Docket No.: 029420.0155-US02 (PATENT) IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re United States Patent of:

Alan J. Korman, et aL.

Patent No.: 7,605,238

Issued: October 20, 2009

For: HUMAN CTLA-4 ANTIBODIES

MS Hatch-Waxman PTE Commissioner for Patents Offce of Patent Legal Administration Room MDW 7D55 600 Dulany Street (Madison Building) Alexandria, VA 223 14

REQUEST FOR EXTENSION OF PATENT TERM UNDER 35 U.S.C. §156

Sir:

Pursuant to 35 U.S.C. §156 and 37 C.F.R. §§1.710-1.791, Medarex, Inc., the current address of which is Route 206 and Province Line Road, Princeton, New Jersey 08540

("Applicant" or "Medarex"), hereby requests an extension of U.S. Patent No. 7,605,238 (the

"'238 patent"). As permitted by 37 C.F.R. §1.785(b) and MPEP §2761, Applicant is concurrently fiing a request for patent term extension of U.S. Patent No. 6,984,720 based upon the same regulatory review period.

Medarex represents that it is the owner and assignee of the entire interest in and to

Letters Patent of the United States No. 7,605,238 (Exhibit 1) granted to Alan J. Korman, Edward

L. Halk, Nils Lonberg, Yashwant M. Deo and Tibor P. Keler (the "inventors") on October 20,

2009, for "Human CTLA-4 Antibodies and Their Uses" by virtue of an assignment from the

DC; 3983891.1 Patent No.: 7,605,238 - 2- Docket No.: 029420.0155-US02

Alan J. Korman, Edward L. Halk and Nils Lonberg to Medarex, recorded in the United States

Patent and Trademark Offce ("PTO") on March 6, 2003 at Reel 013817, Frame 0628 and an assignment from Yashwant M. Deo and Tibor P. Keler to Medarex, recorded on May 23, 2007 at

Reel 019334, Frame 0783 (Exhibit 2). The '238 patent matured from U.S. Patent Application

No. 09/948,939, fied on September 7, 2001, which is a continuation-in-par of U.S. Patent

Application No. 09/644,668, filed on August 24,2000, now U.S. Patent No. 6,984,720, which claims the benefit of U.S. Provisional Patent Application No. 60/150,452, fied on August 24,

1999, now expired.

The approved product that is relevant to this Request is YERVOYTM

(ipilmumab) Injection, for intravenous infusion, referred to herein as "YERVOY" or "Approved

Product."

The Marketing Applicant for YERVOY is Bristol-Myers Squibb Company

("BMS'). Medarex is a wholly-owned subsidiary ofBMS and is authorized to rely upon the activities ofBMS, its predecessors, and affiiates for purposes of this patent term extension application.

The following information is submitted in accordance with 35 U.S.C. § 1 56(d) and the rules for extension of patent term issued by the PTO at 37 C.F.R. Subpart F, §§1.710 to 1.791 and follows the numerical format set forth in 37 C.F.R. § 1.740: Patent No.: 7,605,238 - 3 - Docket No.: 029420.0155-US02

(1) A COMPLETE IDENTIFICATION OF THE APPROVED PRODUCT

AS BY APPROPRIATE CHEMICAL AND GENERIC NAME, PHYSICAL STRUCTURE OR

CHARACTERISTICS:

The approved product is YERVOY, an injection for intravenous infusion of the active ingredient ipilimumab, available in two dosage forms namely, 50 mg/1 0 ml (5 mg/mL) and 200 mg/40 mL (5 mg/mL). YERVOY has been approved for the treatment ofunresectable or metastatic melanoma (approved labeling attached as Exhibit 3). YERVOY is a human cytotoxic T-Iymphocyte antigen 4 (CTLA-4)-blocking antibody that is comprised of 1,326 amino acids. The amino acid sequence for YERVOY is as follows: Patent No.: 7,605,238 - 4 - Docket No.: 029420.0155-US02

Antibody Amino Acid Sequence"''' Segment Heavy Chain FR1 QVQLVESGGGVVQPGRSLRLSCAASGFTFS CDR1 SYTMH FRZ WVRQAPGKGLEWVT CDRZ FISYDGNNKYY ADSVKG FR3 RFTISRDNSKNTL YLQMNSLRAEDTAIYYCAR CDR3 TGWLGPFDY FR4 WGQGTL VTVSS Heavy ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSW Chain NSGAL TSGVHTFPA VLQSSGL YSLSSVVTVPSSSLGTQTYIC Constant NVNHKPSNTKVDKRV Region EPKSCDKTHTCPPCP APELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSL TCL VKGFYPSDIA YEW ESNGQPENNYKTTPPVLDSDGSFFL YSKL TVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Light Chain FR1 EIVL TQSPGTLSLSPGERA TLSC CDR1 RASQSVGSSYLA FRZ WYQQKPGQAPRLLIY CDRZ GAFSRAT FR3 GIPDRFSGSGSGTDFTL TISRLEPEDF A VYYC CDR3 QQYGSSPWT FR4 FGQGTKVEIK Light RTV AAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQW Chain KVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKH Constant KVY ACEVTHQGLSSPVTKSFNRGEC Region

'" '" The one-letter amino acid code used in the table follows the nomenclature developed by the International Union of Pure and Applied Chemistry (IUP AC)

and the International Union of Biochmistry and Molecular Biology (IUB) in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), "Nomenclature and Symbolism for Amino Acids and Peptides," 1983. See http://ww.chem.qmul.ac.ukiupac/AminoAcid/AA1n2.html#AA 1, visited May 13,2011. Patent No.: 7,605,238 - 5 - Docket No.: 029420.01 55-US02

(2) A COMPLETE IDENTIFICATION OF THE FEDERAL STATUTE

INCLUDING THE APPLICABLE PROVISION OF LAW UNDER WHICH THE

REGULA TORY REVIEW OCCURRD:

The Approved Product is a drug product that was approved under section 35i 0.. :: "\ t~ublic Health~Ser.Yice.ActieHS,*) (42 U.S,C. §262).

(3) AN IDENTIFICATION OF THE DATE ON WHICH THE PRODUCT

RECEIVED PERMISSION FOR COMMERCIAL MARKETING OR USE UNDER THE

PROVISION OF LAW UNDER WHICH THE APPLICABLE REGULATORY REVIEW

PERIOD OCCURRED:

The Approved Product received permission for commercial marketing or use by the United States Food and Drug Administration (FDA) pursuant to section 351 (a) of the PHSA

(42 U.S.C. § 262(a)) in a letter dated,~.ch.25,20.l.i ~A copy of the approval letter is attached as Exhibit 4.

(4) IN THE CASE OF A DRUG PRODUCT, AN IDENTIFICATION OF EACH

ACTIVE INGREDIENT IN THE PRODUCT AND AS TO EACH ACTIVE INGREDIENT, A

STATEMENT THAT IT HAS NOT BEEN PREVIOUSLY APPROVED FOR COMMERCIAL

MARKETING OR USE UNDER THE FEDERAL FOOD, DRUG AND COSMETIC ACT,

THE PUBLIC HEALTH SERVICE ACT, OR THE VIRUS-SERUM-TOXIN ACT OR A

STATEMENT OF WHEN THE ACTIVE INGREDIENT WAS APPROVED FOR

COMMERCIAL MARKETING OR USE (EITHER ALONE OR IN COMBINATION WITH Patent No.: 7,605,238 - 6 - Docket No.: 029420.0155-US02

OTHER ACTIVE INGREDIENTS), THE USE FOR WHICH IT WAS APPROVED, AND THE

PROVISION OF LAW UNDER WHICH IT WAS APPROVED: (37 C.F.R. § 1.740(a)(4))

The active ingredient in YERVOY is ipilmumab. Ipilimumab is comprised of

1,326 amino acids and has an amino acid sequence as noted earlier in section (1) of this Request.

YERVOY is a human cytotoxic T-Iymphocyte antigen 4 (CTLA-4)-blocking antibody that has been approved under section 351(a) of the PHS A for the treatment ofunresectable or metastatic melanoma.

Ipilimumab has not been previously approved for commercial marketing or use under the Federal Food, Drug, and Cosmetic Act, the PHSA, or the Virus-Serum-Toxin Act.

(5) A STATEMENT THAT THE APPLICATION IS BEING SUBMITTED

WITHIN THE SIXTY DAY PERIOD PERMITTED FOR SUBMISSION PURSUANT TO

SECTION 1. nO(f) AND AN IDENTIFICATION OF THE DATE OF THE LAST DAY ON

WHICH THE APPLICATION COULD BE SUBMITTED:

This Request is timely fied, pursuant to 35 U.S.C. § 156(d)(1), within the permitted sixty-day (60-day) period that began on March 25, 2011, when the product received permission for commercial marketing or use under section 351(a) of the PHSA and that wil expire on May 24,2011. Applicant understands that, pursuant to 37 C.F.R. § 1 .nO(f), the PTO may deem this period to expire one day earlier, on May 23, 2011.

(6) A COMPLETE IDENTIFICATION OF THE PATENT FOR WHICH AN

EXTENSION IS BEING SOUGHT BY THE NAME OF THE INVENTOR, THE PATENT

NUMBER, THE DATE OF ISSUE, AND THE DATE OF EXPIRATION: Patent No.: 7,605,238 - 7 - Docket No.: 029420.0155-US02

UNITED STATES PATENT NO.: 7,605,238

INVENTORS: ALAN 1. KORMAN EDWARD L. HALK NILS LONBERG Y ASHW ANT M. DEO TIBOR P. KELER

DA TE OF ISSUE: OCTOBER 20, 2009

EXPIRATION DATE: AUGUST 24,2020'

The expiration date of U.S. Patent No. 7,605,238 ("the '238 patent") is August 24,

2020, based on the following information. The patent application that issued as the '238 patent,

U.S. Patent Application No. 09/948,939 ("the '939 application"), was filed on September 7, 2001 as a continuation-in-part or U.S. Patent Application No. 09/644,668 fied on August 24,2000,

now U.S. Patent No. 6,984,720 (the '''720 patent"), and claims the benefit of U.S. Provisional

Patent Application No. 60/150,452, fied on August 24, 1999, now expired. The expiration of the '238 patent pursuant to 35 U.S.C. § 154 is August 24,2020. However, the '238 patent is subject to a terminal disclaimer fied to the '720 patent. The expiration date of the '720 patent is

August 2, 2022, which includes a patent term adjustment pursuant to 35 U.S.C. § 154 of 708 days. Therefore, the expiration date of the '238 patent need not be shortened in view of the patent term of the '720 patent.

i The expiration date identified for U.S. Patent No. 7,605,238 in this Request is based on the assumption that the '238 patent is not entitled to rely on the patent term adjustment that was granted to U.S. Patent No. 6,984,720, despite the language of the terminal disclaimer. However, if the PTO or the courts take the position that, in the circumstance such as the '238 patent, the '238 patent is entitled to rely on the patent term adjustment granted to the '238 patent, then Applicant reserves the right to amend this Request to seek a patent term extension application based on an expiration date of August 2, 2022 for the '238 patent. Patent No.: 7,605,238 - 8 - Docket No.: 029420.0155-US02

(7) A COPY OF THE PATENT FOR WHICH AN EXTENSION IS BEING

SOUGHT, INCLUDING THE ENTIRE SPECIFICATION (INCLUDING CLAIMS) AND

DRAWINGS:

A complete copy of U.S. Patent No. 7,605,238 is attached as Exhibit 1.

(8) A COpy OF ANY DISCLAIMER, CERTIFICATE OF CORRCTION,

RECEIPT OF MAINTENANCE FEE PAYMENT, OR RE-EXAMINATION CERTIFICATE

ISSUED IN THE PATENT:

A terminal disclaimer has been fied in the '238 patent as against U.S. Patent No.

6,984,720, a copy of which is provided at Exhibit 5.

No certificate ofcoITection has been filed in U.S. Patent No. 7,605,238 ("the '238 patent"). Moreover, the '238 patent has not been reexamined, and so no re-examination certificate has been issued in U.S. Patent No. 7,605,238.

No maintenance fees have been paid yet with respect to the '238 patent. The first maintenance fee for the '238 patent is not due until April 20, 2013, as shown by the attached

Patent Bibliographic Sheet obtained from Public PAIR on May 13,2011 and found in Exhibit 5.

Accordingly, there are no unpaid maintenance fees for the '238 patent.

(9) A STATEMENT THAT THE PATENT CLAIMS THE APPROVED

PRODUCT, OR A METHOD OF USING OR MANUFACTURING THE APPROVED

PRODUCT, AND A SHOWING WHICH LISTS EACH APPLICABLE PATENT CLAIM

AND DEMONSTRATES THE MANNER IN WHICH AT LEAST ONE SUCH PATENT Patent No.: 7,605,238 - 9- Docket No.: 029420.0155-US02

CLAIM READS ON THE APPROVED PRODUCT OR A METHOD OF USING OR

MANUFACTURING THE APPROVED PRODUCT:

U.S. Patent No. 7,605,238 claims the Approved Product. At least claims 1-8, 10,

12,15,18,21 and 24-32 read on the Approved Product. These claims are set forth below.

1. An isolated human antibody or antigen binding portion thereof that: (a) is IgG 1 isotype; (b) binds human CTLA-4 with a binding affnity of about 108 MOl or

greater; and (c) inhibits binding of human CTLA-4 to B7-1 and B7-2.

2. The antibody of claim 1, wherein the antibody comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 17, 19 and 23.

3. The antibody of claim 1, wherein the antibody comprises at least one light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOS: 7,9 and 13.

4. An isolated human monoclonal antibody, or antigen-binding portion thereof that is IgG 1 isotype and specifically binds to human CTLA-4, wherein the antibody or antigen-binding portion thereof has a binding affnity that is about 108 M-1 or greater affnity.

5. The antibody of claim 4, wherein the antibody comprises at least one amino

acid sequence selected from the group consisting of SEQ ID NOS:17, 19 and 23.

6. The antibody of claim 4, wherein the antibody comprises at least one amino

acid sequence selected from the group consisting of SEQ ID NOS: 7,9 and 13.

7. An isolated monoclonal antibody, or an antigen binding portion thereof, which is IgG 1 isotype and competes for binding to a human CTLA-4 polypeptide with an antibody comprising the amino acid sequence set forth in SEQ ID NO: i 7, and the amino acid sequence set forth in SEQ ID NO:7, wherein the monoclonal antibody or antigen-binding portion thereof has a binding affnity that is about 108 M-I or greater affnity.

8. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain variable region derived from a human V H 3-30.3 gene; and (b) a light chain variable region derived from a human V K A-27 gene.

* * * *

10. The antibody of claim 4, wherein the antibody comprises: (a) a heavy chain variable region derived from a human V H 3-30.3 gene; and (b) a light chain Patent No.: 7,605,238 - 10- Docket No.: 029420.0155-US02

variable region derived from a human V K A-27 gene.

* * * *

12. The antibody of claim 1, wherein the antibody comprises a the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ IDNO: 7.

* * * *

15. The antibody of claim 4, wherein the antibody comprises the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ IDNO: 7.

* * * *

18. The antibody of claim 1, comprising: (a) a heavy chain variable region comprising CDRl, CDRZ, and CDR3 sequences set forth in SEQ ID NOS:27, 32 and 37, respectively; and (b) a light chain variable region comprising CDR 1, CDRZ, and CDR3 sequences set forth in SEQ ID NOS:24, 29 and 35, respectively.

* * * *

21. The antibody of claim 4, comprising: (a) a heavy chain variable region comprising CDRl, CDR2, and CDR3 sequences set forth in SEQ ID NOS:27, 32 and 37, respectively; and (b) a light chain variable region comprising CDRl, CDRZ, and CDR3 sequences set forth in SEQ ID NOS:24, 29 and 35, respectively.

* * * *

24. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

25. The antibody of claim 4, wherein the antibody is a monoclonal antibody.

26. The antibody of claim 7, wherein the antibody is a monoclonal antibody.

27. The antibody of claim 1, wherein the antibody is a human antibody.

28. The antibody of claim 4, wherein the antibody is a human antibody.

29. The antibody of claim 7, wherein the antibody is a human antibody.

30. A pharaceutical composition comprising the antibody 'of claim 1, and a pharaceutically acceptable carrier. Patent No.: 7,605,238 - 11 - Docket No.: 029420.0155-US02

31. A pharaceutical composition comprising the antibody of claim 4, and a pharaceutically acceptable carrier.

32. A pharmaceutical composition comprising the antibody of claim 7, and a pharaceutically acceptable carrier.

Pursuant to 37 C.F.R. § 1.740(a)(9), a showing which demonstrates the manner in which one claim reads on the Approved Product is set forth herein below.

CLAIM The Approved Product 1. An isolated human antibody or antigen Ipi1imumab (the active ingredient in binding portion thereof that: YERVOy™) is an isolated human antibody. See section 11 "Description" in approved labeling attached as Exhibit 3.

(a) is IgGl isotype; Ipilmumab has an IgG 1 isotype. See section 11 "Description" in approved labeling.

(b) binds human CTLA-4 with a binding Ipilmumab binds human CTLA-4 with a affnity of about 108 MOl or greater; and binding affnity of about 108 M-I or greater. See section 12.1 "Clinical Pharmacology, Mechanism of Action" in approved labeling.

(c) iMibits binding of human CTLA-4 to B7-1 Ipilmumab blocks the interaction of CTLA-4 and B7-2. with its ligands, CD80 and CD86. As noted at

column 2, lines 50-55 of the '238 patent, B7-1 is also called CD80 and B7-2 is also called CD86. Patent No.: 7,605,238 - 12 - Docket No.: 029420.0155-US02

(l0) A STATEMENT BEGINING ON A NEW PAGE OF THE

RELEVANT DATES AND INFORMATION PURSUANT TO 35 U.S.C. §156(g) IN ORDER

TO ENABLE THE SECRETARY OF HEALTH AND HUMAN SERVICES OR THE SECRETARY OF AGRICULTURE, AS APPROPRIATE, TO DETERMINE THE APPLICABLE REGULA TORY REVIEW PERIOD AS FOLLOWS:

(i) FOR A PATENT CLAIMING A HUMAN DRUG, ANTIBIOTIC, OR

HUMAN BIOLOGICAL PRODUCT, THE EFFECTIVE DATE OF THE INVESTIGATIONAL

NEW DRUG APPLICATION (IND) AND THE IND NUMBER; THE DATE ON WHICH A

NEW DRUG APPLICATION (NDA) OR A PRODUCT LICENSE APPLICATION (PLA)

WAS INITIALLY SUBMITTED AND THE NDA OR PLA NUMBER; AND THE DATE ON

WHICH THE NDA WAS APPROVED OR THE PRODUCT LICENSE ISSUED:

An original investigational new drug application ("IND") was submitted by

Medarex, now a wholly-owned subsidiary of Bristol-Myers Squibb Co. ("BMS"), on July 12,

2000 and was received by FDA on July 13,2000. A copy of FDA's acknowledgement letter is provided at Exhibit 6. The FDA assigned BB-IND No. 91S(rtõ"tis IND, which became -- ~_.... effective 30 days after FDA's receipt date, namely, on August 12,2000. 1

A biologics license application ("BLA") was submitted by BMS on.Juae 25i-201Qj and acknowledged as received on this date in a letter from FDA dated July 8, 2010 (Exhibit 7).

The Submission Tracking Number (STN) assigned to this BLA was BL 125377/0. The BLA -- ., was approved on ~h 25, 2011 (Exhibit 4). Patent No.: 7,605,238 - 13 - Docket No.: 029420.0155-US02

(1 I) A BRIEF DESCRIPTION BEGINING ON A NEW PAGE OF THE

SIGNIFICANT ACTIVITIES UNDERTAKEN BY OWNER, THE MARKETING

APPLICANT, DURING THE APPLICABLE REGULATORY REVIEW PERIOD WITH

RESPECT TO THE APPROVED PRODUCT AND THE SIGNIFICANT DATES

APPLICABLE TO SUCH ACTIVITIES:

In accordance with 37 C.F.R. § L.740(a)(l I), a list of significant activities, undertaken by the Marketing Applicant, its predecessors, and affliates, in BB-IND No. 9186 and

BL 125377/0 during the applicable regulatory review period with respect of the approved product is provided at Exhibit 8. Patent No.: 7,605,238 - 14- Docket No.: 029420.0155-US02

(12) A STATEMENT BEGINING ON A NEW PAGE THAT IN THE

OPINION OF THE APPLICANT THE PATENT is ELIGIBLE FOR THE EXTENSION AND

A STATEMENT AS TO THE LENGTH OF EXTENSION CLAIMED, INCLUDING HOW

THE LENGTH OF EXTENSION WAS DETERMINED:

(a) Statement of the eligibility of the patent for extension under 35 U.S.C.

§ 156(a):

Section 1 56(a) provides, in relevant part, that the term of a patent which claims a product, a method of using a product, or a method of manufacturing a product shall be extended

if (i) the term of the patent has not expired before an application for extension is submitted; (ii)

the term of the patent has never been extended under 35 U.S.C. §156(e)(1); (iii) the application

for extension is submitted by the owner of record of the patent or its agent in accordance with 35

U.S.C. §156(d); (iv) the product has been subject to a regulatory review period before its

commercial marketing or use; and (v) the permission for the commercial marketing or use of the product after such regulatory review period is the first permitted commercial marketing or use of the product using the provision of law under which such regulatory review period occurred.

As described below by corresponding number, each of these elements is satisfied here:

(i) Pursuant to 35 U.S.C. §154, the term of United States Patent No.

7,605,238 is currently set to expire on August 24, 2020. This Request is, therefore, being

submitted prior to the expiration of the term of United States Patent No. 7,605,238.

(ii) The term of this patent has never been extended under 35 U.S.c.

§156(e)(1). Patent No.: 7,605,238 - 15 - Docket No.: 029420.0155-US02

(iii) This Request is being submitted by Medarex, the owner of record of

United States Patent No. 7,605,238. (See Exhibit 2). Medarex is the owner of record by virtue of duly recorded assignments discussed above. This Request is submitted in accordance with 35

U .S.C. § 1 56( d) in that it is submitted within the sixty-day period beginning on March 25, 2011, the date the product received permission for marketing under section 351 of the Public Health

Service Act (PHSA) (42 U.S.C. §262), and ending on May 24, 2011. Moreover, this Request contains the information required under 35 U.S.C. §156(d).

(iv) As evidenced by the March 25, 2011 letter from the FDA to BMS (Exhibit

7), the product was subject to a regulatory review period under section 351 of the Public Health

Service Act (PHSA) (42 U.S.C. §262) before its commercial marketing or use.

(v) The permission for the commercial marketing of the YERVOYTM

(ipilimumab) Injection product is the first permitted commercial marketing and use of the product, as defined in 35 U.S.C. §156(f), under section 351 of the Public Health Service Act

(PHSA) (42 U.S.C. §262). (See, e.g., Section (4), above.)

(b) Statement as to length of extension claimed.

The term of U.S. Patent No. 7,605,238, now expiring August 24,2020, should be extended for 398 days, or to September 26,2021, in accordance with 35 U.S.C. §156.

As set forth in 35 U.S.C. § 1 56(g)(l), the regulatory review period equals the length of time between the effective date ofBB-IND No. 9186 of August 12,2000, and the submission of the BL 125377/0 on June 25, 2010 (i.e., the "testing phase"), a period of 3,604

days, plus the length of time between the submission of the BL 125377/0 on June 25,2010 to

BLA approval ori March 25, 2011 (i.e., the "approval phase"), a period of274 days. These two periods added together equal 3,878 days. Patent No.: 7,605,238 - 16 - Docket No.: 029420.0155-US02

Pursuant to 37 C.F.R. § 1 .775(d), the term of the patent as extended is determined by subtracting from the 3,878 day regulatory review period the following:

(i) 3,356 days, which is the number of days in the IND and BLA periods on or before the issuance of U.S. Patent No. 7,605,238 on October 20,2009; and

(ii) 124 days, which is one-half the number of days remaining in the IND period after the subtraction of 3,356 days above (wherein half days are ignored for purposes of this subtraction, as provided by 37 C.F.R. § 1.775(d)(1)(iii)).

From the foregoing calculation, an extension of 398 days results, Le., the remaining period under 35 U.S.C. § I 56(g)(l)(B)(i) (124 days) plus the remaining period under

35 U.S.C. § 156(g)(1 )(B)(ii) (274 days). This length of an extension would provide a new expiration date for U.S. Patent No. 7,605,238 of September 26, 2021. However, this extension period is subject to two further potential limitations under 35 U.S.C. § 156. Neither of these

potential limitations limits the term of the patent.

First, under 35 U.S.C. § 156(g)(6)(A), a maximum extension of five years is

permitted (i.e., 1826 days in this case). Since the current expiry date of U.S. Patent No.

7,605,238 is August 24,2020, no patent term extension could extend the term of the patent beyond August 24,2025. Consequently, this provision does not operate to limit the possible extension available to U.S. Patent 7,605,238.

Second, under 35 U.S.C. § 156(c)(3), the calculated extension period cannot lead to a patent term that would result in a patent term exceeding 14 years after the date of approval, that is, a patent term expiring after March 25, 2025. In this case, 35 U.S.C. §156(c)(3) also does not operate to limit the possible extension available to U.S. Patent 7,605,238. Patent No.: 7,605,238 - 17 - Docket No.: 029420.0155-US02

Accordingly, United States Patent No. 7,605,238 is eligible for a patent term extension of 398 days.

(13) A STATEMENT THAT APPLICANT ACKNOWLEDGES A DUTY TO

DISCLOSE TO THE COMMISSIONER OF PATENTS AND TRADEMARKS AND THE

SECRETARY OF HEALTH AND HUMAN SERVICES ANY INFORMATION WHICH is

MATERIAL TO THE DETERMINATION OF ENTITLEMENT TO THE EXTENSION

SOUGHT (SEE 37 C.F.R. §1.765).

Applicant acknowledges a duty to disclose to the Commissioner of Patents and

Trademarks and the Secretary of Health and Human Services any information which is material to the determination of entitlement to the extension sought.

In accordance with the duty of disclosure described in 37 C.F.R. § 1.765 and acknowledged under 37 C.F.R. § 1.740(13), Applicant wishes to inform the Offce that two patent term extension applications have been fied concurrently with respect to the regulatory review period for YERVOYTM (ipilimumab) Injection. Such patent term extension applications are with respect to U.S. Patent No. 7,605,238 (i.e., the present application) and U.S. Patent No.

6,984,720. It is requested that the Offce examine these extension applications concurrently so that a meaningful election can be made upon the receipt of a Notice of Final Determination and

Requirement of Election as to which patent to ultimately extend in accordance with 37 C.F.R. §

1.785. Patent No.: 7,605,238 - 18 - Docket No.: 029420.0155-US02

(14) THE PRESCRIBED FEE FOR RECEIVING AND ACTING UPON THE

APPLICATION FOR EXTENSION (SEE 37 C.F.R. §1.20U)):

Please charge our Deposit Account No. 50-0740 in the amount of $1,120.00 to

cover the fee for a request for extension of patent term. The Director is hereby authorized to charge our Deposit Account No. 50-0740, under Docket No. 029420.00155, for any deficiency in the fees fied, asserted to be fied or which should have been fied herewith (or with any paper hereafter fied in this application by this firm), to prevent this application from being

inadvertently abandoned. A duplicate of this Request (without Exhibits 1-8) is attached.

(15) THE NAME, ADDRESS, AND TELEPHONE NUMBER OF THE PERSON TO WHOM INQUIRIES AND CORRESPONDENCE RELATING TO THE APPLICATION FOR PATENT TERM EXTENSION ARE TO BE DIRECTED:

Natalie M. Derzko COVINGTON & BURLING LLP 1201 Pennsylvania Avenue, N.W. Washington, DC 20004-2401 Telephone No.: (202) 662-6000 Facsimile No.: (202) 662-6291 Patent No.: 7,605,238 - 19 - Docket No.: 029420.0155-US02

Pursuant to 37 C.F.R. §1.740(b), this Request for Extension of Patent Term Under

35 U.S.C. § 156, including Exhibits 1-8, is accompanied by two additional copies, for a total submission of three copies.

Dated: May 16,2011

By Nata 'e M. Derzko Registration No.: 48,10 Paul J. Berman Registration No.: 36,744 COVINGTON & BURLING LLP 1201 Pennsylvania Avenue, N.W. Washington, DC 20004-2401 (202) 662-6000 Attorneys for Applicant ~~~i'oìt l

111111111111111111111111111111111111111111111111111111111111111111111111111 US007605238B2

(12) United States Patent (10) Patent No.: US 7,605,238 B2 Korman et aI. (45) Date of Patent: *Oct. 20, 2009

(54) HUMAN CTLA-4 ANTIBODIES AND THEIR 5,612,205 A 3/1997 Kay USES 5,625,126 A 4/1997 Lonberg et al. RE35,500 E 5/1997 Rhodes (75) Inventors: Alan J. Korman, Piedmont, CA (US); 5,633,425 A 5/1997 Lonberg et al. Edward L. Halk, Sunnyvale, CA (US); 5,643,763 A 7/1997 Dunn Nils Lonberg, Woodside, CA (US); 5,648,471 A 7/1997 Buttam 5,661,016 A 8/1997 Lonberg et al. Yashwant M. Deo, East Brunswick, NJ 5,693,761 A 12/1997 Queen (US); Tibor P. KeIer, Ottsvile, PA (US) 5,693,792 A 12/1997 Torii 5,697,902 A 12/1997 Goldenberg (73) Assignee: Medarex, Inc., Princeton, NJ (US) 5,703,057 A 12/1997 Johnston 5,714,350 A 2/1998 Co (*) Notice; Subject to any disclaimer, the term of this 5,721,367 A 2/1998 Kay patent is extended or adjusted under 35 5,733,743 A 3/1998 Johnson U.S.c. 154(b) by 0 days. 5,741,957 A 4/1998 Deboer 3,7-0,1'72----/-l998--eade Ths patent is subject to a terminal dis- 5,756,687 A 5/1998 Denman et al. claimer. 5,770,197 A 6/1998 Lisleyet al. 5,770,429 A 6/1998 Lonberg et al. 5,773,253 A 6/1998 Linsleyet al. (21) Appl. No.: 09/948,939 5,777,085 A 7/1998 Co et al. 5,789,215 A 8/1998 Berns et al. (22) Filed: Sep. 7, 2001 5,789,650 A 8/1998 Lonberg et al. 5,811,097 A 9/1998 Allison et al. (65) Prior Publication Data 5,814.318 A 9/1998 Lonberg et al. US 2002/0086014 Al Jul. 4, 2002 5,821,332 A * 10/1998 Godfeyet al...... 530/350 5,827,690 A 10/1998 Meade et al. Related U.S. Application Data 5,844,095 A 12/1998 Linsleyet al. 5,855,887 A 1/1999 Allison et al. (63) Continuation-in-part of application No. 09/644,668, 5,874,299 A 2/1999 Lonberg et al. filed on Aug. 24, 2000, now Pat. No. 6,984,720. 5,877,397 A 3/1999 Lonberg et al. 5,885,796 A 3/1999 Lins1eyet al. (60) Provisional application No. 601150,452, fied on Aug. 5,916,771 A 6/1999 Hori et al. 24, 1999. 5,939,598 A 8/1999 Kucher1apati et al. 5,968,510 A 10/1999 Linsleyet al. (51) Int. CL. 5,977.318 A 11/1999 Linsleyetal. C07K 16/28 (2006.01) 6,051,227 A 4/2000 Allison et ai. A61K 39/395 (2006.01) 6,075,181 A 6/2000 Kucher1apati et al. 6.114,598 A 9/2000 Kucherlapati et al. (52) U.S. Cl...... 530/388.15; 530/388.1; 6,150,584 A 11/2000 Kucher1apati et al. 530/388.22; 530/388.75; 424/141.; 424/142.1; 6,162,963 A 12/2000 Kucherlapati et al. 4241143.1; 4241144.1 6,207,156 Bl'" 3/2001 Kucmoo et al...... 424/154.1 (58) Field of Classification Search ...... 530/391.7, 6,255,458 Bl 7/2001 Loiiherg et al. 530/388.22, 350 6,632,927 B2'" 1 0/2003 Adar et al...... 530/387.3 See application file for complete search history. 6,682,736 Bl * 112004 Hanson et al...... 424/144.1 6,719,972 Bl 4/2004 Gribben et al. (56) References Cited 6,984,720 Bl'" 112006 Korma et al...... 530/388.22 U.S. PATENT DOCUMENTS (Continued) 4,399,216 A 8/1983 Axel FOREIGN PATENT DOCUMNTS 4,681,581 A 711987 Coates 4,683,195 A 711987 Mullis CA 2205680 Bl ll1998 4,683,202 A 7/1987 Mullis 4,735,210 A 4/1988 Goldenberg (Continued) 4,740,461 A 411988 Kaufman OTHER PUBLICATIONS 4,816,397 A 311989 Boss Rudikoffet aI., 1982, Proc. Natl. Acad. Sci. USA, ,79: 1979-1983." 4,921,040 A 5/1990 Ueniendue1 et al. 4,959,455 A 911990 Clark (Continued) 5,101,827 A 411992 Goldenberg 5,151,510 A 9/1992 Stec Primary Examiner-Ila Ouspenski 5,194,594 A 311993 Khawli (74) Attorney, Agent, or Firm-Baker Botts LLP 5,434.131 A 711995 Linsley 5,530,101 A 6/1996 Queen (57) ABSTRACT 5,545,806 A 8/1996 Lonberg et al. 5,545,807 A 811996 Surani The present invention provides human sequence antibodies 5,556,763 A 911996 Ochoa et ai. against human CTLA-4 and methods of treating human dis- 5,569,825 A 10/1996 Lonberg et al. eases, infections and other conditions using these antibodies. 5,585,089 A 12/1996 Queen 5,591,669 A 111997 Krimpenfoit 32 Claims, 24 Drawing Sheets US 7,605,238 B2 Page 2

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FIG.4 u.s. Patent Oct. 20, 2009 Sheet 5 of24 US 7,605,238 B2

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u u E- E- CJ CJ u u C) u CJ E- U i: E- i: i: CJ u E- CJ C) "" "" E- C) CJ E- U u u CJ CJ i: u E- U CJ E- E- () U i: u i: CJ CJ '- u u CJ i: u i: E-i u i i: CJ CJ U U U 0 i: C) i: E- u C' CJ .i .i E- i: CJ CJ CJ CJ E- i: (' i: i: i: E- E- E- U i: CJ u CJ u CJ i: E- (' i: CJ i: CJ i: E- i: .i CJ (' i: u CJ CJ i: u i: ~ E- E- ~ i: u CJ CJ i: E- (' (' CJ u i: ~ CJ CJ CJ i: CJ ~ i: i: 0 rl CJ i: CJ E- rl Q) u CJ i: E- CJ CJ i: CJ CJ Q) 0 i: E- -.-?- ?- i I -.- i: CJ CJ E- CJ i: CJ CJ .¡ U i .¡ CJ i: i: E- i: E- i: CJ CJ I I U CJ 0 OJ E- i: 0 u i: Pi Q) E- C) i: E- U Pi E- CJ U) U) i: il 0 i: C) i: u 0 C) E- E- U 0 i: 1- CJ .i H r- U E- CJ CJ u t' i: E- i: E- (' E- :¡ CJ CJ i: CJ co E- E- i: 0 CJ C\ t' E- 0 ~ E- 10 CJ E- i: i- E- CJ U u lu CJ U CJ CJ E- L( '0 E- i: Ql CJ U U r- E- CJ N I CJ 0 '+ CJ ~ E- (' U CJ iu CJ E- 0 i: 0 i- E- (9 0 C) i~ ti i: 0 0 u ig 8 - T" u i U 0 u il i: Ii: ii; LL u E- CJ 0 8 E- 0 C) ICJ i: I~ i 0 E- i; Z 1 i~ i-i 10 Z I I u i: CJ u ICJ Q CJ 18 CJ I u CJ CJ CJ p-~ ,i: i: i i: i: E- E- i~ H ii: I H i: N 'uCJ u i CJ i: u ICJ u ig CJ CJ U CJ CJ 0 ê5 i: i: E- U I i: 1M 0 i: i~ ¡: O¡~~ 1 E- CJ i: i; 10 ¡i; CI 0 CJ i 8 li-'" CJ (J u C' CJ E- -i CJ 8 i -i E- i: i: I CJ i I~ i: CJ 'M E- i U I 2j (' u CJ i: i i: E- i ,0Ii: i U I i: i: i~ u i i: CJ i i: u Mi: i~ 0 ¡ ~ P: I CJ IE- I~ i U C' 0 U QE- 0 i CJ 18 "" i: u u CJ i: u Q ~ ICJ I i: CJ i: I lg CJ I CJ "" "" 1 E- CJ 18 E- -i 0 CJ E- E- i: CJ ~ ig i ti l~ c: CJ 0 E- 0 r- f- r- E- I i: -i i~ i: 0 I~ E- i i-i i: E- CJ ii: E- i; 1 CJ CJ 0 I CJ 0 10 u E- U E- i: I i: i; i; i: (; u 0 I 0 i: -i ig i: CJ u i: 0 CJ 10 CJ i: (' E- 0 ie i: ICJ E- (' i: 0 ie I u (' 10 0

OJ GJ r- i: r- r- r- e- If. i: i. 11 "'-.- N N N N rl -.- rl rl i,. .. I .. I i .- I .. I,. I I i: s rl .. i: rl .. i: rl .. i:M .. i: rl .. ,. s .. H" ,. .. '" i: I. Q'- i: '- i: \D i: \D ¡"N K OJ a ii N N ~oll :: 0 II ::oi: :: 0 i: :: GJ r, :: Iï :: fi :: C' rl ~ :: rl "" ::H ,. ::H"" ::H.. :: 0 H ::H ::H ~ .lJ "" =f" atD

no :* PN VK L-1S: GAT TTC ACT eTC ACC ATe AGC AGC CTG CAG eCT GAA GAT 1'TT GCA ACT TAT TAe TGC CAÃ-CAGCDR3- TAT AAT N 1E:2 : o \Co ------Ji;l VK L-1S: AGT TAC CCT CC 00 1£2 : ------G ACG TTC GGe CAA GGG ACC AAG GTG GAA ATC AAA C/ =- t' t'- C\ o.. N .¡

FIG. 5 2of2 o ..rL Ô' ti= N~ QO Nc= ~ SEQ ID NOs: 14,16&18 (respecti vely) rJ VH 3-30.3 . Germline: CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCC ~ iaDl: ------~ 4B6 : ------~!" CDRl = VH 3 - 30.3: TCT GGA TTC ACC TTC AGT AGC TAT GCT ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG !" iaDl: ------A-- --- 4B6 : ------A- - - --

CDR2 VH 3 - 30.3: GCA GTT ATA TCA TAT GAT GGA AGC AAT AA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA ~0 lODl: A-- T------A------:- 4B6 : A-- T------C------G------PN N c= VH 3 - 30.3: GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCT GAG GAC ACG GCT GTG TAT TAC TGT c= iaDl: ------A-A - \0 4B6 : ------A-A CDR3 D7-27 J,,4b rz VH 3 - 3 0 . 3: GCG AGA =- tD iaDl: - - - - -G ACC GGC TGG CTG GGG CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA Gj tD- 4B6 : --- --G ------j .. 0.. N SEQ ID NOs:20&22 (respecti vely) .i VH 3-33 Germline: CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCG lB2 : ------

CDRl VH 3-33: TCT GGA TTC ACC TTC AGT AGC TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG lE2 : ------d CDR2 00 VH 3-33: GCA GTT ATA TGG TAT GAT GGA AGT AAT ~~ TAC TAT GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA .. lE2 : ------b- ti= N FIG. 6 ~ 1of2 00 NO: ~ .00 "' ~ ~i- i-=

o :-~ VH 3-33; GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCT GTG TAT TAC TGT ~~ lE2 : ------T------~ ~ CDR3 ~ JH3b VH 3-33: GCG AGA GA lE2 : -CT CCC AAT TAT ATT GGT GCT TTT GAT GTC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA GI íL =- atD QO Q.. .i~ FIG. 6 20f2

d 00 -i Ô\ i.= N~ QO Nc: u.s. Patent Oct. 20, 2009 Sheet 9 of24 US 7,605,238 B2

E- i I U) ~ ~ : i () 0: U) i C\ i- Q t: ~ i: t: u ,: i Q t: I CJ i U i ~ ~ ~ :: H H H Iï ?- Iï i- t: H ~ ~ ~ ~ ¡: E- ti II (' ~ E- i: () i: ()(' a CJ CJ ø ii ~ Ai ii ~ i:Iï :: a E- ~ E- .- a s: () j o. il ?- M Ai .-~ () M o. ~ :s 0: ti i ai ?- i: :: i 'r- Q t: i ~ ~ Q 1I i .w U C! i 'rl U :z i () r: t ?- I .w ?- i il i- i () I ~ Oi i ?- Ç: , () () I il ,. I () i I ri j ~ ~ i- I ~ il ti i u. I ti H i: u. CJ i U i il i- Ul u Q t: i i ?- H ~ H ?- U U) I I ?- Q CJ ?- () l I U Oi E- 0' ti i I ~ en .. i ~ ~ t- i M .. ~ I ~ : Q I ii Q IJ r- Iï r- o. r: .. () !ï ~ U () (9 L( en ,. r- E- i- - ,. ii H en E- ti r- E- Ul LL H . . H ti ~ E- ~ E- Iï i- rn Cl ,. 0 C! E- CJ E- Z i: i: 0 po i: ti Q Z IJ Q H E- ~ E- i ~ u. (' ti C! i- V' Q ~ IJ H E- (' ri (' C! u. H u: IJ II C! i: (' 0 ti ti u: Ul r: QI ri 0 () li E- ¡: ii E- ii CJ ~ i: ~ u. :: i: CI oi II H H H :: IJ (' Q c:

il il C' i: E" lI i: lI ~.i- C\ rl -r- i- i rl .. l .. i rl i: S i- .. i: rl .. i- s .. i-I .. H Cl i. 0\0 H ~ C\ ~ il o¡: ~om ~ il Iï :i Iï po (').-1 -. ?- rl -. po (' r- ¡: rl t .'J SEQ ID NOs:15,17&19 ( respectively) ~ ~ CDRI CDR2 ~ VH 3 - 3 0.3 Germline: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG a 10Dl: ------T------T F- - - - -N------4B6 : ------T------T F------H------o ..l" CDR3 9N N VH 3-30.3: RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAR c: c: lODl: ------1-- --- TGWLGPFDY WGQGTLVTVSS \C 4B6 : -- -V------1- - ---

rL ~=- ii SEQ ID NOs: 21 &23 (respectively) I- c: CDRI CDR2 ..o VH 3 -33 N Germline: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VIWYDGSNKYYADSVKG .¡ lE2 : ------~------~

CDR3 d VH 3 -33 : RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAR r: lE2 : .. ------F- - - - APNYIGAFDV WGQGTMVTVSS Ô' ti= Nw FIG.8 QO Nt: u.s. Patent Oct. 20, 2009 Sheet 11 of 24 US 7,605,238 B2

1.4

1.2 0 LO 1 (--10D1 ~ CO i L( va 0.8 a 0.6 0 0.4 0.2

0

0 0.0001 0.001 0.01 0.1 1 Antibody concentration, IJg/ml

FIG. 9 u.s. Patent Oct. 20, 2009 Sheet 12 of 24 US 7,605,238 B2

~ ~ 700 cen . 1001 -+CD 600 C 500 (.CD c 400 öQ) U) 300 ~C1 :J0 200 LL c 100 CO C1 0 ~ 0 0.01 0.1 1 10 Antibody concentration l Jlg/ml

FIG. 10 u.s. Patent Oct. 20, 2009 Sheet 13 of 24 US 7,605,238 B2

120

100 --1001 c ~hulgG1 "-a 80 :!.. (control) .- 60 ..c '# 40 20

0 0 0.1 1 10 Antibody Concentration, l1g/ml

FIG. 11 u.s. Patent Oct. 20, 2009 Sheet 14 of 24 US 7,605,238 B2

100 --1001 80 c 0 -ahulgG1 .. 60 (control) .-.0 ..c: 40 '* 20

0 0 0.01 0.1 1 10 Antibody Concentration, iig/ml

FIG. 12 u.s. Patent Oct. 20, 2009 Sheet 15 of 24 US 7,605,238 B2

A 9A5 Competition 2 -O3A4 --9A5 1.5 --10D1.3 0io - *' -486.12 -. --5A8 c: --BNI3.1 0.5 ~.. -147 --11E8 0 -.. -1E2 6666 2222 741 247 82 27 9 0 IgG (ng/ml) FIG. 13A

B 3A4 Competition 2.S --9AS 2 -O3A4 ~10D1.3 1.5 l( - *' - 4B6.12 -.0 c: 1 .... --SA8 .. --BNI3.1 -+-147 0.5 -D11E8 0 -"'-1E2 6666 2222 741 247 82 27 9 0 IgG Concentration (ng/ml) FIG. 13B

c 5A8 Competition 2 -O3A4 --9A5 1.5 --10D1.3 0l( - *' -4B6.12 -. --SA8 c: --BNI3.1 0.5 -.. -147 --11E8 0 -.. -1E2 6666 2222 741 247 82 27 9 0 IgG (ng/ml)

FIG. 13C u.s. Patent Oct. 20, 2009 Sheet 16 of 24 US 7,605,238 B2

"¡\09~D:zi-w~~~ N) co ~ ~ M co ~ ô' .. ., U! (0 ~ .. lf1~tl+?~ ffr~nq~ o o ai: OJ c: :e w o en (9 ã) +J c. C" rt E ~ T" ~ r- ~ o ~ 0. C' U . E N coN :€0) o .. .s CJ Ü C) cO - .. ~ i - CD ~ C9 u. C" LL .. " .2 C't¡ .2C9 i:z ~ ~t' ~ ~ N ~ (0 (p æ (p (0 l8 l( N "( o co N o ci ci ~ "' w 9017\f o ci ci (9 9017\f

T"C" Nor 'o M co C" N T" Hnnn~ Hr;nn~ o o c: o m c: ~ :; o 0; :p :p M a.Q) ~ o ~ "' LL E ~ a. ~ o C" E rt Ü ~ ~ '" o ~ ~ ~ .s . o ¡; ~,.s . C' r- t' I' .. (9 "" ~ C9 (9 o ~ .2 .. ~ ~ - .. .. u. t' LL ¡: c; ¡: C' C' C' C' ~ co co (0 co co æ(0 ~ N ~ on o N "( l( o ci ci o 90\1 \f LL soti\f u.s. Patent Oct. 20, 2009 Sheet 17 of 24 US 7,605,238 B2

'lo WithoutAb

Control Lymphocytes o C" HulgG1-FITC

-E 0 147-FfTC ü6 C\

..o 10D1-FITC

o 10° 101 102 103 104 FL2-H

""o WithoutAb PHA activated lymphocytes o (Y HulgG1-FITC

.e 147.nTC ::c C\0 oo 10D1-FITC

..o

102 103 104 FL2-H FIG. 14 u.s. Patent Oct. 20, 2009 Sheet 18 of 24 US 7,605,238 B2

l? Without Complement o With Complement 80

60

Ow(/ 40 .= r¡ 20

o 0.1 1.0 10 0.01 0.1 1.0 10 J.g/ml anti-CD3 mAb l1g/ml anti-CTLA4 mAb -20 1001

FIG. 15 u.s. Patent Oct. 20, 2009 Sheet 19 of 24 US 7,605,238 B2

15

o Anti-CD3 mAb - £l - Anti-CTLA4 mAb 1001

10 .en 3(J '*

5

o 0.001 0.01 0.1 1 10 Antibody Concentration, Jlg/ml

FIG. 16 ~ .rJ 0.2 i- 1 ~ anti-10D1 IgM Response -e Pre-Dose anti-10D1 IgG Response -- Pre-Dose ~ Monkey #1 0.9 Monkey #2 ~ (00.15 ~ -Day4 (! -Day4 ~ o -: Day 7 0 0.8 0"" : 0"" -'Day7 = o ~ - -)E- Day 14 0 0.7 - -)f - Day 14 ~ æ 0.1 - c (j ll 0.6 ....; .. (1 :; :¡ 0.5 0.05 .... aI__ -- 0.4 f 0 0.3 ~ .. . .-.1 :" ~- ...... o 0.2 1 1 """~ -i-i-.~i; = £:~: ~,~~J N o o g o o o 0 0 0 8 0 0 p .. co .. (x 0 co "" 0(x 0 ~ .. IJ .. .. T" .. N .. .. ~ T" ~ .. .. tA 10 C' Q .. .. o T" T" T" 0 Q T" .. .. T" \C Antibody Titer .. Antibody Titer T"

0.2 1 anti-10D1 fgG Response 1J anti-10D1 IgD Response -- Pre-Dose ..Pre-Dose =" 0.9 Monkey #1 i- Monkey #2 -Day4 ~ (! 0.15 -Day4 CD ~.. ~.. -: Day 7 0,8 -'Day7 or; .. oe N .. .. - -)E - Day 14 o 0.7 - -)E - Day 14 Q o .. .. i: o æ 0.1 ~ .. l\ 0.6 .. (j .. (j .. N :; "- ") "'_ :; 0.5 .i 0.05 "'''-~ -.. -- _v______v. .. 0.4 i- 0.3 -- ...... 1 .. o - .. . .. 0.2 o o o 0 o- o o o o o o o o o o 0 o o T" co .~ a: o .. ~ co -= (x .. ~ .. 10 .. T" .. co LO ...... ~ .. ~ d .. .. Antibody Titer .. rL Antibody Titer T" T" .. Ô\= VI Nw 00 FIG. 17 N0= u.s. Patent Oct. 20, 2009 Sheet 21 of 24 US 7,605,238 B2

Month a

Month 7

Month 6 co o Month 5 ~ . .. . , t9 ..0" " Month 4 " - " ..... LL " " " '. "0.. Month 3 , , ø Month 2 " " .' " "

...... 0...... Day 28 .' ..... (l (l .' .' ...... WW " :: :: .p" Day 14 ~~ . , Q.~~ 0- N Ø' b, Day? o00 Cl . uiô ,. i .. . .. :i ç/ g? II Infusion-Day 0 ..c: ;:.. .- i= ..... 0,-Q. 0 . CD c. E lP cS' .- E Screening I- ¡: +9 o It o It Q It o It '' C" (W (' (' "" "" (IWj6u) S3nl'9J\ 'fSd ~ t "0 .rr o 60 .a . Day 1 ~ Group 1 Group 2 o Day 51 ~ ~ ~ Day 64 CO t'~ tn 50 ~ a ai i: 40 -..iiJ. .2 ¡ &! 30 o ..~ Õ N Ul 9 ëi 20 oN ~ I,o cø E 10 th ns rF =- õ: o (D (D- N Vaccine + IgG1 Vaccine + MDX-010 N .. - Q (9 .. '6 Q) N .. .. .i o0, oo ..o 9l o- 0) i.m. X .. )( ~ o Æ c: a: :: :i :2 :i r.d -. Ó'= Day of Study: ---1--- 2-...... _...... 29...30...... -_...... , 51...... __._...... 64 u- N~ 00 FIG. 19 No: u.s. Patent Oct. 20, 2009 Sheet 23 of 24 US 7,605,238 B2

0i. 0T" 0 0 V V 0. a. it it iC

it "- it (j

cCD Q~ - 'ü 0 ~iü ~ (j (0 (0 II CD T" i: C Q ... - "ü 0 o ~ Ü T" ..a ~ :f a .0 it en N c: .. ;: . E ~ 0co t9- LL ~ CD T" (0 l- c Q II () i: ... "üÜ T"0 ,. C i: ro o + ~ :f cr C1i: C1i: .. .0 '0 C, C, ro ro ;: ;:

(J T" .-i: Q0 T 8 T" ~ a. ~ :f 0 0 0 0 0 0 0 o o 0 In 0 In 0 It 0 It "' M M N N ~ ~ 38 -/+ ueew (ewseid lO uo!lnI!P 000 L- L) A:lisualui a:iuÐ:isaJnol.: ueaw u.s. Patent Oct. 20, 2009 Sheet 24 of 24 US 7,605,238 B2

i I i: i II CD I I i o.~ . 0.. I s: I CI t' l I :: .i i 0.. I i CJ- I o ;: I -~ I i .. ~ i I :: ø I i c( '5 ...... _._...... ~. ..._ ...... ~_. _.... ._~.... _._a..... c. I i I II I .I o I c .I II .I :: . o . . æ I o . . . ..Õ I ., I :: I I c( N ..... _M...... _ ..... _.~...... I ...... ~. .~...... __._ _.... . I c. " - II LL ai. ~- I I W :: I '" E I . :¡ . . II I I . -...... -- -... .--... ..--r.I .... - -- - ...----I - .~.. --r .-~..-.-- _.- _..- .-~...- . + CI 00 CO "C (J U - ..Q) ....-o t1:: 0.-C E .0 (. "t i: ::

In In ~ ""o o xapul UO!lelnWllS US 7,605,238 H2 1 2 HUMA CTLA-4 ANTIBODIES AND THEIR Freeman et al. (1987), supra). In addition, expression ofthis USES antigen has been detected on cells of other lineages, such as monocytes (Freeman et aI., supra). REFERENCE TO RELATED APPLICATIONS T helper cell (Th) antigenic response requires signals pro- vided by APC's. The fist signal is initiated by interaction of This application is a continuation-in-par of application the T cell receptor complex (Weiss, J. Clin. Invest. 86:1015 Ser. No. 09/644,668, filed Aug. 24, 2000, now U.S. Pat. No. (1990)) with antigen presented in the context of class II major 6 984 720 claims the benefit of U.S . Provisional Application histocompatibility complex (MHC) molecules on the APC No. 6ÒI150,452, filed Aug. 24,1999. The disclosureofAppli- (Allen, Immunol. Today 8:270 (1987)). This antigen-specific cation No. 60/150,452 is incorporated herein in its entirety. 10 signal is not suffcient to generate a full response, and in the absence of a second signal may actually lead to clonal inac- FIELD OF THE INVNTION tivation or anergy (Schwarz, Science 248: 1349 (1990)). The requirement for a second "costimulatory" signal provided by The present invention relates generally to molecular immu- the MHC has been demonstrated in a number of experimental nology and the treatment of human diseases. In paricular, it 15 systems (Schwartz, supra; Weaver and Unanue, Immunol. relates to novel hwnan sequence antibodies against human Today 11:49 (1990)). The molecular nature of this second CTLA-4 and methods of treating human diseases and infec- signal is not completely understood, although it is clear in tions using these antibodies. some cases that both soluble molecules such as interleuldn (IL )-1 (Weaver and U nanue, supra) and membrane receptors BACKGROUN OF THE INVENTION 20 involved in intercellular adhesion (Springer, Nature 346:425

(1990)) can provide costimulatory signls. The vertebrate imune system requires multiple signals to CD28 antigen, a homodimeric glycoprotein of the immu- achieve optimal immune activation; see, e.g., Janeway, Cold noglobulin superfamily (Arffo and Seed, Proc. Natl. Acad. Spring Harbor Symp. Quant. BioI. 54:1-14 (1989); Paul Wil- Sci. 84:8573-8577 (1987)), is an accessory molecule found liam E., ed. Raven Press, N.Y., Fundamental Immunology, 25 on most mature human T cells (Dame et aI., J. Immunol. 4th edition (1998), paricularly chapters 12 and 13, pages 411 131:2296-2300 (1983)). Current evidence suggests that ths to 478. Interactions between T lymphocytes (T cells) and molecule fìinctions in an alternative T cell activation pathway antigen presenting cells (APC) are essential to the imune distinct from that intiated by the T-cell receptor complex response. Levels of many cohesive molecules found on T (June et aI., Mol. Cell BioI. 7:4472-4481 (1987)). Mono- cells and APC's increase during an immune response 30 clonal antibodies (MAbs) reactive with CD28 antigen can (Springer et aI., A. Rev. Immunol. 5:223-252 (1987); S~aw augment T cell responses initiated by varous polyclonal and Slluzu, Current Opinion in Immunology, Eds. Kindt stimuli (reviewed by June et aI., supra). These stimulatory and Long, 1 :92-97 (1988)); and Hemler, Immunology Today effects may result from MAb-induced cytokine production 9: 1 09-113 (1988)). Increased Ievels of these molecules may (Thompsonetal.,Proc. Natl.Acad. Sci 86:1333-1337 (1989); help explain why activated APC' s are more effective at stimu- 35 and Lindsten et aI., Science 244:339-343 (1989)) as a conse- lating antigen-specific T cell proliferation than are resting quence of increased MRNA stabilization (Lindsten et al. APC's (Kaiuchi et aI., J. Immunol. 131 :109-114 (1983); Kreigeretal.,J. Immunol. 135:2937-2945 (1985); McKenzie, (1989), supra). Anti-CD28 mAbs can also have inhibitory effects, i.e., they can block autologous mixed lymphocyte J. Immunol. 141:2907-2911 (1988); and Hawrylowicz and reactions (Dame et a!., Proc. NaIl. Acad. Sci. 78:5096-6001 Unanue, J. Immunol. 141:4083-4088 (1988)). 40 (1981)) and activation of antigen-specific T cell clones (Les- T cell immune response is a complex process that involves slauer et aI., Eur. J. Immunol. 16: 1289-1296 (1986)). cell-cell interactions (Springer et aI., A. Rev. Immunol. 5:223- 252 (1987)), paricularly between T and accessory cells such Some studies have indicated that CD28 is a counter-recep- as APC's, and production of soluble imune mediators (cy- tor for the B cell activation antigen, B7/BB-1 (Linsley et a!., Proc. Natl. A cad. Sci. USA 87:5031-5035 (1990)). The tokines or Iymphokines) (Dinarello (1987) New Eng!. Jour. 45 B7/BB-1 antigen is hereafter referred to as the "B7 antigen". Med 317:940-945; Sallusto (1997) J. Exp. Med. 179:1109- 1118). Ths response is regulated by several T-cell surface The B7 ligands are also members of the immunoglobulin receptors, including the T-cell receptor complex (Weiss superfamiIy but have, in contrast to CD28, two Ig domains in their extracellular region, an N-terminal varable (V)-like (1986) Ann. Rev Immunol. 4: 593-619) and other "accessory" domain followed by a constant (C)-like domain. surface molecules (Allison (1994) Curr. Opin. Immunol. 50 Delivery of a non -specific costimulatory signal to the T cell 6:414-419; Springer (1987) supra). Many of these accessory molecules are naturally occurrng cell surface differentiation requires at least two homologous B7 family members found on APC's, B7-1 (aIso called B7, B7.1, or CD80) and B7-2 (CD) antigens defied by the reactivity of monoclonal anti- bodies on the surface of cells (McMichael, Ed., Leukocyte (also called B7.2 or CD86), both of which can deliver costimiùatory signals to T cells via CD28. Costimulation Typing III, Oxford Univ. Press, Oxford, N.Y. (1987)). 55 Early studies suggested that B lymphocyte activation through CD28 promotes T cell activation. requires two signals (Bretscher (1970) Science 169:1042- Using genetic fìisions of the extracellular portions of B7 1049) and now it is believed that all lymphocytes require two antigen and CD28 receptor, and Immunoglobulin (Ig) signals for their optimal activation, an antigen specific or C.gana.1 (constant region heavy chains), interactions clonal signal, as well as a second, antigen non-specific signaL. 60 between CD28 and B7 antigen have been characterized (Lin- (Janeway, supra). Freeman (1989) J. Immunol. 143:2714- sley et a!., J. Exp. Med. 173:721-730 (1991)). Immobilized 2722) isolated and sequenced a cDNA clone encoding a B cell B7Ig fìision protein, as well as B7 positive CHO cells, have activation antigen recognized by MAb B7 (Freeman (1987)J. been shown to co stimulate T cell proliferation. Immunol. 138:3260). COS cells transfected with this cDNA T cell stimulation with B7 positive CHO cells also specifi- have been shown to stain by both labeled MAb B7 and MAb 65 cally stimulates increased levels of transcripts for IL-2. Addi- BB-1 (Clark (1986) Human Immunol. 16:100-113; Yokochi tional studies have shown that anti-CD28 MAb inhibited IL-2 (1981) J. Immunol. 128:823; Freeman et aI., (1989) supra; production induced in certain T cell leukemia cell lines by US 7,605,238 B2 3 4 cellular interactions with a B cell leukemia line (Kohno et aI., SUMMAY OF THE INVENTION Cell. Immuno1. 131-1-10 (1990)). CD28 has a single extracellular varable region (V)-like The present invention provides a human sequence antibody domain (Arffo and Seed, supra). A homologous molecule, that specifically binds to human CTLA-4 and a human CTLA-4 has been identified by differential screening of a 5 sequence antibody that specifically binds to human CTLA-4 murine cytolytic- T cell cDNA library (Bruet (1987) Nature which is substantially free of non-immunoglobulin associ- 328:267 -270). ated human proteins. CTLA-4 is a T cell surface molecule that was originally In a related aspect, the invention also provides a therapeu- identified by differential screening of a murie cytolytic T 10 tically-effective human sequence antibody that specifically cell cDNA library (Bruet et-a1., Nature 328:267-270 binds to human CTLA-4. In some embodiments, the thera- (1987)). CTLA-4 is also a member of the immunoglobulin peutically-effective human sequence antibody binds to (Ig) superfamily; CTLA-4 comprises a single extracellular Ig CTLA -4 on the cell surface of normal human T cells. In other domain. CTLA-4 transcripts have been found in T cell popu- embodiments, the T cell subpopulations marked by CD anti- lations having cytotoxic activity, suggesting that CTLA-4 15 gens CD4, CD8, CD25, and CD69 remain stable during and might function in the cytolytic response (Brunet et aI., subsequent to the admstration of the therapeutically-effec- supra; Brunet et aI., Immuno1. Rev. 103-21-36 (1988)). tive human sequence antibody. In other embodiments, the Researchers have reported the clonig and mapping of a gene therapeutically-effective human sequence antibody binds for the human counterpart ofCTLA-4 (Dariavach et aI., Eur. CTLA-4 on the cell surface of normal human T cells. In other J. Immunol. 18:1901-1905 (1988)) to the same chromosomal 20 embodiments, the human sequence antibody well-tolerated in region (2q33-34) as CD28 (Lafage-Pochitaloff et aI., Immu- a patient. In a related embodiment, nogenetics 31:198-201 (1990)). Sequence comparison Also provided is a composition of polyclonal antibodies between this human CTLA-4 DNA and that encoding CD28 comprising a plurality of human sequence antibodies that proteins reveals signficant homology of sequence, with the 25 specifically bind to human CTLA-4. The composition of greatest degree of homology in the juxtamembrane and cyto- polyclonal antibodies can comprise at least about 2, 5, 10, 50, plasmic regions (Bninet et aI., 1988, supra; Dariavach et al., 100, 500 or 1000 different human sequence antibodies that 1988, supra). specifically bind to human CTLA-4. Some studies have suggested that CTLA-4 has an analo- The invention also provides human sequence antibodies gous fìinction as a secondar costimuIator (Linsley et aI., J 30 that specifically bind to human CTLA-4 and which block Exp. Med. 176:1595-1604 (1992); Wu et aI., J Exp. Med. binding of human CTLA-4 to human B7 or do not block 185: 1327-1335 (1997) Lindsley, P. eta1. U.S. Pat. Nos. 5,977, 318; 5,968,510; 5,885,796; and 5,885,579). However, others binding of human CTLA -4 to human B7. have reported that CTLA-4 has an opposing role as a damp- The invention also provides human sequence antibodies ener ofT cell activation (Krel (1995) J. Exp. Med. 182: 35 that bind to human CTLA-4 with an equilibrium association 459-465); Knimmel et aI., In!'l Immunol. 8:519-523(1996); constant (Ka) of at least 108 M-1. Also provided are human Chambers et aI., Immunity. 7:885-895(1997)). It has been sequence antibodies that bind to human CTLA-4 with an reported that CTLA-4 deficient mice suffer from massive equilbrium association constant (Ka) of at least 109 M-1. lymphoproliferation (Chambers et aI., supra). It has been The invention also provides hiunan sequence antibodies reported that CTLA-4 blockade augments T cell responses in 40 that specifically bind to human CTLA-4 that block binding of vitro (Wahinas et al. ,Immunity. 1:405 -413 (1994)) and in vivo human CTLA-4 to human B7 by at least about 10%, 20%, (Kearney (1995) J. Immunol. 155:1032-1036), exacerbates 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. antitumor immunity (Leach (1996) Science. 271: 1734-1736), and enhances an induced autoimmune disease (Luhder The invention also provides human sequence antibodies that specifically bind to human CTLA-4having an antibody (1998) J Exp. Med. 187:427-432). It has also been reported 45 that CTLA-4 has an alternative or additional impact on the heavy chain of either IgG or IgM. The IgG antibody heavy initial character of the T cell immiine response (Chambers chain can be IgGl, IgG2, IgG3 or IgG4. The invention also (1997) Curr. Opin. Immunol. 9:396-404; Bluestone (1997)J. provides human sequence antibodies wherein the antibody Immunol. 158:1989-1993; Thompson (1997) Immunity light chain is a kappa light chain. The human sequence anti- 7 :445-450). Ibs is consistent with the observation that some 50 body can be encoded by human IgG heavy chain and human autoimune patients have autoantibodies to CTLA-4. It is kappa light chain nucleic acids that comprise nucleotide possible that CTLA-4 blocking antibodies have a pathogenic sequences in their variable regions as set forth in SEQ ID role in these patients (Matsui (1999) J. Immunol. 162:4328- NO:2 through SEQ ID NO:23, respectively. 4335). The invention also provides a human sequence antibody Non-human CTLA-4 antibodies have be used in the vari- 55 wherein the human sequence antibody is encoded by human ous studies discussed above. However, one of the major IgG heavy chain and human kappa light chain nucleic acids impediments facing the development of in vivo therapeutic that comprise nucleotide sequences in their variable regions and diagnostic applications for antibodies in humans is the as set forth in SEQ ID NO: 16 and SEQ ID NO: 6, respectively. intrinsic immunogenicity of non-human immunoglobulins. For example, when immunocompetent human patients are 60 The invention also provides a hiinan sequence antibody administered therapeutic doses of rodent monoclonal anti- wherein the human sequence antibody is encoded by human bodies, the patients produce antibodies against the rodent IgG heavy chain and human kappa light chain nucleic acids immunoglobulin sequences; these human anti-mouse anti- that comprise nucleotide sequences in their varable regions bodies (HA) neutralize the therapeutic antibodies and can as set fort in SEQ ID NO: 18 and SEQ ID NO: 8, respectively. cause acute toxicity. These and other deficiencies in the pre- 65 The invention also provides a hiinan sequence antibody vious antibodies are overcome by the provision of human wherein the human sequence antibody is encoded by human antibodies to CTLA-4 by the present invention. IgG heavy chain and human kappa light chain nucleic acids US 7,605,238 H2 5 6 that comprise nucleotide sequences in their variable regions fragment or an analog thereof, whereby the animal expresses as set fort in SEQ ID NO:22 and SEQ ID NO: 12, respec- human sequence antibodies to the human CTLA -4. The trans- tively. genic non-human animal can be a transgenic mouse. The The invention also provides a human sequence antibody transgenic mouse can comprise HC07 or HCo12. wherein the human sequence antibody is encoded by heavy 5 The invention provides a hybridoma cell line comprising a chain and light chain variable region amino acid sequences as B cell obtained from a transgenic non-human animal having set for the in SEQ ID NO: 17 and SEQ ID NO:7, respectively. a genome comprising a human sequence heavy chain trans- The invention provides a human sequence antibody gene and a human sequence light chain trans gene, wherein wherein the human sequence antibody is encoded by heavy the hybridoma produces a human sequence antibody that chain and light chain variable region amino acid sequences as 10 specifically binds to human CTLA-4. In a related embodi- set for the in SEQ ID NO: 19 and SEQ ID NO:9, respectively. ment, the hybridoma secretes a human sequence antibody that The invention also provides a human sequence antibody specifically binds human CTLA-4 or binding fragment wherein the human sequence antibody is encoded by heavy thereof, wherein the antibody is selected from the group chain and light chain variable region amino acid sequences as consisting of: a human sequence antibody comprising heavy set for the in SEQ IDNO:23 and SEQ IDNO:13, respectively. 15 chain heavy chain CDR1, CDR2, and CDR3 sequences, The invention provides a human sequence antibody SYTMH (SEQ ID NO:27), FISYDGNMCYYADSVIZG wherein the human sequence antibody is encoded by human (SEQ ID NO:32) and TGWLGPFDY (SEQ ID NO:37), IgG heavy chain and human kappa light chain nucleic acids respectively, and Iight chain CDR1, CDR2, and CDR3 comprising varable heavy and light chain sequences from V sequences, RASQSVGSSYLA (SEQ ID NO:24), GAFSRAT gene segments VH 3-30.3 and VK A-27, respectively. 20 (SEQ ID NO:29), and QQYGSSPWT (SEQ ID NO:35), The invention also provides a human sequence antibody respectively, and heavy chain and light chain variable region wherein the human sequence antibody is encoded by human amino acid sequences as set forth in SEQ ID NO: 17 and SEQ IgG heavy chain and human kappa light chain nucleic acids ID NO:7, respectively; a human sequence antibody compris- comprising varable heavy and light chain sequences from V ing heavy chain CDR1, CDR2, and CDR3 sequences, gene segments VH 3-33 and VK L-15, respectively. 25 SYTMH (SEQ ID NO:27), FISYDGSNKADSVKG Some human sequence antibodies of the invention com- (SEQ ID NO:33) and TGWLGPFDY (SEQ ID NO:37), prise heavy chain CDR1, CDR2, and CDR3 sequences, respectively, and light chain CDR1, CDR2, and CDR3 SYTMH (SEQ ID NO: 27), FISYDGNNKYYADSVKG sequences, RASQSVSSSFLA (SEQ ID NO:25), GASSRAT (SEQ ID NO:32) and TGWLGPFDY (SEQ ID NO:37), (SEQ ID NO:30), and QQYGSSPWT (SEQ ID NO:35), respectively, and light chain CDR1, CDR2, and CDR3 30 respectively, and heavy chain and light chain variable region sequences, RASQSVGSSYLA (SEQ ID NO:24), GAFSRAT amino acid sequences as set forth in SEQ ID NO: 19 and SEQ (SEQ ID NO:29), and QQYGSSPWT (SEQ ID NO:35), ID NO:9, respectively; or a human sequence antibody of respectively. claim i, comprising heavy chain CDR1, CDR2, and CDR3 Some human sequence antibodies of the invention com- sequences, SYGMH (SEQ ID NO:28) VIWYGSNKYY- prise heavy chain CDR1, CDR2, and CDR3 sequences, 35 ADSVKG (SEQ ID NO:34) and APNYGAFDV (SEQ ID SYTMH (SEQ ID NO:27), FISYDGSNKADSVKG NO:38), respectively, and light chain CDR1, CDR2, and (SEQ ID NO:33) and TGWLGPFDY (SEQ ID NO:37), CDR3 sequences, RASQGISSWLA (SEQ ID NO:26), respectively, and light chain CDR1, CDR2, and CDR3 AASSLQS (SEQ ID NO:3l), and QQYNSYPPT (SEQ ID sequences, RASQSVSSSFLA (SEQ ID NO:25), GASSRA NO:36), respectively, and heavy chain and light chain vari- (SEQ ID NO:30), and QQYGSSPWT (SEQ ID NO:35), 40 able region amio acid sequences as set forth in SEQ ID respectively. NO:23 and SEQ ID NO: 13, respectively. Other human sequence antibodies of the invention com- The invention provides a pharmaceutical composition prise heavy chain CDR1, CDR2, and CDR3 sequences, comprising a human sequence antibody that specifically SYGMH (SEQ ID NO:28), VIWYGSNKYYADSVKG binds to human CTLA-4 and a pharaceutically acceptable (SEQ ID NO:34) and APNYIGAFDV (SEQ ID NO:38), 45 carrier. The pharmaceutical composition can fuher com- respectively, and light chain CDR1, CDR2, and CDR3 prise an agent effective to induce an immune response against sequences, RASQGISSWLA (SEQ ID NO:26), AASSLQS a target antigen. Also provided are chemotherapeutic agents. (SEQ ID NO:3l), and QQYNSYPPT (SEQ ID NO:36), In addition, antibodies to immunosuppressive molecules are respectively. also provided. The invention also provides human sequence antibodies 50 The invention provides a method for inducing, augmenting that specifically bind to human CTLA-4, wherein said human or prolonging an immune response to an antigen in a patient, sequence antibody is produced by a transgenic non-human comprising admiistering to the patient an effective dosage of anmaL. The transgenic non-human animal can be a mouse. a human sequence antibody that specifically binds to human The invention also provides a human sequence antibody CTLA-4, wherein the antibody blocks binding of human that specifically bind to human CTLA-4 that is a Fab frag- 55 CTLA -4 to human B7 . The antigen can be a tumor antigen, or ment. the antigen can be from a pathogen. The tumor antigen can The invention provides a poIyvalent complex comprising also be telomerase. The pathogen can be a vinis, a bacterium, at least two human sequence antibodies each of which spe- a fìingus or a parasite. The pathogen can also be an HN. This cifically binds to human CTLA-4. The two different antibod- method can fuer comprise administering the antigen, or a ies can be linked to each other covalently or non-covalently. 60 fragment or an analog thereof, to the patient, whereby the l1ie invention provides a nucleic acid encoding a heavy antigen in combination with the human sequence antibody chain of a human sequence antibody. The nucleic acid can induces, augments or prolongs the immune response. The comprise a nucleotide sequence as set forth in SEQ ID NO: 1. antigen can be a tumor antigen or a component of an amyloid The invention provides a transgenic non-human anial formation in the patient, such as a patient suffering from having a genome comprising a human sequence heavy chain 65 Alzheimer's disease and the antigen is AB peptide. This trans gene and a human sequence light chain transgene, which method can further comprise admnistering a cytokine to the animal has been immunized with a human CTLA-4, or a patient. US 7,605,238 B2 7 8

The invention provides a method of suppressing an types (e.g., IgG, IgA and/or IgM) of human monoclonal anti- imune response in a patient, comprising admistering to bodies that specifically bind to human CTLA-4. The isolated the patient an effective dosage of a polyvalent preparation 8 cells can be obtained from a trnsgenic non-hiunan animal, comprising at least two human sequence antibodies to human e.g., a transgenic mouse, which has been immunized with a CTLA-4 lined to each other. The invention also provides a purified or enrched preparation of human CTLA -4 antigen method of suppressing an immune response in a patient, (or antigenic fragment thereof) and/or cells expressing comprising admstering to the patient an effective dosage of hiunan CTLA-4. The transgenic non-human animal, e.g., a a polyclonal preparation comprising at least two human transgenic mouse, can have a genome comprising a human sequence antibodies to human CTLA-4. heavy chain trans gene and a human light chain trans gene. The The present invention further provides isolated or recom- 10 isolated 8-cells can be immortalized to provide a source (e.g., binant human sequence antibodies and human monoclonal a hybridoma) of human monoclonal antibodies to human antibodies which specifically bind to human CTLA-4, as well CTLA-4. as compositions containing one or a combination of such Accordingly, the present invention also provides a hybri- antibodies. Some of the human sequence antibodies of the doma capable of producing human monoclonal antibodies invention are characterized by binding to human CTLA-4 15 that specifically bind to human CTLA-4. The hybridoma can with high affty, and/or by blocking the interaction of include a 8 cell obtained from a transgenic non-hiunan ani- human CTLA-4 with its ligand, the human 87-1 and 87-2 mal, e.g. a transgenic mouse, having a genome comprising a molecules. Accordingly, the human sequence antibodies and Iiiunan heavy chain transgene and a human light chain trans- the human monoclonal antibodies of the invention can be gene fused to an imortlized cell. The transgenic non-hu- used as diagnostic or therapeutic agents in vivo and in vitro. 20 man animal can be immunized with a purified or enrched The human sequence antibodies of the invention can preparation of human CTLA-4 antigen and/or cells express- encompass various antibody isotypes, or mixtures thereof, ing human CTLA-4 to generate antibody-producing hybrido- such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, mas. IgD, and IgE. Typically, they include IgGl (e.g., IgGlk) and In yet another aspect, the invention provides a transgenic IgM isotypes. The human sequence antibodies can be fiill- 25 non-human animal, such as a transgenic mouse, which length (e.g., an IgG 1 or IgG4 antibody) or can include only an express human monoclonal antibodies (also referred to herein antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single as a ''HuMAb-Mouse™'') that specifically bind to human chain Fv fragment). Some human sequence antibodies are CTLA-4. The transgenic non-human animal can be a trans- recombinant human sequence antibodies. Some human genic mouse having a genome comprising a human heavy sequence antibodies are produced by a hybridoma which 30 chain trans gene and a human light chain transgene. The trans- includes a 8 cell obtained from a transgenic non-human ani- genic non-hiunan animal can be immunized with a purified or mal, e.g., a transgenic mouse, having a genome comprising a enrched preparation ofCTLA-4 antigen (or antigenic frag- human heavy chain lTansgene and a human light chain trans- ment thereof) and/or cells expressing the human CTLA-4. gene. Thehybridoma can be made by, e.g., fusing the 8 cell to The transgenic non-human animal, e.g., the transgenic an immortalized celL. Some human sequence antibodies of the 35 mouse, can be capable of producing multiple isotypes of invention are produced by hybridomas referred to as 4CS, human monoclonal antibodies to human CTLA-4 (e.g., IgG, 4ElO, 4E10.5, 5AS, 5C4, 5C4.1.3, 507, 507.1, 5ElO, IgA and/or IgM) by undergoing V-D-J recombination and 5ElO.12, 5Gl, 5G1.4, 6AlO, 6C9, 6C9.6, 6D9, 6D9.7, 6G4, isotype switching. Isotype switching may occur by, e.g., clas- 7E4, 7E4.4, 7E6, 7HS, SES, SES.4, SFS, SFS.19, SHl, 9SLO, sical or non-classical isotype switching. 9AlO.1, 989, 9C1, 9G5, 1058, 1085.S, 1089, 1089.2, lODl, 40 In another aspect, the present invention provides methods lOD1., lOEll, lOE4, 10E4.5, 1184, llDlO, llE4, llE4.1, for producing human sequence antibodies and human lIES, 11F10, 11F11, 11F9, 1 1Gl, llG1., 1C7, IHS.S, 2A7, sequence monoclonal antibodies that specifically react with 2A7.6, 2E2, 2E2.7, 2E7, 2E7.2, 2Gl, 2G1., 3C12, 3EIO, human CTLA-4. Some methods of the invention include 3ElO.5, 3E6, 3E6.0, 3FlO, 4A1, 486 and 486.12. Suffxes imunzing a trangenic non-human anmal, e.g., a trans- after the decimal point indicate different clonal isolates of the 45 genic mouse, having a genome comprising a human heavy same hybridoma cell lines. chain transgene and a human light chain transgene, with a Some human sequence anti-CTLA-4 antibodies of the purified or enriched preparation of human CTLA-4 antigen present invention can be characterized by one or more of the and/or cells expressing human CTLA-4. 8 cells (e.g., splenic following properties: a) specificity for human CTLA-4 (spe- 8 cells) of the animal can then be obtained and fused with cifically binding to human CTLA-4); b) a binding affnity to 50 myeloma cells to form immortal, hybridoma cells that secrete human CTLA-4 with an equilibrium association constant human monoclonal antibodies against human CTLA-4. (Ka) of atleast about 107M- 1, or about 10" M-i, or about 1010 Anti -hiunan CTLA -4 human monoclonal antibodies of the M-l to ioll M-l or higher; c) a kinetic association constant invention, or antigen binding portions thereof (e.g., Fab), can (ka) of at least about 103, about 104, or about 105 m-ls-l; be derivatized or linked to another functional molecule, e.g., and/or, d) a kinetic disassociation constant (kd) of at least 55 another peptide or protein (e.g., an Fab' fragment). For about 103, about Hf, or about 105 m-ls-l. example, an antibody or antigen-binding portion of the inven- In another aspect, the invention provides nucleic acid mol- tion can be fl.llctionally linked (e.g., by chemical coupling, ecules encoding the human sequence antibodies, or antigen- genetic fusion, noncovalent association or otherwise) to one binding portions, of the invention. Accordingly, recombinant or more other molecular entities. For example, the human expression vectors that include the antibody-encoding 60 sequence anti -CTLA -4 antibody, or antigen binding fragment nucleic acids of the invention, and host cells transfected with thereof, can be conjugated to a therapeutic moiety, e.g., a such vectors, are also encompassed by the invention, as are cytotoxic dnig, an enzymatically active toxin, or a fragment methods of making the antibodies of the invention by cultur- thereof, a radioisotope, or a small molecule anti-cancer drug. ing these host cells. The antibodies of the invention can also be conjugated to In yet another aspect, the invention provides isolated 65 cytotoxic pharmaceuticals, e.g., radio labeled with a cytotoxic 8-cells from a transgenic non-hiunan anmal, e.g., a trans- agents, such as, e.g., 131 I (e.g. Shen (1997) Cancer SO(12 genic mouse, which are capable of expressing various iso- Suppl):2553-2557), copper-67 (e.g., Deshpande (19SS) J. US 7,605,238 B2 9 10 Nuc!. Med. 29:217-225) or, e.g., conjugation to the ribosome A further understanding ofthe nature and advantages of the inactivating protein gelonin (e.g., Boyle (1996) 1. ofImmu- present invention may be realized by reference to the remain- no!. 18:221-230). ing portions of the specification, the figures and claims. In another aspect, the present invention provides composi- All publications, figures, GenBan Accession references tions' e.g., phannaceutical and diagnostic compositions, (sequences), AICC Deposits, patents and patent applications comprising a pharmaceutically acceptable carrer and at least cited herein are hereby expressly incorporated by reference one human monoclonal antibody of the invention, or an anti- for all purposes to the same extent as if each was so individu- gen-binding portion thereof, which specifically binds to ally denoted. human CTLA-4. Some compositions comprise a combina- 10 tion of the hiian sequence antibodies or antigen-binding BRIEF DESCRIPTION OF THE DRAWINGS portions thereof, preferably each of which binds to a distinct epitope. Compositions, e.g., pharmaceutical compositions, FIG. 1 shows schematics ilustrating the targeted insertion comprising a combination of at least one hiian sequence of a neo cassette into the Sma I site of the ¡.l exon. FIG. 1A) antibodies or at least one human monoclonal antibody of the 15 Schematic diagrani of the genomic structure of the ~l locus. invention, or antigen-binding portions thereof, and at least The filled boxes represent the ¡. exons; FIG. 1B) Schematic one bispecific or multispecific molecule of the invention, are diagram of the CmD targeting vector. The dotted lines also within the scope of the invention. denotes those genomic ¡. sequences included in the construct. For in vivo methods, the antibody, or antigen-binding por- Plasmid sequences are not shown; FIG. 1C) Schematic dia- gram of the targeted ¡.locus in which the neo cassette has been tion thereof (or a bispecific or multi specific molecule of the 20 inserted into ~i1. The box at the lower right shows those invention), can be administered to a human subject suffering RFLP's diagnostic of homologous recombination between from a T-cell-related disease, or a disease that can be amelio- the targeting construct and the ~l locus. The RFLP's were rated or prevented by augmenting or suppressing or prolong- detected by Southern blot hybridization using probe A, the ing an immune response. 25 915 bp Sac I fragment is shown in FIG. 1C. Hiian sequence monoclonal antibody and human FIG. 2 shows the results of experients demonstrating that sequence antibody compositions ofthe invention also can be soluble human sequence antibodies against human CTLA-4 administered in combination with other known therapies, inhbit the binding of recombinant soluble human CTLA -4 to e.g., an anti-cancer therapy. Accordingly, the invention pro- cells expressing mouse B7.1, as described in detail, below. vides a method for treating cancer in a subject comprising 30 administering a therapeutically effective amount of a phar- FIG. 3 shows the results of a competitive binding assay to maceutical composition of a human sequence antibody identify human sequence antibodies of the invention that together with a pharmaceutical carrier to the subject. Some recognize non-overlapping epitopes on human CTLA-4, as such methods include a vaccine. Some such vaccines include described in detail, below. a tumor cell vaccine, a GM -CSF -modified tumor cell vaccine, 35 FIG. 4 shows preliminar nucleotide sequence data for the or an antigen-loaded dendritic cell vaccine. In some such heavy and light chain fragment of the anti-CTLA-4 antibody methods, the cancer is prostate cancer, melanoma, or epithe- 10D1.. lial cancer. FIG. 5 shows the nucleotide sequences of the light chain Hiian sequence antibodies to human CTLA-4 can be variable Regions (V K) of Anti-Human CTLA-4 Antibodies. used in methods oftreatment requirig either stimulation of 40 The anti-CTLA-4 antibodies 10D! (SEQ ID NO: 6) and 4B6 immune responses or suppression. The former indication is (SEQ ID NO:8) derived from the V KA-27 germine sequence treated using antibodies that block binding ofhiian CTLA-4 (SEQ ID NO:4) are depicted at the top of the Figure. The to hiian B7. Diseases amenable to treatment by stimulation, anti-CTLA-4 antibody 1E2 (SEQ ID NO:12) derived from augmentation of prolonging of immune responses including the V K L-15 germline sequence (SEQ ID NO: 10) is shown at cancer, including cancers of the prostate, kidney or colon, 45 the bottom of the Figure. The V K sequences of three anti- pathogenic inections, diseases associated with auto-anti- CTLA-4 antibodies are aligned with their gerinine encoded gens, e.g., amyloidogenic diseases, including Alzheimer's V K gene sequences. The complementar determing resi- disease, and diseases with inflamatory or allergic compo- dues (CDR) are labeled. Dashes denote sequence identity. nents. Immunosuppression is achieved using a polyvalent FIG. 6 shows the nucleotide sequences of the heavy chain preparation comprising at least two different antibodies to 50 variable Regions (V H) of Anti-Human CTLA-4 Antibodies. human CTLA-4 that are lined to each other. Diseases ame- The anti -CTLA -4 antibodies 10D! (SEQ ID NO: 16) and 4B6 nable to treatment include graft versus host disease, host (SEQ ID NO: 18) derived from the V H 3-30.3 germine versus graft disease, autoimune diseases and infammation. sequence (SEQ ID NO:14) are depicted at the top of the In yet another aspect, the present invention provides a Figure. The anti-CTLA-4 antibody 1E2 (SEQ ID NO:22) method for detecting in vitro or in vivo the presence of human 55 derived from the V H 3-33 gerinine sequence (SEQ ID CTLA-4 antigen in a sample, e.g., for diagnosing a human NO:20) is shown at the bottom of the Figure. The V H CTLA-4-related disease. In some methods, this is achieved sequences of three anti-CTLA-4 antibodies are aligned with by contacting a sample to be tested, along with a control their germline encoded sequences. The complementary deter- sample, with a human sequence antibody or a human mono- mining residues (CDR) are labeled. Dashes denote sequence clonal antibody of the invention, or an antigen-binding por- 60 identity. tion thereof (or a bispecific or multispecific molecule), under FIG. 7 shows the predicted amino acid sequences of the conditions that allow for formation of a complex between the light chain Variable Regions of Anti-Hiian CTLA-4 Anti- antibody and hiian CTLA-4. Complex formation is then bodies. The predicted amino acid V K sequences of the anti- detected (e.g. using an ELISA) in both samples, and any CTLA -4 antibodies described in FI G. 5 are shown. The anti -- statistically significant difference in the formation of com- 65 CTLA-4 antibodies 10D! (SEQ 10 NO:7) and 4B6 (SEQ ID plexes between the samples is indicative the presence of NO:9) derived from the V KA-27 germline sequence (SEQ ID human CTLA.4 antigen in the test sample. NO: 5) are depicted at the top of the Figure. The anti -CTLA-4 US 7,605,238 H2 11 12 antibody 1E2 (SEQ ID NO:13) derived from the V K L-15 ies, or antigen-binding portions thereof, which bind to an germline sequence (SEQ ID NO: 11) is shown atthe bottom of epitope present on human CTLA-4. These human sequence the Figure. anti-CTLA-4 antibodies can act as functional antagonists FIG. 8 shows the predicted amno acid sequences of the (e.g., inhbiting the abilty of CTLA-4 to bind ligand or to heavy chain Variable Regions of Anti-Human CTLA-4 Anti- activate the cell, e.g., by inhbiting its ability to transmit a bodies. The predicted amino acid V H sequences of the anti- signal to the cell) or agonists (e.g., to simulate the effect of CTLA-4 antibodies described in FIG. 6 are shown. The anti- ligand). CTLA-4 antibodies 10Dl (SEQ IDNO:17) and4B6 (SEQ ID The human sequence antibodies of the invention can be NO: 19) derived from the V H 3-30.3 germline sequence (SEQ produced in a non-human transgenic anial, e.g., a trans- ID NO:15) are depicted at the top of the Figue. The anti- 10 genic mouse, capable of producing multiple isotypes of CTLA-4 antibody 1E2 (SEQ ID NO:23) derived from the V H human (e.g., monoclonal or polyclonal) antibodies to human 3-33 germline sequence (SEQ ID NO:21) is shown at the CTLA-4 (e.g., IgG, IgA anclor IgE) by undergoing V-D-J bottom of the Figure. recombination and isotype switching. Accordingly, various FIG. 9 shows the results of binding experiments ofMAb aspects of the invention include antibodies and antibody frag- 1 OD1 to recombinant human CTLA-4 by ELISA. MAb 10D1 15 ments' and pharmaceutical compositions thereof, as well as binds with dose-dependent and saturating kinetics to purified non-human transgenic anals, and B-cells and hybridomas recombinant CTLA-4. for making such monoclonal antibodies. Methods of using the FI G. 10 shows the binding of! OD 1 to a CTLA4-expressing antibodies of the invention to detect a cell expressing human T-cell line. These data show that MAb 1 OD 1 binds with dose- CTLA -4 or a related, cross-reactive growth factor receptor, or dependent and saturating kinetics to cells expressing CTLA- 20 to iiùiibit growth, differentiation anclor motility of a cell 4. expressing human CTLA-4, either in vitro or in vivo, are also FIG. 11 shows inhbition of binding of human B7.2 Ig to encompassed by the invention. CTLA4-expressing T-cells. These data show that MAb 1 OD 1 Except when noted, the terms "patient" or "subject" are can effciently block B7.2 binding to CTLA-4 as compared to used interchangeably and refer to mammals such as human a control human MAb. 25 patients and non-human primates, as well as experimental FIG. 12 shows the results for blocking CTLA4-FITC bind- animals such as rabbits, rats, and mice, and other animals. ing to murine B7.1-expressing cells. These data show that The term "treating" includes the administration of the com- MAb 10Dl can effciently block CTLA-4 binding to B7.1 as pounds or agents of the present invention to prevent or delay compared to a control human MAb. the onset of the symptoms, complications, or biochemical FIG. 13 shows competitive ELISAs of anti-CTLA-4 30 indicia of a disease, alleviating the symptoms or arresting or human MAbs demonstrating epitope group classifications. inhbiting further development of the disease, condition, or FIG. 14 shows CTLA-4 expression on PHA-stimulated disorder (e.g., autoimmune disease). Treatment may be pro- T-cells. Activated, but not resting T cells, express low but phylactic (to prevent or delay the onset of the disease, or to detectable levels of CTLA-4 at the cell surface. prevent the manifestation of clinical or subclinical symptoms FIG. 15 shows the results of MAb 1OD1 in Complement 35 thereof) or therapeutic suppression or alleviation of symp- Dependent Lysis of Activated T Cells. No lysis of PHA- toms after the manifestation of the disease. activated T cells is observed. In general, the phrase "well tolerated" refers to the absence FIG. 16 shows the results of MAb lOD1 in Antibody- of adverse changes in health status that occur as a result of the Dependent Lysis of Activated T Cells. No lysis of PHA- treatient and would affect treatment decisions. activated T cells is observed with IOD1 and mononuclear 40 The term "lymphocyte" as used herein has the normal cells. meanng in the ar, and refers to any of the mononuclear, FIG. 17 shows anti-10D1 IgM and IgG responses in cyno- nonphagocytic leukocytes, found in the blood, lymph, and molgus monkeys injected with 1 OD 1 antibody. No significant lymphoid tissues, i.e., Band T lymphocytes. antibody response to IOD1 is observed. The phrse "subpopulations ofT lymphocytes" or "T cell FIG. 18 shows prostate specific antigen (PSA) levels in 45 subset(s)" refers to T lymphocytes orTcells characterized by nglmI in two human patients at various time points after the expression of particular cell surface markers (see Barclay, inusion of an anti-CTLA4 antibody at day O. A. N. et a!. (ees.), 1997, The Leukocyte Antigen Facts Book, FIG. 19 shows the plasma levels of anti-HbsAg antibody in 2nd. edition, Academic Press, London, United Kingdom). primates treated with either a HbsAg vaccine in combination The term "stable" in reference to T cells refers to the fact that with the anti-CTLA4 antibody 10D1 or the vaccine in com- 50 the frequency or percentage of a T cell subset does not change bination with a control IgG1 antibody. over the course or duration of the admnistration of an agent. FIG. 20 shows the level of antibody responses to a mela- The terms "cytotoxic T lymphocyte-associated antigen-4," noma cell vaccine in primates treated with either the vaccine "CTLA -4," "CTLA4," "CTLA -4 antigen" and "CD 152" (see, alone (open circles) or with the vaccine in combination with e.g., Murata (1999) Am. J. Patho!. 155:453-460) are used the anti-CTLA4 antibody 10Dl (closed circles). 55 interchangeably, and include variants, isoforms, species FIG. 21 shows antigen-specific T cell proliferation in a homologs of human CTLA-4, and analogs having at least one primate vaccinated with a melanoma cell vaccine in combi- common epitope with CTLA-4 (see, e.g., Balzano (1992) Int. nation with the anti-CTLA4 antibody 1ODI. J. Cancer Supp!. 7:28-32). The complete cDNA sequence ofhmnan CTLA-4 has the DETAILED DESCRIPTION 60 Genbank accession number Ll5006. The region of amino acids 1-37 is the leader peptide; 38-161 is the ex1racellular The present invention provides novel antibody-based V-like domain; 162-187 is the transmembrane domain; and therapies for treating and diagnosing diseases characterized 188- 223 is the cytoplasmic domain. Variants of the nucleotide by expression, paricularly over-expression, or activation of, sequence have been reported, including a G to A transition at paricularly overactivation, of human CTLA-4 and/or related 65 position 49, a C to T transition at position 272, and anA to G molecules. Therapies of the invention employ human transition at position 439. The complete DNA sequence of sequence antibodies, human sequence monoclonal antibod- mouse CTLA-4 has the EMBL accession number X05719 US 7,605,238 B2 13 14 (Brunet et al. (1987) Nature 328:267-270). The region of be prepared by recombinant techniques or enzymatic or amio acids 1-35 is the leader peptide. chemical cleavage of intact antibodies. The complete DNA sequence of human B7 -I (CD80) has A bispecific antibody has two different binding specifici- the Genban accession number X60958; the accession num- ties, see. e.g., U.S. Pat. Nos. 5,922,845 and5,837,243; Zeilder ber for the mouse sequence is X60958; the accession number (1999) J. Imunol. 163:1246-1252; Soma sundaram (1999) for the rat sequence is U05593. The complete cDNA Hum. Antibodies 9:47-54; Keler (1997) Cancer Res. sequence of human B7-2 (CD86) has the Genban accession 57:4008-4014. For example, the invention provides bispecific number L25259; the accession number for the mouse antibodies having one binding site for a cell surface antigen, sequence is L25606. such as human CTLA-4, and a second binding site for an Fc The genes encoding CD28 have been extensively charac- 10 receptor on the surface of an effector cell. The invention also terized. The chicken mRNA sequence has the Genbank acces- provides multispecific antibodies, which have at least three sion number X67915. The rat mRA sequence has the Gen- binding sites. The term "bispecific antibodies" furter ban accession number X55288. The human mRNA includes diabodies. Diabodies are bivalent, bispecific anti- sequence has the Genban accession number J02988. The bodies in which the VH and VL domains are expressed on a mouse mRNA sequence has the Genban accession number 15 single polypeptide chain, but using a linker that is too short to M34536. allow for pairing between the two domains on the same chain, The term "epitope" means a protein determinant capable of thereby forcing the domains to pair with complementary specific binding to an antibody. Epitopes usually consist of domains of another chain and creating two antigen binding chemically active surface groupings of molecules such as sites (See, e.g., Hollger, P., et al. (1993)Proc. Natl. Acad. Sci. amino acids or sugar side chains and usually have specific 20 USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure thee dimensional structural characteristics, as well as spe- 2: 1121-1123). cific charge characteristics. Conformational and nonconfor- The term "human sequence antibody" includes antibodies mational epitopes are distinguished in that the binding to the having variable and constant regions (if present) derived from former but not the latter is lost in the presence of denaturing human germline i=unoglobulin sequences. The human solvents. 25 sequence antibodies ofthe invention may include amino acid An intact "antibody" comprises at least two heavy (H) residues not encoded by human germline i=unoglobulin chains and two light (L) chains inter-connected by disulfide sequences (e.g., mutations introduced by random or site- bonds. Each heavy chain is comprised of a heavy chain vari- specific mutagenesis in vitro or by somatic mutation in vivo). able region (abbreviated herein as HCVR or VH) and a heavy However, the term "human sequence antibody", as used chain constant region. The heavy chain constant region is 30 herein, is not intended to include antibodies in which CDR comprised of three domains, CHI, CH2 and CH3. Each light sequences derived from the germline of another mamalian chain is comprised of a light chain variable region (abbrevi- species, such as a mouse, have been grafted onto human ated herein as LCVR or VL) and a light chain constant region. framework sequences (i.e., humanzed antibodies). The light chain constant region is comprised of one domain, The terms "monoclonal antibody" or "monoclonal anti- CL. The VH and VL regions can be furher subdivided into 35 body composition" refer to a preparation of antibody mol- regions of hypervariability, termed complementarity deter- ecules of single molecular composition. A monoclonal anti- mining regions (CDR), interspersed with regions that are body composition displays a single binding specificity and more conserved, termed framework regions (FR). Each VH affnity for a particular epitope. Accordingly, the term and VL is composed of three CDRs and four FRs, arranged "human monoclonal antibody" refers to antibodies display- from amino-tenninus to carboxyl-termius in the following 40 ing a single binding specificity which have variable and con- order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thevari- stant regions (if present) derived from human gerrnine able regions of the heavy and light chains contain a binding i=unoglobulin sequences. In one embodiment, the human domain that interacts with an antigen. The constant regions of monoclonal antibodies are produced by a hybridoma which the antibodies may mediate the binding of the i=unoglobu- includes a B cell obtained from a transgenic non-human ani- lin to host tissues or factors, including various cells of the 45 mal, e.g., a transgenic mouse, having a genome comprising a imune system (e.g., effector cells) and the fist component human heavy chain trans gene and a light chain transgene

(CLq) of the classical complement system. The term antibody fused to an i=ortalized cell. includes antigen-binding portions of an intact antibody that The temi "diclonal antibody" refers to a preparation of at retain capacity to bind CTLA -4. Examples of binding include least two antibodies to human CTLA-4. Typically, the differ- (i) a Fab fragment, a monovalent fragment consisting of the 50 ent antibodies bind different epitopes. VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a The term "oligoclonal antibody" refers to a preparation of bivalent fragment comprising two Fab fragments linked by a 3 to 1 00 different antibodies to human CTLA -4. Typically, the disulfide bridge at the hinge region; (iii) a Fd fragment con- antibodies in such a preparation bind to a range of different sisting of the VH and CHI domains; (iv) a Fv fragment epitopes. consisting of the VL and VH domains of a single arm of an 55 The term "polyclonal antibody" refers to a preparation of antibody, (v) a dAb fragment (Ward et aI., (1989) Nature more than 1 (two or more) different antibodies to human 341:544-546), which consists of a VH domain; and (vi) an CTLA-4. Such a preparation includes antibodies binding to a isolated complementarity determining region (CDR). Fur- range of different epitopes. thermore, although the two domains of the Fv fragment, VL The invention provides human sequence antibodies to and VH, are coded for by separate genes, they can be joined, 60 human C1LA-4 which block or antagonize signals trans- using recombinant methods, by a synthetic liner that enables duced by the hiunan CTLA-4 receptor. Some of these anti- them to be made as a single protein chain in which the VL and bodies can bind to an epitope on human CTLA-4 so as to VH regions pair to form monovalent molecules (known as inhibit CTLA-4 from interacting with a human B7 counter- single chain Fv (scFv); See, e.g., Bird et al. (1988) Science receptor. Because interaction of human CTLA -4 wi th human 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 65 B7 transduces a signal leading to inactivation ofT-celis bear- USA 85:5879-5883). Such single chain antibodies are ing the human CTLA-4 receptor, antagonism of the interac- included by reference to the term "antibody" Fragments can tion effectively induces, augments or prolongs the activation US 7,605,238 B2 15 16 ofT cells bearing the human CTLA-4 receptor, thereby pro- comprise more than about 80 to 90 percent by weight of all longing or augmenting an imune response. A "blocking macromolecular species present in the composition. An iso- antibody" refers to an antibody that reduces the binding of lated object species (e.g., antibodies ofthe invention) can aIso soluble human CTLA-4 to cell-expressed human B7 ligand be purfied to essential homogeneity (contaminant species by at least 10%,20%,30%,40%,50%,60%,70%, 80%, 90%, canot be detected in the composition by conventional detec- 99% or 99.9% under conditions in which the ratio of antibody tionmethods) wherein the composition consists essentially of combining site to human CTLA-4 ligand binding site is derivatives of a single macromolecular species. An isolated greater than 1: 1 and the concentration of antibody is greater antibody to human CTLA -4 can be substantially free of other than 10-8 M. antibodies that lack binding to human CTLA-4 and bind to a Other antibody preparations, sometimes referred to as mul- 10 different antigen. An isolated antibody that specifically binds tivalent preparations, bind to human CTLA-4 in such a man- to an epitope, isoform or varant of human CTLA-4 may, ner as to crosslink multiple human CTLA-4 receptors on the however, have cross-reactivity to other related antigens, e.g., same celL. Cross-linking of receptor has the same or similar from other species (e.g., CTLA-4 species homologs). More- effect to binding of human CTLA-4 to human B7. Thus, over, an isolated antibody of the invention be substantially cross-lining of receptors effectively agonizes the human 15 free of other cellular material (e.g., non-immunoglobulin CTLA-4 response resulting in immunosuppression. associated proteins) and/or chemicals. Cross-linng can also be accomplished by combining "Specific binding" refers to antibody binding to a prede- soluble divalent antibodies having different epitope specifici- termined antigen. The phrase "specifically (or selectively) ties. These polyclonal antibody preparations comprise at least binds" to an antibody refers to a binding reaction that is two pairs of heavy and light chains binding to different 20 determinative of the presence of the protein in a heteroge- epitopes on human CTLA -4 such that an immunosuppressing neous population of proteins and other biologics. Typically, signal can be transduced as a result of hmnan CTLA-4 the antibody binds with an association constant (Ka) of at cross linkng. least about lxlO6 M-1 or 107 M-1, or about 108 M-1 to 109 The term "recombinant human antibody" includes all M-1, or about 101OM-1 to 10" M-1 or higher, and binds to the human sequence antibodies of the invention that are prepared, 25 predetermined antigen with an afnity that is at least two- fold expressed, created or isolated by recombinant means, such as greater than its affty for binding to a non-specific antigen antibodies isolated from an animal (e.g., a mouse) that is (e.g., BSA, casein) other than the predetermned antigen or a transgenic for human immunoglobuIin genes (described fur- closely-related antigen. The phrases "an antibody recogniz- ther in Section I, below); antibodies expressed using a recom- ing an antigen" and "an antibody specific for an antigen" are binant expression vector transfected into a host cell, antibod- 30 used interchangeably herein with the term "an antibody ies isolated from a recombinant, combinatorial human which binds specifically to an antigen". antibody librar, or antibodies prepared, expressed, created or The phrase "specifically bind(s)" or "bind(s) specifically" isolated by any other means that involves splicing of human when referring to a peptide refers to a peptide molecule wilch immunoglobulin gene sequences to other DNA sequences. has intermediate or high binding affnity, exclusively or pre- Such recombinant human antibodies have variable and con- 35 dominately, to a target molecule. The phrases "specifically stant regions (if present) derived from human germine binds to" refers to a binding reaction wilch is determinative immunoglobulin sequences. Such antibodies can, however, of the presence of a target protein in the presence of a hetero- be subjected to in vitro mutagenesis (or, when an animal geneous population of proteins and other biologics. Thus, transgenic for human Ig sequences is used, in vivo somatic under designated assay conditions, the specified bindingmoi- mutagenesis) and thus the amino acid sequences of the VH 40 eties bind preferentially to a particular target protein and do and VL regions ofthe recombinant antibodies are sequences not bind in a signficant amount to other components present that, wilIe derived from and related to human germline VH in a test sample. Specific binding to a target protein under and VL sequences, may not naturally exist wit1ur the human such conditions may require a binding moiety that is selected antibody germline repertoire in vivo. for its specificity for a particular target antigen. A variety of A "heterologous antibody" is defied in relation to the 45 assay formats may be used to select ligands that are specifi- transgenic non-human organism producing such an antibody. cally reactive with a paricular protein. For example, solid- Ths term refers to an antibody having an amio acid phase ELISA immunoassays, immunoprecipitation, Biacore sequence or an encoding nucleic acid sequence correspond- and Western blot are used to identifY peptides that specifically ing to that found in an organism not consisting of the trans- react with CTLA-4. Typically a specific or selective reaction genic non-human animal, and generally from a species other 50 wil be at least twice background signal or noise and more than that of the transgenic non-human animaL. typically more than 10 times background. A "heterohybrid antibody" refers to an antibody having a The term "ilgh affty" for an IgG antibody refers to an light and heavy chains of different organsmal origins. For equilbrium association constant (Ka) of at least about example, an antibody having a human heavy chain associated 107M-I, at least about 108M-I, at least about 109M-I, atleast with a murine light chain is a heterohybrid antibody. 55 about IOlOM-1, at least about 10"M-" or at least about Examples of heterohybrid antibodies include chimeric and I 012M-1 or greater, e.g., up to I013M-1 or 1014M-1 or greater. humanzed antibodies, discussed supra. However, "ilgh affty" binding can vary for other antibody The term "substantially pure" or "isolated" means an isotypes. object species (e.g., an antibody of the invention) has been The term "Ka", as used herein, is intended to refer to the identified and separated and/or recovered from a component 60 equilbrium association constant of a particular antibody- of its natual environment such that the object species is the antigen interaction. Tils constant has unts of 11M. predominant species present (i.e., on a molar basis it is more The term "Kd", as used herein, is intended to refer to the abundant than any other individual species in the composi- equilibrium dissociation constant of a paricular antibody- tion); a "substantially pure" or "isolated" composition also antigen interaction. This constant has units ofM. means where the object species comprises at least about 50 65 The term "ka", as used herein, is intended to refer to the percent (on a molar basis) of all macromolecular species kinetic association constant of a paricular antibody-antigen present. A substantially pure or isolated composition can also interaction. Ths constant has units of lIMs US 7,605,238 B2 17 18 The term "k';', as used herein, is intended to refer to the The term "isolated nucleic acid" in reference to nucleic kinetic dissociation constant of a particular antibody-antigen acids encoding antibodies or antibody portions (e.g., VH, VL, interaction. This constant has units of lIs. CDR3) that bind to CTLA-4, is intended to refer to a nucleic "Particular antibody-antigen interactions" refers to the acid in which the nucleotide sequences encoding the antibody experimental conditions under which the equilbrium and 5 or antibody portion are free of other nucleotide sequences kinetic constants are measured. encoding antibodies or antibody portions that bind antigens "Isotype" refers to the antibody class (e.g., IgM or IgG1) other than CTLA-4, which other sequences may naturally that is encoded by heavy chain constant region genes. flan the nucleic acid in human genomic DNA. SEQ ID "Iso type switching" refers to the phenomenon by which NOs:4-23 comprise the nucleotide and anio acid sequences the class, or isotype, of an antibody changes from one Ig class 10 comprising the heavy chain (VH) and light chain (V) vari- to one of the other Ig classes. able regions of the lOD1, 4B6 and 1E2 human anti-CTLA-4 "Nonswitched isotype" refers to the isotypic class of heavy monoclonal antibodies ofthe invention. chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typi- The term "substantially identical," in the context of two cally the fist CH gene immediately downstream from the 15 nucleic acids or polypeptides refers to two or more sequences functionally rearranged VDJ gene. Isotype switching has or subsequences that have at least about 80%, about 90, about been classified as classical or non-classical isotype switching. 95% or higher nucleotide or amino acid residue identity, Classical isotype switching occurs by recombination events when compared and aligned for maxinnun correspondence, which involve at least one switch sequence region in the as measured using the following sequence comparson method and/or by visual inspection. For example, the inven- trans gene. Non-classical isotype switching may occur by, for 20 example, homoIogous recombination between human 01- and tion provides nucleic acids having sequences that are substan- human ~I- (õ-associated deletion). Alternative non-classical tially identical to SEQ ID NO:1, SEQ ID NO:2. Such "sub- switchig mechanisms, such as intertransgene and/or inter- stantially identical" sequences are typically considered to be chromosomal recombination, among others, may occur and homologous. The "substantial identity" can exist over a effectuate isotype switching. 25 region of sequence that is at least about 50 residues in length, The term "switch sequence" refers to those DNA over a region of at least about 1 00 residues, or over a region at sequences responsible for switch recombination. A "switch least about 150 residues, or over the full length of the two donor" sequence, typically a fl switch region, are 5' (i.e., sequences to be compared. As described below, any two anti- upstream) of the constnict region to be deleted during the body sequences can only be aligned in one way, by using the switch recombination. The "switch acceptor" region are 30 numbering scheme in Kabat. Therefore, for antibodies, per- between the constnict region to be deleted and the replace- cent identity has a unique and well-defined meaning. ment constant region (e.g., y, E, etc.). As there is no specific Amno acids from the variable regions of the mature heavy site where recombination always occurs, the fial gene and light chains of immunoglobulins are designated Hx and sequence is not typically predictable from the constnict. Lx respectively, where x is a number designating the position "Glycosylation pattern" is defied as the pattern of carbo- 35 of an amino acid according to the scheme of Kabat, hydrate miits that are covalently attached to a protein, more Sequences of Proteins of Immunological Interest (National specifically to an immunoglobulin protein. A glycosylation Institutes of Health, Bethesda, Md., 1987 and 1991). Kabat pattern of a heterologous antibody can be characterized as lists many anio acid sequences for antibodies for each sub- being substantially similar to glycosylation patterns which group, and lists the most commonly occurring amino acid for occur naturally on antibodies produced by the species of the 40 each residue position in that subgroup to generate a consensus non-human transgenic animal, when one of ordinar skill in sequence. Kabat uses a method for assigning a residue num- the ar would recognize the glycosylation pattern of the het- ber to each amno acid in a listed sequence, and tliis method erologous antibody as being more similar to said pattern of for assigning residue numbers has become standard in the glycosylation in the species of the non-human transgenic field. Kabat's scheme is extendible to other antibodies not animal than to the species from which the CH genes of the 45 included in his compendium by aligning the antibody in ques- 1ransgene were derived. tion with one of the consensus sequences in Kabat by refer- The term "naturally-occurring" as applied to an object ence to conserved amino acids. The use of the Kabat num- refers to the fact that an object can be found in nature. For bering system readily identifies amino acids at equivalent example, a polypeptide or polynucleotide sequence that is positions in different antibodies. For example, an amo acid present in an organism (including vinises) that can be isolated 50 at the L50 position of a human antibody occupies the equiva- from a source in nature and which has not been intentionally lent position to an amino acid position L50 of a mouse anti- modified by man in the laboratory is natually-occurrng. body. Likewise, nucleic acids encoding antibody chains are The term "rearranged" refers to a confguration of a heavy aligned when the amno acid sequences encoded by the chain or light chain immunoglobulin locus wherein a V seg- respective nucleic acids are aligned according to the Kabat ment is positioned immediately adjacent to a D-J or J segment 55 numbering convention. in a confomiation encoding essentially a complete VH or VL The phrase "selectively (or specifically) hybridizes to" domain, respectively. A rearranged imunoglobulin gene refers to the binding, duplexing, or hybridizing of a molecule locus can be identified by comparison to germ1ine DNA; a to a paricular nucleotide sequence under strngent hybridiza- rearranged locus has at least one recombined heptamer/non- tion conditions when that sequence is present in a complex amer homology element. 60 mixtue (e.g., totaI cellular or library DNA or RNA), wherein The term "unearranged" or "germline confguration" in the paricular nucleotide sequence is detected at least at about reference to a V segment refers to the confguration wherein 10 times background. In one embodiment, a nucleic acid can the V segment is not recombined so as to be immediately be determined to be within the scope of the invention (e.g., is adjacent to a D or J segment. substantially identical to SEQ ID NO: 1 or SEQ ID NO:2) by The term "nucleic acid" is intended to include DNA mol- 65 its abilty to hybridize under stringent conditions to a nucleic ecules and RNA molecules. A nucleic acid can be single- acid otherwise determined to be within the scope of the inven- stranded or double-stranded. tion (such as the exemplar sequences described herein). US 7,605,238 B2 19 20 The phrase "stringent hybridization conditions" refers to hybridization complex is washed twice with a solution with a conditions under which a probe wil hybridize to its target salt concentration of about 2xSSC containng 0.1 % SDS at subsequence, typically in a complex mixture of nucleic acid, room temperature for 15 minutes and then washed twice by but not to other sequences in significant amounts (a positive O.lxSSC containig 0.1 % SDS at 68° C. for 15 minutes; or, signal (e.g., identification of a nucleic acid of the invention) is equivalent conditions. See Sambrook, Tijssen and Ausubel about 10 times background hybridization). Stringent condi- for a description of SSC buffer and equivalent conditions. tions are sequence-dependent and wil be different in differ- The nucleic acids of the invention be present in whole cells, ent circumstances. Longer sequences hybridize specifically in a cell lysate, or in a parially purfied or substantially pure at higher temperatures. An extensive guide to the hybridiza- form. A nucleic acid is "isolated" or "rendered substantially tion of nucleic acids is found An extensive guide to the io pure" when purified away from other cellular components or hybridization of nucleic acids is found in e.g., Sambrook, ed., other contanants, e.g., other cellular nucleic acids or pro- MOLECULAR CLONING, A LABORATDRY MANAL (2ND ED.), Vols. teins, by standard technques, including alkaline/SDS treat- 1-3, Cold Spring Harbor Laboratory, (1989); CURNT PROTO- ment, CsCl banding, column chromatogrphy, agarose gel COLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, electrophoresis and others well known in the ar. see, e.g., Inc., New York (1997); LABORATDRY TErnIQUES IN BIOCHEMIS- 15 Sambrook, Tijssen and Ausubel. The nucleic acid sequences TRY AN MOLECUL BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID of the invention and other nucleic acids used to practice ths PROBES, Par 1. Theory and Nucleic Acid Preparation, Tijssen, invention, whether RNA, cDNA, genomic DNA, or hybrids ed. Elsevier, N.Y. (1993). thereof, may be isolated from a varety of sources, genetically Generally, stringent conditions are selected to be about engineered, amplified, anelor expressed recombinantly. Any 5-10° C. lower than the thermal melting point (T m) for the 20 recombinant expression system can be used, including, in specific sequence at a defied ionic strength pH. The T m is the addition to bacterial, e.g., yeast, insect or mammalian sys- temperature (under defined ionic strength, pH, and nucleic tems. Alterntively, these nucleic acids can be chemically concentration) at which 50% of the probes complementary to synthesized in vitro. Technques for the manipulation of the target hybridize to the target sequence at equilibrium (as nucleic acids, such as, e.g., subcloning into expression vec- the target sequences are present in excess, at T m' 50% of the 25 tors, labeIing probes, sequencing, and hybridization are well probes are occupied at equilibrium). Stringent conditions wil described in the scientific and patent literature, see, e.g., be those in which the salt concentration is less than about 1.0 Sambrook, Tijssen and Ausubel. Nucleic acids can be ana- M sodium ion, typically about 0.01 to 1.0 M sodium ion lyzed and quantified by any of a number of general means concentration (or other salts) at pH 7.0 to 8.3 and the tem- well known to those of skil in the art. These include, e.g., perature is at least about 30° C. for short probes (e.g., 10 to 50 30 analytical biochemical methods such as NMR, spectropho- nucleotides) and at least about 60° C. for long probes (e.g., tometry, radiography, electrophoresis, capilar electro- greater than 50 nucleotides). Stringent conditions may also be phoresis, high performance liquid chromatography (HPLC), achieved with the addition of destabilzing agents such as thi layer chromatography (TLC), and hyperdiffusion chro- formamide as described in Sambrook (cited below). For high matography, various immunological methods, such as fluid or stringency hybridization, a positive signal is at least two times 35 gel precipitin reactions, immunodiffision (single or double), background, preferably 10 times background hybridization. immunoelectrophoresis, radioimunoassays (RIAs), Exemplary high stringency or stringent hybridization condi- enzyme-linked immunosorbent assays (ELISAs), imuno- tions include: 50% formamide, 5xSSC and 1 % SDS incu- fluorescent assays,Southern analysis, Northern analysis, dot- bated at 42° C. or 5xSSC and 1 % SDS incubated at 65° c., blot analysis, gel electrophoresis (e.g., SDS-PAGE), with a wash in 0.2xSSC and 0.1 % SDS at 65° C. For selective 40 RT-PCR, quantitative PCR, other nucleic acid or target or or specific hybridization, a positive signal (e.g., identification signal amplification methods, radiolabeling, scintillation of a nucleic acid of the invention) is about 10 times back- counting, and affnity chromatography. ground hybridization. Stringent hybridization conditions that The nucleic acid compositions of the present invention, are used to identifY nucleic acids within the scope of the while often in a native sequence (except for modified restric- invention include, e.g., hybridization in a buffer comprising 45 tion sites and the like), from either cDNA, genomic or mix- 50% formamide, 5xSSC, and 1 % SDS at 42° C., or hybrid- tures may be mutated, thereof in accordance with standard ization in a buffer comprising 5xSSC and 1 % SDS at 65° C., technques to provide gene sequences. For coding sequences, both with a wash of 0.2xSSC and 0.1 % SDS at 65° C. In the these mutations, may affect amno acid sequence as desired. present invention, genomic DNA or cDNA comprising In particular, DNA sequences substantially homologous to or nucleic acids of the invention can be identified in standard 50 derived from native V, D, J, constant, switches and other such Southern blots under stringent conditions using the nucleic sequences described herein are contemplated (where acid sequences disclosed here. Additional stringent condi- "derived" indicates that a sequence is identical or modified tions for such hybridizations (to identifY nucleic acids within from another sequence). the scope ofthe invention) are those which include a hybrid- A nucleic acid is "operably lined" when it is placed into a ization in a buffer of 40% formamide, 1 M NaCl, 1 % SDS at 55 functional relationship with another nucleic acid sequence. 37° C. For instance, a promoter or enhancer is operably linked to a However, the selection of a hybridization format is not coding sequence if it affects the transcription of the sequence. critical-it is the stringency of the wash conditions that set With respect to transcription regulatory sequences, operably forth the conditions which determine whether a nucleic acid linked means that the DNA sequences being linked are con- is within the scope of the invention. Wash conditions used to 60 tiguous and, where necessary to join two protein coding identifY nucleic acids within the scope of the invention regions, contiguous and in reading frame. For switch include, e.g.: a salt concentration of about 0.02 moIar at pH 7 sequences, operably linked indicates that the sequences are and a temperature of at least about 50° C. or about 55° C. to capable of effecting switch recombination. about 60° C.; or, a salt concentration of about 0.15 M NaCl at The term "vector" is intended to refer to a nucleic acid 72° C. for about 15 minutes; or, a salt concentration of about 65 molecule capable of transporting another nucleic acid to O.2xSSC at a temperature of at least about 50° C. or about 55° which it has been linked. One type of vector is a "plasmid", C. to about 60° C. for about 15 to about 20 minutes; or, the which refers to a circular double stranded DNA loop into US 7,605,238 B2 21 22 which additional DNA segments may be ligated. Another release of soluble factors such as cytokines that modulate the type of vector is a virl vector, wherein additional DNA function of other immune cells). segments may be ligated into the viral genome. Certain vec- The term "immune response" refers to the concerted action tors are capable of autonomous replication in a host cell into of lymphocytes, antigen presenting cells, phagocytic cells, which they are introduced (e.g., bacterial vectors having a 5 granulocytes, and soluble macromoIecules produced by the bacterial origin of replication and episomal mamalian vec- above cells or the liver (including antibodies, cytokines, and tors). Other vectors (e.g. non-episomal mamalian vectors) complement) that results in selective damage to, destruction can be integrated into the genome of a host cell upon intro- of, or elimination from the human body of invading patho- duction into the host cell, and thereby are replicated along gens, cells or tissues infected with pathogens, cancerous cells, with the host genome. Moreover, certain vectors are capabIe 10 or, in cases of autoimmunity or pathological infammation, of directing the expression of genes to which they are opera- normal human cells or tissues. tively linked. Such vectors are referred to herein as "recom- Components of an imune response may be detected in binant expression vectors" (or simply, "expression vectors"). vitro by various methods that are well known to those of In general, expression vectors of utility in recombinant DNA ordinary skil in the art. For exampIe, (i) cytotoxic T lym- techniques are often in the form of plasm ids. In the present 15 phocytes can be incubated with radioactively Iabeled target specification, "plasmid" and "vector" may be used inter- cells and the lysis of these target cells detected by the release changeably as the plasmid is the most commonly used form of of radioactivity, (2) helper T lymphocytes can be incubated vector. However, the invention is intended to include such with antigens and antigen presenting cells and the synthesis other forms of expression vectors, such as viral vectors (e.g., and secretion of cytokines measured by standard methods replication defective retroviruses, adenoviruses and adeno- 20 associated vinises), which serve equivalent functions. (Windhagen A; et aI., 1995, Immunity 2(4): 373-80), (3) antigen presenting cells can be incubated with whole protein The term "recombinant host cell" (or simply "host cell") antigen and the presentation of that antigen on MHC detected refers to a cell into which a recombinant expression vector has by either T lymphocyte activation assays or biophysical meth- been introduced. It should be understood that such terms are ods (Harding et aI., 1989, Proc. Natl. Acad. Sci., 86: 4230-4), intended to refer not only to the paricular subject cell but to 25 (4) mast cells can be incubated with reagents that cross-link the progeny of such a cell. Because certain modifications may their F c-epsilon receptors and histamine release measured by occur in succeeding generations due to either mutation or enzyme immunoassay (Siraganian, et aI., 1983, TIPS 4: 432- environmental infuences, such progeny may not, in fact, be 437). identicaI to the parent cell, but are stil included with the scope of the term "host cell" as used herein. 30 Similarly, products of an immune response in either a model organism ( e.g., mouse) or a human patient can also be The term "milocus trans gene" refers to a trans gene that detected by varous methods that are well known to those of comprises a portion of the genomic immunoglobulin locus ordinary skill in the art. For example, (1) the production of having at least one internal (i.e., not at a terminus of the antibodies in response to vaccination can be readily detected portion) deletion of a non-essential DNA portion (e.g., inter- by standard methods currently used in clinical laboratories, venig sequence; intron or portion thereof) as compared to 35 e.g., an ELISA; (2) the migration of imune cells to sites of the naturally-occurring germine Ig locus. infamation can be detected by scratching the surface of A "label" is a composition detectable by spectroscopic, skin and placing a sterile container to capture the migrating photochemical, biochemical, immunochemical, or chemical cells over scratch site (Peters et aI., 1988, Blood 72: 1310-5); means. For example, usefiil labels include 32p, fluorescent 40 (3) the proliferation of peripheral blood mononuclear cells in dyes, electron-dense reagents, enzmes (e.g., as commonly response to mitogens or mixed lymphocyte reaction can be used in an ELISA), biotin, digoxigeni, or haptens and pro- measured using 3H-thymidine; (4) the phagocitic capacity of teins for which antisera or monoclonal antibodies are avail- granulocytes, macrophages, and other phagocytes in PBMCs able (e.g., the polypeptides of the invention can be made can be measured by placing PMBCs in wells together with detectable, e.g. by incorporating a radiolabel into the peptide, 45 labeled paricles (Peters et aI., 1988); and (5) the differenta- and used to detect antibodies specifically reactive with the tion of immune system cells can be measured by labeling peptide). PBMCs with antibodies to CD molecules such as CD4 and The term "sorting" in the context of cells as used herein to CD8 and measuring the frction of the PBMCs expressing refers to both physical sorting of the cells, as can be accom- these markers. plished using, e.g., a fluorescence activated cell sorter, as well 50 As used herein, the phrase "signal transduction pathway" as to analysis of cells based on expression of cell surface or "signl transduction event" refers to at least one biochemi- markers, e.g., FACS analysis in the absence of sorting. cal reaction, but more commonly a series of biochemical The phrase "immune cell response" refers to the response reactions, which result from interaction of a cell with a stimu- of immune system cells to external or internal stimuli (e.g., latory compound or agent. Thus, the interaction of a stimula- antigen, cytokines, chemokines, and other cells) producing 55 tory compound with a cell generates a "signal" that is trans- biochemical changes in the immune cells that resiùt in mitted through the signal transduction pathway, ultimately immune cell migration, killng of target cells, phagocytosis, resulting in a cellular response, e.g., an immune response production of antibodies, other soluble effectors of the described above. immune response, and the like. A signal transduction pathway refers to the biochemical The terms "T lymphocyte response" and "T lymphocyte 60 relationship between a variety of signal transduction mol- activity" are used here interchangeably to refer to the com- ecuIes that playa role in the transmission of a signal from one ponent of immune response dependent on T lymphocytes portion of a cell to another portion of a celL. Signal transduc- (i.e., the proliferation and/or differentiation ofT lymphocytes tion molecules of the present invention include, for example, into helper, cytotoxic killer, or suppressorT lymphocytes, the MAb 147.1 of the invention. As used herein, the phrse "cell provision of signals by helper T Iymphocytes to B Iympho- 65 surface receptor" includes molecules and complexes of mol- cytes that cause or prevent antibody production, the killng of ecuIes capable of receiving a signal and the transmission of specific target cells by cytotoxic T lymphocytes, and the such a signal across the plasma membrane of a cell. An US 7,605,238 H2 23 24 example of a "cell surface receptor" of the present invention 821-830; Tuailon et al. (1994) J. Immunol. 152:2912-2920; is the T cell receptor (TCR) or the B7 ligands ofCTLA-4. Lonberg et aI., (1994) Nature 368(6474): 856-859; Lonberg, A signal transduction pathway in a cell can be initiated by N. (1994) HandbookofExperimentalPharmacology 113:49- interaction of a cell with a stimulator that is inside or outside 101; Taylor, L. et al. (1994)International Immunology 6: 579- of the celL. If an exterior (i.e., outside of the cell) stimulator 591; Lonberg, N. and Huszar, D. (1995) Intem. Rev. Immu- (e.g., an MHC-antigencomplex on an antigen presenting cell) nol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995)Ann. interacts with a cell surface receptor (e.g., a T cell receptor), NY. A cad. Sci764:536-546; Fishwild, D. et al. (1996) Nature a signal transduction pathway can transmit a signal across the Biotechnology 14: 845-851. See further, U.S. Pat. Nos. 5,625, cell's membrane, through the cytoplasm of the cell, and in 126 and 5,770,429, both to Lonberg and Kay, and GenPharm some instances into the nucleus. If an interior (e.g., inside the 10 Intemational; U.S. Pat. No. 5,545,807 to Surani et a1.; Inter- cell) stimulator interacts with an intra cell ular signal transduc- national Publication Nos. WO 98/24884, published on Jun. tion molecule, a signal transduction pathway can resiùt in 11, 1998; WO 94/25585, published Nov. 10, 1994; WO tranmission of a signal through the cell's cytoplasm, and in 93/1227, published Jun. 24, 1993; WO 92/22645, published some instances into the cell's nucleus. Dec. 23,1992; WO 92/03918,publishedMar. 19, 1992. Alter- Signal transduction can occur thrugh, e.g., the phospho- 15 natively, the CMD and HCo12 transgenes, described in rylation of a molecule; non-covalent allosteric interactions; Examples 1 and 2, below, can be used to generate human complexing of molecules; the conformational change of a anti-CTLA-4 antibodies. molecule; calcium release; inositol phosphate production; Detailed procedures to generate fully human monoclonal proteolytic cleavage; cyclic nucleotide production and dia- antibodies to CTLA-4 are described in the Examples below. cylglyceride production. Typically, signal transduction 20 Cumulative experience with varous antigens has shown that occurs through phosphorylating a signal transduction mol- the transgenic mice respond when intially immunized intra- ecule. peritoneally (IP) with antigen in complete Freund's adjuvant, The term "nonspecific T cell activation" refers to the stimu- followed by every other week IP immunizations (up to a total 1ation ofT cells independent of their antigenic specificity. of 6) with antigen in incomplete Freund's adjuvant. However, 25 adjuvants other than Freund's are also found to be effective. Production of Human Antibodies to CTLA-4 The monoclonal antibodies (mAbs) and the human In addition, whole cells in the absence of adjuvant are found sequence antibodies of the invention can be produced by a to be highly immunogenic. The immune response can be monitored over the course of the immunzation protocol with variety of technques, including conventional monoclonal plasma samples being obtained by retroorbital bleeds. The antibody methodology e.g., the standard somatic cell hybrid- 30 plasma can be screened by ELISA (as described below), and ization technique of Kohler and Milstein, Nature 256: 495 mice with suffcient titers of anti-CTLA-4 human immrmo- (1975). Any technque for producing monoclonal antibody globulin can be used for fusions. Mice can be boosted intra- can be employed e.g., viraI or oncogenic transformation ofB venously with antigen 3 days before sacrifice and removal of lymphocytes. One animal system for preparing hybridomas is the spleen. It is expected that 2-3 fusions for each imunza- the murine system. Hybridoma production in the mouse is a 35 tion may need to be performed. Between 6 and 24 mice are very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for typically immunized for each antigen. Usually both HC07 and HCo12 strains are used. In addition, both HC07 and fusion are known in the art. Fusion parners (e.g., murine myeloma cells) and fusion procedures are also known (see, HCo12 transgene can be bred together into a single mouse having two different human heavy chain transgenes. e.g., Harlow and Lane (1988), Antibodies, A Laboratory 40 Manual, Cold Spring Harbor Laboratory Press, Cold Spring To purify human anti-CTLA-4 antibodies, selected hybri- Harbor NY). domas can be grown in two-liter spinner-flasks for mono- Human monoclonal antibodies and human sequence anti- clonal antibody purification. Supernatants can be fitered and bodies directed against human CTLA-4 can be generated concentrated before affnity chromatogrphy with protein using transgenic mice carring a human immune system 45 A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can rather than the mouse system. These transgenic mice, also be checked by gel electrophoresis and high performance liq- referred to herein as ''HuMAb-Mouse™'', contain a human uid chromatography to ensure purity. The buffer solution can immrmoglobulin gene miniloci that encodes unrearranged be exchanged into PBS, and the concentration can be deter- human heavy (fl and y) and K light chain immunoglobulin mined by OD280 using 1.43 extinction coeffcient. The sequences, together with targeted mutations that inactivate 50 monoclonal antibodies can be aliquoted and stored at _800 C. the endogenous fl and K chain loci (Lonberg, N. et al. (1994) To determine if the selected human anti-CTLA-4 mono- Nature 368(6474): 856-859 and U.S. Pat. No. 5,770,429). clonal antibodies bind to unique epitopes, each antibody can Accordingly, the mice exhibit reduced expression of mouse be biotinylated using commercially available reagents IgM or K, and in response to immunization, the introduced (Pierce, Rockford, IlL). Competition studies using unabeled human heavy and light chain transgenes rmdergo class 55 monoclonal antibodies and biotinylated monoclonal antibod- switching and somatic mutation to generate high affnity ies can be performed using CTLA-4 coated-ELISA plates as human IgGK monoclonal (Lonberg, N. et al. (1994), supra; described above. Biotinylated MAb binding can be detected reviewed in Lonberg, N. (1994 ) Handbook of Experimental with a strep-avidin-alkaline phosphatase probe. Pharmacology 113:49-101; Lonberg, N. and Huszar, D. To determine the isotype of purified antibodies, isotype (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. 60 ELISAs can be performed. Wells of microtiter plates can be and Lonberg, N. (1995) Ann. NY. Acad. Sci 764:536-546). coated with 1 flg/ni of anti-human IgG overnight at 40 C. The preparation of transgenic mice is described in detail After blocking with 1% BSA, the plates are reacted with 1 Section II below and in Taylor, L. et al. (1992) Nucleic Acids flg/ni or less of monoclonal antibodies or purified isotype Research 20:6287-6295; Chen, J. et al. (1993) International controls, at ambient temperature for one to two hours. The Immunology 5: 647-656; Tuailon et al. (1993) Proc. Natl. 65 wells can then be reacted with either human IgG I or human Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature IgM-specific alkaline phosphatase-conjugated probes. Plates Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: are developed and anaIyzed as described above. US 7,605,238 H2 25 26 To demonstrate binding of monoclonal antibodies to live rangement is unnecessar, at least for that transgene which is cells expressing the CTLA-4, flow cytometry can be used. already rearranged. For background on molecular inunol- Briefly, cell lines expressing CTLA-4 (grown under standard ogy, See, e.g., Fundamental Immunology, 4th edition (1998), growth conditions) are mixed with various concentrations of Paul, Wiliam E., ed. Lippencott-Raven Press, N.Y monoclonal antibodies in PBS containg 0.1 % BSA and Some transgenic non-human anals used to generate the 10% fetal calf serum, and incubated at 37° C. for 1 hour. After human monoclonal antibodies of the invention contain rear- washing, the cells are reacted with Fluorescein-labeled anti- ranged, unrearranged or a combination of rearranged and human IgG antibody under the same conditions as the pri- unrearranged heterologous inunoglobulin heavy and light mary antibody staining. The samples can be analyzed by chain transgenes in the germline of the transgenic animaL. F ACScan instrument using light and side scatter properties to 10 Each of the heavy chain trans genes comprises at least one CH gate on single cells. An alternative assay using fluorescence gene. In addition, the heavy chain transgene can contain microscopy may be used (in addition to or instead of) the flow functional isotype switch sequences, which are capable of cytometry assay. Cells can be stained exactly as described supporting isotype switching of a heterologous trans gene above and examined by fluorescence microscopy. This encoding multiple CH genes in the B-cells of the transgenic method allows visualization of individual cells, but may have 15 animaL. Such switch sequences can be those which occur diminished sensitivity depending on the density of the anti- naturally in the germline i=unoglobulin locus from the gen. species that serves as the source of the transgene CH genes, or Anti-CTLA-4 human IgGs can be further tested for reac- such switch sequences can be derived from those which occur tivity with CTLA-4 antigen by Western blotting. Briefly, cell in the species that is to receive the transgene constnict (tiie extracts from cells expressing CTLA-4 can be prepared and 20 transgenic animal). For example, a human transgene con- subjected to sodium dodecyl sulfate polyacrylamide gel elec- struct that is used to produce a transgenic mouse may produce trophoresis. After electrophoresis, the separated antigens are a higher frequency of isotype switching events if it incorpo- transferred to nitrocellulose membranes, blocked with 10% rates switch sequences similar to those that occur natually in fetal calf serum, and probed with the monoclonal antibodies the mouse heavy chain locus, as presumably the mouse to be tested. Human IgG binding can be detected using anti- 25 switch sequences are optimized to fìinction with the mouse human IgG alkaline phosphatase and developed with BCIP/ switch recombinase enzyme system, whereas the human NBT substrate tablets (Sigma Chern. Co., St. Louis, Mo.). switch sequences are not. Switch sequences can be isolated and cloned by conventional cloning methods, or can be syn- Production of Transgenic Non -HumanAnmals that Generate thesized de novo from overlapping synthetic oligonucleotides Human Monoclonal Anti-CTLA-4 Antibodies 30 designed on the basis of published sequence information The present invention also provides transgenic non-human relating to i=unoglobulin switch region sequences (Mills et animals, e.g., a trangenic mice, which are capable of aI., Nucl. Acids Res. 15:7305-7316 (1991); Sideras etal.,Intl. expressing human monoclonaI antibodies that specifically Immunol. 1:631-642 (1989). bind to CTLA-4. High affnity human sequence antibodies For each ofthe foregoing transgenic animals, fuctionally are also provided. Some transgenic non-human animals, e.g., 35 rearranged heterologous heavy and light chain imiunoglo- the transgeiuc mice, have a genome comprising a human bulin transgenes are found in a signficant fraction of the heavy chain transgene and a light chain transgene. Some B-cells of the transgenic animal (at least 10 percent). transgenic non-human animals are inunized with a purified The transgenes used to generate the transgenic animals of or enrched preparation of CTLA-4 antigen and/or cells the invention include a heavy chain transgene comprising expressing CTLA-4. Some transgenic non-human animals 40 DNA encoding at least one variable gene segment, one diver- are capable of producing multiple isotypes of human mono- sity gene segment, one joining gene segment and at least one clonal antibodies to CTLA-4 (e.g., IgG, IgA and/or IgE) by constant region gene segment. TIie imiunoglobulin light undergoing V-D-J recombination and isotype switching. Iso- chain transgene comprises DNA encoding at least one vari- type switching may occur by, e.g., classical or non-classical able gene segment, one joinng gene segment and at least one isotype switching. 45 constant region gene segment. The gene segments encoding The design of a transgenic non-human animal that the light and heavy chain gene segments are heterologous to responds to foreign antigen stimulation with a heterologous the transgenic non-human anal in that they are derived antibody repertoire, requires that the heterologous i=uno- from, or correspond to, DNA encoding i=unoglobulin globulin transgenes contained within the transgenic anmal heavy and light chain gene segments from a species not function correctly throughout the pathway ofB-cell develop- 50 consisting of the transgenic non-human animaL. In one aspect ment. In some mice, correct function of a heterologous heavy of the invention, the transgene is constructed such that the chain trans gene includes isotype switching. Accordingly, the individual gene segments are unrearraged, i.e., not rear- trans genes of the invention are constructed so as to produce ranged so as to encode a functional inunoglobulin light or isotype switching and one or more of the following: (1) high heavy chain. Such unrearanged transgenes support recom- level and cell-type specific expression, (2) functional gene 55 bination of the V, D, and J gene segments (functional rear- rearrangement, (3) activation of and response to allelic exclu- rangement) and preferably support incorporation of all or a sion, (4) expression of a sufficient primary repertoire, (5) portion of a D region gene segment in the resultant rearranged signl transduction, (6) somatic hypermutation, and (7) domi- i=iinoglobulin heavy chain within the transgenic non-hu- nation of the trans gene antibody locus during the i=une man animal when exposed to CTLA-4 antigen. response. 60 Such transgenes typically comprise a substantia I portion of Not all of the foregoing criteria need be met. For example, the C, D, and J segments as well as a subset of the V gene in transgenic animal in which the endogenous iniuiiog10- segments. In such transgene constructs, the various regula- bulin loci of the transgenic animals are functionally dis- tory sequences, e.g. promoters, enhancers, class switch nipted, the transgene need not activate allelic exclusion. Fur- regions, splice-donor and splice-acceptor sequences for RNA. ther, in transgenic anmals in which the trans gene comprises 65 processing, recombination signals and the like, comprise cor- a functionally rearranged heavy and/or light chain Immiiio- responding sequences derived from the heterologous DNA. globulin gene, the second criteria of functional gene rear- Such regulatory sequences may be incorporated into the US 7,605,238 B2 27 28 transgene from the same or a related species of the non- gerinine of the mice. Typically, such non-gerinine human animal used in the invention. For example, human sequences (or individual nucleotide positions) cluster in or immunoglobulin gene segments may be combined in a trans- near CDRs, or in regions where somatic mutations are known gene with a rodent immunoglobulin enhancer sequence for to cluster. use in a transgenic mouse. Alternatively, synthetic regulatory 5 The human sequence antibodies which bind to the prede- sequences may be incorporated into the transgene, wherein termined antigen can result from isotype switching, such that such synthetic reguIatory sequences are not homologous to a hlUlan antibodies comprising a human sequence y chain functional DNA sequence that is known to occur naturally in (such as yl, y2, y3, ory4) and a hlUlan sequence light chain the genomes of mammals. Synthetic regulatory sequences are (such as kappa or lambda) are produced. Such isotype- designed according to consensus rules, such as, for example, 10 switched human sequence antibodies often contain one or those specifYing the permissible sequences of a splice-accep- more somatic mutation(s), typically in the variable region and tor site or a promoter/enhancer motif. The transgene may often in or witlùn about 10 residues of a CDR) as a result of comprise a minilocus. affty maturation and selection of 8 cells by antigen, par- Some transgenic animals used to generate human antibod- ticularly subsequent to secondary (or subsequent) antigen ies to CTLA-4 contain at least one, typically 2-10, and some- 15 challenge. Some high affnity human sequence antibodies times 25-50 or more copies of the trans gene described in have equilibrium association constants of at least about I xl 07 Example 37 of U.S. Pat. No. 5,770,429, or the transgene M-I, or at least about IxlO8 M-i, or more than about IxlO9 described in Example 2 below (e.g., HCoI2), at least one M-1, or 5xlO9 M-I to IxlOll M-I or greater. copy of a light chain trans gene described in Examples 38 of Another aspect of the invention pertins to the 8 cells from U.S. Pat. No. 5,770,429, two copies of the Cmu deletion 20 such mice which can be used to generate hybridomas express- described in Example I below, and two copies of the Jkappa ing human monoclonal antibodies which bind with high affn- deletion described in Example 9 of U.S. Pat. No. 5,770,429. ity (e.g., having association constant of greater than 107M-I ) The resultant animals are injected with antigens and used for to CTLA-4. These hybridomas are used to generate a com- production of human monoclonal antibodies against these position comprising an immunoglobulin having an associa- antigens. 25 tion constant (Ka) of at least 107 M-I for binding CTLA-4. Some transgenic anials exhbit immunoglobulin produc- Such iinnunoglobulin contains a human sequence light chain tion with a significant repertoire, ideally substantially similar composed of a light chain variable region having a polypep- to that of a native mouse. Thus, for exampIe, anmals in which tide sequence which is substantially identical to a polypeptide the endogenous Ig genes have been inactivated, the total sequence encoded by a human Vk or Vì. gene segment and a iiunoglobulin levels range from about 0.1 to about 10 30 human Jk or Jì. segment, and a light chain constant region mg/ml of serum. having a polypeptide sequence which is substantially identi- The iinnunoglobulins expressed by the transgenic ince cal to a polypeptide sequence encoded by a human Ck or Cì. typically recognze about one-half or more of higWy anti- gene segment. It also contains a human sequence heavy chain genic proteins, e.g., staphylococcus protein A. Typically, the composed of a heavy chain variable region having a polypep- iinnunoglobulins exhibit an association constant for prese- 35 tide sequence which is substantially identical to a polypeptide lected antigens of at least about 107M-I, 108M-I, 109M-I, sequence encoded by a hlUlan VH gene segment, optionally 10IOM-I, IOllM-I, 10l2M-I, 10l3M-I, or greater. a D region, and a human JH segment, and a constant region The transgenic mice of the present invention can be immu- having a polypeptide sequence which is substantially identi- nized with a purified or enrched preparation of human cal to a polypeptide sequence encoded by a human CH gene CTLA-4 antigen (or antigenic fragment thereof) and/or cells 40 segment. expressing human CTLA-4 as described previously. The ince The invention also provides hlUlan monoclonal antibodies produce 8 cells that undergo class-switching via intratrans- and human sequence antibodies to human CTLA-4 deriva- gene switch recombination (cis-switching) and express tized or lined to another functional molecule, e.g., another iiunoglobulins reactive with CTLA-4. The immunoglobu- peptide or protein (e.g., a cytokie, a cytotoxic agent, an lins can be human sequence antibodies, wherein the heavy 45 immune stimulatory or inhibitory agent, a Fab' fragment, and and light chain polypeptides are encoded by human trans gene the like, as discussed above) to generate a bispecific or mul- sequences, which may include sequences derived by somatic tispecific molecule which binds to multiple binding sites or mutation and V region recombinatorial joints, as well as ger- target epitopes. For example, an antibody or antigen-binding mline-encoded sequences; these human sequence immiilo- portion ofthe invention can be functionally linked (e.g., by globulins can be referred to as being substantially identical to 50 chemical coupling, genetic fusion, noncovalent association a polypeptide sequence encoded by a human VL or VH gene or otherwise) to one or more other binding molecules, such as segment and a human JL or JH segment, even though other another antibody, antibody frgment, peptide or binding non-germline sequences may be present as a result of somatic mimetic. mutation and differential V-J and V-D-J recombination joints. Accordingly, the present invention iiicludes bispecific and With respect to such human sequence antibodies, the varable 55 multispecific composition comprising at least one human regions of each chain are typically at least 80 percent encoded sequence antibody or antigen binding fragment with a first by human gennine V, J, and, in the case of heavy chains, D, binding specificity for human CTLA-4 and a second binding gene segments; frequently at least 85 percent of the variable specificity for a second target epitope. The second target regions are encoded by human gerinine sequences present on epitope can be an Fc receptor, e.g., human FcyRI or a human the trans gene; often 90 or 95 percent or more of the variable 60 F cy receptor. Therefore, the invention includes bispecific and region sequences are encoded by human gerni1ine sequences multispecific molecules capable of binding both to FcyRI, present on the trans gene. However, since non-gennine Fcy R or F cER expressing effector cells (e.g., monocytes, mac- sequences are introduced by somatic mutation and VJ and rophages or polymorphonuclear cells (PMNs)), and to target VDJ joining, the human sequence antibodies frequently have cells expressing hlUlan CTLA-4. These multi-specific (e.g., some variable region sequences (and less frequently constant 65 bispecific or muItispecific) molecules target hlUlan CTLA-4 region sequences) which are not encoded by human V, D, or expressing cells to effector cells, and trigger Fc receptor- J gene segments as found in the human transgene(s) in the mediated effector cell activities, such as phagocytosis of a US 7,605,238 H2 29 30 human CTLA-4-expressing cells, antibody dependent cell- ing specificity is provided by a hiunan sequence antibody or mediated cytotoxicity (ADCC), cytokine release, or genera- a human monoclonal antibody of the present invention. tion of superoxide anion. An "effector cell specific antibody" as used herein refers to The bispecific and multispecific molecules ofthe invention an antibody or fìmctional antibody fragment that binds the Fc can comprise a binding specificity at Ieast one antibody, or an receptor of effector cells. Preferred antibodies for use in the antibody fragment thereof, including, e.g., an Fab, Fab', subject invention bind the Fc receptor of effector cells at a site F(ab'h, Fv, or a single chain Fv. The antibody may also be a which is not bound by endogenous imunoglobulin. light chain or heavy chain dimer, or any minimal fragment As used herein, the term "effector cell" refers to an imune thereof such as a Fv or a singIe chain constnict as described in, cell which is involved in the effector phase of an imnune e.g, Ladner et a!. U.S. Pat. No. 4,946,778. Bispecific and 10 response, as opposed to the cognitive and activation phases of multispecific molecules of the invention can comprise a bind- an imnune response. Exemplar imnune cells include a cell ing specificity for an FcyR or an FcyR present on the surface of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B of an effector cell, and a second binding specificity for a target cells and T cells including cytolytic T cells (CTLs)), killer cell antigen, e.g., human CTLA-4. cells, natual killer cells, macrophages, monocytes, eosino- The binding specificity for an Fc receptor is provided by a 15 phils, neutrophils, polymorphonuclear cells, granulocytes, monoclonal antibody, the binding of which is not blocked by mast cells, and basophils. Effector cells express specific Fc human imnunoglobulin G (IgG). As used herein, the term receptors and carr out specific imune functions. An effec- tor cell can induce antibody-dependent cell-mediated cyto- "IgG receptor" refers to any of the eight y-chain genes located on chromosome 1. These genes encode a total of twelve toxicity (ADCC), e.g., a neutrophil capable of inducing transmembrane or soluble receptor isoforms which are 20 ADCC. For example, monocytes, macrophages, neutrophils, grouped into three Fey receptor classes: FcyRI (CD64), eosinophils, and lymphocytes which express FcaR are FcyRII (CD32), and FeyRlII (CDl6). For example, the Fcy involved in specific killng of target cells and presenting miti- receptor can be a human high affnity FcyRl. The human gens to other components of the imnune system, or binding FcyRI is a 72 kDa molecule, which shows high affnity for to cells that present antigens. An effector cell can also phago- monomeric IgG (108 to 109 M-1). 25 cytose a target antigen, target cell, or microorganism. The expression of a paricular FcR on an effector cell can The production and characterization of these preferred be regulated by humoral factors such as cytokines. For monoclonal antibodies are described by Fanger et a!. in PeT example, expression of FcyRl has been found to be up-regu- application WO 88/00052 and in U.S. Pat. No. 4,954,617. lated by interferongamna (IFN-y). This enhanced expression These antibodies bind to an epitope of FcyRl, FcyRII or 30 increases cytotoxic activity (including, e.g., phagocytosis) by FcyRII at a site which is distinct from the Fcybinding site of FcyRI-bearing cells against target cells. the receptor and, thus, their binding is not blocked substan- "Target cell" shall mean any undesirable cell in a subject tially by physiological levels ofIgG. Specific anti-FcyRI anti- bodies useful in this invention are MAb 22, MAb 32, MAb 44, (e.g., a hiunan or animal) that can be targeted by a composi- tion (e.g., a human sequence antibody or a hiunanmonoclonal MAb 62 and MAb 197. The hybridoma producing MAb 32 is 35 antibody of the invention, a bispecific or a multi specific mol- available from the American Type Culture Collection, ATCC ecule of the invention). The target cell can be a cell expressing Accession No. HB9469. Anti-FcyRl MAb 22, F(ab')2 frag- or overexpressing human CTLA-4. Cells expressing human ments of MAb 22, and can be obtained from Medarex, Inc. CTLA-4 can include tumor cells, e.g. lymphomas. (Annandale, N.J.). In other embodiments, the anti - Fcy recep- In addition to human sequence antibodies and human tor antibody is a humanized form of monoclonal antibody 22 40 monoclonal antibodies of the invention, other antibodies can (H22). The production and characterization of the H22 anti- be also be employed in the bispecific or multispecific mol- body is described in Graziano (1995) J. Imunol 155:4996- ecules of the invention, including, e.g., murine, chimeric and 5002 and PCT/US93/10384. The H22 antibody producing hiunanized monoclonal antibodies. cell line was deposited at the American Type Culture Collec- Chimeric mouse-human monoclonal antibodies (i.e., chi- tion on Nov. 4, 1992 under the designation HA022CLI and 45 meric antibodies) can be produced by recombinant DNA has the accession no. CRL 11177. techniques known in the art. For exmnple, a gene encoding the The binding specificity for an Fc receptor can also be F c constant region of a murine (or other species) monoclonal provided by an antibody that binds to a hiunan IgA receptor, antibody molecule is digested with restriction enzymes to e.g., an Fc-alpha receptor (FcaR (CD89)). Preferably, the remove the region encoding the murine Fc, and the equivalent antibody binds to a human 19A receptor at a site that is not 50 portion of a gene encoding a human Fc constant region is blocked by endogenous IgA. The term "IgA receptor" is substituted. (See, e.g., Robinson et a!., International Patent intended to include the gene product of one a-gene (FcaRl) Publication PCT/US86/02269; Aka, et a!., European Patent located on chromosome 19. 1bs gene is known to encode Application 184,187; Tanguchi, M., European PatentAppli- several alternatively spliced transmembrane isoforms of 55 to cation 171,496; Morrison et a!., European Patent Application 110 kDa. FcaRl (CD89) is constitutively expressed on mono- 55 173,494; Neuberger et aI., International Application WO cytes/macrophages, eosinophilic and neutrophilic granulo- 86/01533; Cabily et al. U.S. Pat. No. 4,816,567; Cabily et cytes, but not on non-effector cell populations. FcaRI has aI., European Patent Application 125,023; Better (1988) Sci- medium affnity (=5x107M-1) for both 19A1 and IgA2, which ence 240:1041-1043; Liu (1987) PNAS 84:3439-3443; Liu is increased upon exposure to cytokines such as G-CSF or (1987) J. Imnunol. 139:3521-3526; Slil (1987) PNAS GM-CSF (Morton (1996) Critical Reviews in Imunology 60 84:214-218; Nishimura (1987) Canc. Res. 47:999-1005; 16:423-440). Four FcaRI-specific monoclonal antibodies, Wood (1985) Nature 314:446-449; Shaw (1988) J. Natl. Can- identified as A3, A59, A62 andA77, which bind FcaRl out- cer Inst. 80: 1553-1559). side the 19A ligand binding domain, have been described by, The chimeric antibody can be fitrther humanized by replac- e.g, Monteiro (1992) J. lmmunol. 148:1764. ing sequences of the Fv variable region which are not directly Bispecific and multispecific molecules of the invention can 65 involved in antigen binding with equivalent sequences from furer comprise a binding specificity which recognzes, e.g., human Fv varable regions. General reviews of hiunanzed binds to, a target cell antigen, e.g. human CTLA-4. The bind- chimeric antibodies are provided by Morrison (1985) Science US 7,605,238 B2 31 32 229: 1202-1207 and by Oi (1986) BioTechniques 4:214. recombinant DNA techniques. Bispecific and multispecific Those methods include isolating, manipulating, and express- molecules of the present invention can also be prepared by ing the nucleic acid sequences that encode all or part of conjugating the constituent binding specificities, e.g., the immunoglobulin Fv variable regions from at least one of a anti-FcR and anti-human CTLA-4 binding specificities, heavy or light chain. Sources of such nucleic acid are well using methods known in the ar and as described herein. For known to those skilled in the art and, for example, may be example, each binding specificity of the bispecific and mul- obtained from 7E3, an anti-GPIIbIIa antibody producing tispecific molecule can be generated separately and then con- hybridoma. TIie recombinant DNA encoding the chimeric jugated to one another. When the binding specificities are antibody, or fragment thereof, can then be cloned into an proteins or peptides, a variety of coupling or cross-linkng appropriate expression vector. Suitable humanized antibod- 10 agents can be used for covalent conjugation. Examples of ies can alternatively be produced by CDR substitution U.S. cross-linlång agents include protein A, carbodiimide, N-suc- Pat. No. 5,225,539; Jones (1986) Nature 321:552-525; Ver- cinimidyl-S-acetyl-thioacetate (SAT A), N-succinimdyl-3- hoeyan et al. 1988 Science 239:1534; and Beidler (1988) J. (2-pyridyldithio )propionate (SPDP), and sulfosuccinimidyl Immunol. 141 :4053-4060. 4-(N-maleimdomethyl) cyclohaxane-1-carboxylate (sulfo- All of the CDRs of a paricular human antibody may be 15 SMCC) (see, e.g., Karovsky (1984) J. Exp. Med. 160:1686; replaced with at least a portion of a non-human CDR or only Liu (1985) Proc. Natl.Acad. Sci. USA 82:8648). Othermeth- some of the CDRs may be replaced with non-hlUiian CDRs. It ods include those described by Paulus (Behring Ins. Mitt. is only necessar to replace the number of CDRs required for (1985) No. 78, 118-132; Brennan (1985) Science 229:81-83), binding of the humanized antibody to the Fc receptor. An Glenne (1987) 1. Immunol. 139: 2367-2375). Other conju- antibody can be humanized by any method, which is capable 20 gating agents are SATA and sulfo-SMCC, both available from of replacing at least a portion of a CDR of a human antibody Pierce Chemical Co. (Rockford, Il. with a CDR derived from a non-human antibody. Winter When the binding specificities are antibodies (e.g., two describes a method which may be used to prepare the human- humanized anti bodies), they can be conjugated via sulfliydry I ized antibodies of the present invention, see UK PatentAppli- bonding of the C-terminus hinge regions of the two heavy cation GB 2188638A, filed on Mar. 26, 1987. The human 25 chains. The hinge region can be modified to contain an odd CDRs may be replaced with non-human CDRs using oligo- number of sulfhydryl residues, e.g., one, prior to conjugation. nucleotide site-directed mutagenesis as described in, e.g., Alternatively, both binding specificities can be encoded in WO 94/1 0332 entitled, Humanized Antibodies to Fe Recep- the same vector and expressed and assembled in tlie same host tors for Immunoglobulin G on Human Mononuclear Phago- celL. This metliod is paricularly useful where the bispecific cytes. 30 and multispecific molecule is a MabxMAb, MabxFab, Fabx Chimeric and humanized antibodies in which specific F(ab')2 or ligandxFab fusion protein. A bispecific and multi- amino acids have been substituted, deleted or added are also specific molecule of the invention, e.g., a bispecific molecule withi the scope of the invention. For example, humanized can be a single chain molecule, such as a single chain bispe- antibodies can have amino acid substitutions in the frame- cific antibody, a single chain bispecific molecule comprising work region, such as to improve binding to the antigen. In a 35 one single chain antibody and a binding determinant, or a humanzed antibody. having mouse CDRs, amino acids single chain bispecific molecule comprising two binding located in the human framework region can be replaced with determinants. Bispecific and multi specific molecules can also the amino acids located at the corresponding positions in the be single chain molecules or may comprise at least two single mouse antibody. Such substitutions are known to improve chain molecules. Methods for preparing bi- and multi specific binding of humanzed antibodies to the antigen in some 40 molecules are described for example in U.S. Pat. Nos. 5,260, instances. Antibodies in which amino acids have been added, 203; ,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; deleted, or substituted are referred to herein as modified anti- 5,013,653; 5,258,498; and 5,482,858. bodies or altered antibodies. Binding of the bispecific and multispecific molecules to Bispecific and multi specific molecules of the invention can their specific targets can be confrmed by enzyme-linked fuher include a third binding specificity, in addition to an 45 immunosorbent assay (ELISA), a radioimimuioassay (RIA), anti-Fc binding specificity and an anti-human CTLA-4 bind- or a Western Blot Assay. Each of these assays generally ing specificity. The third binding specificity can be an aiti- detects the presence of protein-antibody complexes of par- enhancement factor (EF) portion, e.g., a molecule which ticular interest by employing a labeled reagent (e.g., an anti- binds to a surface protein involved in cytotoxic activity and body) specific for the complex of interest. For example, the thereby increases the immiiie response against the target cell. 50 FcR-antibody complexes can be detected using e.g., an The "anti-enhancement factor portion" can be an antibody, enzyme-linked antibody or antibody fragment which recog- functional antibody fragment or a ligand that binds to a given nizes and specifically binds to the antibody-FcR complexes. molecule, e.g., an antigen or a receptor, and thereby results in Alternatively, the complexes can be detected using any of a an enhancement of the effect of the binding determinants for variety of other imiiiuioassays. For example, the antibody the Fc receptor or target cell antigen. The "anti-enhancement 55 can be radioactiveIy labeled and used in a radioimmunoassay factor portion" can bind an F c receptor or a target cell antigen. (RIA) (see, for example, Weintraub, B., Principles of Radio- Alternatively, the anti -enhancement factor portion can bind to imniiiioassays, Seventh Training Course on Radioligand an entity that is different from the entity to which the first and Assay Technques, The Endocrine Society, March, 1986, second binding specificities bind. For example, the anti-en- which is incorporated by reference herein). The radioactive hancement factor portion can bind a cytotoxic T-cell via, e.g., 60 isotope can be detected by such means as the use of a y counter CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other or a scintilation counter or by autoradiography. immiiie cell molecules that are involved in an increased Al so included in the invention are modified antibodies. TIie immiiie response against the target celL. term "modified antibody" includes antibodies, such as mono- Bispecific and multispecific molecules of the present clonal antibodies, chimeric antibodies, and humanized anti- invention can be made using chemical techniques (see, e.g., 65 bodies which have been modified by, e.g., deleting, adding, or Kran (1981) Proc. Natl. Acad. Sci USA 78:5807), "poly- substituting portions of the antibody. For example, an anti- doma" technques (see, e.g., U.S. Pat. No. 4,474,893), or body can be modified by deleting the constant region and US 7,605,238 B2 33 34 replacing it with a constant region meant to increase half-life, (b) the limitations inherent in the art of compounding such an e.g., serum half-life, stability or affty of the antibody. active compound for the treatment of sensitivity in individu- The antibody conjugates of the invention can be used to als. modifY a given biological response or create a biological Examples of pharmaceutically-acceptable antioxidants response (e.g., to recruit effector cells). The drg moiety is include: (1) water soluble antioxidants, such as ascorbic acid, not to be constred as limited to classical chemical therapeu- cysteine hydrocWoride, sodium bisulfate, sodium met- tic agents. For example, the dnig moiety may be a protein or abisulfite, sodium sulfite and the like; (2) oil-soluble antioxi- dants, such as ascorbyl palmitate, butylated hydroxyanisole polypeptide possessing a desired biological activity. Such (BHA), butylated hydroxy toluene (BHT), lecithin, propyl proteins may include, for exampIe, an enzymatically active 10 gallate, alpha-tocopherol, and the like; and (3) metal chelat- toxin, or active fragment thereof, such as abrin, ricin A, ing agents, such as citric acid, ethylenediamine tetraacetic pseudomonas exotoxin, or diphtheria toxin; a protein such as acid (EDT A), sorbitol, tararic acid, phosphoric acid, and the tumor necrosis factor or interferon-alpha; or, biological like. response modifiers such as, for example, Iymphokines, inter- Regardless of the route of administration selected, the leuki-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL- 15 compounds of the present invention, which may be used in a 6"), granulocyte macrophage colony stimulating factor suitable hydrated form, and/or the pharmaceutical composi- ("GM-CSF"), granulocyte colony stimulating factor ("G- tions of the present invention, are formulated into pharma- CSF"), or other growth factors. ceutically acceptable dosage fonns by conventional methods Techniques for conjugating such therapeutic moiety to known to those of skill in the art. antibodies are well known, see, e.g., Amon et aL, "Mono- 20 Actual dosage levels of the active ingredients in the phar- clonal Antibodies For Immunotargeting Of Drugs In Cancer maceutical compositions ofthe present invention can be var- Therapy", in Monoclonal Antibodies And Cancer Therapy, ied so as to obtain an amount of the active ingredient wmch is Reisfeld et aL (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); effective to acmeve the desired therapeutic response for a Hellstrom et aL, "Antibodies For Drug Delivery", in Con- 25 paricular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level trolled Drug Delivery (2nd Ed.), Robinson et aL (eds.), pp. depends upon a variety of pharmacokinetic factors including 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Car- the activity of the paricular compositions of the present riers Of Cytotoxic Agents In Cancer Therapy: A Review", in invention employed, or the ester, salt or amide thereof, the Monoclonal Antibodies '84: Biological And Clinical Appli- route of administration, the time of administration, the rate of cations, PincheraetaL (eds.),pp. 475-506 (1985); "Analysis, 30 excretion of the particular compound being employed, the Results, And Future Prospective Of The Therapeutic Use Of duration of the treatment, other drgs, compounds and/or Radiolabeled Antibody In Cancer Therapy", in Monoclonal materiaIs used in combination with the paricular composi- Antibodies For Cancer DetectionAnd Therapy, Baldwin et aL tions employed, the age, sex, weight, condition, general (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et aL, health and prior medical history of the patient being treated, "The Preparation And Cytotoxic Properties Of Antibody- 35 and like factors. Toxin Conjugates", ImmunoL Rev., 62:119-58 (1982). A physician or veterinarian can start doses of the com- Pharmaceutical Compositions pounds ofthe invention employed in the pharmaceutical com- position at levels lower than that required to achieve the The invention provides pharmaceutical compositions com- desired therapeutic effect and gradually increase the dosage prising one or a combination of human monoclonal antibod- 40 until the desired effect is achieved. In general, a suitable daily ies and/or human sequence antibodies (intact or binding frag- dose of a compositions of the invention is that amount of the ments) formulated together with a pharmaceutically compound which is the lowest dose effective to produce a acceptable carer. Some compositions include a combination therapeutic effect. Such an effective dose generally depends of multiple (e.g., two or more) isolated human antibodies upon the factors described above. It is preferred that adni- and/or human sequence antibody or antigen-binding portions 45 istration be intravenous, intramuscular, intraperitoneal, or thereof of the invention. In some compositions, each of the subcutaneous, or administered proximal to the site of the antibodies or antigen-binding portions thereof of the compo- target. If desired, the effective daily dose of a therapeutic sition is a monoclonal antibody or a human sequence anti- compositions can be administered as two, three, four, five, six body that binds to a distinct, pre-selected epitope of human or more sub-doses administered separately at appropriate CTLA-4. 50 intervaIs throughout the day, optionally, in unit dosage forms. A. Effective Dosages Whle it is possible for a compound of the present invention to Dosage regimens are adjusted to provide the optimum be administered alone, it is preferable to administer the com- desired response (e.g., a therapeutic response). For example, pound as a pharmaceutical formulation (composition). a single bolus may be administered, several divided doses Effective doses of the compositions of the present inven- may be adminstered over time or the dose may be propor- 55 tion, for the treatment of immune-related conditions and dis- tionally reduced or increased as indicated by the exigencies of eases described herein var depending upon many different the therapeutic situation. It is especially advantageous to for- factors, including means of administration, target site, physi- mulate parenteral compositions in dosage unit form for ease ological state of the patient, whether the patient is human or of admstration and uniformity of dosage. Dosage unit form an animal, other medications administered, and whether as used herein refers to physically discrete units suited as 60 treatment is prophylactic or therapeutic. Treatment dosages unitary dosages for the subjects to be treated; each unit con- need to be titrated to optimize safety and effcacy. tains a predetermined quantity of active compound calculated For administration with an antibody, the dosage ranges to produce the desired therapeutic effect in association with from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 the required pharmaceutical carrier. The specification for the mglg, of the host body weight. For example dosages can be dosage unit forms of the invention are dictated by and directly 65 1 mg/kg body weight or 10 mglkg body weight or witmn the dependent on (a) the unique characteristics ofthe active com- range of 1-10 mglg. An exemplar treatment regime entails pound and the particular therapeutic effect to be acmeved, and admnistration once per ever two weeks or once a month or US 7,605,238 H2 35 36 once every 3 to 6 months. In some methods, two or more disease in an amount suffcient to arrest or inbit further monoclonal antibodies with different binding specificities are development or reverse or elimiate, the disease, its symp- adminstered simultaneously, in which case the dosage of toms or biochemical markers. For prophylactic applications, each antibody administered falls within the ranges indicated. the pharmaceutical compositions are admiistered to a Antibody is usually administered on multiple occasions. patient susceptible or at risk of a disease in an amount suff- Intervals between single dosages can be weekly, monthly or cient to delay, inhibit or prevent development of the disease, yearly. Intervals can also be irregular as indicated by measur- its symptoms and biochemical markers. An amount adequate ing blood levels of antibody to CTLA-4 in the patient. In some to accomplish this is defied as a "therapeutically-" or "pro- methods, dosage is adjusted to achieve a plasma antibody phylactically-effective dose." Dosage depends on the disease concentration of 1-1000 fLg/ml and in some methods 25-300 10 being treated, the subject's size, the severity of the subject's fig/mI. Alternatively, antibody can be admstered as a sus- symptoms, and the paricular composition or route of admin- tained release formulation, in which case less frequent admi- istration selected. Specifically, in treatment of tumors, a istration is required. Dosage and frequency vary depending "therapeutically effective dosage" can inhibit tumor growth on the half-life of the antibody in the patient. In general, by at least about 20%, or at least about 40%, or at least about human antibodies show the longest half life, followed by 15 60"10, or at least about 80% relative to imtreated subjects. The humanized antibodies, chimeric antibodies, and nonhuman ability of a compound to inhbit cancer can be evaluated in an antibodies. The dosage and frequency of admnistration can animal model system predictive of effcacy in human tumors. var depending on whether the treatment is prophylactic or Alternatively, this propert of a composition can be evaluated therapeutic. In prophylactic applications, a relatively low by examining the abilty of the compound to inhbit by con- dosage is administered at relatively infrequent intervals over 20 ventional assays in vitro. A therapeutically effective amount a long period of time. Some patients continue to receive of a therapeutic compound can decrease tumor size, or oth- treatment for the rest of their lives. In therapeutic applica- erwise ameliorate symptoms in a subject. tions, a relatively high dosage at relatively short intervals is The composition should be steriIe and fluid to the extent sometimes required until progression of the disease is that the composition is deliverable by syringe. In addition to reduced or terminated, and preferably until the patient shows 25 water, the carrier can be an isotonic buffered saline solution, parial or complete amelioration of symptoms of disease. ethanol, polyol (for example, glycerol, propylene glycol, and Thereafter, the patent can be administered a prophylactic liquid polyetheylene glycol, and the like), and suitable mix- regime. tures thereof. Proper fluidity can be maintained, for example, Doses for nucleic acids encoding iinmunogens range froin by use of coating such as lecithin, by maintenance of required about 10 ng to 1 g, 100 ng to 100 ing, 1 fig to 10 mg, or 30-300 30 paricle size in the case of dispersion and by use of surfac- fig DNA per patient. Doses for infectious viral vectors var tants. In many cases, it is preferable to include isotonic from 10-100, or more, virions per dose. agents, for example, sugars, polyalcohols such as mannitol or Some human sequence antibodies and human monoclonal sorbitol, and sodium chloride in the composition. Long-term antibodies of the invention can be formulated to ensure proper absorption of the injectable compositions can be brought distrbution in vivo. For example, the blood-brain barrer 35 about by including in the composition an agent which delays (BBB) excludes many highly hydrophilic compounds. To absorption, for example, aluniinum monostearate or gelatin. ensure that the therapeutic compounds of the invention cross When the active compound is suitably protected, as the BBB (if desired), they can be formulated, for example, in described above, the compound may be orally administered, liposomes. For methods of manufacturing liposomes, See, for example, with an inert diluent or an assimlable edible e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The 40 carrier. liposomes may comprise one or more moieties which are B. Routes of Adminstration selectively transported into specific cells or organs, thus Pharaceutical compositions of the invention also can be enhance targeted dnig delivery (See, e.g., V. V. Ranade (1989) admnistered in combination therapy, i.e., combined with J. Clin. Pharmacol. 29:685). Exemplary targeting moieties other agents. For example, in treatment of cancer, the combi- include folate or biotin (See, e.g., U.S. Pat. No. 5,416,016 to 45 nation therapy can include a composition of the present inven- Low et a!.); mannosides (Umezawa et aI., (1988) Biochem. tion with at least one anti-tumor agent or other conventional Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloe- therapy, such as radiation treatment. inan et a!. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Pharaceutically acceptable carrers includes solvents, Antimicrob. Agents Chemother: 39: 180); surfactant protein A dispersion media, coatings, antibacterial and antifungal receptor (Briscoe et a!. (1995) Am. J Physiol. 1233:134), 50 agents, isotonic and absorption delaying agents, and the like different species of which may comprise the formulations of that are physiologically compatible. The carrer can be suit- the inventions, as well as components of the invented mol- able for intravenous, intramuscular, subcutaneous, ecules; p120 (Schreieret a!. (1994)J. Bioi. Chem. 269:9090); parenteral, spinal or epidermal admnistration (e.g., by injec- See also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. tion or infusion). Depending on the route of admistration, 346:123; J. 1. Kilion; 1. J. Fidler (1994) Immunomethods 55 the active compound, i.e., antibody, bispecific and multispe- 4:273. In some methods, the therapeutic compOlmds of the cific molecule, may be coated in a material to protect the invention are formulated in liposomes; in a more preferred compound from the action of acids and other natural condi- embodiment, the liposomes include a targeting moiety. In tions that may inactivate the compound. some methods, the therapeutic compounds in the liposomes A "pharmaceutically acceptable salt" refers to a salt that are delivered by bolus injection to a site proximal to the tumor 60 retains the desired biological activity of the parent compound or infection. The composition should be fluid to the extent that and does not impart any imdesired toxicological effects (See, easy syringabilty exists. It should be stable under the condi- e.g., Berge, S. M., et a!. (1977) J. Pharm. Sci. 66:1-19). tions of manufactue and storage and should be preserved Examples of such salts include acid addition salts and base against the contaminating action of microorganisms such as addition salts. Acid addition salts include those derived from bacteria and fimgi. 65 nontoxic inorganc acids, such as hydrochloric, nitrc, phos- For therapeutic applications, the pharaceutical composi- phoric, sulfuric, hydrobromic, hydroiodic, phosphorous and tions are admnistered to a patient suffering from established the like, as well as from nontoxic organic acids such as ali- US 7,605,238 B2 37 38 phatic mono- and dicarboxylic acids, phenyl-substituted tains a basic dispersion medium and the required other ingre- alkanoic acids, hydroxy alkanoic acids, aromatic acids, ali- dients from those emunerated above. In the case of sterile phatic and aromatic sulfonic acids and the like. Base addition powders for the preparation of sterile injectable solutions, the salts include those derived from alkaline earth metals, such as preferred methods of preparation are vacuum diing and sodium, potassium, magnesium, calcium and the like, as well 5 freeze-diing (lyophilization) that yield a powder of the as from nontoxic organic amnes, such as N,N'-dibenzleth- active ingredient plus any additional desired ingredient from ylenediamie, N-methylglucamine, chloroprocaine, choline, a previously sterile-filtered solution thereof. Therapeutic diethanolamine, ethylenediamine, procaine and the like. compositions can also be admnistered with medical devices A composition of the present invention can be adminis- known in the ar. For example, in a preferred embodiment, a tered by a variety of methods known in the ar. The route 10 therapeutic composition of the invention can be administered and/or mode of administration vary depending upon the with a needleless hypodermic injection device, such as the desired results. The active compounds can be prepared with devices disclosed in, e.g., U.S. Pat. Nos. 5,399,163, 5,383, carriers that protect the compound against rapid release, such 851,5,312,335,5,064,413,4,941,880,4,790,824, or 4,596, as a controlled release formulation, including implants, trans- 556. Examples of implants and modules usefuI in the present dermal patches, and microencapsulated delivery systems. 15 invention include: U.S. Pat. No. 4,487,603, which discloses Biodegradable, biocompatible polymers can be used, such as an implantable micro-infìision pump for dispensing medica- ethylene vinyl acetate, polyanydrides, polyglycolic acid, tion at a controlled rate; U.S. Pat. No. 4,486,194, which collagen, polyorthoesters, and polylactic acid. Many methods discloses a therapeutic device for admnistering medicants for the preparation of such formulations are described by e.g., through the ski; U.S. Pat. No. 4,447,233, which discloses a Sustained and Controlled Release Drug Delivery Systems, J. 20 medication infusion pmnp for delivering medication at a pre- R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. cise infsion rate; U.S. Pat. No. 4,447,224, which discloses a Pharmaceutical compositions are preferably manufactured variable flow implantable insion apparatiis for continuous under GMP conditions. dnig delivery; U.S. Pat. No. 4,439,196, which discloses an To admnister a compound of the invention by certain osmotic dnig delivery system having multi-chamber com- routes of administration, it may be necessary to coat the 25 parments; and U.S. Pat. No. 4,475,196, which discloses an compound with, or co-administer the compound with, a mate- osmotic dnig delivery system. Many other such implants, rial to prevent its inactivation. For example, the compound delivery systems, and modules are known. may be administered to a subject in an appropriate carrer, for C. Fonnulation example, liposomes, or a diluent. Pharmaceutically accept- For the therapeutic compositions, formulations of the able diluents include saline and aqueous buffer solutions. 30 present invention include those suitable for oral, nasal, topical Liposomes include water-in-oil-in-water CGF emulsions as (including buccal and sublingual), rectal, vaginal and/or well as conventionalliposomes (Strejan et al. (1984) J. Neu- parenteral administration. The formulations can conveniently roimmunol. 7:27). be presented in unit dosage form and may be prepared by any Pharmaceutically acceptable carriers include sterile aque- methods known in the art of pharmacy. The amount of active ous solutions or dispersions and sterile powders for the 3S ingredient which can be combined with a carrer material to extemporaneous preparation of sterile injectable solutions or produce a single dosage fonn vary depending upon the sub- dispersion. The use of such media and agents for pharmaceu- ject being treated, and the particular mode of administration. tically active substances is known in the art. Except insofar as The amount of active ingredient which can be combined with any conventional media or agent is incompatible with the a carrier material to produce a single dosage form generally active compound, use thereof in the pharmaceutical compo- 40 be that amOlUlt of the composition which produces a thera- sitions of the invention is contemplated. Supplementar peutic effect. Generally, out of one hundred per cent, this active compounds can also be incorporated into the compo- . amount range from about 0.01 percent to about ninety-iune sitions. percent of active ingredient, from about 0.1 percent to about Therapeutic compositions typically must be sterile, sub- 70 per cent, or from about 1 percent to about 30 per cent. stantially isotonic, and stable under the conditions of manu- 45 Formulations of the present invention which are suitable facture and storage. Ibe composition can be formulated as a for vaginal administration also include pessaries, tampons, solution, microemulsion, liposome, or other ordered stnictue creams, gels, pastes, foams or spray formulations containing suitable to high drg concentration. The carrier can be a such carriers as are known in the art to be appropriate. Dosage solvent or dispersion medium containing, for example, water, forms for the topical or transdermal administration of com- ethanol, polyol (for example, glycerol, propylene glycol, and 50 positions of this invention include powders, sprays, oint- liquid polyethylene glycol, and the like), and suitable mix- ments, pastes, creams, lotions, gels, solutions, patches and tiires thereof. The proper fluidity can be maintained, for inhalants. The active compound may be mixed under sterile example, by the use of a coating such as lecithin, by the conditions with a pharmaceutically acceptable carrier, and maintenance of the required paricle size in the case of dis- with any preservatives, buffers, or propellants which may be persion and by the use of surfactants. In many cases, it is 55 required. preferable to include isotonic agents, for example, sugars, The phrases "parenteral administration" and "adminis- polyalcohols such as manntol, sorbitol, or sodium chloride in tered parenterally" mean modes of admnistration other than the composition. Prolonged absorption of the injectable com- enteral and topical administration, usually by injection, and positions can be brought about by including in the composi- includes, without limitation, intravenous, intramuscular, tion an agent that delays absorption, for example, monostear- 60 intraarerial, intrathecal, intracapsular, intraorbital, intracar- ate salts and gelatin. diac, intradermal, intraperitoneal, transtracheal, subcutane- Sterile injectable solutions can be prepared by incorporat- ous, subcuticular, intraaricular, subcapsular, subarachnoid, ing the active compound in the required amount in an appro- intraspinal, epidural and intrasternal injection and infusion. priate solvent with one or a combination of ingredients enu- Examples of suitable aqueous and nonaqueous carriers- merated above, as required, followed by sterilization 65 which may be employed in the pharmaceutical compositions microfiltration. Generally, dispersions are prepared by incor- of the invention include water, ethanol, polyols (such as glyc- porating the active compound into a sterile vehicle that con- erol, propylene glycol, polyethylene glycol, and the like), and US 7,605,238 H2 39 40 suitable mixtures thereof, vegetable oils, such as olive oil, and the variable regions of high-affnity IgG antibodies in IgM injectable organic esters, such as ethyl oleate. Proper fluidity expression vectors or any protein moiety (e.g., polylysine, can be maintained, for example, by the use of coating mate- and the like). Converting a high affnity IgG antibody to an rials, such as lecithin, by the maintenance of the required IgM antibody can create a decavalent complex with very high paricle size in the case of dispersions, and by the use of 5 avidity. IgA2 expression vectors may also be used to produce surfactants. multivalent antibody complexes. IgA2 can form polymers These compositions may also contain adjuvants such as together with J chain and secretory component. IgA2 may preservatives, wetting agents, emulsifYing agents and dis- have the added advantage that it can be additionally persing agents. Prevention of presence of microorganisms crosslinked by the IgA receptor CD89, which is expressed on may be ensured both by sterilization procedures, supra, and 10 neutrophils, macrophages, and monocytes. by the inclusion of various antibacterial and antiningal Agonism can also be obtained using some preparations of agents, for example, paraben, cWorobutanol, phenol sorbic polyclonal antibodies to CTLA-4 comprising antibodies to at acid, and the like. It may also be desirable to include isotonic least two non-overlapping epitopes on CTLA-4. One anti- agents, such as sugars, sodium cWoride, and the like into the body in such a preparation containing two binding sites can compositions. In addition, prolonged absorption of the inject- 15 bind to two molecules ofCTLA-4 to form a small cluster. A able pharmaceutical form may be brought about by the inclu- second antibody possessing different binding sites can then sion of agents which delay absorption such as aluminum link (aggegate) these small clusters to form large clusters, monostearate and gelatin. thereby fonning a compIex ofCTLA-4 (on the cell surface) When the compoiinds of the present invention are admin- that can transduce a signal to the T cell to inhibit, reduce or istered as pharmaceuticals, to humans and animals, they can 20 prevent activation of the T-cell bearing (expressing) CTLA -4. be given alone or as a pharmaceutical composition contain- Thus, some preparations of poly clonal antibodies show simi- ing, for example, 0.01 to 99.5% (or 0.1 to 90%) of active lar agonism to the polyvalent preparations described above. ingredient in combination with a pharmaceutically accept- Therefore, polyvalent or polyclonal preparations of anti able carrer. CTLA-4 antibodies are useful for agonizing the CTLA-4 The phamiaceutical compositions are generally formu- 25 receptor, thereby suppressing immune responses otherwise lated as sterile, substantially isotonic and in full compliance mediated by T cells bearing the CTLA -4 receptor. Some with all Good Manufacturing Practice (GMP) regulations of examples of diseases that can be treated using such polyvalent the U.S. Food and Dnig Administration. or polyclonal preparations of antibodies induce autoinune disease, transplant rejection, and inflammation. Methods and Uses ofthe Invention 30 B. Uses A. Methods 1. Activating Immune Responses The compositions (e.g., human sequence antibodies and a. Cancer human monoclonal antibodies to hiunan CTLA -4 and deriva- Some therapeutic methods treat patients with cancer. tives/conjugates thereof) of the present invention have in vitro Blockade of CTLA-4 by antibodies can enhance the immune and in vivo diagnostic and therapeutic utilities. For example, 35 response to cancerous cells in the patient. Optionally, anti- these molecules can be admnistered to cells in culture, e.g. in bodies to CTLA-4 can be combined with an immunogenic vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent agent, such as cancerous cells, purified tumor antigens (in- or diagnose a varety of disorders. The term "subject" cluding recombinant proteins, peptides, and carbohydrate includes human and non-human animals. Non-human ani- molecules), cells, and cells transfected with genes encoding mals includes all vertebrates, e.g., mammals and non-mam- 40 iiimune stiiiulating cytokines and cell surface antigens such mals, such as non-human primates, sheep, dog, cow, chick- as B7 (see, e.g., Hurwitz, A. et al. (1998) Proc. Natl. A cad. Sci ens, amphibians, and reptiles. The methods are particularly U.S.A. 95, 10067-10071). suitable for treating human patients having a disorder that can In murine experimental systems, implantation of some be treated by augmenting or reducing the T-cell mediated tuors followed by the admstration of anti-CTLA-4 anti- immune response. 45 bodies can result in the rejection of tumors. In some cases When antibodies to CTLA-4 are administered together tumor rejection of established tumors occurs; in other cases with another agent, the two can be admistered in either the growth of the tuiior is slowed by the use of anti-CTLA-4 order or simultaneously. The methods can be used to treat any antibodies. In general CTLA-4 blockade is effective against kind of cancer including melanoma, colon cancer, prostate inunogeiiic tuors. Operationally this is defined as those cancer, and renaI cancer. 50 tumors for which vaccination using the tuiior itself can lead For example, latex microspheres coated with anti-CTLA-4 to immunity to tumor challenge. In humans, soiie tumors (to increase the valency of the antibody) can inhibit T cell have been shown to be immunogenic such as melanoiias. It is proliferation and activation. Agents having the same antibody anticipated that by raising the theshold ofT cell activation by combining site may act as a CTLA-4 antagonist when pre- CTLA-4 blockade, we may expect to activate tumor sentedas anFab or a soluble IgG, and a CTLA-4 agonist when 55 responses in the host. highly cross-linked. Thus multivalent forms of anti-CTLA-4 CTLA -4 blockade is most effective when coiibined with a antibodies are usenil therapeutic agents for down-modulating vaccination protocol. Many experimental strategies for vac- immune responses. cination against tuliOrs have been devised (see Rosenberg, S., In addition to linking to latex microspheres or other 2000, Development of Cancer Vaccines, ASCO Educational insoluble particles, the antibodies can be cross-linked to each 60 Book Spring: 60-62; Logothetis, c., 2000, ASCO Educa- other or genetically engineered to form multimers. Cross- tional Book Spring: 300-302; Khayat, D. 2000, ASCO Edu- linking can be by direct chemical linkage, or by indirect cational Book Spring: 414-428; Foon, K. 2000, ASCO Edu- linkage such as an antibody-biotin-avidin complex. Cross- cational Book Spring: 730-738; see also Restifo, N. and linking can be covalent, where chemical linng groups are Smol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita, employed, or non-covalent, where protein-protein or other 65 V et al. (eds.), 1997, Cancer: Principles and Practice of protein-ligand interactions are employed. Genetic engineer- Oncology. Fifth Edition). In one ofthese strategies, a vaccine ing approaches for Iinkng include, e.g., the re-expression of is prepared using autologous or allogeneic tumor cells. These US 7,605,238 B2 41 42 cellular vaccines have been shown to be most effective when it may be possible to reduce the dose of chemotherapeutic the tumor cells are transduced to express GM -CSF. GM -CSF reagent administered (Mokyr, M. et al. (1998) Cancer has been shown to be a potent activator of antigen presenta- Research 58: 5301-5304). The scientific rationale behind the tion for tumor vaccination (Dranoff et al. (1993) Proc. Nat/. combined use of elLA -4 blockade and chemotherapy is that Acad. Sci U.S.A. 90 (80: 3539-43). cell death, that is a consequence of the cytotoxic action of Anti-CTLA-4 blockade together with the use of GMCSF- most chemotherapeutic compOlUlds, should result in modified tumor cell vaccines has been shown to be effective increased levels of tumor antigen in the antigen presentation in a number of experimental tumor models such as mamar pathway. Other combination therapies that may result in syn- carcinoma (Hurwitz et al. (1998) supra), primary prostate ergy with CTLA -4 blockade through cell death are radiation, cancer (Hurwitz A. et al. (2000)Cancer Research 60 (9): 10 surgery, and hormone deprivation (Kwon, E. et al. (1999) 2444-8) and melanoma (van Elsas, A et al. (1999) J Exp. Proc. Natl. Acad. Sci u.s.A. 96 (26): 15074-9. Each of these Med. 190: 355-66). In these instances, non-i=unogenic protocols creates a source of tuor antigen in the host. Angio- tuors, such as the B 16 melanoma, have been rendered sus- genesis inhibitors iiay also be combined with CTLA-4 block- ceptible to destruction by the imune system. The tumor cell ade. Inhbition of angiogenesis leads to tumor cell death vaccine may also be modified to express other i=une acti- 15 which may feed tumor antigen into host antigen presentation vators such as IL2, and co stimulatory molecules, among oth- pathways. ers. CTLA-4 blocking antibodies can also be used in combina- The study of gene expression and large scale gene expres- tion with bispecific antibodies that target Fc alpha or Fc sion patterns in various tumors has led to the defition of so gama receptor-expressing effectors cells to tumor cells called tumor specific antigens (Rosenberg, SA (1999) Immu- 20 (see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecific nity 10: 281-7). In many cases, these tumor specific antigens antibodies can be used to target two separate antigens. For are differentiation antigens expressed in the tumors and in the example anti-Fc receptor/anti tuor antigen (i.e., Her-2/neu) cell from which the tumor arose, for example melanocyte bispecific antibodies have been used to target macrophages to antigens gp 100, MAGE antigens, Trp-2. More importantly, sites of tumor. This targeting may more effectively activate many of these antigens can be shown to be the targets oftiimor 25 tiimor specific responses. The T cell arm of these responses specific T cells found in the host. CTLA-4 blockade may be would by augmented by the use ofCTLA-4 blockade. Alter- used in conjunction with a collection of recombinant proteins natively, antigen may be delivered directly to DCs by the use and/or peptides expressed in a tumor in order to generate an of bispecific antibodies which bind to tumor antigen and a i=une response to these proteins. These proteins are nor- dendritic cell specific cell suiface marker. mally viewed by the i=une system as self antigens and are 30 Tumors evade host i=une surveilance by a large variety therefore tolerant to them. The tumor antigen may also of mechanisms. Many of these mechanisms may be overcome include the protein telomerase, which is required for the by the inactivation of proteins which are expressed by the synthesis of telomeres of chromosomes and which is tiiiors and which are i=unosuppressive. These include expressed in more than 85% of human cancers and in only a among others Tgfß (Kehrl, 1. et al. (1986) J Exp. Med. 163: limited number of somatic tissues (Kim, N et al. (1994) Sci- 35 1037-1050), IL-lO (Howard, M. & 0' Garra, A. (1992)Immu- ence 266,2011-2013). (These somatic tissues may be pro- nology Today 13: 198-200), and Fas ligand (Hahne, M. et al. tected from i=une attack by various means). Tumor antigen (1996) Science 274: 1363-1365). Antibodies to each of these may also be "neo-antigens" expressed in cancer cells because entities may be used in combination with anti-CTLA-4 to of somatic mutations that alter protein sequence or create counteract the effects of the i=lUlosuppressive agent and fusion proteins between two unrelated sequences (ie. bcr-abl 40 favor tumor i=lUle responses by the host. in the Philadelphia chromosome), or idiotype from B cell Other antibodies which may be used to activate host tiimors. Other tiimor vaccines may include the proteins from i=lUle responsiveness can be used in combination with viruses implicated in human cancers such a Human Papil- anti-CTLA-4. These inchide molecules on the surface of den- loma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and dritic cells which activate DC fl.Ulction and antigen presenta- Kaposi's Herpes Sarcoma Virus (KHSV). Another form of 45 tion. Anti-CD40 antibodies are able to substitute effectively tumor specific antigen which may be used in conjlUlction with for T cell helper activity (Ridge, 1. et al. (1998) Nature 393: CTLA-4 blockade is purfied heat shock proteins (HSP) iso- 474-478). and can be used in conjunction with CTLA-4 anti- lated from the tumor tissue itself. These heat shock proteins bodies (Ito, N. et al. (2000) Immunobiology 201 (5) 527-40). contain fragments of proteins from the tiimor cells and these Activating antibodies to T cell costimulatory molecules such HSPs are highly effcient at delivery to antigen presenting 50 as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160- cells for eliciting tiimor i=unity (Suot, R & Srivastava, P 2169), 4-lBB (Melero, i. et al. (1997) Nature Medicine 3: (1995) Science 269: 1585-1588; Tamura, Y. et al. (1997) 682-685 (1997), and ICOS (Hutloff, A. et al. (1999) Nature Science 278: 117-120. 397: 262-266) may also provide for increased levels ofT cell Dendritic cells (DC) are potent antigen presenting cells activation. that can be used to prime antigen-specific responses. DC's 55 Bone marrow transplantation is currently being used to can be produced ex vivo and loaded with various protein and treat a variety of tuors of hematopoietic origin. While graft peptide antigens as well as tumor cell extracts (Nestle, F. et al. versus host disease is a consequence of this treatment, thera- (1998) Nature Medicine 4: 328-332). DCs may also be trans- peutic benefit may be obtained from graft vs. tiimor duced by genetic means to express these tumor antigens as responses. CTLA-4 blockade can be used to increase the well. DCs have also been fiised directly to tumor cells for the 60 effectiveness of the donor engrafted tumor specific T cells purposes of imunization (Kugler, A. et al. (2000) Nature (Blazar, B. et al. (1999) J Immunoll62: 6368-6377). Medicine 6:332-336). As a method of vaccination, DC i=u- There are also several experimental treatment protocols nization may be effectively combined with CTLA-4 blockade that involve ex vivo activation and expansion of antigen spe- to activate iiore potent anti-tumor responses. cific T cells and adoptive transfer of these cells into recipients CTLA-4 blockade may also be combined with standard 65 in order to antigen-specific T cells against tumor (Greenberg, cancer treatments. CTLA-4 blockade may be effectively R. & Riddell, S. (1999) 285: 546-51). These methods may combined with chemotherapeutic regimes. In these instances, also be used to activate T cell responses to infectious agents US 7,605,238 B2 43 44 such as CMV (see below). Ex vivo activation in the presence C. Promoting Beneficial ''Autoimune'' Reactions for the of anti-CTLA-4 antibodies may be expected to increase the Treatment of Disease and Therapeutic Intervention. frequency and activity of the adoptîvely transferred T cells. The ability of anti-CTLA-4 antibodies to provoke and b. Infectious Diseases amplify autoimmune responses has been documented in a Other methods of the invention are used to treat patients number of experimental systems (EAE~Experimental that have been exposed to particular toxins or pathogens. Autoimune EncephaIomyelitis, a murine model for MS Simlar to its application to tumors as discussed above, anti- (Perrn, P. et a!. (1996) J. Immunol 157 (4): 1333-1336); body mediated CTLA-4 blockade can be used alone, or as an diabetes (Luhder, F. et a!. (1998) supra). Indeed, induction of adjuvant, in combination with vaccines, to stimulate the 10 anti-tuor responses using tumor cell and peptide vaccines iinune response to pathogens, toxins, and self-antigens. reveals that many anti-tuor responses involve anti-selfreac- CTLA -4 blockade has been shown to be effective in the acute tivities (depigtnentation observed in anti-CTLA-4+GM-CSF phase of infections of Nippostrongylus brasiliensis (McCoy, modified B 16 melanoma in van Elsas et a!. supra; depigmen- K. et a!. (1997) 186(2); 183-187) and Leishmania donovani tation in Trp-2 vaccinated mice (Overwijk, W. et a!. (1999) (Murphy, M. et a!. (1998) J. Immunol. 161:4153-4160). 15 Proc. Natl. Acad. Sci Us.A. 96: 2982-2987); autoimmune Examples of pathogens for which this therapeutic approach prostatitis evoked by TRAP tumor cell vaccines (Hurwitz, may be particularly useful, include pathogens for which there A. (2000) supra ), melanoma peptide antigen vaccination and is currently no effective vaccine, or pathogens for which vitilago observed in human clinical trials (Rosenberg, SA and conventional vaccines are less than completely effective. Whte, D E (1996) J. Immunother Emphasis Tiimor Immunol These include, but are not limited to my, Hepatitis (A, B, & 20 19 (1): 81-4). C), Infuenza, Herpes, Giardia, Malaria, Leishmana, Staphy- Therefore, it is possible to consider using anti-CTLA-4 lococcus aureus, Pseudomonas Aeruginosa. CTLA-4 block- blockade in conjunction Witli various self proteins in order to ade is particularly useful against established infections by devise vaccination protocols to effciently generate imune agents such as HIV that present altered antigens over the responses against these self proteins for disease treatment. 25 For example, Alzheimers disease involves inappropriate course of the infections. These novel epitopes are recognized accumulation of Aß peptide in amyloid deposits in the brain; as foreign at the time of anti-human CTLA-4 administration, antibody responses against amyloid are able to clear these thus provoking a strong T cell response that is not dampened amyloid deposits (Schenk et a!., (1999) Nature 400: 173- by negative signals through CTLA-4. 177). Some examples of pathogenic vinises causing infections 30 Other self proteins may also be used as targets such as IgE treatable by methods of the invention include hepatitis (A, B, for the treatment of allergy and asthma, and TNF for rhema- or C), herpes vinis (e.g., VZY, HSV-l, HAV-6, HSV-II, and toid artitis. Finally, antibody responses to various hor- CMV, Epstein Barr vinis), adenovinis, influenza vinis, fla- mones may be induced by the use of anti-CTLA-4 antibody. vivinises, echovinis, rhinovinis, coxsackie vinis, comovinis, Neutralizing antibody responses to reproductive hormones respiratory syncytial vinis, mumps vinis, rotavirs, measles 35 may be used for contraception. Neutralizing antibody VinIS, nibella vinis, parvovinis, vaccinia vinis, HTLV vinis, response to hormones and other soluble factors that are dengue vinis, papilomavinis, molluscum vinis, poliovinis, required for the growth of particular tuniorS may also be rabies vinis, JC virs and arboviral encephalitis vinis. considered as possible vaccination targets. Some examples of pathogenic bacteria causing infections Analogous methods as described above for the use of anti- treatable by methods of the invention include chlamydia, 40 CTLA-4 antibody can be used for induction of therapeutic rickettsial bacteria, mycobacteria, staphylococci, strepto- autoiinune responses to treat patients having an inappropri- cocci, pneumonococci, meningococci and conococci, kleb- ate accumulation of other self-antigens, such as amyloid siella, proteus, serratia, pseudomonas, legionella, diphtheria, deposits, including Aß in Alzheimer's disease, cytokines salmonella, bacili, cholera, tetanus, botulism, anthax, such as TNFa, and IgE. 45 plague, leptospirosis, and Lymes disease bacteria. 2. Inactivating Immune Responses Some examples of pathogenic fungi causing inections Disorders caused by iinune responses are called hyper- treatable by methods of the invention include Candida (albi- sensitivity disease, Diseases caused by failure of self-toler- cans, krusei, glabrata, tropicalis, etc.), Cryptococcus neofor- ance and subsequent iinune responses against self, or mans, Aspergillus (jimigatus, niger, etc.), Genus Mucorales 50 autologous, antigens are called autoiinune diseases. Hyper- (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomy- sensitivity diseases can also result fTom uncontrolled or ces dermatitidis, Paracoccidioides brasiliensis, Coccidioides excessive responses against foreign antigens, such as immitis and Histoplasma capsulatum. microbes. Some examples of pathogenic parasites causing infections Although soluble antibodies to human CTLA-4 have been treatable by methods of the invention include Entamoeba 55 shown to promote the expansion and activation ofT cells (i .e., histolytica, Balantidium coli, Naegleriafowleri, Acan- where CTLA-4 function (e.g., binding to ligand) is inhibited; thamoeba sp., Giardia lambia, Cryptosporidium sp., Pneu- in this scenario the antibodies are antagonists to CTLA-4 mocystis carinii, Plasmodium vivax, Babesia microti, Trypa- function), increasing the valency of these same antibodies nosoma brucei, Trypanosoma cruzi, Leishmania donovani, produces the opposite effect (where now, in contrast, the Toxoplasma gondi, Nippostrongylus brasiliensis. 60 antibodies are acting as agonists of CTLA-4 to suppress the In all of the above methods, a CTLA-4 blockade can be iinune response) (see, e.g., Krinel and Allson, 1996, J. combined with other forms ofiinunotherapy such as cytok- Exp. Med. 183, 2533-2540). For the purposes of inactivating ine treatment (e.g. interferons, GM-CSF, GCSF, IL-2), or antigen specific T cell responses, such as those that are the bispecific antibody therapy, which provides for enhanced targets of pathogenic autoreactive T cells, the target antigen presentation of tuor antigens (see, e.g., Hollger (1993) 65 which is specific for these T cells (ie. antigen and/or MHC/ Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) antigen complexes) must be administered with the polyvalent Structure 2:1121-1123). form of anti-CTLA-4 antibody. US 7,605,238 B2 45 46 a. Inamation transplant (host-versus-graft disease) leading to destniction Inflammation represents the consequence of capilary dila- of the transplanted tissue. CD8+ cel1s, CD4 + cel1s and mono- tion with accumulation of fluid and migration of phagocytic cytes are all involved in the rejection of transplant tissues. The leukocytes, such as granulocytes and monocytes. Infamma- therapeutic agents of the present invention are useful to tion is important in defending a host against a variety of inhibit T-cel1 mediated aIloantigen-induced imune infections but can also have undesirable consequences in responses in the donee thereby preventing such cel1s from inammatory disorders, such as anaphylactic shock, arhrtis, paricipating in the destniction of the transplanted tissue or gout and ischemia-reperfusion. Activated T-cel1s have an organ. important modulatory role in infammation, releasing inter- B. Methods for Detecting/Measuring the Presence of feron y and colony stimulating factors that in tum activate 10 CTLA-4 in a Sample phagocytic leukocytes. TIie activated phagocytic leukocytes The invention fuher provides methods for detecting the are induced to express a number of specific cel1s surface presence of human CTLA-4 antigen in a sample, or measur- molecules termed homingrec€ptors, which serve to attach the ing the amount of human CTLA-4 antigen, comprising con- phagocytes to target endothelial cells. Inamatory tacting tiie sample, and a control sample, with a human mono- responses can be reduced or elimnated by treatment with the 15 clonal antibody, or an antigen binding portion thereof, which therapeutic agents of the present invention. For example, specifical1y binds to hlUnan CTLA-4, under conditions that polyvalent preparations of antibodies against CTLA-4 block al10w for formation of a complex between the antibody or activation of activated T-cel1s, thereby preventing these cells portion thereof and human CTLA -4. The formation of a com- from releasing molecules required for activation of phago- plex is then detected, wherein a difference complex formation cytic cel1 types 20 between the sample compared to the control sample is indica- b. Autoimmune Diseases tive the presence of human CTLA-4 antigen in the sample. A fìirther situation in which immune suppression is desir- C. Kits able is in treatment of autoimune diseases such as insulin- Also within the scope of the invention are kits comprising dependent diabetes mel1itus, multiple sclerosis, stiff man syn- the compositions (e.g., human sequence antibodies, human drome, rheumatoid arthritis, myasthenia gravis and lupus 25 antibodies, multi specific and bispecific molecules) of the erythematosus. In these diseases, the body develops a cellular invention and instnictions for use. The kit can fìirther contain and/or humoral immune response against one of its own a least one additional reagent, or one or more additional antigens leading to destrction ofthat antigen, and potential1y hunian antibodies of the invention (e.g., a human antibody crippling and/or fatal consequences. Activated T-cells are having a complementary activity which binds to an epitope in believed to playa major role in many autoimmune diseases 30 CTLA -4 antigen distinct from the fist human antibody). Kits such as diabetes melltus. Autoimmune diseases are treated typically include a label indicating the intended use of the by administering one ofthe therapeutic agents of the inven- contents of the kit. The tenn label includes any writing, or tion that inhibits activation ofT cel1s. Optional1y, the autoan- recorded material supplied on or with the kit, or which oth- tigen, or a fragment thereof, against which the autoimmune erwise accompanies the kit. disease is targeted can be administered shortly before, con- 35 currently with, or shortly after the immunosuppressive agent. EXALES In this maner, tolerance can be induced to the auto antigen under cover of the suppressive treatment, thereby obviating Example 1 the need for continued immunosuppression. See, e.g., Cob- bold et a!., WO 90115152 (1990). 40 Generation of Cmu Targeted Mice c. Graft Versus Host Disease A related use for the therapeutic agents of the present Constniction of a CMD Targeting Vector. invention is in modulating the immune response involved in The plasmid pICEmu contains an EcoRI/Xhol fragment of "graft versus host" disease (GVHD). GVHD is a potential1y the murine Ig heavy chain locus, spaning the mu gene, that fatal disease that occurs when immunological1y competent 45 was obtained from a Balb/C genomic lambda phage library cells are transferred to an allogeneic recipient. In this situa- (Marcu et a!. Cell 22 : 187, 1980). This genomic fragment was tion, the donor's immunocompetent cel1s may attack tissues sub cloned into the XholiEcoRl sites of the plasmid in the recipient. Tissues of the ski, gut epithelia and liver are pICEMI9H (Marsh et a!.; Gene 32, 481-485, 1984). The frequent targets and may be destroyed during the course of heavy chain sequences included in pICEmu extend down- GVHD. The disease presents an especial1y severe problem 50 stream of the EcoRl site located just 3' of the mu intronic when immune tissue is being transplanted, such as in bone enhancer, to the XhoI site located approximately 1 kb down- marrow transplantation; but less severe GVHD has also been stream of the last transmembrane exon of the mu gene; how- reported in other cases as wel1, including heart and liver ever, much of the mu switch repeat region has been deleted by transplants. The therapeutic agents of the present invention passage in E. coli. are used to inbit activation of donor leukocytes, thereby 55 The targeting vector was constnicted as fol1ows (see FIG. inhibiting their ability to lyse target cells in the host. 1). A 1.3 kb HindII/SmaI fragment was excised from d. Transplant Rejection pICEmu and sub cloned into HindIIISmaI digested pBlue- Over recent years there has been a considerable improve- script (Stratagene, La 1011a, Calif.). This pICEmu fragment ment in the effciency of surgical techniques for transplanting extends from the HindII site located approximately 1 kb 5' of tissues and organs such as skin, kidney, liver, heart, lung, 60 Cmul to tiie SmaI site located witli Cmu1. The resulting pancreas and bone marow. Perhaps the principal outstanding plasmid was digested with SmallS pel and the approximately problem is the lack of satisfactory agents for inducing 4 kb SmaIlbaI fragment from pICEmu, extending from the immune-tolerance in the recipient to the transplanted Sma I site in Cmu! 3' to the XbaI site located just downstream al10graft or organ. When al10geneic cel1s or organs are trans- of the Iast Cmu exon, was inserted. The resulting plasmid, planted into a host (i.e., the donor and donee are different 65 pTAR1, was linearzed at the Sma1 site, and aneo expression individual from the same species), the host immune system is cassette inserted. This cassette consists of the neo gene under likely to mount an immune response to foreign antigens in the the transcriptional control of the mouse phosphoglycerate US 7,605,238 B2 47 48 kiase (pgk) promoter (XbaI/TaqI fragment; Adra et a!. Generation of Mice Bearing the Mutated mu Gene. (1987) Gene 60: 65-74) and containing the pgk polyadenyla- The three targeted ES clones, designated number 264,272, tion site (pvuIIHindII fragment; Boer et a!. (1990) Bio- and 408, were thawed and injected into C57BLl6J blastocysts chemical Genetics 28: 299-308). This cassette was obtained as described by Bradley (Bradley, A. (1987) in Teratocarci- from the plasmid pKJ1 (described by Tybulewicz et a!. (1991) 5 nomas and Embryonic Stem Cells: a Practical Approach. (E. Cell 65: 1153-1163) from which the neo cassette was excised J. Robertson, ed.) Oxford: IRL Press, p. 113-151). Injected as an EcoRI/HindII fragment and subcloned into EcoRI/ HindII digested pGEM-7Zf(+) to generate pGEM-7 (KJ1). blastocysts were transferred into the uteri of pseudopregnant The neo cassette was excised from pGEM -7 (KJ1) by EcoRI/ females to generate chimeric mice representing a mixture of SalI digestion, blunt ended and sub cloned into the SmaI site 10 cells derived from the input ES cells and the host blastocyst. of the plasmid pTAR1, in the opposite orientation of the The extent of ES cell contribution to the chimera can be genomic Cmu sequences. The resulting plasmid was linear- visually estimated by the amount of agouti coat coloration, ized with Not I, and a herpes simplex virus thymidine kinase derived from the ES cell line, on the black C57BLl6J back- (tk) cassette was inserted to allow for enrchment ofES clones ground. Clones 272 and 408 produced only low percentage bearing homologous recombinants, as described by Mansour IS chimeras (i.e. low percentage of agouti pigmentation) but et a!. (1988) Nature 336: 348-352. This cassette consists of clone 264 produced high percentage male chimeras. These the coding sequences of the tk gene bracketed by the mouse chimeras were bred with C57BL/6J females and agouti off- pgk promoter and polyadenylation site, as described by spring were generated, indicative of germine transmission of Tybulewicz et a!. (1991) Cell 65: 1153-1163. The resulting the ES cell genome. Screening for the targeted mu gene was CMD targeting vector contains a total of approximately 5.3 20 carried out by Southern blot analysis of BglI digested DNA kb ofhomoIogy to the heavy chain locus and is designed to from tail biopsies (as described above for analysis ofES cell generate a mutant mu gene into which has been inserted a neo DNA). Approximately 50% of the agouti offspring showed a expression cassette in the unique SmaI site of the fist Cmu hybridizing BglI band of 7.7 kb in addition to the wild type exon. The targeting vector was linearized with PvuI, which band of 15.7 kb, demonstrating a germline transmission of the cuts within plasmid sequences, prior to electroporation into 25 targeted rnu gene. ES cells. Generation and Analysis of Targeted ES Cells. AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62: 1073-1085) were grown on mitotically inactive Analysis of Transgenic Mice for Functional Inactivation of SNL 76/7 cell feeder layers (ibid.) essentially as described 30 mu Gene. (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic To determie whether the insertion of the neo cassette into Stem Cells: a Practical Approach (E. J. Robertson, ed.) Cmul has inactivated the Ig heavy chain gene, a clone 264 Oxford: IRL Press, p. 71-112). The linearized CMD targeting chimera was bred with a mouse homozygous for the JHD vector was electroporated into AB-1 cells by the methods described Hasty et a!. (Hasty, P. R. et a!. (1991) Nature 350: 35 mutation, which inactivates heavy chain expression as a result 243-246). Electroporated cells were plated into 100 in of deletion of the JH gene segments (Chen et a!., (1993) dishes at a density of 1-2x106 cells/dish. Afer 24 hours, Immunol. 5: 647-656). Four agouti offspring were generated. G4l8 (200 micrograms/ml of active component) and FIAU Serum was obtained from these animals at the age of 1 month (5xlO-7 M) were added to the medium, and dnig-resistant and assayed by ELISA for the presence of murine IgM. Two clones were allowed to develop over 8-9 days. Clones were 40 of the four offspring were completely lacking IgM (Table 1). picked, trypsinized, divided into two portions, and nirther Genotyping of the four animals by Southern blot analysis of expanded. Half of the cells derived from each clone were then DNA from tail biopsies by BglI digestion and hybridization frozen and the other half analyzed for homologous recombi- with probe A (FIG. 1), and by StuI digestion and hybridiza- nation between vector and target sequences. tion with a 475 bp EcoRI/StuI fragment (ibid.) demonstrated DNA analysis was carried out by Southern blot hybridiza- 45 that the animals which fail to express serum IgM are those in tion. DNA was isolated from the clones as described Laird et which one allele of the heavy chain locus carres the ilD a!. (Laird, P. W. et a!., (1991) Nucleic Acids Res. 19: 4293). mutation, the other allele the Cmu1 mutation. Mice heterozy- Isolated genomic DNA was digested with SpeI and probed gous for the JHD mutation display wild type levels of senim with a 915 bp Sad fragment, probe A (FIG. 1), which hybrid- Ig. l1iese data demonstrate that the Cmu1 mutation inacti- izes to a sequence between the mu intronic enhancer and the 50 vates expression of the mu gene. mu switch region. Probe A detects a 9.9 kb SpeI fragment from the wild type locus, and a diagnostic 7.6 kb band from a TABLE 1 mu locus which has homologously recombined with the CMD targeting vector (the nea expression cassette contains a Level of serum IgM, detected by ELISA, for mice cariiig both the CMD and JHD mutations (CMD/JHD), for mice heterozygous for SpeI site). Of1132 G418 andFIAU resistant clones screened 55 the JHD mutatiou (+/JHD). forwild type (129Sv x C57BL/6J)FI by Southern blot analysis, 3 displayed the 7.6 kb Spe I band mice (+/+). and for B cell deficient mice homozygous for the JHD indicative of homologous recombination at the mu locus. mutation (lHD/JHD). These 3 clones were furter digested with the enzymes BglI, Senun IgM BstXI, and EcoRI to verify that the vector integratedhomolo- Mouse (micrograms/ml) Ig H chain genotye gously into the mu gene. When hybridized with probe A, 60 Southern blots of wild type DNA digested with BglI, BstXI, 42 ~0.002 CMD/JHD 43 196 +/JHD or EcoRI produce fragments of15.7, 7.3, and 12.5 kb, respec- 44 ~0.002 CMD/JHD tively, whereas the presence of a targeted mu allele is indi- 45 174 +/JHD cated by fragments of7.7, 6.6, and 14.3 kb, respectively. All 129 x BL6 FI 153 +/+ 3 positive clones detected by the SpeI digest showed the 65 JHD ~0.002 JHD/JHD expected BglI, BstXI, and EcoRI restrction fragments diag- nostic of insertion of the neo cassette into the Cmul exon. US 7,605,238 B2 49 50 Example 2 plasmid vector llid the DNA sequence was determined. The cloned insert was then sub cloned into the vector pBABE Generation of HCo 12 Transgenic Mice (which contains a gene encoding for puromycin resistance (Morganstern, J P and Land, H Nucl. Acids Res. 18: 3587-96 The HCo12 human heavy chain transgene. 5 (1990)) to create pBABE-huCTLA-4/CD3z. pBABE- The HCo 12 transgene was generated by coinjection of the huCTLA-4/CD3z was transfected into the retroviral packag- 80 kb insert of pHC2 (Taylor et aI., 1994, lnt. lmmunol., 6: ing line, 1j-2, and a pool of puromycin resistant cells were 579-591) and the 25 kb insert ofp Vx6. The plasmid pVx6 was selected. These cells were co-cultured with the murine T cell constructed as described below. hybridoma BW5l47 (ATCC #TIB-47). After 2 days of co- An 8.5 kb HindII/SalI DNA fragment, comprising the io culture the non-adherent BW5147 cells were removed and gerniine hlUuan VHl-18 (DP-14) gene together with selected for resistance to puromycin. The puromycin resistant approximately 2.5 kb of 5' flankng, and 5 kb of 3' flanking genomic sequence was subcloned into the plasmid vector cell pool was subcloned by limiting dilution and tested for pSP72 (Promega, Madison, Wis.) to generate the plasmid surface expression of human CTLA-4 by FACS. A clone p343.7.l6. A 7 kb BamHI/HindII DNA fragment, compris- 15 expressing high levels of human CTLA-4 at the cell surface ing the germine human VH5-51 (DP-73) gene together with was selected. approximately 5 kb of 5' flankng and 1 kb of 3' flankng Soluble Antigen genOlnic sequence, was cloned into the pBR322 based plas- Recombinant CTLA-4 fusion protein comprismg the mid cloning vector pGP 1 f (Taylor et a!. 1992, Nucleic Acids extracellular domain of human CTLA-4 was purchased from Res. 20: 6287-6295), to generate the plasmid p25lf. A new 20 R&D Systems (Cat. #325-CT-200). Extracellular CTLA-4 cloning vector derived from pGPlf, pGP1k (Seq. ID #1), was fragment was prepared by proteolytic cleavage of the digested with EcoRV/BamHI, and ligated to a 10 kb EcoRV/ CTLA-4 fusion protein at a Factor Xa protease cleavage site BamHl DNA fragment, comprising the germine human located after the C-terminus of the CTLA -4 extracellular VH3-23 (DP47) gene together with approximately 4 kb of 5' domain. Fusion protein was treated with Factor Xa at a ratio flankng and 5 kb OBI flankng genomic sequence. The result- 25 of 50:1 of fiision protein to Factor Xa, and the CTLA-4 ing plasmid, pl12.2RR. 7, was digested with BamHl/SalI and fragment was isolated by passage over protein G-Sepharose ligated with the 7 kb purified BamHl/SalI insert of p251 f. The and Mono Q HPLC. Fractions were tested for the presence of resulting plasmid, p Vx4, was digested with Xhol and ligated human CTLA-4 dimerwere by SDS-PAGE and by binding to with the 8.5 kb XhoI/SalI insert ofp343.7.16. A clone was cells expressing mouse B7 molecules (LtkmB7.1: mouse Ltk obtained with the VHl-18 gene in the same orientation as the 30 (-) cells trllisfected with a mouse B7.1 cDNA clone expres- other two V genes. This clone, designated pVx6, was then sion vector). Positive fractions were pooled and dialyzed into digested with NotI and the purified 26 kb insert coinjected- PBS buffer. together with the purified 80 kb NotI insert of pHC2 at a 1: 1 Transgeiuc Mice molar ratio-into the pronuclei of one-half day (C57BL/6Jx DBA/2J)F2 embryos as described by Hogan et a!. (B. Hogan 35 Two different strains of mice were used to generate et aI., Manipulating the Mouse Embryo, A Laboratory CTLA-4 reactive monoclonal llitibodies. Strain ((CMD)++; Manual, 2nd edition, 1994, Cold Spring Harbor Laboratory (JKD)++; (HC07)11952+/++; (KC05)9272+/++), and strain Press, Plainview N.Y). Three independent lines of transgenic ((CMD)++; (JKD)++; (HCoI2)15087+/++; (KC05) mice comprising sequences from both Vx6 and HC2 were 9272+/ ++). Each of these strains are homozygous for disnip- established from mice that developed from the injected 40 tions of the endogenous heavy chain (CMD) and kappa light embryos. These lines are designated (HCo12)14881, chain (JK) loci. Both strains also comprise a human kappa light chain transgene (KC05), with individual anals either (HCo12)15083, and (HCo12)15087. Each of the three lines were then bred with inice comprising the CMD mutation hemizygous or homozygous for insertion #11952. The two described in Example 1, the JKD mutation (Chen et al. 1993, strains differ in the human heavy chain transgene used. Mice EMBO J. 12: 811-820), and the (KC05)9272 trans gene (Fish- 45 were hemizygous or homozygous for either the HC07 or the wild et al. 1996, Nature Biotechnology 14: 845-851). The HCo12 transgene. The CMD mutation is described above in resulting mice express human heavy and kappa light chain Example 1. The generation of (HCoI2)15087 mice is described in Example 2. The JKD mutation (Chen et a!. 1993, trans genes in a backgrolUidhomozygous for disruption of the endogenous mouse heavy and kappa light chain loci. EMBO J. 12: 811-820) and the (KC05)9272 (Fishwild et al. 50 1996, Nature Biotechnology 14: 845-851) and (HC07)11952 Example 3 mice, are described in U.S. Pat. No. 5,770,429 (Lonberg & Kay, Jun. 23, 1998). Generation of Huian 19G Kappa Anti-Huian Immu1Uzation CTLA-4 Monoclonal Antibodies Transgeiuc mice were initially immu1Uzed i.p. with 1-3x 55 107 cells in PBS, or with 10-50 ug soluble fusion protein in Cell Based Antigen adjuvant (either complete Freund's or Ribi). Immunized mice A DNA segment encoding a fiision protein comprising were subsequently boosted every 2 to 4 weeks i.p. with 1-3x sequences from the huian CTLA-4 and the murine CD3zeta 107 cells in PBS. Anmals were kept on protocol for 2 to 5 genes was constructed by PCR amplification of cDNA clones months. Prior to fusion, animals were boosted Lv. on days-3 together with bridging synthetic oligonucleotides. The 60 and -2 with approximately 106 cells, or with 10-20 ug soluble encoded fiision protein contains the following sequences: i. antigen (fiision protein or fiision protein and extracellular human CTLA -4 encoding amino acids 1-190 (containig the fragment). Some a1Umals also received fiision protein i.v. on signal peptide, the extracellular domain of human CTLA-4 day-4. Successfiil fiisions resulting in CTLA-4 reactive 19G and the entirety ofthe presuied transmembrane sequence of kappa monoclonal antibodies were obtained from mice" human CTLA-4) and ii. murine CD3zeta from amino acid 52 65 immunzed by a variety of different protocols, including cells to the carboxy temiinus (Weissman et al. (1988) Science 239: only, soluble antigen only, and cell immunizations followed 1018-1021). The amplified PCR product was cloned into a by soluble antigen given i.v. prior to fiision. US 7,605,238 B2 51 52 Fusions Approximately 40% of the hybridomas appear to strongly Spleen cells were fused to mouse myeloma cells (line inhbit CTLA-4 binding to the B7 ligand. P3x63 Ag8.6.53, ATCC CRL 1580, or SP2/0-AgI4, ATCC Antibodies from clones lOD1.3, 4B6.12, and llE8, were CRL 1581) by standard procedures (Harlow and Lane, 1988, then assayed by BIAcore (BiacoreAB, Uppsala, Sweden) to Antibodies, A Laboratory Manual, Cold Spring Harbor Labo- determine binding kinetics. Purified recombinant CTLA-4 ratory Press, Cold Spring Harbor N.Y.; Kennett et al. 1980, extracellular fragment was coupled to the CM5 sensor chip (l Monoclonal Antibodies, Hybridomas: A New Dimension in 1200 RU. Binding was measured by adding antibody at con- Biological Analysis. Plenum, N.Y.; Oi and Hertzenberg, centrations of 0.25, 0.5, 1,2.5, and 5 uglml at a flow rate of5 1980, Immunoglobulin Producing Hybrid Cell Lines, in ul/min. The binding curves were fit to a Langmuir binding Selected Methods In Cellular Immunology, ed. Mishell and 10 model using BIAevaluation softare (Biacore AB, Uppsala, Shiigi, pp. 357-372. Freeman, San Francisco; Halk, 1984, Sweden). Antibodies were purified by protein-A Sepharose Methods in Enzymology: Plant Molecular Biology, ed. chromatography. Determined on and off rates are shown in Weissbach and Weissbach, pp. 766-780, Academic Press, Table 2: Orlando, Fla.). Cells were cultured in DMEM, 10% FBS, OPI

(Sigma 0-5(03), BME (Gibco 21985-023), 3% OrigenHybri- 15 TABLE 2 doma Cloning Factor (Igen IG50-0615), and 5% P388d1 (ATCC TIE 63) conditioned media. HAT or HT supplement Kinetics of binding of hiJan IgG kappa antibodies to was added to the medium during initial growth and selection. recombinant CTLA-4 immobilized on a surface.

Hybridoma Screening Hybridoma ka(l/Ms) kd (lIs) Ka(l/M) To identify hybridomas secreting human IgG kappa anti- 20 bodies, ELISA plates (Niilc MaxiSorp) were coated over- IOD1. 4.1 x 10' 1.0 x 10-4 4 x 109 4B6.12 5.1 x 10' 1.3 X 10-4 4x 109 night at 4° C. with 100 ul/well goat anti-human Fcgamma liES 4.3 X 105 I.S X 10-4 2 x 109 specific antibody (Jackson Il1uno Research #109-006-098) at 1 uglml in PBS. Plates were washed and blocked with 100 ul/well PBS-Tween containing 1 % BSA. Fift ul cell culture 25 Serial dilutions of 10 different human IgG kappa anti- supernatant was added followed by a 1-2 hour incubation. human CTLA-4 monoclonal antibodies (3A4, 9A5, 2E2, Plates were washed and then incubated for one hour with 100 2E7, 4B6, 4ElO, 5C4, 5Gl, 11E8, and 1 1Gl) were added to ul/well goat anti-Kappa light chain conjugated to alkaline microtiter wells coated with recombinant CTLA-4 fìision phosphatase or horseradish peroxidase (Sigma #A-3813, or protein. After a 2 hour incubation, biotiny lated antibody 11 E8 #A-7164). Plates were washed three times in PBS-Tween 30 was added to each well at a concentration of 0.1 uglm1. The between each step. An analogous assay was used to identify samples were incubated for 30 miutes, washed, and bound hybridomas that secrete human antibodies reactive with antibody detected with alkaline phosphatase/streptavidin human CTLA-4. This assay was identical except that the conjugate. The titrations are shown in FI G. 3. Antibody 11 E8 ELISA plates were coated with recombinant CTLA-4 fìision binding was blocked by itself and 7 of the other human protein instead of goat anti-human Fcgaia antibody. 35 antibodies. However, binding was not blocked by antibodies Characterization of Monoclonal Antibodies 3A4 or 9A5. Reciprocal binding experiments showed that Seventy two hybridomas that were shown by ELISA to 11E8 binding did not block either 3A4 or 9A5 binding to secrete human IgG kappa binding to human CTLA-4 were CTLA-4. subcloned. Fort seven of these subclones were tested to DNA Sequence determie if the secreted human antibodies bind to CTLA-4 40 RNA was extracted from approximately 2x106 cells of expressing cells, and if the antibodies inhbit soluble CTLA-4 each sub cloned hybridoma cell line and used to synthesize from binding to cells expressing B7. Binding was determined CDNA using reagents and protocols from Invitrogen (Micro- by flow cytometry. To measure inhbition, 50 microliters of FastTrack and cDNA Cycle: Cat. #L131O-0l, and #K1520- each supernatant was incubated with 105 Ltki7.1 cells and 02, Invitrogen, Carlsbad, Calif.). Human il1i11oglobulin 25 ng recombinant CTLA-4 fusion protein. Mean channel 45 heavy and kappa light chain V region fragments were ampli- fluorescence was then determined by flow cytometry. FIG. 2 fied by PCR using pfu polymerase (Stratagene, La Jolla, shows inh bition of soluble CTLA -4 binding to cells express- CaIif.), degenerate FRl primers and imique constant region ing B7.1. Mean channel fluorescence (MCF) of LtkmB7.1 primers. The resulting PCR fragments were cloned into the cells stained with recombinant human CTLA -4 fusion protein pCR-Blunt vector (Invitrogen, Carlsbad, Calif.) and the was determined in the presence of hybridoma supernatant. 50 sequence of the insert determined. The preliminary sequences Hybridomas that secrete blocking antibodies resulted in for the heavy and light chain fragment of hybrid om a 10D1.3 lower MCF values. BNI3.1 (Cat.#34580D, Pharmingen, San are shown in FIG. 4. The determined sequences for the heavy Diego, Calif.) was used as a positive control mouse mono- and light chain fragment of hybrid om a 10D1.3 are shown in clonal antibody that blocks CTLA-4/B7 binding. FIG. 5 through FIG. 8.

TABLE 3

CDR sequences of light and heavy chains for MAbs IODI, 4B6, and IE2.

SEQ il SEQ il SEQ il Chain HuMAb CDRI NO: CDR2 NO: CDR3 NO:

Light IODI RASQSVGSSYLA 24 GAFSRAT 29 QQYGSSPWT 35 Chain 4B6 RASQSVSSSFLA 25 GASSRAT 30 QQYGSSPWT 35 IE2 RASQGISSWLA 26 AASSLQS 31 QQYNSYPPT 36 US 7,605,238 B2 53 54

TABLE 3-continued

CDR seguences of light and heavy chains for MAbs lODI, 4B6, and IE2.

SEQID SEQID SEQID Chain HuMAb CDRI NO: CDR2 NO: CDRJ NO:

Heavy 10m SYTMH 27 FISYDGNNYYADSVKG 32 TGWLGPFDY 37 Chain 4B6 SYTMH 27 FISYDGSNKYADSVKG 33 TGWLGPFDY 37 IE2 SYGMH 28 VDNDGSNKYYADSVKG 34 APNYIGAFDV 38

Example 4 The nucleotide sequences of heavy and light chain tran- scripts from a hybridomas are used to design an overlapping Use of Parial Antibody Sequences to Express Intact 15 set of synthetic oligonucleotides to create synthetic V Antibodies sequences with identical amio acid coding capacities as the natual sequences. The synthetic heavy and kappa light chain Antibodies interact with target antigens predominantly sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are internipted to through amino acid residues that are located in the six heavy and light chain complimentarity determining regions 20 facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according (CDR's). For tils reason, the amino acid sequences within CDR's are more diverse between individual antibodies than to Kozak's niles (Kozak, 1991,1. BioI. Chem. 266, 19867- sequences outside of CDR's. Because CDR sequences are 19870); and, HindII sites are engineered upstream of the responsible for most antibody-antigen interactions, it is pos- translation initiation sites. sible to express recombinant antibodies that mimic the prop- 25 For both the heavy and light chain variable regions, the erties of specific naturally occurring antibodies by constnict- optimized coding, and corresponding non-coding, strand ing expression vectors that include CDR sequences from the sequences are broken down into 30-50 nucleotide segments specific naturally occurring antibody grfted onto framework such that the breaks between nucleotides for the coding strand sequences from a different antibody with different properties sequence occur at approximately the midpoint of the corre- (Jones et al. 1986, Nature 321, 522-525). Such framework 30 sponding non-coding oligonucleotide. Thus, for each chain, sequences can be obtained from public DNA databases that the oligonucleotides can be assemble into overlapping double include gerniline antibody gene sequences. These germline stranded sets that completely span the desired sequence. sequences wil differ from mature antibody gene sequences These oligonucleotides are combined into pools that span because they wil not include completely assembled variable segments of 150-400 nucleotides. The pools are then used as genes, wilch are formed by V(D)J joining during B cell 35 templates to produce PCR amplification products of 150-400 maturation. Germline gene sequences wil also differ from nucleotides. Typically, a single variable region oligonucle- the sequence of a ilgh affty secondar repertoire antibody otide set wil be broken down into two pools wilch are sepa- at individual nucleotides because of somatic mutations. How- rately amplified to generate two overlapping PCR products. ever, somatic mutations are not distributed evenly across the These overlapping products are then combined by PCR variable region. For example, somatic mutations are rela- 40 amplification to form the complete variable region. It may tively infrequent in the amino-terminal portion offramework also be desirable to include an overlapping fragment of the region 1 and in the carboxy-terminal portion of framework heavy or light chain constant region (including the BbsI site region 4. Furthermore, many somatic mutations do not sig- of the kappa light chain, or the Agel site if the ga=a heavy nificantly alter the binding properties of the antibody. For tils chain) in the PCR amplification to generate fragments that reason, it is not necessary to obtain the entire DNA sequence 45 can easily be cloned into the expression vector constnicts. of a paricular antibody in order to recreate an intact recom- The reconstrcted heavy and light chain variable regions binant antibody having binding properties similar to those of are then combined with cloned promoter, translation initia- the original antibody (see PCT/US99/05535 fied on Mar. 12, tion, constant region, 3' untranslated, polyadenylation, and 1999, which is herein incorporated by reference for all pur- transcription termnation, sequences to form expression vec- poses). Partial heavy and light chain sequence spanng the 50 tor constnicts. The heavy and light chain expression con- CDR regions is typically suffcient for tils purpose. The par- stnicts can be combined into a single vector co-transfected tial sequence is used to determine which germline variable serially transfected, or separately transfected into host cell~ and joining gene segments contrbuted to the recombined which are then fused to form a host cell expressing both antibody variable genes. The germine sequence is then used chains. to fill in missing portions of the variable region. Heavy and 55 Plasmids for use in constniction of expression vectors for light chain leader sequences are cleaved during protein matu- human IgGk are described below. The plasmids were con- ration and do not contribute to the properties of the final stnicted so that PCR amplified V heavy and V kappa light antibody. For tils reason it is not necessar to use the corre- chain cDNA sequences could be used to reconstnict complete sponding germline leader sequence for expression constnicts. heavy and light chain ininigenes. These plasmids can be used To add missing sequences, cloned cDNA sequences can be 60 to express completely human, or cilmeric IgGlk or IgG4k combined with synthetic oligonucleotides by ligation or PCR antibodies. Similar plasmids can be constnicted for expres- amplification. Alternatively, the entire variable region can be sion of other heavy chain isotypes, or for expression of anti- synthesized as a set of short, overlapping, oligonucleotides bodies comprising lambda light chains. and combined by PCR amplification to create an entirely The kappa light chain plasmid, pCK7-96 (SEQ IDNO:39), synthetic varable region clone. Tils process has certain 65 includes the kappa constant region and polyadenylation site, advantages such as elimination or inclusion of particular such that kappa sequences amplified with 5' primers that restriction sites, or optimi zation of paricular codons. include HindII sites upstream of the initiator methionine can US 7,605,238 H2 55 56 be digested with HindII and BbsI, and cloned into pCK7- (correlation coeffcient is -0.999). The half-maximal binding 0.96 digested with HindII and BbsI to reconstruct a complete was 190 ng/ml, and saturation was achieved at 2 ~ig/ml. 1 OD 1 light chain coding sequence together with a polyadenylation did not bind to any CTLA4-negative cell lines tested, includ- site. This cassette can be isolated as a HindIIlNot! fragment ing SKBR-3, BT474 and MCFlOA breast epithelial himors and ligated to transcription promoter sequences to create a and L540 Hodgkn's himor cells, nor did it bind to cells fuctional mini gene for transfection into cells. expressing murine CTLA-4. These data indicate the specific- The gama1 heavy chain plasmid, pCG7-96 (SEQ ID ity of10D1 forhumanCTLA. However, lOD1 was shown to NOAO), includes the human gama1 constant region and cross-react with macaque C1LA-4 (see below). polyadenylation site, such that gamma sequences amplified C. Cross-Reactivity of 1 OD 1 with Normal Human Tissues with 5' primers that include HindII sites upstream of the 10 In this study, a fluoresceinated form of the test aricle initiator methionine can be digested with HindII and Agel, (lOD1-FITC) was used to evaluate binding. The objective of and cloned into pCG7 -96 digested with HindII and Agel to the shidy was to evaluate potential cross-reactivity of lOD1- reconstruct a complete gamma 1 heavy chain coding FITC with cryosections of normal human tissues. No unan- sequence together with a polyadenylation site. Ibs cassette ticipated cross-reactivity was observed. can be isolated as a HindII/SalI fragment and ligated to 15 The study was conducted in accordace with the Food and trancription promoter sequences to create a functional mini- Dnig Administration's Good Laboratory Practice (GLP) gene for transfection into cells. Regiùations (21 CFR Part 58). The human tissue panel The gamma4 heavy chain plasmid, pG4HE (SEQ ID included all the tissue on the "suggested list of human tissues NO:41), includes the human gama4 constant region and to be used for immunohistochemical investigations of cross polyadenylation site, such that gamma sequences amplified 20 reactivity" in Anex II ofthe EC CPMP Guideline II/5271/ with 5' primers that include HindII sites upstream of the 94, "Production and quality control of monoclonal antibod- initiator methionine can be digested with HindII and AgeI, ies" and all the tissues recommended in the 1997 US FDA! and cloned into pG4HE digested with HindII and Agel to CBER "Points to Consider in the Manufacture and Testing of reconstrct a complete gamma4heavy chain coding sequence Monclonal Antibody Products for Human Use". together with a polyadenylation site. This cassette can be 25 Using an indirect immunoperoxidase method, 1 OD 1- FITC isolated as a HindII/EcoRI fragment and ligated to transcrip- specifically stained positive control, human CTLA4-express- tion promoter sequences to create a functional minigene for ing, 58aßCTLA4CD3zeta cells as well as positive control transfection into cells. lymphocytes in human tonsiL. 10D1-FITC reactivity was A number of different promoters (including but not lin1Ited moderate to intense and two concentrations of antibody were to CMV, ubiquitin, SRalpha, and beta-actin) can be used to 30 examined (10 ~ig/ml and 2.5 ¡.g/ml). In both positive control express the reconstructed heavy and light chain genes. For 58aßCTLA4CD3zeta and positive contTolhiunan tonsilar exaniple the vector pCDNA3.1+ (Invitrogen, Carlsbad, lymphocytes, lOD l-FITC specifically stained discrete, Calif.), can be cleaved with HindII and either Not!, XhoI, or round, granules at membrane and in the cytoplasm immedi- EcoRI, for ligation with either the kappa, gamma1, or ately below the membrane. Reactivity was observed with gamma4 cassettes described above, to form expression vec- 35 occasional fol1icular, interfollcular, and subepithelial lym- tors that can be directly transfected into mammalian cells. phocytes. Less than 1-2% of al1 tonsilar lymphocytes were reactive with 10Dl-FITC. Example 5 lOD1-FITC did not react with negative control human lOD.1 Binding to CTLA-4 40 brain (cerebel1um). An isotype-matched negative control antibody (Hu1gGl-k-FITC) did not specifically bind to either A. 10D1 Binding to Purified Recombinant Human the positive control human CTLA4-expresing CTLA-4 58aßCTLA4CD3zeta or hiunan tonsil; nor did it bind spe- Binding of 10D1 to purified recombinant human CTLA-4 cifically to negative control human brain (cerebellum). was shown by ELISA using standard methods and procedures 45 To determine cross-reactivity, 1001- FITC was applied to a (FIG. 9 and FIG. 10). Microtiter plates coated with purfied panel of normal human tissues at two concentrations (10 CTLA-4 were incubated with varing concentration of 1 OD1, ¡.g/ml and 2.5 ~ig/ml). Specific 10Dl-FITC reactivity was and then developed with goat anti-human IgG F(ab')2 conju- observed for lymphocytes in the tonsil (3/3 donors), submu- gated to alkaline phosphatase. The data demonstrate dose- cosal lymphoid nodule in the colon (gastrointestinal tract- dependent binding of lOD1 that is well fit to a 4-parameter 50 colon (1/3 donors)), and blood smears (2/3 donors). cure (correlation coeffcient is -1.0). The half-maximal Immunoreactive cells were identified as lymphocytes binding at 15 ng/ml reflects the high binding capacity of 1 OD 1 based on typical morphology (round molecular cells with to CTLA-4. Saturation of binding was observed at approxi- large nucleus:cytoplasm ratio and scant cytoplasm, lack of mately 0.1 ¡.g/m!. dendritic processes, 10-15 ~im in diameter) and location B. lOD.1 Binding to CTLA-4 Expressed on the Plasma 55 within the tissues (e.g., typical location within lymphoid tis- Membrane ofT-Cells sues). In the tonsils from all three donors (test tissues), lym- In order to demonstrate binding of 10D1 to CTLA-4 phocytes, lOD1-FITC specifically stained discrete, round, expressed on the plasma membrane ofT-cells, the results in granules at membrane and in the cytoplasm immediately FIG. 10 from a flow cytometric assay are shown. The flow below the membrane. Reactivity was observed with occa- cytometrc assay was used with a T-cel1 line transfected to 60 sional follicular, interfollcular and subepithelial lympho- express high levels of human CTLA-4 (designated cytes. Less than 1-2% of all tonsillar lymphcyes were reac- 58aßCTLA-4/CD3zeta cells). Varying concentrations of tive with 10Dl-FITC. fluoresceinated lOD1 (lODl-FITC) were incubated with In 1/3 donors examined, 10Dl-FITC also specifically 58aßCTLA-4 cells. The cell associated fluorescence was stained discrete granules in occasional follcular and interfol- determined by flow cytometry. As seen with the purified 65 licular lymphocytes located in submucosal lymphoid nodules CTLA4, 10D1 bound to CTLA4-expressing cells in a dose- in the colon (gastrointestinal tract-colon (large intestine)). dependent manner that was well fit to a 4-parameter equation Again, discrete membrane granules were stained. US 7,605,238 B2 57 58 In peripheral blood smears from two of the three donors brane of rare lymphocytes. The granules were arranged in a examined, 1 aD 1- FITC specifically stained discrete granules ring or in a cured pattern. Less than 1-2% of all peripheral approximately 1 ¡.m in diameter associated with the mem- blood leukocytes were reactive with lOD1-FITC.

TABLE 4

Cross-Reactivity ofMAb 10Dl With Normal Human Tissues

Negative Control Antibody Test Aricle HulgGl-K- 10Dl-FITC FITC Assay Tissue lOfJlml 2.5 fJml 10 flglml 2.5 fJml Contrl' ß,-micl'globulin

Positive Contrl 3-4+ 2- 4+ Neg Neg Neg Pas 58aßCTLA4CD3zeta cells Positive Control Lymphocytes 2- 3+ 2- 3+ Neg Neg Neg Pos in human tonsil Negative Contrl Human bmin - Neg Neg Neg Neg Neg Pos cerebellum Adrenal Neg Neg Neg Neg Neg Pas Blood Pas Neutrophils Neg Neg Neg Neg Neg Pas Lymphocytes 2+ Neg Neg Neg Neg Pos (rare) Eosinophils Neg Neg Neg Neg Neg Pas Monocytes Neg Neg Neg Neg Neg Pos Platelets Neg Neg Neg Neg Neg Pas Blood Vessel (endothelium) Detailed under individual tissues Examined in all tissues Bone Marrow Neg Neg Neg Neg Neg Pas Bmin - Cerebellum Neg Neg Neg Neg Neg Pas Brain - Cerebrum (cortex) Neg Neg Neg Neg Neg Pas Breast (mammar gland) Neg Neg Neg Neg Neg Pas Eye Neg Neg Neg Neg Neg Pas Gastrintestinal Tract - Colon 2- 3+ 2- 3+ Neg Neg Neg Pos (large intestine) Submucosal lymphoid nodule (occasional follicular and interfollicular lymphocytes) Gastrintestinal Tract - Colon Neg Neg Neg Neg Neg Pos (large intestine) Other elements Gastrointestinal Tract - Neg Neg Neg Neg Neg Pas Esophagus Gastrointestinal Tract - Small Neg Neg Neg Neg Neg Pas intestine Gastrointestinal Tract - Neg Neg Neg Neg Neg Pas Stomach Heart Neg Neg Neg Neg Neg Pas Kidney (glomerulus, tubule) Neg Neg Neg Neg Neg Pas Liver Neg Neg Neg Neg Neg Pas Lung Neg Neg Neg Neg Neg Pas Lymph Node Neg Neg Neg Neg Neg Pas Ovar Neg Neg Neg Neg Neg Pos Fallopian Tube (oviduct) Neg Neg Neg Neg Neg Pas Pancreas Neg Neg Neg Neg Neg Pos Parthyroid Neg Neg Neg Neg Neg Pas Peripheral Nerve Neg Neg Neg Neg Neg Pos Pituitar Neg Neg Neg Neg Neg Pas Placenta Neg Neg Neg Neg Neg Pos Prostate Neg Neg Neg Neg Neg Pos Salivar Gland Neg Neg Neg Neg Neg Pas Skin Neg Neg Neg Neg Neg Pas Spinal Cord Neg Neg Neg Neg Neg Pas Spleen Neg Neg Neg Neg Neg Pos Striated (Skeletal) Muscle Neg Neg Neg Neg Neg Pas Testis Neg Neg Neg Neg Neg Pos Thymus Neg Neg Neg Neg Neg Pas Thyroid Neg Neg Neg Neg Neg Pos Tonsil Lymphocytes 2+ 1- 2+ Neg Neg Neg Pas (occasional follicular, interfollicular and subepithelial lymphocytes) Tonsil Other elements Neg Neg Neg Neg Neg Pos Ureter Neg Neg Neg Neg Neg Pas Urinar Bladder Neg Neg Neg Neg Neg Pas US 7,605,238 B2 59 60

TABLE 4-continued

Cross-Reactivity ofMAb 10D1 Will Normal Hmnan Tissues

Negative Control Antibody Test Aricle HulgG1-K- 10Dl-FITC FITC Assay Tissue 10 ¡iglml 2.5 ¡iglml 10 ¡iglml 2.5 ¡iglml Control * flrmicroglobnlin Uterus - Body (endometrium) Neg Neg Neg Neg Neg Pas Uterus - Cervix Neg Neg Neg Neg Neg Pas

* omission of test antibody

15 D. Specific Reactivity of lOD.I with Macaque CTLA-4 Specific reactivity with macaque CTLA-4 was demon- strated using T-cells transfected to express the macaque CTLA-4 at high levels (Table 5). These data suggest that the CTLA-4 epitope for IODI is conserved between macaque 20 and humans, therefore macaque is a good model to evaluate in vivo safety of anti-CTLA4 HuMAb I ODI.

TABLE 5

Species reactivity ofisotye control (MFI) reactivity of ioD1 (MFI) humanCTLA4 3 662 macaqne CTLA4 4 606 murine CTLA4 (negative control) 5 5 MAb ioD1 (10 ¡LgmJ) was incubated with celllines expressing recombinant CTLA-4 from various species. and detected by FITC-aliti human IgG. The cell-associated fluorescence was determined by FACScan and reported as mean fluorescence intensity (MFI). These data show that MAb 10DJ reacts well with macaque and human CTLA-4, but not with miuine CTLA-4.

Example 6 35 ity. Four anti-CTLA-4 epitope binding groups were identified 10m Blocking ofCTLA-4 to B7 Ligands among the human antibodies, and an additional two epitopes were defied by the co=ercial murine monc1onal antibod- In order to show that i OD i binding to CTLA-4 blocks the ies BNI3 (Pharnngen, San Diego, Calif.), and 8H5 (Ancell Corp. Bayport, Min.). FIGS. 3, and 13A-13G show results of interaction ofCTLA-4 with CTLA-4Iigands, B7. I and B7 .2, 40 competitive binding assays that demonstrate differential competition assays were performed by flow cytometry (FIG. 11 and FIG. 12). As shown in FIG. 11, FITC-labeled human competition among the antibodies for binding to CTLA-4. B7.2-Ig fusion protein was incubated with 58ußCTLA4 These results are su=arzed in Table 6. T-cells and various concentrations of lODI MAb. In FIG. 12, FITC-labeled CTLA4-Ig fusion protein was incubated with 45 murine B7.ltransfected cells and various concentrations of 10m MAb. The competition assays demonstrate the ability ofl ODI to Antibodies in anti-CTLA-4 epitope binding groups 4a and effciently inhibit CTLA4-B7 interactions at low concentra- 4b have simlar binding characteristics, and additionally are tions (1-10 ¡.glml). The effective concentration would likely 50 strong blockers of CTLA-4-Ig binding to cell surface be much lower under physiological conditions, which would expressed B7.1 (Table 6). For example, FIG. 3 shows results have far lower concentrations ofCTLA-4 and B7 moleciùes. with biotin labeled I I E8 antibody and i 0 unlabeled antibod- Similar data was obtained using biotinylated reagents in ies (3A4, 9A5, 2E2, 2E7, 4B6, 4ElO, 5C4, 5Gl, lIE8 and ELISA assays. I I G I). Antibody I IE8 binding was blocked by itself and 7 of These in vitro studies demonstrate that MAb lODI binds 55 the other human antibodies in epitope groups 4a and 4b. human CTLA-4 with high affnity and specificity and that However, binding of iI E8 was not blocked by antibodies 3A4 binding of lODI abrogates interaction between B7 costimu- or 9A5(epitope groups I and 2). Reciprocal binding experi- latory molecules and CTLA-4. These data for IODI are con- ments showed that IlE8 binding did not block either 9A5 or sistent with the in vitro activity profies for anti-murine 3A4 binding to CTLA-4 (FIGS. 13A and 13B). Similar CTLA-4 antibodies that have demonstrated effcacy in 60 results are shown for epitope group 4a antibodies IODl and murine tumor models. murine antibody 147 (FIGS. 13D and 13F). Antibodies in epitope group 4b (FIG. BE) are similar to group 4a antibod- Example 6 ies with the exception that the epitope 4b antibodies compete with epitope group 2 antibodies in reciprocal binding experi- Epitope Mapping of IOD.I 65 ments (FIG. 13B). Human antibodies that belong to epitope Competitive ELISAs were done with biotin labeled and groups 3, 4a and 4b are effective blockers ofCTLA-4/B7.l unlabeled antibodies to determine CTLA-4 epitope specific- binding (FIG. 3, and Table 6). US 7,605,238 B2 61 62

TABLE 5

CTLA.4 MAs: Epitope and CTLA-4/B7.1 Blockig Properties

Blocks binding ofCTLA-4-Ig to Monoclonal B7.1 on Ltk Epitope Antibody Competition for CTLA-4 Binding mB7.1

9A5 No competition from groups 3, 4a, 4b, 5, and 6 No Weak Competition form group 2 2 3A4 One way competition from groups i, 4b, 5 and 6 No IE2 No competition with 4a. Weak competition form group 3 5A8 Competes with 4a and 4b. Some competition with 2. Yes No competition form 1 and 5 4a JODI Cross competes with all members of 4b. Yes 147' Competition from 6 (non-reciprocal) lIE8 No competition with 1, 2, and 5. 1IG! Weak competition with 3. 4EJO 5C4 3FlO 4b 4B6 Cross competes with all members of 4a Yes 4AI Competes with 2 2E2 Weak competition with 3. 2E7 No competition with i, and 5. 2Gl Competition from 6 (non-reciprocal) BNI3** Competes with 6, no competition with groups 1 to 4 Yes 8H5'" Competes with 5, no competition with groups 1 to 4 Yes Competition with group 3 not tested ii Murine monoclonal antibody .. Available from Pharmingen. BNI3 Catalog #34580 D, San Diego CA. "'Available from Ancell, ANC l52.2/8H5 Catalog #359-020, Ancell Corp. Bayport, Mn.

Example 7 T-cells were labeled with 51Cr and incubated with varous concentrations of anti -CTLA4 MAb 1 OD 1 or anti -CD3 MAb lOD1 Binds to Human Activated T Cells with or without rabbit serum as a source of complement. After 35 a 1 hour incubation, the 51Cr released by dying cells was The ability oflOD1 antibody to bind to CTLA-4 expressed determined using a gaiia counter. Target cells incubated by normal human T cells was investigated by flow cytometric with 2% SDS served as 100"10 lysis controls. The anti- anlysis of resting and activated T cells (FIG. 14). Freshly CTLA-4 MAb lODI did not mediate CDCC of the activated isolated human peripheral blood mononuclear cells at 2x 1 06/ T-cells (FIG. 15). Under the same conditions, the murine ml were incubated in the presence or absence of2 uglml of the 40 IgG2a anti-CD3 MAb led to signficant CDCC. Both murine T-cell mitogen, phytohemagglutinin (PHA). Afer four days incubation, the cells were washed and stained with the fol- IgG2a and human IgG1 effciently fix rabbit complement; lowing antibodies: 1) no antibody; 2) HuIgG 1-FITC, a human therefore these differences most likely reflect the greatly IgG1 anti EGF receptor antibody; 3) lOD1-FITC, human reduced expression of CTLA -4 as compared to CD3 on acti- IgGl antiCTLA-4 antibody; and 4) 147-FITC-mouse anti- 45 vated T-cells. human CTLA-4 antibody. After incubation for 1 hr., cells Similarly, no ADCC activity was observed for MAb lOD1 were washed and stained with rabbit anti-FITC IgG followed using autologous mononuclear cells as effector cells (FIG. by goat anti-rabbit-PE. Analysis was perfonned on lympho- 16). PHA-stimulated T-cells were labeled with 51Cr andincu- cytes gated by foiward versus side scatter. As shown in FIG. bated with various concentrations ofanti-CTLA4 MAb lOD1 14, resting lymphocytes do not bind lOD1 antibody, wilIe 50 or anti-CD3 MAb and fresh autologous mononuclear cells. PHA-activated T cells express low levels of CTLA-4 at the The effector to target cell ratio was 100: 1. After a 4 hour cell surface incubation, the 51Cr released by dying cells was determied using a gama counter. Target cells incubated with 2% SDS Example 8 served as 100% lysis controls. Although the anti -CD3 MAb is 55 a murine IgG2m which can mediate effcient ADCC with lOD1 Does Not Mediate Complement-Dependent or Antibody-Dependent Lysis of Activated T-Cells human effector cells, only low levels of ADCC were observed. These data are consistent with the requirement of The ability ofMAb 10D1 to mediate complement-depen- ilgh levels of antigen expression on the surface of target cells dent cellular cytotoxicity (CDCC) or antibody-dependent 60 for effcient ADCC. Since MAb lOD1 is a human IgG" an cellular cytotoxicity (ADCC) of CTLA-4 expressing cells isotype generally capable of mediating CDCC and ADCC, was investigated. the lack of these activities is likely due to the very low expres- For CDCC experiments, rabbit serum was used as a source sion ofCTLA-4 on activated T-cells. Furhermore, the obser- of compliment, in order to provide optiial conditions for vation of increased numbers of activated T-cells in the priate. CDCC. Rabbit complement has been shown to be more effec- 65 toxicology studies (see below) is consistent with the lack of tive in mediating CDCC with human IgG1 than human ADCC and CDCC activity of activated T-cells by MAb IOD1 complement (Jurianz, Maslak et a!. 1999). PHA-stimulated in vivo. US 7,605,238 B2 63 64 Example 9 Pharmacokinetic analysis revealed the presence of signifi- cant levels (up to 97.3 /lg/ml) of 1 OD 1 MAb in the plasma of 10D1 Preclincal Toxicity Studies in Cynomolgus both monkeys (see Table 7). Plasma levels of 10Dl were Monkeys determined by a competition assay with FITC-lOD1 using 5 flow cytometry and 58ußCTLA-4 T-cells. Two independent toxicology studies ofl OD1 antibody and macaques were performed. A total of eight monkeys were TABLE 7 analyzed. Four monkeys (two males and two females) toler- ated three bolus i.v. doses of 3 mg/Kg human anti-CTLA4, IODI plasma levels and four monkeys (two males and two females) tolerated 10 three bolus Lv. doses of 10 mg/Kg human anti-CTLA4 with- Time point Monkey #1 Monkey #2 out significant clinical, i=unotoxicology, or histopatho- Pre-l sf dose 0.0 (¡g/ml plasma) 0.0 (¡g/ml plasma) logical fidings. Day 4, pre_2nd dose 17.4 (¡g/ml plasma) 43.6 (¡g/ml plasma) A. 10DI Primate Toxicology Study (3.0 mg/Kg) Day 7, pre-Y" dose 83.6 (¡g/ml plasma) 97.3 (¡g/ml plasma) Day 14 90.2 (fig/ml plasma) 70.9 (fig/ml plasma) To investigate the effects of lOD1 in vivo, a primate toxi- 15 cology study was performed with two macaques. In a mul- tiple dose toxicity study of MAb lOD1, this antibody was Evaluation of the anti-lOD1-antibody response was per- administered via intravenous injection of macaques. The formed by ELISA. No signficant anti-lOD1 response was objective of this study was to determine the tolerability of observed in either animal during the course of study (FIG. MAb JOD1 in two monkeys given at a dose and schedule 20 17). Microtiter plates were coated with lOD1 MAb (for IgM compatible with effcacious treatment in a murne tumor assay) or lOD1 F(ab'), (for IgG assay). Dilutions of plasma regression model and proposed dose in human clincal stud- samples from varous time points were incubated with the ies. Two female cynomolgus monkeys (Macaca fascicilaris) plates, and anti-lOD1 antibodies were detected with either were treated with three intravenous bolus doses of 3.0 mg/Kg anti-IgM or IgG Fc-specific alkaline phosphatase reagents. 10D1 on days 1,4, and 7 to evaluate safety and T-cell activa- 25 IgM anti-lOD1 antibodies appear to have developed by day tion in these anmals. The animals were observed for any adverse reactions, weight loss/gain, and morbidity and mor- 14, however, the titers are very low. IgM anti -1 OD 1 antibodies appear to have developed by day 14, however, the titers are tality up to 14 days post admstration of the fist dose. Seven days after the last dose the animals were sacrificed and very low. These data demonstrate that the monkeys did not necropsiedo to examne their organs individually. Blood 30 develop anti-lOD1 antibody responses after 3 doses of the samples were collected before each dose and before necropsy antibody. for examination ofT-cell populations and expression of acti- These data demonstrate that the animals did not develop a vation markers by flow cytometr. Plasma was also collected significant antibody response against MAb 10D1 during the from blood samples to determine lODl antibody levels and course of this study. anti-lOD1 antibody responses by ELISA. 35 I=unotoxicology was investigated by flow cytometric The animals tolerated three doses of antibody 10D1 with- analysis oflymphocyte populations during the course of the out any clinical symptoms durig the treatment course. The study. The lymphocyte subsets examned included CD3 as a weight of these animals did not change significantly. No gross marker for total T-cells and CD20 as a marker for total findings were documented on 47 organs/tissues examined at B-cells. T-cells, were :firther subdivided for expression of necropsy for either animaL. 40 CD4 (helper T-cell marker) and CD8 (cytotoxic T-cell Histopathology studies were performed at Redfield labo- marker), as well as for activation markers CD25, CD29, ratories, Redfield, Ark. The results from these studies indi- CD69 and HLA-DR. No remarkable changes in T-cell popu- cated that multiple doses ofMAb 1 OD 1 did not produce acute Iations or expression of activation markers was noted. The toxicity in any of the organs and tissues examined. results are sumarzed in Table 8 below.

TABLE 8

Flow cytometric analysis oflymphocyte pOPLÙations

Time point Monkey #1 Monkey #2

51 dose Pre-l O/CD3 = 61. O/CD20 ~ 16 O/CD3 ~ 54, O/CD20 = 22 O/CD4 - 43, O/CD8 = 50 O/CD4 = 59, O/CD8 ~ 36 %CD25", i, O/CD29 -41 O/CD25 ~ I. O/CD29 - 29 O/CD69 ~ i, %HLA-DR~ 4 O/CD69 ~ I. %HLA-DR~ i Day 4, pre_2nd dose O/CD3 ~ 58, O/CD20 ~ 13 O/CD3 ~ 56, O/CD20 ~ 16 %CD4 = 38. O/CD8 ~ 52 O/CD4 = 62. O/CD8 ~ 37 O/CD25 ~ i, O/CD29 = 52 O/CD25 ~ i, O/CD29 ~ 36 O/CD69 '" 1.%HLA-DR~2 O/CD69 ~ I. %HLA-DR '" i Day 7, pre- 3rd dose O/CD3 ~ 59, O/CD20 = 15 O/CD3 ~ 51, O/CD20 ~ 17 O/CD4 ~ 47, O/CD8 ~ 59 O/CD4 ~ 51, O/CD8 = 39 O/CD25 - 2, %CD29 = 44 O/CD25 - i, O/CD29 ~ 39 O/CD69 ~ i, %HLA-DR- 4 O/CD69 ~ i, %HLA-DR~ 2 Day 14 %CD3 = 64, O/CD20 - 14 O/CD3 = 59, O/CD20 - 20 O/CD4 ~ 49, O/CD8 - 44 O/CD4 ~ 60, O/CD8 ~ 35 %CD25 = i, O/CD29 ~ 44 O/CD25 '" i, O/CD29 = 34 O/CD69 '" I. %HLA-DR= 15 %CD69", i, %HLA-DR~ i US 7,605,238 B2 65 66 Heparinized bIood samples were analyzed fresh by flow The results of the analysis of test article concentrtion in cytometry using FITC- or PE-labeled anti-lymphocyte seru samples (i.e., trough levels measured in samples reagents. % CD3 and % CD20 are based on a lymphocyte obtained prior to dosing on Days 4 and 7, and prior to gate. The additional T-cel1 markers and activation markers are necropsy on Day 14) indicated dose-dependent exposure to al1 based on CD3-positive cel1s. These data indicate that the test article. On Day 7, predose meaii concentrations were multiple doses of MAb 10DI does not have a significant approximately 84 and 240 ¡.g/ml for the 3 - and 1 O-mglkg dose effect on B and T-cel1 populations or T-cel1 activation mark- groups, respectively. ers. A potential for accumulation of the test aricle in serum B. 10DI Prinate Toxicology Study (3.0 and 10.0 mg/Kg) with the every-three-day dosing schedule in monkeys was Six cynomolgus monkeys (four males and two females), 10 evident from the difference between the Day 4 and Day 7 experimental1y non-naïve and weighing 2.4 to 3.8 kg at the trough levels (i.e., means concentrations on Day 7 were outset of the study, were assigned to treatment groups as approximately twice as high as on Day 4), as wel1 as from the shown in Table 9 below. high residual levels on Day 14 (one week after the last dose), which were similar to the Day 7 trough levels. Evidence of TABLE 9 15 antibody formation against the test aricle was detected in two of the six study anals (one from Group 1 and another from Number of Dose Level Dose Vol. Dose Solution Group 2). In the former case, it appeared that the antibody Group No. Males/Females Cone. mglml) (mglkg) (mllkg) response might have affected the clearance of the test aricle 1 2/0 3 0.6 5.0 from circulation. Flow cytometric analysis of lymphocyte 2 2/2 10 2.0 5.0 20 subsets revealed a modest increase in total CD3-positive cel1s between Days 1 and Day 14, which correlated with an Each animal received a dose of human aiiti-CTLA4 (5 increase in CD3/CD4-positive cel1s, and a respective mg/ml concentration) by intravenous injection (i.e., "slow- decrease in CD3/CD8-positive cel1s (Group 2 only). The per- push" bolus injection) every three days for one week (i.e., on centage ofCD3 cel1s expressing CD29 and HLA-DR moder- Days 1, 4 and 7). Detailed clinical observations were con- 25 ately increased over the course of the study, which was con- ducted at least twice daily ("cages ide observations"), and a sistentwith previous findings thatanti-CTLA4 antibodies can thorough physical examination was performed on each ani- enhance antigen -specific T-cel1s. mal prior to the study and on Day 12. Body weights were In conclusion, apart from the minor changes in circulating measured weekly (prestudy and Days 7 and 14), and ophthal- lymphocyte subpopulations, the highest dose level tested in moscopic examination was conducted on al1 animals prior to 30 this study (i.e., three doses of 10 mg/kg given at three-day the study aiid on Day 12. Blood samples for evaluation of intervals) was an absolute no-effect dose level in cynomolgus senun chemistry, hematology and coagulation parameters monkeys. were col1ected from all animals prestudy and on Day 14. Additional samples for selected hematology parameters (total Example 10 and differential white blood cel1s only) were col1ected prior to 35 dosing on each dosing day (Days 1,4, and 7). Urine samples A Phase I Human Clinical Trial ofMAb 10DI in for standard urinalysis were obtained by drainage from spe- Prostate Cancer (MDXCTLA4-0l) and Melanoma cially designed cage-pans prior to dosing and on Day 13. (MDXCTLA4-02) Blood samples were also col1ected prior to each dose (Days 1, 4 and 7) aiid prior to termination (Day 14) for various analy- 40 MDXCTLA4-0l is an open-label study of anti-cytotoxic ses conducted by Medarex. These included analysis of test T-lymphocyte-associated antigen-4 (anti-CTLA-4) mono- aricle concentration (pharnacokinetics), determnation of the clonal antibody 10DI (MAb 1ODl) in patients with progres- presence of antibodies to the test article, and flow cytometr sive, metastatic, honnone-refractory prostate cancer. Treat- analysis. Al1 animals were euthanized on Day 14, at which ment is a single dose of MAb 10DI that is administered time, a complete gross necropsy was conducted, major organs 45 intravenously, as an iiision, at a dosage of3.0 mg/Kg. were weighed, and a standard complete set of tissues was The objectives of ths tral are to determine if i. adminis- col1ected from each animal and processed for examination by tration of MAb 10D 1 causes nonspecific T-cel1 activation, ii. Iight microscopy. to establish a safety/tolerability profile for MAb 10DI in Intravenous adminstration of human anti-CTLA4 at dose these patients and, iii. to determine the pharmacokinetic pro- levels of 3 mglkg and 10 mg/g given every three days for a 50 fie of MAb 10D1 and assess the development of a host total of thee doses was very wel1 tolerated by cynomolgus iniune response to MAb 10Dl. In addition the study wil1 monkeys. There were no clincal signs of toxicity from the attempt to identify prelimnar evidence of effcacy. TIie cageside observations and physical exaniations, and no study is a multicenter, open-label study of a single dose of effects on body weight, ocular examination findings, clinical MAb 10DI in 14 subjects. The study consists offourphases: pathology parameters, gross necropsy fidings, organ 55 Screening, Insion, Post-infsion, and Follow-up (see Table weights or tissue histomorphology. 10 below).

TABLE 10

Follow- Phase Screen Infusion Post-infusion up

Time days -30 to 130 145 160 190 250 370 24 48 72 day day day day monthly -14toO min min min min min min hrs hrs hrs 7 14 21 28 US 7,605,238 B2 67 68 Patients with histologic diagnosis of priary adenocarci- noma of the prostate, and progressive metastatic carcinoma of TABLE 12-continued the prostate after androgen deprivation and at least one sys- temic non-hormonal manipulation, are being screened for Flow cytometric analysis ofT cell activation markers in prostate participation in this study. Subjects must have progressive cancer subjects treated with 3.0 mg/Kg MAb 10Dl. measurable disease, progressive PSA, PSA;,5 ng/ml, test- Patient osterone.:50 ng/dl, primar gonadal androgen suppression, Number Time Point CD(4 + 25 + 69) % CD (8 + 25 + 69) % life expectancy;,12 weeks, and Kamofsky Performance Sta- 3 40 MIN 2.5 0.7 tus~60%. 3 130 MIN 1.9 0.9 Subjects undergo physical examnation, ECG, chest radi- 10 3 145 MIN 1. 0.5 ography, diagnostic imaging, and blood sanipling for hema- 3 160 MI 1.7 1 tological, biochemical, and i=une function assessments, 3 190 MIN 1. 1. 3 250 MI 2.1 1. and have vital signs monitored. Monthly telephone inter- 3 370 MI 1. 0.9 views are used to collect and record information on a subset of 3 24HR 1.6 1.6 adverse events, including autoi=une adverse events after 15 3 48HR 2.7 3 disease progression, until six months after treatment. PSA 3 72HR 0.9 0.5 3 Day 7 0.9 0.1 (decline, duration of decline, progression, time to progres- 3 Day 14 0.4 0.5 sion) and disease response (complete, parial, stable, progres- 3 Day 21 2.3 1.9 sive) are monitored. Plasma concentrations ofMAb 1 OD 1 are 4 Screen 1.4 0.8 4 -30 MIN (Pre- 0.5 0.3 being assessed immediately prior to, during, and up to two 20 Inion months after, infusion. 4 40 MIN 0.3 0.1 Data from four prostate cancer subjects that have been 4 130 MIN 0.3 0.1 treated are shown in Table 11. No adverse events have been 4 145 MIN 0.4 0.2 4 160 MI 0.2 0.2 recorded. For all of the subjects treated, Mab IOD1 appears to 4 190 MIN 0.8 0.3 be well tolerated. 25 4 250 MI 0.1 0 Because of the importance of monitoring the i=une sta- 4 370 MIN 0.3 0.1 tus of patients in the trial and the specific goal of monitoring 4 24HR 0.2 0.3 generalized effects on T cell activation by anti-CTLA-4 anti- 4 48HR 0.4 0.6 4 72HR 0.8 0.3 body, the entr criteria in this study included minimum levels 4 Day 7 I 0.7 of CD4 and CDS T cells of ~500/ml and ~500/ml respec- 30 4 Day 14 1. 0.8 tively. However, it was observed during the initial accrual in the study that prostate cancer patients have significantly reduced T cell numbers although CD4 and CDS T cells are A second clincal trial (MDXCTLA4-02) using MAb 10D1 in subjects with Stage IV malignant melanoma has also clearly present. Many patients were initially rejected based on the above entry criteria (see Table 11). The apparent reduced 35 been initiated. A single dose of MAb 1 OD 1 wil be Adminis- T cell counts observed is a previously undocumented obser- tered intravenously, as an infsion, at a dosage oD.O mg/Kg. vation in prostate cancer patients that may have relevance in This study also consists Of four phases (Screening, Infusion, treatments involving cancer vaccination in these patients. Post-Infusion and Follow-up) as described in Table 9, above. Subsequent to these observations, the entry criteria were The goals of tils study are as those regarding the above- amended to include patients having CD4 and CDS count of 40 described study in prostate cancers as well as to specifically ~300/ml and ~200/ml respectively. establish a safety/tolerability profile for MAb 10D1 in In order to evaluate whether administration of MAb 1 OD 1 patients with Stage iv malignant melanoma. One patient has can induce undesirable non-specific T cell activation, periph- been treated in this study (see Table 13). As in the prostate eral blood lymphocytes from the prostate cancer subjects cancer study, MAb 10D1appears to be well tolerated. Flow were analyzed by flow cytometry for each of the following 45 cytometric analysis of T cell activation markers in this sub- markers: CD4, CDS, CD25, CD44, CD69 and HLA-DR. ject, analogous to that performed for the prostate tumor trial, Blood samples were taken at time points indicated in Table also showed no evidence of non-specific T cell activation. 10. No significant change in the frequency of any of these Ongoing results from the MDXCTLA4-01 and MDX- markers was observed durig the course of the treatment for CTLA4-02 clinical trials have demonstrated that the infu- each of the prostate cancer subjects treated thus far. An so sions are tolerable with only minor reactions. Prolonged example of this analysis is shown in Table i 2 which shows the plasma half-life of the antibody was seen, with the antibody frequency of CD4, CD25, CD69-positive cells and CDS, remaining in the plasma for approximately 3 to 4 months. CD25, CD69-positive cells at times prior to, during, and Clear evidence ofi=une effects was observed without overt subsequent to MAb 10DI administration in two of the sub- non -specific T cell activation. Symptomatic relief and reduc- jects. These data demonstrate that MAb lOD1 does not result 55 tions in prostate specific antigen (PSA) levels have been in non-specific T cell activation. observed in prostate cancer patients treated with the anti- CTLA-4 antibody. Representative results for reductions in TABLE 12 PSA levels are shown in FIG. 18, which shows PSA levels (in ng/ml) in two patients (one represented by the closed circles, Flow cytometric analysis ofT cell activation markers in prostate 60 the other by the open circles) at various time points after cancer subjects treated with 3.0 mg/Kg MAb lOD1. inusion of 3 mg/kg anti-CTLA-4 antibody at day O. The Patient results demonstrate that PSA levels decreased after infusion Number Time Point CD(4+25+69)% CD (8 + 25 + 69) % of the antibody and remained suppressed for approximately 3-4 months after treatment, correlating with the presence of 3 Screen 1.7 0.8 3 -30 MIN (Pre- 2.6 0.8 65 the anti-CTLA-4 antibody in the plasma. Other examples of Infusion i=une effects observed included i=une-mediated rash and pruritis, transient seroconversion to positive autoantibod- US 7,605,238 B2 69 70 ies, melani pigment changes in melanoma patients and six animals in each group, +/-SE. Results from the animals inflammatory reactions at tumor sites. Except for the rash and treated with the vaccine alone are depicted with the open pniritis, all potentially adverse immune effects were subclini- circles, whereas results from anals treated with both the caL. In summary, the ongoing results from human clinical anti-CTLA-4 antibody and the vaccine are depicted with trials with anti-CTLA-4 antibody treatment demonstrate that closed circles. Use of the anti-CTLA-4 antibody in combina- the antibody is well-tolerated and stimulates immune effects tion with the vaccine led to a significantly greater antibody in recipients. response against the melanoma cells than use of the vaccine alone. These results demonstrate that a human anti-CTLA-4 Exaniple 11 antibody of the invention is capable of enhancing antibody 10 responses to a tuor cell vaccine in vivo in primates. Anti-CTLA-4 Treatment Enhances Antibody The effect of anti-CTLA-4 treatment on antigen-specific T Responses to a Hepatitis B Surface Antigen cell proliferation was also examined. Prior to vaccination of (HBsAg) Vaccine the animals, blood was drawn and monocytes from the ani- mals were differentiated in vitro into dendrtic cells (DC) to The abilty of a human anti -CTLA -4 antibody of the inven- 15 provide a population of autologous dendrtic cells for use in T tion to enhance antibody responses to a hepatitis B surface cell proliferation studies. A portion of theses autologous DCs antigen (HBsAg) vaccine was examned in cynomolgus mon- were incubated with the SK-mel-3 cells to provide a popula- keys. Test groups of four monkeys each (two males, two tion of autologous DCs that had been pulsed with melanoma females) were treated with either 1) the HBsAg vaccine in antigens. At various time points after vaccination, polymor- combination with a control IgG 1 antibody (a humanized anti- 20 phonuclear cells (PMNC) were obtained from the animals RSV antibody, Synagis™, commercially available from and incubated in vitro either 1) alone (as a negative control), MedImmune) or 2) the HbsAg vaccine in combination with 2) with Staphylococcus enterotoxin B (SEB, a non-specific the anti -CTLA -4 anti body 1 OD 1. The anti-CTLA -4 antibody activator of certain T cell populations, as a positive control), or control IgG 1 were administered intravenously at a dosage 3) with autologous dendritic cells or 4) with autologous den- of 10 mg/kg in a volume of 2.0 inkg. The HBsAg vaccine 25 dritic cells that had been pulsed with melanoma antigens. T (Engerix-BTM, commercially available from GlaxoSmith- cell proliferation was assessed using a quantitative flow Kline) was administered intramuscularly at a dosage of 10 ~ig cytometry assay that allowed for a quantitative measure ofthe in a volume of 0.5 ml. The anti-CTLA-4 or control IgG1 total number ofT cells per well (through the use of an anti- antibody was administered on days 1 and 29, whereas the CD3 antibody), as well as the number of CDS- vs. CDS+ cells HBsAg vaccine was administered on days 2 and 30. Plasma 30 (through the use of an anti-CDS antibody). The results from levels of anti - HBsAg antibody were measured on days 1, 51 an aiumal treated with SK-mel-3 in combination with anti- and 64 using a radioiiunoassay kit (commercially avail- CTLA-4, assessed at day 41 after vaccination, are summa- able from Abbott). Results presented represent the mean of rized in FIG. 21, wherein T cell proliferation is expressed as the four anmals in each group, +/-SE. The results are shown a stimulation index relative to the number of control unstimu- in the bar grphs of FIG. 19, wherein Group 1 was treated 35 lated cells (set at a stimulation index of one). As ilustrated in with vaccine and control IgG 1 and Group 2 was treated with FIG. 21, stimulation with the non-specific activator SEB vaccine and anti-CTLA-4. The left bar for each group repre- increased the stimulation index at least 5 fold in both CDS+ sents day 1, the middle bar represents day 5 1 and the right bar and CDS- cells, whereas incubation with autologous den- represents day 64. Use of the anti-CTLA-4 antibody in com- dritic cells alone increased the stimulation index only very bination with the vaccine led to a significantly greater anti- 40 slightly. Incubation with autologous dendritic cells pulsed HBsAg antibody response than use of the vaccine with a with melanoma cell antigens also increased the stimulation control IgGL. These results demonstrate that a human anti- index at least 5 fold in both CDS+ and CDS- cells (the latter CTLA-4 antibody of the invention is capable of enhancing essentially corresponding to the CD4+ T cell population), antibody responses to a viraI antigen vaccine in vivo in pri- thereby indicating that vaccination with the melanoma cell mates. 45 vaccine in combination with anti-CTLA-4 results in antigen- specific T cell proliferation of both CDS+ and CD4+ T cells. Example 12 Further evidence of antigen-specific T cell proliferation was obtained from delayed type hypersensitivity (DTH) Anti-CTLA-4 Treatment Enhances Antibody and T experiments. Anmals treated with either the melanoma vac- Cell Responses to a Melanoma Cell Vaccine 50 cine alone or with the melanoma vaccine in combination with the anti-CTLA-4 antibody were tested for a DTH reaction to The ability ofa human anti-CTLA-4 antibody of the inven- either SK-mel-3 or to a saline control using standard DTH tion to enhance antibody and T cell responses to a melanoma assay methods. The results demonstrated that 3 of 6 of the cell vaccine was examed in cynomolgus monkeys. Test animals treated with the combination of the vaccine and the groups of six monkeys each (three males, thee females) were 55 anti-CTLA-4 antibody exhibited a specific DTH response to treated with either 1) a melanoma cell vaccine alone (SK - mel- the SK-mel-3 cells, whereas only one of the 6 animals treated 3, a hmnan melanoma tumor cell line transfected to express with the vaccine alone exhbited a specific DTH response to GM-CSF) or 2) both SK-mel-3 and the anti-CTLA-4 anti- the SK-mel-3 cells. These results futher demonstrate the body 1 OD 1. The antibody was administered intravenously at ability of anti-CTLA-4 antibody treatment to enhance anti- a dosage of 1 0 mg/kg in a volume of 1.3 ml/kg. The SK-meI-3 60 gen-specific T cell responses in vivo in primates. cells were administered subcutaneously in a fixed amount Although the foregoing invention has been described in (5x106 cells/animal at 0.5 ml/anmal). The appropriate anti- detail for puroses of clarity of understanding, it wil be body and/or vaccine were administered on days 0, 2S, 56 and obvious that certain modifications may be practiced within S4. Antibody responses to the melanoma cell vaccine were the scope of the appended claims. All publications and patent assessed on days 13,41, 69 and 97. The results are shown in 65 documents cited herein are hereby incorporated by reference the graph of FIG. 20, in which a 1/1000 dilution of plasma in their entirety for all purposes to the same extent as if each was examined. Results presented represent the mean of the were so individually denoted. US 7,605,238 B2 71 72

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~ ~ 0\o 0\i: 00~ \0 \0 \0 l' -n\0 0\,. 001 0 ,.tr i: 0\ i: 0 r- tr 0 0\ r- VN \0 -n 00 00 r- \0 00 l' 0\0\ \0r- ~V -n"' \0r- i:V N\0 l-00 0\ 0 -n \001 0.. l'\0 .. 0\ 0 0\\0 \001 0\ 0\ \0 01 l' ,. :50 i~ rr ~ -. -. -. r- r- rr oô rr ~ i. ., ~ M N N ~ ~ ~ ~ ~ rr rr rr 0¡~ rr"' rrrr rrrr~ ~.. r. ,. ¡. ~ ~x ~ " j 000000000000000000000000 a 0000000000000 ~ ~~ ~ a a 0 0 0 0 0\ 0 0 V 0.. i: i: 0 0 0 0 0 0 0 0 000 l-l-\O..OOOOOOO-nO ó j ~~ $ ~ ~~ ~~æ ~ $~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ $~ ~ ~ z." ~æ~æ~~~~~~;:~~ ;" '" '3 01 ,. .. 0\ l' 0 r- 01 00 0 0\ \0 ~ tr 00 i: 0 01 ~ -n 0\ 00 00 01 tr -g õ u ;; .. i: l' tr -n i: 00 i: l' \0 "' N 00 ~ i: \0 i: 0\ l' 0 i: l' \0 00 00 0001\O-nVl'-noo",O\O\..OO l' 0\ \0 -n \0 00 l- tr 00 l' 00 i: II 0 cx i. \Ó \Ó \Ó \Ó \Ó 0\ i. ~ i. \Ó \Ó r- i. ~ rr rr rr ,. rr rr rr ,. ,.,.,.~ "'oö"' ~ in ~,. r- M ~ ~~rI :s x õt ) Oi:O\O\OO01i:\Ol'O\\OO tj 3 01NNNN01l'Nl'N..NN..NN_____æ~~~~~ ~~~ ~~i~~8~~ ~~~~ $õ~~ _.... trvtro\NN..lNN..O-n...... 01 01 01 01 01 01 .. .. ¡¡ x 00 0 0 0 00 '" 00'" '" 0000000000o l'i:O"'OOi:\D 0\ '"00 00 '" 0\000000 ..1 i: tr 0 0 -. l-' M ..oo~o\Nrr-.doô\Ó or- 0\ M r- -0 r- rI ~ V) '" -n01_-n0\00\l-l'l' ~ $ ~ 0\0"'..01 V) "" " !~ '" '" '" "'.. tr l' '" '" '" '" - '" 00 - "'- '" '" tj Q -:.g '§ ~ GO..01l'i:,...OO G G G G G G G GO..01l'i:,... G..01l'i:V G GO..01l' Q 00 ..01010000000000000000 ..NOO ..0000 rItJ

'I '" OJ ~ ~ '¡ § 1 "0 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ry~ ~ ~ II II II II II II II II Il:i ff:i:i:i:i ~ rI rI rI rI rI ~ ~ ~~~~~~~~~~~~:s~~ö~~~~~~~~ôuôôôô~~~~~~Ó

'" g "'''888888888888 N01NNN;§ Bg §§§§§§§§§ 88000000000000 88888~ a .S g 000000õ õ õ õ õ õ õ000 õ Õ 80000 8;g ~ ~0000 b ~ ~ ~0000 ~ ~ ~ ~00000000 8 ~ ~ ~ ~ ~ ~ 0000000~ ~ ~ ~ ~ ~ ~ 0 ~ 0000000000000000NN NNNN NNN ...... _...... a 0 a a a 0 000 a 0 0 0 ....NN0 00 0 NNN..000 0 rI~ US 7,605,238 B2 73 74

TABLE 13

Study No. MDXCTLA4-02 Selected Lab Values Summar

Platelets WBC Neiits Lymphs

Screen no. Subject no. Intials Amendment # Day Date xlO'/ul x103/ui % xlO'/ul % x103/ul

02001 001 SAH 0 Scr 216 6.28 56.60 3.52 35.60 2.23 02001 001 SAH 0 0 230 5.58 59.70 3.33 32.30 1.80 02001 001 SAH 0 1 202 5.12 61.80 3.16 30.20 1.5 normal range low 150 3.80 40.50 1.96 15.40 0.80 high 11111111111111110.70 75.00 7.23 48.50 3.00

Monos Eos CD41 CD81 ESR Hgb Hcrit

Screen no. % x103/ul % xlO'/ul ul ul rnhr g1dl %

02001 5.90 0.37 1.80 0.11 1189 631 14.4 39 02001 5.70 0.32 1.80 0.10 1039 502 111I1I11111111114.9 43 02001 5.00 0.26 2.30 0.12 957 40711111111111111113.4 37 2.60 0.12 1111111111111111111111111404 220111111111111111 10.10 0.92 6.80 0.57 1612 1129 30

SEQUENCE LISTING

~160~ NUMBER OF SEQ ID NOS, 41

~2 10~ SEQ ID NO 1 ~2 ll~ LENGTH, 3159 ~2l2~ TYPE, DNA ~2i3~ ORGAISM: Artificial Sequence ~220~ FEATURE, ~223~ OTHER INFORMATION, Description of Artificial Sequence, cloning vector pGPlk

~4 OO~ SEQUENCE, 1 aattagcggc cgctgtcgac aagct tcgaa t tcagtatcg atgtggggta cctactgtcc 60 cgggattgcg gatccgcgat gatatcgttg atcctcgagt gcggccgcag tatgcaaaaa 120 aaagcccgct cattaggcgg gctcttggca gaacatatcc atcgcgtccg ccatctccag lBO cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt 240 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga 300 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc 360 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc 420 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac 480 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg 540 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc 600 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa 660 cagaaattcc cccttacacg gaggcatcaa gtgaccaaac aggaaaaaac cgcccttaac 720 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac 7BO gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc B40 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 900 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 960 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg 1020 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt lOBO US 7,605,238 B2 75 76 -continued gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 1140 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 1200 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 1260 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 1320 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 1380 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 1440 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 1500 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 1560 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 1620 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccaggcgc gccttggcct 1680 aagaggccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1740 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 1800 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1860 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1920 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 1980 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 2040 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 2100 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 2160 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 2220 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 2280 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 2340 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 2400 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2460 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 2520 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 2580 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 2640 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 2700 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 2760 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 2820 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 2880 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 2940 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 3000 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 3060 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 3120 ataaaaatag gcgtatcacg aggccctttc gtcttcaag 3159

~210,. SEQ ID NO 2 ~21b LENGTH: 349 ~212" TYPE: DNA ~213,. ORGAISM: Homo sapiens ~220" FEATURE: ~223,. OTHER INFORMATION: preliminary sequence for heavy chain fragment 10D1.3 US 7,605,238 B2 77 78 -continued

",4 OO~ SEQUENCE, 2

tgggggaggc gtggtccagc ctgggaggtc cctgagactc tcctgtgcag cctctggatt 60 caccttcagt agctatacta tgcactgggt ccgccaggct ccaggcaagg ggctggagtg 120 ggtgacattt atatcatatg atggaaacaa taaatactac gcagactccg tgaagggccg 180 attcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag 240 agctgaggac acggctatat attactgtgc gaggaccggc tggctggggc cctttgacta 300 ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc accaagggc 349 ",2l0~ SEQ ID NO 3 ",2ll~ LENGTH, 321 "'2l2~ TYPE, DNA ~2i3~ ORGAISM: Homo sapiens "'220~ FEATURE, ~223~ OTHER INFORMATION: preliminary sequence for light chain fragment 10D1.3

",400~ SEQUENCE, 3

ctccaggcac cctgtctttg tctccagggg aaagagccac cctctcctgc agggccagtc 60 agagtgttgg cagcagctac ttagcctggt accagcagaa acctggccag gctcccaggc 120 tcctcatcta tggtgcattc agcagggcca ctggcatccc agacaggttc agtggcagtg 180 ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat tttgcagtgt 240 attactgtca gcagtatggt agctcaccgt ggacgttcgg ccaagggacc aaggtggaaa 300 tcaaacgaac tgtggctgca c 321 ",2l0~ SEQ ID NO 4 ",2ll~ LENGTH, 287 ",2l2~ TYPE, DNA ~2i3~ ORGAISM: Homo sapiens ",220~ FEATURE, ",223~ OTHER INFORMATION, Vk A- 27 germline sequence

",400~ SEQUENCE, 4

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga. aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacc 287

",2l0~ SEQ ID NO 5 "'2 ll~ LENGTH, 95 ",2l2~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ",220~ FEATURE, ",223~ OTHER INFORMATION, light chain variable region predicted sequence for Vk A-27 germline

",4 OO~ SEQUENCE, 5 Glu1 Ile Val Leu5 Thr Gln Ser 10Pro Gly Thr Leu Ser15 Leu Ser Pro Gly Glu Arg Ala Thr20 Leu Ser Cys Arg25 Ala Ser Gln Ser30 Val Ser Ser Ser Tyr Leu Ala35 Trp Tyr Gln Gln40 Lys Pro Gly Gln45 Ala Pro Arg Leu Leu Ile Tyr50 Gly Ala Ser Ser55 Arg Ala Thr Gly60 Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu US 7,605,238 H2 79 80 -continued

65 70 75 BO Pro Glu Asp Phe AlaB5 Val Tyr Tyr Cys90 GIn GIn Tyr Gly95 Ser Ser

~210~ SEQ ID NO 6 ~211~ LENGTH, 325 ~212~ TYPE, DNA ~213~ ORGAI SM: Homo sapiens "2 2 O~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region (Vk), 10Dl from Vk A-27

~400~ SEQUENCE,

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttggc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcattca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgtg gacgttcggc 300 caagggacca aggtggaaat caaac 325

~210~ SEQ ID NO 7 ~211~ LENGTH, 108 ~212~ TYPE, PRT ~2 i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region predicted sequence for 10Dl from Vk A- 27

~4 OO~ SEQUENCE, 7 Glu1 lIe Val Leu5 Thr GIn Ser 10Pro Gly Thr Leu Ser15 Leu Ser Pro Gly Glu Arg Ala Thr20 Leu Ser Cys Arg25 Ala Ser GIn Ser30 Val Gly Ser Ser Tyr Leu Ala Trp Tyr GIn GIn Lys Pro Gly GIn Ala Pro Arg Leu Leu 35 40 45

lIe Tyr Gly Ala Phe Ser Arg Ala Thr Gly lIe Pro Asp Arg Phe Ser 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lIe Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala85 Val Tyr Tyr Cys90 GIn GIn Tyr Gly95 Ser Ser Pro Trp Thr Phe Gly100 GIn Gly Thr105 Lys Val Glu lIe Lys

~210~ SEQ ID NO 8 ~211~ LENGTH, 325 ~212~ TYPE, DNA ~213~ ORGAISM: Homo sapiens ,,220~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region (Vk) 486 from Vk A- 27

~400~ SEQUENCE, 8 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 etctcctgca gggccagtca gagtgttagc agcagcttct tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 US 7,605,238 H2 81 82 -continued cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgtg gacgttcggc 300 caagggacca aggtggaaat caaac 325

~2 10~ SEQ ID NO 9 ~2 1 l~ LENGTH, 108 ~2 l2~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION: light chain variable region predicted sequence for 4B6 from Vk A-27

~4 OO~ SEQUENCE, 9 Glu1 lIe Val Leu5 Thr GIn Ser 10Pro Gly Thr Leu Ser15 Leu Ser Pro Gly Glu Arg Ala Thr20 Leu Ser Cys Arg25 Ala Ser GIn Ser30 Val Ser Ser Ser Phe Leu Ala35 Trp Tyr GIn GIn40 Lys Pro Gly GIn45 Ala Pro Arg Leu Leu lIe Tyr50 Gly Ala Ser Ser55 Arg Ala Thr Gly60 lIe Pro Asp Arg Phe Ser Gly65 Ser Gly Ser Gly70 Thr Asp Phe Thr75 Leu Thr lIe Ser 80Arg Leu Glu Pro Glu Asp Phe Ala85 Val Tyr Tyr Cys90 GIn GIn Tyr Gly95 Ser Ser Pro Trp Thr Phe Gly100 GIn Gly Thr105 Lys Val Glu lIe Lys

~2l0~ SEQ ID NO 10 ~2ll~ LENGTH, 287 ~2l2~ TYPE, DNA ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, Vk L-15 germline sequence

~400~ SEQUENCE: 10 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatagtt accctcc 287

~2l0~ SEQ ID NO 11 ~2ll~ LENGTH, 94 ~2 l2~ TYPE, PRT ~2l3~ ORGAISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region predicted sequence for Vk L-15 germline

~4 OO~ SEQUENCE, 11 Aspi lIe GIn Met5 Thr GIn Ser 10Pro Ser Ser Leu Ser15 Ala Ser Val Gly Asp Arg Val Thr20 lIe Thr Cys Arg25 Ala Ser GIn Gly30 lIe Ser Ser Trp Leu Ala Trp35 Tyr GIn GIn Lys40 Pro Glu Lys Ala45 Pro Lys Ser Leu lIe Tyr Ala50 Ala Ser Ser Leu55 GIn Ser Gly Val60 Pro Ser Arg Phe Ser Gly US 7,605,238 B2 83 84 -continued Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lIe Ber Ber Leu GIn Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys GIn GIn Tyr Asn Ser Tyr 85 90

~210~ SEQ ID NO 12 ~211~ LENGTH, 322 ~212~ TYPE, DNA ~2i3~ ORGAISM: Homo sapiens ~2 2 O~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region Vk lE2 from Vk L-15

~400~ SEQUENCE, 12

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatagtt accctccgac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

~210~ SEQ ID NO 13 ~211~ LENGTH, 107 ~2 12~ TYPE, PRT ~213~ ORGANISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain variable region predicted sequence for lE2 from Vk L-15

.:400;: SEQUENCE, 13

Asp lIe GIn Met Thr GIn Ser Pro Ber Ser Leu Ber Ala Ber Val Gly 1 5 10 15

Asp Arg Val Thr lIe Thr Cys Arg Ala Ser GIn Gly lIe Ser Ser Trp 20 25 30 Leu Ala Trp35 Tyr GIn GIn Lys40 Pro Glu Lys Ala45 Pro Lys Ser Leu lIe Tyr Ala50 Ala Ser Ser Leu55 GIn Ser Gly Val60 Pro Ser Arg Phe Ser Gly Ser65 Gly Ser Gly Thr70 Asp Phe Thr Leu75 Thr lIe Ser Ser 80Leu GIn Pro Glu Asp Phe Ala ThrB5 Tyr Tyr Cys GIn90 GIn Tyr Asn Ser95 Tyr Pro Pro Thr Phe Gly GIn100 Gly Thr Lysios Val Glu lIe Lys

~210~ SEQ ID NO 14 ~21b LENGTH, 294 ~212~ TYPE, DNA ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, VH 3-30.3 germline sequence

~400~ SEQUENCE, 14 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac 1BO gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gaga 294 US 7,605,238 B2 85 86 -continued

~210~ SEQ ID NO 15 ~211~ LENGTH, 98 ~212~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region predicted sequence for VH 3-30.3 gerrnline

~4 OO~ SEQUENCE, 15 GIn1 Val GIn Leu5 Val Glu Ser 10Gly Gly Gly Val Val15 GIn Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met His Trp Val Arg GIn Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys65 Gly Arg Phe Thr70 lIe Ser Arg Asp75 Asn Ser Lys Asn 80Thr Leu Tyr Leu GIn Met Asn Ser85 Leu Arg Ala Glu90 Asp Thr Ala Val95 Tyr Tyr eys Ala Arg

~210~ SEQ ID NO 16 ~211~ LENGTH, 355 ~212~ TYPE, DNA ~213~ ORGAISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region VH 10D1 from VH 3- 30.3

~400~ SEQUENCE, 16

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatacta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtgacattt atatcatatg atggaaacaa taaatactac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctatat attactgtgc gaggaccggc 300 tggctggggc cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355

~210~ SEQ ID NO 17 ~211~ LENGTH, 118 ~212~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region predicted sequence for 10D1 from VH 3-30.3

~4 OO~ SEQUENCE, 17 GIn1 Val GIn Leu5 Val Glu Ser 10Gly Gly Gly Val Val15 GIn Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Thr Met His Trp Val Arg GIn Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr lIe Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr US 7,605,238 B2 87 88 -continued 65 70 75 80 Leu Gln Met Asn Ser85 Leu Arg Ala Glu90 Asp Thr Ala Ile95 Tyr Tyr Cys Ala Arg Thr Gly100 Trp Leu Gly 105Pro Phe Asp Tyr 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115

~210~ SEQ ID NO 18 ~211~ LENGTH, 355 ~212~ TYPE, DNA ~213~ ORGAISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region VH 4B6 from VH 3- 30.3

~400~ SEQUENCE, 18 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatacta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtgacattt atatcatatg atggaagcaa taaacactac 180 gcagactccg tgaagggccg attcaccgtc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctatat attactgtgc gaggaccggc 300 tggctggggc cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355

~210~ SEQ ID NO 19 ~2 11~ LENGTH, 118 ~2 12~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region predicted sequence for 4B6 from VB 3-30.3

~400~ SEQUENCE, 19 Gln1 Val Gln Leu5 Val Glu Ser 10Gly Gly Gly Val Val15 Gln Pro Gly Arg Ser Leu Arg Leu20 Ser Cys Ala Ala25 Ser Gly Phe Thr30 Phe Ser Ser Tyr Thr Met His35 Trp Val Arg Gln40 Ala Pro Gly Lys45 Gly Leu Glu Trp Val Thr Phe50 Ile Ser Tyr Asp55 Gly Ser Asn Lys60 His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val 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 Ile Tyr Tyr Cys 85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

~210~ SEQ ID NO 20 ~211~ LENGTH, 296 ~212~ TYPE, DNA ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, VI1 3-33 germline sequence

~4 OO~ SEQUENCE, 20 US 7,605,238 B2 89 90 -continued caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaga 296

~210~ SEQ ID NO 21 ~211~ LENGTH, 98 ~2 12~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ,,220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain variable region predicted sequence for VH 3 -33 germline

,,400~ SEQUENCE, 21 Gln1 Val Gln Leu5 Val Glu Ser 10Gly Gly Gly Val Val15 Gln Pro Gly Arg Ser Leu Arg Leu20 Ser Cys Ala Ala25 Ser Gly Phe Thr30 Phe Ser Ser Tyr Gly Met His35 Trp Val Arg Gln40 Ala Pro Gly Lys45 Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys 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

~210~ SEQ ID NO 22 ~211~ LENGTH, 358 ~212~ TYPE, DNA ~2 13~ ORGAISM, Homo sapiens ~2 2 O~ F.EATURE, ~223~ OTHER INFORMATION, heavy chain variable region VH 1E2 from VH 3-33

,,400~ SEQUENCE, 22 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt tttactgtgc gagagctccc 300 aattatattg gtgcttttga tgtctggggc caagggacaa tggtcaccgt ctcttcag 358

~210~ SEQ ID NO 23 ~211~ LENGTH, 119 ~212~ TYPE, PRT ~2 13~ ORGA I SM, Homo sapiens ~220~ FEATURE, ,,223~ OTHER INFORMATION, heavy chain variable region predicted sequence for 1E2 from VH 3-33

~400~ SEQUENCE, 23

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 US 7,605,238 B2 91 92 -continued Gly Met His35 Trp Val Arg GIn40 Ala Pro Gly Lys45 Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr lIe Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu GIn Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Ala Pro100 Asn Tyr lIe 105Gly Ala Phe Asp 110Val Trp Gly GIn Gly Thr Met Val Thr Val Ser Ser 115

~210~ SEQ ID NO 24 ~2 11~ LENGTH, 12 d 12~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~2 2 O~ FEATURE, ~223~ OTHER INFORMATION, light chain CDR1 (HuMab 10D1)

~400~ SEQUENCE, 24 Arg1 Ala Ser G1n5 Ser Val Gly10 Ser Ser Tyr Leu Ala

~210~ SEQ ID NO 25 ~21b LENGTH, 12 ~212~ TYPE, PRT ~213~ ORGANISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain CDRI (HuMab 486)

~4 0 O~ SEQUENCE, 25 Arg1 Ala Ser GIn5 Ser Val Ser10 Ser Ser Phe Leu Ala

~210~ SEQ ID NO 26 ~2 11~ LENGTH, 11 ~212~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain CDR1 (HuMab 1E2)

~4 OO~ SEQUENCE, 26 Arg1 Ala Ser GIn5 Gly lIe Ser10 Ser Trp Leu Ala

~210~ SEQ ID NO 27 ~2 1b LENGTH, 5 ~212~ TYPE, PRT ~2 13~ ORGAISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain CDR1 (HuMab 10D1. 486)

~4 OO~ SEQUENCE, 27 Ser1 Tyr Thr Met5 His

~210~ SEQ ID NO 28 ~21b LENGTH, 5 ~2 I2~ TYPE, PRT ~213~ ORGAISM, Homo sapiens ~2 2 O~ FEATURE, ~223~ OTHER INFORMATION, heavy chain CDRI (HuMab 1E2)

~4 OO~ SEQUENCE, 28 US 7,605,238 B2 93 94 -continued Ser1 Tyr Gly Met5 His

~210~ SEQ ID NO 29 "'21b LENGTH, 7 ",212~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ",223~ OTHER INFORMATION, light chain CDR2 (HuMab 10D1)

",4 OO~ SEQUENCE, 29 Gly1 Ala Phe Ser5 Arg Ala Thr

",210~ SEQ ID NO 30 "'21b LENGTH, 7 "'2 12~ TYPE, PRT ~2 i3~ ORGAI8M: Homo sapiens ~220~ FEATURE, ",223~ OTHER INFORMATION, light chain CDR2 (HuMab 4B6)

",4 OO~ SEQUENCE, 30 Gly1 Ala Ser Ser5 Arg Ala Thr

~210~ SEQ ID NO 31 ",211~ LENGTH, 7 ~212~ TYPE, PRT ~213~ ORGAISM: Homo sapiens ~220~ FEATURE, "'223~ OTHER INFORMATION, light chain CDR2 (HuMab 1E2)

",4 OO~ SEQUENCE, 31 Ala1 Ala Ser Ser5 Leu GIn Ser

~210~ SEQ ID NO 32 ",211~ LENGTH, 17 "'2 12~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens "'220", FEATURE, ~223", OTHER INFORMATION, heavy chain CDR2 (HuMab 10Dl)

~400", SEQUENCE, 32 Phe1 lIe Ser Tyr5 Asp Gly Asn 10Asn Lys Tyr Tyr Ala15 Asp Ser Val Lys Gly

~210~ SEQ ID NO 33 "'211'" LENGTH, 17 "'212'" TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ",220", FEATURE, ",223", OTHER INFORMATION, heavy chain CDR2 (HuMab 4B6)

"'400'" SEQUENCE, 33 Phe1 lIe Ser Tyr5 Asp Gly Ser 10Asn Lys His Tyr Ala15 Asp Ser Val Lys Gly

"'210'" SEQ ID NO 34 ",211", LENGTH, 17 "'212'" TYPE, PRT "'213'" ORGAISM, Homo sapiens ",220~ FEATURE, US 7,605,238 B2 95 96 -continued ~223~ OTHER INFORMATION, heavy chain CDR2 (HuMab 1E2)

~4 OO~ SEQUENCE, 34 Val1 I1e Trp Tyr5 Asp G1y Ser 10Asn Lys Tyr Tyr Ala15 Asp Ser Val Lys G1y

~210~ SEQ ID NO 35 ~21b LENGTH, 9 ~2 12~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain CDR3 (HuMab 10D1, 4B6)

~4 OO~ SEQUENCE, 35 GIn1 GIn Tyr Gly5 Ser Ser Pro Trp Thr

~210~ SEQ ID NO 36 ~21b LENGTH, 9 ~2 12~ TYPE, PRT ~213~ ORGAISM, Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, light chain CDR3 (HuMab 1E2)

~4 OO~ SEQUENCE, 36 GIn1 GIn Tyr Asn5 Ser Tyr Pro Pro Thr

~210~ SEQ ID NO 37 ~21b LENGTH, 9 ~2 12~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain CDR3 (HuMab 10D1, 4B6)

~4 OO~ SEQUENCE, 37 Thr1 Gly Trp Leu5 Gly Pro Phe Asp Tyr

~210~ SEQ ID NO 38 ~211~ LENGTH, 10 ~2 12~ TYPE, PRT ~2i3~ ORGAISM: Homo sapiens ~220~ FEATURE, ~223~ OTHER INFORMATION, heavy chain CDR3 (MuMab 1E2)

~4 OO~ SEQUENCE, 38 Ala1 Pro Asn Tyr5 lIe Gly Ala10 Phe Asp Val

~210~ SEQ ID NO 39 ~211~ LENGTH, 3881 ~2 12~ TYPE, DNA ~213~ ORGAISM, Artificial Sequence ~220~ FEATURE, ~223~ OTHER INFORMATION, Description of Artificial Sequence, kappa light chain plasmid pCK7-96

~4 OO~ SEQUENCE, 39 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240 US 7,605,238 H2 97 98 -continued

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc age agee act 540

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 660

accttcggaa .aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840

gtcatgagat tatcaaaaag gatct tcacc tagatccttt taaat taaaa atgaagttt t 900

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560

cgggataata ccgcgccaca tagcagaact t taaaagtgc tcatcat tgg aaaacgttct 1620 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920

aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100 cgtcagggcg cgtcagcggg tgt tggcggg tgtcggggct ggcttaacta tgcggcatca 2160 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc 2400 aagctagcgg ccgcggtcca accaccaatc tcaaagcttg gtacccggga gcctgttatc 2460 ccagcacagt cctggaagag gcacagggga aataaaagcg gacggaggct ttccttgact 2520 cagccgctgc ctggtcttct tcagacctgt tctgaattct aaactctgag ggggtcggat 2580 US 7,605,238 B2 99 100 -continued gacgtggcca ttctttgcct aaagcattga gtttactgca aggtcagaaa agcatgcaaa 2640 gccctcagaa tggctgcaaa gagctccaac aaaacaattt agaactttat taaggaatag 2700 ggggaagcta ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg 2760 ctataattat ctgggataag catgctgttt tctgtctgtc cctaacatgc cctgtgatta 2820 tccgcaaaca acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc 2880 tttcctcagg aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt 2940 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca 3000 aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 3060 agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag 3120 actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 3180 tcacaaagag cttcaacagg ggagagtgtt agagggagaa gtgcccccac ctgctcctca 3240 gttccagcct gaccccctcc catcctttgg cctctgaccc tttttccaca ggggacctac 3300 ccctattgcg gtcctccagc tcatctttca cctcaccccc ctcctcctcc ttggctttaa 3360 ttatgctaat gttggaggag aatgaataaa taaagtgaat ctttgcacct gtggtttctc 3420 tett tcctca atttaataat tattatctgt tgtttaccaa ctactcaatt tctcttataa 3480 gggactaaat atgtagtcat cctaaggcgc ataaccattt ataaaaatca tccttcattc 3540 tattttaccc tatcatcctc tgcaagacag tcctccctca aacccacaag ccttctgtcc 3600 tcacagtccc ctgggccatg gatcctcaca tcccaatccg cggccgcaat tcgtaatcat 3660 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3720 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3780 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3840 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg c 3881 c:210:: SEQ ID NO 40 ~211" LENGTH, 4723 -:212" TYPE, DNA -:213" ORGAISM, Artificial Sequence ~220" FEATURE, c:223:: OTHER INFORMATION, Description of Artificial Sequence:gammal heavy chain plasmid pCG-96

-:400" SEQUENCE, 40 gaactcgagc agctgaagct ttctggggca ggccaggcct gaccttggct ttggggcagg 60 gagggggcta aggtgaggca ggtggcgcca gccaggtgca cacccaatgc ccatgagccc 120 agacactgga cgctgaacct cgcggacagt taagaaccca ggggcctctg cgccctgggc 180 ccagctctgt cccacaccgc ggtcacatgg caccacctct cttgcagcct ccaccaaggg 240 cccatcggtc ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct 300 gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc 360 cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct 420 cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt 480 gaatcacaag cccagcaaca ccaaggtgga caagaaagtt ggtgagaggc cagcacaggg 540 agggagggtg tctgctggaa gccaggctca gcgctcctgc ctggacgcat cccggctatg 600 cagccccagt ccagggcagc aaggcaggcc ccgtctgcct cttcacccgg aggcctctgc 660 ccgccccact catgctcagg gagagggtct tctggctttt tccccaggct ctgggcaggc 720 acaggctagg tgcccctaac ccaggccctg cacacaaagg ggcaggtgct gggctcagac 780 US 7,605,238 H2 101 102 -continued

ctgccaagag ccatatccgg gaggaccctg cccctgacct aagcccaccc caaaggccaa 840

actctccact ccctcagctc ggacaccttc tctcctccca gattccagta actcccaatc 900

ttctctctgc agagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccaggta 960

agccagccca ggcctcgccc tccagctcaa ggcgggacag gtgccctaga gtagcctgca 1020

tccagggaca ggccccagcc gggtgctgac acgtccacct ccatctcttc ctcagcacct 1080

gaactcctgg ggggaccgtc agtct tcctc t tccccccaa aacccaagga caccctcatg 1140

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 1200

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1260

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1320

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1380

gagaaaacca tctccaaagc caaaggtggg acccgtgggg tgcgagggcc acatggacag 1440

aggccggctc ggcccaccct ctgccctgag agtgaccgct gtaccaacct ctgtccctac 1500

agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa 1560

gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga 1620

gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc 1680

cgacggctcc ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg 1740

gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag 1800

cctctccctg tctccgggta aatgagtgcg acggccggca agcccccgct ccccgggctc 1860

tcgcggtcgc acgaggatgc ttggcacgta ccccctgtac atacttcccg ggcgcccagc 1920

atggaaataa agcacccagc gctgccctgg gcccctgcga gactgtgatg gttctttcca 1980

cgggtcaggc cgagtctgag gcctgagtgg catgagggag gcagagcggg tcccactgtc 2040

cccacactgg cccaggctgt gcaggtgtgc ctgggccccc tagggtgggg ctcagccagg 2100

ggctgccctc ggcagggtgg gggat ttgcc agcgtggccc tccctccagc agcacctgcc 2160

ctgggctggg ccacgggaag ccctaggagc ccctggggac agacacacag cccctgcctc 2220

tgtaggagac tgtcctgttc tgtgagcgcc cctgtcctcc cgacctccat gcccactcgg 2280

gggcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa ttcatcgatg 2340

atatcagatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac 2400

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 2460 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2520 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 2580 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 2640 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2700 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2760 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 2820 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 2880 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2940 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3000 US 7,605,238 B2 103 104 -continued

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3060

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3120

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3180

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3240

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3300

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3360

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3420

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3480

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 3540

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 3600

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 3660

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 3720

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 3780

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 3840

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 3900

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 3960

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 4020

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 4080

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 4140

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 4200

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4260

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4320

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4380

ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 4440

acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 4500

ctcccggaga cggtcacagc t tgtctgtaa gcggatgccg ggagcagaca agcccgtcag 4560

ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 4620

attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac 4680

ataaccttat gtatcataca catacgattt aggtgacact ata 4723

~210~ SEQ ID NO 41 ~211~ LENGTH, 4694 ~212~ TYPE, DNA ~213~ ORGAISM, Artificial Sequence ~220~ FEATURE, ~223~ OTHER INFORMATION, Description of Artificial Sequence,gamma4 heavy chain plasmid pG4HE

~4 OO~ SEQUENCE, 41 gaactcgagc agctgaagct ttctggggca ggccgggcct gactttggct gggggcaggg 60 agggggctaa ggtgacgcag gtggcgccag ccaggtgcac acccaatgcc catgagccca 120 gacactggac cctgcatgga ccatcgcgga tagacaagaa ccgaggggcc tctgcgccct 180 US 7,605,238 B2 105 106 -continued

gggcccagct ctgtcccaca ccgcggtcac atggcaccac ctctcttgca gcttccacca 240

agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag agcacagccg 300

ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag 360

gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca ggactctact 420

ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc tacacctgca 480

acgtagatca caagcccagc aacaccaagg tggacaagag agttggtgag aggccagcac 540

agggagggag ggtgtctgct ggaagccagg ctcagccctc ctgcctggac gcaccccggc 600

tgtgcagccc cagcccaggg cagcaaggca tgccccatct gtctcctcac ccggaggcct 660

ctgaccaccc cactcatgct cagggagagg gtcttctgga tttttccacc aggctccggg 720

cagccacagg ctggatgccc ctaccccagg ccctgcgcat acaggggcag gtgctgcgct 780

cagacctgcc aagagccata tccgggagga ccctgcccct gacctaagcc caccccaaag 840

gccaaactct ccactccctc agctcagaca ccttctctcc tcccagatct gagtaactcc 900

caatcttctc tctgcagagt ccaaatatgg tcccccatgc ccatcatgcc caggtaagcc 960

aacccaggcc tcgccctcca gctcaaggcg ggacaggtgc cctagagtag cctgcatcca 1020

gggacaggcc ccagccgggt gctgacgcat ccacctccat ctcttcctca gcacctgagt 1080

tcctgggggg accatcagtc t tcctgttcc ccccaaaacc caaggacact ctcatgatct 1140

cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc 1200

agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag ccgcgggagg 1260

agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc 1320

tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga 1380

aaaccatctc caaagccaaa ggtgggaccc acggggtgcg agggccacat ggacagaggt 1440

cagctcggcc caccctctgc cctgggagtg accgctgtgc caacctctgt ccctacaggg 1500

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1560

caggtcagcc tgacctgcct ggtcaaaggc t tctacccca gcgacatcgc cgtggagtgg 1620

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1680

ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1740

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1800

tccctgtctc tgggtaaatg agtgccaggg ccggcaagcc cccgctcccc gggctctcgg 1860

ggtcgcgcga ggatgcttgg cacgtacccc gtctacatac ttcccaggca cccagcatgg 1920

aaataaagca cccaccactg ccctgggccc ctgtgagact gtgatggttc tttccacggg 1980

tcaggccgag tctgaggcct gagtgacatg agggaggcag agcgggtccc actgtcccca 2040

cactggccca ggctgtgcag gtgtgcctgg gccacctagg gtggggctca gccaggggct 2100 gccctcggca gggtggggga t ttgccagcg tggccctccc tccagcagca gctgccctgg 2160 gctgggccac gggaagccct aggagcccct ggggacagac acacagcccc tgcctctgta 2220 ggagactgtc ctgtcctgtg agcgccctgt cctccgaccc cccatgccca ctcgggggga 2280

tccccgggta ccgagctcga attcatcgat gatatcagat ctgccggtct ccctatagtg 2340 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg 2400 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2460 US 7,605,238 H2 107 108 -continued

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2520

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 2580 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 2640

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2700

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2760

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 2820

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2880

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2940 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3000

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 3060

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3120

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3180 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3240

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3300 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3360

tgacagttac caatgcttaa tcagtgaggc acetate tea gcgatctgtc tatttcgttc 3420 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3480

tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3540

aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaact t tatccgcctc 3600

catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 3660

gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 3720 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 3780

aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 3840 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 3900

cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 3960

gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 4020

agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4080 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4140

caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4200 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4260

tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4320 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 4380 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 4440 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 4500 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 4560 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat 4620 attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt 4680 taggtgacac tata 4694 US 7,605,238 B2 109 110 What is claimed is: 16. The antibody of claim 4, wherein the antibody com- 1. An isolated human antibody or antigen binding portion prises the amino acid sequence set forth in SEQ ID NO: 19 thereof that: (a) is IgGl isotype; (b) binds human CTLA-4 and the amino acid sequence set forth in SEQ ID NO: 9. with a binding affnity of about 108 M-1 or greater; and (c) 17. The antibody of claim 4, wherein the antibody com- inbits binding of human CTLA-4 to B7-1 and B7-2. 5 prises the amino acid sequence set forth in SEQ ID NO: 23 2. The antibody of claim 1, wherein the antibody comprises and the amino acid sequence set forth in SEQ ID NO: 13. at least one amino acid sequence selected from the group 18. The antibody of claim 1, comprising: (a) a heavy chain consisting of SEQ ID NOS: 17, 19 and 23. variable region comprising CDR1, CDR2, and CDR3 3. The antibody of claim 1, wherein the antibody comprises sequences set forth in SEQ ID NOS:27, 32 and 37, respec- at least one light chain variable region amino acid sequence 10 tiveIy; and (b) a light chain varable region comprising CDRI, selected from the group consisting of SEQ ID NOS: 7, 9 and CDR2, and CDR3 sequences set forth in SEQ ID NOS:24, 29 13. and 35, respectively. 19. The antibody of claim 1, comprising: (a) a heavy chain 4. An isolated human monoclonal antibody, or antigen- variable region comprising CDRI, CDR2, and CDR3 binding portion thereof that is IgG 1 isotype and specifically 15 sequences set forth in SEQ ID NOS:27, 33 and 37, respec- binds to human CTLA-4, wherein the antibody or antigen- binding portion thereofhas a binding affnity that is about 108 tively; and (b) a light chain varable region comprising CDR 1 , CDR2, and CDR3 sequences set forth in SEQ ID NOS:25, 30 M-1 or greater affty. and 35, respectively. 5. The antibody of claim 4, wherein the antibody comprises 20. The antibody of claim 1, comprising: (a) a heavy chain at least one amino acid sequence selected from the group 20 variable region comprising CDR1, CDR2, and CDR3 consisting of SEQ ID NOS: 17, 19 and 23. sequences set fort in SEQ ID NOS:28, 34 and 38, respec- 6. The antibody of claim 4, wherein the antibody comprises tively; and (b) a light chain variable region comprising CDR1, at least one amino acid sequence selected from the group CDR2, and CDR3 sequences set forth in SEQ ID NOS:26, 31 consisting of SEQ ID NOS: 7, 9 and 13. and 36, respectively. 7. An isolated monoclonal antibody, or an antigen binding 25 21. The antibody of claim 4, comprising: (a) a heavy chain portion thereof, which is IgG1 isotype and competes for variable region comprising CDR1, CDR2, and CDR3 binding to a human CTLA-4 polypeptide with an antibody sequences set forth in SEQ ID NOS:27, 32 and 37, respec- comprising the amno acid sequence set forth in SEQ ID tively; and (b) a light chain variable region comprising CDR1, NO:17, and the amino acid sequence set fort in SEQ ID CDR2, and CDR3 sequences set forth in SEQ ID NOS:24, 29 NO:7, wherein the monoclonal antibody or antigen-binding 30 and 35, respectively. portion thereof has a binding affty that is about 108 M-1 or 22. The antibody of claim 4, comprising: (a) a heavy chain greater affnity. variable region comprising CDR1, CDR2, and CDR3 8. The antibody of claim 1, wherein the antibody com- sequences set forth in SEQ ID NOS:27, 33 and 37, respec- prises: ( a) a heavy chain variable region derived from a human tively; and (b) a light chain variable region comprising CDR1, V H3-30.3 gene; and (b) a light chain variable region derived 35 CDR2, and CDR3 sequences set forth in SEQ ID NOS:25, 30 from a human V K A-27 gene. and 35, respectively. 9. The antibody of claim 4, wherein the antibody com- 23. The antibody of claim 4, comprising: (a) a heavy chain variable region comprising CDR1, CDR2, and CDR3 prises: (a) a heavy chain variable region derived from a human V H 3-33 gene; and (b) a light chain variable region derived sequences set forth in SEQ ID NOS:28, 34 and 38, respec- from a human V K L-15 gene. 40 tively; and (b) a light chain variable region comprising CDR1, 10. The antibody of claim 4, wherein the antibody com- CDR2, and CDR3 sequences set fort in SEQ ID NOS:26, 31 prises: ( a) a heavy chain variable region derived from a human and 36, respectively. V H3-30.3 gene; and (b) a light chain variable region derived 24. The antibody of claim 1, wherein the antibody is a monoclonal antibody. from a human V K A-27 gene. 45 25. The antibody of claim 4, wherein the antibody is a 11. The antibody of claim 1, wherein the antibody com- monoclonal antibody. prises: (a) a heavy chain variable region derived from a human 26. The antibody of claim 7, wherein the antibody is a V H 3-33 gene; and (b) a light chain variable region derived monoclonal antibody. from a human V K L-15 gene. 27. The antibody of claim 1, wherein the antibody is a 12. The antibody of claim 1, wherein the antibody com- 50 human antibody. prises a the amno acid sequence set forth in SEQ ID NO: 17 28. The antibody of claim 4, wherein the antibody is a and the amino acid sequence set forth in SEQ ID NO: 7. human antibody. 13. The antibody of claim 1, wherein the antibody com- 29. The antibody of claim 7, wherein the antibody is a prises the amino acid sequence set fort in SEQ ID NO: 19 human antibody. and the amino acid sequence set forth in SEQ ID NO: 9. 55 30. A pharmaceutical composition comprising the anti- 14. The antibody of claim 1, wherein the antibody com- body of claim 1, and a pharmaceutically acceptable carrier. prises the amo acid sequence set forth in SEQ ID NO: 23 31. A pharmaceutical composition comprising the anti- and the amno acid sequence set forth in SEQ ID NO: 13. body of claim 4, and a pharmaceutically acceptable carrier. 15. The antibody of claim 4, wherein the antibody com- 32. A pharmaceutical composition comprising the anti- prises the amino acid sequence set forth in SEQ ID NO: 17 60 body of claim 7, and a pharmaceutically acceptable carrier. and the amino acid sequence set forth in SEQ ID NO: 7. * * * * * q¿~ÙJì+z. _

I ~.' UNITED STATES ' OOdJlCl0 ~ J-" .- r PATENT AND · .f* TRAEMA OFFICE LJ2.8D !IM3¿IUS~2,

JULY 23, 2003 Under Secretary of Commerce For Intellectual Property and PTAS Director of the United States Patent and Trademark Office DARBY & DARY P. C . Washington, DC 20231 POST OFFICE BOX 5257 ww.uspto.gov NEW YORK, NEW YORK 10150-5257

1/11111111111111111111 11111111111111111111 11111111111111/111 *102386272A*

UNITED STATES PATENT AN TRADEMARK OFFICE NOTICE OF RECORDATION OF ASSIGNMENT DOCUMENT THE ENCLOSED DOCUMNT HAS BEEN RECORDED BY THE ASSIGNMENT DIVISION OF THE U. S. PATENT AND TRADEMARK OFFICE. A COMPLETE MICROFILM COpy IS AVAILABLE AT THE ASSIGNMENT SEARCH ROOM ON THE REEL AND FRAE NUMBER REFERENCED BELOW. PLEASE REVIEW ALL INFORMTION CONTAINED ON THIS NOTICE. THE INFORMTION CONTAINED ON THIS RECORDATION NOTICE REFLECTS THE DATA PRESENT IN THE PATENT AND TRADEMARK ASSIGNMENT SYSTEM. IF YOU SHOULD FIND AN ERRORS OR 1JVE QUESTIONS CONCERNING THIS NDTICE, YOU CONTACT THE EMPLOYEE WHOSE NAME APPEARS ON THIS NOTICE AT 703-308-9723.MAY PLEASE SEND REQUEST FOR CORRECTION TO: U. S. PATENT AND TRAEMAK OFFICE, ASSIGNMNT DIVISION, BOX ASSIGNMENTS, CG-4, 1213 JEFFERSON DAVIS HW, SUITE 320, WASHINGTON, D. C. 20231.

RECORDATION DATE: 03/06/2003 REEL/FRAE: 013817/0628 NUER OF PAGES: 4 BRIEF: ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS) . ASSIGNOR:KORM, ALAN J. DOC DATE: 02/26/2003 ASSIGNOR:HALK, EDWARD L. DOC DATE: 02/24/2003 ASSIGNOR:LONBERG, NILS DOC DATE: 02/25/2003 ASSIGNEE: MEDAREX, INC. 707 STATE ROAD PRINCETON, NEW JERSEY 08540 PATENTSERIAL NUMBER: NUBER: 09948939 ISSUE FILING DATE: DATE: 09/07/20.01

.t.;, .

" 013817/0628 PAGE 2

STEVEN POST, EXAINER ASSIGNMENT DIVISION OFFICE OF PUBLIC RECORDS , fii~159 5 U.S. DEPARTMENT OF COMMERCE (Rev. 6-93l .:CORDATION FORM COVER SH OMS No. 0651-0011 (exp. 41941 Patent and Trademark Office PATENTS ONLY Docket No.: 4280/1M321.US2

To the Honorable Commissioner of Patents and Trademarks: Please record the attached original documents or copy thereof.

1 . Name of conveying party(ies): 2. Name and address of receiving party(ies): Alan J. KORMAN, Edward L HALK, Nils LONBERG Name: MEDAREX, INC. Additional name(s) of conveying party(ies) attached? ii Yes (xl Internal Address: 707 State Road No 3. Nature of conveyance: Street Address: City: Princeton State: New Jersey Zip: 08540 IX) Assignment IJ Merger .. IJ Security Agreement (J Change of Name Additional name(s) & address(esl attached? (J Yes (XJ No (J Other - Execution Date: _February 26, 2003, February 24, 2003 and February 25, 2003 ¥~lMA~1'94TØ 3 6 G 2 5 -().£ .. Date Label No. 4. Application number(s) or patent number(s): IherebyCerttft9nlhedatelndicledaaove.thisliapeor fe was depOsited wit th U.S. Potal Seivic & that It was - t isIf ocument h'IS ein9 ied toget . er witb' .a new fi appto ication, d th eA~~¡ess' exec. h l' h '~1i.ort~~.~ommissionerfor serv.uti . Sil," ¡a1D l_MaîlPostOf e~ , A. Patent ApphcatJon No.(s) B. Patent No.(s(TY~OrprlnteNa ~ 09/948,939, filed September 7, 2001 ~f"" . SIgnalUre r MI ~ Dale -,~ Additional numbers attached? II Yes (xl No 5. Name and address of party to whom correspondence 6. Total number of applications and patents concerning document should be mailed: involved: Name: Darby & Darby P.C. CZ ; Internal Address: 7. Total fee (37 CFR 3.41):...... $40

IXj Enclosed Street Address: Post Office Box 5257 ri Authorized to be charged to deposit account

City: New York State: New York Zip: 10150-5257 8. Deposit account number: 04-0100 (Attach duplicate copy of this page if paying by deposit account)

DO NOT USE THIS SPACE

.. 9. Statement and signature. To the best of my knowledge and belief, the foregoing information is true and correct and any attached copy is a true copy of the original document. ,r í2al r

Paul F. Fehlner (35,135) March 5, 2003 Name of Person Signing Si&nature ?:&ec. Date Total number of pages including cover sheet, attachments, and document: ~ Mail documents to be recorded with required cover sheet information to: Commissioner of Patents & Trademarks, Box Assignments Washington, D.C. 20231 M:\4280/1 M321 US2\REK9563. WPD

;l 4280/1 M321 US2 Page 1 of3

ASSIGNMENT

WHEREAS, We,

ALAN J. KORMAN, a citizen of USA, residing at 301 El Cerrto Avenue, Piedmont, CA 94611

EDWARD L. HALK, a citizen of USA, residing a.t 1004 Edmonds Court, Sunnyvale, CA 94087; and

NILS LONBERG, a citizen of USA, residing at 780 W. Californa Way, Woodside, CA 94062,

hereinbelow called "Assignors" have made a certain in invention in

HUMAN CTLA-4 ANTIBODIES AND THEIR USES

described in the Specification filed in the U.S. Patent and Trademark Office on September 7, 2001 and assigned Serial No. 09/948,939; and

WHEREAS, MEDAREX, INC. , a corporation organized under the laws of New Jersey, located at 707 State Road, Princeton, NJ 08540, hereinbelow called the "Assignee", is desirous of securng the entire right, title and interest in to the said invention, application and Letters Patent, when granted, and in and to any divisions, continuations, improvements, reissues or extensions that may be made or granted thereon anywhere throughout the world, including the right of priority;

NOW, THEREFORE, BE IT KNOWN, that for good and valuable consideration, the receipt of which is hereby acknowledged, we, the said Assignors, have sold, assigned, transferred and set over, and by these presents do hereby sell, assign, transfer and set over, unto the said Assignee, its successors and assigns, the entire right, title and interest throughout the world in and to the said invention, application and Letters Patent, when granted, and in and to any divisions, continuations, improvements, reissues or extensions that may be made or granted on any of them for the United States and its terrtorial possessions, and in all foreign countries, including all rights to claim priority;

TO HAVE AN TO HOLD the same to the full end of the term or terms for which said Letters Patent may be granted, as fully and completely as the same might be held by us had this sale and assignent not been made. ¡M:'4280'lm32Ius2'SG2767.DOC;IJ 4280/IM32 i VS2 Page 2 of3

For the consideration aforesaid, we hereby covenant and agree to and with the said Assignee, its successors and assigns, that whenever its counselor representative, or the counsel or representative of its successors or assigns, shall advise that an amendment to, or a division of, or any other proceeding or action in connection with said application or invention, including interference proceedings, is lawful and desirable, or that a reissue or continuation or extension of said Letters Patent is lawful and desirable, we wil sign all papers and drawings, take all rightful oaths and affidavits, and do all acts necessary or required to be done for the procurement of valid Letters Patent for said invention, or for the reissue or continuation or extension of the same, and wil do all acts necessary or required to secure to the said Assignee, its successors and assigns, the title to and full benefit of all rights hereby assigned, without charge to said Assignee or its successors or assigns, but at its or their expense;

AND the Commissioner of Patents and Trademarks is requested to issue the said Letters Patent, when granted, in accordance with this sale and assignent.

For the consideration aforesaid, we have sold, assigned, transferred and set over and by these presents do sell, assign, transfer and set over unto the said Assignee, its successors and assigns, or the nominees of any of them, the entire right, title and interest in and to any and all Letters Patent for said invention which may be granted in countries foreign to the United States, and in and to any applications for Letters Patent which may be fied for said invention In countries foreign to the United States, and in and to the invention described in said applications including the right to claim priority; and we hereby authorize and empower said Assignee and its successors, assigns or nominees, to apply for Letters Patent or other form of protection on said invention in Assignor's name or in its own name or in the name of its successors, assigns or nominees, In any or all countries where it may desire to file such application, and where said application may be filed; and we hereby covenant and agree to sign all papers and drawings, take all rightful oaths, execute all rightful affdavits, and do all acts necessary or required to be done for the procurement and maintenance of Letters Patent or other form of protection for said invention in countries foreign to the United States, and for fuher investing or confirming the right and title thereto in the Assignee, its successors, assigns or nominees, without charge to said Assignee, its successors, assigns or nominees, but at its or their expense.

We hereby covenant that no assignment, sale, agreement or encumbrance has been or wil be made or entered into which would conflct with this assignent.

(M:\4280\ I m321 us2\SG2767 .DOC; 1 L 4280llM32 1 US2 Page 3 of3

We declare under penalty of peijury under the laws of the United States of America that we have signed this document as our own free act and that all of the foregoing is true and correct.

Dated: ~J (i- (,I (ø 1

Dated: ;)/!)~ i1./03 EDWAR~&? L. HALK, Inventor

2/7 ~(O'5 Dated: - ~~~ ~ NILS LONBERG, Inven~

(M:\4280\1 m321 us2\SG2767.DOC; 1 J

USPTO 5/25/2007 7: 13: 56 AM PAGE 1/005 Fax Server TO: BAKER BOTTS L. L. P. COMPANY: 30 ROCKEFELLER PLAZA

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UNI STATE PATE ANTRDEMi OFFCE UNOI S~CRêI\V OF COMERCE FO 1N'Li.ti PROPERT AND DIRECTOR OF THE UNITED STATES PATENT ANO TIWEM OffICE MAY 23, 2007 PTAS BAKER BOTTS L.L. P. *500282575A* 30 ROCKEFELLER PLAZA .. fJ5A 44TH FLOOR NEW YORK, NY 10112-4498

UNITED STATES PATENT AND TRADEM OFFICE NOTICE OF RECORDATION OF ASSIGNMENT DOCUMENT

THE ENCLOSED DOCUMENT HAS BEEN RECORDED BY THE ASSIGNMENT DIVISION OF THE U.S. PATENT AND TRADEMRK OFFICE. A COMPLETE MICROFILM COPY is AVAILALE AT THE ASSIGNMENT SEACH ROOM ON THE REEL AND FRAE NuaBER REFERENCED BELOW. PLEASE REVIEW ALL INFORMTION CONTAINED ON THIS NOTICE. THE INFORMTION CONTAINED ON THIS RECORDATION NOTICE REFLECTS THE DATA PRESENT IN THE PATENT AND TRADEMK ASSIGNMENT SYSTEM. IF YOU SHOULD FIND ANY ERRORS OR HAVE QUESTIONS CONCERNING THIS NOTICE, YOU MAY CONTACT THE EMPLOYEE WHOSE NAME APPEAS ON THIS NOTICE AT 571-272-3350. PLEASE SEND REQUEST FOR CORRECTION TO: U.S. PATENT AND TRADEM OFFICE, MAIL STOP: ASSIGNMENT SERVICES BRACH, P.O. BOX 1450, ALEXADRIA, VA 22313.

RECORDATION DATE: OS/23/2007 REEL/FRAE: 019334/0783 NUMBER OF PAGES: 5

BRIEF: ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS). DOCKET NUBER: 077375.0135

ASSIGNOR: DEO, YASHWANT M. DOC DATE: 04/24/2007

ASSIGNOR: KELER, T IBOR P. DOC DATE: 04/26/2007

ASSIGNEE: MEDARX, INC. 7 07 STATE ROAD PRINCETON, NEW JERSEY 08540 PATENTSERIAL NUMBER: NUMBER: 09948939 ISSUE FILING DATE: DATE: 09/07/2001 TITLE: HUM CTLA-4 ANTIBODIES AND THEIR USES

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019334/0783 PAGE 2

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PATENT ASSIGNMENT II ~

Electronic Version v1.1 05/23/2007 Styesheet Version v1. 1 500282575

i SUBMISSION TYE: II NEW ASSIGNMENT I

I NATURE OF CONVEANCE: II ASSIGNMENT i CONVEYING PARTY DATA

Name II Execution Date I I IYashwant M. Deo 110412412007 I

ITibor P. Kelor 1104/2612007 1

RECEIVING PARTY DATA

Name: IMEDAREX. INC. I stet Addre 1707 State Road i ICIly IIPrinceton i ISlateunt: ¡INEW JERSEY I IPOsll Code: IloB540 I

PROPERTY NUMBERS Totl: 1

Number lI ApplIcatin _erTyp Number: I110998939 II I CORRESPONDENCE DATA -. .. Fax Numbe (212)259-2482 . Coponc will be sen vi US MilI when the fa attmpt is unsl Phane: 212-48-2539 Email: davld.schalk~bakerbott.com COrrespondent Name: Baker Bott L.LP. Address Line 1: 30 Rockefeller Plaza Adre Line 2: 44th Floor Addre Line 4: New York, NEW YORK 10112-498

I ATTORNEY DOCKET NUMBER: II 077375.0135 I

Schalk I NAE OF SUBMmER: II David i

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II II sourc=0135#page3.tif 077375.0135 PATENT

ASSIGNMENT

WHREAS, WE, YASHWANT M. DEO, a citizen of the United States, residing at 35

Cortland Drive, East Brunswick, New Jersey 08816; and TIBOR P. KELER, a citizen of the United States, residing at 30 Park Road, Ottsvile, Pennsylvania 18942 (hereinafter "ASSIGNORS"), have made an invention entitled "HUMA CTLA-4 ANTIBODIES AND THEIR USES" as set forth and described in application for Letters Patent of the United States, Application Serial No. 09/948,939, fied September 7,2001; and

WHREAS, MEDAREX. INC. (hereinafter "ASSIGNEE"), having an offce for the transaction of business at 707 State Road, Princeton, New Jersey 08540, is desirous of acquiring ASSIGNORS' entire right, title and interest in and to said application for Letters Patent, and any continuations, divisions, extensions, substitutions, reissues and reexaminations thereof;

NOW, THEREFORE, TO ALL WHOM IT MAY CONCERN, BE IT KNOWN, that

WE, the said ASSIGNORS, for and in consideration of the sum of One Dollar ($1.00), lawful money of the United States, to us in hand paid by said ASSIGNEE, and other valuable considerations unto us moving from said ASSIGNEE, at or before the ensealing and delivery of these presents, the receipt of which is hereby acknowledged, have sold, assigned, transferred and conveyed and by these presents do sell, assign, transfer and convey, unto said ASSIGNEE, its successors and assigns, ASSIGNORS' entire right, title and interest in and to the said invention entitled "HUMAN CTLA-4 ANTIBODIES AND THEIR USES" and any and all improvements thereon, and in and to said application and any division, continuation or \continuation~in-part thereof, and in and to any Letters Patent ofthe United States which may be issued on any of said applications, and any reissues thereof, and in and to any and all applications for Letters Patent fied in foreign countries for said invention or improvements including all priority rights, and any and all Letters Patent wruch may be granted in foreign countries therefor, TO HA VB AN TO HOLD THE SAM to the full end of the term or terms for which any and all said Letters Patent may be granted;

AN WE, the said ASSIGNORS, do hereby authorize and request the Commissioner of Patents and Trademarks to issue the said Letters Patent of the United States to said ASSIGNEE, as the assignee of ASSIGNORS' entire right, title and interest in and to the same, for the sole use and benefit of said ASSIGNEE, its successors and assigns;

AN WE, the said ASSIGNORS, for the considerations aforesaid, do hereby covenant and agree to and with said ASSIGNEE, its successors and assigns, that we have the full power to make this assignment, and that the rights assigned are not encumbered by any grant, license or right heretofore given, and that we, our executors or administrators, shall and will do all lawful acts and things and make, execute and deliver without further compensation, any and all other instruments in writing, further applications, papers, affdavits; powers of attorney, assignments, and other documents which, in the opinion of counsel for said ASSIGNEE, its successors and assigns, may be required or necessary more effectively to secure to and vest in said ASSIGNEE, its successors and assigns, ASSIGNORS' entire right, title and interest in and to said invention and improvements, applications, Letters Patent, rights, titles, benefits, privileges; and advantages

NY02:580899.1 077375.0135 PATENT hereby sold, assigned, transferred and conveyed, and that we wil sign any applications for reissue which may be desired by the owner of the patent or patents which may be issued for the said invention or improvements.

IN WITNSS WHREOF, I, the said YAS hand and seal on the date below written.

STATE OF ) :58: COUNTY OF )

BE IT KNOWN, that on this 21"tlday of Ap/(( , 2007, before me personally came YASHWANT M. DEO, to me knownlãnd known to me to be the person mentioned in and who executed the foregoing assignment, and he acknowledged to me that he executed the same as his free act and deed for the use and purposes therein mentíoned. Not~~

NOTARYPUBUC ATHEN MY COMMISS/ON EXP¡RE~~~2~~~'; GEORGIA

NY02:580899.1 .2. 077375.0135 PATENT andIN sea WITNSS on WHREOF, the date T, the belowsaid TmOR P.writen. KELER have ~ hereunto ~úì set~ my hand

TffOR P. KELER

STATE OF ) l-e.¡. :Te...'! 'r :ss: COUNTY OF ) r+u..,"¡ ..o ,,\

BE IT KNOWN, that on this :;" .... day of it".) , 2007, before me personally

came TID OR P. KELER, to me known and known to me to be the person mentioned in and who executed the foregoing assignment, and he acknowledged to me that he executed the same as his free act and deed for the use and purposes therein mentioned. ~~ t). ~-A~ NotarPu lC

lC D. REER NOTA PUBLIC OF NEW JERS MY COMMISSION EXPIRES FEBR 7, 2012

NY02:580899.1 .3- ~h,¡'o;1 .3 HIGHLIGHTS OF PRESCRIBING INFORMATION ------DOSAGE FORMS AND STRENGTHS------These highlights do not include all the information needed to use . 50 mg/IO mL (5 mg/mL) (3) YERVOY safely and effectively. See full prescribing information for 200 mg/40 mL (5 mg/mL) (3) YERVOY. ------CO NTRAIND I CA TI 0 NS------YERVOY"" (ipilmumab) None. (4) Injection, for intravenous infusion Initial U.S. Approval: 2011 ------WARNINGS AND PRECAUTIONS------Immune-mediated adverse reactions: Permanently discontinue for severe reactions. Withhold dose for moderate immune-mediated adverse reactions WARNING: IMMUNE-MEDIA TED ADVERSE REACTIONS until return to baseline, improvement to mild severity, or complete resolution, See full prescribing information for complete boxed warning. and patient is receiving less than 7.5 mg prednisone or equivalent per day. YERVOY can result in severe and fatal immune-mediated adverse Administer systemic high-dose corticosteroids for severe,. persistent, or reactions due to T-cell activation and proliferation. These recurring immune-mediated reactions. (5.1, 5.2, 5.3, 5.4, 5.5) immune-mediated reactions may involve any organ system; however, the · Immune-mediated hepatitis: Evaluate liver function tests before each most common severe immune-mediated adverse reactions are dose ofYERVOY. enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), · Immune-mediated endocrinopathies: Monitor thyroid function tests and neuropathy, and endocrinopathy. The majority of these immune- clinical chemistries prior to each dose. Evaluate at each visit for signs mediated reactions initially manifested during treatment; however, a and symptoms of endocrinopathy. Institute hormone replacement minority occurred weeks to months after discontinuation ofYERVOY. therapy as needed. Permanently discontinue YERVOY and initiate systemic high-dose ------AD VERSE REA CTI 0 NS------corticosteroid therapy for severe immune-mediated reactions. (2.2) Most common adverse reactions (2'5%) are fatigue, diarrhea, pruritus, rash, and colitis. (6.1) Assess patients for signs and symptoms of enterocolitis, dermatiis, neuropathy, and endocrinopathy and evaluate clinical chemistries To report SUSPECTED ADVERSE REACTIONS, contact Bristol-Myers including liver function tests and thyroid function tests at baseline and Squibb at 1-800-721-5072 or FDA at 1-800-FDA-I088 or before each dose. (5.1, 5.2, 5.3, 5.4, 5.5) www.fda.gov/medwatch.

------INDICATIONS AND USA GE------USE IN SPECIFIC POPULATIONS------Pregnancy: Based on animal data, YERVOY may cause fetal harm. (8. I) YERVOY is a human cytotoxic T-Iymphocyte antigen 4 (CTLA-4)-blocking . Nursing mothers: Discontinue nursing or discontinue YERVOY. (8.3) antibody indicated for the treatment of unresectable or metastatic melanoma. (I) See 17 for PATIENT COUNSELING IN~'ORMATION and Medication Guide ------DOSAGE AND AD MINISTRA TI ON ------Revised: March 2011 . YERVOY 3 mg/kg administered intravenously over 90 minutes every 3 weeks for a total offour doses. (2. I) . Permanently discontinue for severe adverse reactions. (2.2)

FULL PRESCRIBING INFORMATION: CONTENTS* FULL PRESCRIBING INFORMATION 8 USE IN SPECIFIC POPULATIONS WARNING: IMMUNE-MEDIATED ADVERSE REACTIONS 8.1 Pregnancy 1 INDICATIONS AND USAGE 8.3 Nursing Mothers 2 DOSAGE AND ADMINISTRATION 8.4 Pediatric Use 2.1 Recommended Dosing 8.5 Geriatric Use 2.2 Recommended Dose Modifications 8.6 Renal Impairment 2.3 Preparation and Administration 8.7 Hepatic Impairment 3 DOSAGE FORMS AND STRENGTHS 10 OVERDOSAGE 4 CONTRAINDICATIONS 11 DESCRIPTION 5 WARNINGS AND PRECAUTIONS 12 CLINICAL PHARMACOLOGY 5.1 Immune-mediated Enterocoliis 12.1 Mechanism of Action 5.2 Immune-mediated Hepatitis 12.3 Pharmacokinetics 5.3 Immune-mediated Dermatitis 13 NONCLINICAL TOXICOLOGY 5.4 Immune-mediated Neuropathies 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 5.5 Immune-mediated Endocrinopathies 13.2 Animal Toxicology and/or Pharmacology 5.6 Other Immune-mediated Adverse Reactions, Including 14 CLINICAL STUDIES Ocular Manifestations 16 HOW SUPPLIED/STORAGE AND HANDLING 6 ADVERSE REACTIONS 17 PATIENT COUNSELING INFORMATION 6.1 Clinical Trials Experience 6.2 Immunogenicily * Sections or subsections omitted from the full prescribing information 7 DRUG INTERACTIONS are not listed FULL PRESCRIBING INFORMATION

WARNING: IMMUNE-MEDIATED ADVERSE REACTIONS

YERVOY can result in severe and fatal immune-mediated adverse reactions due to T-cell activation and proliferation. These immune-mediated reactions may involve any organ system; however, the most common severe immune-mediated adverse reactions are enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrinopathy. The majority of these immune-mediated reactions initially manifested during treatment; however, a minority occurred weeks to months after discontinuation of YERVOY.

Permanently discontinue YERVOY and initiate systemic high-dose corticosteroid therapy for severe immune-mediated reactions. ¡See Dosage and Administration (2.2))

Assess patients for signs and symptoms of enterocolitis, dermatitis, neuropathy, and endocrinopathy and evaluate clinical chemistries including liver function tests and thyroid function tests at baseline and before each dose. ¡See Warnings and Precautions (5.1, 5.2, 5.3, 5.4,5.5))

1 INDICATIONS AND USAGE

YERVOY (ipilimumab) is indicated for the treatment ofunresectab1e or metastatic melanoma. 2 DOSAGE AND ADMINISTRATION

2.1 Recommended Dosing

The recommended dose of YERVOY is 3 mg/kg administered intravenously over 90 minutes every 3 weeks for a total of four doses. 2.2 Recommended Dose Modifications

· Withhold scheduled dose ofYERVOY for any moderate immune-mediated adverse reactions or for symptomatic endocrinopathy. For patients with complete or partial resolution of adverse reactions (Grade 0-1), and who are receiving less than 7.5 mg prednisone or

2 equivalent per day, resume YERVOY at a dose of 3 mg/kg every 3 weeks until administration of all 4 planned doses or 16 weeks from first dose, whichever occurs earlier.

. Permanently discontinue YERVOY for any of the following: o Persistent moderate adverse reactions or inability to reduce corticosteroid dose to 7.5 mg prednisone or equivalent per day.

o Failure to complete full treatment course within 16 weeks from administration of first dose.

o Severe or life-threatening adverse reactions, including any of the following: . Colitis with abdominal pain, fever, ileus, or peritoneal signs; increase in stool frequency (7 or more over baseline), stool incontinence, need for intravenous hydration for more than 24 hours, gastrointestinal hemorrhage, and gastrointestinal perforation . Aspartate aminotransferase (AST) or alanine aminotransferase (ALT)::5 times the upper limit of normal or total bilirubin ::3 times the upper limit of normal . Stevens-Johnson syndrome, toxic epidermal necrolysis, or rash complicated by full thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations . Severe motor or sensory neuropathy, Guilain-Barré syndrome, or myasthenia gravis . Severe immune-mediated reactions involving any organ system (eg, nephritis, pneumonitis, pancreatitis, non-infectious myocarditis) . Immune-mediated ocular disease that is unresponsive to topical immunosuppressive therapy

2.3 Preparation and Administration

· Do not shake product.

· Inspect parenteral drug products visually for particulate matter and discoloration prior to administration. Discard vial if solution is cloudy, there is pronounced discoloration (solution may have pale yellow color), or there is foreign particulate matter other than translucent-to- white, amorphous particles.

3 Preparation of Solution

· Allow the vials to stand at room temperature for approximately 5 minutes prior to preparation of infusion.

. Withdraw the required volume ofYERVOY and transfer into an intravenous bag.

. Dilute with 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP to prepare a diluted solution with a final concentration ranging from 1 mg/mL to 2 mg/mL. Mix diluted solution by gentle inversion.

· Store the diluted solution for no more than 24 hours under refrigeration (2°C to 8°C, 36°F to 46°F) or at room temperature (20°C to 25°C, 68°F to 77°F).

· Discard partially used vials or empty vials ofYERVOY.

Administration Instructions

· Do not mix YERVOY with, or administer as an infusion with, other medicinal products.

. Flush the intravenous line with 0.9% Sodium Chloride Injection, USP or 0.5% Dextrose Injection, USP after each dose.

· Administer diluted solution over 90 minutes through an intravenous line containing a sterile, non-pyrogenic, 10w-protein-binding in-line filter. 3 DOSAGE FORMS AND STRENGTHS

50 mg/io mL (5 mg/mL). 200 mg/40 mL (5 mg/mL). 4 CONTRAINDICATIONS

None. 5 WARNINGS AND PRECAUTIONS

YERVOY can result in severe and fatal immune-mediated reactions due to T-cell activation and proliferation. (See Boxed Warning)

4 5.1 Immune-mediated Enterocolitis

In Study 1, severe, life-threatening, or fatal (diarrhea of 7 or more stools above baseline, fever, ileus, peritoneal signs; Grade 3-5) immune-mediated enterocolitis occurred in 34 (7%) YERVOY-treated patients, and moderate (diarrhea with up to 6 stools above baseline, abdominal pain, mucus or blood in stool; Grade 2) enterocolitis occurred in 28 (5%) YERVOY-treated patients. Across all YERVOY-treated patients (n=511), 5 (1%) patients developed intestinal perforation, 4 (0.8%) patients died as a result of complications, and 26 (5%) patients were hospitalized for severe enterocolitis.

The median time to onset was 7.4 weeks (range 1.6-13.4) and 6.3 weeks (range 0.3-18.9) after the initiation of YERVOY for patients with Grade 3-5 enterocolitis and with Grade 2 enterocolitis, respectively.

Twenty-nine patients (85%) with Grade 3-5 enterocolitis were treated with high-dose (240 mg prednisone equivalent per day) corticosteroids, with a median dose of 80 mg/day of prednisone or equivalent; the median duration of treatment was 2.3 weeks (ranging up to 13.9 weeks) followed by corticosteroid taper. Of the 28 patients with moderate enterocolitis, 46% were not treated with systemic corticosteroids, 29% were treated with ~40 mg prednisone or equivalent per day for a median duration of 5.1 weeks, and 25% were treated with high-dose corticosteroids for a median duration of 10 days prior to corticosteroid taper. Infliximab was administered to 5 of the 62 patients (8%) with moderate, severe, or life-threatening immune-mediated enterocolitis following inadequate response to corticosteroids.

Of the 34 patients with Grade 3-5 enterocolitis, 74% experienced complete resolution, 3% experienced improvement to Grade 2 severity, and 24% did not improve. Among the 28 patients with Grade 2 enterocolitis, 79% experienced complete resolution, 11 % improved, and 11 % did not improve.

Monitor patients for signs and symptoms of enterocolitis (such as diarrhea, abdominal pain, mucus or blood in stool, with or without fever) and of bowel perforation (such as peritoneal signs and ileus). In symptomatic patients, rule out infectious etiologies and consider endoscopic evaluation for persistent or severe symptoms.

Permanently discontinue YERVOY in patients with severe enterocolitis and initiate systemic corticosteroids at a dose of 1 to 2 mg/kg/day of prednisone or equivalent. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least one month. In

5 clinical trials, rapid corticosteroid tapering resulted in recurrence or worsening symptoms of enterocolitis in some patients.

Withhold YERVOY dosing for moderate enterocolitis; administer anti-diarrheal treatment and, if persistent for more than one week, initiate systemic corticosteroids at a dose of 0.5 mg/kg/day prednisone or equivalent. ¡See Dosage and Administration (2.2))

5.2 Immune-mediated Hepatitis

In Study 1, severe, life-threatening, or fatal hepatotoxicity (AST or ALT elevations of more than 5 times the upper limit of normal or total bilirubin elevations more than 3 times the upper limit of normal; Grade 3-5) occurred in 8 (2%) YERVOY-treated patients, with fatal hepatic failure in

0.2% and hospitalization in 0.4% ofYERVOY-treated patients. An additional 13 (2.5%) patients experienced moderate hepatotoxicity manifested by liver function test abnormalities (AST or AL T elevations of more than 2.5 times but not more than 5 times the upper limit of normal or total bilirubin elevation of more than 1.5 times but not more than 3 times the upper limit of normal; Grade 2). The underlying pathology was not ascertained in all patients but in some instances included immune-mediated hepatitis. There were insuffcient numbers of patients with biopsy-proven hepatitis to characterize the clinical course of this event.

Monitor liver function tests (hepatic transaminase and bilirubin levels) and assess patients for signs and symptoms of hepatotoxicity before each dose of YERVOY. In patients with hepatotoxicity, rule out infectious or malignant causes and increase frequency of liver function test monitoring until resolution.

Permanently discontinue YERVOY in patients with Grade 3-5 hepatotoxicity and administer systemic corticosteroids at a dose of 1 to 2 mg/kg/day of prednisone or equivalent. When liver function tests show sustained improvement or return to baseline, initiate corticosteroid tapering and continue to taper over 1 month. Across the clinical development program for YERVOY, mycophenolate treatment has been administered in patients who have persistent severe hepatitis despite high-dose corticosteroids. Withhold YERVOY in patients with Grade 2 hepatotoxicity. ¡See Dosage and Administration (2.2))

5.3 Immune-mediated Dermatitis

In Study 1, severe, life-threatening, or fatal immune-mediated dermatitis (eg, Stevens-Johnson syndrome, toxic epidermal necrolysis, or rash complicated by full thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations; Grade 3-5) occured in 13 (2.5%)

6 YERVOY -treated patients. One (0.2%) patient died as a result of toxic epidermal necro1ysis and one additional patient required hospitalization for severe dermatitis. There were 63 (12%) patients with moderate (Grade 2) dermatitis.

The median time to onset of moderate, severe, or life-threatening immune-mediated dermatitis was 3.1 weeks and ranged up to 17.3 weeks from the initiation ofYERVOY.

Seven (54%) YERVOY-treated patients with severe dermatitis received high-dose corticosteroids (median dose 60 mg prednisone/day or equivalent) for up to 14.9 weeks followed by corticosteroid taper. Of these 7 patients, 6 had complete resolution; time to resolution ranged up to 15.6 weeks.

Of the 63 patients with moderate dermatitis, 25 (40%) were treated with systemic corticosteroids (median of 60 mg/day of prednisone or equivalent) for a median of 2.1 weeks, 7 (11 %) were treated with only topical corticosteroids, and 31 (49%) did not receive systemic or topical corticosteroids. Fort-four (70%) patients with moderate dermatitis were reported to have complete resolution, 7 (11%) improved to mild (Grade 1) severity, and 12 (19%) had no reported improvement.

Monitor patients for signs and symptoms of dermatitis such as rash and pruritus. Unless an alternate etiology has been identified, signs or symptoms of dermatitis should be considered immune-mediated.

Permanently discontinue YERVOY in patients with Stevens-Johnson syndrome, toxic epidermal necrolysis, or rash complicated by full thickness dermal ulceration, or necrotic, bullous, or hemorrhagic manifestations. Administer systemic corticosteroids at a dose of 1 to 2 mg/kg/day of prednisone or equivalent. When dermatitis is controlled, corticosteroid tapering should occur

over a period of at least 1 month. Withhold YERVOY dosing in patients with moderate to severe signs and symptoms. (See Dosage and Administration (2.2))

For mild to moderate dermatitis, such as localized rash and pruritus, treat symptomatically. Administer topical or systemic corticosteroids if there is no improvement of symptoms within 1 week.

5.4 Immune-mediated Neuropathies

In Study 1, one case of fatal Guilain-Barré syndrome and one case of severe (Grade 3) peripheral motor neuropathy were reported. Across the clinical development program of

7 YERVOY, myasthenia gravis and additional cases of Guilain-Barré syndrome have been reported.

Monitor for symptoms of motor or sensory neuropathy such as unilateral or bilateral weakness, sensory alterations, or paresthesia. Permanently discontinue YERVOY in patients with severe neuropathy (interfering with daily activities) such as Guilain-Barré-like syndromes. Institute medical intervention as appropriate for management of severe neuropathy. Consider initiation of systemic corticosteroids at a dose of 1 to 2 mg/kg/day prednisone or equivalent for severe neuropathies. Withhold YERVOY dosing in patients with moderate neuropathy (not interfering with daily activities). ¡See Dosage and Administration (2.2))

5.5 Immune-mediated Endocrinopathies

In Study 1, severe to life-threatening immune-mediated endocrinopathies (requiring hospitalization, urgent medical intervention, or interfering with activities of daily living; Grade 3-4) occurred in 9 (1.8%) YERVOY-treated patients. All 9 patients had hypopituitarism and some had additional concomitant endocrinopathies such as adrenal insufficiency, hypogonadism, and hypothyroidism. Six of the 9 patients were hospitalized for severe endocrinopathies. Moderate endocrinopathy (requiring hormone replacement or medical intervention; Grade 2) occurred in 12 (2.3%) patients and consisted of hypothyroidism, adrenal insufficiency, hypopituitarism, and one case each of hyperthyroidism and Cushing's syndrome. The median time to onset of moderate to severe immune-mediated endocrinopathy was 11 weeks and ranged up to 19.3 weeks after the initiation ofYERVOY.

Of the 21 patients with moderate to life-threatening endocrinopathy, 17 patients required long-term hormone replacement therapy including, most commonly, adrenal hormones (n=lO) and thyroid hormones (n=13).

Monitor patients for clinical signs and symptoms of hypophysitis, adrenal insuffciency (including adrenal crisis), and hyper- or hypothyroidism. Patients may present with fatigue, headache, mental status changes, abdominal pain, unusual bowel habits, and hypotension, or nonspecific symptoms which may resemble other causes such as brain metastasis or underlying disease. Unless an alternate etiology has been identified, signs or symptoms of endocrinopathies should be considered immune-mediated.

Monitor thyroid function tests and clinical chemistries at the start of treatment, before each dose, and as clinically indicated based on symptoms. In a limited number of patients, hypophysitis was diagnosed by imaging studies through enlargement of the pituitary gland.

8 Withhold YERVOY dosing in symptomatic patients. Initiate systemic corticosteroids at a dose of I to 2 mg/kg/day of prednisone or equivalent, and initiate appropriate hormone replacement therapy. ¡See Dosage and Administration (2.2))

5.6 Other Immune-mediated Adverse Reactions, Including Ocular Manifestations

The following clinically significant immune-mediated adverse reactions were seen in less than I % of YERVOY-treated patients in Study I: nephritis, pneumonitis, meningitis, pericarditis, uveitis, iritis, and hemolytic anemia.

Across the clinical development program for YERVOY, the following likely immune-mediated adverse reactions were also reported with less than i % incidence: myocarditis, angiopathy, temporal arteritis, vasculitis, polymyalgia rheumatica, conjunctivitis, blepharitis, episcleritis, scleritis, 1eukocytoclastic vasculitis, erythema multiforme, psoriasis, pancreatitis, arthritis, and autoimmune thyroiditis.

Permanently discontinue YERVOY for clinically significant or severe immune-mediated adverse reactions. Initiate systemic corticosteroids at a dose of i to 2 mg/kg/day prednisone or equivalent for severe immune-mediated adverse reactions.

Administer corticosteroid eye drops to patients who develop uveitis, irtis, or episcleritis. Permanently discontinue YERVOY for immune-mediated ocular disease that is uilesponsive to local immunosuppressive therapy. ¡See Dosage and Administration (2.2)) 6 ADVERSE REACTIONS

The following adverse reactions are discussed in greater detail in other sections of the labeling.

· Immune-mediated enterocolitis ¡see Warnings and Precautions (5.1)). · Immune-mediated hepatitis ¡see Warnings and Precautions (5.2)). · Immune-mediated dermatitis ¡see Warnings and Precautions (5.3)). · Immune-mediated neuropathies ¡see Warnings and Precautions (5.4)). · Immune-mediated endocrinopathies ¡see Warnings and Precautions (5.5)). · Other immune-mediated adverse reactions, including ocular manifestations ¡see Warnings and Precautions (5.6)).

9 6.1 Clinical Trials Experience

Because clinical trials are conducted under widely varying conditions, the adverse reaction rates observed cannot be directly compared with rates in other clinical trials or experience with therapeutics in the same class and may not reflect the rates observed in clinical practice.

The clinical development program excluded patients with active autoimmune disease or those receiving systemic immunosuppression for organ transplantation. Exposure to YERVOY 3 mg/kg for four doses given by intravenous infusion in previously treated patients with unresectable or metastatic melanoma was assessed in a randomized, double-blind clinical study (Study 1). ¡See Clinical Studies (14)) One hundred thirt-one patients (median age 57 years, 60% male) received YERVOY as a single agent, 380 patients (median age 56 years, 61% male) received YERVOY with an investigational gp100 peptide vaccine (gp100), and 132 patients (median age 57 years, 54% male) received gplOO peptide vaccine alone. Patients in the study received a median of 4 doses (range 1 to 4 doses). YERVOY was discontinued for adverse reactions in 10% of patients.

The most common adverse reactions (~5%) in patients who received YERVOY at 3 mg/kg were fatigue, diarrhea, pruritus, rash, and colitis.

Table 1 presents selected adverse reactions from Study 1, which occurred in at least 5% of patients in the YERVOY-containing arms and with at least 5% increased incidence over the control gp 100 arm for all-grade events and at least 1 % incidence over the control group for Grade 3-5 events.

10 Table 1: Selected Adverse Reactions in Study 1

a Percentage (%) of Patients YERVOY YERVOY 3 mg/kg 3 mg/kg+gp 100 gpl00 n=131 n=380 n=I32 System Organ Class/ Any Grade Any Grade Any Grade Preferred Term Grade 3-5 Grade 3-5 Grade 3-5 Gastrointestinal Disorders

Diarrhea 32 5 37 4 20

Colitis 8 5 5 3 2 0 Skin and Subcutaneous Tissue Disorders

Pruritus 31 0 21 -cl 11 0

Rash 29 2 25 2 8 0 General Disorders and Administration Site Conditions

Fatigue 41 7 34 5 31 3 a Incidences presented in this table are based on reports of adverse events regardless of causality.

Table 2 presents the per-patient incidence of severe, life-threatening, or fatal immune-mediated adverse reactions from Study i.

11 Table 2: Severe to Fatal Immune-mediated Adverse Reactions in Study 1 YERVOYPercentage (%) of YERVOYPatients 3n=I3I mg/kg 3 mg/kg+gpn=380 roo Any Immune-mediated Adverse Reaction 15 12 Enteroco I. . itiSa,b 7 7 Hepatotoxicitl 2

Dermatitis a 2 3 a Neuropathy ,1 Endocrinopathy 4 Hypopituitarism 4 Adrenal insuffciency o Other Pneumonitis o ,1 Meningitis o ,1

Nephritis 1 o

Eosinophilia C 1 o

Pericarditis a,c o ,1 a Including fatal outcome. b Including intestinal perforation.

C Underlying etiology not established.

Across clinical studies that utilized YERVOY doses ranging from 0.3 to 10 mg/kg, the following adverse reactions were also reported (incidence less than 1 % unless otherwise noted): urticaria (2%), large intestinal ulcer, esophagitis, acute respiratory distress syndrome, renal failure, and infusion reaction.

Based on the experience in the entire clinical program for melanoma, the incidence and severity of enterocolitis and hepatitis appear to be dose dependent.

12 6.2 Immunogenicity

In clinical studies, 1.1 % of 1024 evaluable patients tested positive for binding antibodies against ipilimumab in an e1ectrochemiluminescent (ECL) based assay. This assay has substantial limitations in detecting anti-ipilimumab antibodies in the presence of ipilimumab. Infusion-related or peri-infusional reactions consistent with hypersensitivity or anaphylaxis were not reported in these 11 patients nor were neutralizing antibodies against ipi1imumab detected.

Because trough levels of ipilimumab interfere with the ECL assay results, a subset analysis was performed in the dose cohort with the lowest trough levels. In this analysis, 6.9% of 58 evaluable patients, who were treated with 0.3 mg/kg dose, tested positive for binding antibodies against ipilimumab.

Immunogenicity assay results are highly dependent on several factors including assay sensitivity and specificity, assay methodology, sample handling, timing of sample collection, concomitant medications, and underlying disease. For these reasons, comparison of incidence of antibodies to YERVOY with the incidences of antibodies to other products may be misleading. 7 DRUG INTERACTIONS

No formal drug-drug interaction studies have been conducted with YERVOY.

8 USE IN SPECIFIC POPULATIONS

8.1 Pregnancy

Pregnancy Category C

There are no adequate and well-controlled studies of YERVOY in pregnant women. Use

. YERVOY during pregnancy only if the potential benefit justifies the potential risk to the fetus.

In a combined study of embryo-fetal and peri-postnatal development, severe toxicities including increased incidences of third-trimester abortion, stillbirth, premature delivery, low birth weight, and infant mortality occurred following intravenous administration of ipilimumab to pregnant cynomolgus monkeys every 2 1 days from the onset of organogenesis through partrition at doses of 2.6 or 7.2 times the recommended human dose of 3 mg/kg (by AUC). (See Nonclinical Toxicology (13.2))

13 In genetically engineered mice in which the gene for CTLA-4 has been deleted (a "knockout mouse"), offspring lacking CTLA-4 were born apparently healthy, but died within 3-- weeks due to multi-organ infiltration and damage by lymphocytes.

Human IgG i is known to cross the placental barrier and ipilimumab is an IgG i; therefore, ipilimumab has the potential to be transmitted from the mother to the developing fetus.

8.3 Nursing Mothers

It is not known whether ipi1imumab is secreted in human milk. Because many drugs are secreted in human milk and because of the potential for serious adverse reactions in nursing infants from YERVOY, a decision should be made whether to discontinue nursing or to discontinue YERVOY, taking into account the importance ofYERVOY to the mother.

8.4 Pediatric Use

Safety and effectiveness ofYERVOY have not been established in pediatric patients.

8.5 Geriatric Use

Of the 511 patients treated with YERVOY at 3 mg/kg, 28% were 65years and over. No overall differences in safety or efficacy were reported between the elderly patients (65 years and over) and younger patients (less than 65 years).

8.6 Renal Impairment

No formal studies of YERVOY in patients with renal impairment have been conducted. ¡See Clinical Pharmacology (12.3))

8.7 Hepatic Impairment

No formal studies of YERVOY in patients with hepatic impairment have been conducted. ¡See Clinical Pharmacology (12.3)) 10 OVERDOSAGE

There is no information on overdosage with YERVOY.

14 11 DESCRIPTION

YERVOY (ipilimumab) is a recombinant, human monoclonal antibody that binds to the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Ipilimumab is an IgG1 kappa immunoglobulin with an approximate molecular weight of 148 kDa. Ipilimumab is produced in mammalian (Chinese hamster ovary) cell culture.

YERVOY is a sterile, preservative-free, clear to slightly opalescent, colorless to pale yellow solution for intravenous infusion, which may contain a small amount of visible translucent-to- white, amorphous ipilimumab particulates. It is supplied in single-use vials of 50 mg/lO mL and 200 mg/40 mL. Each mililiter contains 5 mg ofipilimumab and the following inactive ingredients: diethylene triamine pentaacetic acid (DTPA) (0.04 mg), mannitol (10 mg), polysorbate 80 (vegetable origin) (0.1 mg), sodium chloride (5.85 mg), tris hydrochloride (3.15 mg), and Water for Injection, USP at a pH of7. 12 CLINICAL PHARMACOLOGY

12.1 Mechanism of Action

CTLA-4 is a negative regulator ofT-cell activation. Ipilimumab binds to CTLA-4 and blocks the interaction of CTLA-4 with its ligands, CD80/CD86. Blockade of CTLA-4 has been shown to augment T-cell activation and proliferation. The mechanism of action of ipi1imumab's effect in patients with melanoma is indirect, possibly through T-cell mediated anti-tumor immune responses.

12.3 Pharmacokinetics

The pharmacokinetics of ipilimumab was studied in 499 patients with unresectable or metastatic melanoma who received doses of 0.3, 3, or 10 mg/kg administered once every 3 weeks for four doses. Peak concentration (Cmax), trough concentration (Cmin), and area under the curve (AUC) of ipilimumab were found to be dose proportional within the dose range examined. Upon repeated dosing of YERVOY administered every 3 weeks, ipilimumab clearance was found to be time- invariant, and minimal systemic accumulation was observed as evident by an accumulation index of 1.5-fold or less. Ipilimumab steady-state concentration was reached by the third dose. The following mean (percent coeffcient of variation) parameters were generated through population pharmacokinetic analysis: terminal half-life of 14.7 days (30.1 %); systemic clearance (CL) of 15.3 mLih (38.5%); and volume of distribution at steady-state (Vss) of 7.21 L (10.5%). The

I5 mean (:tSD) ipilimumab Cmin achieved at steady-state with the 3-mg/kg regimen was 21.8 mcg/mL (:tll.2).

Specifc Populations: Cross-study analyses were performed on data from patients with a variety of conditions, including 420 patients with melanoma who received single or multiple infusions of YERVOY at doses of 0.3, 3, or 10 mg/kg. The effects of various covariates on ipilimumab pharmacokinetics were assessed in population pharmacokinetic analyses.

Ipilimumab CL increased with increasing body weight; however, no dose adjustment of YERVOY is required for body weight after administration on a mg/kg basis. The following factors had no clinically meaningful effect on the CL ofipilimumab: age (range 26 to 86 years), gender, concomitant use of budesonide, performance status, HLA-A2*0201 status, positive anti-ipilimumab antibody status, prior use of systemic anticancer therapy, or baseline lactate dehydrogenase (LDH) levels. The effect of race was not examined as there were insuffcient

numbers of patients in non-Caucasian ethnic groups.

Renal Impairment: Creatinine clearance at baseline did not have a clinically important effect on ipi1imumab pharmacokinetics in patients with calculated creatinine clearance values of 29 mL/min or greater.

Hepatic Impairment: Baseline AST, total bilirubin, and ALT levels did not have a clinically important effect on ipi1imumab pharmacokinetics in patients with various degrees of hepatic impairment. 13 NONCLINICAL TOXICOLOGY

13.1 Carcinogenesis, Mutagenesis, Impairment of Fertilty

Carcinogenesis

The carcinogenic potential of ipilimumab has not been evaluated in long-term animal studies. Mutagenesis

The genotoxic potential of ipilimumab has not been evaluated. Impairment of Fertilty

Fertility studies have not been performed with ipilimumab.

I6 13.2 Animal Toxicology and/or Pharmacology

The effects of ipi1imumab on prenatal and postnatal development in monkeys have not been fully investigated. Preliminary results are available from an ongoing study in cynomolgus monkeys. Pregnant monkeys received ipilimumab every 21 days from the onset of organogenesis in the first trimester through delivery, at dose levels either 2.6 or 7.2 times higher than the clinical dose of 3 mg/kg of ipi1imumab (by AUC). No treatment-related adverse effects on reproduction were detected during the first two trimesters of pregnancy. Beginning in the third trimester, the ipilimumab groups experienced higher incidences of abortion, stilbirth, premature delivery (with corresponding lower birth weight), and higher incidences of infant mortality in a dose-related manner compared to controls.

Genetically engineered mice heterozygous for CTLA-4 (CTLA-4+/-), the target for ipilimumab, appeared healthy and gave birth to healthy CTLA-4+/- heterozygous offspring. Mated CTLA-4+/- heterozygous mice also produced offspring deficient in CTLA-4 (homozygous negative, CTLA-4-/-). The CTLA-4-/- homozygous negative offspring appeared healthy at birth, exhibited signs of multiorgan lymphoproliferative disease by 2 weeks of age, and all died by 3-4 weeks of age with massive 1ymphoproliferation and multiorgan tissue destruction.

14 CLINICAL STUDIES

The safety and efficacy of YERVOY were investigated in a randomized (3:1:1), double-blind, double-dummy study (Study 1) that included 676 randomized patients with unresectable or metastatic melanoma previously treated with one or more of the following: aldes1eukin, dacarbazine, temozolomide, fotemustine, or carboplatin. Of these 676 patients, 403 were randomized to receive YERVOY at 3 mg/kg in combination with an investigational peptide vaccine with incomplete Freund's adjuvant (gplOO), 137 were randomized to receive YERVOY at 3 mg/kg, and 136 were randomized to receive gplOO alone. The study enrolled only patients with HLA-A2*020l genotype; this HLA genotype facilitates the immune presentation of the investigational peptide vaccine. The study excluded patients with active autoimmune disease or those receiving systemic immunosuppression for organ transplantation. YERVOY/placebo was administered at 3 mg/kg as an intravenous infusion every 3 weeks for four doses. Gp 1 OO/placebo was administered at a dose of 2 mg peptide by deep subcutaneous injection every 3 weeks for four doses. Assessment of tumor response was conducted at weeks 12 and 24, and every 3 months thereafter. Patients with evidence of objective tumor response at 12 or 24 weeks had assessment for confirmation of durability of response at 16 or 28 weeks, respectively.

17 The major efficacy outcome measure was overall survival (OS) in the YERVOY+gplOO arm compared to that in the gp 1 00 arm. Secondary efficacy outcome measures were OS in the YERVOY+gplOO arm compared to the YERVOY arm, OS in the YERVOY arm compared to the gplOO arm, best overall response rate (BORR) at week 24 between each of the study arms, and duration of response.

Of the randomized patients, 61%, 59%, and 54% in the YERVOY+gplOO, YERVOY, and gplOO arms, respectively, were men. Twenty-nine percent were 265 years of age, the median age was 57 years, 71% had Mlc stage, 12% had a history of previously treated brain metastasis, 98% had ECOG performance status of 0 and 1, 23% had received aldesleukin and 38% had elevated LDH leveL. Sixty-one percent of patients randomized to either YERVOY-containing arm received all 4 planned doses. The median duration of follow-up was 8.9 months.

The OS results are shown in Table 3 and Figure 1.

Table 3: Overall Survival Results

YERVOY YERVOY+gpIOO gplOO n=137 n=403 n=136 Hazard Ratio (vs. gpl00) 0.66 0.68 (95% CI) (0.5 I, 0.87) (0.55,0.85) a p-value p=0.0026 p=0.0004 Hazard Ratio (vs. YERVOY) 1.04

(95% CI) (0.83, 1.30) Median (months) 10 10 6 (95% CI) (8.0, 13.8) (8.5, 1 I.) (5.5,8.7) a Not adjusted for multiple comparisons.

18 Figure 1: Overall Survival

1.0 0.9 w 0.8 , L_, :: L, 1 -- 0.7 , oc i z 0.6 ,, o ,\ ~ 0.5 '.. L~ o 0.4 ,, a. " ~ 0.3 1',., ,~ a. 0.2 "Ð_ ---~-x-~ L.. ¡g""-'--L-_-"' - - - - - ~)&- ~* -+ - - -x- - - -)E)o ------x 0.1 '--_ -G- - - -.g - -G- - -- Ð 0.0

o 4 8 12 16 20 24 28 32 36 40 44 48 52 56

SUBJECTS AT RISK MONTHS Ipi+gp100 403 297 223 163 115 81 54 42 33 24 17 7 6 4 0 Ipi 137 106 79 56 38 30 24 18 13 13 8 5 2 1 0 gp100 136 93 58 32 23 17 16 7 5 5 3 1 0 o 0 - Ipi+gp100 - - - - Ipi --- gp100 (' (' (' CENSORED x x x CENSORED o 0 0 CENSORED

The best overall response rate (BORR) as assessed by the investigator was 5.7% (95% CI: 3.7%, 8.4%) in the YERVOY+gp100 arm, 10.9% (95% CI: 6.3%, 17.4%) in the YERVOY arm, and

1.5% (95% CI: 0.2%, 5.2%) in the gp100 arm. The median duration of response was 11.5 months in the YERVOY+gp100 arm and has not been reached in the YERVOY or gp100 arm. 16 HOW SUPPLIED/STORAGE AND HANDLING

YERVOY is available as follows:

Carton Contents NDC One 50 mg vial (5 mg/mL), single-use vial NDC 0003-2327-I 1 One 200 mg vial (5 mg/mL), single-use vial NDC 0003-2328-22

Store YERVOY under refrigeration at 2°C to 8°C (36°F to 46°F). Do not freeze. Protect vials from light.

I9 17 PATIENT COUNSELING INFORMATION

See MEDICATION GUIDE.

· Inform patients of the potential risk of immune-mediated adverse reactions.

· Advise patients to read the YERVOY Medication Guide before each YERVOY infusion.

· Advise women that YERVOY may cause fetal harm.

· Advise nursing mothers not to breast-feed while taking YERVOY.

Manufactured by: Bristol-Myers Squibb Company Princeton, NJ 08543 USA U.S. License NO.1 713

20 MEDICATION GUIDE

YERVOy™ (yur-voi) (ipilimumab)

Read this Medication Guide before you start receiving YERVOY and before each infusion. There may be new information. This Medication Guide does not take the place of talking with your healthcare provider about your medical condition or your treatment.

What is the most important information I should know about YERVOY?

YERVOY can cause serious side effects in many parts of your body which can lead to death. These side effects are most likely to begin during treatment; however, side effects can show up months after your last infusion.

These side effects may include: 1. Inflammation of the intestines (colitis) that can cause tears or holes (perforation) in the intestines. Signs and symptoms of colitis may include: · diarrhea (loose stools) or more bowel movements than usual · blood in your stools or dark, tarry, sticky stools · stomach pain (abdominal pain) or tenderness

2. Inflammation of the liver (hepatitis) that can lead to liver failure. Signs and symptoms of hepatitis may include: · yellowing of your skin or the whites of your eyes · dark urine (tea colored) · nausea or vomiting · pain on the right side of your stomach · bleeding or bruise more easily than normal

3. Inflammation of the skin that can lead to severe skin reaction (toxic epidermal necrolysis). Signs and symptoms of severe skin reactions may include: · skin rash with or without itching · sores in your mouth · your skin blisters and/or peels

21 4. Inflammation of the nerves that can lead to paralysis. Symptoms of nerve problems may include: · unusual weakness of legs, arms, or face . numbness or tingling in hands or feet

S. Inflammation of hormone glands (especially the pituitary, adrenal, and thyroid glands) that may affect how these glands work. Signs and symptoms that your glands are not working properly may include: . persistent or unusual headaches · unusual sluggishness, feeling cold all the time, or weight gain · changes in mood or behavior such as decreased sex drive, irritability, or forgetfulness . dizziness or fainting

6. Inflammation of the eyes. Symptoms may include: . blurry vision, double vision, or other vision problems . eye pain or redness Call your healthcare provider if you have any of these signs or symptoms or they get worse. Do not try to treat symptoms yourself.

Getting medical treatment right away may keep the problem from becoming more serious. Your oncologist may decide to delay or stop YERVOY.

What is YERVOY? YERVOY is a prescription medicine used in adults to treat melanoma (a kind of skin cancer) that has spread or cannot be removed by surgery.

It is not known if YERVOY is safe and effective in children less than 18 years of age.

What should I tell my healthcare provider before getting YERVOY?

Before you are given YERVOY, tell your healthcare provider about all your health problems if you: · have an active condition where your immune system attacks your body (autoimmune disease), such as ulcerative colitis, Crohn's disease, lupus, or sarcoidosis . had an organ transplant, such as a kidney transplant . have liver damage from diseases or drugs . have any other medical conditions · are pregnant or plan to become pregnant. YERVOY may cause stillbirth, premature delivery, and/or death of your unborn baby . are breast-feeding

22 Tell your healthcare provider about all the medicines you take, including all prescription and non-prescription medicines, steroids or other medicines that lower your immune response, vitamins, and herbal supplements. Know the medicines you take. Keep a list to show your doctors and pharmacists each time you get a new medicine. You should not start a new medicine before your talk with the healthcare provider who prescribes you YERVOY.

How wil I receive YERVOY? You will get YERVOY through an intravenous line in your vein (infusion). It takes about 90 minutes to get a full dose. · YERVOY is usually given every 3 weeks for up to 4 doses. Your healthcare provider may change how often you receive YERVOY or how long the infusion may take. · Your healthcare provider should perform blood tests before starting and during treatment with YERVOY.

It is important for you to keep all appointments with your healthcare provider. Call your healthcare provider if you miss an appointment. There may be special instructions for you. What are the possible side effects of YERVOY? YERVOY can cause serious side effects. See "What is the most important information I should know about YERVOY?" The most common side effects of YERVOY include: . tiredness . diarrhea . itching . rash

These are not all of the possible side effects of YERVOY. For more information, ask your healthcare provider.

Call your healthcare provider for medical advice about side effects. You may report side effects to FDA at 1-800-FDA-1088.

You may also report side effects to Bristol-Myers Squibb at 1-800-721-5072.

23 General information about the safe and effective use of YERVOY. Medicines are sometimes prescribed for purposes other than those listed in a Medication Guide.

This Medication Guide summarizes the most important information about YERVOY. If you would like more information, talk with your healthcare provider. You can ask your healthcare provider for information about YERVOY that is written for healthcare professionals.

For more information, call 1-800-321-1335.

What are the ingredients of YERVOY?

Active ingredient: ipilimumab Inactive ingredients: diethylene triamine pentaacetic acid (DTPA), mannitol, polysorbate 80, sodium chloride, tris hydrochloride, and Water for Injection, USP This Medication Guide has been approved by the U.S. Food and Drug Administration.

Manufactured by: Bristol-Myers Squibb Company Princeton, NJ 08543 USA U.S. License Number 1713

Bristol-Myers Squibb Company Princeton, NJ 08543 USA

1281558 Issued: March 2011

24 ~~\\iìt4 ~si'Wc:.

( g~t DEPARtivf'Nt OF lIALta AND HUlAN SERVICES or'. '" ,....l Food and Drug Adiniùstration Silver Spring MD. 20993

Our STN: BL 125377/0 BLA APPROVAL March 25,2011

Bristol-Myers. Squibb Company Attention: A. Heather Knight-Trent, Pharm Director-Onco10 gy 5 Research Parkway Wallingford, CT 06492-7660

Dear Dr. Knight-Trent:

Please refer to your Biologics LicenseApplication (BLA) dated June 25,2010, received June 25, 2010, submitted under section 351 of the Public Health Service Act for YERVOY (ipilimumab).

We acknowledge receipt of all subsequent amendments received through March 24, 2011.

We have approved your BLA for ipilmumab effective this date. You are hereby authoried to introduce or deliver for introduction jnto interstate commerce, ipi1imumab, under your existing

Department of Health and Human Services U.S. License No. 1713. Ipilimumab is indicated for the treatment ofunresectable or metastatic melanoma.

Under this lic~nse, you are approved to manufactureipilmuinab.clrug.substance at Lonza Biologics, Incorporated at Portsmouth,N ew Hampshire. The fial formulated product will be

inanufactured, filed, labeled and packaged at B~xter Pharmaceutical Solutions,LLC at B1oominRì0n, Indiana. You may labeLyour productwith the proprietary name YERVOY and will market it in 50mg/10 roand 200 mgl40 rnL single-use vials.

Yourapplicationforipilmumabwas not referred to an FDA advisory committee because outside expertise was not necessary; tberewere no controversial issues that'Youldbenefit from advisory cotnittee. discussion.

The dating period for ipilmumab shall be 36 months from the date of manufacture when stored at 2-8 DC, but should not exceed 48 months from the date of drug substance manufacture. The date of drug product manufacture shall be defied as the date of fial sterile filtration of the formulated drug product. The dating period for your drug substance shall be 36 months from the date of manufacture when stored at 2-8 DC. The expiration date for the packaged product, ipilimumab single-use vials, shall be dependent on the shortest expiration date of any component.

We have approved the stability protocols in your license application for the purpose of extending

the expiration dating period of your drug substance and drug product under 21 CFR 601.12. BL 125377/0 Page 2

You are not currently required to submit samples of future lots of ipilimumab to the Center for

Drug Evaluation and Research (CDER) for release by the Director, CDER, under 21 CFR 610.2. We wil contînue to monitor compliance with 21 CFR 610.1, requiring completion oHests for conformty with standards applicable to each product prior to release ofeach lot.

Any changes in the manufacturing,.testing, packaging, or labeling ofipilmumab, or in the manufacturing facilties, wil require the submission of information to your biologics license application for our review and written approval, consistent with 21 CFR 601.12.

We are approving this application for use as recommended in theenclos.ed agreed-upon labeling text.

CONTENT OF LABELING

As soon as possible, but flO later than 14 days from the date of this letter, submít, via the FDA automated drug registration and listing system (eLIST), the content oflabeling (21 60L.14(b)) in structured product labeling (SPL) format, as described at http://www.fda.gov/ForIndustry/DataStandards/StructuredProductLaheling/default.htm. that is identical totheenclosed labe1in& (text forthe package insert, MedicationGuide). Information on submitting SPLfiles using eLIST may be found in the guidance for industry titled "SPL

Standard for Content of Labeling TechnicalQs and As" at http://www . fda.gov/down10ads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/U CM072392.pdf. For administrative purposes, please designate this. submission "Product Correspoiidence- Final SPL for approvedBLA STN 125377/0."

The SPL wil be accessible via publicly available labeling repositories.

CARTON AND IMMEDIATE CONTAINER LABELS

Submit fial printed caron and container labels that are identical to the enclosed caron and immediate container labels and carton and immediate container labels submitted on March 1 1, 2011 as soon as they are available, but no more than 30 days after they are printed. Please submit these labels electronically according to the guidance for industry titled "Providing Regulatory Submissions in Electronic Format - Human Pharmaceutical Product Applications and Related Submissions Using the eCTD Specifications (June 2008)". Alternatively, you may submit 12 paper copies, with 6 0 f the copies individually mounted on heavy- weight paper or similar materiaL. For admiistrative purposes, designate this submission "Product Correspondence - Final Printed Carton and Container Labels for approved BLA STN 125377/0." Approval of this submission by FDA is not required before the labeling is used.

Marketing the product with fial printed labeling (FPL) that is not identical to the approved labeling text may render the product misbranded and an unapproved new drug. BL 125377/0 Page 3

Under the Pediatric Research Equity Act (PREA)(21 U.S.C. 355c), all applications for new active ingredients;.new indications, new dosage form, new dosing regimens, or new routes of admiistraIionare required to contain an assessment of the safety and effectiveness of the product for the claimed indication in pediatric patients unless this requirement is :waived, ... deferred, or iìapplicable.

Because this drug product for this indication has an orpha.drug de~ignation, You are exempt from this requirement.

POSTMARKETING.REQUIREMENTS. UNDERSOS(o)

Section 505(0)(3) of the FederalFood, Drug, and Cosmetic Act (FDCA) authories FDA to require holders ofapproved drug and biological product applications to cönduct pOstmarketii studies aiid.clinical trials for certain purposes, if FDA makes certain. fidings required hythe

. statute.

We have deterned that an analysis 0 f spoiitC\eousPQstmarketing adverse events reported. . under subsection 505(k)(1) of the FDCA wil not be suffcient to identìfy an unexpected serious risk of embryo-fetal toxicity or anti:.drug antibody responses.

. Furthemore, the neW pharnüicovigi1ance system that FDA is requifëd to establish undèr sectio:i 505(k)(3) of the FDCA is not yet suffcient to assess these serious risks. .

Therefore, based on appropriate.scientific.data, FDA has determined that you are required to. conduct the following: .

1. . To subint the final report for study DN120Q20(Intravenous StlclY of Pre-and Post~natal Developmentàl in Cynomolgus Monkeys with a 6-Month Post-natal Evaluation). .

The. timetable you sübmitted on March 14; 2011, states that you wilconductìhis study acoordingtÖthe follöwing schèdule:. ..

. . Fiìal Report Submission: December 31, 2011

2. To develop a validated, sensitive, and açcurate assay for the detection ofbmding antibodies to ipilimuma1J, including procedures foqiccurate detection of antibodies to ipiliniumabinthe presence of ipilimumab levels that are expected to be present in the serum or plasma at the time of patient sampling.

The tìmetable you submitted on Mätch 14,2011, statèS that yoU wil conduct this assay

according to the following schedule: .

Final Report Submission (Assay and Methodology): December 2;2011 BL 125377/0 Page 4

3. To develop a validated, sensitive, and accurate assay for the detection of neutralizing antibodies to ipilmumab, including procedures for accurate detection of neutralizing antibodies to ipilimumab in the presence ofipilmumab levels that are expected to be present in the serum or plasma at the time of patient sampling. In the event such an assay can not be

developed, evidence of due diligence in attempting to develop the assay wil be provided.

The timetable you submitted on March 14, 2011, states that you wil conduct this assay according to the following schedule:

Final Report Submission (Assay and Methodology): February 20,2012

Finally, we have determed that only a clinical trial (rather than a noncliical or observational study) wil be sufficient to address the following:

. Identify unexpected serious risk of anti-drug antibody responses; · Assess a signal of serious risk of immune-mediated adverse reactions associated with CD86 gene po1ymorphisms;

. Assess a known serious risk of fatal and life-threatenig imune-mediated adverse reactions Therefore, based on appropriate scientific data, FDA has determed that you .are requied, to conduct the following:

4. To conduct an assesSment of anti-drug antibody (ADA) response and neutralizing ADA responses to ipilimumab with a validated assay (required in PMR 2 and 3) capable of sensitively detecting ADA responses in the presence of ipilimumab levels that are expected to be present at the time ofpatient sampling. The ADA response wil be evaluated in at least 300 ipilimumab-treated patients enrolled in the required postmarketing trial (PMR 6) comparing 3 ing versus 10 mgkg of ipilimumab monotherapy. The fial report wil include information on the level ofipilimumab in each patient's test sample at each sampling time point.

The timetable you submitted on March 14, 2011, states that you will conduct this assessment from clinical trial data according to the following schedule:

Final Protocol Submission: September 30, 2011

Patient Accrual Completed December 31, 2014 Trial Completion Date: August 31, 2017 Final Report Submission: December 29, 2017 BL 125377/0 Page 5

5. During the conduct of the required postmarketing trial comparing 3mg/g vs. 10mg/kg ipilimumab mono therapy (PMR 6), you wil obtain comprehensive baseline DNA sample acquisition ( ~5% ofITT) and conduct pharmacogenomic association analyses to assess the potential clinical utility ofCD86 gene polymorphisms as genetic determants of imune mediated adverse events. You wil provide a protocol that addresses SNP selection, data analyses approaches, and other methodological issues. You wil provide a Final Report including electronic datasets.

\ The timetable you submitted on March 14, 201 1, state~ that you wil conduct this assessment from clinical trial data according to the following schedule:

Draft Protocol Submission: November 30,2011

Final Protocol Submission: May 30, 2012 Final Report Submission: December 29,2016

6. Following the assessment of data from Trial CA184024, you wil design and conduct a trial to compare the effcacy, with the primar endpoint of overall survival and the safety of ipilimumab at doses of 3mg/kg versus 10mg/g given as monotherapy every three weeks for four doses in patients with unesectable Stage III or Stage iv melanoma. (

The timetable you submitted on March 14, 2011, states that you wil conductthis trial according to the following schedule:

Preliinar CA184024 Data Submission: June 30, 2011 Draft Protocol Synopsis Submission: June 30, 2011 Final Protocol Submission: September 30, 2011 First Patient Accrued to Trial: March 30, 2012 Last Patient Accrued to Trial: December 31, 2014 Trial Completion: August, 31,2017 Final Report Submission: December 31, 2017

Submit protocols to your IND, with a cross-reference letter to this BLA. Submit all fial reports to your BLA. Promiently identity the submission with the following wording in bold capital letters at the top ofthe fist page ofthe submission, as appropriate:

. REQUIRED POSTMARKETING PROTOCOL UNDER 505(0) . REQUIRED POSTMARKETING FINAL REPORT UNDER 505(0) . REQUIRED POSTMARKTING CORRSPt?NDENCE UNDER 505(0)

Section505(0)(3)(E)(ü) of the FDCAtequires you to report perodically on the status of any study or clinical trial required under this section. This section also requires you to periodically reprt to FDA on the status of any study or clinica1 trial otherwise undertaken to investigate a safety issue. Section 506B ofthe FDCA, as well as 21 CFR 601.70 requires you to report anually on the status of any postmaketing coniitments or required studies or clincal trials. BL 125377/0 Page 6

. FDA wil consider the submission of your anual report under section 5068 and 21 CFR 601.70

to satisfy the periodic reporting requirement under section 505(0)(3)(E)(ii) provided that you include the elements listed in 505(0) and 21 CFR 601.70. We remid you that to comply with 505(0), your anual report must also include a report on the status of any study or clinical trial otherwise undertaken to investigate a safety issue. Failure to subipt an anual report for studies or clinical trials required under 505(0) on the date required wil be considered a violation of FDCA section 505(0)(3)(E)(ii) and could result in enforcement action.

POSTMARKETING COMMITMENTS SUBJECT TO THE REPORTING REQUIRMENTS UNDER SECTION 506B

We remid you of your postmarketing comritments:

7. To identify further genetic determants ofimnune-mediated adverse events caused by ipilimumab. DNA samples from the required postmarketing study comparing 3mg/kg vs. 10 mg/g ipilmumab mono therapy wil be used to conduct genome-wide association analyses.

The design of these analyses wil be reviewed by FDA and a fial report with e1ectroriic datasets wil be provided.

The timetable you submitted on March 14, 2011, states that you wil conduct thi study according to the following schedule:

Draft Protocol Submission: December 29,2016 Final Protocol Submission: July 31,2017 Final Report Submission: December31,2018

POSTMARKETING COMMITMENTS NOT SUBJECT TO THE REPORTING REQUIRMENTS UNDER SECTION506B

We remid you of your postmarketing commitments:

8. To develop and validate a semi-quantitative assay to evaluate visible particulates in drug product. The assay wil be incorporated into the drug product release and stability testing programs. The fial validation report with the specifications and method validation wil be submitted as a CBE-30 supplement by May 30, 2011.

The timetable you submitted on March 14, 201 1, states that you wil conduct this study according to the following schedule:

Final Report Submission as a CBE-30 supplement: May 30,2011 BL 125377/0 Page 7

9. To replace the IEF assay with the CEX assay for the release of drug product after suffcient data has been acquired to support establishment of CEX acceptance criteria. The fial study report wil be submitted as a CBE-30 by June 30, 2011.

The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

Final Report Submission as a CBE-30 supplement: June 30, 2011

10. To discontinue the IEF method as a specification for charge in the drug substance and drug product stabilty programs after three years of market life data are collected for the CEX assay on three batches of drug substance and three batches of either presentation of drug

product. The fial results and proposed CEX specification wil be submitted as a CBE-30 supplement by March 31, 2014.

The timetable you submitted on March 14,2011, states that you wil conduct this study according to the following schedule:

Final Report Submission as a CBE-30 supplement: March 31,2014

11. To perform studies to confirm that clearance of Antifoam C is well controlled by the manufacturing process and provide a risk assessment for residual amounts that may be present in the drug product. The fial report wil be submitted as a CBE-O supplement by July 29, 2011.

The timetable you submitted on March 14,2011, states that you wil conduct this study according to the followlig schedule:

Final Report Submission as a CBE-30 supplement: July 29, 2011

12. To develop and validate a process-specific host cell protein (HCP) ELISA. This assay wil replace the current Cygnus Kit ELISA being used in the drug substance release program. The fial study and validation reports wil be submitted as a CBE-30 supplement by November 30, 2011. .

The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

Final ReportN alidatIon Report Submission as a CBE-30 supplement: November 30, 2011 BL 125377/0 Page 8

13. To reassess release and stabilty specifications for ipilmumab drug substance and drug product through April 30, 2013. The assessment wil be submitted in the 2013 Anual Report.

The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

Final Report Submission (Annual Report): May 2013

14. To submit the fial study reports for studies performed to confi product stability over the course bfthein-process hold times of14 days at 2-8°C and 72 hours at 22-28°C. Final study results wil be submitted in the 2012 Anual Report.

The timetable you submitted on March 14, 2011, states that you wil conduct thi study according to the following schedule:

Final Report Submission (Animal Report): May 2012

15. To submit the fial concurrent column life-time study reports for the Poros 50RS, Q- AnualSepharose and CRT Type II columns.Report. The fial report wil be submitted - in the 2013 The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

Final Report Submission (Anual Report): May 2013

16. To submit the fial study reports for the drug substance storage container leachate studies to assess the volatile organic compounds (VOC), semi- VOC, non- VOC and trace metals in drug substance and formulation buffer samples held at 2 to 8°C for up to 3 years and under accelerated aging conditions 0 f 40°C to simulate 3 years at 2 to 8°C. Final reports wil be submitted in the 2013 Anual Report.

The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

FinalReport Submission (Anual Report): May 2013

17. To re-assess the bioburden action limits for the purification in-process internediates based on the manufacturing scale data from 30 lots using a 10 ni sample volume and submit the summary report in a CBE-O supplement by March 31, 2013. BL 125377/0 Page 9

The timetable you submitted on March 14, 2011, states that you wil conduct this study according to the following schedule:

Final Report Submission as a CBE-O supplement; March 31, 2013

18. To develop and implement a container closure integrity test to replace the sterility test in the stabilty program. The ability of a container closure system to maintain the integrity

of its microbial barier and hence the sterility ofa drug product throughout its shelf-life should be demonstrated. Submit the summary report and data in a CBE-O supplement by December 201 L.

The timetable you submitted on March 14,2011, states that you wil conduct this study according to the following schedule:

Final Report Submission as a CBE-O supplement: December 31,2011

Submit clinical protocols to your IND 9186 for this product. Submit nonclinica1 and chemistry, manufacturing, and controls protocols and all fial reports to this BLA. In addition, under 21 CFR 601.70 you should include a status summary of each commitment in your anual progress report ofpostmarketing studies to this BLA. The status summary should include expected summary completion and fial report submission dates, any changes in plans since the last anual report, and, for clinical studies/trials, number of patients entered into each study/triaL. All submissions, including supplements, relating to these postmarketing commitments should be promiently labeled "Postmarketig Commitment Protocol," "Postmarketing Commtment Final Report," or "Postmarketing Commitment Correspondence."

RISK EVALUATION AND MITIGATION STRATEGY REQUIREMENTS

Section 505-1 of the FDCA authories FDA to require the submission ofa risk evaluation and mitigation strategy (REMS), if FDA determes that such a strategy is necessary to ensure that the benefits of the drug outweigh the risks (section 505-1 (a)). . In accordance with section 505-1 ofFDCA, we have determed that a REMS is necessary for YERVOY (ipi1imumab) to ensure the benefits ofthe drug outweigh the risks ofsevere and fatal immune-mediated adverse reactions such as fatal immune-mediated enterocolitis (including gastrointestinal perforation), fatal immune-mediated hepatitis (including hepatic failure), fatal immune-mediated toxicities of the skin (including toxic epidermal necroiysis), fatal nervous system toxicity, and endocrinopathies, associated with the use ofYERVOY (ipi1imurnab).

We have determed that a communication plan targeted to healthcare providers is necessary to support implementation of the REMS. BL 125377/0 Page 10

Your proposed REMS, submitted on June 25, 2010, as amended, and appended to this letter, is approved. The REMS. consists of a communication plan and a timetable for submission of assessments ofthe REMS. The REMS assessment plan should include but is not limited to the following:

a. An evaluation of health care providers' (HCPs) understanding of the serious risks of YERVOY (ipi1imumab) and the management ofthe imune-mediated adverse reactions caused byYERVOY.

b. With regard to assessment ofthe communication plan:

i The date ofproduct launch and the launch of the communication plan.

ii The date(s) of mailing and number of recipients of the Dear Hea1thcare Provider (DHCP) letter and the communication package. iii The number of mailings returned. iv The sources of the recipient lists.

v The number of new prescrbers prescribing YERVOY (ipilmumab) /new facilities purchasing YERVOY (ipilmumab) durig the reporting period. Of the new prescribers/purchasers, the number supplied with the communication materials withi the required timeframe;the number not supplied with communication materüils within the required time frame; the reasons for the failure to deliver communication materials within the required timeframe.

c. Based on the information submitted, an assessment of and conclusion regarding whether the REMS is meeting its goals, and whether modifications' to the REMS are needed.

d. Specification of measures that would be taken to increase awareness ifsurveys ofHCPs indicate that provider awareness is not adequate. e. An analysis ofpost-marketing cases of imune-mediated adverse events reported for YERVOY that result in the patient's death, including an analysis ofthe length and

reasons for any reported delay in recognition and treatment of the events.

f Information on the status of any post-approval study or clinical trial required under section 505(0) or otherwise undertaken to investigate a safety issue. With respect to any such post-approval study, you must include the status of such study, inchiding whether any difficulties completing the study have been encountered. With respect to any such post-approval c1inicà1 trial, you must include the status of such cliical trial, including whether enrollment has begun, the number of paricipants enrolled, the expected completion date, whether any diffculties completing the clinical trial have been encountered, and registration information with respect to requirements under subsections (i) and CD ofsection 402 ofthe Public Health Service Act. You can satisfy these. requirements in your REMS assessments by refering to relevant information included in the most recent anual report required under section 506B and 21 CFR 601.70 and including any material or significant updates to the status information since the anual

report was prepared. Failure to comply with the REMS assessments provisions in section 50S-I(g) could result in enforcement action. . BL 125377/0 Page 11

Submit thé methodology and survey instrument(s) for review at least 90 days before the next evaluation is conducted. Submit both methodsand intruments together.

We remind. you that in addition to the assessments submîtted according to the timetable included

in the approved REMS,you must submit aREMS assessment and may propose a modification to theapprovedREMS when you submit a supplemental application for anew indication for use as described in section 505-1 (g)(2)(A) of the FDCA.

Proinent1yidentifythe subinssion containing the REMS assessments or proposed

. modifications with the following wording in bold capital letters atthe top ofthe fist page ofthe submission:

BLA125377 REMS ASSESSMENT

NEW SUPPLEMENT FORBLA 125377 PROPOSED REMS MODIFICATION REMS ASSESSMENT

NEW SUPPLEMENT (NEW INDICATION FOR USE) . FORBLA 125377 . .REMS ASSESSMENT PROPOSED REMS MODIFICATION (ifiiCllided) .

If you do not submit electronically, please send 5 copies ofREMS-reiated submissions.

REPORTING REQUIREMENTS

You mustsiibmit adverse exp~rience reports underthe adverse experìence reporting requirements for licensed biological products (21 CFR 600.80). You should submit postmarketing adverse experience reports to:

Food and Drug Adinistratioii Center for Drg Evaluation and Research Central Document Roòm. 5901-BAmmendale Road. . Beltsvile, MD 20705-1266 Promiently identify all adverse experience. reports as '..described ìn 21 CFR. 600.80~ The MedWatch:'to-Manufacturer Program provides manufacturers with copies of serious adverse event reports that are received directly by the FDA. New.mo1ecular.entities.and important new. · biologics qualify for inclusion for three years after approval: Youtfi is eligible to receive

copies of reports for this product. To participate in the program, please seethe enrollment

instructions and program description details at http://ww.fda.gov/SafetylMedWatchIowToReportucm166910.htm. . BL 125377/0 Page 12

Yon must submit distribution reports under the distribution reporting requìrements for licensëd bio10gicalproducts(21 CFR600.81). .

. You must submit reports of biological product deviations under 21 CFR 600.14. You should

promptly identify and investigate all manufacturing deviations, including those associated with

processing, testin, packing, labellg, storage, holding and distribution. Ifthe deviation involves a distributed product, may affect the safety, purity, or potency of the product, and meets the other

criteria in the regulation, you must submit a report önForm FDÁ-3486 to:

Food and Drug Admiistration

Center for Drg Evaluation and Research

Division of Compliance Risk Management and Surveilance 5901-B Airêndale Road Be1tsvile,'MD 20705~1266

Biological product deyiations,sent by courier orovemight mail, should be addressed to:

. Food andprug Admiistration CeJiter for Drug Evaluation aId Research Division of Compliance Risk Management and Surveilance

10903 New Hampshire Avenue, Bldg. 51; Room 4206 .Silver.Sprig, MD. 20903.

PROMOTIONAL MATERIALS

You may request advisory comments on proposed introductory advertising and promotional labeling. To do so, submit, in triplicate, a cover letter requesting advisQry comments, the. . proposed materials in draft or mock-up form with anotated references, and the package msert. to:

Food and Drug Admiistration Center tor Drug Evaluation and Research Divisionöf Drug Matketing; Adverisìig, and Cöìnurueations 5901-B"Amendale Road . Beltsvile,MD 20705-1266

You must submit :fal promotional materials, and the package inert, at the time of initial dissemiation or publication, accompanied by a FOrm FDA 2253.' For .ìntrtction on' completing the Fomi FDA2253, seepage 2 ofthe Fonn.Formor~inforiation aboiitsubmi~sion of promotional materials to the Division of Drug Marketing, Advertising, and Communications (DDMAC), see.http://Ww.fda.govl AboutFDNCentersOffces/CDERlucm090 142:htm.

. . All promotiönal chi.ims must be consistëiit with and not COiitrar to. approved 1a:beling~ . You

should I)ot make a comparative promotional claim or c1aimofsuperiority oVer other products.

unless you have substantial evidence to support that claim. BL 125377/0 Page 13

LETTERS TO HEALTH CARE PROFESSIONALS

We acknowledge that you wil issue a letter communicating imortant safety-related information about this drug product (i.e., a "Dear Health Care Professional" letter); we request that you submit, at least 24 hours prior to issuing the letter, an electronic copy ofthe letter to thi BLA to the following address:

MedWatch Program Office of Special Health Issues Food and Drug Admiistration 10903 New Hampshire Ave Building 32, Mail Stop 5353 Silver Sprig, MD 20993

POST -ACTION FEEDBACK MEETING

New molecular entities and new biologics qualify for a post-action feedback meeting. Such meetings are used to discuss the quality of the application and to evaluate the communication process durig drug development and marketing application review. The purpose is to learn from successful aspects of the review process and to identify areas that could benefit from improvement. If you would like to have such a meeting with us, call the Regulatory Project Manager for this application.

If you have any questions, call Erik S. Laughner, M.S., RAC (US), Senior Regulatory Health Project Manager, at (301) 796-1393.

!RicharK~.r¡.~ Pazur/ ~ Richard Pazdur, M.D. Director, Offce of Oncology Drug Products Center for Drug Evaluation and Research

ENCLOSURES: Content of Labeling Carton and Container Labeling REMS REMS Materials

'\ t4 ~~i'o ì + ~ USPTO - Patent Bibliographic Data (Patent Number: 7605238) Page 1 of 1

- i Patent and . Trademiuk Offce . (l United States Patent Bibliographic Data 05/13/2011 04:27 PM Patent Number: 7605238 Application Number: 09948939 II Issue Date: 10/20/2009 Filng Date: 09/07/2001 Title: HUMAN CTLA-4 ANTIBODIES AND THEIR USES Status: 4th year fee window opens: 10/20/2012 Entity: Large Window Opens: 10/20/2012 Surcharge Date: 04/23/2013 Expiration: N/A Window Window Fee Amt Due: Surchg Amt Due: Window not open Total Amt Due: not not open open Fee Code: 1551 MAINTENANCE FEE DUE AT 3.5 YEARS Surcharge Fee Code: Most recent events (up to 7): No Maintenance History Found -- End of Maintenance History -- Address for fee purposes: BAKER BOTTS L.L.P. 30 ROCKEFELLER PLA 44th Floor NEW YORK, NY 101124498 ;,,:-.:: ~ " .,-;,,:::: ':., ~.,'-, - ".-. ". . ...: -.-. ',,'.: , '., ,Run AIotnt, Quêrý I

Need Help? I U.sET-Q Home Page I flnçlil!it1QJg-.e.1 AlertsPage

https://ramps.uspto.gov/eram/getMaintFeeslnfo.do;jsessionid=OOOOMu_ WU-3rzF66YQhft... 5/13/2011 077375.0135 PATENT

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

Applicant Korman, et aL. Serial No. 09/948,939 Examiner i. Ouspenski

Filed September 7, 2001 Group Art Unit 1644

For Human CTLA-4 Antibodies and Their Uses

TERMINAL DISCLAIMER

Mail Stop Amendment . Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313- 1 450

Sir:

Medarex, Inc., the Assignee of record of the entire right, title and interest

in and to the above-identified application, which is a continuation-in-part of United States

Patent Application Serial No. 09/644,668, now United States Patent No. 6,984,720 (the

'720 patent), by virtue of Assignment recorded at reel/frame 013 817/0628, recorded on

March 6, 2003 and 019334/0783, recorded May 23, 2007, hereby waives and disclaims the terminal portion of the term of any patent to be granted on the above-identified

application subsequent to the expiration date of the '720 Patent, whereby any patent to be granted on the above-identified application wil expire on the same day, provided any patent granted on the above-identified application shall be enforceable only for and during such period that said patent is commonly owned with the '720 Patent. 077375.0135 PATENT

In making the above disclaimer, the owner does not disclaim the terminal part of any patent granted on the instant application that would extend to the expiration date of the full statutory term as defined in 35 U.S.C. 154 and 173 of the prior patents,

"as the term of the prior patents is presently shortened by any terminal disclaimer," in the event that said prior patents later: expire for failure to pay a maintenance fee, are held unenforceable, are found invalid by a court of competent jurisdiction, are statutorily disclaimed in whole or terminally disclaimed under 37 CFR 1.321, have all claims canceled by a reexamination certificate, are reissued, or are in any manner terminated prior to the expiration of its full statutory term as presently shortened by any terminal disclaimer.

The undersigned is an attorney of record.

Please charge the required fee for this Terminal Disclaimer to Deposit

Account No. 02-4377. A copy of this paper is enclosed.

Respectfully submitted,

Lisa D. Tyner Patent Office ego No. 51,619 Attorney for Applicants

BAKER BOTTS L.L.P. Customer No. 21003 30 Rockefeller Plaza New York, New York 10112-0228 (212) 408-2628

-2- ~k\ bif /. (~ DEPARMENT OF HETH &. HUMA SERVICES Public Health Service .. '~.-"-'---- f,I' and Drug Administation \¡'rf::.:, :t ~ Rockville Pike ) ila MD 2086-144 rI f;.",. i , i '1..=,; ,f~ '\ i..fj§fl U: Ou Reference: BB-IND 9186 ,.4tt ...... _7_ '~~'l:'f~ ¡:

i."/ " . JUL 2 O~li \ r Medarex, Incorporated 1 8 ..'nnn.u JJ Attntion: RadaUT. Curow, M.D. Senior Vice President and Chief Medical Offce · ~ I 67 Beaver Avenue Andale, NJ 08801

Dear Dr. Curnow:

'J The Center for Biologics Evaluation and Research has received your Invesigational New Drug Application (IN). The following product name and BB-IND number have been assigned to ths application. They serve only to identi it an do not imply that ths Center either endorses or does not endorse your application.

BB-IN #: 9186

SPONSOR: Medarex, Incorporated

PRODUCT NAM: Human Monoclonal Antibody (MX-CTLA4) to CTLA4

DATE OF SUBMISSION: July 12, 2000

DATE OF RECEIP: July 13, 2000

This BB-IND number should be used to identi all future correspondence and submissions, as well as telephone inquiries concern ths IND. Please provide an origial and two copies of every submision to ths tile. Please include thee originals of all ilustrations which do not reproduce well.

It is understood that studies in hwn wil not be intiated unti 30 days after the date of recipt show above. If ths offce notifies you, verbally or in writing, of serious deficiencies that require correction before human studies can begin, it is understood that you wil continue to withold such studies unti you are notified tht the material you have submittd to correct the deficiencies is satisfactory. If such a clincal hold is placed on th fie, you wil be notified in writing of the reasons for placing the IND on hold.

) Page 2 - BB-IND 9186 )

You are responsible for compliance with applicable portions of the Public Health Service Act, :2.) the Federal Food, Drug, and Cosmetic Act, and the Code of Federal Regulations (CFR). A copy of 21 CFR Par 312, perting to INDs, is enclosed. Copies of other pertinent regulations are available from ths Center upon request. The following points regarding beobligations comprehensive. of an IND sponsor are included for your - information only, and are not intended to Progress reports are required at intervals not exceeding one year and are due withn 60 days of the anversar of the date that the IND went into effect (21 CFR 312.33). Any unexpected, '1 fatal or imediately life-theatening reaction associated with use of this product must be reported to this Division by telephone or facsimle transmission no later than seven calendar days after intial receipt of the inormation. All serious, unexpected adverse experiences, as well as results from anal studies that suggest significant clincal risk, must be reported, in writing, to ths Division and to all investigators with fifteen calendar days after initial receipt of ths information (21 CPR 312.32).

Chaging for an investigational product in a clinical trial under an IND is not permtted without the prior written approval of the FDA.

Prior to use of each new lot of the investigational biologic in clincal trials, please submit the lot number, the results of all tests performed on the lot, and the specifications when established (Le., the range of acceptable results).

If not included in your submission, please provide copies of the consent forms for each clincal study. A copy of the requirements for and elements of informed consent are enclosed. Also, please provide documentation of the institutional review board approval(s) for each clincal study.

All laboratory or anial studies intended to support the safety of this product should be conducted in compliance with the regulations for "Good Laboratory Practice for Nonclincal Laboratory Studies" (21 CFR Part 58, copies available upon request). If such studies have not been conducted in compliance with these regulations, please provide a statement describing in detail all differences between the practices used and those required in the regulations.

:~~ Item 7a of form FDA 1571 requests tht either an "environmental assessment," or a "claim for categorical exclusion" from the requirements for environmental assessment, be included in the IND. If you did not include a response to ths item with your application, please submit one. See the enclosed inormation sheet for additional inormtion on how these requirements may be addressed.

) Page 3 - BB-IND 9186 )

Telephone inquiries concernig this IND should be made directly to me at (301) 827-5101. J Correspondence regarding ths fie should be addressed as follows:

Center for Biologics Evaluation and Research Att: Offce of Therapeutics Research and Review HFM -99, Room 200N 1401 Rockvile Pike Rockvile, MD 20852-1448

'1 If we have any comments after we have reviewed ths submission, we wil contact you.

Sincerely yours, , SÎCMO n &. kolWU Sharon Sickafuse. M.S. Regulatory Project Manager Division of Application Review and Policy Offce of Therapeutics Research and Review Center for Biologics Evaluation and Research

Enclosures (3): 21 CFR Part 312 21 CFR 50.20, 50.25 Information sheet on 21 CFR 25.24

--.;.. ..~

) " . §310.5a7 21 CFR Ch. , (4-1-99 Edition) Food and Drug Adminlsfrollon. HHS for marketing. In the absence of an ap- tlon or abbreviated new drug appllca. §312.2 proved new drug appHcatlon or abbre- 312.44 Tennlnatlon. tlon, such product Is also misbranded 312.45 In-.i.tlve status. Subpart G-Drugs for Investigal1miol Use In viated new drug application, such prod- under section 502 of the act. Laboratory Research Anliiuis or In uct Is also misbranded under section 312.47 Ml'ctlnR"S. (c) Clinical Investigations designed 312.48 Dispute resolution. Vllro Tests 502 of the act. to obtain evidence that any dru!: prod- (c) CHnical Investigations designed 312.160 Drugs for Investigational use In lab- uct labeled, repreRented, or promoted Subpart D-Responslbillles of Sponsors to obtain evidence that any drug prod- llr:tory research ariimii1s or In vitro uct labeled, represented. or promoted for OTC use for the treat.ment and/or and Investigators tests. for OTC use for the treatment and/or prevention of malarIa is safe and effec- tive for the purpose intended must 312.50 General responiilbllltleii of sponsol". AUTllOniTY; 21 U.S.C. 321, 331. 351, 352. 353. prevention of noctumal leg muscle comply with the requirements and pro- 312.52 Transfer of obliltatlons to a co"'.ract 355.371: 42 U.S.C. 262. cramps is sa.fe and effective for the pur-. research oriranlz:itlon. pose Intended must comply with the re-" cedures governing the use of Investlga. SOURCE: 52 FR 881, Mar. 19, 1987, unless tlonal new drugs set forth In part 312 of 312.53 Selectlnir Invelitlltatol" and monItors. otherwise noted. qulrements and procedures governing 312.54 Emf!riiency research under 150.24 of the use of Investigational new drugS this chapter. (d) After April 20, 1998, any such OTC this chapter. Subpart A-General Provisions set forth in part 312 of this chapter. drug product initially Introduced or 312.55 Inrormlng Investigators. Cd) After February 22, 1995, any such . 312.56 Review of ongolnir Investliratlons. §312.1 Scope. GTC drug product Initially Introduced Initially delivered for Introduction into 312.57 Recordkeeplng and record retention. or Initially delivered for introduction interstate commerce that is not In 312.58 Inspection of sponsor's records and (a) This part contain" procedures and Into Interstate commerce that is not In compliance with this section Is subject report.~. requirements governiiig the use of In- compliance with this section Is subject to regulatory action. 312.59 Diiipositlon of unused supply of Inves- vestigational new drugs, Including pro- to regulatory action. 1G3 FR 13520, Mar. 20. 19901 tl/latlonnl drull. cedures and requirements for the sub- 312.60 General responiilhilltles of Investlga. mission to. and review by. the Food (59 FR 43252. Au¡r. 22. 1994) PART 312-INVESTlGATlONAL NEW torii. and Drug Administration of investiga- 312.61 Control of the Invelitllll\tlonRI drug. §310.547 Dnisr products containing DRUG APPLICATION tional new drug applications (lNO's). quinine offered over-the.counter 312.62 Inveitlirator recordkeeplng and An Investigational new drug for which record retention. (GTe) Cor the treatment and/or pre- Subpart A-Gneral ProVisions an IND Is In effect In accordance with vention DC malaria. 312.64 Imieiitl/lator reports. this part is exempt from the premar- Sec. 312.66 Al''1urance of IRB rl'vll'w. ketlng approval requIrements that are (a) Quinine and quinine salts have 312.68 Iniipectlon of Investlgator's records been used GTC for the treatment and/or 3i2.1 Scope. otherwise applicable and may be 312.2 Applicability. and reporti. shipped lawfully for the purpose of con- prevention of maIarla. a serIous and 312.3 DefinItions and Interpretations. 312.69 Handling of controlled Rubstiinces. potentially life-threatening dIsease. ducting cHnical InvestigatIons of that 312.6 Labeling of an Investigational new 312.70 DIsqualification of a clinIcal Inveatl. drug. Quinine Is no longer the drug of choice drult. irator. for the treatment and/or prevention of . 312.7 Promotton antI charlllnir for Investlira. (b) References in this part to regula- most types of malaria. In additloii. tiona! druKs. Subpart E-Drugs Intended to Trea Ute- tions in the Code or Federal Regula- there are serious and complicating as- 312.10 Waivers. threaening and Severely.debllltalng tions are to chapter I of title 21, unless pects of the disease itself and some po- Ilnesses otherwIse noted. tentially serious and Ufe-threatenlng Subpart B-Invesllgatlonai New Drug risks associated with the use of quinine Application (INO) 312.80 Pu rpose. §312.2 ApplicabiUty. at doses employed for the treatment of 312.20 RequIrement for an IND. 312.81 Scope. (a) Applicabilty. Except as provided malaria. There Is a lack of adequate 312.21 Phases of an Investigation. 312.82 Early conRultatlon. In this section, this part applies to all data to establish general recognItIon of 312.22 General principiI'S of tbe IND submls- 312.83 Treatment protocols. clinical investIgations of products that the safety of quinine drug products for $Ion. 312.81 R1sk.beneClt anlllYSls In review of are subject to section 505 of the Federal OTC use Iii the treatment apùlor pre- 312.23 IND content and format. marketing applications for dniirs to treiit Food, Drug, and Cosmetic Act or to the' .vention of malaria. Therefore, quinine 312.30 Protocol amendments. 'iire-threatenlng and severely-debiltating llcensl'ng provisions of the Public 312.31 Information iimenrlments. Illneiiiies. or quinine salts cannot be safely and 312.32 INO safety rr.ports. 312.85 Phase 4 studies. Health Service Act (58 Stat. 632, as effectIvely used for the treatment and/ 312.33 Annuiil reportfl. 312.M Focufled FDA reirulat.f': amended (42 U.S.C. 201 et seq.)). or prevention of malaria except under ""rcb. (b) Exemptions. (1) The clinical inves- 312.31 Trl'atment use of an inveiitlllational 312.87 Active monltorinR" ..,luet and the care and supervision of a doctor. new dl'u¡r. 'evaluation of clinical trials. tlgaUon of a drug product that is law-. (b) Any GTC drug product containing 312.3.5 Suhmll'iilonflJor treiitment Ul'e. 312.88 Sareguards for patlent.sii.'~ty. fully marketed in the United States is. quinine or quinine salts that Is labeled, 312.36 Emerirency use of an Investlltatlonal exempt from the requirements of this new ilriiir. represented, or promoted for the treat- Subpart F-Mjscen('~Ii~US part If all the following apply: ment and/or prevention of malaria is 312.38 Withdrawal of an IND. (I) The investigation is not intended 312.110 Import and (:.tport requirements. rei;arded as a new drug within the Subpart C-Admlnlsfatlve Acllons to be reported to FDA as a well-con-" meaning of section 20lrp) of the act, for 312.120 Forelim clinical studll'~ ". t. con- trolled study in support of a new indi- which an approved application or ab- 312.40 General reriulrements for UM of an In. rlncted under an IND. cation for use nor intended to be used breviated application under section 505 vestiiriiLionii1 new drug In a cllnlca1'ID' 312.130 Availabilty ror puhllc dliiclosure of to support any other slgnlfcant change of the act and~rt 314 of this chaptcr vestiiratlon. 'data and. Information In an IND. in the labeling for the drug; 312.41 Comment Rnd advice on an INn. ;u40 Address for correspondence. Is require(l fr arketlng. In the ab- 312.42 ClinIcal holds and requeiits ror modI. . (II) If the druK that Is unù~lng in- sence of an ap~ ,'Jed ne~drug applica- 15 GuIdelines. vest.gation Is lawful1y rna, id as a fjcati~ l /,r"~l ,I . ~ . . §312.3 21 CFR Cn. I (4-1-99 Editon) Food and Drug Adminlslratlon. HHS prescription druir product. the Inves- §312.7 tliratlon III n'it Intended to RUppOI.t a (d) Unlaliclrrl indication. This part driig or blologleal drill\ that ¡Ii used in docs not apply to the usc In the prac- rfF'F.CTVE DATF: NOTF:: At 64 FR ~01. Jan. sl¡;nificant cliange in tIie advertising tice of medicine for an unlabeled indi- a clinical investl~atlon. The term algo 5. 1999. §312.3 wiiii nmenùed by remcvlnR' ", for the product; i. Judes a biologica ì product that Is antlhl'll.lc druR'," (rom the piiragrapb defin- cation of a new drug product approved used in vitro for dIagnostic purposes. ing "lii':estlgatlona1 new drug" and by re- (111) The investigation does not in- under part 314 or of a licensed biologi- volve a route of adminIstration or dos- The terms "in.. - ..tlgational drug" and moving the phrase ", a request to provIde (or age level or use in a patient population cal product. "Investigational new drug" are deemed c!!rt,lflcotlon o( an antibiotic. submitted (e) Guidance. FDA may, on its own to be synonymous for purposes of this u ¡ ,- r~ction 50 of the Act," from the para- or other factor that significantly in- gmi'li ùP.rinJnJ! "Marketing applicatIon". ef- creases the risks (or decreases the ac- Initiative. Issue ~uldance on the appli- part. cabilty of this part to particular in- fective May 20, 199. ceptabilty of the rIsks). associated Investigator means an Incl1vidual who vestigational use~ ..r drugs In request, actually conducts a clinical investiga- with the use of the drug product; FDA wil advise OJ¡ the applicabllty oC §312.6 Labeling or an investigRtiona (Iv) The investigation is conducted In tion (l.e.. under whose immediate dl- new drug. this part to a planned clinical Inves- rectlDII the drug 19 administered or dis- compllance with the requirements for tigation. (a) The Immediate package of an in- instItutional review set forth In part 56 pensed to a subject). In the event an In- vestIgational new drug Intended for lind with the requirements for informed (52 FR 8831. Miir. 19, 1987. M iimended iit 61 vestigation Is conducted by .~ team of human use shall bear a label with the consent set forth In part 50; and FR 51529. Oct. 2. 1996; 64 FR 401. Jan. 5. 1991 Individuals, the investigator is the re- sp(jj~;;ible leader of the team. "Sub- statement "Caution; New Drug-Lim- (v) The Investliration Is conducted in F.1'1'F.CTIVr. DATE Nl)TE: At 64 FR 401. Jan. investiirator" includes any othei' Indi- ited by Federal (or United States) law compliance with the requirements of 5. 1999, §3J2.2 WM iiinentled by rf'movlnR' "or to investigational use." §312.7. 507" from parnirriiph (ii) iind hy reinovlnir "or \'jdual member of that team. MaT', 'ing application means an appll- (b) The label or labeling of an inves- (2)(1) A cllnlcal investliratlon Involv- niitlilotlc ùi'urr" from paTlgraph (d). effec. tli,"-"lonaJ new drul' shall not bear any tlve Miiy 20. 1999. cal.if;;. rrir a new drug submItted under Inir an In vi tro dlairnostic bioloirical. statement that is false or mi:ileadlng in product listed in paragraph (b)(2)(1) of sp.ctloii .i()5(b) of the Act or a product li- §312.3 Definition" and interpretations. cense application for a bIoloirlcal prou- any particular and shall not represent this section is exempt from the re- that the investigational new drug Is quirements of this part if (a) it is in- (a) The deflnl tlons and interpreta- uct submitted under the Public Health Service Act. safe or effective for the purposes for tended to be used in a diagnostic proce- tions of terms contained in section 201 whIch It Is being Investigated. dure that confirms tlie diagnosis made of the Act apply to those terms when Sponsor means a person who takes re- by another, medically established. di- used in this part: sponsibilty for and initiates a clinical §312.7 Promotion Rnd chargiDg for in- agnostic product or procedure and (b) it (b) The following definitions of tt.rms Investigation. The sponsor may be an vestigational drugs. is shipped in compliance with §312.160. also apply to this part: individual or ph'lrmaceutlcal company, govemmental agency, academic Insti- (a) Promotion of an investigational new . (iil In accordance with paragraph Act means the Federal Food, Drug. tution. private oriranlzatlon. or other drug. A sponsor or investigator, or any (b)(2)(1) of this section, the following and Cosmetic Act (secs. 201-902. 52 person acting on behalf of a sponsor or products are exempt from the require- Stat. 1010 et seq., as amended (21 U.S.C. organization. The sponsor does not ac- ments of this part: (a) blood irrouplnir tually conduct the investigation unless Investigator, shall not represent in a 301-392)). the sponsor is a sponsor-Investigator. A promotional context that an investiga- serum; (b) reagent red blood cells; and Clinical inveMlgation meanß any ex- (c) anti-human globulin. person other than an Individual that tIonal new ,I~',g Is safe or effective for periment In which a drug Is adminis- uses one or more of Its own employees the P!i-roseii for which It is under in- (3) A drug intended solely for tests in tered or dispensed to, or used Involv- vestlgat.Jon or otherwise promote the vitro or in laboratory research animals ing, one or more human subjects. For to conduct an investigation that it has Is exempt from the requirements of initiated Is a sponsor, not a sponsor-in- drug. This provision is not Intended to the purposes of this part, an experi- vestigator, and the employees are in- restrict the fuii exchange of scientific this part if shipped in accordance wIth ment is any use oC a drug except for the 1nforration concerning the drug, In- § 312.160. vestigators. use of a marketed drug in the course of Sponsor-Investigator means an Indi- cluding dissemination of scientific (4) FDA wil not accep~ an applica- medical practice. findings In scientific or lay media. tion for an investigation that is ex- vidual who both initiates and conducts Contract research organization means a Rather, its Intent Is to restrict pro- empt under the provisions of paragraph an InvestIgation. and under whose Im- person Liiat a~iiumes. as an independent mediate direction the investigational motional claims of safety or effective- (b)(l) of this section. contractor wnli the sponsor. one or drug iß administered or dispensed. The ness of the drug fl)r a use for which it (5) A clinical investliration involving more of the obligations of a sponsor. term docs not Include any person other Is under investigation and to preclude lise of a placebo Is exempt from the re- e.I'., design of a protocol, selection or commerclallzatio'n of the drug before it quIrements of this part if the inves- than an individuaL. The requirements monitoring of Investigations, evalua- applicable to a sponsor-investigator Is approved for commercial dIstribu- tliration does not otherwise require tion of reports. ani! preparation of ma- submission of an IND. under this part include both those ap- tion. terials to be ßuli'nltted to the Food and plicable to an Investigator and a spon- (b) Commercial distribution of an inves- (6) A clinical investigation Involving Drug Admlnlsti.ation. tigational new drug. A sponsor or inves- an exception from Informed consent sor. FDA means the Food and Drug Ad- tIgator shall not commercially dis- under § 50.24 of this chapter Is not ex- Subject means a human who partici- empt from the requirements of ~Ills ministration. pates In an investl~atjon, either as a tribute or test market an Investiga- IND mf'anR an investigational new reclT'lent oC the Investigational new tional new drug. part. drug ,. - ication. For purposes oC this (c) Biorwa)Jilt.1/ studies. The appllca- drug or as a control. A subject may be (c) Prolongin.C1 an investi.qation~ A hllity of part · ,. in vivo blo- part, ". ;'D" is synonymous with "No- a healthy human or a patient with a sponsor shall not unduly prolong an In- tice of Claimed Investlgatlonf\l Exemp- dlsp.ase. vegtlgatlon after flndj ~hat the re- av:iil:ibl1lt;i ..iiiip.s~, humans is sub- tion ~ ,. New Driig'." RUlt.s of the Inve~tlgati ,ppear to es- ject to Lhp. pl'oviglons bf § 320.31. 't71:rt:lirrnfinnnl ".0111 nr.,n --...... (lo2 F'R 0°11. ~f;ir. HI. 19f1, aii amended nt 64 tabi1.S1 suffclp.I1t data to Buooort a §312.1O 21 CrR Ch. I (4-1--/9 Editon) Food and Drug Adrninislrolion, HHS § 312.22 (d) Cliargin.Q for mid cOllimercicilization j of iiwcstigcr!ioncrl drlips-(1) Clinical §312.10 Waivers. l Such a clinical i nvpi;tiimt.ion Is nnt (c) rlia.m 3. Pha¡;e 3 studies are p.x- trials undcr an IND. ChargIng for an In- (a) A sponsor may reciuc!lt FDA to . permitted t.o proceeu wlt.hout the J" lor panded controlled and uncontrolled vestigational drug in a clinical '.trial waive appllc.able requIrement under i wrItten authorIzation from FDA. :¡DA trials. They are performed after pre- under an IND is not permItted without this part. A waIver request may be sub- . shall provide a written det.ermlnatlon liminary evidence sui;gesting effective- the prior written approval of FDA. In mltted either In an IND or ;.: ~n InfoI" 30 days after FDA receives the IND or ness of the drug has been obtained, and requesting such approval. the Sponsor mation amendment to an iND. In an earlier. are Intended to gather the additional shall provide a full written explanation emerg-ency. a request may ùe made by 152 fR 8831. MRr. l!l. IP87. II ampndl'd lit GI information abou t effectiveness and t!!lephone or other rapid communica- of why charging is necessary in order FR 51529. Oct. 2. 1996: 62 fIt 32179. June 16. safety that Is needed to evaluate the for the sponsor to undertake or con- tion means. A waiver request Is reo 1997) overall benefit-risk relationship of the quirerl to contain at least oiie of the tinue the clinical trial, e.g.. why dis- drug and to provide an adequate basis folJowlnir: 1312.21 Phaiieii of an invcaligBtion. for physician labeling. Phase 3 studies tribution of the drug to test subjects (1) An explanatIon why the sponsor's should not he comildcred part of the An IND may be submitted for one or usually 1nclude from sevE'n,; hundred compllanee with the requirement Is un- more phases of an InvE'i;tllmtlon. TIie to several thousand suhj"cts. normal cost of doing business. necessary or cannot be achIeved; cllnlcal.lnvestliratfon of a previously (2) Treatmcnt protocol or treatment (2) A description of an alternative untested drug Is generally divided into 1312.22 Generiil principles of the IND IND. A sponsor or h1vestlgator may submission or course or action that three phases. Although In general the submiliiiion. chargc for an Investigational drug for a satiiifies the purpoiie of the requIre- phases are conducted sequentially, treatment use undcr a treatment pro- ment; or they may overlap. 'rhesc three phases (a) FDA's primary objectives In re- tocol or treatment IND provided: (1) (3) Other information justifyIng a viewing an IND are, in all phases of the of an investigation are a follows: Investigation. to assure the safety and There is adequate enrollment in the waiver. (a)l'ha.ee 1. 0) Ph aRe 1 Includes the ongoinir clinical investIgations under (b) FDA may R'rant a waiver if It Inlt.ial Introduction of an InvestIga- rights of subjects. and, In Phase" and the authorized IND; (i) charginir does finds that the Sponsor's noncompliance tional' new drug Into humans. Phase i 3, to help assure that the quality 01 the not constitute commercial marketing wouid not pose a siimificant anrl unrt'n- St".l";P.~ are typlcaHy closely monitored scientific evaluation of drugs is ade- of a new drug for which a marketing sonable risk to human subjects of the and inay be conducted In patients or quate to permIt an evaluation of the investigation and that one of the fol- normal volunteer subjects. These stud- drug's effectIveness and safety. There- application has not been approved; (ill) fore. although FDA's review of Phase 1 the drug is not being cQmrnercially lowing is met: ir.s are deslg"ned to detp.rmlne the me. (1) The sponsor's compliance with the tabolism and pharmacologic actions of suhmlsslons wil focus on asscssinir the promoted or advertised; and (iv) the requiremen i, is unnecessary for the safety of Phase 1 Investigations, FDA's sponsor of the drug Is actively pursuing the drug in humans, the side effects as- agency to evaluat.e the application, or sociated with increasing. doses, and, If review of Phases 2 a.nd 3 submissions niarkt~ .~ approval with due diligence. compliance cannot be achieved; wil also include an assessment of the FpA must. be notified in wrillnir In ad- possible, to gain early evidence on ef- (2) The sponsor's proposc(i 1\1 fectivene!ls. Durlnir Phase I. suffclent scientific (¡uality of the clinIcal inves- vaiice of commencIng aiiy such native satisfie!l I,he requlremfmt.: or ter- t,lp,atlonii ali(l the lIkp.Ilhood that the InformatIon ahout. the drug's ph ar- chantes. In an Information amendment (ai The applilants submission other- niacol:liietlcs and pharniacoloidcal ef- investlimtlons wil yield data capahle Ruhriltt.I".1 unil..r §312.3L. AuthorIzation wise just.ifies a waiver. of meeting statutory standards for foi' ch:iixlng' go('s into effect automati- fect~ should be oIJI,ainl"d t.o permit the (Co)Jl'cUrin or Inr"rmAt.lon rp'iulremp.nt!l np- de:-Iim of well-controlle!!. scientifically marketing approvaL. cally 30 days after receipt by FDA of provt'il hy the Orrce or Man;igenient and valid. Phase 2 stuuips. The total num- .(bl TIie amount of information on.a the Information amendment. unless the Builllct under control number 0910-0014) ber of subjects and patients included in particular drug that must be submitted Sponsor Is notifi('d to the contrary. 152 FR Bn~i. Mnr. 19. 1987. "' Rmen'led at 52 Pha!:e 1 studIes varies with the drug, in an IND to assure the accomplish- (3) Noncommcrcialization of investiga- . FR 23031. June 17. 1907) but. Is generally In the range of 20 to 80. ment of the objectIves described in tim/(l driig. Under t,/iis sect.on. t,he (2) Phase 1 studies also include stud- paragraph (a) of this section depends sponsor may not commerciallze an in- Ips of dluir metabolism, st.ructure-ac- upon such fod,ors as the novelty of the vestii;at lonal drug by cliarg'nir a price Subpart ß-Investigational New tivit.y relationshIps. and mechanism of drug. the exi.ent to which it has been larger than that necessary to recover Drug Applic-.,'.Jn (INO) action in hiimanii. aii w~ll as studies in studied previously, the .known or sus- costs of manufacture. research. devel- which invp.stigatíonal dnigii are used as pected risks. and the developmental opment, and lianullng of the investIi;a- §312.20 Iiequirl'mcnt (or an IND. research tools to explore biologIcal phasc of the druir. tional drug'. (a) A sponflor shall flubmit an IND to phenomena or dlseasp. processes. (c) The centi'al fOCUR of t.he Ini tlal (4) Witidmli:al of arilhorization. Au- FDA if the sponsor intends t.o conduct (hi l'llUs/! 2. Phai:e 2 includes the con- INO submission should be on the gen- thorization \0 chargn for an Investiga- a clinl.cal investiiration with an inves- trolled cli nlcal st,uilies conducted to eral investigational plan and the proto- tional drug under this section may be tigational iiew drug that is .suhject to evaluate the effectiveness of the drug cols for l!pecific human studies. Subse- withdrawn by FDA if t.he agency finds §312.2(a). for a particular Inuication or Indlca- quent amendments to the IND that that the conditions underlyinir the au- (b) A sponsor sh;iH not begin a clin- f.onii In patients with the disease or contain new or revised. protocols should thol'lzatlon are no long-nr saLisfiell. ical investigation suhject to §3I2.2(a) condition under stuuy and to deter- build 10Jiically on previous submtssions until the iiivest.gatinn is subject. to an mine the common short-term side ef- and should be supported by addl tional (Coll..cllon or i nrormaLion r!!iiulremenl!l ap. IND which Is in effect in accordance fects and risks a!l:'ociated with the Information, including the results of provc,! loy th!! orn!!!' or Man:il:t'nient and with §3I2.10. drug. Phase 2 studies are typically well animal toxicoloR'Y studies or other lludg'Cl uii.lc!' c~!l0l numher 091G-00l1) ((') A Spon:-or shaH stlhmlt a separate controlled. closely monitored, and con- human studies as appropriatp. Annual IND for any clini~al Iiivestiitatlon In- i.-.iuctl"ù in a relatively ¡¡mall number of (52 FR 8831, M' \. 1987. aR ameiided nt 52 volving an ex~pption from informed reporLs to the IND iihoul'-"i'" .. as the Fit !!l7G. ~!n~' 7... "'07) -. .. )atient,ii. uflually involviiiir no more cons!'!' '~nunr § 50.21 of this chapter. focus for reporl.hig the st of studIes " than sevcral/iundred sl1lije~ts. bcing/;Ç'bnducteù under Lhe IND and §312.23 21 CFR Ch. i (4-1-19 Editon) Food and Drug Administration, HHS i;hould Upd:i.e I.he J!enernl investiiw.- § J 12.23 poi;eil clinical 1/l...~1 igatlon aiiil tional plan for tli~ eoniinir year. t/i e investigator wll report 10 the I1B i lie rr.levant to thci saf-:!.'. of the pro- not suhmit.ted initially In the IND Id) The IN)) foriiat Silt fort.h Iii po~p.ù clinical Investigat.iuii(s). §312.23 should be followed routinely by Jlropm:eil ch:uIJles hi t.ir. I'l'sr.:irch act.lv. ) should be submitted In accordance with ity in accoriJ:i.nce ".-ith the require-l I .(11) If the drul? has bep.n wlthi!rawn § 312.30(a).) In geiieml, protocols for SPOil SOl'S in the int.erest of fostering an from Investigation or marketing in any Phaiie i studies may be less detalled , effcient revieiv of applications. Spon- ments of part 56. country for any reason related to safe- sors are expectcil to exercise consider- (v) A commitment to conduct the In. . and more flexible than protocols for ty or .effectiveness, Identification of Phase 2 and 3 studies. Phase 1 protocols able discretion. however, reg'ardiiig the vestlgation In accordance with all I the country(ies) where the drug' waii content of information slibmltLeil in other aJlPli~"Jlle regulatory requlre- should be directed primarily at pro- withdrawn and the reason ii for the vldlng- nn outline of the investliiatlon- each i:ection, depending UPO!, 'ìie kInd men\,s. wIthdrawaL. of drug beinir stuilll!l anil the nat.ure of (vi) The name and title of the person I an estimate of the number of patients (Iv) A brief description of the overall to be Involved, a description of safety the avallahle information. Sllcl.lon respOTlsl1iI"! for monltOl'liiir thr. conduct plan Cor invest.iratliill t.hf' ilriig' produet 312.23 ou 1,lIl1es the In forinaLi on iieeded and proi:rr.sii of the clinical hlvesLi¡nl.- : exclusions. and a description of the for the folJowinR' year. The plan iihould doiiliiK plan Incluilhig' dur:tion, ilose. or for a commercially Sponsoi'ed IND for a include the following: (a) The rationale new molecular entity. A spomior-Inves- tlons. : metJioil to he uii..d In dete/'minlng (vii) The name(ii) and tltle(s) of the ! for the druir or the research study; (b) dose-and should specify In detail only !.Ig-ator who uiles, as a rei:iml'ch 1'001. ii n p.l:rson(s) reiipon!lihle under §3l2.32 for I' the .lmlir:atlon(s) to be studied; (e) the investigational Jlew drug that 'is al- review and evaluatIon of iliConnation thoiie elements of the study that are ireneral approach to be followcil 111 crltfcal to safety, iiuch as iiece~sary ready subject to a manufacturer's IND relevant to thp. safety of the drug. evaluatinl' the drul1: (d) the kinds of moni torinir of vi tal signs ancl biood or marketing appJlcatloti should follow (viII) If a sponsor has transferred any l clinical trials to be conduct~d In the the same general format. hut ordi- chemistries. Modifcatjon~ iif the ex- obligations for the conduct of any clin. j first year following the submlsiiion (if perimental design of Phai;e 1 studies narily may. if au thorlz.ed by the manu- Jcal .study to :i contract research orlla. i plans are not developed for the entire fac\'urer. refer to the manufac\,urer's ni7.aUon. a st.iitement conl.alnlnll the " that do iiot affect crl tical safpt:v as- IND or marketIng application iii pro- i-ear. the sponsor should l'O indicate): 5es~'inents are required to be ,eported name and aililre!:ii of the contract reo ie) the estimated nunilicr of patients to to PDA only III the annual report. vidIng the technlcai information sup- search orimnl7.:itioll. identification of i be iriven the drug iii tho!'c studies: and porting the proposed clinical liivestlga- (il) In Phases 2 and 3. deta¡ ..,1 proto- the clinical study. and a listing of the I If) any risks of particular lIeve/'ity or cois describing all aspects of the tion. A spoiii;or-investigatol' wiio uses obliiratlons traniiferred. If ali ohllga- I sl'iousness anticipal.e(l on the baRls of study an Investigational drug not suhject to should be submitted. A .protocol for a tionsP.overninP. the conduct of the I the toxicological data In animals or Phnse 2 or 3 Investiiratlon should be de- a manufacturer's IND or marketing aii- study have br.en transfèrred, a general I prior stuilles In humans with the drug signed in such a way that. If the spon- plication is ordinarily required to sub- statement of this t,raiisfer-fn Heu of a I or relateil drug'S. mit all technical Iiiformatlon sup- sor anticipates that some deviation listing of the specific obligations .trans- I m (Rei;erved) from the study deiiig'n may become nec- port.ing the IND, unless such informa- ferred-may be submitted. (5) In¡'esli.aalur's broe/nac. If required tion may be referenceil from the sci- ess:iry as the investig'atlon progresses. (Ix) The iilgnature of the sponsor or under' § 312.55. a copy of the investlira- alternatives or continllencies to pro- entific literature. the sponsor's au thori7.ed representa- I tor's brochure. containing the fol- vide for such deviatIon are built Into § 312.23 IND content and Connat, . tlve. If the p..rson signing- the applica- loi\'iiil! Informal.ion: the protocols at the outset. For exam- tion does not reshIp. or have a place oC (i) A brief description of the drug ple. a protocol for a controlled iihort- (a) A sponsor who Intends to conduct business withIn the UnIted States, the suli!\t.:ince and the forniulal,ion. Includ- term study might include a plan for an 1\ cUnical investIgation subject to this IND is required to contain the niime InK the structural formula. if known. part shall submit an "Invest.gational early crossover of 1l0nresponders to an and address of, and be countersigned Ii) A summary of the pharma- altp.rnapve therapy. New Drug Application" (IND) includ- colog'ICal nricl toxiiolol!ical effects of by, an attorney, agent. or ot.her au- (ii) A protocol is required to contain Ing, hi the followIng order: thorized official wJio res.iiles. or main- the drug In animals and. to the extent the following, with the specIfic ele- (1) Coiier slieet (Form FDA-1571). A tains a place of business within the known. in humans. cover sheet for the applicatIon con- ments and detail of the protocol re- United Statei¡. (iii) A summary of the pharmaco- flecting the above distinctions depend- t:iIiiin~ t.he followiiig-: f (2) A table of contents. kinetics and bloloirical diiipositlon of ing on the phase of iituily: (i) The n:iiie. :iddress. 1I.nd telephone the dlliir in animals and, if known, In number of the sroiisor. the ilate of the (3) lntroductorll .tliitemenl ii7lri r¡eneral ((71 A statement of t.he objectives and illt'esti.aiitioiiall'lan. Ii) A brief introduc- humans. purpose of the stuily. a¡iplicatioii. iind the name of the ¡iives- (iv) A summary of Information relat- tlg-:ülol1al new druir. tory statement giving the name of I.he (11 The name anil arlrlresii and a Rtate- dl'l! anil :ill active Inirredlr.nts. the ing' to safety and effectiveness in hu- ment of the qualificatIons icurriculum (in Idp.utiflcaUoii of the phiise or mans obtained from prior clinical iitud- pha~es of l.he clinical investigation to di'ug"s pharmacologIcal class, the vitae or other st.aternent of qualifica- structural formula of the druir ur les. (Rp.prlnt,s of publiiihed articles on tions) of each invest.lgator. and the be coiiducted. siich studies may be appended when liIi) A commltmiint not to b..g-in clin- known), the formulation of the iJosa~e name of each suhlnvestiirator (e.g'.. re- formis) to he used, the route of admin- useful. search fellow, resident) working under Ical III"estlg-ations until an IND cov- Iv) A rleRcrlption of possi hIe risks and ering' thii Investlirations Is In effect. Istration. ani! the broail objectives and the supervision of the investigator; the planned duration of the proposed. clin- side effects t.f) he anticipateil on the name and address of the research fa- (iv) lir:onimit.lTeiit that an Instil :1- ical investlgat.on(s). basis oC prior experience with the drug cilities to be used; and the name and tiiiii:il IlI!vil!\Y Bo:ird (iiUI) I.li:t com- under investip,al,i. or wit.h related plies with the reauirements Silt fort.h In (il) A 1,,'lef i'Immary of previous aclrlreiiii of each reviewing Institutional p:irt 5G will h~iipom;ible for \'he Inl- human exp~rieiii:p. wi th the druir. with drul!s. and of pJ'~caut.ons or special Itevlew Board. referp.ncp. I "Uier IND's If pp.rtinent. ..(lnitorlnK to Ire don.. as part of t.he In- ti:l and co' iIng i'evicw and lip- i:t.ÌJmt.ional use of the rlrug'. (e) The criteria for pntie.-.selectlon pro'::il of each ... the st.~ies In the pro- an,1 to Investiinl.t.lonal or markel.ng eii. amI for excJiiiilon of p;i\,ler. HI :in es- perienLerln otlii;r countries that miiy i6) l'ro(ui:ols. ti) A pro\'ocol for eadi timat.e/.oJ the number of p1h.."nts to be planned study. (Proto(;ols for iil.uilies i;L.ii ,Ii ed,: , §312:23 21 CFR Cii. I (4-1-97 Edifon) . iood and Drug J\dminislralion, HHS "1) A description of I.he deiihm of thii §312.23 study. Including the kind of control tlg-al.on. If vpry !lhf\rt-tcrm t.,.st.s are proposeil. the RUI'I'ortfiip, stability data : rlruir product.: anil lnform:if.lon suff. group to be used. if any, and ;. clescrlp- : cicnt to assui'e t.hf' product's 'itabillt.y tpsts of the driig's effects on reproduc- tlon of rnethodii to be uiied to mlnlmi7.e can he cOlTiispondjngly lflllt.l'd. tion and the developlii,. '"t.us: any spe- (j it) lis drug- rlevelopment procpr,ls "luring- the planned clinical stuilles. bias ot. '.he part of suhjects, hivestlga- Hefprence to thp current eilll,ion of the cial toxicity tei;t relal.e"i to t.he drug's tors. and analysts. and ll!l t.he scale or production Is particular moile of administration. or chn.::. .,el from the pilot.-scale Jirorluc- 'United Sta.tes Pharm1\copeia-~htional (e) TIie rni:thod for det.ermlnlng the : Formulary may satisf.v certain require. coiiditlonii of usc (e.g-., Inhalation, der- dosees) to be administ.ered. the planned Lion apPl'priat.e for t.he IImll.prl Ini tlal maL. or ocular toxicology); and any in clinical Invest.lgatlons to the larger- ; ments In this paragrn.ph. rn:ixlmum dORage. and the duration of Ie) A bl'lef R'enernl df':"criptlon of the vi Lro studies intended to evaluate drug scalc productIon needed for expa.nded toxicity. IndIvidual p:itlent eXposure to the clinical trials. the sponsor should sub- composition, manuf;ic:ture, anil control driig. of lIny placebo useû in a controlled (b) For each toxicology study that is ef) A desi:rlpt.lon of the o!iiierval.ions mit. Infonnation arnendment,ii to sup- plement the liilI.i:i1 Informai.lon Ruh- clinical triaL. Intended primarily to support the safe- and meMurements to ue made to fulfill idl 1.ribdjn9. A copy of all labels and ty of the prOII(lgell clinicn.l Investiga- the objectives of the study. mltted 011 the chemistry. maiiifac- tion. " '1111 tabulation of data suItable t.uring, and control procp.siies with 111- labe!!n" to be provided to p.ach Investi- (9) A description of clinical proce- giitor. for detailed review. dures. lahoratory tests, or other meas- formation appropriat.e to the expanded scope of the investigation. leI Ent'ironmentul anal.1Jsjs require- i 1Ii) For each nonclinical laboratory Ures to be taken to monl tor the effects /IC1/t.~.. A claim for cal,cJ!orical exclu- st.iitly subject to the g-ood lahoratory of the drug in human subjects and to (iv) Reflec;ting the distinctions de- scrlhetl Iii this p:mlg'raph (a)(7) and sion under §25.30 or 2~.JI or an envil'n- practice regulat.ions under part 58, a minimize risk. mental assessmiint under §25.40. statement that the study waii con- (7) CJiemi.çlry. manlifacliir;n,Q. and COlI- baRed on the pliaseis) to be studied. t.he siihrrisslon Is required to contain the (8) lJwmlfr;olo.Q.1J and tuxicology info ducted in compliance ,.\'ith the good tTO/ inr(lTna/ion. (i) As npproprlate for mati'in. Adequate inform:it1on about1'- laboratory practice regulat.ions In part the parl.cli1ar Invm,l.igatlonR covereil foiiowlii~; (II) DTU.Q .tii/¡.çliinct'. A rleRr.rlpl,lon of ph:i'lwl.CoICJdcal iinri \.i)xieoloirical 58, or. If the study was not conducted by the IND. a sectIon ilescrllilng the st.udies of the iiruir involving lahora- In compliance wit.h thoiic regulations. a composit.ion. manufact.ui'ii. and control the druir suliRtance. incluililiP. its phys- IcaL. chl'micaJ. or birilogir;al character- tory animals or in vitro, on the basis of brief statement, of the reason for the of t.he druir siili"tance and I.he ilnig which the sponsoi' h;is concludeii that .noncompliance. product. Alt.holll!h in p:ich phase of the isties; the name and address of Its mn.n- ufacturer; t.he gPllf!r:l lJetholl of prepa- it is rcnsonably safe to Cfln'!uct the (9) l'revious human p.x¡JcriC'nre with the investigation suffcient Information is proposed clinical lnvestlgatiuiis. The int'tsligritianri/ rJrU9. A summary of pre- required . (I lie sul.rni tted 1.0 assure the ratloT' rir the drug- substance; the iiC- ceptahle liinitii anti analyt.lcal methods kin.1, ilumtion. and scope of animal amI vious human experience known to the propel' iùeiitification. quality, purity, used to assure the identit.y. ~tren¡rth, other tests riir¡uireil varies wit.h the du- applicant, if any, wit.h the investiga- and strength of the investigational ration and nature of the proposed cliii- tional drug. The iiifol'inatlon is re- dnig-. th'. ;iinount of information need- ((uallt,y. and purity of tlui drug iiub- eeL. to make thaI. assurance will vary stance; and Iiiformii.tion suffcient to leal investigal ioiis. Ou i,j"lines are i¡uired to Include the following: iiupport stahllity òf the drug Ruhstance a\'al1able from FDA t.hn.t d""I.Tihc ways (i) If the invcstiR'ational drug has with the phase of the iiivefi.iiration. the In which these requireril'ints may he proposed duration of the investlg-a.t.ion, durin!\ the toxicological stu(lIl's anel ùeen investigat.ed or marketed pre- the planned clinical st.udies. Hef~reiii:l' met. Such informaU'In is required to viously, either in the United States or the dosage form. IUHI the amount of in- include the identification and quali- formation ol.herwlse available. FDA .to I.he current edition of the Unltrd other countries, detailed information States Phannacripeiil-Nat.lon:il For- fieat ions of thii iiiúiviiluals ..ho eva111- ahout such experience that is releva.nt reeo~ni7,t'~ that modifications to the mulary may satisfy relevant require- at"'cI the resuits of such st-udieR and 1,0 thc.safet.y of the propo!;pd investlga- method ol preparation of the new drug- cnnclurled t.h" i. it is reasonn.bly safe to i;uli.~tance amI riosage form and changes ment!' in this parairi'aph. t.ion or to the investi¡ration', . ''t.lonale. (bi DTU.Q product. A lI!'t of loll compo- i hei!in the pr"rosed investigations and a If the durg has been the suhJ~ct of con- in the dosng-e forin \t~elf are likely a~ nen til. which niiw j nclude rea~.,niilile st.;i t.eml'n t of whei'e the Investigations tlolJed trials. detailed information on th" invesL.igation prOP.I'eSRCII. There- werc conducted aiict where the recordii alte1'atlves for inact.ive compoiinils. such trials that i~ rei evan t to an as- for... t.he ("Ilphnsis iii lin initial Phasii 1 URci/ iii tJw mamifiicture of thii ill'es- are avnilable for inRpec\.ion. As drUg" seiiiinient of the drug's effectiveness for submissj on i;liould gcnci'all.'í bc placed ,Ipvelopmr-nt prociiprl!l. LhC! sponsor Is the proposed investlg-ational USe(s) on I.he iiliint.fical.ioii and coi\t.rol of t,he tig'lltional drug' product. Incluiling hoth li'quirf'11 to submit informational raw miil-erlais alHI the ncwtlrug suh- t.hORC! compolIl'n ts In tcnrlerl r'l IippC!ar should n.lso be provided. Aiiy published In t.he dniir product anil those wlllch amr.iidiiiints. as al'propri:tp.. with addi- niaterla.1 that Is I'elevnnt to the safety sl.ance, Final specifications for the tional information pertinent to safety. drug subst.ance a:irl dl'uR' iirorluct ai'e. may 1I0t appf'ar but which arp used In of the proposeû Investigation or to an tJi~ 'n:inufacturinrr procpss, anil. where (i) I'licmnacalo,QY and drug dispo.~itiun. assessment of the drug-'s effecUveness not expect.ed until the end of th~ invcs- A section describing- the pharma- tlimtlonal proC'ess. llI'J.¡Ii;n.ble. I.he quantitative coinpoRI- for Its proposed investig"ational use tion of the Invest,lP.ational drug prod- colt¡g-lcal effects and ineehanism(s) of should be provided In fulL. Published iill It shoulil be iiinphasi7.ed thn.t the uct. Iiieluiling- any reasonahle viiri- action of the dru/r in animals. amI in- materIal that Is less directly relevant amount of informat.ion to be submitted formation on the absorption. dIstribu- depends upon t.he scope of the proposerl atioiis that may he expectp.rl during- thp may be 'supplied by a bibliography. InvestiP.atlonal sf ~.,p; t.he name anil ad-. tion. metabolism, and excretion of the cllnie:il liiviist.iirrit.ion. Foi' exn.l1ple. .al- drui;, if known. (i) If the drug is a combination of I.hou~h sl-abili ty ilrit.:i are required iii c1reRs of L.he dniir product mamifac- drug-s previousiy Investigated or mar- tUlcr; a hrf,.f g'f'neral .lescrlpt,ion of the (ii) Tnxicolo9.l/. (II) An integrated gum- a II phases of the IND t.o demonstrate mary of the toxicological pffects of the keted. the information required under thrit the new di'u,, subsl.ancc and drug mn.iifacturing and packag-iiil. proce- parag"raph (a)(9HI) of this sect.lon d,' .' 11.S a¡iprop'" . t.f! for the product: :' . drug in animalii and in \'it.ro~ Depend- should be proviiled for cach active drug Ploùuct ar(' wit.hiii ac~eptable chcmlcii1 acccptalile liiii." and analytical Inr-I.h- in~ on \.ie nature of the rlriig and the lind physic;iI''' il.s for th(' planned du- : -, ph:ise of the Investigation. the r1cscrip- component. However. if ~ component ration of Ih, ()JlOSer~Iini('ai inves- od!; lIllCrl to assure t.lie irlent.ily. Iii Ruch combination ir¡ject to an gtrenir~.ljualit.y, and purity of the ' tiOlI Is 10 include th~ results of acute. approvc(1 marketing ai., .aLi.on or Is iniliacute. and chi'oiiic toxicity tests: ot.hcfW'hie lawfully markete(1 iii the §312.'3 21 CFR (.i. I (4-1-99 r"';ii..,) Uniterl Stat('i;. t.he !iprini;oi' Is not. I'- Food end Drug ¡\':''Tinh:lofion, HHS qUlled to submit puh!isheiI material sl'on"or III rcqiili'p,l 1.0 emil,aiii ii writ- §312.30 ten statemP.nt that authorlies the ref- (a) Ncw protocol. Whflnrvpl' a spon!lor concerninrr that active druir component Intllllis to conduct a study that Is not IHB is notified in accordance with unles!liiuch mat.erial relate!l directly 1.0 erence aliil that is !lhmeil hy t.he person §5G.l01(c). the proposed invei;tigational use lip- who iHlbmit.t,P.ill.he infolinatlon.. covered by a protocol already con- tained In Uie IND, thl iiponsor !lhall (c) New invcstigCJlor. A sponsor shall cluiliriir publications relevant to coni- (c) Material in n foreign langii(l.qe. The submit to FDA a protocol amendment !lulmilt a prot.ocol amendment when a pon.,;¡t.-componcnt interaction). i;ponsor shall iHlbmit nn acciiral.e and new investigator Is added to carry out coiiplete F:ni::ili;h tr:inslatlon of C'ach containlii¡r the protocol for the iitudy. (iii) If the dlull has been marketeiI Such !It-udy may bl'g'iii JirovldeiI two a plevlously submltt.ed protocol, except ou tside the Uiii ted States, a list of the part of the IND that is 1I0t In Enirllsh. that, a protocol amenilment is not re- countries in which the ùrug has been Thp. sponsor !lhall aho submit a coiiy of conditfons are niet: il) The :"ponsor lias quired when a licensed practitioner is marketed anti a li~t of the count.rles In each . iirinal litp.i'a\.urfl puh!lcal.ioii for 8ubmitted the protocol to FDA for Its review: and (2) the Protocol has been added in the case ola treatment pro- which the tlnlK has heen wIthdrawn which an gn~iisli t.l'tn!llatlon lii iiub- tocol under § 312.34. Once t.he lnvesti- 1I1i Lt.pil. llpproveil hy th" Iniit.i l.ut.ioiial Review' from marketing for reasons pot,entially Board (11tH) with re!ip(lrii;iliillt.y for re- g'ator I~ added to the study. the inves- relateiI to i:afety or effectlvene!is. (i1) Nuiiber of copic.ç. The spon!lor view and approval of the sturly in ac- t.lgational druR maybe shippeù to t.he (10) Additiol/al iiifomialion. Tn ccrl.ain shall suliinit an oliginal anil two copies inv()sl.iJ:ator and the invesL.ig'a.tor may of all iiiihiils!lïollfi t.o the IND fie. In. con!ance with the requli'ements of part applications. as descrlbeil below, infor- 56. The sponsor may comply with these begin part.iclpatirig- in the study. 111e mation on special topics ma,v be need- cludinir the original f1uhmlssion and all iiponsor shall notify FDA of the new In- eiI. Such infolmation i;liaU be sub- amendments and reports. two conditions in either order, (b) CJiIJngp.s in a protocol. (1) A sponsor vestil!ator within 30 days of the Inves- mltl.ed in this Sf'ction :is folI'ows: (c) Nlliibcrin.Q of IND sUIJmi.~.Çiolis. tlKator being added, T~:ich !llibllissioli relal.iiiir to an IND Is shall sulimit a pl'tocol amendment c1e- (I) LJrllt¡ depen.clci,. :iiid abiise polel!- sci'binir any chanire in a Phase 1 pro- (ill Content :ind format, A protocol liiii. If t.he di'arr is a lmychot,l'oplc !lub- 1l!llulr~(1 to hr. niimhr.rrd !lerialli' usIng tocol .that iiig'nificantly affect,,! the :imr.ndmp.nt Is required t.o be promi- stiuice or othenv iiie haii ahu!le pol.eii- it !il ng-Ie. t.hrC'e-lliglt i;~r'i:-.i nuinbr.r. The i;afety or !luhjpcts or any chan¡re In a nently Identlfiell as sudi (I.c., "Pi'O- tlal. a iif'ction descrihiiii¡ relevant clin- IiliUitl IND l!i rl'quli'cd to he nuniherr.1! Phai;P. 2 or 3 protocol that siirnlficaiitl,y l.ocoj Ameni1ment: New Protocol". ical studies and experience and studies 000; each sub!lequellt siilimii;sion (e.g.. affects the iiafety of sUIJjcctii. the scope "lrotocoIAmendinent: Chang-e In Pro- in test animals, ameiiilinent. report. or corresponilence) of the investigation. or the scientific tocol", or "Protocol Amendment: New (i1) Radioactive dTlt¡.ç, If the drug Is a is reriuircil to be numbereu chroiio- Investigator"). and to contain the fol- radio:ir:tive iInlg', suffcient data from qualiy of the study. F:xam¡iles of 10¡rically In sequence. chang'es requiring an amendment under lowing: animal or human stull1es to allow a (f) IrJel!tificalion of e.ri:tptioii from in- reasonable caiculation of radiatioii-ab- this paragraph Inchide: (1)(1) In the case of a new protocol, a formed consciit. If tlii: investigation in- (i) Any increase in druir do!l:i¡re or du- copy of the new protocol and a brief ue- iiorhcil dose to the whole body and crit- volves an exception from inform!!il con- ration of exposure of inilivii\ual sub- scription of t.he most clinically signif- Ical ,org-ans upon administration to a sent undp.r §!jO.21 of this ch:ipter. the human subject. Phase 1 stUdies of ra- sponsor shall prominently iuentify on jects to the druir beyond that in the cant differences between it and pre- dioar:tive druirs must include studies current protocol. or any significant in- vious Plotocols. the Cover sllfJp.t that l.he invesl.iir:ition crease in the number of i;ubjects under (ill In the case of a change in pro- iI doiiimetr.vwhich wil calculations. obtain suffcient dat.a for is siibject to the requiremrmts in §50,24 of this chapter. study. tocol, a brief description of the chang-e (iii Pediatric ,çtiidics, Plans for assess- (ii) Any ioi.imlfkant chanire in the de- and reference (date and number) to the in~ peiliatric s;iety and effecth. 'iiess, (ColIl'ct.ion or InrOl'm:itlon rf'Quircmp.nLo; np- si~n of a protocol (such as tbp. adllition submission t.hat contained the pro- (Iv) Other infoTiialilln. A brieC state- pi'oved hy the Offce or Mnnnl!emnnt anii or ilriil'ping of a controi RlOUp), tocol. DullR'r- uiider contl'oJ numher 091lH11) (iii) The addition of a new tC!it or (ii) In the case of a new investigator, ment of any other iiiformation that procedure t.hat is IntendfJd to improve i would ail! evaluation of the proposed ¡r'Z FR 11031. Mar. 19. 1987. :Ill am",nrl..,. l\t 52 the investigator's name, the quallfica- cliiiical Investigations w1th I'uspect to FR Z:1031. ,JIIM 17. 19R7: 53 FR 1918. ,lnn. l!.'i. monitorin¡r Cor. or i'educe the risk of, a tioi:s to conduct t !!e investigation, ref- their safety or their uesign and poten- I9IJ8: r. FR 51529. 0..,. 2. WOO: 62 Fl' 10.')99. side effect or adverse event; or the erence to the previously submItted pro- July 29. 1997; 6:1 FH. 6(jIi69. Dec. 2. 199111 dro¡iping of a test intended to monitor tocol, aiid all additional Information tial as controlled çllnical trial!f to sup- safety. pOlt ni:irketiiir. of the dlllg'. § J12.:iO Protocol amendment!l. about the fnveiitig-atol"S study as is re- (11) lll'C'i'ant information. If n'fjuestpd (2)( i) A protocol l'inn~e unller para- quired under §312.23(a)(6)(iIiHb). by FDA, any othel lclevant informa- Oner. an IND Is in cffect. a 1'l'on!Ìor l!riiph (bHl) of t.his :"cction mity be (2) Reference. if necessary. to i;peclfic tion npeded for review of the applica- !lhall ameiiit it as nl.idp.il to eii!lure that made prr.'vidcd two condition~ are met: technical information in the TND or in tion. the clfnical Investigations are con- ((I) The sponsor haii submi tted t.he a. i:oncurrently submlt.Lcij liillii'mation (b) Inhnnatinn prcviously sulJmitfccl. ducteil accorilinir to J.rotocols Included chanize to FDA Cor its review; and amendment to the IND that the spon- The spon!\r ordinarily is not refjuireù in t,he applkat.ion. '1...'- sel;ticJl sets (b) The chang-ehas been a¡iproved by !lor rplies on to !lupport any clinically 'to rpsubmit infolmatlon pleviously Corth the provisions \indcr which new the IltH with rei;pon!iiliility for review siimificant change in thP. new or !lubmil.cil. hut may hicorporatp. 1.lle iii- Pl'OtOCOlR may bri submltt."lI and and ap"i'oval of thc study. TI!' ¡'onRor aml'lIdcd protocol. If the reference Is formatilJ/ by reference. A i'efercnce to chanl!c!l in previolJ!lJ.v submltterl proto- may comply wit.h these t.wo conditions made t,o supporLinp, information al- infol'ml.tion imhrnl tteil Plf'\'iOllSh' IlI1!lt cols may be made. Whmiever a sponsor In either order. ready in the IND. the i;ponsor shall Identify Ulfl file by naiTic. r('fCrp.lice intenc!s to condur:t 'l cllnkal Investl¡ra- Iii! Not.withstantlin,, "~rairraph Identify by name, referciice number, lIumber, volume, anil piig-c. number tloii wit,h an exerpt.ion f!'ln !nformell (h)(21(j) of thl!l SCCt.iIJl. pi'otocol volume. ::nd page number the location whelP the iiiron~ion eiin he fOlllllI. A consent for emen"r.ney rf'!l~al''h aii iiet chiinge iiitenùp.d to eliiiinate aii a¡'par- of I.he Information. reference to Inf ;it,lon !lubmltted to forth In §50.21 of this ch'lpt.er, t.hp. !lpon- tnt iiiineiliate haiard 1.0 !llihjp.ctR may iJ) If the sponsor ùesii'eii ~A t.o com- the ag'ency by a .'son o~r than the iior shall submit a separate IND for ~-~ implr.iip.nt.eri irniillr1iat.r.I,y ¡irovl,If'r1 ment. on the i;uhiilsslon, iuest for !luch inveii*:iioii. JA iii suhsequp.ntly notified b.v pro- sud! r,oinment anil the 5,. .flc ques- t ocol amend men t and the reviewing tions FßA's response should addrei;s. §312.31 21 CFR Cil. I (4-1-99 Edilon) food and Drug Admlnjotrotion. tilS (e) When siilimited. A sponi:or shall mitteil as necessary but. to the (lxt~nt §J12.32 submit a proLocol amendment ior a fC'aiihlc. not more I.han every 30 days. hl'''''!iurii: or. if ;i ii Invp.stIRat.cir bro- new protocol or a chanKe In proLocol ch.. e Is not required or available. the mlt.tcir clt.her on an FDA Form 3500A before Its Implementation. Protocol (Collec\.oii of inLi.. "1"lIon rcqulrr.mpnts IIp- øpeclflcit.y or iip.verity of which Is not or. If preferre(l. on a CIOMS I form: re- amendments to add a new InvestIgator plo\'l'l h.v .the Offi;e of M~n:iP.eni!'iit nnd consistent with the risk Information porl.s from anImal or epideinIoIoglcn.1 Dudllct. undel' contl'OI niiiilicr 0910.0014) or to provide adùl tional information described In t.he I!eneral lnvl''ltl¡ra- studIes shall be submitted In a nar- about investigators may be irrouped ¡52 Fn ~831. Mar. 19. 198ï. M 'Iml'ndpil at 52 tiona) plan or elllt'where in the current rative format) and shall bear promi- and submitted at 30-ùay intervals. FH. 2303J, June 11. 1981; 53 FR 19J8. Jan. 25. appllcatlon, as amended. For example. nent Identification of its contents. i.e.. When severiil submii:i:ions of new proto- J!lRRI under. this deflnlLion. hepatic necrosis "IUD Sarcty Report." Each written no- would be unp.xpect.c,1 (by virtue of tificaLlon to FDA shall be transmitted cols or protocol changes are anticI- § 312.32 INO safety reports. to t.ie FDA new drug review division in pated during a short period. the spon- greater severity) If the Investlirator (a) J)l'filiilions. Thp. followi .. deflni- brochure only referred t.o elevated lie: tho Cent.er for Drug Evaluation and Re- sor is encouz'aned. to the cxLent fea- tlonii of i.erms apply 1.(. . Iils iil.cl,lon:- iiearch or the pi'oilnct z'evleiv ùlvislon sible; to Include these all in a single paLIc rn7.yiicii 01' hl'pal.1 tiii. Similarly. A.~sociatl!d wit/¡ tlie use of /lie drUQ. ¡ cerehral thl'mliocmholiiim and cere- in'the Center for Blolog-Ics I!valuatlon submission. There ls a reasonable possibility that i bral vasculitis would ue unexpecteù (by anù H.ciiearch that has responsiblIity (Collpctlon of informat.loii rPQlilrpmcnt.s IIp- the. cxperience may ha.ve been ciiused virtue of great,er iipeciflcity) if the In- for review .of the IND. If FDA deter- by the drug. prov!'d hy l.he Orfce of Maii"i:ement and í veiitiipl.tor brochure only listed cere- iriineii that additional data are needed. Bud¡i.." under coiit.rol number 0910-011) Di.eribilil.l/. A suhst.antlal ùli:rupt.on of bral vaiicular açcldent.R. "Ullxpected." the :t~ency may requIre furLher ùata to a peliloii's abillL.v to conùuct normal bc submitted. ¡r'2 pn Rfll1. Mar. 19. 1901. All nrnl'iidi'd at 52 ì as useù In this ùeflriition. refers to an Fit 2~ii3i. Jnnr. 11, J901: .'~ Fit J918. Jan. 25. Ilfe fiint'Lioml. ailvers'! drug- experlenr.e that has not (ii) In each written IND iiafety re- /.'.' .'lirr.atpiiing "rfi1r.r.ee drug experi- ber.1l pre\riously obiiervpil (e.g.. lneluiled port. thc 8ponsor sliall1ilentlfy all safe- J91111: 6J Pit r'J5:iO. Oct. 2. J9961 ence. Any aÙVl'li'H! ,¡rug' expp.rience that I in the inve8tigator brochure) rather l.y rcporLs prevlouiily f1ed with Lhe § 312.31 Information amendmenfs. places the patient or subject. in the , . than from the perspective of such expe- IND concerning a similar aùverse expe- vicw of the invei:i.lgator, at ImmedIate i rlence noL helnir aiit.iclpatcù from the rience. and shC\1l aniilyze the slgnifi- (ii) Rl'qztirement for information amend- risk of death from the react.ion as It oc- ment. A sponsor shall report in an in- phnrinn.colop,ical propertie8 of the caiice of the aùversc experieni'f' in I1ght curreil. i.e.. It ùoes not Include a reac- pliarmllceutical product. of Lhe pleviouos. similar reports. formation amendment essimtial infor- tion tli:i.t. hail It occurred In a more se- mation on the IND Lhat is not within vere form, mii;lit have caused ùeath. (1)) ReviclQ of safetr¡ information. The (2) Telephone and facsimile trans- I sponiior shall promptly review all infor- mission safety reports. The sponsor i:hall the scope of a proLocol amendment, . Serious adverse drll,a experience: Any i mation relevant to Lhe i::iety of thc alr,o notif.v FDA by telephone or by fC\c- IND safety reports. 01' annual rp.port. aùverse ùrug experience occurri ng at I i ùrug obtalneù or otherwise received by simile' 'ransmission of any unexpected Examples of Inforina' .:1 requiring an any (lose that reRllltii in any of the fol- the sponsor from :tny soii'ce. foreign or fat.al or life-threatening expelicncc as- infoi'hi:ition amenùment includc: lowing outcomes: Death. a llfe-threat- domp.stlc. Includlnir Information de- Bocin.Leù with the use of the ùrug. as i (1) Ncw toxicology. chemistry, or ening adv.erse ùrug experience. Inpa- rIveù from any clinical or epiclemlolog- soon as possible but in no event later ¡ other t,echnical inforiiiition; or tient hoi:pit,ali7.at.on or prolongation of leal InvestiJ!atlons. animal investl~a- than 7 calendar ùays after the spon- (2) A report reg;u'dliiir the discontinu- e)(ii:ting hospiL.illiz:ition. a perslstenL or tionii. ; ciimmercial marketinJ! experl- Ror'ii Initial receipt of the information. ance of:l clinical investigation. significant disabilit,Y/ilicapaçity. or a congenital anomaly/birth defect. Im- encc. 'reports in Lhe scientific lIt- Each t.elephone call or facsimile trans- (b) Content and format of an informa- eraLure. and unpublil'hp.ù scientific pa- mission to FDA shall be IT'aiismitted to tion amendment. An informaLion a'''v:nù- portant medical events that may not pers. aii well il8 reports from foreil,n Lhe FDA new drug review division In ment is required to bear promInent rcsult in deaLh. hc life-threatenlnir. or reglll:'lory allt:horities t.hat have not the Center for Drug- Evaluation anù Re- iiùentification of Its contents (e.g.. "In- require hO!;pitall7iiLivn may be consid- already been previously rCJlorLed to the scarçh or the proiluct review division ered a i:crious ailverst! ùruir experience agency h.v the sponsor. formation Amenùment: ChemisLl'Y. when. bn.¡:eil upon appropriatemeùlcal In Lhe Center for B10lQgics EvaluatIon Manufa.cturinir, anù Control", "Infor- 'C) IN lJ safety reIJr71;. (1) Writen re- anù Research that has responsiblli ty judimient. they may jeopardize the pa- porIS-(j Thc sponsor shall notify FDA mation Amenilinent: Plin.rmÅcolo¡;y- Lient 01' iiubjeet anù may require med- fol' review of Lhe IND. 'loxicohi!:y", "lnforllaLi(lii Amenù- ani! all p'!rLlclpating- invf?stlg'ators In a (3) R~porting format ar freqiltncy. FDA Ical oz' ¡;Ur~ieal Int.ervenLioli Lo i,rev!'nt wrll.l,en IND safety rciiort i)f: ment: Clinical"). aiid to contain the one of Lhc outcomes llsteù In tliiii iIer¡- ma.v roquest a sponsor to submit IND (A) Any ailverse expiirience alillocl- safety reporLs in a format or .:it a fre- folloiving: ni tion. Exarrpl C1l of Slic)Z incrlical atcù wll.h Lhe use of Lhe drug' that is (1) A stat.cment of the nat.ure and event1l inchide allerJ!!r bronchoiipaiim quency diffei'ent than that required purpose of the amendment. l'eiiuii'tng- inteniilvc tri~atment in aii bot.h sp.rIous and uiiexpected: or under this paz'agraph. The sponsor may 11) Any f1nrlliil! from t.r.l't.ll In labor:- aliio proiiose anù adopt a different re- (2) An or¡;ani7.ed submission of the eniel"~enr;y room or at home, hlood tory anlma.ls that iiul:!i-ciits a sj~nir- daLa in a foz'ma:t appropriate for sci- dyscz'asias or convllll'ionii Lhat do not ('''''t, risk for human subjectsincluùlnir porting format or frequency if the entific rp.vlew. result in InpaLienL iir)¡:pit,alii:at.ion. or rej'orts of mut:ig-enlclty. change is agreeù to in advance by the (3) If the SPOil,,'lr desires FDA to com- the development of drug depenùency or teratoJ(eniclty. or carcinog'enlclty. iilr'lctor of the np.w drug review divi- ment on an information amenùment, a drug- ahuse. Each notIfication shall he made as soon sion In the Centiir for Dz'ug Evaluation request for such comment. Unl'.Tpccll'd adt'crse cirug e:rpp.rience: aii pos8ilile iln'l In no ('vent later than and Research or the director of the (c) Whl'n .tlClmiiti'd. Information An.v adverse tlrug expcrip.nce. the iipecl- 15 calendar ilays aft'll' the i:poniior's inl- Pl'oducts review ilivliiion in the Cent.er amendments to V-rND shoulù be sub- ficiLy or i:everll.y of whi':h Is not con- fol" Biologics Evaluation anti H.esearch sistenL with the current investigator -lal receipt of the Information. Each wlilch is responsible for rt'~ of the itten notiflcilt.lrin may hI! submit.t.ed FDA Form 350iJA or In a narrative IND. "' ~ (.1) A ..8flnnsor of a cllnicai titudy of a format (foreIgn events mn.v hp. iiiih_ ""'l,~trd"n,~l ,1...... _ ..._.. ~__.,_.t . ~ .. § 3 12..33 21 qR Ch. , (4-1-99 Edilion) Food and Drug Adminislralion, HHS a safety report for any adverse experI- ence associated with use of the drug protocol numher). its purpose.. a hrief § 3 12.34 that Is not from the clinical study st.atimient idenl.ifYin~ the Jl:'l.limt pop. (el A description of lIny Ri~nificant ulatlon, and a statement as li, whether PJiase 1 pi'otocol moilifications made the Use of a drug for diai:mostic pi'r. Itself. (\urlnlf the previous year and not pre- poses. If a protocol for an investl~a- (d) Followiip. (1) The sponsor shall the study is completed. (2) The total nu¡r"'~r of subjects Inl- vlouslJ' reported to the IND in a pro- tlonal drug meets the criteria of thIs promptly investigate all safety infor- section. the protocol tialIy planned for inclusion hi the tocol ameniIment. Is to be submitted mation received by it. en A brief summaiy of slg-nincant as a treatment protocol under the pro- (2) Followup information to a safety study; the number entered Into the foreign marketin~ developments with visions of this section. report shall be !lubmltted as Boon as study to date. tabiilat.ed by itrre ¡rroiip. the drug during the past year, such as (b) Criteria. (1) FDA shaIl permit an the relevant Information is available. genilr.r, and race: the niimb!)r whoioe approval of rnarki;ting In any country investlR'ational drug'to be used for a (3) If the results of a sponsor's inves- participation in the study Wfl'l com. or withdrawal or Suspension from mar- treatment use under a treatment pro- tigat.ion show that an adverse drul: ex- pletr.d as planned; and thr. n. f!r who i keting In ;lny country. ; tocol or treatment IND if; perience not initially determined to he dropped out of the study fur any rea. I son. (g) if des,rl~II by the sponsor. 1\ lOR' of (I) TIie druir Is Intendec! 1;0 treat a se- r.eportable under paragraph (c) of this I any outstanding buslne!"s w..:! .:.spect rious or Immediately We-threatening section is so reportable, the sponsor (3) If the study has been completed, to the IND for which the sponsor re- disease: shall report such experience In a writ- or if interim results are known, a brief queiits or expect.s a reply. comment, or (II) There is no comparable or satis- ten safety report as soon as possible, description of any available st.udy re- meeting. factory alternative drug or other ther- but in no event later than 15 calendar sults. apy available to treat that stage of the . days after the detennlnat!on Is made. (b) SummnrJl inlOT1lJltion. Information (Collection of Inrormatlon r~i¡ulremenls np- disease In the intended pa.tient popu- (4) Results of a sponsor's Investl~'1\- obtained duri nil the previou!l year's I pro'.ed )¡y the Offce Q( M~ na~emeiit and clinical and noncllnlcal Investigations, Duil¡¡et under controlllUmlier 091~14) lation; tloii of othp.i' safety information shall (ill) The druir Iii under Inve.'itlgatlon be submitted, as appropriate, In an in- includlllg": Ir'2 Fit Rß3L, Mar. 19. 1987. M nmendrd ll 52 In a controlIed clinical trial under an formation amendment or annual re- (1) A narrative or tabular IHlmmary Fit 2.1031, Julie 17. 1987; 6: FR 6862, Feb. 11, INO In effect for the trial, or all clin- port. showing the most frequent and most i 1998) seril)\s adverse experiences by body ical trials have becii completed; and (e) Disclaim~T. A safety report or i §312.34 Treiitment U'le or an investign- (Iv) The sponsor of the controlled other information suhmitted by a spon- system. tional Dew dng, Clinical trial is actively pursuing mar- sor under this part (and any release by (2) A summary of all IND safety re- keting- approval of the Investigational ports submitted during the pa!lt year. (a) General. A druir that is not ap. FDA of that report or information) proved for marketing may be under drug with due dl1gence. does not necesSfl.rlIy reOeet a conclu- (3) A list of subjects who died durln~ I participation In the invelitiR'atlon, with clinical Investliratlon for a serious or (2) Serious disease. For a drug in- sion by t.he spomior or FDA that the re- i Immediately life-threatening dlseaie tended to treat a serious dIsease. the port or Information constitutes an ad- the cause of death for each subject. Commissioner may deny a request for mii:sion that the drug- caused or con- (1) A list of subjects who dropped out condition In patients for whom no COIl- parable or satlsfaGtory alternii.OIve treatment use under a treatment pro- trÙ:mted to an adverse experience. A during the course of the Investigation tocol or treatment IND If there is in- sponsor need not admit, and may deny. in association with any ailverse experi- drug or other therapy III available. Dur- liig the clinkal InvE'sl.lgatlon of the sufficient evidence of safety and effec- that the report or Information sub- ence. whether or not thought to be tiveness to support such use. drug- related. druir. It may he appropriate to use the mitted by the sponsor constitutes an (3) Immediately life-threateninr¡ disease. admission that the drug caused or con. (5) A hrief description of'what. If any- ".g in the treatment of patIents not tribut.ed to an adverse experience. thing, was obtained t.hat is pertinent to . IL the clinIcal trials, iii accordance (I) For a drug intended to treat an im- an undersl.anding of the drug's actions. with a treatment protocol or treat- mediately life-threatening disease, the (Collection of Information l'equll'ement.s ap- ment IND. The purpose of this section Commissioner may deny a request for prove.. by t.hc Offce of Miiiini:enicnt ami includiiig, for example. information about dose response, information from Is to faclitate the avaIlabilty of treatment use of an investigational fluili:et uiiler contl'oJ nuiiber 0910-0014) drug under a treatment protocol or controlled trails, and Information promisIng new drugs to desperately II patients as early in the drug develop- treatment IND if the available scI- 152 FIt 80:i1. Mar. 19. 1987. flll amend!!rl nt 52 abou t bioa vail Fit 2~O;lJ. June 17, 1987: 55 FR I1f79. Mar. 29, ali i Ii t:\.. ment process as Po~sible, before gen- entific evidence, taken as a whole, falls 199; 62 FR 52250. Oct. 7. 1997) (6) A list of the preclinical studies eral marketing bei;in~, and to obtain. to provide a reasonable basis for con- (includinir animal studies) completed cluding t.hat the drug: §312.3:i Annunl reports. or In progress dui'ing the past year and additional data on the drug's safety a summary of the major preclinical and effectiveness. In the case of a seri- (A) May be effective for its Intended A sponsor shall within 60 days of the ous disease, a drug ordinarily may he use in its intended patient population; annivcniary date that the IND went finil':'h!l.(7) A summary of any sillnIficiint . ml\de avaIlable for treatment use under or into effect. suhmit a lirief report of the manufacturing or microbiological this section during Phase 3 investiga_ (E) Would not expose the patients to pi'oirrei:s of the investigation that in- tions or after all clinical trials have whom the drug is to be acIminIstered to cludes: changes made during the past year. an unreasonable and significant addi- (C) A description of the general hives- ; be~n completed; however. In appro- (a) Iiidi¡;icluul stiid.ii informatinn. A tirratlonal plan for t.he coming year to i prIate circumstances. a druir may be tional risk of ilness or injury. brIef summary of the status of each ¡ , made avallabJp. for treatment use dur- (II) For the purpose of this section, stuily in proirress and each study com- replace. t.hat submitted 1 year earlIer. The general Invesl.gational plan shaH : inll Phase 2. In the case of an Imme- an "immediately life-threatening" dis- pIE-ted during the previous year. The diately life-tlireatenlriir dliieaiie, a druir ease means a stai;e of a dlseMe in llllnlmary is rl"c¡ulrt'd to include (,he fol- contain the information required under ; may be made avallable for treatment which there Is a reasonable likelihood lowlii!~ iiifonmi.t.ion for ('\ch study: § 312.23(a)(3)(iv). . use under thl" section earUer than that death wil occur within a matter (1) The t1tV- f the !\tuily (wit.h any (d) If the Inv~stlA'ato¡' brochure has L : Phase 3, but ordinarIli-' not earlier than of months or In which ~ature death aPPlopriate. ,y ide~,Ciers such as lief!n revised. a dC$crlptiiii of the r~vl. sian an~ copy of the new brochure. Phase 2. For purposes of this section, Is likely without early tment.. " , the ':'treatment uiie" of a drug Includes (cl,,/~aft!r¡'Lards. Treati.."nt use of an inve.'jÎ:'lllatlonal drip' tq "n",IlHn~n" __ I ;312.35 21 CFR Ch. I (~-1-99 Edilion) Food and Drug Administration. HHS he sponsor and invrstiirators com- (2) A I.reatmi'lt. prot.ocol is t.o be sup- §312.40 'Iyln¡r with the saff'Jruards of I.he IND of thl! technical Informal.lon contained rocess. IllCludin~ the regulations gov- ported by the fOllowing: H.ockvlle. MD 20857. 301-4.1:i-4320. After (i) Informational brochure for sup- in the sponsor's IND into the medical rning' informed consent (21 CFH. part practltlonei"s treatment IND. normal working hours. e;i, .~(!rn stand- 0) and institutional review boards (21 plying to each treating' physician. ard time, the request shonl'¡ be di- (ii The technicai information that Is (Jil) A statement of t.he steps taken ~FR part 56) and the applicable provl. by the practitioner to obtain the drug rected to the FDA Dlvlslon ,,¡ Emer- ri"levant. to safety and effl'ltiveness of gency and Epidemioiogical OperatIons, ions of part 312, 1iicludlnl¡ distribution the dru¡r for the intenderl t.reai.iir.nt under a treatment protocol from the f the driiir throuidi (¡ualifind experts, druir Sponiior. . 202-57-8400. Except in extraordinary ialntrnance of adequate manufac- purJlOII~. Iiifoi'matfon cont.alri..d In the (Iv) A t.reatment prot')eol containing clrcumiitances, sucli aut.horization will urlnJ; facilities, and suumission of IND spoiisoi"s INO may be incorporated by the same information IIl1ted in para- be conditioned. on the sponsor making '\feLy reports. reference, an appropriate IND submlsiilon as soon (iii) A commitnip.iit h:v til.. sponiior to graph (a)(l) of this section. (d) Clinical hold. FDA may place on (v) A iitatement of the Practitioner's as practica1ile after receiving the au- ::f1f1ure comJiliancn of all pai'tir.patlng thorization. IInlcal hold a proposed or onirolng'. fnvesl,l!!at.ol:i wii.h the lnforinp.d con- quaiiricatlonii to ullp. the Inveiitlga- :'eaLmcnt protocol 01' l.n~aLmr.llt IND sent requli'('ni('nts of 21 Ci'l! pai't !i0. tlonal drug for the intended treatment (Collp.ctlon of Inform:\l.lon r..qulrements RJ) i accordance wi th § 312.42. use. l'rovp.d by t.lC Orrce of Miiiiureinent and (3) A Ilcensrd pract.ioner who i'ecelves (vi! The pract,itioner's iitatement of Ducliiet under control number 0910-14) 2 Fit 1!J17r.. M:iy n. 1~87, AS liii('llclctl at 57 an Invl'stliratlonal (1rui: for t.reatment familiarity with Information on the It 132.18. A pI'. 15, 1992) Uf1e under a treat.ment protocol is an drug-'s safety and effectivrDl'lIl\ derived (!i2 Fit 11831, Mar. 19. 1987. ni nmr.ndpd at 52 "lnvef1tig-atol'" under the prot.ocol anil FR 23031. June 17, 1987; 55 Ffi 11579. Mar. 29, from prevlouii clinical and iionclinicai 1!I90J 312.35 Subniissions for trentment Is reRI)Onf1ible for mt~ellng' all aplllÎla- u~c. experIence with the rJI'Uir. hie InvesLh:at.or I'l'sponsihilfties iiiiiier (vII) Agreement to report t.o FDA § 312.38 Withilrawnl of nn IND, (a) Trratment Protocol wlmiilcri b.ii this part. aiid 21 CFIt parts 50 and 56. iiafet.y Informat.ion in accordance with (a) At any tIme a sponsor may with- I/D spuiisor. Any f1ponsor of a clinical (b) 1'rrClt,iw71t IN/) .~ulmiillr.d hi, li- §312.32. IvesLlgaLion of a drug' who InLcnds to cmi.~r.d pricfititmcr. (1) If a licensed meil- (2) A iic('ii.~eii pract.ltioner who /luh- draw an' effective IND without prrju- Jonsor a treatmf'nt use for the drug ical pi'actitioner want.s to o1itain an In- mlt.s a tr~atmp.iil, IND unùer tIlis sec- dicp.. iall submit to FDA a t,reatment pm- vef1t.i¡rational drug' i:uhj~ct, 1.0 a con- (b) If an IND is withdrawn, FDA shall tion iii tIie sponsor-inv~!ltiimtor for be so notifed, all clinical invei:t.lla- icol under §312.31 if the sponsor be- trolled: clinical trial for a ti'eatmtnt sucli IND and is reRponf1ibJe for mep.t- eves the criteria of §312.31 are satls- use, the practil.ioner should first at- t.ions conclucted under the IND shall be Ing aU appllcahle sponsor and lnvestl- cniled. aU current InvestlJ;aLors not.l- eiL. If a protocol is not submitted tempt to obtain the i1ru~ from the I!lIor r~!lponslliiitles under this part rirler § 312.31, but FDA brlieves that iiponsor of I.he controlled trial uniler a and 21 CFR parts 50 and 56, fied, and all stocks of the drug re- ie protocol shoulil Jiave heim sub- treatment prol,ocoJ. If the spollitor of turned to the sponsor or otherwise dis- .ittedunurr this section, FDA may the coni.rolll'd cllnical investigation of CColJp~t1on or Information reriulrern"nts IIp- posed of at the request of the sponsor !rm the protocol to be submitted pro\'f't! hy the OHke or Man:iiieii~nt and In accordance wi th § 312.59. the uru:. '.'ii not establii:h a treatment Butlltp.t uniJer control nurnber 119l().JOl1) PlOtocol for thE! ùrug' unilr:r parag-raph (e) If an IND Is withdrawn be~"'1Re of ìder §312.34. A treatment usc under a a safety reason, the sponsor shall 'eatni~nl. protocol may be¡rin 30 clays (a) of this f1ection, the lic~nsed rnedi('al ¡52 FH I!li;;. M:iy 22. 1987. n~ :imrmilcd at 57 'tel' FDA receivef1 the Pl'tocol or on practitioner may i:~l'k to ohl.aln I.he FIl1321'l. Apr. 15. 1992) promptly so inform FDA, all partlcl-, irller notification by FDA that thE! drug- frorri the S¡iOllf10r anil suhmit a patiii¡r investigators, aiid all reviewing 1312.:i6 EmerR'ency mie of ltD inves. Institutional Review Boai'cls. toget.her tatment use described in the protocol treatment IND to FDA requesting- au- tigational new dl"g. ay begin. thorization 1,0 use the inveiitiKational with the reasons for such withilrawal. Need for an Investigational drug may (1) A treatment protocol Is required drug for treatment use. A treatment CColI!'ct.lon of Informiitlon i'equlrements ap. , contain the following: use under a treatment IND may hr.irin arli;e in an emerJ;ency ¡iit.ulIl.on that proved by the Offce of MiinallE'inent and 30 day!! after I~DA receivr.it the INO or doE'S not allow time for submission of Biidiret u nihir control number 0910-0014) (j) The intended use of th.. dl'UK. an IND In acconlani:e wllh § 312.23 or OJ) An explanation of the ratibnale on earlier noti ficatioTl b.v FDA that t.he §3i2.31.In sudi a cas~. FDA may au- r!i:i FIl "R:l1. Mar. 19. 1987. M amended at 52 i' use of the drug'. including. as appi'o- treatment use unùer I.he IND may FIl23U31, June 17. 19871 ueKin, A I.reatment INO Is required to thorize shipment of the flruir for a spec- 'iate. eit.her a list of what available iflp.d use in advance of subrnli:lIlon of an ¡¡Imens orrlinarily should be tried be- contain the following: IND. A request for such authorization Subpart C-Administrative Actions. i'e using the investigatiomi.I drug or (I) A cover sheet (Form FDA 1571) i explanation of why the use of the meeting- §312.23iiO(1). may "be; transmltterl to FDA by telce §312.40 Gr.neral requirementR for use: vestiJ;ational drul¡ is preferable to (11) Information (when not provided phonp. or ol.her rapid communication or nn inveøtiiintilJnnl new dnig in a by the sponsor) on th~ rlrug-'s chem- means, ,For Ill'cstlimtlonal biological clinical in\'c!lti~ntion, . e use of availaule marketed treat- drugs. thp. requ.t9t i;hou1c1 be dIrected to ents. istry, manufacturinJ;, a. ..(int.rols. and (a) An Investig-at.lonal new drug may pi'ior clinical and nonclinical experi- the Division of Blolollical hivestiga- (iii) A briE'f description of the criteria tlonlll New Druiri; CHFU-230), Center for hp' ui:ed in a clinical In'" ~t,iJration if" i' patient f1election. ence with the drug' submltteù In ac- Dlolorics F:\"aluatlon lilI'! It'.iiearcli. the followl",: conult.lon!! arc met: ¡iv) The method of administration of corùance with §312.23, A sponsor of a (1) The sponsor of the investigation clinical Investigation subject to an INO 8800 Hockvilp. Pi ke, Ilethf':"I~. MD e druJ( and the dosaires, 20892. 301-413-1861, For all oth'~i ¡nves- iiulmilts an IND for the drug to FDA: ~v) A des('ri¡it.on of c:linical proce- who f1uppllef1 an Iiivm¡tiinl.t.ional drul: to t.he IND Is In effect under parai;raph Cb) a licensed med ¡cal practitioner for pur- tlJ!ational drug'S. the request for aii- res. I:homt.oi'y t~ or oLlil'r lIe:\s- i.riziit.ion iihoiilrl hp. i1irectf'.f to I.hp. of this sect.ion; and the ap~or con¡- os to monit.or t.hi ~ct.s of the drug poseii of a R~Jlarate trr:alm"'nt clinical ¡ilirs wit.1i all appllcalile lf 'em~iits invf!stig-at.ion iihall hE' decmed to au- Ulll'llt Mn.iiaii..m~nl. anil Hcportiriir d to ii;iilmize risk. -: -...1lr:h.'HFU-531. Cf"nt.nr f'ir Drug- Bval- In tli iii part and ¡iarts 50 anú \V I th re- t.horize t.he ~orporatlon-hy-refei'ence uat.fnn :lrHl n",~ol)p",.h r.r.lln r.:_l. _6._ .. Rp~ct to/X'fie conduct of the clInical in- §J I2.41 2 I CFR Ch. I (4- 1-99 ,.. ':1.....,\ Food and nrug Adminisfralion, HilS (2) Each p:irt.iip:ii.in/! invest.igator cliniial investigations or reiipon!'e to §J 12.42 conducts his or her invcsttgatlon in the agency. (Ii) The pl:i11 Clr Jlrrtoc0l for L.he fri- coiipliance with the requirements of vesthmtlnii is clp.arly dl'ficlrint iii i!e- (\iH4)(i) t.hrough (b)(1)(viii) of this sec- th i s part and parts 50 aiil fiG. IColIl'ctifln of Inrol'l1:\t.on rl''1l1lrl'nil'nt s IIp- siini tQ II. ;~t it.s s(.at.cd ohjiict.ives, tl(m aJI)lIY. (ù) An IND goes into effcct: provl'll Iiy Oie Ollil,C' or l\anAI~l'liil'1i1 Rliil (31 CJI"lcal hol.l of a treatmeiit 'IND' (1) Clinicrl ¡wid of an.ii sturl.v tliat is 1111'll(l't un,lpr cont rol numb!'!, 0910 OUHI (I) Thirty days after FDA receives Cir t.reatiieiit protocol. not desi!lIcd to be adequate and well-con- the IND. unless FDA notifies the spon- (!i2 Fn ß831. 1-nr. l!l. J987. :\~ amended at 52 Ii) l'roposed Use. FDA may place a trolled. FDA may place a proposed or sor th:it, the investigations desci'lhcil In FR 23031. Julie 17. J!lß71 . proposeil treatment INn or treat.ment ongoing investigatIon that Is not de- the IND ai'e subject to a clinical hold PJ'tocol on clinical 11l",1 If I i. Is ùeter- Shwed to be ailequate and well-con- § 312.42 Cjinif'AJ holds and request!! for mlneil that: trolle,l on clinical hold If It finds that: under § 312.42: or modification. (2) On earlier notiflcation liy FDA (A) The pertinent criteria. in m Any of the conditions in para- t.h:it the clinical inVf~stlKatlomi In t.hc (a) G('ncrnl, A dinlcalh')ltl Is an or,lp.r §:i12.31(b) for piirniitl.inl! t.he treatmeiit graph (b)(1) or (b)(2) of thIs section INIJ may 1,,'I~lli. FDA wIll notify the issued h.v FDA to the spon~IJI' t.1J delay u!'e to hei:in an) not satiMil'll: or apply: or a llJOPO"C'11 cllnk:i! lrivl'stli.:iI.lnn or to (0) Thp treatlll'nt prot.ocol or trp.:it.- 'Iill There Is reasonable evidence the sJlonsor in wi'it.lng of Lhc date It rc- i-ti;)Jciiil an oiii:oi ii~ hl\'cst.g-:it.loii. The UJl'nt IND doe!' not 1."lI(:iln t.he fiifOl'- fiivl'!'tlmi.lon t.hat 1': \lot ilp.ll!imed to ùe ceives t.he IND. elinicaJ hoI,) order may apply to oiie or maL.I on required under § 312,35 (a) or (h) ade(iuate anil well-controlled Is imped- (e) A sponsor m:i:v ship an iiivclItiga- more of the investi¡!aLioiiS covcred by .1.0 make the specified determInation Ing- eiirollment In, or otherwise inter- tional new drug to investigators named .an JND. \\'hen a Pl'oposcil ~'l' .IV is uniler §312.31Iù). fering with the coiiduct or completion In the INO: iil:i.eccl on clinic:i1 h('ld. !'uhje~,., may tli) OIl.Qoinr¡ iac, FDA may place :\11 of. a study that Is de~iR'ned to be an (1) Thirty days after FDA receives not. lie ¡dveii I,he im'est.igational drug-, ongoiiiir treatment protocol or treat- ailequate and well-controlled investiga- thc INO; or When aii ong-oiiig st.udy is pl:iced on ment IND on clinical hold If it Is ileter- tion of the same or another Investiga- (2) On earlier FDA authorl7.:ition to cllnk:i1 hold. no new !'ulJjccts may he m I lied tha i.: . tiunal ilrug: or ship the drug-. recriiiteil to the study and plac!'l on (A) 'lhp.re becom~s available a com- (iii) InSuffcient quantities of the In- (d) An investii::itoi' may !lot ailinin- the invest.ig-ational dl'g-: patieiit.!; al- paralile or satisfactory al ternatlve vesti¡:ational drug exist to aileQuately ister an investii:ational new drug to ready in the st,udy should be tak~n off ~ dnlK òr oLhcr therapy to treat that conduct both the investiKation that Is human subjects unl,¡¡ I.he IND goes into thel'aJlY involvin¡r the inve!lthmtional st.a~e of the disea!'e in t.hn Intended pa- not designed to be adequate and well- effect under paragraph (b) of this sec- ilruK unless specifically permitt.ed by ,tIei;;. population for which the inves- eonti'olled anil the investigations that tion. FDA in the interest. of p:itie~it lIafpty. I tl.!atlonal ilrug I!!\ieing ulIed; are dei:lgneil to be ailequate and well- (h) Grounds for il1po.~iloil of I:. .Iiciil Ol, The Im'estli¡atioiial ilru¡: is not controlleil; or §312.41 Comment and advice on an liolrf-(1) Clinical hold uf /I l'ic.~c 1 stud,V : unilr.r investli¡ation In a controlled (Iv) 'I'he drug has been st,udieil in one IND. unci'!r an IND. FDA may place a pro- ,db !.d trial under an IND iii effect for or more aileauate an'! well-controlled posed or oii¡roin¡: Phase 1 investii,ation ; the 'Üial anil not all contl'lIeil clinIcal inviistlgations that :nrongly suggest (a) FDA may at any time ilurinla the on clilliçal hold if it finds that: 't.rlals neceiis::r'l to RUpport a mar- lack of effectiveness; or !cour~e of the investigation Commu- (I) Human subjects arc or would he 'ket.iiil¡. appllc;il;nn have been com- (v) Another drug uniler investlg-atlon 'nicate with the sponsor orally or in or approved for the same inillcation writ.lni: ahriut deficiencies in the IND expoi;ed to an unl'~a~onahle anilsigiiifi. .pltte!J.. or a clinical stiidy under the caiiI. risk of illness 01' injury:. i INfJ has heen placed on clinical hold: and available t.o the saine patient popu- or :iIJOU t FDA's neecl for more data or (i) The clinical ili..e~l;il!ators naineil I (e). The spom'wr of the control1ed' )at.on lia~ dl'monst.r:teil a better po- Iiiformat.on. In the IND are not l(uallfied by reason clinical trial Is. not puri:uing- marketing tential benefiVrisk ùalance; or th) On the :.ponsor's request, FDA of their scientific t!'ainin¡: and experi- 'apPi'oval wiLh due dlligenco;; (vi) The dru¡: has received marketlnir iwill provide advice on ~peclflc mat.tf!rs ence to conduct the im"estigation ile- ! (Di If the treatment IND or treat- approval for the ~aine inilicatlon in the , relating to an IND. Examples of such scribeil in the IND: ¡ ment prot,ocol Is iiiten1lp.il for a i:erious same patient POpulation; or ad..ice m:iy include :iilvicl' on the aile- (iiI) The invest.ii:atoi' brochure is ijisea!'o;. there is Insiiffici~nt evidence (vii) The sponsor of the study that Is desig-ned to be an adequate and well- qu:i('~' of t('l'hnkal d:it.a t.o sulfport. an misll'adini:. (,ITO Ill' 'lIo;. or m:iterially of i:afety and effectiveness to support im'l'sl.i¡mtioiial plan. un the design of a incoiiplete: or such U!1('; or . controlled Investii;atloii is not actively clinic:i1 triaL. and 011 whether Jlroposf"l (iV) The IND ilo~s not contaiii suff . lEi If the treal,rt(lnt protocol or pursuing market.lng approval of the in- investigations ai'e likely 1.0 produce thi: cient infonn:iLioll requil'l'l) undei' treat,menL IND was hasp'J 011 an Imme- vestigational drug wi th due diligence; data :ind infurmation that Is needed to §:i12.23 to a~se~s the rl!lks to suùjecl. of iliately IIfe-threateniilir diseMe. the or meet requii'ements for a marketing ap- the proposeil stuilies. available scientific evidence, taken as (viII) The Commissioner determines pllcat.ion. (2) Clinical hold of a l'liasc 2 or J stlid.1I a whole, fall!! Lo pro\'i(le a reasonable that It woulil not be In the public inter- (c) Unless the communication is ac- iind(!r an IND, FDA may placl' a pro- basis for concluilinl! that the dru¡:; est for the ~tudy to be conducted or cQntinueil. FDA ordinarily intenils that companied b.. a clinical hold posed or ong-oinK l'!i,\se 2 ur 3 Iiives- (1), May be effective for it.s intended order tii,àtion on clinical holil if it finils Use fn ILs Intended population; or clinIcal holds under paragraphs under §:ii2.12. FDA communications (b)(1)(l). tb)(1)(\i) and (b)(4)(v) of this wif.h a SJ.onsor under this section are thil.: (2) Would not expOse l.he patients to (i) Aiiy uf the conditions in para- whol) the drug is to be li'lminlstnred to section would only apply to additional solely auvisory anil do iiot require aiiy enrOllment iii nonconcurreiitly con- moilification in t.he planned or oni,oing graph (!))(1l(i) throug-h liv) of this sec- an unreason:ible anù slirnlfirant aildl- tion apply; or tional risk of iInp.~s or injui.\. trolled trials rathei' th:in eUmin~tfiig -- (iii) FDA may plar:e a proposeil or 011- . continued access to lnillv~als already -"'OlllR treat.miint INO or t.reatment pro- receiving the investlllati 'drull. "oco) on cliniial hold if it finrls that (6) Clinical/in¡. of an!l L tÍ,Qfl/ion in- -i -!I aiiy of the conditions In naral!r:inh t'(Jll'in~"~Jin e.f( ..¡ltìo71 from informed COll- lllIn' ..+01...._ r rn. .. i ..... '§3'12.42 21 CFR Ch. 1(4-1-99 Edifion) may place a proposed or ongoing Inves- Food and Drug Administration, l1HS tigation Involving an exception from endar day~ of receipt of t.he rec¡uc!lt ;\. §312.44 the complete response. FDA's response informed consent under §50.24 of this 1312.44 Termination, (viiI) The sponsor fails to submit an chapter on.clinical hold if it is deter- wil either remove or maintain the clinical hold, and wil state the reasons (a) General. This section describes the accurate annual report of to""! inves- mined tha.t: procedures under whIch FDA may ter- tigations in accordance with §312.33. for such determination. Notwith- (i) Any of the coniIitions in para- minate an IND. If an lND is termi- (Ix) The sponsor fails to comply with graphs (b)(l) or (b)(2) of this section standing the 30-calendar day response nated, the sponsor shall end all clinical any other applicable requirement of apply; or time. II sponsor may not proceed with a investigations conducted under the this part, piirt 50, or part 56. (U) The pertinent criteria in §50.24 of clinical trial on which a cllnh11 hold IND and recall or otherwise provIde for (x) The IND has remained on inactive this chapter for such an investigation has been Imposert until the sponsor has the disposition of all unused supplies o( status for 5 years or more. _ to begin or continue are not submitted been notified by FDA that the bold bas the drug. A termination action may be (xi) The sponsor falls to delay a pro- or not satisfied. been lifted. based 00 deficiencies in the IND or hi posed investigation under the IND or (c) Discussion of deficiency. Whenever (0 Appeal. If the sponsor dls:grees the conduct of an investigatIon under to suspend an ongoing iov~~t.igatlon FDA concludes that a deficiency exists with the reasons cited for the clinical an IND., Except as provided In para- that has been placed 00 clinical hold In a clinical investigation that may be bold, the sponsor may request recon- graph (d) of this section, a termInation under § 312.42(b)(1). grounds for the Imposition of clinical sideration of the decision in accord- shall be preceded by a proposal to ter- (2) Phase 2 aT 3. FDA may propose to hold FDA wil, unless patients are ex- ance wi th § 312.48. minate by FDA and an opportunIty for terminate an IND during Phase 2 or posed to immediate and serious risk, (g) Conversion of iN" on clinical hold the sponsor to respond. FDA wil, in Phase 3 if FDA finds that: attempt to discuss and satlsfactorlly to inactive status. If all investigations general, only initiate an action under (! Any of the conditIons in para- resolve the matter with the sponsor be- covered by an IND remain on clinIcal this section after first attemptio!\ to graphs (b)(l)(l) through (b)(l)(xi) of this fore Issuing the clinical hold order. hold for 1 year or morl!, the IND miiy resolve differences Informally or, when section apply; or (d) Imposition of clinical hold. 'I'he approT"rlate. through the cllnlcai 'hold clinicai hold order may ùe made by be pJacl!d on Inactive status by FDA (ll) The investl¡;atlonal. plan or pro- under § 312.45. prucedliles descrIliedln §312.'2. toC01(8) is not re:\...~onable as a bona fide telephone or other means of rapid com- (b) Grounds for terminatian-H) Phase scientific plan to determine whether or munication or in writing. The clinical (52 FR 8831. Mar. 19. J987. as amended at 52 1. FDA may propose to termlna.te an not the drug i8 safe and effective for hold order wil identify the 8tudles FR 19-17. Ma.y 22. 1987; 57 FR 132'19. Apr. 15, IND during Phase I if it finds that: use; or under the IND to whIch the hold ap- 1992; 61 FR 51530. Oct. 2. 1996; 63 FR 68õ18. ,(I) Human subjects would be exposed (11) There is convincing evidence plies. and wil briefly explain the basIs Dec. 14, 1998) to an unreasonable and significant risk that the drug is not effective for the rorthe action. The clinIcal hold order EFFEGTI\'E DATE NOTE: At 63 FR 68678, Dec. of llness or unjury. purpose for which it is being inves- wil be made by or on behalf of the Di- 14. 1998. §312.42 wa.'\ amended by revising (I) The IND does not contain 8uff- tigated. vision Director with responsibilty for pnraKraph (e). effective Apr. 28, 199. For the clent InformatIon rE!quired under (3) FDA may propose to terminate a revIew-of the IND. As soon as possible, convenience of the user. the superseded text §3l2.23 to assess the safety to subjects treatment IND if it finds that: :ind no more th.. 30 days after irnposi- follows: of the clinical investiirations. (I Any of the condItions in para- tlon of the clinical hold, the DivIsion graphs (b)(l)(l) through (x) of this sec- DIrector wil provide the sponsor a 1312.12 CliniC'AJ holdø And requeRtØ for (Ii) The methods. facilties. and con- modificAtion. trols used for the manufacturing, proc- tion apply; or .vritten explanation of the basis for the essing, and packing of the investiga- (i) Any of the conditions in hold. § 312.42(b)(3) apply. * * tional drug are inadequate to establish (e) Resumption of clinical investiga- * * * and maintain appropriate standards of (c) Opportunity for sponsor response. 'ions. An investigation may only re- (el Resumption of clinical Ini'estigalions. Jr, identity, strength, quality, and purity (1) If FDA proposes to terminate an ~ume after .FDA (usually the Division by the terms of the clinical -hold order. re- as needed for subject safety. IND, FDA wUl notify the sponsor in LJirect.or. or the Director's designee. sumption of the affected Investliratlon Is ppr- (Iv) The clinical investigations are writing, and invite correction or expla- Nith rcsponsibilty for review of the rnittecl without prior notification by FDA being. conducted in a manner substan- natIon within a period o( 30 days. :ND) has notified the sponsor tl¡at the onee a stat.ed correct.on or modlflr.atlon Is tially different than that described In (2) On such notllcation, the sponsor nvestl!!ation may proceed. Rbump- made. the Invl'stlirat.ton m~:v pi'oce~d A" F.oon the protocols suhmitted in the IND. may provide a written explanation or ;Ion of the affected investigatlon(s) as the corrl'ction or modification IR made. In (V) The drug Is being promoted or dis- correction or may reque8t a conference Nil be authorized when the sponsor all ot.her case!!. an Inve!!tliratlon may only tributed (or commercial purposes not with FDA to provide the requested ex- :orrects the deficiency(les) previously resume after the Division IJlrector tor t.he planation or correction. If the sponsor :1ted or otherwise satisfies the agcncy Dfr'ecl.or'l' c1eslirnee) with J'Pl'Jlonl'lhiiity for justified by the requirements of the in- review of t.he IND ha. notified the iiponsor vestigation or permitted by §3l2.7. does not respond to the notification ;hat the investigation(s) can proceed. withIn the allocated time, the IND "DA may notify a sponsor of Its deter- that t.he Im'estlira.tion may proceed. In theiie (vi) The'IND, or any amendment or cases resumption of the affected Inve"Uiia- report to the IND. cont!\ins an untrue shall be terminated, I1lnatlon regarding the clinical hold ùy tlont!!) wil be authorlz~d whp.n the spiiniior (3) If the sponsor responds but FDA ,elephonc Dr othcr means of rapid com- statement of a material fact or omits does not accept the explanation or cor- nunlcatlon. If a sponsor of an lND that corrl'cl,/I t.he i1eClclencY(ieiil pre,'lously cited material information requircd by this or OUwl'wl~e satisfieù the allency that the In- part. rection Rubmitted. FDA 8hall inform ias bcen placed on clinical hold re- VCl'lliralionis) can prClceed. Resumption of a: the sponsor In wrIting of the reason for iuests In wrltin" that the clinical hold iituil.y may he alithol'i7.ed by telephone or (vII) The sponsor fails promptly to in- the nonacceptance and provide the ie removcd and suumlts a complete re- oLher means or rapid communicatIon. vestigate and inform the Food and sponsor with an opportunity for a regu- 'ponse to the issu~i identified in the Drug Administration and all Investiga.- tors of serious and unexpected adverse latory hearIng before FDA under part :lInical hold ordr-'DA shall respond * * * * * -tp~rl~nces In accordance with §312.32 1G on the question of whe~ the IND n writing to the, _ .isor w~ln 30-cal- should be terminated. .TIil nsor's re- ~. .. falls to make any other report re- queiit Wr, a regulatory Iieai ."g must be quired under this pa.rt. m~rip w.~tlil" 1n ..l..nn ,.ç ..i... __..____1_ § 312.45 f 2: ,. fR Ch. I (4-1-99 Edi!c. . ì i rI~~ ..J"H~ Drug /,.,.Iminisli(ji;~n. HHS receipt of FDA's notification of non- , ! §312.47 acceptance. ((l) A iiponiior who !nt.ends to resume 'i clinical In\'I":! iiratlon unclei' an IND moiif'\' ~:nil thi'" In spef'eliii¡r the drug nal Gn/de 4850.7 that Iii publicly avail- (d) Immediate termillafirm of IND. Not-. placed on Inactive sLatus shall i;uhnilt devcloi.i'ient anel f!vll.ll!'1t.fon PI". .1::. In withstandlnR' pllrag-raphs (a) throug-h pa~' c('iilar, FD.\ '..\S J'!ind thai' meet- able under FDA's public informatIon a protocol amendment under §312.30 re¡ruIatlons in part. 20. (c) of this section. if at any timp. FDA containing the proposed R'encr:il inves- in/!" ,.¿ the end of Phase 2 of an Invés- concludes that continuation of the In- tIgatlon (end-of-Phase 2 ml:I'tlngs) arc (v) (;rJ;Hlltct of meetiiig. Arrangements tigational plan for the coming year and for aii end-of-Phase 2 meeting are to be vestigation presents an Immediate and approprial,e protocols. If the protocol of consldf'rable assistance in planning substantial danger to the health of In- amendment relies on Informatlnn pre- ,.~ter àtudles ai:ù: that meetings held made with the division In FDA's Center dividuals. the a¡;ency shall imme- viously submitted. the plan iihall ref- near complf!tlon óf Phase 3 and before for Dru' Evaluation ani~ Research or diately. by written notice to the spon- erence such information. Additional In- 8ubmiiiliion of a marketlnK application the Center for BIologics Evaluation sor from the Director of the Center for formation ilUpportlnK the proposed .In- ("rre-NDA" meetlnirs) are helpful In and Res(~:';..:h which Is responsible for Drug gvahiatlon anù Research or the vestll!at.on, If any, iihall he siibmlttr.d developing methods of preRentatlon review of the IND. The meetIng wil be DIrector of the Center for BiologIcs In an Information amenùment. Not- an,l submission of dill-a In the mar- scheduled by FDA at a time convenIent Evaluation and Research, terminate wlthstandlnir the provisions of §3J2.30. ketlnir application that faclUtate re- to both FDA :in\. ¡.he sponsor. Both the the IND. An IND so terminated Is sub- clinical invest.lgations under an IND on vIew and allow timely FDA response. sponsor and FDA may bring consu1t- ject to rclristatemf!nt hy the Dii.ector inactive status may only resume (1) 30 (1) End-or-Phase 2 meetings-W PUT- anLs to the meeting. The meeting on the basis of ii.ilillt,ional submissions iJayii after FDA receives the protocol po.ve. The purpose of an end-oC-phase 2 should be directed primarily at estab- that eliminate such danger. If an IND amendment. unlp.ss FDA notifies tlie meeting- Is to determine the safety of lishing agreement between FDA and 1s tcrmiri:it.ed un((er t.hls par:igraph. the sponiior tliat the invest.irat.lons de- proceedIng to Phase 3, to evaluate the t.he sponsor of the overall plan for agency will afford the sponsor an op- scribed in the amenclml"nt arc iiihject Ph Me 3.plan and protocols and the ade- Phase 3 and the objectives and desIgn portunity for a regulatory Iiearing to a clinical hold under §312.42. or (2) quacy of current stuilies and plans to of particular sLudies. The adequacy of under p:irf. 16 on the question of wheth- on earlier notification by FDA that the Ilsess pedIatric safety and effective- thc technical information to support er the IND should be reinstated. clinical investigations described in the ness. and to Identify any additional in- Phase 3 studies anùJor a marketing ap- (Collection oC inronnaLion requIrements ap- protocol amenilment may beirln. formation necessary to support a mar- plication may also be discussed. FDA proved by Lhe OCfce oC ManaR'ement and (e) An IND Lhat remains on inactive ketinir application for the uses under wil also provide its best judgment, at DudR'et under control number 0910-H) status for 5 years or more may be ter- investiiratiim. that time. of the pediatric studies that minated under §312.44. (I) .Eliqibilit,1/ faT meeting. While tl,n wil be required for the drug product 152 FR 8831. Mar. 19. 1987. as amended at 52 Fit 2.1031. June 17. 1987: 55 Fit 11579. Mar. 29. (ColJpctlon or Infol'm;\tion rr.'llill'pm('nls IIp- end-of-Phase 2 mep.ting i5 designed pu- and whether their submission wlI be 199; 57 FR 13219. Apr. 15. 19921 pl'l)veil 1iy t.he Offce or M:inflFrPnlr.nt and marily for IND's involving new molec- deferred until after approvaL. Agree- Dudllct unller conLrol numbrr 0910-0011) ular pntlties or major new uscs of mar- ments reached at the meeting on these keteù druirii, a iiponsor of any IND may §312.45 Inactive iitatus. Ifi2 Frt R8:iI, Mar. 19. 1987. ai nmrnded at 52 matLers wil be rcp-rirclp.rl In minutes of FR 23031. June 17. 19871 request and obtain an end-of-Phase 2 the conference th;, ,'il be taken by ¡i clinical(a) If studiesno subjects for a periodare entered of 2 years into meeting. FDA in accordance with § 10.65 and pro- or more under an IND, or If all inves- § 312.47 Meetin~R. (\i) Timin.Q. To bp. most useful to the vided to the sponsor. The minutes ti¡;ations under an IND remain on clln- (a) General. Meetings between a spon- sponsor. end-of-Phase 2 meetings along with any other written material should be held before major commit- proviùed to the sponsor will serve as a ical hold for 1 year or more, the IND SOl' and. the agency are freq'uently use- m",nts of effort and resources to spe- may be placed by FDA on I.nactive sta- . ful in resolving qut"stlons ani: issues permanent record of any agreements raised during the course of a clinical clfic Phase 3 tests are made. The sched- reached. Barring a significant sci- tus. This action may be taken by FDA uling of an end-of-Pha.'le 2 meeting Is I eIther on request of the sponsor or on inveiiLlgation. FDA encourages such entific development that requires oth- FDA's own Inltlat.ve. If FDA seeks to meetings to the extent that they aid in not. however, Intended to delay the erwise, studies conducted in accord- the evaluation of the dru~ and in the transition of an investigation from ance with the agreement shall be pre- act on its own Inltlat.lve under thIs sec- Phase 2 to Phase 3. tion. it shall first notify the sJfollsor in solution of scient.fic problems con- sumed to be suffcient in objective and wrlt.ing of t.he proposed inactive status. cernln~ the driir. to the extent that (iv) Adi:ance iiiformatinn. At least 1 deslirn for the purpose of obtaining FDA's reiiourcr.s perml t. The R'enera1 month In ai1vance oI . ii end-of-Phase 2 marketing approval for the drug. . Upon receipt of such notification, the meeting. the sponsor should submit sponsor shall have 30 days to respond principle underlying the conduct of (2) "Pre-NDA" and "pre-BLA" meet- as to why the IND shoult! continue to such meetings fs that there should be background Infrirmatlon on the spon- ings. FDA has found that delays associ- remain active. . free. full, and open communication sor's plan for Phase 3. including sum- ated with the initial review of a mar- about any scIentific or medical ques- marIes of the Phase 1 and 2 investiga- keting application may be reduced by (hI If an IND is placet! on Inactive tions. the specific protocols for Phase 3 , stat.ns. all investil!ators shall be so no- tion t.hat may arise durfnir the cllnical . exchange:" rif information about a pro- lnvest.iiration. These meetings shall be clinical studies, plans for any addi- posed marketing application. The pri- tifiet! and all stocks of the drug sha!! tional nonclinical stui1ieii. plans for pe- be retumetl or othp.rwfse disposed of in coni!t,ded and ik ...iiiented in accord- mary purpose of this kind of exchange ance with part 10. dii\t.rlc iih"lIes, includinir a time line is to uncover any major unresolved accorilancl" with § 312.59. COJ protocol finalization, enrollment, (c) A Sponsor is not required to sub- (b) "End-of-PJiase 2" meelin.Q.v and problems. to Identify those studies that meetings held before stlbmi.esion (,r a mar- completion, and data analyslfl. or infor- the spoiisor is relying on as adequate mit annual reportii to an rND on inac- keti ii" application. A t spec/ fic times mation to support any planned request tive !'f:itus. An inactive IND Is, how- r"r waiver or deferral of peiliatrjc stud- and well-controlled to establliih the evpr. stil in eff) for purposes of the during the drug Investigation pi'oce!'s. drn¡r's effectiveness, to Identify the meet.inirs between FDA and a sponsor A~ and, If available, tentative labellng status of ongoing or ne~ stud1es public i1ii:closUl data and informa. r the drug. The recommend~d con- tion under §312.1.)v. -: can he especially helpful In m/nlml,.ing adequate to assess pedlat~ ""!tyand wai;l.cfUI!'penditures of time and ..mts of su~h a submission are de- effectly:e.,ess. to acquaint!' vA revlew- scribed more fullv In FDA ~t,,,rr M,,"_ ~,'a nr.i+i.l +i... ______1 i_,._. .t ... . §J12AB 21 CFR (;h. i (A-?-'~9 Edilon) i-iil'iiiir.teii In the markel.ilig appllca- Food or-" Dru~ l.dminisfrai¡on, HHS 1.1011 i fncl udinR' technical Information), Tmii int'ullc rpsolvinir IliHicult,ieli In §312.53 schi:dullng 11 thi:m wll.h the Iliformat!r'l t.hey need 1.C) iliscuss appropi'iate met.hods for iita- eetliiio' i\lid obtaining to conduct an 1ilvcsti!latli.ii properly, ¡rator to begIn participation In an In- tlS/.ica I aniilysis of the data, and to dls- timely rf"vlil's to imluÍl'ics. Fui'I.lwr de. VesLI¡rlttlon. the sponsor shaii obtain t.ails on tJiiii procedure a enllUI'!iI/f prover inonltorinir of t.ii,. In. CUSH the best approach to the presen- i',, conl.ained In the following: FDA St.aff Manual Giil,l(' '1820.7 t.hat III veiitlgation(s). en!liirln¡r that the ln~es- tation and formatting of data in the (1) A 1I1gned investIgator sta.tement marketing application. Arrangements pulilicly availalile under FDA's public thtatlon(s) iii ~onducted In accordance with the gèll\l'al Investigational plan (Form FDA-1572) containinir: for sucl1 a meeting ;i:',, to be inftlai-'.,d information re¡nilatioiis in part 20. (i) The name and addresB of the In- by the sponsor with t.he division 1'e- (c) Scitntific nnd lI('dicnl dispiites. () and protocols contained In the IND. vestigator; sponslhle for review of thc IND. To per- When scientific or mcilical dl!'putes maintaining an effective IND with re- 1!pect to the Imrestiirations. :intl eniiur- (i) TIie name and code number, if mit FDA t.o pi-vlrle the sponsor with alise durin" the lini/. ili~.."t.iKation i any. of the protocol(s) In the IND Iden- thc inost usefu I "'Ivice on preparin¡: a PlOC(!!lll. RpOnROI'S !lhouJ(1 dli-i;Ul's Oie I Ing that FDA and all part.lclpat.lng- In- mat,ter i1trect,ly wi tli the l'eRponiiible vestil!'at.ors are prOmli!.'", !nrormed of tifyiiiir the stucly(ies) to be conducted market.in~ apPlil:i.t.lon. t.he Sponsor by the Investl~ator; shoulil Ruhmit to FDA's reviewing divi- reviewing offcials. If necessary. i;pon- ! Bhmlflcant new arlveri-!. "'¡I'I;tS 01' risks SOl'S may reqii..i-t a Tn/Jctinll with the I with respect to the druir. Additional (il) The name and address of any sion at least 1 niont.h in advance of the medical school. hospit,aL. or otiìp.r re- meeting- the followin¡r Information: appropriate reviewing- offi:lals and specific re:iponsllii!ities or !lponsors are described elsewhere in t.hi.' part. search facllty whei'e the clinical inves- (i A hrlef i:umiml.'y of the clinical manag-ement rep.l'eiient.;üi\'Cll in order I tigat.ion(s) wil be conducted; studies to be suùmittcd in t.he applica- t.o !'p.k a reiiohit.ion. It. 'i,;"sts for such ineetln¡rs shall b(! directpi1 to t.he direc-¡ §312.!'2 Trnniifcr or ohligiiti'.Jii to a (iv) The name and address of any contract research organization. clinical laborat,ory facilities to be used tion.(ti) A ¡i ')sed fMmat for. orR'anizln¡r t.or of t.he division in FDA's Center for in the study; the suhl1is~;¡uii. including methods for DI'!: gvaluation and Hriiearch CH' Cen- (a) A sponsor ma.y transfer responiil- ter foi' Illol"l.ÌlR l'valiiatfo!l anil He. hllil..v for any or all of the olillicatioiis iV) The name and address of the IRB prl'Sl'litiiiir t.lic dil ~:i. that Is re:oponliiP~ for review and ap- (iii) Tnfornm\.ion on the statiis of si!an:h which Is I''RprmliilJle for review set forth In thlii part. 1,0 a contract re- proval of t.he st.udy(ies); nc('(led or on/wiiig' pediatric studlp.s. of the IND. FDA wil make evei'.\' at. search onmnlzatlon. Aiiy such t.ransfer (iv) Any ol.her iiiformation for discus- tempt 1.0 g'lant requeRt.!l for nieet.inirs l shall IJ" 'i~scribed in writliiL" '!ot all (vil A commitment ùy the 1iivesti- sion at the meeting. that involve import.ant issueR aiid that i' obliirIIt.lonli are transferr~ii. the writing gator that he or she: can he seheduleù at mutually conven- I is rer¡uii'pd to describe each of the obli- (a) Wil conduct t.Jie study(fes) In ac- ICollr,ction or inrol'm"Uon rC(jnlrernl'nt.s ap. cord:ince wi th the relevant. current ieiit t,imes. gatiOns being assumed by the contract ¡lrovl'i1 h.v I.hl' om,.1' or Man:ii!l'mpiil, alHI . protocoUs) anti wll only make changes Bnil""i. nndl'r cont 1'01 nniihci' 0910-0014) (2) The "(',iil-of-Phase 2" antI "pre. research organlzation. If all oblii:-atloiis NDA" nieeti nirs descri bed In § 312.47(b) I are tlanMelTcd, a gr.neral stat.emeiit in a protocol after not.ifying the spon- 152 PR OA31. Mar. 19. I!107. as ainpnilcJ iii. r'2 wil also Plovirlp. a timely foi-in¡ for I that all obligatIoiis have been trans- sor, except when necessary to protect PH 2:1031. Juiiø 17. 190i: .~r, PR 11580. M:ir. 29. d. isclI!'Rinir and I'e:oolvlni¡ scientifk ami, . ferreiHs acceptable. Any oblii;ationnot the safety, the rights, or welfare. of 1990:'fj I'n 66669. !)('C. 2. 19981 mei1ical issues on whkh t.be sponsor covered hy the wri tten description suhject.s: i §312.48 Dispute resolution. . dlsag-rces wi1h t.h,' :izl"ricy. . lIhall be deemed not to have been trans- (1)) Wil comply with all requirements \3l Iii reqiiesti:.. a meetlnK dpsl¡rned í ferred. reg-alding the obllgat.lons of cliiiical in- (a) ('"ncTnl. The Food and Druir Ad- to resolvp. a seieiit.jii: 01' inedical dis- i vestir,at.ors and all other pertinent re- miiiii:tra.tioli '.; commi t.ted to resolving Ib) A contract resp.arch organization put.e. applicants may siiggest. I.hat FDA i t.hat. assumes any o1ililllItion of a spon- qUllenients in this part; rlifferenccs between SPOnsors and FDA sp.ek the advice of out-side experts. in (rl Wil personally conduct or super- reviewinK rliviRioiis wi th respect to i'e- sor i:hall comply with the speci!ii: regu- which ciise FDA ria)'. in It.R rJji:crctlon. lat,ionil in t.his chapt.p.r ~ppllca1iie t.o vif'e the described investlgation(s): quiremf'iits for IND'R as ciulckly aiid invite to t.he mcet,¡ iiir one or more rif its (dl Wil Inform :i.ny potential subjp.cts , amicahly as P(iRsible throuirh the coop- this ohliirat.lon anti shall he subject to advisory coinini Uee membels or other the same i...irulal,ory action as a spon- that the druirs are being used for inves- . erative exchaiiire of information and consultants. liS dC'iiii;mlted by t.he agen- sor for failure to comply with any ohli- tigational purposes and wil ensure vipws. cy. Applicants may i.ely on. anrl may gation assumed under t.hese regula- that the requirements relating to ob- (b) Arliiinistl'rltii'e and J1'Occdliral blinir to any incp.tini:. their own con- tions. 'rhUlI, all rP.erences to "sponsor" talning- informerl conRPnt (21 CFTt part issues. When administrative or proce- sult.anLs. For major scientific and med- in th is part apply to ii contract re- 50) and insti tu tional review Iic¡¡.ril re- dural disputE'S arise. t.hc sponsor should ical policy issues not resolved by infor- vIew and approval (21 CFR part 56) are firsl. :i I.tempt 1,0 resolve the matter sp.arch oriranization to the extent that mal meet.inirs. FDA may I'cfer the mat- it a!'.'!umes one or rnor~ obllgiitions of met: with't.he division iii FDA's Center for ter to on.. of its st.anrliiill aùvisor.v com- (ei Wil rp.port to the sponsor adveriie Dri!! gvalllation and ltE'search or Cp.n- mit.tees for its coiisidemtion and rec. the sponsor. experiences that or;cur In thp. course of tel' for Biolog-Ics ¡'~valiiation and Re- ommendations. §3J2.i;3 Sclcctin¡r investigators lind the Investlgatlon(s) In accordance with search whkh is rp.sponslble for review rnunitOl'I. § 312.;1: of the IND. ùeglnning with t.he con- rr'2 Fit nu:ii. Mar. 19. 1!1P.7. lI amp.iillp.,1 :it 55 Fit 115fll). Mal'. 29. I9l"lJ If) Has read and understands the In- (:i) Splcctin.Q in t'e.t I I '1r!1. A sponsor sumer safety officer aRsIgn('d to the ap- i,Q" formation In the Invefltlgator's bro- pllclltion. If the dlRpute is not reSolved, shall i-r.lel't only Invesl.il(ators qualified chuip.. Includlnir t,he potential risks I.he Sl"lIRor may raise the matter with . Subparl D-Responsibillies of by I.rainillir aii'l r.xpi:rience a.'! appro- priate exp~rt.s to investiKiitP. the druir. and side effects of the druii: iind the pei .:on desi¡mat.ed as omhudsman. Sponsors and Invesligalors (Q) Will ensure that all ai;soclates. whoRc function shall be to Invcsti¡rate (11) Control of rir"Q. A i-ponRor sha ii coIJeairlies. and employees a!lqlstliig In what hai: happ('~ and 1,0 facilitate a § 31 2.r'O G('nerRI reRl'oni;iliiltieii of ship investii;atioiiiil il~w IITlIE:R only to Blllllsorli, .-IVC!~til!ators participating- In the In- the conduct of the study(Jp.s) ai-e in- timely lind equi ~ rci-oliitlo!l. Appro- fonneij about their I)I~tions in prial.e issiics t.o. .oe with~ie ombiids- ::pon:ooi'!l aI''' rl'.~Jlonsibile for i-p.II"ct_ !~til!at.i on. Ie) ()/¡lrlinin.Q inrrmnnlimi from Ilie in- meet.Îl1!! t.he above coinml its. ing r¡ualifil invcstiir:it.ors. providing (vll) ,A-.,coinlnll.l1rnt by l.il"! Invest!- tpsliqrrlor. Before ii~niiit.ti"L" an liivl'st.i- fT~ln.' fh'~" f"'...... :..u....'~__..,___ § 312.54 21 CFU '.11. I (.i-1-99 Editon) Food and Drug i\dminislralion. llHS §312.57 to an ini;titul.ional rpview rl'C'uir"!np.nt (i1) Sr./ccIiIlQ IlHmit"r.~. A SI'0IlSol' !'hall ilist.l'iliul"ll 1.0 in\'~stk:itorii li:v mP-lIlIS unilcr iiirt fiG. an m.B that complirs 51'1("'" a monitoi' C'lI:ilifip.11 1,::' . ;¡Ing- of . "I'ioilically revi~erl Invl'stfg-atOl' th" investigation as soon :l.S possible. with the requlrr.iiciil.s of t.h:ü p:it wil anI! cxpcril'llce I ri moiilt.or t.IC l!l O¡:I'CSS Iilodiul'l's. ("'pl'iilt!' or pulilisJieil ~tiirl. ii nil in 110 even t latl'l' th:Ul ~i work inli be responsible for the ini tial and con- of tlie invest.igation. lei;. rcports or lettel':" to clinical iIl;es- days after iiakinK t.he det.ermlnation tinuing review and approval of the clin- (Ciillc('lloll or inriirin'ltinn rl'qlilri:lI"nls lip, tiirators. or ot.her appropriate means. that the Investigation should be dis- ical investig-ation and that the investi- proye'l hy t.he Oflii:e of M..n"EiPIIl"n' ~ .,.1 Important Mf('ty information Is re- continued. Upon request. FDA wil con- gator wil promptly report to the IRB Buitg"t unùcr cont.rol nurnlier O~IO 00111 qiiircil to be rp.layp.il to Investigators in fer with a sponsor on the need to dis- all changes in the reseai'ch activ...y and accordance with § 312.32. continue an investigation. all \I iiantici pated problems I iivolvi nir 152 FIl nn:ii. Mar. l!l. 19n7. ai- ampli'lcil at 52 risks to human subjects 01' othen~, :uid FIt i:iu:ii. .hii", 17. 1!11l1; r.i FIt r.niiii. Nfl\". r.. ce~nll"~l Inn or i"fnrm" t Inn .""'111 (Gollpctlon i,r !nrormnr.fon r~'1ulr~m'!nts ap- '1!!l; G:I Fil r'~r,~. 10,,". ~, 19!ilil J1r"flV'"iI 11.\ the (Jrii(~~ (Jr l\1:ilinl.r''''l'lItIrrnlfUi i ~anti nr- pr"",,,1 h.v Ihl! om,:" or MnnnP.l!inl"nt and will not make any changes In the rri- ßul!::ct uiid~r control number 0910-0014) . IhliJi,et uniler contl'oJ number 09JO..H¡ scalch without inB apPloval. exeept § 3J2.!í4 Emf'rl!"llry r('Renrch under where neeesi¡ary to eliminate apparent § 50.24 of this chnpter. i~? F'H ßn~i. M"r. 1'1. Hlß7. a" amended at li? 152 ¡;n nii~i. M:ir. 19. 1987, M nmendCtl ii 52 FR 23031. June 17.1987) FR2;lU3J. Juri,. 17. 1987) immediate hazards to t.he human .sub- (a) The sponsor sh:i1I monitor the jects. proglesii of all invcst.ig-atiom¡ im'olvinir §312.5G He\'iew of ongoing investiga- §:iii.ã7 Rccordkeeping and record reo (vi 11 i A lIi:t, of t.he names of t.hr. sub- an except.ion from informei! coiisimt tions. tentioii. InvesUinil.ols (e.I'.. rr.i;carch ft.lIows. uncleI' §!i.:1, of this chnptl'r. Wh~n t.he la) 'Th. "ponsor iih:iJl monitor \'he (a) A sponsor sliall m:iintaln ade- l('i;iilciits) who wi)1 he a:"sisl.ing t,1ie in- sponsor receivei; from i.lip. lItn iiiforl1a- prog-res!! of all cllnJc:l.1 Investigations qii:ite records i:howlnir the receipt, vci;tiimtur in t.1ic coniluet of the inves- t.ion coneel'ing t.hc pulilic tlist'usures beliig conducted under its IND. iihlpiicnt. or o\'her dispoiiltion of the tiimtion(s). re1iuircd li:v §50.21(aH7Hii) anil (,a1l7)(iil) (lil A sponsor. who discovers ¡.hat an inveiitigational drug. 'lliei:i; i'ecords are (2) Curricuiiim ¡¡¡¡nc. A curriculum of this chapter. tlip. I'Jlt)T1SOl' iiroinptl.v invf'ii\'Iir:itor Is not compl.vlnir with the required to include, as appropriate, the vi t:ie or other st:itemeii' of qualifica- sh:i1l f'uhmit. to t.he IND fi~ :ind to siimerlairreement (Form FDA-1572), ~.Jie name ¡f i.he inveiitlgator to whom the tiOIlS of the invest.ig-ator showin~ the Docket Number 95S-01fi8 iii the Dor.ke\'s i:enelal investigatIonal plan. or the re- drug is shipped. and the date. qUOl ¡¡tlty. educ:ition. tnii ningo. and expi:rif'nçe r.ianag-ement Branch (II FA-:iOfii. Fooi! quirements of this p:ii.t or o\,hcr appli- and batch or coùe mark of each such t.hat C'ualifies l,he investigatur :is an €'x- and Drug Ailministratiun. 12120 Park- cahle partii shall promptly either se- shipment. pp.rt, iii t.he clinic:i1 investig-ation of tIie lawn Dr.. rm. 1-23. HO(;kvillli. MD 20857. cure compliance or discontinue ship- (b) A sponsor shall maintain com- drug- fur the use under invt.st.gation. copies of the lnforina\,ion that was dis- ments of t.he Invciitlirat.ioiial new drug plete aniI accurate reco.rds iiJiowlng- any (3) Clinical protiicol. (j For Phase i in- closed. iilen \,i fi ed )JY t.he IND numher. to lhe investiga.tor anrl end the iiives- finii nclal interest in § 54.1(a)(3)(i). vC'i;t.ig-:iI,ions. a ~enp.ral out.ine of the (h) The iiponi;or also sha1l monilor tiR'ator'ii partlci¡iat.on in the investii;a- (a)(3)(il), (a)(3)(1Il), and (a)(3)(Iv) of this pl:riineil investiimtion Inchiilhig the es- such investig-al.ions to identify wh~n lin t.lnii. If the Investig-ator'ii piirtlcipatlon cJiiip\'er paid to clinical investIgators tin~~tcd dUl'tlon of the sl,udy anil the litH iletr.nninr.i: t,hat it cannot apiirove L. ilP. Investigation iii endeil. the iipon- by Ic!ie sponiior of the covered study. A l1:ixinitll numbel of su1iject.s that will. 1.1.. rei:e:iri;h hecniisp. it rloes not meet SOl' shall require th:it thp. invpiil,lg-ator sponsor shall also m:iin taln complete l.e involved. the i;riteria in the (,xep,¡ition in dliipl)s'! of 01' return the invPol.li:aLlonal and accurate records concernlnir all (ij) POl' Ph:i~e 2 or 3 iiivest.ii:a\'ions. §!i1l.21(a) of t.hii¡ chapt.pr or 1iecausp. of druir i n accoril.~ 'i~e wi th thE' reC'IiIl1~- oi-,her financial Interestii of Investiira- :in'out.liiie of t.he st,uily prol.oi:ol includ. ot.lif,r re1cv:int c\,hie:il concp.nis.. The Inl'iits of §312.!i9 and shaH not.iCv FDA. tors suhject to p:irt 51 of this chapter. (C) The sponsor shall review and ing- an approxi'mat.on of t.he number of Sponsor promptly sh;:ll pl'oviilp. this in- (c) A Sponsor shall retain the records subjects tu he treattld wlt,h the drug formal.inn in writ.ing- to FDA. invf's- evaluate the evidence relal,in~ to the and reports required by this p:irt for 2 ami the numher to be employe.1 as con- tiirators who :ire a!:keil to participate safet.y and effectivrinei:ii of t,he drug as years iifter a marketing- application is trols. If lI.ny; the clinical uses to be In- in this or a siihst.:intially eiiuivnlent It iii obtaineù from the investlirator. appro\'ei1 for the drul!; or. If a II applica- vC'st.ig:iI,r.cI; char:icl.cri~tics of siiliject., clinical Investj¡r:ition. and oth'lr IItH's Thp. sponsors shall piake such reports tion iii not approved for the drug. Until I.h:il, ;:1'C as!tp.fl '.0 rl!vicw tliii: 01' a !'ub- to FDA reiranlinir informal,lon relevant 2 years after shipment anil delivery of I'Y a.i~p,. i;l'x. nJlI l'oiiclil.ioii: \'lir. Itiiiil of to the ~:tety of l.hp. drug- ii!! arr. rp,- c\ ink-a i olii;crv:it.lon~ ai1l1 l:iliOl':iL.or.v stantinll... .'i¡iiivall'iit inv¡,i;ti/!atluii. tliii ilniir fiir Invl's1.Ig-:itlunal liRe Is diii. qllil'~'1 uiii!l'r §3JZ.:i2. Th!! i;linniioi' shall tl'l't.s t.o he concluct.cd: the el'l.imated 161 Fit r,15:1O. Ol:. 2. I!I!J6) coiitiiiued and FDA has been so noti- dura\'iun of t.he i;t.uily; antI copies or a make annu:il.rf'pol.tii on the prog'rcsii of fied, ilescrip\'ion of case report forms to be §:i i 2.55 Informing invc!ltii:ntor!l. the investlg-:ilion in aCl.ol'dance with (r1) A Sponsor shall retain reserve §312.;1:i. usp.!. (a) Befm'p, th" invp.sLiR'atioli b"l!liiS. a samples .if any teiit ¡u'I.lcle and ref- (i!1 A iiJlonsor who dl't"rmines that its ereiice standard 1i1r.ntiflp.i1 In. and used i') Finiiiicilil disc/lIslirc illrlIwiilioli. f'Jltlnsol rot.hp.r than :i sponsor-lrl\'C'stl- Invp.sl,jiral.lniiaiilruir pi'ei:i~nlii an unrea- Suffcient accUl'ate financial informa- In any of t.he IiloCrlulvalence or hio- gal.ol' shall give ..açh p:lJtiei Jiatinl( soiiable anii ,iiimifi~ant risk to subjectii tion t.o allow the sponsor' to sulimit availaliilty studies deRcl'bed in. cliii ii:al Investigator an invp.sliirat.or sh:ill dl~contlilUe thciiie ilivP,ilLlg-ationii compleie and ac('ur;\ic ccr\,lficat.oli 01' §320.31l or §320.63 of this chapter, :ind J'l(l(:liulP. eoiit.airiiiil( i.he information thaI. prp!'l!ril. t.lP. risk. notify FIJA. all rell'ase the reserve sampleii to FDA disclosurc stat.enieiits reljulred uniler ,Ip.si;ril,r.') in §312.23cal(!i). Instltutlonil; rr.view hoarrlii. and all In- part 5.' of I.his chapt.l'l". The sponsor ni) 'Thp. S¡ioii!;(ir shall. a.q t.!ie overall vest.iRators wl¡ hiive at any 1.lme par- Upon request. In accordance wlt.h. and shall obtain a coiiiiiitiiient frum the invcst.imi.Ioii )ll'(il'~edii. Itl'p.p 1':\(:Ii' pai'. i t1t:l'atl'd in the Invl'sthmtion 'of the for the lPI'lod specified In § 320.3B. clinical invc~tiimt.or to promptly up- ticipa\,ini: invp.st,ii:at.oi informeil of np.w e1lscon!;' 'nce, ili;ure the iilspoiiitlon (coii~.." .., of Iiiform~tloii re'lllll''ment.'l np- I!ate t.hls Inf~:iii(ln If any rl'levant ()lii;~rvat.ioiiii rI iseovcl~d liy or rl'ported of all slocks of t.he drui, out.stanrllnir ai pro,'rrl h.y th() Office or Mnn"IH'ml"nt and Ch:iili:('!' occu ri ng- t.he course of the t.o the I'ponSlJr on t.he ilru~. particu- ; -riiiired hy §312.59. anr/ furnish FDA !liic1iil!t under control nuiibei' ~HI invl'st.~:it.ion .1 ft" 4e:ir following larly wit.h rp.!'pp.ct to ilfl\'''I'SP. errect.s . ,th a full repoi.t of t.he sponslJr's ar;- r~2 FR 8R31. r.lnr. 19, 19117. "q iiM,1 l\t. !i2 the complet.ion or t.he Sl'lliiy. aiii sal~use. l:ut:Ji iiiforin:iUon rna:v be iil1rJs. . The s¡.",lIS01' i-Iiall discnii!.¡IlIl" Frt 2.103,f.~'.qiin" 17. Wn7: !iR FIt 25926. Apr. 28. lOn'). r.,. T:~'t ...~...... §JI2.58 21 CFR eii I (.4-1-99 Edilon) Food and Drug Admin!i;!ralicn, HHS § 312.58 Insp(".tion of i;ponRor's recoi'ùs of an.v iIlsposltli," of I.hr. di'ug §J12.69 !ecords and reportR. in accordance with §312.57. l.:nJlI all observat(on~ and ot.hcr da.ta tlflcatlon or disclo!iire statemr./' .1 as (a) FDA inspcction. A sponsor shall pertinent to the investi~atlon on. eal'1 upon request from any properly au- (Collect.Jon of informatIon rpr¡lilr..ml'nt.. ~p. required under part 54 of .. ',chapter. proved h.v t.he OfCr.e of Miiniil(l'm"'nt. and In1lividual administered thc inveiitig'l- 'I'he clinical investl¡rator shall prompt- thorized officer or employee of the Duil¡;et under cont.rol number 0910-011) tlonal drug or employed as l\ cont-IT. . n Food and Drug Administration. at rea- the investigatIon. Case hIstories In- ly update this illforrnation if any rel- evant changes occur during the course sonable times. permit such offcer or 1:;2 FR 8831. MRr. 19. 1987. as amended at 52 clude the case report forms and sup- employee to have access to and copy FIt 23031. June 17. 19U7) of the investl¡ration and for 1 year fol- porting data Including'. for example, lowing the completion of the study. and verify any records and reports re- §312.60 General responsibilties or in. sl/!ned and dated consent forms ani1 lating to a clinical Investigation con- vestigators. mediial records Ineluiliiig. for example. (Collp.ctlon of Information reriulrp.mp.nts .ap- ducted under this part. Upon written prO¡rlc:;s notes of the physIcian, the In- proved by the Offce of MnnalZement and request by FDA. the sponsor shall sub- An Investigator 19 rpspoi,s¡lile for en- dividual's hospital chutes). and the Budp,p.t under control number OtliO-14) ml i. I.he recOI'i1s or reports (or cople~ of !iirln¡r that an In1iesti¡rn tlon Is con- nurses' notes. The case iiist.",.y for each them) to FDA. .The sponsor i;hall dis- ducted accordlnJr t.o t.he si¡rn..il Irivest.. IndivIdual shall document that In- .rr'2 Fn 8831. MRr. 19. 1987. Rll Rml'ncleij Rt 52 gator gtatement. the Invest.¡~ational FIt 2:IU31. June 17. 1987: 63 Fit 5252. Feb. 2. conl.inue shipments of the drug to any formed consent was obtained prior to 19981 investigator who has failed to maintain plan. and applicable regulations; for participation in the stui1y. protect,ing the right.~. safety. ani1 weI- (c) Record retention. An investigator § 312.66 Assurance or IRB review. or make ;wal1able rC'cords or reporti; of fare of subjects under the InvC'stlf¡a- the investigation as required by this shall retain records required to be A n I nvestlgator shall assure that an part. tor's care; and for the control of il ':5 maintained under this part for a perioi1 under investigation. An Investli;ator of 2 years followinir the date a mar- IRB that compIles with the rcqulre- (0) Controlled substanccs. If an inves- ment.'3 set forth 1n part 56 wil b.. re- thratlonal new drug Is a substance list- . shall. in accordance with the provi- ketlnR' application is approved for the sions of part 50 of thl.s chaptcr. obtain dru~ for the Indication for which It is sponsible for the initial and contInuing ed In any schedule of the Conl.rolled the informed consent of cach human review and approval of the proposed Substances Act (21 U.S.C. 801; 21 CPR belnR' investigated; or, if no application subject to whom' the drug Is adminis- Is to oe fled or If the application Is not clinical study. The Investigator shall part 1308). records concerning i;hip- tered, except as providpil In §§ 50.23 or also as!lure that lie or she wil prompt- ment. delivery. receipt, and disposition approvcil for such Indication. until 2 50.24 of this chapter. ArJiIltional spe- years .after the invcst.igat.ion is discon- ly report to the BloB all changes In tile of the dniir. which are required to be clClc respoiislblli t.ies of clinical invcs- research activity and all unanticipated kept under this part or other applica- tinued and FDA Is notlCied. tigators are set forth In t.hls part and problems involving- risk to human sub- ble parts of this chapter shalL. unon the Iii parts 50 ani1 56 of this chapter. (Collectfon of Information rpi¡ulrernents ap- jects or others, and that he or she wil request of a properly authorized em- provpil hy the Offce of Maniil(pmr.nt and not make any chani;es in the research ployee of the Dru~ Enforcement Ad- rfi2 ron llB31, Mar. 19. 1987, as amcnd..,! at 61 Dud~p.t under control number 0910-14) without IRB approval. except where ministration of the U.S. Department of FIt 51530. Oct. 2. 19!1G) r~2 FR' 8831. Mar. 19. 1987. II!! am!'nderl at 52 necessary to eliminate apparent imme- JusticP.. be made availaùle by the In- § 312.61 Control of the investigational Fit 23031. June 17. 1987; 61 FH 57280. Nov. 5, dIate hazards to human subjects. i vestiimtor or sponsor to whom the re- dl"Ug. . 1996) , quest is made. for Inspection anil copy- (Coii..ctlon of Inforniat.on rl'qulrements ap- inR'. In :uldi tlon, the sponsoi' shall as- An InveRtlgat.or iihall ailmlnlster the 1312.64 Investigator reports. provp.il by the Offce of Mana~ement and sure that adE'quate precautions are ,Iru~ only to 11lhj('cts undnr the Inves. (l\) Pro.qrc.~s n~ports. 'J'h", Investl¡rator nucl~ct under control number 0910-14) taken. inclui1ing storaire of the inves- t.igator's personal SIlPp.rvls¡'on 01' l'r1(ler ~. i furnish all report.s to the sponsor (52 Fn 81131, MRl. 19. 1987. as amended at 52 I.i¡.ational (\t'UK in a seciirely locked, t.he supervision of a suhinvci;tlgai.or re- of the druir who is rt'sporislble for col- FR 23031. June 17, 1987) suhstantlally constiuctei1 cabinet. or spiinsible to the InvestiKHtor. The In- Iecting and evaluatlnir the results ob- other securely locked. suhstanl.ally vestl¡rator shall not supply. the Inves- talnerI. The sponi:r is requIred unùer § 312.68 InRpection or investigator's thmtional 'Irul¡ to any ¡iei:;oii not au- records and reports. com:tructcd enclosure. access to which §312.33 to submit an J1! al reports to is limited. t.o prevent theft or diversion tllOlized under t.hls part to receive it. FnA on t.he progress of t.he clinical In- An Investi,gator shall upon request vesti¡ratlons. from any propp.rly authorized officer or of the sulii:l.ance Into i1e~ai channels §312.62 Invl'iitiJ!ator rccordkecping of illsi.rlbu tion. nnd record retention. (h) Safety reports. An Investigator employee of FDA. at reasonable t.lmes. shall promptly report to the sponsor . permit such offcer or employee to § 312.59 Dii;poi:ition of unuRed supply (a) J)i.~po.~it()n of dru.a. An Investl- any adv.erse effect that may re:l"onably have access to. and copy and verify any of inve~tig8tionni drug. intl.or is requlreil to maintain arlequate be rr.~ari1ed as. caused by. 01' probably records or reports made by the investi- The sJloni:ol' sh:i II asgure the return r('coldg of the iJil'position of the drug. caused liy. the di'u~. If the adverse ef- gnt.or pursuant to § 312.62. The Invest1- of all unused supplies of t.he investiga. incluilin~ dal,es. qu;intity. and use by fect Is alarming-, the invcstir,ator shall ¡mtor Is not required to divulge subj~ct tional druK from each Ini1lvidual inves- suhjccl.a. If the invesl.lg-atioli is termi- report the adverse effect. ImmedIately. names unless the records of particular ti¡rator whose jlitrticipation in the In- nateil. sui;pp.nde,i. ilisclJntlnul'il. or (e) Final report. An invp.sti¡rator shall ini1ividuals require a more detailed vesti¡ration is dli:contlnued or terini- completed. t.he invest.l¡rat.or shall re- provide the 8pOnSQr wi th an adequate study of the cases. or unless there is nal.l'ù. The gpom:or may authorize al- turn the unused suppllcs of the drug to reason to believe that the records do tClIative ùispm:ii.ioii of UllUScù supjJlles the :iponsor. or oL1iel'wise pro\'iile for Investi¡rator's participation In the In- not represent actual case studIes. or do of the investi¡rational drug provided ùlspr;i¡itlon of ".~ uiiused supplies of I reportvesthmtlon. !lhortly after completion of the not represent actuall'esults obtained. this alternative dii;posit.ion does not the di'ug under § ,'. J.~9. I (d) Financial disclosure reports. The expose humaW-. rislts from the dru¡r. (1)) Case histories. An Invest.i¡¡ator Is § 312.69 Handling or eOEloIJed sub- The 1'ponsorlI maillt.ain written rf!riuired t.o pl'l'pal'c and maIntain ~,ie- .i clinical-"ponsr,r invp.stfirator wíth sufficient shall accurate provide f1nan- the stances. fiuate a~accurate case hist.ories that If the Investlirational H. " Is subject -:. .' to !'l1bmit. complete and ar;curate cer- I, ~ial information to allow an applicant to thrff!ontrolled Substances Act, the §312.70 21 CF¡: Ch. I (4-'1-99 Edition) . food and Drug Administration, HilS Jnvl'~l.jl:at.oi' I"h:i1I t:ike arler¡u:itl) prl'- §312.82 cau I.ionl", iiicliiilliig' stora~e of t.he lii- liil'lll!ihll' \./) lf'Ct"Í\'P Invesl.ii~:il.lrin:il &IIIRI:r.: 5:1 r'R 1152:1. (Jct. 21, 191'". unl('~ii driil!s will 1.'J examiii~d 1.0 i11....lmnine (1-) ~I'. ,;, ii :'. are encourag-eil to con- vei:tiini.tJolial driiir In a securely lockecl, c~bprwi~c noted. Subl"t:iiitl:ilJ.v const.nicteù cahln!'t. 01' whether the inve!!tlgator has !!iibmltt~d ! !llli wit,h I~DA on the :ipplicaùillty of oLller securely locked. suhstant.lally Iinrl'llahll' d:ita that are ('iiscliLlal to 1312.110 Purose, these jJrocedures tii ~pecific products. construct.ed enclosul'E', acces!' to which the ".!'~..iiu:ition of the iil\e.~t.l:atloD ¡ The pUl'Jl08e of thiii !'~çt.ion is to r.s- Ir.:1 FR 11!i?:i. Or.I.. 21, 1988. as rißlclilictl aL 64 Is ¡¡mited. to prevent theft 01' diversion or (:s:;ential to the approval of anr 1 tAblish procedures d(!!'ii;ned to expeilite FIt '10i. .Jan. 5. 1999) of the suùstance hito illegal channels marketing application. l the development. evaluatIon. and mar- of distriùution. (ù) If the Coinmis!'ioiier dE't.l!rmlnf'~. ! ErrF.c,.", :.. n.~TF NOTE: At 61 FR 101. Jan. aft,er the IinrE'li:iòle dat.a Suùmit,ted b; i keting' of new therapips Intp.ideil to 5. 1999. §.11:.:..i¡ was amended h.\' 'invln~ ". §:JJ 2.70 Di"'iunlìficntion of n "linicnl the investll!ator art" p.llmlnatrd from i treat pf.rsons with iife-t.hrp.ateninl! and ant.hiotk." fi'om Ihe Intl'oductoiy Lext. ef- ~~\'er~ly-debili tatinl! . ill nes!!c!'. espe- fcut.ve May 20. 1999. inveRtif!ntor. en/lslrli!ratlon, i'hal. 1.h" el:it.:i l',inalnlnv: I cllilly wh'!re 110 Ral.Rfactory alter- (a) If FDA 1ias informatIon Inrllcatlnir al'l) Iil:ilr.'lu:ttp. 1'0 !!upprirL a conclUsIon j :i.live therapy exllil~. A!! st.at.'!il § 3J2.82 Enrly conAultntion. that it is reasonahly !!afe t.o continue : that an Investiirator OncludInir a spon- l3i~.~ri:,(c) "r Ihis chapt...r. while the For products Intended to treat IIfe- sor-iiivesLiimtor) has repeatcdly or de- .the liivrstil!al,ioii. the Coininissloner I statutory staiidai'ds of safety and erfec- liberately fRUed to comply witli the re- wll notify the sponsor who shall h:we ¡ threateninir or severely-debllì tating 1l- an opportunity for a reg-uJatol'y hear. i' 1I\'!ßess apply to all drilllS, the many nesses. sponsors may rr.quest to meet quirements of this part, part 50, or part kinds or drugs that are subject to wIth FDA-revIewing offcials early in 56 of this chapter. or ha!' submitted to ing under part 16. If a dang-~r to the thpiT. and t.he \vide rang-p. of uses for FDA or to the !!¡ionsor fal!!e liiforma- public hcal\h "x¡iits. hO\vevr.r. thp Com. . the drug rlevelopment process to review those dnigii. IlemaJlI f!exihllll,.y in ap- and reach arrreemeiit "11 t.hp. r1csig-n of tion in aiiy i'eqiiired report, the C"ntei'. mls!'ioiii'r shall tel'nln:. ell t.he IND Im- l plying the st.andai'ds. 'I'hp. Foorl and for Di'ii,; Rvaliiri tioii and Rese:trch or mr.rjiat~ly find notify t.hl' I"ponsor of l'ie neceiisary preclinical and clinical st.ud- Druir Admini!!tration !FDA) hIl dp.t.er- iI'S. Where appropriate, FDA will invlt.e thc Ceiit!'r for DioloirIcs Evalu:itloii rletcorrniii:il.loli. In ~ul'h caM, t.he ~pon- J minl'll t.1flL. it. Is apl1l0IIi'iatp. \.0 ",xl'rci!'c and Hel"('fll'ch will fUl'lish the invr.sl.I- SOl' shall have an O¡iPOI.t.I.I/l i ty for a re!!- / to i'H('i inl'eLiriv.s one or more outside ulatory hearlii,; hefore FDA un/1m' part j the 1.roõlllest f1p.xlhiilLy tii applylnl! t.he expelt I"clent,lflc consult.ant.!! or advi- g-at.or written not.ice of the maLLer Sl:tf.iitory st.andanl!'. while pre~ervlnir sory commIttee members. To the ex- complained of and offer l.he Inveiitl- 16 on the r(uest.lon of whether the IND Rtl¡iroJlrlate g-uara IJ tee!! for i;afcty and shn'llr1 he reiniitatcd. teril~ FDA rciiources permit. agency re- g-ator an op¡iortiini ty to explain the errectivenf'sii. Thel"p. plocedures reflect viewing offcials wil honor requests matter in writing-, or. at the option of 'f t.he Commislliont"r rlct(,i'inlnp.s. the lf'cog-nitlon t.haL phY!'iclans and pa- t.he inv('stigoatcir. in an Informal con- afi.ui' the u/I'elllilile data i;iihmitted by I for such meetings tien\.s are generally willinl! to accept (a) lrC.illlJesli,QCltinnal nell drug (iND) ference. If an eii:il:natiou is offererl but the investi~ator are elimiii:\Ied from I greater risks or side effect.s from wod- not accept.ed hy the Center for Drug considerat.ion. t.h:ü the continued ap- I mcdin.Qs. Prior to the submission of t,he uctii that t.l'f'at life-t.hl'cat!'JJ1IJR and se- iiiitial IND, t.he sponsor may reljUest a Evaluatioii and nesearch or tlie Center pi'oval of the tlnil! product for which , verelY-debilitating ililJess~ii. than they for Bioloirjrs Evaluation 1I111 H.esearch. the ilat,a were suhmittf.d cannot he jU$. ¡ meeting- wit.h FDA-reviewing offcials. tined. t.he Coinirif,sJoncr wil riroceed I would a';cept from proiliicts thaI, treat The primary purpo!;e of this meeting is i tiinithe investig-atoi.ty for a r('g'ulatory wil be givenhearin¡r an underOppor- If'~s sl!rious ilnes!;cii, Thp.!'e procp.dures to withdraw approval of t.he dnii; proil- r also reflect the recognition that the to review ánrl reach agreem('nt on t.he part 16 on the question of whether the uct in accortlaJi('r. with the applicable , desil!n of animal studies needed to ini- provisions of the act.. i bf'nefi.s of t11f' driignep.d to lie evalu- tiate huinaii testing. The ineetin¡r may Jnvestiimtor is entltleil to receive in- ated In light of the i;~verily of the di!'- also pi:ovide an opportunity for dis- ve!!t.iK:ttional new drug-so (f) An invest,i/.al.or who has bc!'n rle- i eaM iJeinl! I.reateil. The procedure out- (h) After evaluaLIng all avaIlaùll: in- tpr"itiil'i\ to 1m i/lell/.ihle to receive In- ! IlnE'd in thl!! s!:ction should he inLer- cussing- the scope anrlilp~il:n of phase i formation, incllidlnK nn:,' explan'lt.lon vesi.i~ational druKs may he relnRt.at.ed i preted consistent with that purpose. testing-. plans for sLudying- the drug plesf'nted hy the investigator. if the as eJig-iJile when tIie Coinmis!!ionp.r de. i product in pediatric populations. anrl COlilniil"sioner determine!! that the in- tcrmiiies t.hat the inve!'tlgator h¡is pre- §3r2.111 Scope. the bl!st :ipproach for preiient.ation and i formatLin" of data iii the IND. vest.I(:n.tor has rep('aLf!IIY or delib- seiitp.d adequate agsuraiices I.hat the in- Thi!! section appli('!' to new dl'lll! and vl'stiirator will employ Invp.st.liratloal (h) Enri-of-pha.ee J meetin.Qs. When ùata ei-i-cl.v f:iill'll 1.0 cnrnp!.v wil.I' I.he rp.- bIological prorhict.s tlmt arp. bcinK iitud- '1l1ilt'nienLs of this part. Ilal'l, ;,0. 01' part 1!i'IiI!!! solely in C'Jllplia ncr. wi t.h the ¡ from phase 1 clinical testliiir are avail- 56 of t.iifl chapt.er. or ha!! cI('lliJerately Plovl!'Ions of thi.~ part and of parts 50 led for 1.11111', l"afl'Ly aiil crr"cLivl~IiI,!'" In ahle. Llie sponsol' may ag-ain re'lul'iil. l\ and 56. trr~.linl! . llfe-threatenlnir or severely- meet.Ìli!; wi th FDA-reviewlnir offcials. or repeat.t"dly submi tt.ed false informa- I delil! Ital i 01: :diseal"c!!.; tion. 1.0 FDA 01' to The .pliinary purpose of. this meetIng is the I"POi/flor In any (CnJlI"'I.inn or inronri:i.(rin rrlllilr'l'lil'ntii ni' ! Ill) ¡"or purposes of thl!! ~cct.lon, the to revtew and reach airreeinent on thp. reiI" i :,cd report. t.he (;oiilJ IssIoiier will Pl()'I'111,y Uii: OCf"p or l\rin:iii:IlPIIL Rill! f tl'rin "iif('.-I.hreaLenlrig" IIp.anii: notify the inve"~LIg-ator and t.he i-llonsor DudKeL uiider coiitrol numbei' 09100(11) dcsll!ii of ph Me 2 conl.rollprl "lliilc:i1 of any im'csLiiratio/l in which tile iii- 0): Diseal"r.!' or conditions ~'here the trlalR, with the gon.1 that 8Ui. ',~iitinir vesli¡mtor has been /IalJed aii a partici- (52 Ffl Rß3I, jI.lar. 19, 1!il;7. n" iirn"IIiJ('rl at S2 I likelihood of death is iiig-h iirless t.he wll he ii.ilequate to provide suffcient Fli 2~n'ii. .¡unn 17, i:ij17: 55 Fit 1l581). Mar. 29. ¡ course 'of' the dIsease Is interrnrited; data on the i1rul!'s safety and effE'ctive- pant. t.hri \. the i/l'estiimtor is not enLi- 19!J0; 1i2 Fit '16876, Si:pl.. 5. 199711 anil tIed t.o I'l'c('lve iin'rstclimtional dnig-ii. nel's to support a ,Ip.cision on its ap- Th!' notification will Jli'ovidl' a statl!- (2) Diiieasel" or condit.ions wll,li p/)t'!n- provability for marketelng-. aiiri to dis- SuiJpart E-Drugs Infended to (.ally fa t.fl mE'nt of hast!! foi' such dl'Lcl'nJnatioli. I /)ui.com'!!!, whe/"~ I hp. end cu!lg thp. /Iced for. as well as the dp.sig-n lreaf life-threatening and Se- i point of cl' ..''1 trli\1 analysis is siir- and tlminl! of. sLudir.s of the druir in pe- Ie) gric1i IND aiid coach approvp.tl ap- \'Í\'al. plie:it.ion Slllimi.JI 1I!Ji!"r part 314 con- verelY-debiltating /Inesses diatric piitlp.nts. For rlrugs for IJfe- tail/inK data r teil h.v an hl"l'st.i- i .-l For plirJlos~!! of I.his secLion. the. threaten!nl! disease!'. FDA w~rovide ì i "sevelely dcIiIJILat.ing" m~ans Its be~t jUflgment. at that ti ivhel.h- gat,or who h:iii ....,~n dete"Yinell to he . lll"l1IIl/lll'Y: 21 u.~.c. 3:;1. 1.S2. 3:¡~. 1.)~. 371: i .~!'''s or conrllt.onio t.haL cause major "12 U.S.C. 2r.~ er pc,lhi;i;i- stuille!' will hI. .e'l,,¡"il j frrrv"'l'~ible morùidity. :inr1 whf\t.hhr t,hoi',. C"nl....l....;,.'ñ ...OJ I... "§3'i.83 21 CFR Ch. I (4-1-99 Edilon) Food and Drug Administration, mis deferred until after apI'rov:i.i. The pm- §312.110 ketlng- approv:il. FDA wil iii!H11l (for a cedures outlned In §3J2.17(b)(I) wIth trials and he Involved In facilitating clatf!. Commissioner for Health Affairs. respect to end-of-pha~c 2 conferences. druin a not approvable letter pursuant their appropriate progress. lncludin¡r document:itlon oC agree- to §311.120 of this chapter. or ((or a blo- Food and Drug Admlnlstratton. 5600 loiclc) ll deflchmclcs letter consistent §312.88 Safeguards for pilti('nt surety. Fiiiher!! Lane, Rockvile. MD 20857, of a. ments reached. would also be used for written request from the person that end-oC-phase 1 meetings. with the biolol\lcal product licensing All of the safeguards incorporated procedures. Such letter. In describing \vithin parts 50, 56. 312, 314, and 600 of seeks tC' ""i:ort the drug. A request (!i FR 4152.1. Oct. 21. 1988. as amended at 63 the deflclenCI"3 In the application, wil must pn,i:ide adequate informa.tlon FR 669. Vec. 2. 19981 this chapter designed to ensure the. address why the results of the research safety of clinIcal testing and the safety about the drug to satisfy FDA that the §312.83 Tratment protocols. design agreed to under §312.82. or in of products following marketing ap- drug is appropriate for the proposed in- subsequent meetinirs. have not pro. vestigational use In humans, thllt the If the prelimInary analysis of phR.e 2 proval apply to drugs covered by this vlded suffcient evi.lence for markf!tlnir sect.Ion. This Includes the requlriiments drug wil be used for Investigational test results appears prornllilnK. FDA approvaL. Hiieh let.tei' wi ii 1\)1'0 dr.1'cl'lbe may ask the IIPODSor to submIt a treat- for Inform/'il conRimt (part 50 of this purposei; only. and that the drul? may ment protocol to be reviewed under the l".ny recommen,1:.t.!onR JlI\.le by the iiil- chapt.er) and Instlt.ut.lonal rl'vlew be legally used by that cOJlsJg-nee In the vlsory commit~. . regarding the appli- boards (part 56 of t.hls chapt.er). These ImportIng country for the proposed In- procedures and criteria listed I,ll cation. safeguards further Include the review vestigatIonal use. The request shall §§ 312.31 and 312.35. Such a treatment Cd) Marketln~ applications submitted protoco' if requcsted and granted. of animal studiiis Jirlor to Iiiitlal specify the quantity of the drug to be under the procedures contained In l.his human testing (§312.23). and the monl- s)','''ped per shipment and the fre- would normally remain in ef(ect while section wil be subject to the require. t.he complete data necessary for a mar- torin!! of adverse drug experiences quency of expected shipments. If FDA ments aiid procedures contained In part through the requirement!! of IND safe- ketinir R.pplication are behiir Msembled 314 or part 600 of th is chapter. a.s well authorize!! exportation unùer thl!! para- by the sponsor and reviewed by FDA .as thoiie In this subpart. ty reports (§312.32). safety update re- graph. the agency shall concurrently (un)r,~s irl'ounds exist for clinical hold ports .durinir ag-ency..review of a mar- not.iy the irovernment of the Importing of oiiiroin¡¡ protocols. as provIded In §312.85 Phase 4 ¡¡tudic". keting application (§314.50 of this chap- country of iiuch authorization. § 312.42(b)(3)(Ii)). ter). anil postmarketlng adverse reac- Concurrent witli market.lng approval. tion reportlnir (§ 314.80 of this chapter). (II) Throuirh submission to the Inter- UI2.R4 n¡Rk.bent'fit anAh'RiR in review FDA may seek ag-reement from the national Affairs Staff (HFY-50). Asso- of mnrketing npplientionii for drli~!' Sponsor to conduct cert:iln post. Subpart F-Miscellaneous ciate Commissioner for Healt.h Affairs. to trent Iire.threnlcmiii~ nnd see market.iliA' (phase ..1) studies to ùelln. Food and Druir Administration. 5600 verely-dcbiltating ilnesses. eate aùditional information about the §312.iio Import and export rl'quire. Fishers I..ane. Rockvlle, MD 20857, of a (a) FDA's llpplication of the statu- drug's i'lsks. beiicfi t.!!. anil optlmll1 use. mentir. formal request from an aut.horized off- ~ory standards for marketing approval These studies COUld Include. but would (al bll1JOTt.ç. An Invest,igatlonal new cial of t,he R'overnment of the country ihall recognlze the need for a medical not be limited to. studyinir different drug offereil tor Import, into the United to which the driig is proposed to be 'isk-benefit judgment In maklnir the doses or sclie(lulcs of admlniiit.ration Stat.eil complies wil.h the J'er¡uirements shipped. A request must specify that ïlnal decision ,on approvllhlIty. Aii part than were uiied In phase 2 studle!!. use . the forehm government has adequate of the c1ru!! In other patient popu- of this par~ if it is subject to an IND lr this evaluation. consistent wIth the that Is In erfiict for It iindp.r § 312.40 and: Informat.on about the drug and the itatcment of purpose In §312.80. FDA lations or other sta¡:es of t.he. disease. (1) The conslKnee In the United St.ates propo~cil investiglltlonal USf!, that the vil consider whether the benefits of or use of the drug over a longer period Is the spo~sor of the IND; (2) the con- . drug wil be used for Investigational .he .Irug outweiirh the known and po- of time. siirnee Is, a r¡uallflecl Investigator . purposes only, and that the foreign .entlal risks of the drug and the need §:J12.116 Focused FDA regulatory reo named In t1ie IND; or (3) thp. consignee . governmen i. Is satisfied t1iat tIie druir .~ aniiwer remaining- Questions about seArch. Is the domestic agent. of a foreiKn spon. : may legally be used by the intended 'isks and benefitii of the drng-.t.akln,i conslgniie In that country. Such a re- At thii diRr.I'r.l,lon of t.!tr. al!('lIr.y. FDA SOl'. is reRponillbJe for the coiitrol and nto conilldf'ratlon the iieverity of the dis.trlhution. of the. Inve!lI.Ig-at.lonal quest i;h:i1I specify the quan 1.1 t:v of drug :ii;caiie and \.ie alii;cnce of satisf:ictory ma.y undcrtl\lm foclisl'll l'l'p.ull\tory reo drug-.an,1 the IND Identifies the con- ,to be shipped per shipment and the fre- search on critical rate-limiting aspf'cts .\ternaUve therapy. i;ji'nce and describes what" If any. IIC- . quency of expected shipment.s. (b) In making decisions on whether of the preclinical. chemical/manufac. tioii!l the consignee wil tllke with re- o grant marketlnir llpproval for prod- turin!!. and clinical phMes of druK de. (ii) Authorization to export an In- velopmf'nt and evaluation. When Initi- spect to the investi!!atlonal drug-. vestigational drug under paragraph cts I.hllt havr. li('en the suhject of an (bl EXJlorts. An Invesl.igation?.l new (b)(2)(i) or (il of this section may be nd-oC-phase 1 meetinir under §312.82. ated. FDA wil undertake iiuch re- druir liit.enl1eil for export from the 'DA wil usually seck the advice of search efforts aii a ineanii for meeUni¡- a revoked by FDA If the agency finds United States complies with the re- that the conditions underlying its au- iitside expert scientific consultants or public health need In faclJtating the qulremeiits of this part. as follows: dvisory committees. Upon the flinir development of therapies to treat life. (I) If l\n, IND 1s in effect for the drug tliorl7.ation are not longer met. f such a markel.inir application under tlire"":lIing or severely debllltat.lng il. undl"r §3l2.40 and each perl!on who re- (3) Thiii pai'airraph applir.s only where 311.101 or part GOl of this chapter. FDA nesses. the drug is to be used for the purpose of ceivc!! the druir is an Invest.igator clinIcal investigatIon. .11 notify the members of the relevant § 312.87 Artive monitorin", of l'onduct named in t,he appliclItion; or ;llndlnir advisory committee of the flP, iind eVAluntion of clinical trials. (2) If FDA authorizes i;hipment of the (4) 1'1ii!! paragraph does not apply to f1cat.ion's fiing anil its availability drug for use In a clinical investi¡mtlon. t.he export of new druirs (IncludIng bio- Ii' review. For dnJ~s covererl under this siictlon. Aut,hori7.atlon may be obtain!!'1 as fol. logical products. antibiotIc drugs, and (c) If FDA con~)11 that t.he dat.a t.he Coinrnislllonl'" ;irid ot.ier airency of- 1- Insulin) approved or authorl7.eoor ex- ~esenk'~ are not. ificif'n'"0r mllr- ficials wll moni lor U:e progress of the lhroul?h "ilbmli;i;lon tri the Inter. port uniler sect.inn 802 of i.: :1. (21 coiiduct aii~evaluation of clinical na~ioiial Affairs Staff CHFY-50), Asso- U.S.C. 382)"o)i section 351(h)(1)t.., of the ,i § 3 1.7.120 2 i CFR Ch. I (-1-1-99 Edilivll) Food and Drug Adminislralion, BHS Public Health Service Act (12 U.S.C. §312.120 262( h)(l )(A)). shouJrI FDA reque~t. i::i" . r"r:onls maln- Laliieil hy t.he il1v~s\.Kal.or or adlll- IlF:"ri~""F.:-ri,\TIOSS (::~"ii~ri Plir~ICIA~I.~ IN 2. Thl! li"iill1n an') ,.I'rfni'mance of each ex- (ColI('ction of liiformatJon rcqiiirp.ments ap. . tlonaI background dat.a such as hos- iiIO~IF.JIlCIlL 1t~;S~:IIItl'li IUVOI.VINO HUMAN p"J'lrnr.lital proc('(hir" Iii\'olvlriir human 8uh- prov('i1 by the Offce of Mllnnircment and S'Jii.a;ITS pital or other Instit.utlonaI records; j..cts shOUld he clt'arl.v formulated I n an ex- Budget uiider control number 09J D- 11) (1) A description of the drug sub- Introduc/ion perlm..ntal protocol which should be trans- i~2 FR 8R31. Mar. 19. 1987, as amended at fi2 miLted for consideration, Cfliiment and guid- FR 2331. June 11. 1907; 61 FR 401, Jail. 5. 1999) stance and drug product used in the rt I'" lh~ ml",!'lon of the ¡ihy!'lcl~ n to 8afe- ance to a specially Ilppolnt-C'd committee sLudy. including a description of com- ¡rii~ril the heallh of Ihe "'Jople. HIs or h"r Inrl",iiE'nilent of the tnveRti¡¡ator and the F.FFF.cn: OIlTF. Non:: At i;1 FR 401, Jan. knowlp.i1icc and con,;clciii:!" al'e dedicated to S. 1!I. §312.1io wn. amr.ii.¡,.,. hy revl!'ln~ ponen til. formu lation. speciflea.tIons. I'poiisor provided I.hiit this Inde¡wnd1mt com- anil bioavallahllity of t)ie specific drul: th~ fuirlliment of t.hl,; ml"'''lon. mll-ee Is In conformity wIth the laws and plllairrlll'h (h)(4) and by rp.movln~ parii~i'aph product used In the clinical study, If The llrc:'al'at.oJl of Gi:n",'a or thp. World rrKul:itlon!' of the coiint.r.v In which the re- (h)(S). effective May 20, 199. For the conven. Mp.i1lcl\l Assor.laLlon hind!' i.ie phy"'lclan wIth sE'arch expel'lment Is perrorm('d. lenre of the II ior. r. the iouperneded text fol. available; and thp. wonla. "The h~alth of m:v paLlent wil he 3. Blomedlciil research Involvlnir huma.n Iowa: (5) If the atuily Iii Inl.i:mileil to Rupport mv firM. (innioldrrnllon:' nnd the Int.rr. !'uhJp.el!' should be con,!iictcd only by scl- 1312.110 Import Rnd export reuirementA. the effectIveness of a ilnlK product.. In- niil.luna' (;",Ir. of M~.Iii:i1 F.l.hIt8 i1cclar~ii I!nt.lfleally 'iuallfled peT!'oml n",1 under the formation Showing that the Rtudy is 'Uiat....A I'hY81c1an sh:ill ;i':t only In the pa- "lIpr.r\'lslon of II cllnlcnll.v conipel('nt med- adequate and well controlled under .llenl's Inlprrst wh('n pro\'ldlnir medical care Ical perion. The re8ponslbillty for the humRn * * * * . ivhlch mi¡¡ht ha\'e I.he cffect Of weakenlnir subject must always re8t with a medlciilly * §311.126. (h). . . Ihl! ph:v~ical and mental condItion of the pa- qualified pernon and never rest on the sub- (1) 'niia pnra,miph 110l!8 not appl.v to thii (e) Conformance with el1licnlllrinciples. ' 'tl"ni.:. , ject of the J'e!'enrch. p.ven t.houirh thp !!ubject export nf an iintihiol.le I1nlR' prorliict shipped (1) ForeiKn clinical resea.rch Is requIred , The JllirJlr)~e uf hiomeillcal re!,"arch Im'olv- haii R'1\'en hIs or hE'r con8ent. In iiccolllnncc with the provialona of aectlon to have been conducted In accordance 11Il( human !'lihJE'cts iiui;t he 10 Improve dlair- 4. Ilornrdlcal rel'enrch Involvlnir human ROICd) of I.he nct. with Uie ethical principles stat.erl In : no",1 lc. I h"rnp'!utfc anr! prophylactic proce- " !!uhjp.cts c;innot IE'R'ltmat('ly he carrIed out (5) Thla paragraph dol!a not apply to the the "Declaration of Helsinki" (see ,dlil.P!'; a n.1 Ihe iiniirirnt nn.lInir of the iieUolo~y iinles,; th' :inporl.ance of the ohJectlve Is In I!xpnrt of new dnil,s (including bloloR'lcal i: an'lIn eurr~nt pntli"l!pne~ls m('dlcal of ,lisE.ns~ iiiac:; ir:e mo!'t dla¡r- pl'lJ1ortlon to the Inherent rl"k to the sub- prodiici.~) approved for expnrt uncleI' section paragraph (C)(4) of this sectlnn) or the JI'ct.. laws and regulations of the country in ,no"t1i:. Iherapeutlc or prr¡ihyladlc prrce. 5. Every blompdlcnl reiif'arch proJect' In- 802 of Ihe ar.t or section 351(h)(l)(A) of the ! i1ur~". iii\'o\ve hil?:\rilii. This applies cspe- Public Health Service Act. which the research was conclucted, vo1vlnir human 8ubject..i !'hould he preceded wliichever represent.s t.he greater pro- . clnll\; to hiompdlcal ri:s('~ri:h. by cueful asse!'!'mcnt of predictable risks In comparliion wll ,. rrire!'eeahle benefit! to the * tection of the indivIduaL. Meilical pro¡rresa 18 Iin~ed on rei;E'arch * * * * whkli ullimalel.v mu.t rest In part on ex- ~iihjei:t or to others. Concern for the Inter- (2) For each forelirn clinical atudy ppr¡ii~nlat.on In\'olvlnp, human i;uhjecI8. I'iit.s Of thii 811hJect must alwaya prevail over §:J12.12() FOn'iim clinicnl Iltudies not suhmitted under this sect.ion, the spon- In Ih(' (l,,!rl of hloli('(IIc:al rc!'carch II fUlll:i- thf' inlcrpsta Of aclcnce and .'ocfp.ty.. conducted under nn IND. sor i;hall explain how t.he re!learch con- 'm'!litnl,(IIi;linct!on must he recoimlze.1 be. 6. The rlR'ht or the le"E'arch 8uhJect 1.0 eafe- (ll) Introduction. This section de- formed to the ethical prIncIples COD- tWl'pn nip'lI('al rp."'eai'ch in whlrh the aim 18 J!uard hl8 or her InteRl'It.y must alwa:;s be reo scrIbes the criteria for acceptance by tained in the "Declaration of Helsinki" e",,;pntlnlly dla¡¡iio~tlc or lhempeutlc for :i "iiected. 8very precaution ~hould he tAken to FDA of foreig-n clinical studies not con- patl",nt. lind mrirllcai re"i:arch. the es!'Pllt,inl re"pect. t.he privacy of the subject and to or the fn"~;'!n country's standard8, ohj~ct of whlrh Is piirely "'cientlflc and wit.h. mlriimlze the Impact of I.he study on the sub- ducted under an IND. In general. FDA whichever were used. If the foreign oiit Iniplyln¡r .lirect dinimostlc or thera- Jiicts phy!!lcal aiid mental integrity and on accept.'l siich studies provIded they are count.ry's standards were used, the prutie ,'alu,! to the person subjected to the t.he pcrsonaJ!t..v of the aubjcct.. we1l desi~necl, well conducted, per- Rponsor shall explain in detail how l('~I';i..ch. 7. Phn.lclans iihould ah"tain from en¡rirlng formed by qualified Investigators, and those standard8 differ from the "Dec- ~¡iecial c:iullon mii"t he iixiircli;fJll In L.he Iii i'r."'eareh projer.l" Invol"in~ humnn !'lIb- conducted iii accordance with ethical laration of Helsinki" and how they coiidui:t of re"eareh whh:h may nffeet the en. jriel.,; unless they ¡no: ".'.Liiirip.d that the haz- principles acceptable to t.he world com- "Ironm'!nt. anrl the w"if:ire of animals ui;ed ants involved are believed to be predictable. offer irreater protection. for rei;earch must h", rpiipectp(l. Phv",lcl;ini; "hould cpase iiny Inve8t-Ig:atlon If munity. Stuùlea meeting these criteria the h;izRrils are found to outweigh t.he poten- may be utilized to support clinIcal In- (3) When the research has been ap- ßt'iau"e It la e"8enllRJ thnt the reiiulta of proved by an Iiidp.pendent review com- lahorntory (,Xlli:I'lrneiit" he all,lIci! to humaii tial hlmeCit!'. vesLlgatLons in the United ~tes and! hp.lrilts tl) furlher 8c1entlfic knnwlerlKe iin'l to 8. In puhllcatlon of the re8uJt8 of hl8 or her or marler-tin!: approvaL. Marketln!: llp- mittee, the sponsor shall submit to research. the phyiilclan tii ohii~ed to preserve FDA documentation of !luch re\'Ip'w and h!"lp aiifferlo~ hum~ril!)'. thri WorJrl Medlcnl Jlroval of a /lew druir hased solely 011 l\s"tÌr,l;i I inn hl\ prcpal'l't1 thp. followlnR' ri:c- the accurar;y of the re.'Ult8. Report.8 of ex- forclim clinical data is governed by approval.. Inelurlliiir the names and ommrnil;it.lon8 as n J!uhle i 0 e"~ry phyl!lclnn ",.rlrn~ni.atlon not In accor,lnncp. with the § 3I1.106. .qualificat1onR of the memberR of the in hlomrir.ical re!'earch Illvol,,.lnl' human auh. prlnclpl~8 laid down In l.hl8 Declaration (b) Data sii/¡missions. A sponsor who committee. In thIs regard. a "i'evlew je':t!;. They l!hould hE' k"pt uiid"l' review In !'houlrl not h"l Accepted for piibllc:itloTl. wiahes to rely on a foreign clinIcal committee" meiins a committee com- the future. It must he slrril!sed that the 9. In an.v rei;earch on hiiman hiiinirii. each study to support. an IND or to Suppor- posed of sclp.nt.ists and, where prac- I "'tanllanl", as lIrnfliid aI''! only a ¡wiele to phy- poip.nfJal suhject must he adequately In- ticahlr., Individuals who arp. otherwlRe i "Ii:iaiis 1\11 o"er the w(Jrhl. Ph."sici;i.ns are not f()l'i~il of the ahn". methoi:!'. anl.!clpated an application for marketinll llpproval reIiP"p"1 fnim rrimlnn!. c:I\'1I aii') rthii:al re- heliiiflL~ and poi.ential h;izards of the study shall submit to FDA t.he following In- qualified (e.ir., other health prof~s- i spoli"lhllitles under the lnws of their -own and the dls('onifort It. mn.v ent.lI. He or she formation: sionala or laymen). The InvestIgator I couiitrlea. shoiiid hI! liiform~d that hI' or !'he Is at IIb- (1) A ilescriptlon of the Investigator's may not vote on 8.ny a8pect of the're- ert.:v to ali"t-aln from ¡iai.tlclpatlon in the "i ,.iy and that he or !'hp. I" frep, to withdraw qunllfications; view of hIs or her protocol by a revIew I i. Bu.tic rrinciples (2) A de~cription of the research fa-- committee. i i. nlomedlr,IlI rp.",parrh In'''ol\'ln£ human hl~ or her consent to i.arl.lclplltlon at any cilltle~; ,. ¡ time. The physlcil\n "hoiiid then ohl.alo the (1) The "D~c1aration of Helsinki" ",uhjeel" must conform t.o gr'nørnlly accpptr.d :;uhJpcl'io fl'epIY-P.lven Inrori~ consent. pref- (3) A cletail uininary of the pro- :--.. ~ri"ni in,: pl'lnclpJe" and shoiil!! he h:i8..1 on states as follows: ~'!"'luat~I.\' p"rrorm'!.l l~brii'at.'Jr)' and animal emhl.v In wrilihll. tocol and resi..vs of t~. study. and, .~ 10. Wli'!n ohtnlnln¡¡ lnf, J consent for pxp"rtinenl~t1on ~!1,i 0n a thorough know!. thr. l'.!Varch projpi;t the physlciaii iihouJd be p.fh!p. of I hi. C:t'¡".nrifir' Iífr....,tlll.p .... ~.ii: ,.~. i _ _1.. _. _ __. r _.. . I' ...... '. . §JI2.IJO 21 CFR Ch. I (4-1-Ç''' ~':1itjon) dpii('iid('nt rlJlationi;hfp to him or her or miiy Foed and Drug Administration, HHS con~"nt un(ler dlin'lOll. hi thiit CMe the In. th(' (,:'p"rlnipnlnl dcslirn Iii not rplatl'd to the §J 12. 145 formed COll!'ent lIhould he obtained by il phy. pal.ll'nl:s IlIn"qii. submit a request under the Freedom of Slclan who Is not en~a¡red In the Investiga_ 3. The Inv('~tl~ator or the Inv('!!L1itnting Inforiniitlon Act. (C) All correspondence relating to bl-. tion iind who III completely Independent of tPRm should dliicontlnue the r/!search If In ologlc:i1 products for human use which this offcial relationship. hl!'hiir ur their jUIIKment It mRY. If contln. 152 FR 883L Mnr. 19. I!lR7. n~/Jpql".natlJd at !i3 are also raiUoact.ve dru.iii shall be sub- 11. In caiie of le¡ral Incompetence. Informed ued. be harmrul to t.he IndivIduaL. FIt 415i1. Oct. 21. 1988. as am. ..i~d at 61 FR mltt.ed to the DIvJsion oC Oncology and coniient should be obtained from the leiral 4. In re!learch on mn.n. the Interp.st of 51530. Oct. ~. 199; 61 FR 401. Jan. 5. 1999) iruardlan In accordance "vlth natloniil leirls- aclence and 80clety lOhoulo: never t.ake preee- Radiopharmaceutical Drug Products latlon. Where ph:v"lcal or mental Incapacity d"ncc over coniiideratfoiis reliited to the EF'FF;CTI\'F. DATE NOTF.: At 6~ FR 401. .Inn. (HFD-150), Center for Drug Evaluation makes It Impossible to obtain Inrorme4 con- well-h('inR' of the liuhJr.ct. 5. 199. 1312.130 WAS aiiend"t1 b.v rcmovliiK' and. Rcseai'ch, Food and Drug Adminis- sent. or when the subject Is a minor. perm!s- (Collt'ctlon of Inrormatlon reQuiremt'nt.'l ap. "or ant.hlaLic drn¡r" (¡'om ¡iarnKrnph (b). ef- tration. 5GOO Fishers Lane. Rockvile, Rlon from the rei;pon!'lhle rel:i.tlve rpplacIJ9 proved Ii:v thii orr"p' or Man~p;pm"nt and fective May 20. 1m. MD 20857, except that applications for. t.i:it or t.ir lOlitijlict In Acconl:ince wIth na- BiulKet uiiilcr e(Jlit!'ol niirnhrr 0910-0(11) i t1111al ler.19Iatlon. § 312.1-10 AddreR/l for eorrcl'pondence. ' coupled antibodl!!s shall be submlttE"d Whenpver the minor chlld 19 In fact ahle to 152 Fn RB31, M:ir. 19. 1987. II!! Rnleiid"" iit 52 in accordanco with pai'agriiph (b) of give a COiisent. t.he mlnor's consent must be lo'n 2:i1l:J1. June 17. 1987; r, Fit 22113. Miiy 14. Ca) Ex'cept as provided in paragraph 1991; 61 FIt 401. Jan. 5, 199) this section. ' ObL.lllned In addition to t.he consent of the (b) of thl~ section, a sponsor shaH send (d) All correspondence relating to ex-. mlnor'9 legal J:uarillan. F.F'FECTIVE DATE NOTE: At 61 FR 401. Jan. an. initial IND submission to the Cen- port of an Investigational drug under 12. The rp.sparch protocol shOUld ill 5, 19'19. §312.1iO was arnt'ncled by rpmovhill tral Document Room. Center for Drug conLRln II stat.pment or the ethical con!llder_ways "or a.ntlbiotlc dnijt" from the laiit !'l'ntence Evaluation and Research. Food and §312.1I0(ù)(2) shall be submitted to the Iltlons InVolved aiiii i;hould Indicate t.hat the of pnl'airrnph (a), ..rfectlve May 20. 1999. Int.ernat.onaI Affairs Sta.ff CHFY-50), prliiclpl('!1 eniiiclat.ed III the preseiit Decll\rn- Druir AdministratIon. Park Bldg.. Rm. Offce of Health Affairs, Food and Drug: tloii are complI"i1 with. 214. 12120 Pai'klawn Dr.. Rockville. MD § :i12.I:¡O Avnilnbilty for piihlir diRcJo_ 20852. On receiving the IND. FDA wll Admlnlst.ratJon. SGOO Fishers Lane" fllIre 0(. diitn and informntion in an Rockvile, MD 20857. If. Medicol Rp'.tearch Comhined with IND. Inform the sponsor whteh one of the di- I'ro!lSsioiial Cure (Clinical Research) visIons in the Cent.er for Dl1lir Evalua- (a) The exliitence of an Inve~Llga. (Collection of InrorrnLloli rt'qulrement.'l ap- 1. III the trt'ntment of the sick person. the tion and Research or the Center for proved by the Ornce or ManaR'ernent. nnd' phY~lclan must he free to ui;e a new diag- tlonal new drug applfcatlon wl1 not be nostic and therapeutic measure. If In hla or disclosed by FDA unl('ss It has pre- BiologIcs Evaluation and Research Is Dudi:et uni1er control number 0910-11) her JudP.ment It oHcrs hope of savinI; IirP.. reo VIOusly becn publlc)y dISclosed or ac- responsiiile for the IND. Amendment.s, 152 FR 11831. Mar. 19. 1987. as amended at 52 cst,i.hlli;hin¡r healt.h or aJlevlntlnir suHerln~. knowleiiged. reports; and other correspondence re- FR 23031. June 17. 1987; 55 FR 11580. Mar. 29. 2. The potential benerii.. haz:irds and dls- (IJ) The avallalJIlty for puhllc dliiclo- lating to matters Covered by the IND 1991 comfort or.. new method should be welirhed Sure of all data and Information In 'an should be directed to the appropriate al;alnst. the advanl.l'es of the be~t current division. The outside wrapper of each § 312.145 Guidelines. dlagnoi;tlc and l.herapeutic mf!thods. Investigational new drug application I 3. In any medical study. every piiLlt'nt-ln_ for a new drug wil be handled in ac- submlsiiion shall stat.e what is con- (a) FDA has made available guide- cludln~ those of a control irroup. If Rny- cordance with the provisIons estab- tained in the submisiolon. for example, lines under § 10.90(b) to help persons to should be as.~nred or the h'l!'t proven diag. llshed in §314.430 for the confldent.ally "IND Application". "Protocol Amend- comply with certain requirements of nostle iind l.ieni.peiitlc method. of data and information in applications ment". etc. this part. 4. The I't'fui;al of the patient to participate sulJmit.ted in part 314. The availability . (h) Applications foi' the products list- In a sturly must never Interfere with the phy- for public di~closure of ail data and in- ed below shoulù be submitt.ed to the DI- (h) The Center for Drug Evaluation' Slclan-pat.ient relationship. and Research and the Center for BJo- . formation In an investi~ational new vision of Biological Investigational logics Evaluation and Research maIn- notI 5. to If obtain the phyi;lclnn Informed conslilers coni;'mt. Itthe e~i;enLial speciric drug application for a biological prod- New Drugs CHFB-230). Center for Bio- uct wl1 be governed by the provliiions tain lists of guIdelines that apply to r"iiqons for r.hls propoi;al shoulel be st.atecl In I logIes Evaluation and Reseiirch. Food the exp(,l'lmental protocoi for tmns¡iilssion of §§ 601.50 and 60Ul. and Drug Administration. 8800 Rock- the Centers' regulations. The lists i state how a person can obtain a copy of to the inllpl'('niIpnt conimll.t!'e (I. 2). r (C) Notwiih~tanding the provisions of i vile Pike. Bethesda. MD 20892. (1) 6. Thc physklan C:iii comhln!! medical re- §314.430. FDA shall rliiiclose upon re- I Products subject to the Iicensln¡r provi- each guideline. A request for a copy of sf'arch wil.h ilror('i;lOlon:i1 cnre. the objective quest t.o an indIvidual to whom aii In- I sions of the Publ1c Health Service Act the lists should be directed to the hiiin!: the aC'luilOiiion or new medical knowl- vest1¡mtional new drug lias been g-Iven edire. only t.o the extent \.int m!'illcai re- of July 1, 1944 (58 Stat. 682. as amended CDF.R Executive Secretariat Staff seareh Is JUl"i.lned b.v il.s IlOtenl.inl diairnostic a copy of any IND safcty report relat. (42 U.S.C. 201 et seq.)) or subject to part (HFD-8). Center for Drug Evaluation or lh('mpeutlc \'alue for the pntieiit. Ing t,o the use In the IndividuaL. 600; (2) Ingredients pack~.ged together and Research. Food and Drug AdmJnls- (d) Tlie aV'lI/:i.billt.v of information with containers Intended for the collec- tratlon. 5600 Fishers Lane. Rockvile, ILL. Nnn-7'1('07'"litic ßiomcdica/ llc.ttmch In. required t.o be publicly disclosed for In- MD 20857, for drug products, and the t'n/ping /lumun SlihjeCls (Non-Clinical Bio- vestigations inVOlving an exception tion. processing. or iit.oraire of blood or medical Rcseareh) blood components: (3) urokinaiie prod- Congressiona.l. Consumer. and Inter- from Informed consent under §50.24 oC n:- ~ional Affairs Staff (HF~142), Cen- 1. In the purel.v sClt'ntific aJlpllc:i.ion or this chapter ..ni he hand led as follows: I ucts; (4) plasma volume expanùers and m("lic:i1 1'''Scarch c:irried oul. on a hnmiin Persons wlsliiuK to i'equest the publicly hydroxyethyl starch for lriikaphereiiis; ter for Biologics. Evaluation and Re. lit'ln¡r. I t is t.he clut.y or i he ph~'sicinn to re- disclosable information in t.he IND that and (S) coupled antibodies. I.e.. prod- search, Food and Drug Administration, main the i'rott'ct,'ir of the Ii re and health of ucts t.hat consist of an antIbody com- 8800 Hockvlle Pike. Dethesda. MD ~hat 1'('l"on nn \\hn~"II"llical r('i;'!arch Is was required to be Cied In Docket Number 9r'S~158 in the Dock'?ts Man- i ponent coupled with a drug or radio- 20892. for biological products. ietnr. ('nri'lC'd au t. -lide component In which both CfJm- 2. 1'he lOUhj"Ci.ii ll, .1 he v~nlt'el's-(,i_ agement Branch (JlFA-305). I'ood and 152 FR 111m. M:ir. 19. 1987. a.q a' 'cd at 55 f .- .h('r h('l\li.hy P"''Ron,; or I'at.leiil's.' foi' whom Drug Adml~tration. 12.120 Park .~nts provide a pharmacological ef- FIt i J~RO. Mar. 29. 1900: 56 FR . Jan. 31. Dr.. nn. 1 23: HOl'kvi lit' ~fn ?nD~"7 _I.lawn _" leet hilt the biolo.ilcalcornponent de- . .._~- =- - - ~. 1991: 57 FR't?814. Mar. 31. 1992) . . " l §J; .'.lúO l ! 21 CFR Ch. I (4-1-99 (dilion) I Food 'and Drug Adiniiiislralion, UHS SubFJort G-Drugs for Investiga- Pf.314 (1) TIII sponsor of the i\l'cs(.igatloD ¡ tional Use in laboratory Re- has failp.ù to r.omply with any or the 314.60 I Amendments to an unapproved appli- 311.151 Withdrawal of Approval of nn abhre- search Animals or In Vitro f cation. . \ ;" Lf'i1 m'w driiir appllca1.0n under sec- conclit.ons for Shlpmcnt est.alilJshed 3ll.6.'i ¡ \\'lthiÌrawalby thp. appliiaiit of nn un. Tests under this section: or tion 505(j)(5) of the act. nlprovl'1 applicatIon. 314.152 Not.lce of withdrawal of approval Of §312.160 Dni¡r.. for invt'!'tigationnl wie (2) The continuance of the investiga_ 311.70 . Supplements and other chanires to aD lin iippllcation or abbreviated application in laborntory research animals or tion is unsafe or otherwise contrary to npproved Application. 314.71 Procedures for ,siibmliiiiion of -a sup- for a new:druK. in vitro tests. the pulilic intereiit or the dru¡r is used 314.153 SUlll'enslon of approval of an abbre- for purposeR other than bona fide scl- plement to an Appro\'p"1 application. (a) Autliori:znlion to ship. (1)(1) A per~ 31l.i2 Chani¡e In owii(!r~hlp of l\n Ai/Pllca- vll\teil new druir application. son may ship a ill'U~ Intcnilr.d solely for ent.lfe invr.stlirlltlon. FDA wil notify Unn. 314.1r.1 Approval of an application or iihhre- test!! In vitro or In animals USllU only. the rip.rsoii IlhlriPlni. the i1ruJl of IlR find. 311.80 T'n"t.rn:irk,.tlnK 1'p.pol.t.nl1 of Adverse vlated Aiiplh'atlon Cor which approval W"LL pl'evlously refused. suspended. or for laborator:: raseai'ch purposes 1C 1t 15' hlK and Invite Immediate correction. U drul1 rxl'erlrlH'!'II. correction is not immediately made. 3R81 Othpr I'o"tinarketlnll rl'portii. wlthdrnwn. labeled as follows: 311.161 IletJ'rmln"i Ion of reasons for vol- the periion lIhall have an opportunity' 314090 Wiiivern. CAUTION: Contains a np.w druir for Inves- for a regulatory hearing before FDA untary wlthdm Hal of R IIst!,rl drul\. tliratlonal use onl.v In lithomtory research pursuant to part 16. Subpart'C-Abbreviated Appirealions 314.162 Removal of a druir product from the animalii. or for tests In vitro. Not for use In list. humans. (c) Disposition of unused drug. The 3H.92 Druir product.s for which abhreviated 314.170 Adiiltpratlon and misbranding of an pcrson who ships the ùrug under para- applications mny he imhmittctl. approved drug, . (I) A person may ship a' bloIog-Jcal g-raph (a) of thIs section shall allRUre 3H.93 PetItion to request a chanl1p. from a. product fo!' Investl~ational In' vitro dl- the return of all unused supplip.s of t,he Iiiited drull.. Subpart E-Hearlng Procedures for New airnoiit.lc lise that Is listed in dnill from inrllvlilual Investil!ators 311.91 Content and forrrat of nn abhrrvlatcd Drugs §312.2(b)(2)(1l) if it is labeled as follows: wheneviir the hlvcstlKlltlon discon- nppl h:a t.lon. tinues or the inve!'tlgatlon is termI- 314.95 Noller of ('('rtlflcatlon of Invallility 311.?OO Not.lce of opportunity for heRrinI': CAUTION: ContaIns a bioloirlcal product or nonlnrl'lnl1ement of a patent. notice of participation nnd request for for lm'eiitiimtlonnl in vitro dlngnostlc tests nated. 'lhe person who ships the drug only. may authorize In wrltln¡r alternative 3H.9G Am.." ':ii"ntii to an unapproved abbre- hearlnir: l1rant or denial of hearing. disposition of unused supplies of the vlat.E'd appliC'atlon. 314.201 Procedure for heal'lngs. (2) A person shippinir a drug under 314091 i:"piileml'ntiiiinrl oth..r ch"nires to an 311.235 JiidicliiJ review. paragraph (a) of this section shall use drug 'provided this alternative disposi- Appro\'cd abhrevlated iippliciitlon. . due diligence to aSSure that the con- tion docs not expose humans to risks 314.98 l'rlstmarkJ't1n~ rpporLs. Subpart F-Admlnlstratlve Procedures for signee is regularly eniraged in con- from the ùrug, either directly or indi- 314.99 Oth'!r reiiponlilbllIUes of lin applicant Anllblolics ductinl! such tests and that the shlp- rectly (e.r-. through food-producing of an abbreviated application. animals). The shipper shall maintain 311.300 Procedure for the liisuance, amend- mentof the new drug wil actually be Subpart D-FDA Acllon on Applicallons ment..or repeal of rpgulations. used for test.s in vitro or In animals records of any alternative disposition. and Abbfevlaled Appficalions iiis~d (3) onlyA persoii for laboratory who ships rescarch. a drug undp.r (Coller-tlnn of Inr')I'mat.lon r"'llilr..m"ntsAIl Subpart G-Mlscellaneous ProvIsIons pmveil hy t.le' Offce of M"nR~p.rnpnt and 311.100 Tlm"fr""'l"~ C',r revl",,'lny. iippllca. paragraph (a) of this section shall Dudi¡eL under control numher 0910-011) t Inn" anil abhl'evlat.cil alipllc"Unns. 311.1!0 ),IlJJOrtli and exports of new druirs. 31UOI Filing lin appllciitlon and rJ'cl'lvlng 311.420 Druir master fles. maintain adequate records shO\ving the 152 FR 8631. Mar. 19. 1!181. aii amended at fi2 name and post offce address of the ex- lin IihhrcvlatE'rl new ,Iriiir application. 314.430 Availahlllt.v for public disclosure of FR 23031. June 17. 1981. Redesignated at 53 da.ta anillnforinatloii In an application pert to whom the drug iii shipped and FR 1J523. Oct. 21. 19881 314.102 Communications between FDA and ~he date, quantity, and batch or code iippllcant". or abhrcviated appllcat.lon. 314.103 Dispute resolution. 314.410 Addresses for applications "..d ab- mark of each shipment and delivery. PART 314-APPlICATi::NS FOR FDA Records of shipments under parairraph 314.101 JJrul!s with potr.ntiaJ for ahuse. brevi a ted. applications. APPROVAL TO MARKET A NEW 314.105 Appro,'al of an Application and an 311.145 Guidelines. (A.)(1(l of this lIection arc to bq main- DRUG Ahhrp.viated ;ippllcation. tained for a. period of 2 years an.er the i 314.106 Foreiim data. Subpart H-Acceleraled Approval 01 New shipment. Rp.corùs and reports of data 314.107 F:rfect.ve date of Approval of a Drugs for Serious or Ufe-Threatenlng il- and shipments under paragraph Subpart A-General ProvisIons 505rhl(21 iippll('ation III' nhhr!''''al....1 new nesses (a)(1)(li) of thIs section are to he mnln- S..c. drup. applicatIon under sectIon 5050) of t.ninl'd in accordaiil'c with §312.57(bL. .:H.I s,'opn of thlii part. the Rct. 311.MO Scope. .The person who ships the ùrug- shall 311.2 l'ul'POIIC. ~11.I08 New ,Inil( proiluct pxcluslvlty. 314.r.10 Approval has..,! lin a iiurrol'ate !'nd- UPOIl rei¡ue!'t from any properly au- 311.3 Dcl1nltionii. I 3l1.1J0 Approvable lPotter t.il l.he nppllriint. point or on an effri;t on a cllnltal end. thorized offcer or employee of the 314.120 Not approvahle letter to the appll. point. other than iiurvivalor Irrevernlble Fooel and Drug Admlnlst,ratlon, at rea- Subpart B-APplicatlons clint. morblrli ty. sonable tinip.s. permit siich officer or 314.122 !'uhmlttlni¡ an lIhlirp.vlall'd iippllca- 314 .520 Approv:i, wi th restrIctions to assure 311.50 Contpnt and r'ii'rnat of "n "ppJicat.on. 1 lion f')r. or a 505Cjl(2,icl p..tlUon that re- i"afe uiie. eniployl'fl to have aCCfl!'R to and COJIY 311.52 Nol.fce of çprtlkatlon of InvalidIty IIp.s (in. n Iliited drug that Is no longer 314.fi'l0 Wllhdr:iwal procediireii. anil verify r('eorùs required to be main- markelI'lL. or noii infi'lnireii"n t. of a patent. ¡ 311.r,10 f'o~t.marketlnir ii"fety reportinir. tained under I.hls section. 314.S3 Suhmliision of patent 'nformaUnn. 311.125 ii,.rulIaJ to approv,. "n appllratlon. 314.fi50 Prr:Jnot.lonal materials. (b) 1'r.rmiiialion of iiitliori;;ation to 311.fi1 Procedure for ~IJhinl~slon of an appll- 314.125 Adequate and well-controlled stud- 311.ræO 'lcrrnination of rer¡ulrements. slii/l. FDA inay "'-ninat.e authOl'iza- CR.liml "iiiliini¡ Inv"Hil1atlons for aii l I..". ~iO~l to ship a ùru ..ùp.r th~,seetlon if Ilrov,,1 of n new Indkatlon for. or other 3- Høfii~al to approve an abhrevlatcd A llTJlOP.TY: 21 U.S.C. 321. 331. :i~2. 353, it finùs that: " chau~e fr.. a liiitNI dru~. i w ilru~ appllciit Ion. 355.371. 371.3ïge. 311.55 1'E'lliat.ric u~1) Iiifnr'","i ;nn i 311.15Q Withdrawal of aI'PT'v,,1 of an nppll- SOURC¡:: 5l;"íiR 7493. Feb. 22. 19R5, unless i i"!'tinn nr .,h"rn..I...... _..r:.....r__ §50.20 Genera reuiments for in. planation as to whether any medic:i care to the ei:ten t the physician is per- fonned consent. t:ea.¡;men¡;s are avaible if injur oc- mitted to do so under applicable Fed- \~cept as provided in § 50.23. no in~ cur and. if so. what they consist of. or eral. State. or local law. &tiga.tor ma.y involve a hum being where further inormtion may be ob- as a. subject in research covered by tained. these reguations uness the investiga.~ (7) An explana.tion of whom to con- tor ha obtaned the legay effective ta.ct for anwers to pertinent questions 'J inormed consent of the subject or the a.bout the research and research sub- subject's legaly authoried re-pesenca- jects' rights. and whom to contat.in tive. An investigator sha sea suel the event of a. researh-related injur consene only under c1wntaces tht to the subject. provide the prospective subject or the (8) A statement tht pacipation Is representative sufficient opportuty voluntary, that refusal to paicipate to consider wlieeher or not to paci- will involve no penaty or loss of ÒèD.e- pate and that minie the possibilty fits to which the subject is otherwse of coercion or. undue infuence. The in- entitled. and tht the subject ma di formation that is given to the subject continue paticipation a.t an t1e or the representative shal be in ia~ without penaty or loss of benefits to guage underStandable to the subject or which the subject ia otherwe entitled. " the representative. No 1normed con- (b) Additional elemts of infor con- sent. whether oral or wrtten. may in- sent. When approprte. one or more of clude any exculpatory lae the followin elements of inormtion through which the subject or the rep- shl also be provided to eac subject: resentative is made to waive or appear (1) A. statement tht the pacular to waive any of the subject's lega.l treatment or proceur may involve rights. or. releases or a.ppea. to release risks to the subject (or to the embryo the investigator. the sponsor. the insti- or fetus. if the subject 15 or may be- tution. or its agents from liabilty for come prt) which ar curentl negligence. 'unoreseea.ble. . --...,-~- (2) Anticipated ciumtaces under which the subject's pacipation may be termted by the investigator w1thoutrega to the subject'S con- sent. (3). AnY a.ditiona costs to the sub- 51)0.2 . Elements of liormed const. ject that may result frm pacipation Ba elemts of informed consent. In the researh. .. l)seeki . inormed.'consent. the fol-- _. H)..The_consequences of a. subject's lowig inormtion sha be provided to decision to withdrw from the resea.h e&Csubject: and . procedurs .Io.t_orderly termnation __ __ (1). A. statement that the study in- of participation by the subject. volves research, an explanation of the (5) A statement tht signcat new puoses of the research and the ex- findings develope dur the coure of pete duration of the subject's parici- the research which may relate to the pation. a. description of the procedures subject's w111iess to contiue par- - to be followed. and identifcation of ticipation wi be provided to the sub- any procedures which are expri- ject. menta. (6) The a.pprox1te number of sub- . (2) A description of an reasona.bly jects involved in the study. foreseeable risks or discomforts to the. (c) The informed consent requi subject. . ments in these reguations ar not in- (3) A descrption of any benefi ts to tended to preempt any applicable Fed- the subject or to others which may rea- era. Sta.te. or loca laws which require ;ionably be expted from the research. a.ddltional informa.tion to be disclosed (4) A disclosure of appropriate alter- for informed consent to be legally ef- native procedures or coures of treat- fective. ment. if any. that might be advan- Cd) Nothing in these regua.tions is in- tageous to the subject. tended to limt the authority of a phy- (5) A statement describing the ex- sician to provide emergency medical tent. if an. to which confdentiality of ::.J. records identifying the subject will be ïrtanèd and tht notes the possibil- ity tha.t the Food and Drug Adminis- tration may inspect the records. (6) For research involving more than mini risk. an explanation as to whether any compensation and a.n ex-

)

,; Information Sheet for a Claim of Categorica ). .Exclusion for an INO Under 21 CFR 25.24 For those wases generated in the producton and use of the produd which wil be :J contUed, please include documentaon that such wase .storage or disposal is in compliace wi federal, ste and loca requirements for hazdous wae producton. As an alrnate. identi an genera recognized, scientificaly sound control proceures which have been. Implemented to reduce the likelihood of inadvertent release of potential toxic materials into tte environment (e.g., compliance wi the NIH GuideDnes for Research InvoMng Recombinant ONA Molecules (51 FR 16958 (1886)) and/or compDace wi the EPA Effuent Guidelines and Stadards for Pharaceutca Manufctring (40 CFR 439)). If these alternaties are not applicale~ " a descption of the control procedures actally used to prevent wåste from entering the environment should be submited.

For 'those ,waes' generated in the producton and use of the produd which will not be contlled, please list the potentially toxic wae compounds, including the quanties and cocentations which mey be expeded to enter the environment from both productons of the produd and from the intended clinica studies~ and briefly describe the imediate environment into which such release will occur. Furter. provide the appropriate references or experimenta data from which it may be reasonably concluded that such release Is non-toxic.

) If the wase to be generated during the producton and proposed investgational use of this produci, Is eiter riot 'ëëiñtIo/led or Is ,'not' reãsonàbly expedëd to be non-toxic in . - the' environment to whiph it wil. be released. please submit an environmental åssessment using the format described in 21' CFR 25.31. If actons under proposed amendments to this INO substntially alter the quanti, qualit or conditons of wase r'elease'ln'süch"à way as to alter the basis for eiter a claim of categorica exclusion or an environmental assessment. then such . . amendments should be supported by'the appropriate data for a claim of categorical exclusion or an amended environmental assessment for wastes generated under the proposed amendments. to this INO.

An investgator sponsored INo for which-no additonal produd manufaduring is intended will ordinarly have addressed'these environmental issues by incorporating the manufactrer's INO or MF by cross reference. However, if the use of the produd ~~.. ~~ during clinica investgation is expected to result in the uncontrqlled release of toxic submited.materials into the environment then an environmenta assessment . should be

3/30/94 )

_.. .-..--.-"-.----.--.. - _._.. 0 _. _ _ . ....

- . -.. 0 . ._...__ _.__ __0_ _. .____ . ~~ìb~t 1-

DEPARTMENT OF HEALTH AND HUMAN SERVICES

(j. Food and Drug Administration Silver Spring MD 20993

Our STN: BL (125377/0) BLA ACKNOWLEDGEMENT July 8, 2010

Bristol-Myers Squibb. Company Attention: A. Heather Knight-Trent, Phar D. Director-Oncology 5 Research Parkway Wallingford, CT 06492-7660

Dear Dr. Knight-Trent:

We have received your biologics license application (BLA) submitted under section 351 of the Public Health Service Act (PHS Act) for the following:

Name of Biological Product: Ipilmumab

DRte of Application: JUNE 25,2010

Date of Receipt: JUE 25, 2010

Our Submission Trackig Number (STN): BL 125377/0

Proposed Use: Pretreated Advanced Melanoma

We wil notify you within 60 days of the receipt date if the application is sufficiently complete to permit a substantive review.

The BLA Submission Tracking Number provided ve should be cited at the top of the first page of all submissions to this application. If you e any questions, contact Erik S. Laugher, Senior Regulatory Health Project Manager, at (301) 96-1393. Sincerely,æ,.~.. ~ /Patricia Keegan Patricia Keegan, M.D. Director Division of Biologic Oncology Products Offce of Oncology Drug Products Center for Drug Evaluation and Research ~hi~)Lf

Exhibit 8

DESCRIPTION OF SIGNIFICANT ACTIVITIES OF APPLICANT DURING REGULATORY REVIEW

!Date ¡Event 11 2-JUL- 2000 ¡Submission of initial IND under IND8937.005 by Medarex 11 3-JUL- 2000 ¡FA receipt of IND (transferred by FDA to IND 9186.000) 131 -JUL- 2000 ISubmission of protocol amendment 124-AUG-2000 ¡Submission of protocol IRB approval 127-NOV-2000 ISubmission of protocol amendment 123-AUG-2001 ISubmission of protocol amendments and investigator brochure 104-SEP-2001 ISubmission of annual report Submission of protocol amendment, IRB approval, approved informed consent, and toxicity 111 -MAR-2002 report 127-JUN-2002 ISubmission of pharacokinetic data 127-JUN-2002 ¡Submission of protocol, DTIC, and informed consent 120-AUG-2002 ISubmission of protocol amendment 17 -OCT -2002 ISubmission of annual report containing revised investigator brochure 120-JAN-2003 ¡Response to FDA request for monit?ring information 128-FEB-2003 ISubmission of new protocol 121 -MAR-2003 ISubmission of protocol amendment 113-MAY-2003 !Request for mid-phase II teleconference 119-MAY-2003 ISubmission of protocol 101 -JUL-2003 ¡Submission of new protocol 103-JUL-2003 ISubmission of protocol amendment 109-JUL-2003 ISubmission of protocol amendment 105-AUG-2003 ¡Submission of mid-phase II follow-up questions ¡1 1 -SEP-2003 IResponse to FDA request regarding patient death ¡Submission of protocol amendment 116-SEP-2003 I

106-0CT - 2003 ¡ISubmission of annual report 123-DEC-2003 IISubmission of investigator brochure, version 4

DC: 3970774-4 Page 1 of 14 Inate oovent 129-DEC-2003 !Request for EOP2 meeting 110-MAR-2004 ISubmission of protocol amendment 117-MAY-2004 ISubmission of protocol amendment 103-JUN-2004 ISubmission of protocol amendment 122-JUN-2004 ISubmission of protocol amendment 122-JUN-2004 ISubmission of protocol amendment 120-AUG-2004 ISubmission of annual report 118-0CT-2004 ISubmission of investigator brochure, version 5 127-0CT-2004 ISubmission of protocol amendment 101-JAN-2005 ISubmission of annual report supplement and investigator brochure, version 6 104-MAR - 2005 ISubmission of protocol amendment 122- MAR - 2005 ISubmission of quarterly safety report 128- MAR - 2005 ISubmission of rational for protocol arm 120-JUL-2005 INotification of study closure for Canadian protocol 128-JUL-2005 ISubmission of quarterly safety report 108-AUG-2005 !Transfer of IND from Medarex to Bristol Myers Squibb (BMS) 111 -AUG-2005 ¡FA acceptance of BMS sponsorship 123-AUG-2005 ISubmission of investigator brochure 130-AUG-2005 ¡Notification of orphan drug designation 112-SEPT-2005 ISubmission of new investigator information 113-SEP-2005 ISubmission of data summary 122-SEP-2005 ISubmission of quarterly safety report 126-SEP-2005 IRequest for type B meeting ¡03-0CT - 2005 ISubmission of protocol amendment

105-0CT - 2005 ISubmission of preliminary safety data, protocol synopsis, and draft protocol 106-0CT - 2005 IReceipt of Orphan Drug Application letter from FDA 113-0CT-2005 ¡FA acceptance of sponsorship 114-0CT-2005 IConfirmation from FDA of EOP1 meeting

120-0CT -2005 ISubmission of annual report

120-0CT - 2005 ISubmission of investigator brochure, version 8 128-0CT-2005 ¡FA comments on briefing document submitted for EOP1 meeting

Page 2 of 14 Inate !Event I

103-NOV-2005 IRequest for SPA I

118-NOV-2005 ISubmission of protocol amendments, revised protocol, and new investigator information I

122-NOV-2005 ISubmission updated safety data I

122-NOV-2005 !preliminary comments from FDA regarding BMS questions I

129-NOV-2005 ISubmission of CMC amendment I

107-DEC-2005 ¡FA minutes of 11/28/05 EOP1/pre-phase 2 meeting I

109-DEC-2005 I~orrespondencecomments on SPA regarding sponsor minutes from 11/28/05 EOP1 meeting and FDA i

113-DEC-2005 ISubmission of protocol amendments and new investigator information I

114-DEC-2005 ISubmission of quarterly safety report I

122-DEC-2005 I~ynchronizationSubmission of annual of oncology integrated annual summary reports of safety I

111-JAN-2006 ISubmission of protocol amendments I

126-J AN - 2006 IRequest for FDA feedback and guidance on revision of protocol I ¡FDA responses to BMS questions regarding protocol revisions 130-JAN-2006 I

106- FEB- 2006 IRequest for SPA I ~;otocois for studies to investigate monotherapy in solid tumors or 113- FEB- 2006 ematologic malignancies can be included under IND 8937. I 120- FEB- 2006 IsP A submission 121 -FEB-2006 ISubmission of new protocol, revised protocol, and protocol amendments 121-FEB-2006 ISubmission of request for SPA 112-MAR-2006 !Discussion of SPA with FDA 114-MAR-2006 ISubmission of new investigator information 114-MAR-2006 ¡Letter to investigators regarding protocol 115-MAR-2006 ¡FA request for a formal type C meeting 116-MAR-2006 IRequest for SPA 116-MAR-2006 IFDA rejection of request for SPA 120- MAR - 2006 ISubmission of draft statistical analysis plan 123- MAR - 2006 ICorrespondence from FDA regarding SPA 124- MAR - 2006 fDA internal meeting regarding SPA 124- MAR - 2006 ¡FA rejection of request for SPA

129-MAR - 2006 ISubmission of new investigator information

Page 3 of 14 !Date I ¡Event ¡Request for SPA for revised protocol 111 -APR-2006 1 13-APR-2006 ransfer of obligations to CROs I I Submission of protocol amendments and revised protocol

120-APR-2006 I ¡Submission of new investigator information

120- APR - 2006 I Submissionransfer of obligationsof new protocol to CROs lio-MAY-2006 ¡ISubmission of new investigator information lii-MAY-2006 !FA comments on SPA 115-MA Y -2006 !FA grant of SPA 118-MAY-2006 tteleconference regarding reproductive toxicology studies 118-MAY-2006 ¡Request for FDA clarification on SPA 118-MAY-2006 ¡Request for FDA guidance on international Phase 3 triaL. 119-MAY-2006 !Withdrawal of protocol 124- MAY - 2006 IResponse to FDA regarding reproductive toxicology studies 130-MAY-2006 IResubmission of request for SPA 131- MAY - 2006 ISubmission of administrative letter, protocol amendment, and new investigator information I 102-JUN - 2006 ISubmission of administrative letter 114-JUN-2006 ¡Submission of revised protocol 116-JUN-2006 IFDA comments on SPA 127-JUN-2006 ISubmission of BMS proposal regarding reproductive toxicology studies 128-JUN-2006 ¡Submission of annual report 129-JUN-2006 ¡Submission of new investigator information 112-JUL-2006 IRequest for FDA advice regarding proposed modifications to IRC charters 117-JUL-2006 ¡Submission of protocol amendment and new investigator information 121 -JUL-2006 ¡Request for FDA feedback on retreatment recommendation 126-JUL-2006 ISubmission of new investigator information 103-AUG-2006 ¡Submission of protocol amendment and new investigator information 105-AUG-2006 ¡FDA review of retreatment recommendation ¡io-AUG-2006 ISubmission of proposal regarding DMC review 114-AUG-2006 ¡Submission of new investigator information 122-AUG-2006 ISubmission of new investigator information 125-AUG-2006 IRequest for CMC type B meeting

Page 4 of 14 IDate I !Event 128-AUG-2006 IISubmission of protocol amendment and new investigator information

131 -AUG-2006 I !Receipt of FDA feedback regarding BMS proposal for IRC charters 131-AUG-2006 I tleleconference regarding a site-specific amendment ¡Request for EOP1 type B meeting 131-AUG-2006 I

107 -SEP- 2006 ISubmission of protocol amendment, informed consent form, and draft case report forms 114-SEP-2006 ISubmission of new investigator information and investigator brochure 118-SEP-2006 ISubmission of background information for 10/19/06 CMC type B meeting 126-SEP-2006 ISubmission of new investigator information IOJ-OCT - 2006 IBMS request for FDA review of cardiovascular safety and assessments. 106-0CT - 2006 IBMS request for fast-track designation

112-0CT -2006 !FA comments on site-specific amendment 126-0CT - 2006 ISubmission of protocol amendment and revised protocol 127-0CT-2006 ¡Submission of new investigator information for protocols CA184-022, -025 127 -OCT - 2006 ISubmission of new investigator information and revised protocol 103-NOV-2006 ISubmission of protocol amendments and revised protocols 107-NOV-2006 !Follow-up from 10/19/06 CMC type B meeting 108.,NOV-2006 ¡Submission of new investigator information 11O-NOV-2006 ISubmission of new investigator information 121-NOV-2006 ISubmission of clinical study report 121-NOV-2006 ¡Submission of new investigator information 128-NOV-2006 /FA Grant of fast track designation 128-NOV-2006 ¡FA support for site-specific protocol amendment 107-DEC-2006 Ilpilimumab presentation to DMC 113-DEC-2006 'Submission of IND SN352 115-DEC-2006 ISubmission of new investigator information 119-DEC-2006 ISubmission of new investigator information 12l-DEC-2006 ¡Submission of new investigator information

109-JAN-2007 I Requestesponse to FDA regarding wording for amendments and regarding patients with mixed 112-JAN-2007 IISubmission of new investigator information 118-JAN-2007 IIBriefing of new FDA reviewer on recent expedited safety reports.

Page 5 of 14 !Date I !Event 102-FEB-2007 IISubmission of SPA, draft revised protocol, protocol amendment, and draft DMC charter

105-FEB-2007 I SubmissionMC charter of SPA, draft revised protocol, informed consent form, case report form, and 108-FEB-2007 IISubmission of new investigator information 108-FEB-2007 ¡ISubmission of protocol amendment and revised protocol

106- MAR - 2007 lIs sue of investigator letter regarding collecting blood samples. 107 - MAR -2007 ISubmission of administrative letter and investigator letter 115-MAR-2007 ISubmission of addendum to investigator brochure, version 9 116-MAR-2007 ¡Amendment to Drug Master File for CMC variation for a new presentation 122- MAR - 2007 ¡Submission of protocol amendment and revised protocol 127 -MAR - 2007 ISubmission to FDA of minutes from 11/2/06 meeting 130- MAR - 2007 ISubmission of administrative letter, protocol amendment, and revised protocol 103-APR-2007 ISubmission of new investigator information 104-APR-2007 ISubmission of new protocol lio-APR-2007 ¡Submission of administrative letter 113-APR-2007 ¡Submission of CMC information amendment

120-APR -2007 IProposal of addendum to IRe charter 125-APR-2007 ¡Notice to FDA of a safety event ¡30-APR - 2007 ISubmission of new investigator information

101 - MA Y - 2007 ISubmission of initial written report for expedited safety report Ill-MAY-2007 ¡Submission of protocol amendment 115-MAY-2007 ¡Response to FDA request for synopsis for ipilmumab treatment use protocol 116-MAY-2007 ¡Submission of new investigator information 122-MAY-2007 ISubmission of new protocol 124- MAY -2007 ¡Submission of request for review of proposed tradename

125-MA Y -2007 ¡Submission of addendum to IRC charter 130-MAY-2007 ISubmission of new investigator information 104-JUN-2007 ISubmission of new investigator information 105-JUN-2007 !Discussion of timelines for submission of treatment protocol with FDA 111 -JUN-2007 ¡Response to FDA request for information 115-JUN-2007 ISubmission of addendum to investigator brochure

Page 6 of 14 pate oovent 115-JUN-2007 ISubmission of draft treatment protocol

120-JUN - 2007 ¡Submission of administrative letters and revised protocol

120-JUN - 2007 ISubmission of protocol amendment and revised protocol 122-JUN-2007 Wre-BLA meeting discussion with FDA

126-JUN - 2007 ¡Submission of annual report 127-JUN-2007 IResponse to FDA request for information 128-JUN-2007 ¡Submission of new investigator information BMS to provide Core Statistical Analysis Plan for Clinical Study Reports of Protocols 129-JUN-2007 CA184-004, 007, -008, -022 and -024 in Unresectable Stage III or iv Melanoma 103-JUL- 2007 ISubmission of administrative letters 112-JUL-2007 ¡Submission of new investigator information ¡Submission of revised draft treatment protocol 113-JUL-2007 117-JUL-2007 ISubmission of new investigator information 117-JUL-2007 ISubmission of protocol amendment, revised protocol, and informed consent form 117-JUL-2007 ¡Correspondence with FDA regarding SPA 118-JUL-2007 !FA receipt of development meeting background document 118-JUL-2007 !FA approval of draft treatment protocol 118-JUL-2007 ILetter from FDA regarding treatment use protocol Response to FDA request for submission of development meeting background document as /18-JUL-2007 an informal amendment to IND 119-JUL-2007 ¡Response to FDA request for redline of revised protocol 120-JUL- 2007 ¡Submission of new protocol 131 -JUL-2007 ISubmission of protocol amendment and revised protocols 108-AUG-2007 ISubmission of new investigator information 108-AUG-2007 ¡Submission of new investigator information 120-AUG-2007 ISubmission of protocol amendment and revised protocols

120- A UG- 2007 ISubmission of new investigator information 129-AUG-2007 IRequest for a face-to-face CMC pre-BLA type B meeting

106-SEPT -2007 ¡Submission of new investigator information

107 -SEPT - 2007 ¡Letter from FDA confirming 10/31107 type B meeting

125-SEPT -2007 ¡ISubmission of CMC pre- BLA type B meeting background document

102-0CT - 2007 1 ¡Submission of investigator brochure

Page 7 of 14 !Date ¡Event 104-0CT - 2007 ISubmission of new investigator information 105-0CT-2007 ¡Communication of plans regarding access program 115-0CT-2007 ¡Submission of minutes from 8/28/07 meeting and update on status of communication plan 115-0CT-2007 IRequest for a type B meeting 116-0CT-2007 ¡Submission of new investigator information 123-0CT-2007 ISubmission of new investigator information 123-0CT-2007 ISubmission of communication plan for treatment protocol 129-0CT-2007 !Letter from FDA regarding type B meeting 129-0CT - 2007 ¡FA comments on briefing package for 10/31107 CMC pre- BLA meeting

13 1 -OCT - 2007 /Type B CMC pre-BLA meeting with FDA 105-NOV-2007 ISubmission of new investigator information and administrative letter 114-NOV-2007 ISubmission of new investigator information 114-NOV-2007 ¡Submission of updated consent forms 116-NOV-2007 ISubmission of protocol amendment and revised protocols 116-NOV-2007 ISubmission of background document for type B meeting 103-DEC-2007 ISubmission of new investigator information 105-DEC-2007 ISubmission of clinical study reports 112-DEC-2007 ¡Response to FDA questions

114-DEC-2007 I ¡Submission of new investigator information 119-DEC-2007 IIResponse to FDA questions received on 12/18/07 120-DEC-2007 1 /Type B meeting at FDA

107-JAN-2008 1 !Notification of new BMS contact ¡Response to FDA request for hepatotoxicity analysis 124-JAN-2008 I 125-JAN-2008 ¡¡Submission of new investigator infonnation 131-JAN-2008 IIFDA response to hepatotoxicity analysis ¡FDA feedback on review of tradename 131-JAN-2008 1 104- FEB- 2008 IISubmission of change in investigator information 106-FEB-2008 I ¡Email correspondence with FDA regarding hepatotoxicity management 108-FEB-2008 IISubmission of administrative letter 120- FEB- 2008 IIBMS request for type C meeting 121-FEB-2008 I !Withdrawal of application for type C meeting.

Page 8 of 14 !Date I !Event ¡Request for a face-to-face pre-BLA type B meeting 121 -FEB-2008 I 125-FEB-2008 IISubmission of final clinical study report 126-FEB-2008 IISubmission of change in investigator information 127-FEB-2008 IIRequest for SPA 103-MAR-2008 IILetter from FDA confirming 4/25/08 pre-BLA meeting 111 -MAR-2008 i ISubmission of protocol amendment and revised protocols

121 -MAR-2008 IISubmission of background document for type B pre-BLA meeting.

128-MAR-2008 1 !Notification of new BMS contact

128- MAR - 2008 1 ¡FA Comments on SPA ¡01-APR-2008 ¡!Response to FDA comments regarding SPA 102-APR-2008 ¡Submission of new investigator information and change in investigator information 111 -APR-2008 ¡FA request regarding expanded access protocol 114-APR-2008 ISubmission of new protocol and new investigator information 114-APR-2008 !Acknowledgement acceptance letter for SPA ¡Submission of changes in investigator information 115-APR-2008 1 ¡Response to FDA comments 118-APR-2008 I

118-APR-2008 ISubmission of new investigator information I

125- APR - 2008 Wre-BLA meeting I

129-APR-2008 IFDA request for teleconference I

130- APR - 2008 IResponse to FDA request regarding changes to protocol I

130-APR-2008 ¡Submission of SN 061 I ¡Submission of new investigator information 108-MAY-2008 I

113-MA Y-2008 ¡Submission of protocol amendment and revised protocol I 13-MAY-2008 ¡Submission of new protocol and new investigator information

I Irransfer of obligation I ¡Addendum to investigator brochure, version 10 115-MAY-2008 i ¡Responses to FDA comments 129-MAY-2008 I

130- MAY - 2008 ISubmission of protocol amendment and revised protocol I

106-JUN - 2008 IResponse to 6/5/08 FDA request regarding amount of site activity and status I

109-JUN - 2008 ISubmission of new investigator information I

126-JUN - 2008 ISubmission of annual report I

Page 9 of 14 pate I ¡Event 101-JUL-2008 IISubmission of documents for 7/9/08 teleconference 109-JUL-2008 I tteleconference with FDA 104-AUG-2008 IISubmission of protocol amendment 107-AUG-2008 IISubmission of new investigator information 121-AUG-2008 IISubmission of prostate meeting information 126-AUG-2008 ¡¡Submission of new investigator information

127-AUG-2008 I ¡Response to FDA request to provide information for 7/912008 teleconference 128-AUG-2008 IISubmission of CMC-DMF amendment ¡Submission of investigator brochure, version 11 128-AUG-2008 I ¡Submission of new investigator information 104-SEP-2008 I 110-SEP-2008 IISubmission of protocol amendment

119-SEP-2008 I ¡Discussion on prostate meeting with FDA 125-SEP-2008 IISubmission of protocol amendment and revised protocol 130-SEP-2008 IResponse to FDA request for revisions to informed consent form 101 -OCT - 2008 ISubmission of new investigator information 107-0CT-2008 ¡Submission of protocol amendment 108-0CT-2008 ISubmission of protocol amendment and revised protocol 117-oCT-2008 ISubmission of new investigator information 117-0CT-2008 ¡Request for SPA 123-0CT-2008 ISubmission of request for review of proposed trade name 128-0CT - 2008 IDiscussion regarding SPA request, safety issues, and DMC 129-0CT - 2008 ISubmission of new investigator information 130-0CT - 2008 ¡Submission of final clinical study report 107-NOV-2008 ISubmission of amendment to SPA 107-NOV-2008 ¡Submission of revised case report form 118-NOV-2008 ISubmission of clinical report protocol 119-NOV-2008 ISubmission of new investigator information 119-NOV-2008 ¡ISubmission of slides for 1 1/20/08 teleconference ¡Submission of new investigator information 118-DEC-2008 I 119-DEC-2008 IIFDA questions concerning revisions to study ¡Submission of new investigator information 106-JAN-2009 I

Page 10 of 14 Inate !Event I 122-JAN-2009 ISubmission of addendum to investigator brochure, version 11 I 127-JAN-2009 IRequest for FDA assistance on SPA I 130-JAN-2009 ISubmission of new investigator information I 118-FEB-2009 ¡Email correspondence with FDA regarding DMC I 119-FEB-2009 ISubmission of new investigator information I ¡Submission of new protocol, protocol amendment, new investigator information, and I 124-FEB-2009 kransfer of obligation 125- FEB- 2009 IRequest for SPA 1 126-FEB-2009 ISubmission of protocol amendments and revised protocols 1 103-MAR-2009 ¡Letter from FDA regarding SPA comments 1 117-MAR-2009 IResponse to FDA regarding SPA comments 1 120-MAR - 2009 IRequest for type C meeting 1 120-MAR-2009 ¡Meeting request regarding SN694 I 123- MAR - 2009 ¡IResponse to FDA request for additional information I 126- MAR - 2009 IISubmission of archival copy of approved protocols and amendments approved under SPA I 127-MAR-2009 1 ¡FDA response regarding logistics for type C meeting 1

101 -APR - 2009 1 ¡Submission of new investigator information 1 103-APR-2009 IICorrespondence with FDA regarding converting to eCTD 1 ¡¡Submission of summary of changes, revised protocol, protocol amendments, abbreviated I 106- APR - 2009 ¡statistical analysis plan, IRC charter and data monitoring committee information ¡Submission of change in investigator information I 106- APR - 2009 1 122-APR-2009 IISubmission of protocol amendment and revised protocol I 130- APR - 2009 ¡ISubmission of background document for type C meeting I 130- APR - 2009 1 ¡Safety teleconference with FDA 1 101-MAY-2009 I ¡Teleconference regarding Type C questions 1 127-MAY-2009 IISubmission of protocol amendment and revised protocol 1

128- MAY - 2009 I ¡Submission of changes in investigator information I 128-MAY-2009 IIFDA comments regarding upcoming teleconference I

103-JUN - 2009 I ¡Teleconference with FDA regarding comparability process I

¡1 1 -JUN-2009 I ¡Submission of protocol amendment and revised protocol 1 125-JUN-2009 IISubmission of annual report 1 114-JUN-2009 IISubmission of change in investigator information I

Page 11 of 14 !Date I ¡Event 114-JUL-2009 IISubmission of change in investigator information 128-JUL-2009 I ¡Response to FDA request for meeting minutes from teleconference 107-AUG-2009 I 'Submission of amendment to CMC information by reference to DMF amendment

107-AUG-2009 I informationSubmission of new protocol, protocol amendment, revised protocol, and new investigator 124-AUG-2009 IISubmission of investigator brochure, version 12 ¡Submission of administrative letter 125-AUG-2009 I 114-SEP-2009 IIRequest for review of proposed tradename ¡Submission of addendum to investigator brochure, version 12 129-SEP-2009 I 114-0CT-2009 IISubmission of protocol amendment, revised protocol, administrative letter 123-0CT-2009 ¡¡Submission of new investigator information and change in investigators 106-NOV-2009 ISubmission of protocol amendment and revised protocol

I 13-NOV-2009 ISubmission of preliminary data J03-DEC-2009 IRequest for type C meeting

107-DEC-2009 Wurchase of MDS Pharma Services's central lab by Clears tone Central Laboratories 109-DEC-2009 JSubmission of change in investigator information 111 -DEC-2009 ISubmission of background document for type C meeting 114-DEC-2009 IRequest for type B pre- BLA meeting 112-JAN-201O ¡Submission of administrative letters and protocol amendment 119-JAN-201O ¡Request for waiver of requirements regarding supportive documentation

125-J AN - 2010 ISubmission of clarification questions to FDA regarding BLA Correspondence with FDA regarding topics for background document discussion on 102- FEB- 2010 3/4/2010 J22-FEB-201O ¡Submission of new investigator information and change of investigator information 102-MAR-2010 I ¡FDA comments on ipilimumab pre-BLA 112-MAR-201O IIRequest for type C meeting ¡Submission of protocol amendment and revised protocol /23-MAR-201O I 123-MAR-201O ¡ WDA grant of type C meeting request 124-MAR-2010 ¡ ICorrespondence with FDA regarding informal meeting request with CDRH 124-MAR-2010 I ¡FA grant of waiver for studies not conducted under IND 109-APR-2010 ¡IRequest for proprietary name review 126-APR-2010 1 ISubmission of comparability type C meeting background document

Page 12 of 14 ijate I !Event

105-MAY-201O I ¡Submission of clinical study report 105-MAY-201O IIFDA request for information on Al monkey toxicity 107-MAY-2010 ¡!Response to FDA request for information regarding patients with brain metastases 118-MAY-201O IIResponse to FDA request for information regarding pre-license inspections esponse to FDA comments on enhanced pre- and post-natal development protocol in 116-JUN-201O onkeys 122-JUN-201O IISubmission of annual report 125-JUN-201O ¡ISubmission of initial BLA application for ipilimumab injection (5mg/1mL) 107 -JUL- 2010 ¡Submission of administrative letter, revised protocol, and new investigator information 108-JUL-201O ¡FA acknowledgement of BLA receipt 112-JUL-201O tELA monthly update teleconference !14-JUL-201O ISubmission regarding expanded access program 102-AUG-2010 IResponse to FDA request for information regarding statistics 11O-AUG-201O ISubmission of proposal for providing high-level OS 113-AUG-201O IResponse to FDA regarding annotated package insert 116-AUG-201O ¡Response to FDA questions 116-SEP-201O IResponse to FDA regarding protocol and SPA 120-SEP-201O 'Response to FDA nonclinical requests 128-SEP-2010 ¡Response to FDA requests regarding ECGs 128-SEP- 2010 ¡¡FDA grant of tradename YERVOY 130-SEP-2010 ¡Response to FDA request to provide study I04-0CT - 2010 ISubmission of investigator brochure, version 13 119-0CT-201O ¡Response to FDA questions 112-NOV-201O ISubmission of administrative letter, protocol amendment, and new investigator information 116-NOV-201O IResponse to FDA requests from 11/4/2010 I02-DEC-201O ISubmission of statistical analysis plan 113-DEC-201O ¡Response to FDA's request for information regarding safety review I06-JAN-2011 IResponse to FDA package insert revisions 12-JAN-201 1 I ¡IResponse to FDA request for CMC information ¡18-JAN-2011 IIResponse to FDA request regarding post marketing requirement request

120-JAN-201 1 I IResponse to FDA request regarding proposed REMS comments

Page 13 of 14 pate I ijvent 107-FEB-2011 IIResponse to FDA request regarding immunogenicity 108-FEB-2011 ¡¡Response to FDA request regarding proposed labeling and cases for further examination

108-FEB-201 1 ¡IResponse to FDA request regarding business card in packaging 122-FEB-2011 IIResponse to FDA request for additional CMC information

124-FEB-201 1 I !Response to FDA request regarding package insert revisions ¡Response to FDA request regarding post-marketing requirements and medication guide ¡01 -MAR-201 1 I

101 -MAR-201 1 ¡IResponse to FDA request for CMS and PMC information 102-MAR-2011 IResponse to FDA request regarding postmarketing commitments Ill-MAR-2011 IResponse to FDA request regarding carton/container labels 114-MAR-2011 !Response to FDA request regarding package insert revisions 114-MAR-201 1 ¡Response to FDA request regarding REMS and supporting document assessment revisions 114-MAR-201 1 IResponse to FDA request regarding postmarketing commitments 115-MAR-201 1 ISubmission of revised version of REMS materials 124-MAR-2011 IResponse to FDA package insert revisions and REMS material 125-MAR-2011 ¡Response to FDA revisions to BMS REMS webpage ¡25-MAR-2011 ¡Approval of BLA for YERVOY (ipilimumab)

101-APR-2011 ISubmission of final product label/structured product label management guide submission

Page 14 of 14