predic- erated a panel of anti-ErbB2 antibodies, injected them i.p. 5 tor of a poor prognosis, especially in patients with primary into nude mice and evaluated their effect on tumor growth of disease that involves axillary lymph nodes (Slamon et al., murine fibroblasts transformed by overexpression of the [1987] and [1989], supra; Ravdin and Chamness, Gene erbB2 gene. Various levels of tumor inhibition were detected 159:19-27 [1995]; and Hynes and Stem, Biochim Biophys for four of the antibodies, but one of the antibodies (N28) Acta 1198:165-184 [1994]), and has been linked to sensi- consistently stimulated tumor growth. Monoclonal antibody 10 tivity and/or resistance to hormone therapy and chemothera N28 induced significant phosphorylation of the ErbB2 peutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga receptor, whereas the other four antibodies generally dis et al., Oncology 11(3 Suppl 1):43-48 [1997]). However, played low or no phosphorylation-inducing activity. The despite the association of ErbB2 overexpression with poor effect of the anti-ErbB2 antibodies on proliferation of 15 prognosis, the odds of HER2-positive patients responding SKBR3 cells was also assessed. In this SKBR3 cell prolif clinically to treatment with taxanes were greater than three eration assay, two of the antibodies (N12 and N29) caused times those of HER2-negative patients (Ibid). rhuMab a reduction in cell proliferation relative to control. The HER2 was shown to enhance the activity of paclitaxel ability of the various antibodies to induce cell lysis in vitro (TAXOL®) and doxorubicin against breast cancer xeno- via complement-dependent cytotoxicity (CDC) and anti 20 grafts in nude mice injected with BT-474 human breast body-mediated cell-dependent cytotoxicity (ADCC) was adenocarcinoma cells, which express high levels of HER2 assessed, with the authors of this paper concluding that the (Baselga et al., Breast Cancer, Proceedings of ASCO, Vol. inhibitory function of the antibodies was not attributed 13, Abstract 53 [1994]). significantly to CDC or ADCC. Bacus et al. Cancer Research 52:2580-2589 (1992) fur- 25 SUMMARY OF THE INVENTION ther characterized the antibodies described in Bacus et al. (1990) and Stancovski et al. of the preceding paragraphs. The present invention concerns the discovery that an early Extending the i.p. studies of Stancovski et al., the effect of attainment of an efficacious target trough serum concentra the antibodies after i.v. injection into nude mice harboring tion by providing an initial dose or doses of anti-ErbB2 mouse fibroblasts overexpressing human ErbB2 was 30 antibodies followed by subsequent doses of equal or smaller amounts of antibody (greater front loading) is more effica assessed. As observed in their earlier work, N28 accelerated cious than conventional treatments. The efficacious target tumor growth, whereas N12 and N29 significantly inhibited trough serum concentration is reached in 4 weeks or less, growth of the ErbB2-expressing cells. Partial tumor inhibi preferably 3 weeks or less, more preferably 2 weeks or less, tion was also observed with the N24 antibody. Bacus et al. 35 and most preferably 1 week or less, including 1 day or less. also tested the ability of the antibodies to promote a mature The target serum concentration is thereafter maintained by phenotype in the human breast cancer cell lines AU-565 and the administration of maintenance doses of equal or smaller MDA-MB453 (which overexpress ErbB2) as well as MCF-7 amounts for the remainder of the treatment regimen or until (containing low levels of the receptor). Bacus et al. saw a suppression of disease symptoms is achieved. correlation between tumor inhibition in vivo and cellular 40 The invention further concerns a method for the treatment differentiation; the tumor-stimulatory antibody N28 had no of a human patient susceptible to or diagnosed with a effect on differentiation, and the tumor inhibitory action of disorder characterized by overexpression of ErbB2 receptor the N12, N29 and N24 antibodies correlated with the extent comprising administering a therapeutically effective amount of differentiation they induced. of an anti-ErbB2 antibody subcutaneously. Preferably, the Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a 45 initial dose (or doses) as well as the subsequent maintenance panel of anti-ErbB2 antibodies for their epitope binding dose or doses are administered subcutaneously. Optionally, specificities, as well as their ability to inhibit anchorage where the patient's tolerance to the anti-ErbB2 antibody is independent and anchorage-dependent growth of SKBR3 unknown, the initial dose is administered by intravenous cells (by individual antibodies and in combinations), modu infusion, followed by subcutaneous administration of the late cell-surface ErbB2, and inhibit ligand stimulated 50 maintenance doses if the patient's tolerance for the antibody anchorage-independent growth. See also W094/00136 pub is acceptable. lished Jan. 6, 1994 and Kasprzyk et al. Cancer Research According to the invention, the method of treatment 52:2771-2776 (1992) concerning anti-ErbB2 antibody com involves administration of an initial dose of anti-ErbB2 binations. Other anti-ErbB2 antibodies are discussed in antibody of more than approximately 4 mg/kg, preferably Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver 55 more than approximately 5 mg/kg. The maximum initial et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. dose or a subsequent dose does not exceed 50 mg/kg, Cancer Res. 54:3758-3765 (1994); and Harwerth et al. J. preferably does not exceed 40 mg/kg, and more preferably Biol. Chem. 267:15160-15167 (1992). does not exceed 30 mg/kg. Administration is by intravenous A recombinant humanized anti-ErbB2 monoclonal anti or subcutaneous administration, preferably intravenous infu body (a humanized version of the murine anti-ErbB2 anti- 60 sion or bolus injection, or more preferably subcutaneous body 4D5, referred to as rhuMAb HER2, HERCEPTIN®, or bolus injection. The initial dose may be one or more HERCEPTIN® anti-ErbB2 antibody) has been clinically administrations of drug sufficient to reach the target trough active in patients with ErbB2-overexpressing metastatic serum concentration in 4 weeks or less, preferably 3 weeks breast cancers that had received extensive prior anti-cancer or less, more preferably 2 weeks or less, and most preferably therapy (Baselga et al., J. Clin. Oneal. 14:737-744 [1996]). 65 1 week or less, including one day or less. The recommended initial loading dose for HERCEPTIN® is According to the invention, the initial dose or doses is/are 4 mg/kg administered as a 90-minute infusion. The recom- followed by subsequent doses of equal or smaller amounts Appx112 GNE-HER_002953490 Case: 19-2156 Document: 19 Page: 184 Filed: 07/26/2019 US 10,160,811 B2 5 6 of antibody at intervals sufficiently close to maintain the small-cell lung cancer, non-small cell lung cancer, gastro trough serum concentration of antibody at or above an intestinal cancer, pancreatic cancer, glioblastoma, cervical efficacious target level. Preferably, an initial dose or subse cancer, ovarian cancer, liver cancer, bladder cancer, hepa- quent dose does not exceed 50 mg/kg, and each subsequent toma, colon cancer, colorectal cancer, endometrial carci dose is at least 0.01 mg/kg. Preferably the amount of drug 5 noma, salivary gland carcinoma, kidney cancer, liver cancer, administered is sufficient to maintain the target trough serum prostate cancer, vulva! cancer, thyroid cancer, hepatic car- concentration such that the interval between administration cinoma and various types of head and neck cancer. The cycles is at least one week. Preferably the trough serum method of the invention may further comprise administra concentration does not exceed 2500 µg/ml and does not fall tion of a chemotherapeutic agent other than an anthracy below 0.01 µg/ml during treatment. The front loading drug 10 cline, e.g. doxorubicin or epirubicin. The chemotherapeutic treatment method of the invention has the advantage of agent preferably is a taxoid, such as TAXOL® (paclitaxel) increased efficacy by reaching a target serum drug concen or a TAXOL® derivative. tration early in treatment. The subcutaneous delivery of Preferred anti-ErbB2 antibodies bind the extracellular maintenance doses according to the invention has the advan domain of the ErbB2 receptor, and preferably bind to the tage of being convenient for the patient and health care 15 epitope 4D5 or 3H4 within the ErbB2 extracellular domain professionals, reducing time and costs for drug treatment. sequence. More preferably, the antibody is the antibody Preferably, the initial dose (or the last dose within an initial 4D5, most preferably in a humanized form. Other preferred dose series) is separated in time from the first subsequent ErbB2-binding antibodies include, but are not limited to, dose by 4 weeks or less, preferably 3 weeks or less, more antibodies 7C2, 7F3, and 2C4, preferably in a humanized preferably 3 weeks or less, most preferably 1 week or less. 20 form. In an embodiment of the invention, the initial dose of The method of the present invention is particularly suit anti-ErbB2 is 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by able for the treatment of breast or ovarian cancer, charac intravenous or subcutaneous administration, such as intra terized by the overexpression of the ErbB2 receptor. venous infusion or subcutaneous bolus injection. The sub The present application also provides a method of therapy sequent maintenance doses are 2 mg/kg delivered once per 25 involving infrequent dosing of an anti-ErbB2 antibody. In week by intravenous infusion, intravenous bolus injection, particular, the invention provides a method for the treatment subcutaneous infusion, or subcutaneous bolus injection. The of cancer (e.g. cancer characterized by overexpression of the choice of delivery method for the initial and maintenance ErbB2 receptor) in a human patient comprising administer doses is made according to the ability of the animal or ing to the patient a first dose of an anti-ErbB2 antibody human patient to tolerate introduction of the antibody into 30 followed by at least one subsequent dose of the antibody, the body. Where the antibody is well-tolerated, the time of wherein the first dose and subsequent dose are separated infusion may be reduced. The choice of delivery method as from each other in time by at least about two weeks (e.g. disclosed for this embodiment applies to all drug delivery from about two weeks to about two months), and optionally regimens contemplated according to the invention. at least about three weeks (e.g. from about three weeks to In another embodiment, the invention includes an initial 35 about six weeks). For instance, the antibody may be admin dose of 12 mg/kg anti-ErbB2 antibody, followed by subse istered about every three weeks, about two to about 20 times, quent maintenance doses of 6 mg/kg once per 3 weeks. e.g. about six times. The first dose and subsequent dose may In still another embodiment, the invention includes an each be from about 2 mg/kg to about 16 mg/kg; e.g. from initial dose of 8 mg/kg anti-ErbB2 antibody, followed by 6 about 4 mg/kg to about 12 mg/kg; and optionally from about mg/kg once per 3 weeks. 40 6 mg/kg to about 12 mg/kg. Generally, two or more subse In yet another embodiment, the invention includes an quent doses (e.g. from about two to about ten subsequent initial dose of 8 mg/kg anti-ErbB2 antibody, followed by doses) of the antibody are administered to the patient, and subsequent maintenance doses of 8 mg/kg once per week or those subsequent doses are preferably separated from each 8 mg/kg once every 2 to 3 weeks. other in time by at least about two weeks (e.g. from about In another embodiment, the invention includes initial 45 two weeks to about two months), and optionally at least doses of at least 1 mg/kg, preferably 4 mg/kg, anti-ErbB2 about three weeks ( e.g. from about three weeks to about six antibody on each of days 1, 2 and 3, followed by subsequent weeks). The two or more subsequent doses may each be maintenance doses of 6 mg/kg once per 3 weeks. from about 2 mg/kg to about 16 mg/kg; or from about 4 In another embodiment, the invention includes an initial mg/kg to about 12 mg/kg; or from about 6 mg/kg to about dose of 4 mg/kg anti-ErbB2 antibody, followed by subse- 50 12 mg/kg. The invention additionally provides an article of quent maintenance doses of 2 mg/kg twice per week, manufacture, comprising a container, a composition within wherein the maintenance doses are separated by 3 days. the container comprising an anti-ErbB2 antibody, and a In still another embodiment, the invention includes a package insert containing instructions to administer the cycle of dosing in which delivery of anti-ErbB2 antibody is antibody according to such methods. 2-3 times per week for 3 weeks. In one embodiment of the 55 The presently described dosing protocols may be applied invention, each dose is approximately 25 mg/kg or less for to other anti-ErbB antibodies such as anti-epidermal growth a human patient, preferably approximately 10 mg/kg or less. factor receptor (EGFR), anti-ErbB3 and anti-ErbB4 anti This 3 week cycle is preferably repeated as necessary to bodies. Thus, the invention provides a method for the achieve suppression of disease symptoms. treatment of cancer in a human patient, comprising admin In another embodiment, the invention includes a cycle of 60 istering an effective amount of an anti-ErbB antibody to the dosing in which delivery of anti-ErbB2 antibody is daily for human patient, the method comprising administering to the 5 days. According to the invention, the cycle is preferably patient an initial dose of at least approximately 5 mg/kg of repeated as necessary to achieve suppression of disease the anti-ErbB antibody; and administering to the patient a symptoms. plurality of subsequent doses of the antibody in an amount The disorder preferably is a benign or malignant tumor 65 that is approximately the same or less than the initial dose. characterized by the overexpression of the ErbB2 receptor, Alternatively, or additionally, the invention pertains to a e.g. a cancer, such as, breast cancer, squamous cell cancer, method for the treatment of cancer in a human patient Appx113 GNE-HER_002953491 Case: 19-2156 Document: 19 Page: 185 Filed: 07/26/2019 US 10,160,811 B2 7 8 comprising administering to the patient a first dose of an body may be an intact antibody (e.g., an intact IgG1 anti anti-ErbB antibody followed by at least one subsequent dose body) or an antibody fragment (e.g., a Fab, F(ab')2 , diabody, of the antibody, wherein the first dose and subsequent dose and the like). The variable light chain and variable heavy are separated from each other in time by at least about two chain regions of humanized anti-ErbB2 antibody 2C4 are weeks. The invention additionally provides an article of 5 shown in FIGS. SA and SB. manufacture, comprising a container, a composition within the container comprising an anti-ErbB antibody, and a BRIEF DESCRIPTION OF THE DRAWINGS package insert containing instructions to administer the antibody according to such methods. FIG. 1 shows epitope-mapping of the extracellular In another aspect, the invention concerns an article of 10 domain ofErbB2 as determined by truncation mutant analy manufacture, comprising a container, a composition within sis and site-directed mutagenesis (Nakamura et al. J. of the container comprising an anti-ErbB2 antibody, optionally Virology 67(10):6179-6191 [October 1993]; Renz et al. J. a label on or associated with the container that indicates that Cell Biol. 125(6):1395-1406 [June 1994]). The anti-prolif the composition can be used for treating a condition char erative MAbs 4D5 and 3H4 bind adjacent to the transmem- acterized by overexpression of ErbB2 receptor, and a pack 15 brane domain. The various ErbB2-ECD truncations or point age insert containing instructions to avoid the use of anthra mutations were prepared from cDNA using polymerase cycline-type chemotherapeutics in combination with the chain reaction technology. The ErbB2 mutants were composition. According to the invention, the package insert expressed as gD fusion proteins in a mammalian expression further includes instructions to administer the anti-ErbB2 plasmid. This expression plasmid uses the cytomegalovirus antibody at an initial dose of 5 mg/kg followed by the same 20 promoter/enhancer with SV40 termination and polyade or smaller subsequent dose or doses. In another embodiment nylation signals located downstream of the inserted cDNA. of the invention, the package insert further includes instruc Plasmid DNA was transfected into 293S cells. One day tions to administer the anti-ErbB2 antibody subcutaneously following transfection, the cells were metabolically labeled for at least one of the doses, preferably for all of the overnight in methionine and cysteine-free, low glucose subsequent doses following the initial dose, most preferably 25 DMEM containing 1% dialyzed fetal bovine serum and 25 for all doses. µCi each of 35S methionine and 35S cysteine. Supernatants In a further aspect, the invention provides a method of were harvested either the ErbB2 MAbs or control antibodies treating ErbB2 expressing cancer in a human patient com were added to the supernatant and incubated 2-4 hours at 4 ° prising administering to the patient effective amounts of an C. The complexes were precipitated, applied to a 10-20% anti-ErbB2 antibody and a chemotherapeutic agent. In one 30 Tricine SDS gradient gel and electrophoresed at 100 V. The embodiment of the invention, the chemotherapeutic agent is gel was electroblotted onto a membrane and analyzed by a taxoid including, but not limited to, paclitaxel and doc autoradiography. SEQ ID NOs:8 and 9 depict the 3H4 and etaxel. In another embodiment, the chemotherapeutic agent 4D5 epitopes, respectively. is an anthracyline derivative including, but not limited to, FIG. 2 depicts with underlining the amino acid sequence doxorubicin or epirubicin. In still another embodiment of the 35 of Domain 1 of ErbB2 (SEQ ID N0:1). Bold amino acids invention, treatment with an anti-ErbB2 antibody and an indicate the location of the epitope recognized by MAbs 7C2 anthracycline derivative further includes administration of a and 7F3 as determined by deletion mapping, i.e. the "7C2/ cardioprotectant to the patient. In still another embodiment, 7F3 epitope" (SEQ ID N0:2). an anthracycline derivative is not administered to the patient FIG. 3 is a graph of anti-ErbB2 antibody (HERCEP with the anti-ErbB2 antibody. One or more additional che- 40 TIN®) trough serum concentration (µg/ml, mean±SE, dark motherapeutic agents may also be administered to the circles) by week from week 2 through week 36 for ErbB2 patient. The cancer is preferably characterized by overex overexpressing patients treated with HERCEPTIN® anti pression of ErbB2. ErbB2 antibody at 4 mg/kg initial dose, followed by 2 mg/kg The invention further provides an article of manufacture weekly. The number of patients at each time point is comprising a container, a composition within the container 45 represented by "n" (white squares). comprising an anti-ErbB2 antibody and a package insert FIG. 4A is a linear plot of tumor volume changes over instructing the user of the composition to administer the time in mice treated with HERCEPTIN® anti-ErbB2 anti anti-ErbB2 antibody composition and a chemotherapeutic body. FIG. 48 is a semi-logarithmic plot of the same data as agent to a patient. In another embodiment, the chemothera in FIG. 4A such that the variation in tumor volume for the peutic agent is other than an anthracycline, and is preferably 50 treated animals is observed more readily. a taxoid, such as TAXOL®. In still another embodiment, the FIGS. SA and SB depict alignments of the amino acid chemotherapeutic agent is an anthracycline, including but sequences of the variable light (VL) (FIG. SA) and variable not limited to, doxorubicin or epirubicin. In yet another heavy (VH) (FIG. SB) domains of murine monoclonal anti embodiment, the chemotherapeutic agent is an anthracycline body 2C4 (SEQ ID Nos. 10 and 11, respectively); VL and V H and the package insert further instructs the user to administer 55 domains of humanized Fab version 574 (SEQ ID Nos. 12 a cardioprotectant. and 13, respectively), and human VL and V H consensus The methods and compositions of the invention comprise frameworks (hum Kl, light kappa subgroup I; humIII, heavy an anti-ErbB2 antibody and include a humanized anti-ErbB2 subgroup III) (SEQ ID Nos. 14 and 15, respectively). antibody. Thus, the invention further pertains to a compo Asterisks identify differences between humanized Fab ver sition comprising an antibody that binds ErbB2 and the use 60 sion 574 and murine monoclonal antibody 2C4 or between of the antibody for treating ErbB2 expressing cancer, e.g., humanized Fab version 574 and the human framework. ErbB2 overexpressing cancer, in a human. The invention Complementarity Determining Regions (CDRs) are in also pertains to the use of the antibody for treating EGFR brackets. Humanized Fab version 574, with the changes expressing cancer. Preferably the antibody is a monoclonal ArgH71 Val, AspH73Arg and IleH69Leu, appears to have antibody 4D5, e.g., humanized 4D5 (and preferably 65 binding restored to that of the original chimeric 2C4 Fab huMAb4D5-8 (HERCEPTIN® anti-ErbB2 antibody); or fragment. Additional FR and/or CDR residues, such as L2, monoclonal antibody 2C4, e.g., humanized 2C4. The anti- L54, L55, L56, H35 and/or H48, may be modified (e.g. Appx114 GNE-HER_002953492 Case: 19-2156 Document: 19 Page: 186 Filed: 07/26/2019 US 10,160,811 B2 9 10 substituted as follows-IleL2Thr; ArgL54Leu; TyrL55Glu; Harbor Laboratory, Ed Harlow and David Lane (1988), can ThrL56Ser; AspH35Ser; and Va!H48Ile) in order to further be performed. Alternatively, epitope mapping can be per refine or enhance binding of the humanized antibody. Alter formed (see FIG. 1) to assess whether the antibody binds to natively, or additionally, the humanized antibody may be the 4D5 epitope of ErbB2 (i.e. any one or more residues in affinity matured in order to further improve or refine its 5 the region from about residue 529, e.g. about residue 561 to affinity and/or other biological activities. about residue 625, inclusive). The "epitope 3H4" is the region in the extracellular DETAILED DESCRIPTION OF THE domain of ErbB2 to which the antibody 3H4 binds. This PREFERRED EMBODIMENTS epitope is shown in FIG. 1, and includes residues from about 10 541 to about 599, inclusive, in the amino acid sequence of I. Definitions ErbB2 extracellular domain. The "epitope 7C2/7F3" is the region at the N-terminus of An "ErbB receptor" is a receptor protein tyrosine kinase the extracellular domain of ErbB2 to which the 7C2 and/or which belongs to the ErbB receptor family and includes 7F3 antibodies (each deposited with the ATCC, see below) EGFR, HER2, ErbB3 and ErbB4 receptors as well as 15 bind. To screen for antibodies which bind to the 7C2/7F3 TEGFR (U.S. Pat. No. 5,708,156) and other members ofthis epitope, a routine cross-blocking assay such as that family to be identified in the future. The ErbB receptor will described in Antibodies, A Laboratory Manual, Cold Spring generally comprise an extracellular domain, which may bind Harbor Laboratory, Ed Harlow and David Lane (1988), can an ErbB ligand; a lipophilic transmembrane domain; a be performed. Alternatively, epitope mapping can be per conserved intracellular tyrosine kinase domain; and a car- 20 formed to establish whether the antibody binds to the boxyl-terminal signaling domain harboring several tyrosine 7C2/7F3 epitope on ErbB2 (i.e. any one or more ofresidues residues which can be phosphorylated. The ErbB receptor in the region from about residue 22 to about residue 53 of may be a native sequence ErbB receptor or an amino acid ErbB2; SEQ ID N0:2). sequence variant thereof. Preferably the ErbB receptor is The term "induces cell death" or "capable of inducing cell native sequence human ErbB receptor. 25 death" refers to the ability of the antibody to make a viable The terms "ErbB1", "epidermal growth factor receptor" cell become nonviable. The "cell" here is one which and "EGFR" are used interchangeably herein and refer to expresses the ErbB2 receptor, especially where the cell native sequence EGFR as disclosed, for example, in Car overexpresses the ErbB2 receptor. A cell which "overex penter et al. Ann. Rev. Biochem. 56:881-914 (1987), includ presses" ErbB2 has significantly higher than normal ErbB2 ing variants thereof ( e.g. a deletion mutant EGFR as in 30 levels compared to a noncancerous cell of the same tissue Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 type. Preferably, the cell is a cancer cell, e.g. a breast, refers to the gene encoding the EGFR protein product. ovarian, stomach, endometrial, salivary gland, lung, kidney, Examples of antibodies which bind to EGFR include MAb colon, thyroid, pancreatic or bladder cell. In vitro, the cell 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL may be a SKBR3, BT474, Calu 3, MDA-MB-453, MDA HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC 35 MB-361 or SKOV3 cell. Cell death in vitro may be deter CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) mined in the absence of complement and immune effector and variants thereof, such as chimerized 225 (C225) and cells to distinguish cell death induced by antibody dependent reshaped human 225 (H225) (see, WO 96/40210, Imclone cellular cytotoxicity (ADCC) or complement dependent Systems Inc.). cytotoxicity (CDC). Thus, the assay for cell death may be "ErbB3" and "HER3" refer to the receptor polypeptide as 40 performed using heat inactivated serum (i.e. in the absence disclosed, for example, in U.S. Pat. Nos. 5,183,884 and of complement) and in the absence of immune effector cells. 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 To determine whether the antibody is able to induce cell (1989), including variants thereof. Examples of antibodies death, loss of membrane integrity as evaluated by uptake of which bind HER3 are described in U.S. Pat. No. 5,968,511 propidium iodide (PI), trypan blue (see Moore et al. Cyto (Akita and Sliwkowski), e.g. the 8B8 antibody (ATCC HB 45 technology 17:1-11 [1995]) or 7AAD can be assessed rela 12070) or a humanized variant thereof. tive to untreated cells. Preferred cell death-inducing anti- The terms "ErbB4" and "HER4" herein refer to the bodies are those which induce PI uptake in the "PI uptake receptor polypeptide as disclosed, for example, in EP Pat assay in BT474 cells". Appln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. The phrase "induces apoptosis" or "capable of inducing USA, 90:1746-1750 (1993); and Plowman et al., Nature, 50 apoptosis" refers to the ability of the antibody to induce 366:473-475 (1993), including variants thereof such as the programmed cell death as determined by binding of amiexin HER4 isoforms disclosed in WO 99/19488. V, fragmentation of DNA, cell shrinkage, dilation of endo The terms "HER2", "ErbB2" "c-Erb-B2" are used inter plasmic reticulum, cell fragmentation, and/or formation of changeably. Unless indicated otherwise, the terms "ErbB2" membrane vesicles (called apoptotic bodies). The cell is one "c-Erb-B2" and "HER2" when used herein refer to the 55 which overexpresses the ErbB2 receptor. Preferably the human protein, and "erbB2," "c-erb-B2," and "her2" refer to "cell" is a tumor cell, e.g. a breast, ovarian, stomach, human gene. The human erbB2 gene and ErbB2 protein are, endometrial, salivary gland, lung, kidney, colon, thyroid, for example, described in Semba et al., PNAS (USA) pancreatic or bladder cell. In vitro, the cell may be a SKBR3, 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230- BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or 234 (1986) (Genebank accession number X03363). ErbB2 60 SKOV3 cell. Various methods are available for evaluating comprises four domains (Domains 1-4). the cellular events associated with apoptosis. For example, The "epitope 4D5" is the region in the extracellular phosphatidyl serine (PS) translocation can be measured by domain of ErbB2 to which the antibody 4D5 (ATCC CRL annexin binding; DNA fragmentation can be evaluated 10463) binds. This epitope is close to the transmembrane through DNA laddering as disclosed in the example herein; region of ErbB2. To screen for antibodies which bind to the 65 and nuclear/chromatin condensation along with DNA frag 4D5 epitope, a routine cross-blocking assay such as that mentation can be evaluated by any increase in hypodiploid described in Antibodies, A Laboratory Manual, Cold Spring cells. Preferably, the antibody which induces apoptosis is Appx115 GNE-HER_002953493 Case: 19-2156 Document: 19 Page: 187 Filed: 07/26/2019 US 10,160,811 B2 11 12 one which results in about 2 to 50 fold, preferably about 5 variable domain of the heavy chain. Particular amino acid to 50 fold, and most preferably about 10 to 50 fold, induction residues are believed to form an interface between the light of annexin binding relative to untreated cell in an "annexin and heavy-chain variable domains. binding assay using BT474 cells" (see below). The term "variable" refers to the fact that certain portions Sometimes the pro-apoptotic antibody will be one which 5 of the variable domains differ extensively in sequence blocks HRG binding/activation of the ErbB2/ErbB3 com among antibodies and are used in the binding and specificity plex (e.g. 7F3 antibody). In other situations, the antibody is of each particular antibody for its particular antigen. How one which does not significantly block activation of the ever, the variability is not evenly distributed throughout the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2). Further, variable domains of antibodies. It is concentrated in three the antibody may be one like 7C2 which, while inducing 10 segments called complementarity determining regions apoptosis, does not induce a large reduction in the percent of (CD Rs) or hypervariable regions both in the light-chain and cells in S phase (e.g. one which only induces about 0-10% the heavy-chain variable domains. The more highly con reduction in the percent of these cells relative to control). served portions of variable domains are called the frame The antibody of interest may be one like 7C2 which binds work region (FR). The variable domains of native heavy and specifically to human ErbB2 and does not significantly 15 light chains each comprise four FR regions, largely adopting cross-react with other proteins such as those encoded by the a ~-sheet configuration, connected by three CDRs, which erbB1, erbB3 and/or erbB4 genes. Sometimes, the antibody form loops connecting, and in some cases forming part of, may not significantly cross-react with the rat neu protein, the ~-sheet structure. The CDRs in each chain are held e.g., as described in Schecter et al. Nature 312:513 (1984) together in close proximity by the FRs and, with the CDRs and Drebin et al., Nature 312:545-548 (1984). In such 20 from the other chain, contribute to the formation of the embodiments, the extent of binding of the antibody to these antigen-binding site of antibodies (see Kabat et al., NIH proteins (e.g., cell surface binding to endogenous receptor) Pub!. No. 91-3242, Vol. I, pages 647-669 [1991]). The will be less than about 10% as determined by fluorescence constant domains are not involved directly in binding an activated cell sorting (FACS) analysis or radioimmunopre antibody to an antigen, but exhibit various effector func- cipitation (RIA). 25 tions, such as participation of the antibody in antibody "Heregulin" (HRG) when used herein refers to a poly dependent cellular cytotoxicity. peptide which activates the ErbB2-ErbB3 and ErbB2-ErbB4 Papain digestion of antibodies produces two identical protein complexes (i.e. induces phosphorylation of tyrosine antigen-binding fragments, called "Fab" fragments, each residues in the complex upon binding thereto). Various with a single antigen-binding site, and a residual "Fe" heregulin polypeptides encompassed by this term are dis- 30 fragment, whose name reflects its ability to crystallize closed in Holmes et al., Science, 256:1205-1210 (1992); WO readily. Pepsin treatment yields an F(ab')2 fragment that has 92/20798; Wen et al., Mal. Cell. Biol., 14(3):1909-1919 two antigen-combining sites and is still capable of cross (1994); and Marchionni et al., Nature, 362:312-318 (1993), linking antigen. for example. The term includes biologically active frag "Fv" is the minimum antibody fragment which contains a ments and/or variants of a naturally occurring HRG poly- 35 complete antigen-recognition and -binding site. This region peptide, such as an EGF-like domain fragment thereof (e.g. consists of a dimer of one heavy- and one light-chain HRG~ l 177_244). variable domain in tight, non-covalent association. It is in The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 this configuration that the three CDRs of each variable protein complex" are noncovalently associated oligomers of domain interact to define an antigen-binding site on the the ErbB2 receptor and the ErbB3 receptor or ErbB4 recep- 40 surface of the VH-VL dimer. Collectively, the six CDRs tor, respectively. The complexes form when a cell express confer antigen-binding specificity to the antibody. However, ing both of these receptors is exposed to HRG and can be even a single variable domain (or half of an Fv comprising isolated by immunoprecipitation and analyzed by SDS only three CDRs specific for an antigen) has the ability to PAGE as described in Sliwkowski et al., J. Biol. Chem., recognize and bind antigen, although at a lower affinity than 269(20):14661-14665 (1994). 45 the entire binding site. "Antibodies" (Abs) and "immunoglobulins" (Igs) are The Fab fragment also contains the constant domain of glycoproteins having the same structural characteristics. the light chain and the first constant domain (CHI) of the While antibodies exhibit binding specificity to a specific heavy chain. Fab' fragments differ from Fab fragments by antigen, immunoglobulins include both antibodies and other the addition of a few residues at the carboxy terminus of the antibody-like molecules which lack antigen specificity. 50 heavy chain CHI domain including one or more cysteines Polypeptides of the latter kind are, for example, produced at from the antibody hinge region. Fab'-SH is the designation low levels by the lymph system and at increased levels by herein for Fab' in which the cysteine residue(s) of the myelomas. constant domains bear a free thiol group. F(ab')2 antibody "Native antibodies" and "native immunoglobulins" are fragments originally were produced as pairs of Fab' frag usually heterotetrameric glycoproteins of about 150,000 55 ments which have hinge cysteines between them. Other daltons, composed of two identical light (L) chains and two chemical couplings of antibody fragments are also known. identical heavy (H) chains. Each light chain is linked to a The "light chains" of antibodies (immunoglobulins) from heavy chain by one covalent disulfide bond, while the any vertebrate species can be assigned to one of two clearly number of disulfide linkages varies among the heavy chains distinct types, called kappa (K) and lambda(!,), based on the of different immunoglobulin isotypes. Each heavy and light 60 amino acid sequences of their constant domains. chain also has regularly spaced intrachain disulfide bridges. Depending on the amino acid sequence of the constant Each heavy chain has at one end a variable domain (VH) domain of their heavy chains, immunoglobulins can be followed by a number of constant domains. Each light chain assigned to different classes. There are five major classes of has a variable domain at one end (VL) and a constant domain immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several at its other end; the constant domain of the light chain is 65 of these may be further divided into subclasses (isotypes), aligned with the first constant domain of the heavy chain, e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy- and the light-chain variable domain is aligned with the chain constant domains that correspond to the different Appx116 GNE-HER_002953494 Case: 19-2156 Document: 19 Page: 188 Filed: 07/26/2019 US 10,160,811 B2 13 14 classes of immunoglobulins are called a, Ii, E, y, and µ, some instances, framework region (FR) residues of the respectively. The subunit structures and three-dimensional human immunoglobulin are replaced by corresponding non configurations of different classes of immunoglobulins are human residues. Furthermore, humanized antibodies may well known. comprise residues which are found neither in the recipient The term "antibody" is used in the broadest sense and 5 antibody nor in the imported CDR or framework sequences. specifically covers intact monoclonal antibodies, polyclonal These modifications are made to further refine and maximize antibodies, multispecific antibodies (e.g. bispecific antibod antibody performance. In general, the humanized antibody ies) formed from at least two intact antibodies, and antibody will comprise substantially all of at least one, and typically fragments so long as they exhibit the desired biological two, variable domains, in which all or substantially all of the activity. 10 CDRs correspond to those of a non-human immunoglobulin "Antibody fragments" comprise a portion of an intact and all or substantially all of the FRs are those of a human antibody, preferably the antigen binding or variable region immunoglobulin sequence. The humanized antibody opti of the intact antibody. Examples of antibody fragments mally also will comprise at least a portion of an immuno- include Fab, Fab', F(ab')2 , and Fv fragments; diabodies; globulin constant region (Fe), typically that of a human linear antibodies (Zapata et al., Protein Eng. 8(10):1057- 15 immunoglobulin. For further details, see Jones et al., Nature, 1062 [1995]); single-chain antibody molecules; and multi 321 :522-525 (1986); Reichmann et al., Nature, 332:323-329 specific antibodies formed from antibody fragments. (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 The term "monoclonal antibody" as used herein refers to (1992). The humanized antibody includes a PRIMA an antibody obtained from a population of substantially TIZED™ antibody wherein the antigen-binding region of homogeneous antibodies, i.e., the individual antibodies 20 the antibody is derived from an antibody produced by comprising the population are identical except for possible immunizing macaque monkeys with the antigen of interest. naturally occurring mutations that may be present in minor "Single-chain Fv" or "sFv" antibody fragments comprise amounts. Monoclonal antibodies are highly specific, being the V Hand V L domains of antibody, wherein these domains directed against a single antigenic site. Furthermore, in are present in a single polypeptide chain. Preferably, the Fv contrast to conventional (polyclonal) antibody preparations 25 polypeptide further comprises a polypeptide linker between which typically include different antibodies directed against the V H and V L domains which enables the sFv to form the different determinants ( epitopes ), each monoclonal antibody desired structure for antigen binding. For a review of sFv see is directed against a single determinant on the antigen. In Pluckthun in The Pharmacology ofMonoclonal Antibodies, addition to their specificity, the monoclonal antibodies are vol. 113, Rosenburg and Moore eds., Springer-Verlag, New advantageous in that they are synthesized by the hybridoma 30 York, pp. 269-315 (1994). culture, uncontaminated by other immunoglobulins. The The term "diabodies" refers to small antibody fragments modifier "monoclonal" indicates the character of the anti with two antigen-binding sites, which fragments comprise a body as being obtained from a substantially homogeneous heavy-chain variable domain (VH) connected to a light population of antibodies, and is not to be construed as chain variable domain (VL) in the same polypeptide chain requiring production of the antibody by any particular 35 (VH-V L). By using a linker that is too short to allow pairing method. For example, the monoclonal antibodies to be used between the two domains on the same chain, the domains are in accordance with the present invention may be made by the forced to pair with the complementary domains of another hybridoma method first described by Kohler et al., Nature, chain and create two antigen-binding sites. Diabodies are 256:495 (1975), or may be made by recombinant DNA described more fully in, for example, EP 404,097; WO methods (see, e.g., U.S. Pat. No. 4,816,567). The "mono 40 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, clonal antibodies" may also be isolated from phage antibody 90:6444-6448 (1993). libraries using the techniques described in Clackson et al., An "isolated" antibody is one which has been identified Nature, 352:624-628 (1991) and Marks et al., J. Mal. Biol., and separated and/or recovered from a component of its 222:581-597 (1991), for example. natural environment. Contaminant components of its natural The monoclonal antibodies herein specifically include 45 environment are materials which would interfere with diag "chimeric" antibodies (immunoglobulins) in which a portion nostic or therapeutic uses for the antibody, and may include of the heavy and/or light chain is identical with or homolo enzymes, hormones, and other proteinaceous or nonpro gous to corresponding sequences in antibodies derived from teinaceous solutes. In preferred embodiments, the antibody a particular species or belonging to a particular antibody will be purified (1) to greater than 95% by weight of class or subclass, while the remainder of the chain(s) is 50 antibody as determined by the Lowry method, and most identical with or homologous to corresponding sequences in preferably more than 99% by weight, (2) to a degree antibodies derived from another species or belonging to sufficient to obtain at least 15 residues of N-terminal or another antibody class or subclass, as well as fragments of internal amino acid sequence by use of a spinning cup such antibodies, so long as they exhibit the desired biologi- sequenator, or (3) to homogeneity by SDS-PAGE under cal activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. 55 reducing or nonreducing conditions using Coomassie blue Natl. Acad. Sci. USA, 81:6851-6855 [1984]). or, preferably, silver stain. Isolated antibody includes the "Humanized" forms of non-human (e.g., murine) anti antibody in situ within recombinant cells since at least one bodies are chimeric immunoglobulins, immunoglobulin component of the antibody's natural environment will not be chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or present. Ordinarily, however, isolated antibody will be pre other antigen-binding subsequences of antibodies) which 60 pared by at least one purification step. contain minimal sequence derived from non-human immu As used herein, the term "salvage receptor binding noglobulin. For the most part, humanized antibodies are epitope" refers to an epitope of the Fe region of an IgG human immunoglobulins (recipient antibody) in which resi molecule (e.g., IgGi, IgG2 , IgG3 , or IgG4 ) that is responsible dues from a complementarity determining region (CDR) of for increasing the in vivo serum half-life of the IgG mol the recipient are replaced by residues from a CDR of a 65 ecule. non-human species (donor antibody) such as mouse, rat or "Treatment" refers to both therapeutic treatment and rabbit having the desired specificity, affinity, and capacity. In prophylactic or preventative measures. Those in need of Appx117 GNE-HER_002953495 Case: 19-2156 Document: 19 Page: 189 Filed: 07/26/2019 US 10,160,811 B2 15 16 treatment include those already with the disorder as well as caminomycin, carzinophilin, chromomycins, dactinomycin, those in which the disorder is to be prevented. daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, "Mammal" for purposes of treatment refers to any animal doxorubicin, epirubicin, esorubicin, idarubicin, marcello classified as a mammal, including humans, domestic and mycin, mitomycins, mycophenolic acid, nogalamycin, farm animals, and zoo, sports, or pet animals, such as dogs, 5 olivomycins, peplomycin, potfiromycin, puromycin, que horses, cats, cows, etc. Preferably, the mammal is human. lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, A "disorder" is any condition that would benefit from ubenimex, zinostatin, zorubicin; anti-metabolites such as treatment with the anti-ErbB2 antibody. This includes methotrexate and 5-fluorouracil (5-FU); folic acid analogues chronic and acute disorders or diseases including those such as denopterin, methotrexate, pteropterin, trimetrexate; pathological conditions which predispose the mammal to the 10 purine analogs such as fludarabine, 6-mercaptopurine, thia disorder in question. Non-limiting examples of disorders to miprine, thioguanine; pyrimidine analogs such as ancit be treated herein include benign and malignant tumors; abine, azacitidine, 6-azauridine, carmofur, cytarabine, dide leukemias and lymphoid malignancies; neuronal, glial, oxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; astrocytal, hypothalamic and other glandular, macrophagal, androgens such as calusterone, dromostanolone propionate, epithelial, stromal and blastocoelic disorders; and inflam 15 epitiostanol, mepitiostane, testolactone; anti-adrenals such matory, angiogenic and immunologic disorders. as aminoglutethimide, mitotane, trilostane; folic acid replen The term "therapeutically effective amount" is used to isher such as frolinic acid; aceglatone; aldophosphamide refer to an amount having antiproliferative effect. Preferably, glycoside; aminolevulinic acid; amsacrine; bestrabucil; the therapeutically effective amount has apoptotic activity, bisantrene; edatraxate; defofamine; demecolcine; diazi- or is capable of inducing cell death, and preferably death of 20 quone; elformithine; elliptinium acetate; etoglucid; gallium benign or malignant tumor cells, in particular cancer cells. nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; Efficacy can be measured in conventional ways, depending mitoxantrone; mopidamol; nitracrine; pentostatin; phe on the condition to be treated. For cancer therapy, efficacy namet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; can, for example, be measured by assessing the time to procarbazine; PSK®; razoxane; sizofiran; spirogermanium; disease progression (TTP), or determining the response rates 25 tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; (RR) (see Example 1, below). Therapeutically effective urethan; vindesine; dacarbazine; mannomustine; mitobroni amount also refers to a target serum concentration, such as tol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara a trough serum concentration, that has been shown to be C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel effective in suppressing disease symptoms when maintained (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, for a period of time. 30 N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, The terms "cancer" and "cancerous" refer to or describe Antony, France); chlorambucil; gemcitabine; 6-thioguanine; the physiological condition in mammals that is typically mercaptopurine; methotrexate; platinum analogs such as characterized by unregulated cell growth. Examples of can cisplatin and carboplatin; vinblastine; platinum; etoposide cer include, but are not limited to, carcinoma, lymphoma, (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincris- blastoma, sarcoma, and leukemia. More particular examples 35 tine; vinorelbine; navelbine; novantrone; teniposide; dauno of such cancers include squamous cell cancer, small-cell mycin; aminopterin; xeloda; ibandronate; CPT-11; topoi lung cancer, non-small cell lung cancer, gastrointestinal somerase inhibitor RFS 2000; difluoromethylornithine cancer, pancreatic cancer, glioblastoma, cervical cancer, (DMFO); retinoic acid; esperamicins; capecitabine; and ovarian cancer, liver cancer, bladder cancer, hepatoma, pharmaceutically acceptable salts, acids or derivatives of breast cancer, colon cancer, colorectal cancer, endometrial 40 any of the above. Also included in this definition are carcinoma, salivary gland carcinoma, kidney cancer, pros anti-hormonal agents that act to regulate or inhibit hormone tate cancer, vulva! cancer, thyroid cancer, hepatic carcinoma action on tumors such as anti-estrogens including for and various types of head and neck cancer. example tamoxifen, raloxifene, aromatase inhibiting 4(5) The term "cytotoxic agent" as used herein refers to a imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, substance that inhibits or prevents the function of cells 45 LYll 7018, onapristone, and toremifene (Fareston); and anti and/or causes destruction of cells. The term is intended to androgens such as flutamide, nilutamide, bicalutamide, leu 131 125 90 186 include radioactive isotopes (e.g. I , I , Y and Re ), prolide, and goserelin; and pharmaceutically acceptable chemotherapeutic agents, and toxins such as enzymatically salts, acids or derivatives of any of the above. active toxins of bacterial, fungal, plant or animal origin, or A "growth inhibitory agent" when used herein refers to a fragments thereof. 50 compound or composition which inhibits growth of a cell, A "chemotherapeutic agent" is a chemical compound especially an ErbB2-overexpressing cancer cell either in useful in the treatment of cancer. Examples of chemothera vitro or in vivo. Thus, the growth inhibitory agent is one peutic agents include alkylating agents such as thiotepa and which significantly reduces the percentage of ErbB2 over cyclosphosphamide (CYTOXAWM); alkyl sulfonates such expressing cells in S phase. Examples of growth inhibitory as busulfan, improsulfan and piposulfan; aziridines such as 55 agents include agents that block cell cycle progression (at a benzodopa, carboquone, meturedopa, and uredopa; ethylen place other than S phase), such as agents that induce G 1 imines and methylamelamines including altretamine, trieth arrest and M-phase arrest. Classical M-phase blockers ylenemelamine, trietylenephosphoramide, triethylenethio include the vincas (vincristine and vinblastine), TAXOL®, phosphaoramide and trimethylolomelamine; nitrogen and topo II inhibitors such as doxorubicin, epirubicin, mustards such as chlorambucil, chlornaphazine, cholophos 60 daunorubicin, etoposide, and bleomycin. Those agents that phamide, estramustine, ifosfamide, mechlorethamine, arrest Gl also spill over into S-phase arrest, for example, mechlorethamine oxide hydrochloride, melphalan, novem DNA alkylating agents such as tamoxifen, prednisone, dac bichin, phenesterine, prednimustine, trofosfamide, uracil arbazine, mechlorethamine, cisplatin, methotrexate, 5-fluo mustard; nitrosureas such as carmustine, chlorozotocin, rouracil, and ara-C. Further information can be found in The fotemustine, lomustine, nimustine, ranimustine; antibiotics 65 Molecular Basis of Cancer, Mendelsohn and Israel, eds., such as aclacinomysins, actinomycin, authramycin, azaser Chapter 1, entitled "Cell cycle regulation, oncogenes, and ine, bleomycins, cactinomycin, calicheamicin, carabicin, antineoplastic drugs" by Murakami et al. (WB Saunders: Appx118 GNE-HER_002953496 Case: 19-2156 Document: 19 Page: 190 Filed: 07/26/2019 US 10,160,811 B2 17 18 Philadelphia, 1995), especially p. 13. The 4D5 antibody (and phase can comprise the well of an assay plate; in others it is functional equivalents thereof) can also be employed for this a purification column (e.g., an affinity chromatography col purpose. unm). This term also includes a discontinuous solid phase of "Doxorubicin" is an athracycline antibiotic. The full discrete particles, such as those described in U.S. Pat. No. chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2, 5 4,275,149. 3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]- 7,8,9, 1O-tetra A "liposome" is a small vesicle composed of various hydro-6,8, 11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5, types of lipids, phospholipids and/or surfactant which is 12-naphthacenedione. useful for delivery of a drug (such as the anti-ErbB2 The term "cytokine" is a generic term for proteins antibodies disclosed herein and, optionally, a chemothera released by one cell population which act on another cell as 10 peutic agent) to a mammal. The components of the liposome intercellular mediators. Examples of such cytokines are are commonly arranged in a bilayer formation, similar to the lymphokines, monokines, and traditional polypeptide hor lipid arrangement of biological membranes. mones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic growth hormone, and bovine growth hormone; parathyroid 15 hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; products, that contain information about the indications, glycoprotein hormones such as follicle stimulating hormone usage, dosage, administration, contraindications and/or (FSH), thyroid stimulating hormone (TSH), and luteinizing warnings concerning the use of such therapeutic products. hormone (LH); hepatic growth factor; fibroblast growth The term "serum concentration," "serum drug concentra- factor; prolactin; placental lactogen; tumor necrosis factor-a 20 tion," or "serum HERCEPTIN® anti-ErbB2 antibody con and -~; mullerian-inhibiting substance; mouse gonadotro centration" refers to the concentration of a drug, such as pin-associated peptide; inhibin; activin; vascular endothelial HERCEPTIN® anti-ErbB2 antibody, in the blood serum of growth factor; integrin; thrombopoietin (TPO); nerve an animal or human patient being treated with the drug. growth factors such as NGF-~; platelet-growth factor; trans Serum concentration of HERCEPTIN® anti-ErbB2 anti- forming growth factors (TGFs) such as TGF-a and TGF-~; 25 body, for example, is preferably determined by immunoas insulin-like growth factor-I and -II; erythropoietin (EPO); say. Preferably, the immunoassay is an ELISA according to osteoinductive factors; interferons such as interferon-a, -~, the procedure disclosed herein. and -y; colony stimulating factors (CSFs) such as macro The term "peak serum concentration" refers to the maxi phage-CSP (M-CSF); granulocyte-macrophage-CSF (GM mal serum drug concentration shortly after delivery of the CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) 30 drug into the animal or human patient, after the drug has such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, been distributed throughout the blood system, but before IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-a significant tissue distribution, metabolism or excretion of or TNF-~; and other polypeptide factors including LIF and drug by the body has occurred. kit ligand (KL). As used herein, the term cytokine includes The term "trough serum concentration" refers to the proteins from natural sources or from recombinant cell 35 serum drug concentration at a time after delivery of a culture and biologically active equivalents of the native previous dose and immediately prior to delivery of the next sequence cytokines. subsequent dose of drug in a series of doses. Generally, the The term "prodrug" as used in this application refers to a trough serum concentration is a minimum sustained effica precursor or derivative form of a pharmaceutically active cious drug concentration in the series of drug administra- substance that is less cytotoxic to tumor cells compared to 40 tions. Also, the trough serum concentration is frequently the parent drug and is capable of being enzymatically targeted as a minimum serum concentration for efficacy activated or converted into the more active parent form. See, because it represents the serum concentration at which e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Bio another dose of drug is to be administered as part of the chemical Society Transactions, 14, pp. 375-382, 615th treatment regimen. If the delivery of drug is by intravenous Meeting Belfast (1986) and Stella et al., "Prodrugs: A 45 administration, the trough serum concentration is most pref Chemical Approach to Targeted Drug Delivery," Directed erably attained within 1 day of a front loading initial drug Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana delivery. If the delivery of drug is by subcutaneous admin Press (1985). The prodrugs of this invention include, but are istration, the peak serum concentration is preferably attained not limited to, phosphate-containing prodrugs, thiophos in 3 days or less. According to the invention, the trough phate-containing prodrugs, sulfate-containing prodrugs, 50 serum concentration is preferably attained in 4 weeks or less, peptide-containing prodrugs, D-amino acid-modified prod preferably 3 weeks or less, more preferably 2 weeks or less, rugs, glycosylated prodrugs, ~-lactam-containing prodrugs, most preferably in 1 week or less, including 1 day or less optionally substituted phenoxyacetamide-containing prod using any of the drug delivery methods disclosed herein. rugs or optionally substituted phenylacetamide-containing The term "intravenous infusion" refers to introduction of prodrugs, 5-fluorocytosine and other 5-fluorouridine prod- 55 a drug into the vein of an animal or human patient over a rugs which can be converted into the more active cytotoxic period of time greater than approximately 5 minutes, pref free drug. Examples of cytotoxic drugs that can be deriva erably between approximately 30 to 90 minutes, although, tized into a prodrug form for use in this invention include, according to the invention, intravenous infusion is alterna but are not limited to, those chemotherapeutic agents tively administered for 10 hours or less. described above: 60 The term "intravenous bolus" or "intravenous push" By "solid phase" is meant a non-aqueous matrix to which refers to drug administration into a vein of an animal or the antibodies used in accordance with the present invention human such that the body receives the drug in approximately can adhere. Examples of solid phases encompassed herein 15 minutes or less, preferably 5 minutes or less. include those formed partially or entirely of glass (e.g., The term "subcutaneous administration" refers to intro- controlled pore glass), polysaccharides (e.g., agarose), poly 65 duction of a drug under the skin of an animal or human acrylamides, polystyrene, polyvinyl alcohol and silicones. In patient, preferable within a pocket between the skin and certain embodiments, depending on the context, the solid underlying tissue, by relatively slow, sustained delivery Appx119 GNE-HER_002953497 Case: 19-2156 Document: 19 Page: 191 Filed: 07/26/2019 US 10,160,811 B2 19 20 from a drug receptacle. The pocket may be created by present invention. The ErbB2 antigen to be used for pro pinching or drawing the skin up and away from underlying duction of antibodies may be, e.g., a soluble form of the tissue. extracellular domain ofErbB2 or a portion thereof, contain The term "subcutaneous infusion" refers to introduction ing the desired epitope. Alternatively, cells expressing of a drug under the skin of an animal or human patient, 5 ErbB2 at their cell surface (e.g. NIH-3T3 cells transformed preferably within a pocket between the skin and underlying to overexpress ErbB2; or a carcinoma cell line such as tissue, by relatively slow, sustained delivery from a drug SKBR3 cells, see Stancovski et al., PNAS (USA) 88:8691- receptacle for a period of time including, but not limited to, 8695 [1991]) can be used to generate antibodies. Other 30 minutes or less, or 90 minutes or less. Optionally, the forms of ErbB2 useful for generating antibodies will be infusion may be made by subcutaneous implantation of a 10 apparent to those skilled in the art. drug delivery pump implanted under the skin of the animal (i) Polyclonal Antibodies or human patient, wherein the pump delivers a predeter mined amount of drug for a predetermined period of time, Polyclonal antibodies are preferably raised in animals by such as 30 minutes, 90 minutes, or a time period spanning multiple subcutaneous (sc) or intraperitoneal (ip) injections the length of the treatment regimen. of the relevant antigen and an adjuvant. It may be useful to The term "subcutaneous bolus" refers to drug adminis 15 conjugate the relevant antigen to a protein that is immuno tration beneath the skin of an animal or human patient, genic in the species to be immunized, e.g., keyhole limpet where bolus drug delivery is preferably less than approxi hemocyanin, serum albumin, bovine thyroglobulin, or soy mately 15 minutes, more preferably less than 5 minutes, and bean trypsin inhibitor using a bifunctional or derivatizing most preferably less than 60 seconds. Administration is agent, for example, maleimidobenzoyl sulfosuccinimide preferably within a pocket between the skin and underlying 20 ester (conjugation through cysteine residues), N-hydroxy tissue, where the pocket is created, for example, by pinching succinimide (through lysine residues), glutaraldehyde, suc 1 1 or drawing the skin up and away from underlying tissue. cinic anhydride, SOC1 2 , or R N=C=NR, where R and R The term "front loading" when referring to drug admin are different alkyl groups. istration is meant to describe an initially higher dose fol Animals are immunized against the antigen, immuno- lowed by the same or lower doses at intervals. The initial 25 genie conjugates, or derivatives by combining, e.g., 100 µg higher dose or doses are meant to more rapidly increase the or 5 µg of the protein or conjugate (for rabbits or mice, animal or human patient's serum drug concentration to an respectively) with 3 volumes ofFreund's complete adjuvant efficacious target serum concentration. According to the and injecting the solution intradermally at multiple sites. present invention, front loading is achieved by an initial dose One month later the animals are boosted with 1/s to 1/io the or doses delivered over three weeks or less that causes the 30 original amount of peptide or conjugate in Freund's com animal's or patient's serum concentration to reach a target serum trough concentration. Preferably, the initial front plete adjuvant by subcutaneous injection at multiple sites. loading dose or series of doses is administered in two weeks Seven to 14 days later the animals are bled and the serum is or less, more preferably in 1 week or less, including 1 day assayed for antibody titer. Animals are boosted until the titer or less. Most preferably, where the initial dose is a single plateaus. Preferably, the animal is boosted with the conju- dose and is not followed by a subsequent maintenance dose 35 gate of the same antigen, but conjugated to a different for at least 1 week, the initial dose is administered in 1 day protein and/or through a different cross-linking reagent. or less. Where the initial dose is a series of doses, each dose Conjugates also can be made in recombinant cell culture as is separated by at least 3 hours, but not more than 3 weeks protein fusions. Also, aggregating agents such as alum are or less, preferably 2 weeks or less, more preferably 1 week suitably used to enhance the immune response. or less, most preferably 1 day or less. To avoid adverse 40 (ii) Monoclonal Antibodies immune reaction to an antibody drug such as an anti-ErbB2 Monoclonal antibodies are obtained from a population of antibody (e.g., HERCEPTIN® anti-ErbB2 antibody) in an substantially homogeneous antibodies, i.e., the individual animal or patient who has not previously been treated with antibodies comprising the population are identical except for the antibody, it may be preferable to deliver initial doses of possible naturally occurring mutations that may be present the antibody by intravenous infusion. The present invention 45 in minor amounts. Thus, the modifier "monoclonal" indi includes front loading drug delivery of initial and mainte cates the character of the antibody as not being a mixture of nance doses by infusion or bolus administration, intrave discrete antibodies. nously or subcutaneously. For example, the monoclonal antibodies may be made Published information related to anti-ErbB2 antibodies using the hybridoma method first described by Kohler et al., includes the following issued patents and published appli cations: PCT/US89/00051, published Jan. 5, 1989; PCT/ 50 Nature, 256:495 (1975), or may be made by recombinant US90/02697, published May 18, 1990; EU 0474727 issued DNA methods (U.S. Pat. No. 4,816,567). Jul. 23, 1997; DE 69031120.6, issued Jul. 23, 1997; PCT/ In the hybridoma method, a mouse or other appropriate US97/18385, published Oct. 9, 1997; SA 97/9185, issued host animal, such as a hamster, is immunized as hereinabove Oct. 14, 1997; U.S. Pat. No. 5,677,171, issued Oct. 14, 1997; described to elicit lymphocytes that produce or are capable U.S. Pat. No. 5,720,937, issued Feb. 24, 1998; U.S. Pat. No. 55 of producing antibodies that will specifically bind to the 5,720,954, issued Feb. 24, 1998; U.S. Pat. No. 5,725,856, protein used for immunization. Alternatively, lymphocytes issued Mar. 10, 1998; U.S. Pat. No. 5,770,195, issued Jun. may be immunized in vitro. Lymphocytes then are fused 23, 1998; U.S. Pat. No. 5,772,997, issued Jun. 30, 1998; with myeloma cells using a suitable fusing agent, such as PCT/US98/2626, published Dec. 10, 1998; and PCT/US99/ polyethylene glycol, to form a hybridoma cell (Goding, 06673, published Mar. 26, 1999, each of which patents and 60 Monoclonal Antibodies: Principles and Practice, pp. 59-103 publications is herein incorporated by reference in its [Academic Press, 1986]). entirety. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or II. Production of Anti-ErbB2 Antibodies more substances that inhibit the growth or survival of the 65 unfused, parental myeloma cells. For example, if the paren A description follows as to exemplary techniques for the tal myeloma cells lack the enzyme hypoxanthine guanine production of the antibodies used in accordance with the phosphoribosyl transferase (HGPRT or HPRT), the culture Appx120 GNE-HER_002953498 Case: 19-2156 Document: 19 Page: 192 Filed: 07/26/2019 US 10,160,811 B2 21 22 medium for the hybridomas typically will include hypox Technology, 10:779-783 [1992]), as well as combinatorial anthine, aminopterin, and thymidine (HAT medium), which infection and in vivo recombination as a strategy for con substances prevent the growth of HGPRT-deficient cells. structing very large phage libraries (Waterhouse et al., Nuc. Preferred myeloma cells are those that fuse efficiently, Acids. Res., 21:2265-2266 [1993]). Thus, these techniques support stable high-level production of antibody by the 5 are viable alternatives to traditional monoclonal antibody selected antibody-producing cells, and are sensitive to a hybridoma techniques for isolation of monoclonal antibod- medium such as HAT medium. Among these, preferred ies. myeloma cell lines are murine myeloma lines, such as those The DNA also may be modified, for example, by substi derived from MOPC-21 and MPC-11 mouse tumors avail tuting the coding sequence for human heavy- and light-chain able from the Salk Institute Cell Distribution Center, San 10 constant domains in place of the homologous murine Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells avail sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. able from the American Type Culture Collection, Rockville, Natl. Acad. Sci. USA, 81:6851 [1984]), or by covalently Md. USA. Human myeloma and mouse-human heteromy joining to the immunoglobulin coding sequence all or part of eloma cell lines also have been described for the production the coding sequence for a non-immunoglobulin polypeptide. of human monoclonal antibodies (Kozbor, J. Immunol., 15 Typically such non-immunoglobulin polypeptides are 133:3001 (1984); Brodeur et al., Monoclonal Antibody Pro substituted for the constant domains of an antibody, or they duction Techniques and Applications, pp. 51-63 [Marcel are substituted for the variable domains of one antigen Dekker, Inc., New York, 1987]). combining site of an antibody to create a chimeric bivalent Culture medium in which hybridoma cells are growing is antibody comprising one antigen-combining site having assayed for production of monoclonal antibodies directed 20 specificity for an antigen and another antigen-combining site against the antigen. Preferably, the binding specificity of having specificity for a different antigen. monoclonal antibodies produced by hybridoma cells is (iii) Humanized and Human Antibodies determined by immunoprecipitation or by an in vitro binding Methods for humanizing non-human antibodies are well assay, such as radioimmunoassay (RIA) or enzyme-linked known in the art. Preferably, a humanized antibody has one immunoabsorbent assay (ELISA). 25 or more amino acid residues introduced into it from a source The binding affinity of the monoclonal antibody can, for which is non-human. These non-human amino acid residues example, be determined by the Scatchard analysis of Mun are often referred to as "import" residues, which are typi son et al., Anal. Biochem., 107:220 (1980). cally taken from an "import" variable domain. Humaniza After hybridoma cells are identified that produce antibod- tion can be essentially performed following the method of ies of the desired specificity, affinity, and/or activity, the 30 Winter and co-workers (Jones et al., Nature, 321 :522-525 clones may be subcloned by limiting dilution procedures and (1986); Riechmann et al., Nature, 332:323-327 (1988); grown by standard methods (Goding, Monoclonal Antibod Verhoeyen et al., Science, 239:1534-1536 [1988]), by sub ies: Principles and Practice, pp. 59-103 [Academic Press, stituting rodent CDRs or CDR sequences for the correspond 1986]). Suitable culture media for this purpose include, for ing sequences of a human antibody. Accordingly, such example, D-MEM or RPMI-1640 medium. In addition, the 35 "humanized" antibodies are chimeric antibodies (U.S. Pat. hybridoma cells may be grown in vivo as ascites tumors in No. 4,816,567) wherein substantially less than an intact an animal. human variable domain has been substituted by the corre The monoclonal antibodies secreted by the subclones are sponding sequence from a non-human species. In practice, suitably separated from the culture medium, ascites fluid, or humanized antibodies are typically human antibodies in serum by conventional immunoglobulin purification proce- 40 which some CDR residues and possibly some FR residues US 10,160,811 B2 23 24 programs are available which illustrate and display probable laid, ricin A chain, methotrexate or radioactive isotope three-dimensional conformational structures of selected can hapten). Bispecific antibodies can be prepared as full length didate immunoglobulin sequences. Inspection of these dis antibodies or antibody fragments (e.g. F(ab')2 bispecific plays permits analysis of the likely role of the residues in the antibodies). functioning of the candidate immunoglobulin sequence, i.e., 5 Methods for making bispecific antibodies are known in the analysis of residues that influence the ability of the the art. Traditional production of full length bispecific anti candidate immunoglobulin to bind its antigen. In this way, bodies is based on the coexpression of two immunoglobulin FR residues can be selected and combined from the recipient heavy chain-light chain pairs, where the two chains have and import sequences so that the desired antibody charac different specificities (Millstein et al., Nature, 305:537-539 teristic, such as increased affinity for the target antigen(s), is 10 [1983]). Because of the random assortment of immuno achieved. In general, the CDR residues are directly and most globulin heavy and light chains, these hybridomas (quadro- substantially involved in influencing antigen binding. mas) produce a potential mixture of 10 different antibody Alternatively, it is now possible to produce transgenic molecules, of which only one has the correct bispecific animals (e.g., mice) that are capable, upon immunization, of structure. Purification of the correct molecule, which is producing a full repertoire of human antibodies in the 15 usually done by affinity chromatography steps, is rather absence of endogenous immunoglobulin production. For cumbersome, and the product yields are low. Similar pro- example, it has been described that the homozygous deletion cedures are disclosed in WO 93/08829, and in Traunecker et of the antibody heavy-chain joining region (JH) gene in al., EMBO J., 10:3655-3659 (1991). chimeric and germ-line mutant mice results in complete According to a different approach, antibody variable inhibition of endogenous antibody production. Transfer of 20 domains with the desired binding specificities (antibody the human germ-line immunoglobulin gene array in such antigen combining sites) are fused to immunoglobulin con germ-line mutant mice will result in the production of stant domain sequences. The fusion preferably is with an human antibodies upon antigen challenge. See, e.g., Jako immunoglobulin heavy chain constant domain, comprising bovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); at least part of the hinge, CH2, and CH3 regions. It is Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann 25 preferred to have the first heavy-chain constant region et al., Year in Immuno., 7:33 (1993). Human antibodies can (CHI) containing the site necessary for light chain binding, also be derived from phage-display libraries (Hoogenboom present in at least one of the fusions. DNAs encoding the et al., J. Mal. Biol., 227:381 (1991); Marks et al., J. Mal. immunoglobulin heavy chain fusions and, if desired, the Biol., 222:581-597 [1991]). immunoglobulin light chain, are inserted into separate (iv) Antibody Fragments 30 expression vectors, and are co-transfected into a suitable Various techniques have been developed for the produc host organism. This provides for great flexibility in adjusting tion of antibody fragments. Traditionally, these fragments the mutual proportions of the three polypeptide fragments in were derived via proteolytic digestion of intact antibodies embodiments when unequal ratios of the three polypeptide (see, e.g., Morimoto et al., Journal of Biochemical and chains used in the construction provide the optimum yields. Biophysical Methods 24: 107-117 (1992) and Brennan et al., 35 It is, however, possible to insert the coding sequences for Science, 229:81 [1985]). However, these fragments can now two or all three polypeptide chains in one expression vector be produced directly by recombinant host cells. For when the expression of at least two polypeptide chains in example, the antibody fragments can be isolated from the equal ratios results in high yields or when the ratios are of antibody phage libraries discussed above. Alternatively, no particular significance. Fab'-SH fragments can be directly recovered from E. coli 40 In a preferred embodiment of this approach, the bispecific and chemically coupled to form F(ab')2 fragments (Carter et antibodies are composed of a hybrid immunoglobulin heavy al., Bio/Technology 10:163-167 [1992]). According to chain with a first binding specificity in one arm, and a hybrid another approach, F(ab')2 fragments can be isolated directly immunoglobulin heavy chain-light chain pair (providing a from recombinant host cell culture. Other techniques for the second binding specificity) in the other arm. It was found production of antibody fragments will be apparent to the 45 that this asymmetric structure facilitates the separation of the skilled practitioner. In other embodiments, the antibody of desired bispecific compound from unwanted immunoglobu- choice is a single chain Fv fragment (scFv). See WO lin chain combinations, as the presence of an immunoglobu 93/16185. lin light chain in only one half of the bispecific molecule (v) Bispecific Antibodies provides for a facile way of separation. This approach is Bispecific antibodies are antibodies that have binding 50 disclosed in WO 94/04690. For further details of generating specificities for at least two different epitopes. Exemplary bispecific antibodies see, for example, Suresh et al., Methods bispecific antibodies may bind to two different epitopes of in Enzymology, 121:210 (1986). the ErbB2 protein. For example, one arm may bind an According to another approach described in W096/ epitope in Domain 1 ofErbB2 such as the 7C2/7F3 epitope, 27011, the interface between a pair of antibody molecules the other may bind a different ErbB2 epitope, e.g. the 4D5 55 can be engineered to maximize the percentage of heterodi epitope. Other such antibodies may combine an ErbB2 mers which are recovered from recombinant cell culture. binding site with binding site(s) for EGFR, ErbB3 and/or The preferred interface comprises at least a part of the CH3 ErbB4. Alternatively, an anti-ErbB2 arm may be combined domain of an antibody constant domain. In this method, one with an arm which binds to a triggering molecule on a or more small amino acid side chains from the interface of leukocyte such as a T-cell receptor molecule (e.g. CD2 or 60 the first antibody molecule are replaced with larger side CD3), or Fe receptors for IgG (FcyR), such as FcyRI chains (e.g. tyrosine or tryptophan). Compensatory "cavi (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus ties" of identical or similar size to the large side chain(s) are cellular defense mechanisms to the ErbB2-expressing cell. created on the interface of the second antibody molecule by Bispecific antibodies may also be used to localize cytotoxic replacing large amino acid side chains with smaller ones agents to cells which express ErbB2. These antibodies 65 ( e.g. alanine or threonine). This provides a mechanism for possess an ErbB2-binding arm and an arm which binds the increasing the yield of the heterodimer over other unwanted cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alka- end-products such as homodimers. Appx122 GNE-HER_002953500 Case: 19-2156 Document: 19 Page: 194 Filed: 07/26/2019 US 10,160,811 B2 25 26 Bispecific antibodies include cross-linked or "heterocon To select for antibodies which induce cell death, loss of jugate" antibodies. For example, one of the antibodies in the membrane integrity as indicated by, e.g., PI, trypan blue or heteroconjugate can be coupled to avidin, the other to biotin. 7 AAD uptake is assessed relative to control. The preferred Such antibodies have, for example, been proposed to target assay is the "PI uptake assay using BT474 cells". According immune system cells to unwanted cells (U.S. Pat. No. 5 to this assay, BT474 cells (which can be obtained from the 4,676,980), and for treatment of HIV infection (WO American Type Culture Collection [Rockville, Md.]) are 91/00360, WO 92/200373, and EP 03089). Heteroconjugate cultured in Dulbecco's Modified Eagle Medium (D-MEM): antibodies may be made using any convenient cross-linking Ham's F-12 (50:50) supplemented with 10% heat-inacti methods. Suitable cross-linking agents are well known in the vated FBS (Hyclone) and 2 mM L-glutamine. (Thus, the art, and are disclosed in U.S. Pat. No. 4,676,980, along with 10 assay is performed in the absence of complement and a number of cross-linking techniques. immune effector cells). The BT474 cells are seeded at a Techniques for generating bispecific antibodies from anti density of3xl06 per dish in 100x20 mm dishes and allowed body fragments have also been described in the literature. to attach overnight. The medium is then removed and For example, bispecific antibodies can be prepared using replaced with fresh medium alone or medium containing 10 chemical linkage. Brennan et al., Science, 229: 81 (1985) 15 µg/ml of the appropriate MAb. The cells are incubated for a describe a procedure wherein intact antibodies are pro 3 day time period. Following each treatment, monolayers are teolytically cleaved to generate F(ab')2 fragments. These washed with PBS and detached by trypsinization. Cells are fragments are reduced in the presence of the dithiol com then centrifuged at 1200 rpm for 5 minutes at 4° C., the 2 plexing agent sodium arsenite to stabilize vicinal dithiols pellet resuspended in 3 ml ice cold Ca + binding buffer (10 and prevent intermolecular disulfide formation. The Fab' 20 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaC12 ) and fragments generated are then converted to thionitrobenzoate aliquoted into 35 mm strainer-capped 12x75 tubes (1 ml per (TNB) derivatives. One of the Fab'-TNB derivatives is then tube, 3 tubes per treatment group) for removal of cell reconverted to the Fab'-thiol by reduction with mercapto clumps. Tubes then receive PI (10 µg/ml). Samples may be ethylamine and is mixed with an equimolar amount of the analyzed using a FACSCAWM flow cytometer and FAC- other Fab'-TNB derivative to form the bispecific antibody. 25 SCONVERT™ CellQuest software (Becton Dickinson). The bispecific antibodies produced can be used as agents for Those antibodies which induce statistically significant levels the selective immobilization of enzymes. of cell death as determined by PI uptake are selected. Recent progress has facilitated the direct recovery of In order to select for antibodies which induce apoptosis, Fab'-SH fragments from E. coli, which can be chemically an "annexin binding assay using BT474 cells" is available. coupled to form bispecific antibodies. Shalaby et al., J. Exp. 30 The BT474 cells are cultured and seeded in dishes as Med., 175: 217-225 (1992) describe the production of a fully discussed in the preceding paragraph. The medium is then humanized bispecific antibody F(ab')2 molecule. Each Fab' removed and replaced with fresh medium alone or medium fragment was separately secreted from E. coli and subjected containing 10 µg/ml of the MAb. Following a three day to directed chemical coupling in vitro to form the bispecific incubation period, monolayers are washed with PBS and antibody. The bispecific antibody thus formed was able to 35 detached by trypsinization. Cells are then centrifuged, resus bind to cells overexpressing the ErbB2 receptor and normal 2 pended in Ca + binding buffer and aliquoted into tubes as human T cells, as well as trigger the lytic activity of human discussed above for the cell death assay. Tubes then receive cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific labeled annexin (e.g. annexin V-FTIC) (1 µg/ml). Samples antibody fragments directly from recombinant cell culture may be analyzed using a FACSCAWM flow cytometer and have also been described. For example, bispecific antibodies 40 FACSCONVERT™ CellQuest software (Becton Dickin have been produced using leucine zippers. Kostelny et al., J. son). Those antibodies which induce statistically significant Immunol., 148(5):1547-1553 (1992). The leucine zipper levels of annexin binding relative to control are selected as peptides from the Fos and Jun proteins were linked to the apoptosis-inducing antibodies. Fab' portions of two different antibodies by gene fusion. The In addition to the annexin binding assay, a "DNA staining antibody homodimers were reduced at the hinge region to 45 assay using BT474 cells" is available. In order to perform form monomers and then re-oxidized to form the antibody this assay, BT474 cells which have been treated with the heterodimers. This method can also be utilized for the antibody of interest as described in the preceding two production of antibody homodimers. The "diabody" tech paragraphs are incubated with 9 µg/ml HOECHST 33342™ nology described by Hollinger et al., Proc. Natl. Acad. Sci. for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flow USA, 90:6444-6448 (1993) has provided an alternative 50 cytometer (Coulter Corporation) using MODFIT LT™ soft mechanism for making bispecific antibody fragments. The ware (Verity Software House). Antibodies which induce a fragments comprise a heavy-chain variable domain (VH) change in the percentage of apoptotic cells which is 2 fold connected to a light-chain variable domain (VL) by a linker or greater (and preferably 3 fold or greater) than untreated which is too short to allow pairing between the two domains cells (up to 100% apoptotic cells) may be selected as on the same chain. Accordingly, the V H and V L domains of 55 pro-apoptotic antibodies using this assay. one fragment are forced to pair with the complementary V L To screen for antibodies which bind to an epitope on and V H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific ErbB2 bound by an antibody of interest, a routine cross antibody fragments by the use of single-chain Fv (sFv) blocking assay such as that described in Antibodies, A dimers has also been reported. See Gruber et al., J. Immu Laboratory Manual, Cold Spring Harbor Laboratory, Ed nol., 152:5368 (1994). 60 Harlow and David Lane (1988), can be performed. Alter Antibodies with more than two valencies are contem natively, epitope mapping can be performed by methods plated. For example, trispecific antibodies can be prepared. known in the art. Tutt et al., J. Immunol. 147: 60 (1991). To identify anti-ErbB2 antibodies which inhibit growth of (vi) Screening for Antibodies with the Desired Properties SKBR3 cells in cell culture by 50-100%, the SKBR3 assay Techniques for generating antibodies have been described 65 described in WO 89/06692 can be performed. According to above. Those antibodies having the characteristics described this assay, SKBR3 cells are grown in a 1:1 mixture ofF12 herein are selected. and DMEM medium supplemented with 10% fetal bovine Appx123 GNE-HER_002953501 Case: 19-2156 Document: 19 Page: 195 Filed: 07/26/2019 US 10,160,811 B2 27 28 serum, glutamine and penicillinstreptomycin. The SKBR3 pretargeting wherein the antibody-receptor conjugate is cells are plated at 20,000 cells in a 35 mm cell culture dish administered to the patient, followed by removal of unbound (2 mls/35 mm dish). 2.5 µg/ml of the anti-ErbB2 antibody is conjugate from the circulation using a clearing agent and added per dish. After six days, the number of cells, com then administration of a "ligand" (e.g. avidin) which 1s pared to untreated cells are counted using an electronic 5 conjugated to a cytotoxic agent (e.g. a radionucleotide). COULTER™ cell counter. Those antibodies which inhibit (ix) Immunoliposomes growth of the SKBR3 cells by 50-100% are selected for The anti-ErbB2 antibodies disclosed herein may also be combination with the apoptotic antibodies as desired. formulated as immunoliposomes. Liposomes containing the (vii) Effector Function Engineering antibody are prepared by methods known in the art, such as It may be desirable to modify the antibody of the inven 10 described in Epstein et al., Proc. Natl. Acad. Sci. USA, tion with respect to effector function, so as to enhance the 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, effectiveness of the antibody in treating cancer, for example. 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544, For example, cysteine residue(s) may be introduced in the Fe 545. Liposomes with enhanced circulation time are dis- region, thereby allowing interchain disulfide bond formation closed in U.S. Pat. No. 5,013,556. in this region. The homodimeric antibody thus generated 15 Particularly useful liposomes can be generated by the may have improved internalization capability and/or reverse phase evaporation method with a lipid composition increased complement-mediated cell killing and antibody comprising phosphatidylcholine, cholesterol and PEG-de dependent cellular cytotoxicity (ADCC). See Caron et al., J. rivatized phosphatidylethanolamine (PEG-PE). Liposomes Exp Med. 176: 1191-1195 (1992) and Shapes, B. J. Immunol. are extruded through filters of defined pore size to yield 148:2918-2922 (1992). Homodimeric antibodies with 20 liposomes with the desired diameter. Fab' fragments of the enhanced anti-tumor activity may also be prepared using antibody of the present invention can be conjugated to the heterobifunctional cross-linkers as described in Wolff et al. liposomes as described in Martin et al., J. Biol. Chem. 257: Cancer Research 53:2560-2565 (1993). Alternatively, an 286-288 (1982) via a disulfide interchange reaction. A antibody can be engineered which has dual Fe regions and chemotherapeutic agent is optionally contained within the may thereby have enhanced complement lysis and ADCC 25 liposome. See Gabizon et al., J. National Cancer Inst. capabilities. See Stevenson et al. Anti-Cancer Drug Design 81(19)1484 (1989). 3:219-230 (1989). (x) Antibody Dependent Enzyme Mediated Prodrug (viii) Immunoconjugates Therapy (ADEPT) The invention also pertains to immunoconjugates com The antibodies of the present invention may also be used prising the antibody described herein conjugated to a cyto 30 in ADEPT by conjugating the antibody to a prodrug-acti toxic agent such as a chemotherapeutic agent, toxin (e.g. an vating enzyme which converts a prodrug (e.g. a peptidyl enzymatically active toxin of bacterial, fungal, plant or chemotherapeutic agent, see WO 81/01145) to an active animal origin, or fragments thereof), or a radioactive isotope anti-cancer drug. See, for example, WO 88/07378 and U.S. (i.e., a radioconjugate ). Pat. No. 4,975,278. Chemotherapeutic agents useful in the generation of such 35 The enzyme component of the immunoconjugate useful immunoconjugates have been described above. Enzymati for ADEPT includes any enzyme capable of acting on a cally active toxins and fragments thereof which can be used prodrug in such a way so as to covert it into its more active, include diphtheria A chain, nonbinding active fragments of cytotoxic form. diphtheria toxin, exotoxin A chain (from Pseudomonas Enzymes that are useful in the method of this invention aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, 40 include, but are not limited to, alkaline phosphatase useful alpha-sarcin, Aleurites fordii proteins, dianthin proteins, for converting phosphate-containing prodrugs into free Phytolaca americana proteins (PAPI, PAPII, and PAP-S), drugs; arylsulfatase useful for converting sulfate-containing momordica charantia inhibitor, curcin, crotin, sapaonaria prodrugs into free drugs; cytosine deaminase useful for officinalis inhibitor, gelonin, mitogellin, restrictocin, pheno converting non-toxic 5-fluorocytosine into the anti-cancer mycin, enomycin and the tricothecenes. A variety of radio 45 drug, 5-fluorouracil; proteases, such as serratia protease, nuclides are available for the production of radioconjugated thermolysin, subtilisin, carboxypeptidases and cathepsins anti-ErbB2 antibodies. Examples include 212Bi, 131I, 131In, (such as cathepsins B and L), that are useful for converting 9oy and is6Re. peptide-containing prodrugs into free drugs; D-alanylcar Conjugates of the antibody and cytotoxic agent are made boxypeptidases, useful for converting prodrugs that contain using a variety of bifunctional protein coupling agents such 50 D-amino acid substituents; carbohydrate-cleaving enzymes as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), such as ~-galactosidase and neuraminidase useful for con iminothiolane (IT), bifunctional derivatives of imidoesters verting glycosylated prodrugs into free drugs; ~-lactamase (such as dimethyl adipimidate HCL), active esters (such as useful for converting drugs derivatized with ~-lactams into disuccinimidyl suberate), aldehydes (such as glutarelde free drugs; and penicillin amidases, such as penicillin V hyde ), bis-azido compounds (such as bis(p-azidobenzoyl) 55 amidase or penicillin G amidase, useful for converting drugs hexanediamine), bis-diazonium derivatives (such as bis-(p derivatized at their amine nitrogens with phenoxyacetyl or diazoniumbenzoy1 )-ethy lenediamine ), diisocyanates ( such phenylacetyl groups, respectively, into free drugs. Alterna as tolyene 2,6-diisocyanate), and bis-active fluorine com tively, antibodies with enzymatic activity, also known in the pounds (such as 1,5-difluoro-2,4-dinitrobenzene). For art as "abzymes", can be used to convert the prodrugs of the example, a ricin immunotoxin can be prepared as described 60 invention into free active drugs (see, e.g., Massey, Nature in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 328: 457-458 [1987]). Antibody-abzyme conjugates can be 1-isothiocyanatobenzy 1-3-methyldiethylene triaminepen prepared as described herein for delivery of the abzyme to taacetic acid (MX-DTPA) is an exemplary chelating agent a tumor cell population. for conjugation of radionucleotide to the antibody. See WO The enzymes of this invention can be covalently bound to 94/11026. 65 the anti-ErbB2 antibodies by techniques well known in the In another embodiment, the antibody may be conjugated art such as the use of the heterobifunctional crosslinking to a "receptor" (such streptavidin) for utilization in tumor reagents discussed above. Alternatively, fusion proteins Appx124 GNE-HER_002953502 Case: 19-2156 Document: 19 Page: 196 Filed: 07/26/2019 US 10,160,811 B2 29 30 comprising at least the antigen binding region of an antibody intracellularly, as a first step, the particulate debris, either of the invention linked to at least a functionally active host cells or lysed fragments, is removed, for example, by portion of an enzyme of the invention can be constructed centrifugation or ultrafiltration. Carter et al., Bio/Technology using recombinant DNA techniques well known in the art 10:163-167 (1992) describe a procedure for isolating anti- (see, e.g., Neuberger et al., Nature, 312: 604-608 [1984]). 5 bodies which are secreted to the periplasmic space of E.coli. (xi) Antibody-Salvage Receptor Binding Epitope Fusions Briefly, cell paste is thawed in the presence of sodium In certain embodiments of the invention, it may be acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride desirable to use an antibody fragment, rather than an intact (PMSF) over about 30 min. Cell debris can be removed by antibody, to increase tumor penetration, for example. In this centrifugation. Where the antibody is secreted into the case, it may be desirable to modify the antibody fragment in 10 medium, supernatants from such expression systems are order to increase its serum half life. This may be achieved, preferably first concentrated using a commercially available for example, by incorporation of a salvage receptor binding protein concentration filter, for example, an Amicon or epitope into the antibody fragment ( e.g. by mutation of the Millipore Pellicon ultrafiltration unit. A protease inhibitor appropriate region in the antibody fragment or by incorpo- such as PMSF may be included in any of the foregoing steps rating the epitope into a peptide tag that is then fused to the 15 to inhibit proteolysis and antibiotics may be included to antibody fragment at either end or in the middle, e.g., by prevent the growth of adventitious contaminants. DNA or peptide synthesis). The antibody composition prepared from the cells can be A systematic method for preparing such an antibody purified using, for example, hydroxylapatite chromatogra- variant having an increased in vivo half-life comprises phy, gel electrophoresis, dialysis, and affinity chromatogra- several steps. The first involves identifying the sequence and 20 phy, with affinity chromatography being the preferred puri- conformation of a salvage receptor binding epitope of an Fe fication technique. The suitability of protein A as an affinity region of an IgG molecule. Once this epitope is identified, ligand depends on the species and isotype of any immuno- the sequence of the antibody of interest is modified to globulin Fe domain that is present in the antibody. Protein A include the sequence and conformation of the identified can be used to purify antibodies that are based on human yl, binding epitope. After the sequence is mutated, the antibody 25 y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth. variant is tested to see if it has a longer in vivo half-life than 62: 1-13 [1983]). Protein G is recommended for all mouse that of the original antibody. If the antibody variant does not isotypes and for human y3 (Guss et al., EMBO J. have a longer in vivo half-life upon testing, its sequence is 5:15671575 [1986]). The matrix to which the affinity ligand further altered to include the sequence and conformation of is attached is most often agarose, but other matrices are the identified binding epitope. The altered antibody is tested 30 available. Mechanically stable matrices such as controlled for longer in vivo half-life, and this process is continued pore glass or poly(styrenedivinyl)benzene allow for faster until a molecule is obtained that exhibits a longer in vivo flow rates and shorter processing times than can be achieved half-life. with agarose. Where the antibody comprises a CH3 domain, The salvage receptor binding epitope being thus incorpo- the BakerbondABX™ resin (J. T. Baker, Phillipsburg, N.J.) rated into the antibody of interest is any suitable such 35 is useful for purification. Other techniques for protein puri- epitope as defined above, and its nature will depend, e.g., on fication such as fractionation on an ion-exchange column, the type of antibody being modified. The transfer is made ethanol precipitation, Reverse Phase HPLC, chromatogra- such that the antibody of interest still possesses the biologi- phy on silica, chromatography on heparin SEPHAROSE™ cal activities described herein. chromatography on an anion or cation exchange resin (such The epitope preferably constitutes a region wherein any 40 as a polyaspartic acid column), chromatofocusing, SDS- one or more amino acid residues from one or two loops of PAGE, and ammonium sulfate precipitation are also avail- a Fe domain are transferred to an analogous position of the able depending on the antibody to be recovered. antibody fragment. Even more preferably, three or more Following any preliminary purification step(s), the mix- residues from one or two loops of the Fe domain are ture comprising the antibody of interest and contaminants transferred. Still more preferred, the epitope is taken from 45 may be subjected to low pH hydrophobic interaction chro- the CH2 domain of the Fe region (e.g., of an IgG) and matography using an elution buffer at a pH between about transferred to the CHI, CH3, or V H region, or more than one 2.5-4.5, preferably performed at low salt concentrations (e.g. such region, of the antibody. Alternatively, the epitope is from about 0-0.25M salt). taken from the CH2 domain of the Fe region and transferred to the CL region or VL region, or both, of the antibody 50 III. Determination of Anti-ErbB2 Antibody fragment. Concentration in Serum In one most preferred embodiment, the salvage receptor binding epitope comprises the sequence (5' to 3'): PKNS The following non-limiting assay is useful for determin SMISNTP (SEQ ID N0:3 ), and optionally further comprises ing the presence of and to quantitate the amount of specific 2 a sequence selected from the group consisting ofHQSLGTQ 55 rhuMAb HER2 (humanized anti-pl 85HER monoclonal anti (SEQ ID N0:4), HQNLSDGK (SEQ ID N0:5), HQNIS body, including HERCEPTIN® anti-ErbB2 antibody) in a DGK (SEQ ID N0:6), or VISSHLGQ (SEQ ID N0:7), body fluid of a mammal including, but not limited to, serum, particularly where the antibody fragment is a Fab or F(ab )2 . amniotic fluid, milk, umbilical cord serum, ocular aqueous In another most preferred embodiment, the salvage receptor and vitreous liquids, and ocular vitreous gel. binding epitope is a polypeptide containing the sequence(s) 60 Plate Binding Activity Assay for rhuMAb HER2 (Human (5' to 3'): HQNLSDGK (SEQ ID N0:5), HQNISDGK (SEQ ized Anti-pl 85HER 2 Monoclonal Antibody ID N0:6), or VISSHLGQ (SEQ ID N0:7) and the sequence: The method of assaying rhuMAb HER2 described herein PKNSSMISNTP (SEQ ID N0:3). is meant as an example of such a method and is not meant (xii) Purification of Anti-ErbB2 Antibody to be limiting. A standardized preparation of rhuMAb HER2 When using recombinant techniques, the antibody can be 65 (Genentech, Inc., South San Francisco, Calif.), controls, and produced intracellularly, in the periplasmic space, or directly serum samples were diluted with Assay Diluent (PBS/0.5% secreted into the medium. If the antibody is produced BSA/0.05% Polysorbate 20/0.01 % Thimerosal). The dilu- Appx125 GNE-HER_002953503 Case: 19-2156 Document: 19 Page: 197 Filed: 07/26/2019 US 10,160,811 B2 31 32 tions of standardized rhuMAb HER2 were prepared to span anti-ErbB2 antibody formulations are described in WO a range of concentrations useful for a standard curve. The 97/04801, expressly incorporated herein be reference. samples were diluted to fall within the standard curve. The formulation herein may also contain more than one An aliquot of Coat Antigen in Coating buffer (recombi active compound as necessary for the particular indication nant pl85HER2 (Genentech, Inc.) in 0.05 M sodium carbon 5 being treated, preferably those with complementary activi ate buffer) was added to each well of a microtiter plate and ties that do not adversely affect each other. For example, it incubated at 2-8° C. for 12-72 hours. The coating solution may be desirable to further provide antibodies which bind to was removed and each well was washed six times with EGFR, ErbB2 (e.g. an antibody which binds a different water, then blotted to remove excess water: epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial An aliquot of Assay Diluent was added to each well and 10 growth factor (VEGF) in the one formulation. Alternatively, incubated for 1-2 hours at ambient temperature with agita or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules tion. The wells were washed as in the previous step. are suitably present in combination in amounts that are Aliquots of diluted standard, control and sample solutions effective for the purpose intended. were added to the wells and incubated at ambient tempera- 15 The active ingredients may also be entrapped in micro ture for 1 hour with agitation to allow binding of the capsules prepared, for example, by coacervation techniques antibody to the coating antigen. The wells are washed again or by interfacial polymerization, for example, hydroxym with water as in previous steps. ethylcellulose or gelatin-microcapsules and poly-(methyl Horse radish peroxidase-conjugate (HRP-conjugate, Goat methacylate) microcapsules, respectively, in colloidal drug anti-human IgG Fe conjugated to horseradish peroxidase; 20 delivery systems (for example, liposomes, albumin micro Organon Teknika catalog #55253 or equivalent) was diluted spheres, microemulsions, nano-particles and nanocapsules) with Assay Diluent to yield an appropriate optical density or in macroemulsions. Such techniques are disclosed in range between the highest and lowest standards. An aliquot Remington's Pharmaceutical Sciences 16th edition, Osol, A. of the HRP-conjugate solution was added to each well and Ed. (1980). incubated at ambient temperature for 1 hour with agitation. 25 The formulations to be used for in vivo administration The wells were washed with water as in previous steps. must be sterile. This is readily accomplished by filtration An aliquot of Substrate Solution (o-phenylenediamine through sterile filtration membranes. (OPD) 5 mg tablet (Sigma P6912 or equivalent) in 12.5 ml Sustained-release preparations may be prepared. Suitable 4 mM H2 0 2 in PBS) was added to each well and incubated examples of sustained-release preparations include semiper- for a sufficient period of time (approximately 8-10 minutes) 30 meable matrices of solid hydrophobic polymers containing in the dark at ambient temperature to allow color develop the antibody, which matrices are in the form of shaped ment. The reaction was stopped with an aliquot of 4.5 N articles, e.g. films, or microcapsules. Examples of sustained sulfuric acid. Optical density was read at 490-492 nm for release matrices include polyesters, hydrogels (for example, detection absorbance and 405 nm for reference absorbance. poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), The standard curve data are plotted and the results for the 35 polylactides (U.S. Pat. No. 3,773,919), copolymers ofL-glu controls and samples are determined from the standard tamic acid and y ethyl-L-glutamate, non-degradable ethyl curve. ene-vinyl acetate, degradable lactic acid-glycolic acid copo lymers such as the LUPRON DEPOT™ (injectable IV. Pharmaceutical Formulations microspheres composed of lactic acid-glycolic acid copoly- 40 mer and leuprolide acetate), and poly-D-(-)-3-hydroxybu Therapeutic formulations of the antibodies used in accor tyric acid. While polymers such as ethylene-vinyl acetate dance with the present invention are prepared for storage by and lactic acid-glycolic acid enable release of molecules for mixing an antibody having the desired degree of purity with over 100 days, certain hydro gels release proteins for shorter optional pharmaceutically acceptable carriers, excipients or time periods. When encapsulated antibodies remain in the stabilizers (Remington's Pharmaceutical Sciences 16th edi 45 body for a long time, they may denature or aggregate as a tion, Osol, A. Ed. [1980]), in the form of lyophilized result of exposure to moisture at 37° C., resulting in a loss formulations or aqueous solutions. Acceptable carriers, of biological activity and possible changes in immunoge excipients, or stabilizers are nontoxic to recipients at the nicity. Rational strategies can be devised for stabilization dosages and concentrations employed, and include buffers depending on the mechanism involved. For example, if the such as phosphate, citrate, and other organic acids; antioxi 50 aggregation mechanism is discovered to be intermolecular dants including ascorbic acid and methionine; preservatives S-S bond formation through thio-disulfide interchange, (such as octadecyldimethylbenzyl ammonium chloride; hex stabilization may be achieved by modifying sulfhydryl resi amethonium chloride; benzalkonium chloride, benzetho dues, lyophilizing from acidic solutions, controlling mois nium chloride; phenol, butyl or benzyl alcohol; alkyl para ture content, using appropriate additives, and developing bens such as methyl or propyl paraben; catechol; resorcinol; 55 specific polymer matrix compositions. cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, V. Treatment with the Anti-ErbB2 Antibodies such as serum albumin, gelatin, or immunoglobulins; hydro philic polymers such as polyvinylpyrrolidone; amino acids It is contemplated that, according to the present invention, such as glycine, glutamine, asparagine, histidine, arginine, 60 the anti-ErbB2 antibodies may be used to treat various or lysine; monosaccharides, disaccharides, and other carbo conditions characterized by overexpression and/or activa hydrates including glucose, mannose, or dextrins; chelating tion of the ErbB2 receptor. Exemplary conditions or disor agents such as EDTA; sugars such as sucrose, mannitol, ders include benign or malignant tumors ( e.g. renal, liver, trehalose or sorbitol; salt-forming counter-ions such as kidney, bladder, breast, gastric, ovarian, colorectal, prostate, sodium; metal complexes (e.g. Zn-protein complexes); and/ 65 pancreatic, lung, vulva!, thyroid, hepatic carcinomas; sarco or non-ionic surfactants such as TWEEWM, PLURON mas; glioblastomas; and various head and neck tumors); ICS™ or polyethylene glycol (PEG). Preferred lyophilized leukemias and lymphoid malignancies; other disorders such Appx126 GNE-HER_002953504 Case: 19-2156 Document: 19 Page: 198 Filed: 07/26/2019 US 10,160,811 B2 33 34 as neuronal, glial, astrocytal, hypothalamic and other glan treatments, the initial dose or initial doses are followed at dular, macrophagal, epithelial, stromal and blastocoelic dis daily or weekly intervals by maintenance doses. Each main orders; and inflammatory, angiogenic and immunologic dis tenance dose provides the same or a smaller amount of orders. antibody compared to the amount of antibody administered The antibodies of the invention are administered to a 5 in the initial dose or doses. human patient, in accord with known methods, such as Depending on the type and severity of the disease, about intravenous administration as a bolus or by continuous 1 µg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an infusion over a period of time, by intramuscular, intraperi initial candidate dosage for administration to the patient, toneal, intracerobrospinal, subcutaneous, intra-articular, whether, for example, by one or more separate administra intrasynovial, intrathecal, oral, topical, or inhalation routes. 10 tions, or by continuous infusion. A typical daily dosage Intravenous or subcutaneous administration of the antibody might range from about 1 µg/kg to 100 mg/kg or more, is preferred. depending on the factors mentioned above. For repeated The treatment of the present invention involves the administrations over several days or longer, depending on administration of an anti-ErbB2 antibody to an animal or the condition, the treatment is sustained until a desired human patient, followed at intervals by subsequent doses of 15 suppression of disease symptoms occurs. The progress of equal or smaller doses such that a target serum concentration this therapy is easily monitored by conventional techniques is achieved and maintained during treatment. Preferably, and assays. maintenance doses are delivered by bolus delivery, prefer According to the invention, dosage regimens may include ably by subcutaneous bolus administration, making treat an initial dose of anti-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 ment convenient and cost-effective for the patient and health 20 mg/kg delivered by intravenous or subcutaneous infusion, care professionals. followed by subsequent weekly maintenance doses of 2 Where combined administration of a chemotherapeutic mg/kg by intravenous infusion, intravenous bolus injection, agent ( other than an antracycline) is desired, the combined subcutaneous infusion, or subcutaneous bolus injection. administration includes coadministration, using separate for Where the antibody is well-tolerated by the patient, the time mulations or a single pharmaceutical formulation, and con- 25 of infusion may be reduced. secutive administration in either order, wherein preferably Alternatively, the invention includes an initial dose of 12 there is a time period while both (or all) active agents mg/kg anti-ErbB2 antibody, followed by subsequent main simultaneously exert their biological activities. Preparation tenance doses of 6 mg/kg once per 3 weeks. and dosing schedules for such chemotherapeutic agents may Another dosage regimen involves an initial dose of 8 be used according to manufacturers' instructions or as 30 mg/kg anti-ErbB2 antibody, followed by 6 mg/kg once per determined empirically by the skilled practitioner. Prepara 3 weeks. tion and dosing schedules for such chemotherapy are also Still another dosage regimen involves an initial dose of 8 described in Chemotherapy Service Ed., M. C. Perry, Wil mg/kg anti-ErbB2 antibody, followed by subsequent main liams & Wilkins, Baltimore, Md. (1992). The chemothera tenance doses of 8 mg/kg once per week or 8 mg/kg once peutic agent may precede, or follow administration of the 35 every 2 to 3 weeks. antibody or may be given simultaneously therewith. The As an alternative regimen, initial doses of 4 mg/kg antibody may be combined with an anti-estrogen compound anti-ErbB2 antibody may be administered on each of days 1, such as tamoxifen or an anti-progesterone such as onapris 2 and 3, followed by subsequent maintenance doses of 6 tone (see, EP 616 812) in dosages known for such mol- mg/kg once per 3 weeks. ecules. 40 An additional regimen involves an initial dose of 4 mg/kg It may be desirable to also administer antibodies against anti-ErbB2 antibody, followed by subsequent maintenance other tumor associated antigens, such as antibodies which doses of 2 mg/kg twice per week, wherein the maintenance bind to the EGFR, ErbB3, ErbB4, or vascular endothelial doses are separated by 3 days. growth factor (VEGF). Alternatively, or additionally, two or Alternatively, the invention may include a cycle of dosing more anti-ErbB2 antibodies may be co-administered to the 45 in which delivery of anti-ErbB2 antibody is 2-3 times per patient. Sometimes, it may be beneficial to also administer week for 3 weeks. The 3 week cycle is preferably repeated one or more cytokines to the patient. The ErbB2 antibody as necessary to achieve suppression of disease symptoms. may be co-administered with a growth inhibitory agent. For The invention further includes a cyclic dosage regimen in example, the growth inhibitory agent may be administered which delivery of anti-ErbB2 antibody is daily for 5 days. first, followed by the ErbB2 antibody. However, simultane- 50 According to the invention, the cycle is preferably repeated ous administration, or administration of the ErbB2 antibody as necessary to achieve suppression of disease symptoms. first is also contemplated. Suitable dosages for the growth Further information about suitable dosages is provided in the inhibitory agent are those presently used and may be low Examples below. ered due to the combined action (synergy) of the growth inhibitory agent and anti-ErbB2 antibody. 55 VI. Articles of Manufacture In addition to the above therapeutic regimens, the patient may be subjected to surgical removal of cancer cells and/or In another embodiment of the invention, an article of radiation therapy. manufacture containing materials useful for the treatment of For the prevention or treatment of disease, the appropriate the disorders described above is provided. The article of dosage of anti-ErbB2 antibody will depend on the type of 60 manufacture comprises a container, a label and a package disease to be treated, as defined above, the severity and insert. Suitable containers include, for example, bottles, course of the disease, whether the antibody is administered vials, syringes, etc. The containers may be formed from a for preventive or therapeutic purposes, previous therapy, the variety of materials such as glass or plastic. The container patient's clinical history and response to the antibody, and holds a composition which is effective for treating the the discretion of the attending physician. The antibody is 65 condition and may have a sterile access port (for example, suitably administered to the patient at one time or over a the container may be an intravenous solution bag or a vial series of treatments. Where the treatment involves a series of having a stopper pierceable by a hypodermic injection Appx127 GNE-HER_002953505 Case: 19-2156 Document: 19 Page: 199 Filed: 07/26/2019 US 10,160,811 B2 35 36 2 needle). At least one active agent in the composition is an pl8SHER , induces antibody-dependent cellular cytotoxicity anti-ErbB2 antibody. The label on, or associated with, the (ADCC), and has been found clinically active, as a single container indicates that the composition is used for treating agent, in patients with ErbB2-overexpressing metastatic the condition of choice. The article of manufacture may breast cancers that had received extensive prior therapy. further comprise a second container comprising a pharma- 5 HERCEPTIN® anti-ErbB2 antibody is produced by a ceutically-acceptable buffer, such as phosphate-buffered genetically engineered Chinese Hamster Ovary (CHO) cell saline, Ringer's solution and dextrose solution. It may line, grown in large scale, that secretes the antibody into the further include other materials desirable from a commercial culture medium. The antibody is purified from the CHO and user standpoint, including other buffers, diluents, filters, culture media using standard chromatographic and filtration needles, and syringes. In addition, the article of manufacture 10 methods. Each lot of antibody used in this study was assayed may comprise a package inserts with instructions for use, to verify identity, purity, and potency, as well as to meet including, e.g., a warning that the composition is not to be Food and Drug Administration requirements for sterility and used in combination with anthacycline-type chemotherapeu safety. tic agent, e.g. doxorubicin or epirubicin. 15 Eligibility Criteria Deposit of Materials Patients had to fulfill all of the following criteria to be The following hybridoma cell lines have been deposited eligible for study admission: with the American Type Culture Collection, 12301 Parklawn Metastatic breast cancer Drive, Rockville, Md., USA (ATCC): Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ 20 as determined by immunohistochemistry or fluores cence in situ hybridization (FISH). [Tumor expression Antibody Designation ATCC No. Deposit Date of ErbB2 can be determined by immunohistochemical 7C2 ATCC HB-12215 Oct. 17, 1996 analysis, as previously described (Slamon et al., [ 1987] 7F3 ATCC HB-12216 Oct. 17, 1996 and [1989], supra), of a set of thin sections prepared 4D5 ATCC CRL 10463 May 24, 1990 25 from the patient's paraffin-archived tumor blocks. The 2C4 ATCC HB-12697 Apr. 8, 1999 primary detecting antibody used is murine 4D5 MAb, which has the same CDRs as the humanized antibody Further details of the invention are illustrated by the used for the treatment. Tumors are considered to over following non-limiting Examples. express ErbB2 if at least 25% of tumor cells exhibit 30 2 characteristic membrane staining for pl 85HER ]. EXAMPLES Bidimensionally measurable disease (including lytic bone lesions) by radiographic means, physical examination, Example 1: Preparation and Efficacy of or photographs HERCEPTIN® Anti-ErbB2 Antibody Measurable disease was defined as any mass reproducibly 35 Materials and Methods measurable in two perpendicular diameters by physical Anti-ErbB2 Monoclonal Antibody examination, X-ray (plain films), computerized tomography (CT), magnetic resonance imaging (MRI), ultrasound, or The anti-ErbB2 IgG1K murine monoclonal antibody 4D5, specific for the extracellular domain ofErbB2, was produced photographs. as described in Fendly et al., Cancer Research So: 1550-1558 40 Osteoblastic metastases, pleural effusions, or ascites were (1990) and W089/06692. Briefly, NIH 3T3/HER2-3400 cells not considered to be measurable. Measurable lesions must (expressing approximately lxl05 ErbB2 molecules/cell) be at least 1 cm in greatest dimension. Enumeration of produced as described in Hudziak, et al., Proc. Natl. Acad. evaluable sites of metastatic disease and number of lesions Sci. (USA) 84:7159 (1987) were harvested with phosphate in an evaluable site (e.g. lung) had to be recorded on the buffered saline (PBS) containing 25 mM EDTA and used to 45 appropriate Case Report Form (CRF). If a large number of immunize BALB/c mice. The mice were given injections i.p. pulmonary or hepatic lesions were present, the six largest of 107 cells in 0.5 ml PBS on weeks, 0, 2, 5 and 7. The mice lesions per site were followed. with antisera that immunoprecipitated 32P-labeled ErbB2 The ability to understand and willingness to sign a written were given i.p. injections of a wheat germ agglutinin informed consent form Sepharose (WGA) purified ErbB2 membrane extract on 50 Women s18 years weeks 9 and 13. This was followed by an i.v. injection of0.1 Suitable candidates for receiving concomitant cytotoxic ml of the ErbB2 preparation and the splenocytes were fused chemotherapy as evidenced by screening laboratory with mouse myeloma line X63-Ag8.653. Hybridoma super assessments of hematologic, renal, hepatic, and meta natants were screened for ErbB2-binding by ELISA and bolic functions. radioimmunoprecipitation. MOPC-21 (IgGl), (Cappell, 55 Exclusion Criteria Durham, N.C.), was used as an isotype-matched control. Patients with any of the following were excluded from The treatment was performed with a humanized version study entry: of the murine 4D5 antibody (HERCEPTIN® anti-ErbB2 Prior cytotoxic chemotherapy for metastatic breast cancer antibody). The humanized antibody was engineered by Patients may have received prior hormonal therapy (e.g. inserting the complementarity determining regions of the 60 tamoxifen) for metastatic disease or cytotoxic therapy murine 4D5 antibody into the framework of a consensus in the adjuvant setting. human immunoglobulin IgG1 (IgG1) (Carter et al., Proc. Concomitant malignancy that has not been curatively Natl. Acad. Sci. USA 89:4285-4289 [1992]). The resulting treated humanized anti-ErbB2 monoclonal antibody has high affin- A performance status of <60% on the Karnofsky scale ity for pl85HER2 (Dillohiation constant [Kd]=0.1 nmol/L), 65 Pregnant or nursing women; women of childbearing markedly inhibits, in vitro and in human xenografts, the potential, unless using effective contraception as deter growth of breast cancer cells that contain high levels of mined by the investigator Appx128 GNE-HER_002953506 Case: 19-2156 Document: 19 Page: 200 Filed: 07/26/2019 US 10,160,811 B2 37 38 Bilateral breast cancer ( either both primary tumors must minimum period of 4 weeks. No new lesions may have have 2+ to 3+ HER2 overexpression, or the metastatic appeared, nor may any lesions have progressed in size. site must have 2+ to 3+ HER2 overexpression) Minor Response Use of investigational or unlicensed agents within 30 days A reduction of 25% to 49% in the sum of the products of prior to study entry 5 the perpendicular diameters of all measurable lesions. No Clinically unstable or untreated metastases to the brain new lesions may have appeared, nor may any lesions have (e.g. requiring radiation therapy) progressed in size. Based upon the foregoing criteria, 469 patients were Stable Disease chosen, and enrolled in the study. Half the patients (stratified No change of greater than 25% in the size of measurable by chemotherapy) were randomized to additionally receive 10 lesions. No lesions may have appeared. the HERCEPTIN® anti-ErbB2 antibody (see below). Time to disease progression (TTP) was calculated from Administration and Dosage the beginning of therapy to progression. Anti-ErbB2 Antibody Confidence limits for response rates were calculated using On day 0, a 4 mg/kg dose of humanized anti-ErbB2 the exact method for a single proportion. (Pleiss, J L, antibody (HERCEPTIN®, H) was administered intrave- 15 Statistical Methods for Rates and Proportions (ed. 2), New nously, over a 90-minute period. Beginning on day 7, York, N.Y., Wiley, 1981, pp 13-17). patients received weekly administration of2 mg/kg antibody Results (i.v.) over a 90-minute period. At a median follow-up of 10.5 months, assessments of Chemotherapy time to disease progression (TTP in months) and response The patients received one of two chemotherapy regimens 20 rates (RR) showed a significant augmentation of the che for a minimum of six cycles, provided their disease was not motherapeutic effect by HERCEPTIN® anti-ErbB2 anti progressing: a) cyclophosphamide and doxorubicin or epi body, without increase in overall severe adverse events rubicin (AC), if patients have not received anthracycline (AE): therapy in the adjuvant setting, or b) paclitaxel (T, TAXOL®), if patients have received any anthracycline 25 TABLE 1 therapy in the adjuvant setting. The initial dose of the HERCEPTIN® anti-ErbB2 antibody preceded the first cycle HERCEPTIN ® Anti-ErbB2 Antibody Efficacy of either chemotherapy regimen by 24 hours. Subsequent doses of the antibody were given immediately before che Enrolled TTP (montbs) RR(%) AE(%) motherapy administration, if the initial dose of the antibody 30 CRx 234 5.5 36.2 66 was well tolerated. If the first dose of the antibody was not CRx + H 235 8.6* 62.00** 69 well tolerated, subsequent infusions continued to precede AC 145 6.5 42.1 71 chemotherapy administration by 24 hours. Patients were AC+ H 146 9.0 64.9 68 T 89 4.2 25.0 59 permitted to continue receiving chemotherapy beyond six T+H 89 7.1 57.3 70 cycles if, in the opinion of the treating physician, they were 35 *p < 0.001 by log-rank test; continuing to receive treatment benefit. 2 2 **p < 0.01 by X test; Cyclophosphamide (600 mg/m ) was given either by iv CRx: chemotherapy; push over a minimum period of 3 minutes or by infusion AC: anthracycline/cyclophosphamide treatment; over a maximum period of 2 hours. H: HERCEPTIN ® anti-ErbB2 antibody; 2 2 T: TAXOL® Doxorubicin (60 mg/m ) or epirubicin (75 mg/m ) were 40 given either by slow iv push over a minimum period of 3-5 A syndrome of myocardial dysfunction similar to that minutes or by infusion over a maximum period of 2 hours, observed with anthracyclines was reported more commonly according to institutional protocol. with a combined treatment of AC+H (18% Grade 3/4) than 2 Paclitaxel (TAXOL®) was given at a dose of 175 mg/m with AC alone (3%), T (0%), or T+H (2%). over 3 hours by intravenous administration. All patients 45 These data indicate that the combination of anti-ErbB2 receiving paclitaxel were premedicated with dexamethasone antibody treatment with chemotherapy markedly increases (or its equivalent) 20 mgx2, administered orally 12 and 6 the clinical benefit, as assessed by response rates and the hours prior to paclitaxel; diphenhydramine (or its equiva evaluation of disease progression. However, due to the lent) 50 mg, iv, administered 30 minutes prior to paclitaxel, increased cardiac side-effects of doxorubicin or epirubicin, and dimetidine (or another H2 blocker) 300 mg, iv, admin- 50 the combined use of anthracyclines with anti-ErbB2 anti istered 30 minutes prior to paclitaxel. body therapy is contraindicated. The results, taking into Response Criteria account risk and benefit, favor treatment with HERCEP Progressive Disease TIN® anti-ErbB2 antibody and paclitaxel (TAXOL®) Objective evidence of an increase of 25% or more in any where a combined treatment regimen is desired. measurable lesion. Progressive disease also includes those 55 instances when new lesions have appeared. For bone lesions, Example 2: Pharmacokinetic and Pharmacodynamic Properties of Anti-ErbB2 Antibody progression is defined as a 25% increase in objective mea (HERCEPTIN®) surement by plain film, CT, MRI; symptomatic new lesions not due to fracture; or requirement for palliative radio HERCEPTIN® anti-ErbB2 antibody was administered by therapy. 60 intravenous infusion to human patients selected according to Complete Response the criteria provided in Example 1. An initial dose of 4 Disappearance of all radiographically and/or visually mg/kg HERCEPTIN® anti-ErbB2 antibody was delivered apparent tumor for a minimum of 4 weeks. Skin and chest by intravenous infusion, followed by subsequent i.v. infu wall complete responses had to be confirmed by biopsy. sions of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody Partial Response 65 weekly for several weeks. Two hundred thirteen patients A reduction of at least 50% in the sum of the products of began this treatment regimen and serum drug concentration the perpendicular diameters of all measurable lesions for a was obtained beyond 8 weeks for fewer than 90 patients as Appx129 GNE-HER_002953507 Case: 19-2156 Document: 19 Page: 201 Filed: 07/26/2019 US 10,160,811 B2 39 40 selective discontinuation of patients with rapidly progress Committee). The increase in serum concentration between ing disease occurred. Of the 213 patients who began treat Weeks 2 and 8 appeared to be greater in responders than in ment, serum trough concentration data were available for 80 nonresponders, suggesting that there is a relationship patients at Week 12, for 77 patients at Week 16, for 44 between response status and HERCEPTIN® anti-ErbB2 patients at Week 20, for 51 patients at Week 24, for 25 5 antibody serum concentration. A statistical analysis ( analysis patients at Week 28, for 23 patients at Week 32, and for 37 of variance) of trough serum concentration values at Week patients at Week 36. 2 and an average of Weeks 7 and 8 in relation to response HERCEPTIN® Anti-ErbB2 Antibody Trough Serum status indicated a highly significant relationship between Concentrations for Weeks 0-36 response status and average trough of Weeks 7 and 8 The HERCEPTIN® anti-ErbB2 antibody trough serum 10 (p<0.001). The results indicated that there was a significant concentrations (µg/ml, mean±SE) from Week 2 through difference between the trough serum concentration (average Week 36 are plotted in FIG. 3 (dark circles). The number of troughs of Weeks 7 and 8) in the responders and nonre patients was fairly constant because data from patients sponders: trough concentrations were 60±20 µg/ml in the discontinued from the program due to rapidly progressing responders versus 44±25 µg/ml in the nonresponders disease were excluded from this analysis. Trough serum 15 (mean±SD). HER2 overexpression level and type of meta concentrations tended to increase through Week 12 and static sites were associated with significant differences in tended to plateau after that time. trough serum concentrations. At Week 2, patients with 2+ HERCEPTIN® Anti-ErbB2 Antibody Trough and Peak Serum Concentrations for Weeks 1-8 HER2 overexpression had significantly higher trough serum Some HERCEPTIN® anti-ErbB2 antibody serum con concentrations (n=40, mean=28.8 µg/ml, SD=l0.4) com centration data were available for 212 of the original 213 20 pared with patients with 3+ HER2 overexpression (n=155, patients. Trough and peak serum concentration data reflect mean=24.1 µg/ml, SD=13.1). This difference in the average ing the first HERCEPTIN® anti-ErbB2 antibody infusion trough serum concentrations for Weeks 7 and 8 was no were available for 195 of the 212 patients. For the seventh longer statistically significant. Further, at Week 2, patients infusion, trough serum concentration data were available for with superficial disease had significantly higher trough 137/212 patients and peak serum concentration data were 25 serum concentrations (n=12, mean 34.1 µg/ml, SD=12.0) available for 114/212 patients. Table 2 presents a summary compared with patients with visceral disease (n=183, of statistics from trough and peak serum concentrations for mean=24.4 µg/ml, SD=12.6). This difference in the average the first 8 weeks of treatment. Peak samples were drawn trough serum concentrations for Weeks 7 and 8 was signifi shortly after the end ofHERCEPTIN® anti-ErbB2 antibody cant. These data indicate that the rise in trough serum administration; trough samples were drawn prior to the 30 concentrations between Weeks 2 and 7 /8 occurs for human subsequent dose (i.e., 1 week later). Serum concentrations of patients with various disease profiles. HERCEPTIN® anti-ErbB2 antibody were determined as In a subsequent, similarly designed study, human breast disclosed herein. cancer patients were treated with a loading dose of 8 mg/kg followed by maintenance doses of 4 mg/kg weekly. The TABLE 2 35 results of this preliminary human study indicated that an 8 mg/kg load:4 mg/kg weekly maintenance regimen was effi HERCEPTIN ® Anti-ErbB2 Antibody Trough and Peak cacious in reducing tumor volume in the patients. Serum Concentrations for tbe First 8 Weeks of Treatment (µg/ml) The data disclosed in this Example indicate that front Dose loading of antibody, such that a target serum concentration Number n Mean SD Minimun Maximum is reached more quickly, may be associated with improved 40 outcomes. Peak 195 100.3 35.2 30.7 274.6 Trough 195 25.0 12.7 0.16 60.7 Peak 2 190 74.3 31.3 20.8 307.9 Example 3: I.V. Bolus Delivery and Subcutaneous Trough 167 30.4 16.0 0.2 74.4 Infusion of HERCEPTIN® Anti-ErbB2 Antibody Peak 3 167 75.3 26.8 16.1 194.8 Effectively Decrease Tumor Volume in the Mouse Trough 179 33.7 17.9 0.2 98.2 45 Peak 4 175 80.2 26.9 22.2 167 The efficacy of infusion or bolus delivery of humanized Trough 132 38.6 20.1 0.2 89.4 anti-ErbB2 antibody (HERCEPTIN® see Example 1 for Peak 5 128 85.9 29.2 27.8 185.8 Trough 141 42.1 24.8 0.2 148.7 preparation), either by intravenous injection or subcutaneous Peak 6 137 87.2 32.2 28.9 218.1 injection, was examined. The purpose of the study was to Trough 115 43.2 24.0 0.2 109.9 50 ask whether subcutaneous delivery was feasible and whether Peak 7 114 89.7 32.5 16.3 187.8 the convenient subcutaneous bolus delivery was useful in Trough 137 48.8 24.9 0.2 105.2 treating metastatic breast cancer in animals inoculated with Peak 8 133 95.6 35.9 11.4 295.6 a cell line that overexpresses the HER2 gene. The results, detailed below, show that i.v. and s.c. infusion and bolus The data in Table 2 suggest that there was an increase in 55 delivery are feasible treatment methodologies. trough serum concentration over time. Of the many patients A study in a nude mouse xenograft model, which incor studied, there were 18 patients for whom the trough con porates a human breast cancer cell line that naturally over centrations did not exceed 20 µg/ml from Week 2 through expresses the HER2 gene (BT-474Ml, derived from BT-474 Week 8. A HERCEPTIN® anti-ErbB2 antibody trough cells, ATCC Accession number HTB-20), comparing tumor serum concentration of 20 µg/ml was nominally targeted for volume as a function of i.v. bolus versus s.c. infusion was these studies based on prior pharmacologic studies in ani 60 performed as follows. In the first study athymic nude nu nu mals and exploratory analyses in clinical trials. 7-9 week old female mice were obtained from Taconic Inc Patient response status was evaluated relative to serum (Germantown, N.Y.). To initiate tumor development, each concentration of HERCEPTIN® anti-ErbB2 antibody. For mouse was inoculated subcutaneously with 3xl06 BT474M this purpose, mean serum concentration (an average of 1 cells suspended in Matrigel™. When tumor nodules 3 troughs and peaks) was calculated for various times and 65 reached a volume of approximately 100 mm , animals were patient response status (where the patient response status randomized to 4 treatment groups. The groups were treated was determined by an independent Response Evaluation according to Table 3. Appx130 GNE-HER_002953508 Case: 19-2156 Document: 19 Page: 202 Filed: 07/26/2019 US 10,160,811 B2 41 42 TABLE 3 Animal Groups and Doses for Comparison of I.V. Bolus and S.C. Infusion Group, Target Dose, Serum Cone. Route of Loading Dose Maintenance Antibody µg/ml Administration (mg/kg) Dose !--Control, rhuMAb 20 N LD and 2.20 0.250 mg/ml E25 SC infusion (infusate) 2-Low Dose SC N LD and 0.313 0.050 mg/ml rhuMAb HER2 SC infusion (infusate) 3-High Dose SC 20 N LD and 6.25 1.00 mg/ml rhuMAb HER2 SC infusion (infusate) 4-N Multi-Dose 20 (trough) N LD and MD 4.00 2 mg/kg/week rhuMAb HER2 (IV bolus) Serum Cone. = concentration in serum. LD = loading dose. 1v1D = maintenance dose. Infusate concentration was calculated to achieve targeted serum concentration using Alzet ® osmotic minipumps (Alza Corp., Palo Alto, CA). Animals were exposed to estrogen by subcutaneous sus- 20 The results tabulated above indicate that maintenance of tained release estrogen pellet 9 days before the start of a serum concentration of approximately 2 µg/ml was as dosing to promote growth of grafted tumor cells. The effective as a concentration of 20 µg/ml in this study. The results indicated that dosing by subcutaneous infusion was animals were inoculated with the BT474Ml cells 8 days as effective as intravenous bolus dosing and achieved similar before the beginning of treatment and tumors were allowed trough serum concentrations. The results also indicate that to grow. The animals were then treated with nonrelevant 25 the dose levels studied are at the top of the dose-response antibody E25 (non-specific for HER2 receptor, but a mem curve in this model and that subcutaneous dosing is effective ber of the monoclonal IgG class) or test antibody HERCEP in treating breast cancer tumors. Thus, subcutaneous admin TIN® anti-ErbB2 antibody as indicated in Table 3. The istration of maintenance doses is feasible as part of a dosage levels were selected to achieve target serum concen- HERCEPTIN® anti-ErbB2 antibody treatment regimen. 30 trations of HERCEPTIN®, either 1 µg/ml or 20 µg/ml, by subcutaneous pump infusion or by i.v. bolus delivery. The Example 4: I.V. Bolus and Subcutaneous Bolus study groups were treated until day 35. The serum concen Deliveries of HERCEPTIN® Anti-ErbB2 Antibody tration of HERCEPTIN® anti-ErbB2 antibody was mea Effectively Decrease Tumor Volume in the Mouse sured weekly (just prior to dosing for Group 4) using 3 35 Subcutaneous bolus delivery is convenient and cost mice/group/time point. The anti-ErbB2 antibody concentra effective for the patient and health care professionals. The tion was determined according to the method disclosed results of the study disclosed in this example indicate that herein involving standard techniques. Tumor volumes were subcutaneous bolus delivery was as effective as intravenous measured two days before dosing began and twice per week bolus delivery in reducing breast cell tumor size in a mouse. from day 6 to day 35 in the study for which data is tabulated 40 This study was set up as disclosed herein in Example 3 for below. Tumors were measured in three dimensions and the comparison of intravenous bolus and subcutaneous infu 3 volumes were expressed in mm • Efficacy was determined sion delivery. A sustained release estrogen implant was by a statistical comparison (ANOVA) of tumor volumes of inserted subcutaneously one day before tumor cell inocula test animals relative to untreated control animals. tion as described in Example 3. Six days after tumor cell As shown in Table 4, below, treatment of the BT474Ml 45 inoculation, the initial tumor measurement was performed. tumor-bearing mice with HERCEPTIN® anti-ErbB2 anti Seven days after tumor cell inoculation, the first dose of body by the indicated dosage methods significantly inhibited control antibody or HERCEPTIN® anti-ErbB2 antibody the growth of the tumors. All HERCEPTIN®-treated groups was delivered. The animal groups, type of delivery, loading showed similar inhibition of tumor growth relative to the dose and maintenance doses are provided in Table 4. Ani control group. No dose-response was observed. mals were dosed once weekly for 4 weeks. TABLE 4 Comparison of S.C. Infusion and I.V. Bolus Delivery Twnor Volume HERCEPTIN® (area under curve) Serum Cone. 3 Tumor Volume (mm ), Day 6-Day 35 (µg/ml), Day 27, (n - Treatment Group Day 35, (n - 14) (n - 13) 3) control s.c. infusion 764 ± 700 5650 ± 4700 4.16 ± 1.94 s.c. infusion (low dose) 80.6 ± 158 1610 ± 1250 2.11 ± 1.74 s.c. infusion (high dose) 31 ± 75.6 1440 ± 1140 22.1 ± 5.43 i.v. bolus dose* 49.7 ± 95.7 2150 ± 1480 21.7±17.1** s.c. = subcutaneous delivery; i.v. = intravenous delivery. *4.0 mg/kg Loading Dose and 2.0 mg/kg/week Maintenance Dose. **at predose (trough serum concentration immediately prior to a maintenance dose) Appx131 GNE-HER_002953509 Case: 19-2156 Document: 19 Page: 203 Filed: 07/26/2019 US 10,160,811 B2 43 44 TABLE 5 TABLE 6-continued Animal Groups and Doses for Comparison of N versus SC Bolus Delivery: Serum HERCEPTIN ® Anti-ErbB2 I.V Bolus and S.C. Bolus Delivery Antibody Concentration Serum Concentration, µg/ml Loading Route of Dose Maintenance Dose Day 21 Group Administration (mg/kg) (mg/kg/week) n Treatment Group Day 0 Day 7 Day 14 Mean (delivery, MD) Mean (SD) Mean (SD) Mean (SD) (SD) !--Control N 8 4 10 rhuMAb E25 4--rhu MAb HER2 0 29.6 37.7 46.2 2-rhuMAb HER2 N 2 10 10 (N, 4mg/kg) (0) (13.5) (14.4) (13.8) 3-rhuMAb HER2 N 4 2 10 5-rhu MAb HER2 0 12.5 16.9 17.6 4-rhuMAb HER2 N 8 4 10 (SC, 2 mg/kg) (0) (7.33) (10.2) (10.7) 5-rhuMAb HER2 SC 4 2 10 n = 10 for time points Days 0, 7 and 14. N = intraveneous; N=9forDay21. SC = subcutaneous; 15 n = number of animals per group. Table 7 shows the relative efficacy of intravenous bolus The mice were treated according to the information in delivery and subcutaneous bolus delivery for Groups 1-5 Table 4 and using the techniques disclosed in Example 3. having achieved the serum antibody concentrations pre The serum concentration of HERCEPTIN® anti-ErbB2 sented in Table 6. For this study, efficacy was measured as antibody was measured weekly before each weekly i.v. 20 a decrease in tumor volume. Tumor volume was measured maintenance dose according to the procedure described twice weekly. TABLE 7 Efficacy of HERCEPTIN ® Anti-ErbB2 Antibody Measured as a Change in Tumor Volume Comparing Intravenous Bolus and Subcutaneous Bolus Delivery, Mean (SD) Treatment Tumor Tumor Vol. Day 6-Day 31 * Tumor Growth Group Tumor Vol. Vol. Day Day 31, Area Under Curve Rate on (Delivery, MD) Day 6, mm3 28, mm3 mm3 Twnor Vol., mm3 Log (TM+ 1) 1-IV Control 321 1530 1630 13600 0.0660 (190) (1040) (1170) (7230) (0.0200) 2-IV Herceptin 297 175 151 4690 -0.0505 1 mg/kg (130) (215) (188) (1400) (0.142) 3-IV Herceptin 269 75.7 73.6 3510 -0.0608 2 mg/kg (129) (92.4) (84.5) (1220) (0.110) 4--IV Herceptin 272 25.3 25.8 2880 -0.0810 4 mg/kg (117) (75.9) (72.9) (1230) (0.0859) 5-SC Herceptin 268 76.2 90.4 3230 -0.0304 2 mg/kg (117) (98.8) (105) (1440) (0.104) N = 10 for each data point. TM = tumor measurement. N = intravenous. SC= subcutaneous. 1v1D = maintenance dose. 3 Tumor Vol. = tumor volume, mm . *Day 17 excluded due to measurement error. Tumor growth rate calculated on Day 21-Day 31 Log(TM + 1). Area under the curve is the area beneath a plot of tumor volume versus time. herein and using standard techniques. The control E25 FIGS. 4A and 48 are graphical plots of changes in tumor antibody serum concentration was determined according to volume over time, some of which data is found in Table 7. standard immunoassay techniques. Table 6 shows the FIG. 4A is a linear plot of tumor volume versus time. FIG. increase in HERCEPTIN® anti-ErbB2 antibody serum con 50 48 is a semilogarithmic plot of the same data, allowing the centrations with time. test points be viewed more clearly. The data in Table 7 and FIGS. 4A and 48 indicate that, although a dose-related TABLE 6 response was not observed between HERCEPTIN-treated groups, dosing by subcutaneous bolus was as effective as N versus SC Bolus Delivery: Serum HERCEPTIN ® Anti-ErbB2 55 Antibody Concentration intravenous bolus dosing and achieved similar trough serum Serum Concentration, µg/ml concentrations. Day 21 Treatment Group Day 0 Day 7 Day 14 Mean Example 5: Regimens for Intravenous and (delivery, MD) Mean (SD) Mean (SD) Mean (SD) (SD) 60 Subcutaneous Delivery of Anti-ErbB2 Antibody !--Control 0 25.9 34.6 38.5 rhu MAb E25 (0) (8.29) (11.2) (14.4) According to the invention, methods of anti-ErbB2 anti (N, 4 mg/kg) body (e.g., HERCEPTIN®) delivery comprise greater front 2-rhu MAb HER2 0 4.96 8.55 8.05 loading of the drug to achieve a target serum concentration (N, 1 mg/kg) (0) (3.79) (5.83) (4.67) 3-rhu MAb HER2 0 13.4 18.9 22.6 65 in approximately 4 weeks or less, preferably 3 weeks or less, (N, 2 mg/kg) (0) (9.24) (12.0) (9.21) more preferably 2 weeks or less, and most preferably 1 week or less, including one day or less. According to the inven- Appx132 GNE-HER_002953510 Case: 19-2156 Document: 19 Page: 204 Filed: 07/26/2019 US 10,160,811 B2 45 46 tion, this initial dosing is followed by dosing that maintains administration of 8 mg/kg per week or 8 mg/kg per 2-3 the target serum concentration by subsequent doses of equal weeks to maintain a trough serum concentration of HER or smaller amount. An advantage of the methods of the CEPTIN® anti-ErbB2 antibody of approximately 10-20 invention is that the maintenance dosing may be less fre µg/ml. Maintenance doses are delivered by intravenous quent and/or delivered by subcutaneous injection, making 5 infusion or bolus injection, or preferably by subcutaneous the treatment regimens of the invention convenient and infusion or bolus injection. cost-effective for the patient and medical professionals In another method, the front loading initial dose is a series administering the antibody. In addition, a subcutaneous of intravenous or subcutaneous injections, for example, one maintenance dose regimen may be interrupted by intrave on each of days 1, 2, and 3 of at least 1 mg/kg for each nous dosing (such as infusion) when the patient's chemo- 10 injection (where the amount of anti-ErbB2 antibody deliv therapy requires delivery of other drugs by intravenous ered by the sum of initial injections is more than 4 mg/kg), injection. followed by maintenance doses of 6 mg/kg once each 3 To test the following dosage regimens, human subjects are week interval to maintain a target trough serum concentra selected according to the criteria disclosed in Example 1, tion (for example, approximately 10-20 µg/ml) ofHERCEP above. The number of initial doses is one or more doses 15 TIN® anti-ErbB2 antibody. The maintenance doses are sufficient to achieve an efficacious target serum concentra delivered by intravenous infusion or bolus injection or by tion in approximately 4 weeks or less, preferably 3 weeks or subcutaneous infusion or subcutaneous bolus injection. less, more preferably 2 weeks or less, and most preferably 1 In yet another method, the front loading is by intravenous infusion of at least 1 mg/kg, preferably 4 mg/kg on each of week or less, including 1 day or less. The number of five consecutive days, followed by repeats of this cycle a maintenance doses may be one or more doses sufficient to 20 sufficient number of times to achieve suppression of disease achieve suppression of disease symptoms, such as a symptoms. Following the initial dose or doses, subsequent decrease in tumor volume. The maintenance doses are equal doses may be delivered by subcutaneous infusion or bolus to or smaller than the initial dose or doses, consistent with injection if tolerated by the patient. Such subcutaneous an object of the invention of administering HERCEPTIN® delivery is convenient and cost-effective for the patient and anti-ErbB2 antibody by regimens providing greater front 25 administering health care professionals. loading. The specific drug delivery regimens disclosed In still another method, HERCEPTIN® anti-ErbB2 anti herein are representative of the invention and are not meant body is delivered initially as at least 2 intravenous infusions to be limiting. per week for three weeks, followed by repeats of this cycle In one trial, an initial dose of 6 mg/kg, 8 mg/kg, or 12 to maintain an efficacious trough serum concentration of mg/kg of HERCEPTIN® anti-ErbB2 antibody is delivered 30 HERCEPTIN® anti-ErbB2 antibody. The dose is at least 4 to human patients by intravenous or subcutaneous injection. mg/kg of anti-ErbB2 antibody, preferably at least 5 mg/kg. Initial doses (loading doses) are delivered by intravenous The maintenance drug deliveries may be intravenous or infusion or bolus injection or preferably subcutaneous bolus subcutaneous. injection. Preferably a target trough serum concentration of Where the animal or patient tolerates the antibody during HERCEPTIN® anti-ErbB2 antibody of approximately 35 and after an initial dose, delivery of subsequent doses may 10-20 µg/m 1 is achieved (averaged for all patients in the be subcutaneous, thereby providing greater convenience and treatment group) and maintained by subsequent doses of cost-effectiveness for the patient and health care profession als. anti-ErbB2 antibody that are equal to or smaller than the In animal studies, an initial dose of more than 4 mg/kg, initial dose. In one method, a target trough serum concen preferably more than 5 mg/kg delivered by intravenous or tration is achieved and maintained by once-per-week deliv- 40 subcutaneous injection, is followed by subcutaneous bolus eries of 2 mg/kg HERCEPTIN® anti-ErbB2 antibody by injections of 2 mg/kg twice per week (separated by 3 days) intravenous or subcutaneous injection for at least eight to maintain a trough serum concentration of approximately weeks. Alternatively, for this or any dosage regimen dis 10-20 µg/ml. In addition, where the animal or patient is closed herein, subcutaneous continuous infusion by subcu known to tolerate the antibody, an initial dose of HERCEP taneous pump is used to delivery subsequent maintenance 45 TIN® anti-ErbB2 antibody is optionally and preferably doses. deliverable by subcutaneous bolus injection followed by In another method, an initial (front loading) dose of 8 subcutaneous maintenance injections. mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by While target serum concentrations are disclosed herein for intravenous injection (infusion or bolus injection) or by the purpose of comparing animal studies and human trials, subcutaneous bolus injection. This is followed by intrave- 50 target serum concentrations in clinical uses may differ. The nous bolus injections, intravenous infusion, subcutaneous disclosure provided herein guides the user in selecting a infusion, or subcutaneous bolus injection of 6 mg/kg at front loading drug delivery regimen that provides an effi 3-week intervals to maintain a trough serum concentration cacious target trough serum concentration. of approximately 10-20 µg/ml, averaged for an entire treat The methods of the invention disclosed herein optionally include the delivery ofHERCEPTIN® anti-ErbB2 antibody ment group. 55 In another method, an initial (front loading) dose of 12 in combination with a chemotherapeutic agent ( other than an anthrocycline derivative) to achieve suppression of disease mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by symptoms. The chemotherapeutic agent may be delivered intravenous injection (infusion or bolus injection) or by with HERCEPTIN® anti-ErbB2 antibody or separately and subcutaneous bolus injection. This is followed by intrave according to a different dosing schedule. For example, nous bolus injections, intravenous infusion, subcutaneous 60 subcutaneous delivery of HERCEPTIN® anti-ErbB2 anti infusion, or subcutaneous bolus injection of 6 mg/kg at body with TAXOL® is included in the invention. In addi 3-week intervals to maintain a trough serum concentration tion, intravenous or subcutaneous injection of 8 mg/kg of approximately 10-20 µg/ml. HERCEPTIN® anti-ErbB2 antibody, followed by intrave- In yet another method, an initial (front loading) dose of 8 nous or subcutaneous injection of 6 mg/kg HERCEPTIN® mg/kg HERCEPTIN® anti-ErbB2 antibody is delivered by 65 anti-ErbB2 antibody every 3 weeks is administered in com intravenous infusion or bolus injection, or preferably by bination with a chemotherapeutic agent, such as a taxoid subcutaneous bolus injection or infusion. This is followed by (e.g. paclitaxel 175 mg/m2 every 3 weeks) or an anthracy- Appx133 GNE-HER_002953511 Case: 19-2156 Document: 19 Page: 205 Filed: 07/26/2019 US 10,160,811 B2 47 48 cline derivative (e.g. doxorubicin 60 mg/m2 or epirubicin 7 5 6) No prior taxane therapy in any treatment setting. mg/m2 every 3 weeks). Optionally, where an anthracycline 7) Any of the following abnormal baseline hematologic derivative is administered, a cardioprotectant (e.g. 600 values: mg/m2 cyclophosphamide every 3 weeks) is also adminis Hb less than 9 g/dl 9 tered. In another combination therapy, anti-ErbB2 antibody 5 WBC less than 3.0xl0 /l is administered in a loading dose of more than 4 mg/kg, Granulocytes less than 1.5x109/l preferably more than 5 mg/kg, and more preferably at least Platelets less than 1OOx109 /1 8 mg/kg. The loading dose is followed by maintenance doses 8) Any of the following abnormal baseline liver function of at least 2 mg/kg weekly, preferably 6 mg/kg every 3 tests: weeks. The combination therapy includes administration of Serum bilirubin greater than 1.5xULN (upper normal 10 a taxoid during treatment with anti-ErbB2 antibody. Accord limit) ing to one embodiment of the invention, the taxoid is ALT and/or AST greater than 2.5xULN (greater than 2 paclitaxel and is administered at a dose of 70-100 mg/m / 4.0xULN if liver or bone metastasis) week. According to another embodiment of the invention, Alkaline phosphatase greater than 2.5xULN (greater than the taxoid is docetaxel and is administered at a dose of30-70 4.0xULN if liver or bone metastasis) mg/m2 /week. 15 9) The following abnormal baseline renal function tests: serum creatinine greater than 1.5xULN Example 6: HERCEPTIN® Administered 10) History of other serious medical conditions that would Intravenously Every Three Weeks in Combination preclude patient participation in an investigational study. with Paclitaxel HERCEPTIN® 20 Loading dose and schedule: 8 mg/kg for first dose. Currently, the recommended dose of HERCEPTIN® is 2 Maintenance dose and schedule: 6 mg/kg every 3 weeks. mg/kg once weekly. Patients will be administered HERCEP Paclitaxel- TIN® every three weeks instead of weekly, along with 175 mg/m2 IV every 3 weeksx6 cycles as a 3-hour paclitaxel (17 5 mg/m2 every three weeks). Simulation of the infusion. proposed treatment regimen suggests that the trough serum 25 NOTE: On the first cycle of treatment, paclitaxel will be concentrations will be 17 mcg/ml, in the range (10-20 dosed 8 hours prior to HERCEPTIN® to determine the PK mcg/ml) of the targeted trough serum concentrations from of paclitaxel alone. HERCEPTIN® will be administered 8 previous HERCEPTIN® IV clinical trials. After the first 12 hours post-paclitaxel for the 1st cycle only. In subsequent patients the PK parameters will be assessed, if exposure is treatment cycles, HERCEPTIN® will be administered prior felt inadequate, then the dose will be increased to 8 mg/kg to paclitaxel. every three weeks for the remaining 12 patients. 30 The total duration of this study is 18 weeks. Study Inclusion Criteria subjects will receive up to 6 total HERCEPTIN® doses. 1) Females s18 years of age After the last subject has received the last cycle of paclitaxel, 2) Histologically confirmed ErbB2 over-expressing meta data collection for safety and pharmacokinetic analysis will static breast cancer stop, and the study will close to protocol specified treatment. 3) Patients who have been newly diagnosed with metastatic 35 Study subjects may continue to receive the HERCEP disease TIN®+/-paclitaxel at the discretion of the investigator. 4) Have a Kamofsky performance status of s70% It is believed that the above treatment regimen will be 5) Give written informed consent prior to any study specific effective in treating metastatic breast cancer, despite the screening procedures with the understanding that the patient infrequency with which HERCEPTIN® is administered to has the right to withdraw from the study at any time, without 40 the patient. prejudice. While the particular aspects and embodiments of the Exclusion Criteria invention as herein shown and disclosed in detail is fully 1) Pregnant or lactating women capable of obtaining the objects and providing the advan 2) Women of childbearing potential unless (1) surgically tages herein before stated, it is to be understood that it is sterile or (2) using adequate measures of contraception such 45 merely illustrative of some of the presently preferred as oral contraceptive, intra-uterine device or barrier method embodiments of the invention and that no limitations are of contraception in conjunction with spermicidal jelly. intended to the details of methods and articles of manufac 3) Clinical or radiologic evidence of CNS metastases. ture shown other than as described in the appended claims. 4) History of any significant cardiac disease The disclosures of all citations in the specification are 5) LVEF s50% expressly incorporated herein by reference. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS, 15 <210> SEQ ID NO 1 <211> LENGTH, 166 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 1 Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr 1 5 10 15 His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val Val Gln 20 25 30 Appx134 GNE-HER_002953512 Case: 19-2156 Document: 19 Page: 206 Filed: 07/26/2019 US 10,160,811 B2 49 50 -continued Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe 35 40 45 Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn 50 55 60 Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr 65 70 75 80 Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp 85 90 95 Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu 100 105 110 Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val 115 120 125 Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp 130 135 140 Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp 145 150 155 160 Thr Asn Arg Ser Arg Ala 165 <210> SEQ ID NO 2 <211> LENGTH, 32 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 2 Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala 1 5 10 15 Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys 20 25 30 <210> SEQ ID NO 3 <211> LENGTH, 11 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic salvage receptor binding epitope <400> SEQUENCE, 3 Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro 1 5 10 <210> SEQ ID NO 4 <211> LENGTH, 7 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic salvage receptor binding epitope <400> SEQUENCE, 4 His Gln Ser Leu Gly Thr Gln 1 5 <210> SEQ ID NO 5 <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic salvage receptor binding epitope Appx135 GNE-HER_002953513 Case: 19-2156 Document: 19 Page: 207 Filed: 07/26/2019 US 10,160,811 B2 51 52 -continued <400> SEQUENCE, 5 His Gln Asn Leu Ser Asp Gly Lys 1 5 <210> SEQ ID NO 6 <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic salvage receptor binding epitope <400> SEQUENCE, 6 His Gln Asn Ile Ser Asp Gly Lys 1 5 <210> SEQ ID NO 7 <211> LENGTH, 8 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic salvage receptor binding epitope <400> SEQUENCE, 7 Val Ile Ser Ser His Leu Gly Gln 1 5 <210> SEQ ID NO 8 <211> LENGTH, 59 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 8 Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn 1 5 10 15 Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly 20 25 30 Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala 35 40 45 His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 50 55 <210> SEQ ID NO 9 <211> LENGTH, 65 <212> TYPE, PRT <213> ORGANISM, Homo sapiens <400> SEQUENCE, 9 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 1 5 10 15 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 20 25 30 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 35 40 45 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 50 55 60 Pro 65 Appx136 GNE-HER_002953514 Case: 19-2156 Document: 19 Page: 208 Filed: 07/26/2019 US 10,160,811 B2 53 54 -continued <210> SEQ ID NO 10 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Mus musculus <400> SEQUENCE, 10 Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> SEQ ID NO 11 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Mus musculus <400> SEQUENCE, 11 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> SEQ ID NO 12 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic humanized VL polypeptide sequence <400> SEQUENCE, 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Appx137 GNE-HER_002953515 Case: 19-2156 Document: 19 Page: 209 Filed: 07/26/2019 US 10,160,811 B2 55 56 -continued Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> SEQ ID NO 13 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic humanized VH polypeptide sequence <400> SEQUENCE, 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 14 <211> LENGTH, 107 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic VL consensus sequence <400> SEQUENCE, 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 Appx138 GNE-HER_002953516 Case: 19-2156 Document: 19 Page: 210 Filed: 07/26/2019 US 10,160,811 B2 57 58 -continued <210> SEQ ID NO 15 <211> LENGTH, 119 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Description of Artificial Sequence, Synthetic VH consensus sequence <400> SEQUENCE, 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 The invention claimed is: method compnsmg: administering intravenously to the 1. A method for the treatment of a human patient diag patient an initial dose of 8 mg/kg of anti-ErbB2 huMAb nosed with breast cancer characterized by 2+ or 3+ overex 4D5-8 antibody; and administering intravenously to the pression of ErbB2 receptor as determined by immunohisto 35 patient a plurality of subsequent 6 mg/kg doses of the chemistry or fluorescence in situ hybridization (FISH), antibody, wherein the initial dose is separated in time from comprising the steps of administering to the patient an initial the first subsequent dose by three weeks, and the subsequent dose of8 mg/kg ofanti-ErbB2 huMAb 4D5-8 antibody; and doses are separated from each other in time by three weeks. administering to the patient a plurality of subsequent doses 8. The method of claim 7, wherein the intravenous admin of 6 mg/kg of the antibody, wherein all doses are separated 40 istration is an intravenous infusion. in time from each other by three weeks. 9. The method of claim 8, wherein the subsequent doses 2. The method of claim 1, further comprising administer maintain a trough serum concentration of the anti-ErbB2 ing an effective amount of a chemotherapeutic agent. huMAb 4D5-8 antibody at or above 10 µg/mL. 3. The method of claim 2, wherein said chemotherapeutic 10. The method of claim 8, wherein the subsequent doses agent is a taxoid. 45 4. The method of claim 3, wherein said taxoid is paclitaxel maintain a trough serum concentration of the anti-ErbB2 or docetaxel. huMAb 4D5-8 antibody at or above 20 µg/mL. 11. The method of claim 7, wherein the subsequent doses 5. The method of claim 4 wherein said taxoid is paclitaxel. maintain a trough serum concentration of the anti-ErbB2 6. The method of claim 1, wherein said antibody is huMAb 4D5-8 antibody at or above 10 µg/mL. administered by intravenous injection. 50 7. A method for the treatment of a human patient diag 12. The method of claim 7, wherein the subsequent doses nosed with breast cancer characterized by 2+ or 3+ overex maintain a trough serum concentration of the anti-ErbB2 pression of ErbB2 receptor as determined by immunohisto huMAb 4D5-8 antibody at or above 20 µg/mL. chemistry or fluorescence in situ hybridization (FISH), the * * * * * Appx139 GNE-HER_002953517 Case: 19-2156 Document: 19 Page: 211 Filed: 07/26/2019 CERTIFICATE OF SERVICE I hereby certify that, on this 26th day of July, 2019, I filed the foregoing Non-Confidential Brief for Plaintiffs-Appellants Genentech Inc. with the Clerk of the United States Court of Appeals for the Federal Circuit via the CM/ECF system, which will send notice of such filing to all registered CM/ECF users. /s/ William F. Lee WILLIAM F. LEE WILMER CUTLER PICKERING HALE AND DORR LLP 60 State Street Boston, MA 02109 (617) 526-6000 Case: 19-2156 Document: 19 Page: 212 Filed: 07/26/2019 CERTIFICATE OF COMPLIANCE Pursuant to Fed. R. App. P. 32(g), the undersigned hereby certifies that this brief complies with the type-volume limitation of Federal Circuit Rule 32(a). 1. Exclusive of the exempted portions of the brief, as provided in Fed. R. App. P. 32(f) and Fed. Cir. R. 32(b), the brief contains 13,006 words. 2. The brief has been prepared in proportionally spaced typeface using Microsoft Word 2010 in 14 point Times New Roman font. As permitted by Fed. R. App. P. 32(g), the undersigned has relied upon the word count feature of this word processing system in preparing this certificate. /s/ William F. Lee WILLIAM F. LEE WILMER CUTLER PICKERING HALE AND DORR LLP 60 State Street Boston, MA 02109 (617) 526-6000 July 26, 2019