(12) United States Patent (10) Patent No.: US 7,994,135 B2 Doronina Et Al

US007994.135B2

(12) United States Patent (10) Patent No.: US 7,994,135 B2 Doronina et al. (45) Date of Patent: Aug. 9, 2011

(54) MONOMETHYLVALINE COMPOUNDS EE R 358, si al. CAPABLE OF CONUGATION TO LIGANDS 6,569,834W B1 5/2003 Pettitarter et et al. al. 6,620,911 B1 9, 2003 Pettit et al. (75) Inventors: Svetlana O. Doronina, Snohomish, WA 6,639,055 B1 10/2003 ETA. (US); Peter D. Senter, Seattle, WA (US); 6,884,869 B2 4/2005 Senter et al. Brian E. Toki, Shoreline, WA (US); 2.92. R. 39. sn' et al. Allen J. Ebens, San Carlos, CA (US), 7.553,816 B2 62009 senter et al. Toni Beth Kline, Seattle, WA (US); Paul 7,659,241 B2 2/2010 Senter et al. Polakis, Burlingame, CA (US); Mark X. 2S3, E: 23.9 E. et al. Sliwkowski, San ETR CA (US); 7,829,531- I B2 11/2010 Sentereng et al. Susan D. Spencer, Tiburon, CA (US) 7,837,980 B2 11/2010 Alley et al. 7.851,437 B2 12/2010 Senter et al. (73) Assignee: Seattle Genetics, Inc., Bothell, WA (US) 2002/0001587 A1 1/2002 Erickson et al. 2003/0083263 A1 5/2003 Doronina et al. (*) Notice: Subject to any disclaimer, the term of this 3.s: A. 3.3. She s al patent is extended or adjusted under 35 2004/0018194, A1 1/2004 Francisco et al. U.S.C. 154(b) by 812 days. 2004/0235068 A1 11/2004 Levinson 2005/OOO9751 A1 1/2005 Senter et al. (21) Appl. No.: 11/833,954 2005/0106644 A1 5/2005 Cairns et al. 2005/0107595 A1 5/2005 Cairns et al. (22) Filed: Aug. 3, 2007 2005.0113308 A1 5/2005 Senter et al. 2005/0232929 A1 10/2005 Kadkhodayan et al. O O 2005/0238650 A1 10/2005 Crowley et al. (65) Prior Publication Data 2005/0256030 A1 1 1/2005 Feng 2005/0272665 A1 12/2005 Schmid et al. US 2009/OO47296A1 Feb. 19, 2009 2006, OO73152 A1 4, 2006 Dennis Related U.S. Application Data (Continued) (62) Division of application No. 10/983,340, filed on Nov. FOREIGN PATENT DOCUMENTS 5, 2004, now Pat. No. 7,498,298. CA 211415.6 A1 7, 1994 (60) Provisional application No. 60/622,455, filed on Oct. (Continued) 27, 2004, provisional application No. 60/598,899, filed on Aug. 4, 2004, provisional application No. OTHER PUBLICATIONS 60/557,116, filed on Mar. 26, 2004, provisional application No. 60/518.534, filed on Nov. 6, 2003. Doronina et al., “Enhanced Activity of Monomethylauristatin F through Monoclonal Antibody Delivery: Effects on Linker Technol (51) Int. Cl. ogy of Efficacy and Toxicity.” Bioconjugate Chem., 17:114-124 A6 IK38/00 (2006.01) (2006). A6 IK38/02 (2006.01) Doronina et al., “Immunoconjugates comprised of drugs with C07K 2/00 (2006.01) impaired cellular permeability: A new approach to targeted therapy.” (52) U.S. Cl...... S14/193: 514/19.4: 514/19.5 SciFinder search result, abstract of paper from 228th ACS National 514/19.6: 424/178.1: 424/179 1: 424/181 1. Meeting held in Philadelphia, PA, Aug. 22-26, 2004. • Yus • us • us 530?391 Gaertner & Offord, “Site-specific attachment of functionalized poly (ethylene glycol) to the amino terminus of proteins.” Bioconi. Chem. (58) Field of Classification Search ...... None 7(1): 38-44 (1996). See application file for complete search history. Genet, J. P. “Recent studies on asymmetric hydrogenation. New catalysts and synthetic applications in organic synthesis.” Pure Appl. (56) References Cited Chem., 74(1):77-83 (2002). U.S. PATENT DOCUMENTS (Continued) 4,753,894 A 6, 1988 Frankel et al. 4,943,628 A 7, 1990 Rosen et al. 4,978,744 A 12/1990 Pettit et al. Primary Examiner — Andrew D. Kosar 5,169,774 A 12/1992 Frankel et al. (74) Attorney, Agent, or Firm — Kilpatrick Townsend & 5,286,637 A 2f1994 Veronese et al. Stockton LLP 5,410,024 A 4/1995 Pettit et al. 5,629, 197 A 5/1997 Ring et al. 5,635,483. A 6, 1997 Pettit et al. (57) ABSTRACT 5,654,399 A 8, 1997 Sakakibara et al. 5,767,237 A 6, 1998 Sakakibara et al. Auristatin peptides, including MeVal-Val-Dil-Dap-Norephe 5,780,588 A 7, 1998 Pettit et al. drine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were 5,821,337 A 10, 1998 Carter et al. 5,840,699 A 11/1998 Sakakibara et al. prepared and attached to Ligands through various linkers, 6,004,934. A 12/1999 Sakakibara et al. including maleimidocaproyl-val-cit-PAB. The resulting 6,048,720 A 4/2000 Dalborg et al. ligand drug conjugates were active in vitro and in vivo. 6,054,297 A 4/2000 Carter et al. 6,054,561 A 4/2000 Ring 6,124,431 A 9, 2000 Sakakibara et al. 51 Claims, 40 Drawing Sheets US 7,994,135 B2 Page 2

U.S. PATENT DOCUMENTS Carter, “Improving the Efficacy of Antibody-Based Cancer Thera 2006, OO740O8 A1 4/2006 Senter et al. pies,” Nature Reviews 1:118-129 (2001). 2006.012897O A1 6, 2006 Bliss et al. Dillman, "Monoclonal Antibodies for Treating Cancer.” Annals of 2006, O182751 A1 8, 2006 Gazzard et al. Internal Medicine 1 11:592-603 (1989). 2006/0233794 A1 10, 2006 Law et al. Doronina et al., “Development of potent monoclonal antibody 2007/0092.520 A1 4/2007 Dennis et al. auristatin conjugates for cancer therapy.” Nature Biotechnology, 2007/0134243 A1 6, 2007 Gazzard et al. 21(7):778-784 (2003) + Erratum, Nature Biotechnology, 21 (8):941 2007/0212356 A1 9, 2007 Chen et al. (2003). FOREIGN PATENT DOCUMENTS Emery et al., “Humanized monoclonal antibodies for therapeutic applications.” Exp. Opin. Invest. Drugs 3(3):241-251 (1994). JP 06-234790 A 8, 1994 Francisco et al., “cAC10-veMMAE, an anti-CD30-monomethyl JP O9-77791 A 3, 1997 WO WO99,35164 A1 7, 1999 auristatin E conjugate with potent and selective antitumor activity.” WO WO 01/18032 A2 3, 2001 Blood, 102(4): 1458-1465 (2003). WO WO O2/O88172 A2 11/2002 Hamblett et al., “Effect of drug loading on the pharmacology, WO WOO3,OO8378 A1 1, 2003 pharmacokinetics, and toxicity of an antiCD30 antibody-drug con WO WOO3,O34903 A2 5, 2003 jugate.” Proceedings of the AACR. vol. 45, abstract # 624 (2004). WO WO 03/043583 A2 5, 2003 Kline et al., “Novel Antitumor Prodrugs Designed for Activation by WO WO 2004/O10957 A2 2, 2004 Matrix Metalloproteinases-2 and -9.” Molecular Pharmaceutics, WO WO 2004/032828 A2 4/2004 1(1):9-22 (2004). WO WO 2004/0736.56 A2 9, 2004 Klussman et al., “Secondary mAb—vcMMAE conjugates are highly WO WO 2006/034488 A3 3, 2006 sensitive reporters of antibody internalization via the lysosome path WO WO 2006/083936 A3 8, 2006 way,” Bioconjug Chem., 15(4):765-773 (2004). WO WO 2007 OO1851 A3 1, 2007 Law et al., “CD70 is expressed on renal cell carcinoma and is a WO WO 2007/109567 A1 9, 2007 potential target for tumor cell elimination by antibody-drug conju OTHER PUBLICATIONS gates.” Proceedings of the AACR. vol. 45, abstract # 625 (2004). Inada et al., “Modification of proteins with polyethylene glycol Mao et al., “EphB2 as a Therapeutic Antibody Drug Target for the derivatives.” Methods Enzymol. 242: 65-90 (1994). Treatment of Colorectal Cancer.” Cancer Research, 64:781-788 Miyazaki et al., “Synthesis and Antitumor Activity of Novel (2004). Dolastatin 10 Analogs.” Chem. Pharm. Bull., 43(10): 1706-1718 Meyer et al., “Recent Advances in Antibody Drug Conjugates for (1995). Cancer Therapy.” Annual Reports in Medical Chemistry, 38(chapter Natsume et al., “Characterization of the Interaction of TZT-1027, a 23):229-237 (2003). Potent Antitumor Agent, with Tubulin.” Jpn. J. Cancer, 91:737-747 Pettit et al., “The Absolute Configuration and Synthesis of Natural (2000). (-)-Dolastatin 10.” J. Am. Chem. Soc., 111:5463-5465 (1989). Petit et al., “Antineoplastic agents 337. Synthesis of dolastatin 10 Pettit et al., "Dolastatins 24. Synthesis of (-)-dolastatin 10. X-Ray structural modifications.” Anti-Cancer Drug Design, 10:529-544 molecular structure of N,N-dimethylvalyl-valyl-dolaisoleuine tert (1995). butyl ester.” J. Chem. Soc. Perkin Trans. 1, 5:859-863 (1996). Petit et al., “Specific Activities of Dolastatin 10 and Peptide Deriva Pettit et al., “Antineoplastic agents 365. Dolastatin 10 SAR probes.” tives against Cryptococcus neoformans,' Antimicrobial Agents and Anticancer Drug Des. 13(4): 243-277. Chemotherapy, 42(11):2961-2965 (1998). Press Release, “Seattle Genetics, Inc. (SGEN) to Present Advances in Petit et al., “A Cobalt-Phosphine Complex Directed Reformatsky Preclinical Research. At American Cancer Research Annual Meet Approach to a Stereospecific Synthesis of the Dolastatin 10 Unit ing.” Mar. 24, 2004, downloaded from intemet on Aug. 31, 2004. Dolaproine (Dap)".” J. Org. Chem. 66:8640-8642 (2001). Schoffski et al., “Phase I and pharmacokinetic study of TZT-1027, a Thornber, “Isosterism and molecular modification in drug design.” novel synthetic dolastatin 10 derivative, administered as a 1-hour Chem. Soc. Rev. 8(4): 563-580 (1979). intravenous infusion every 3 weeks in patients with advanced refrac Vippagunta et al., “Crystalline solids.” Adv. Drug Delivery Rev. 48: tory cancer.” Annals of Oncology, 15:671-679 (2004). 3-26 (2001). Senter et al., “Immunoconjugates comprised of drugs with impaired Woyke et al., “Effect of auristatin PHE on microtube integrity and cellular permeability: A new approach to targeted therapy.” Proceed nuclear localization in Cryptococcus neoformans.” Antimicrobial ings of the AACR. vol. 45, abstract # 623 (2004). Agents and Chemotherapy, 46(12):3802-3808 (2003). Toki et al., “Protease-Mediated Fragmentation of p-Amidobenzyl International Search Report of Oct. 2, 2006 for PCT application Ethers: A New Strategy for the Activation of Anticancer Prodrugs.”J. PCT/US04/38392, which corresponds to the parent application, U.S. Org. Chem., 67: 1866-1872 (2002). Appl. No. 10/983,340. Woyke et al., “In Vitro Activities and Postantifungal Effects of the Written Opinion of Oct. 2, 2006 for PCT application PCT/US04/ Potent Dolastatin 10 Derivative Auristatin PHE.' Antimicrobial 38392, which corresponds to the parent application, U.S. Appl. No. Agents and Chemotherapy, 45(12):3580-3584 (2001). 10/983,340. Carl, P.L. et al., “A novel connector linkage applicable in prodrug Alley et al., “Controlling the location of drug attachment in antibody design.” Journal of Medicinal Chemistry, May 1981, vol. 24. No. 5, drug conjugates.” Proceedings of the AACR. vol. 45, abstract # 627 pp. 479-480. (2004). Garteiz, D.A. et al., “Quantitation of dolastatin-10 using HPLC/ Afar et al., “Preclinical validation of anti-TMEFF2-auristatin E-con electrospray ionization mass spectrometry: application in a phase I jugated antibodies in the treatment of prostate cancer. Molecular clinical trial.” Cancer Chemother: Pharmacol., 1998, vol. 41, pp. Cancer Therapeutics, 3(8):921-932 (2004). 299-306. Bhaskar et al., “E-Selectin Up-Regulation Allows for Targeted Drug Jansen, F.K. et al., “Immunotoxins: hybrid molecules combining Delivery in Prostate Cancer.” Cancer Research, 63:6387-6394 high specificity and potent cytotoxicity.” Immunol. Rev., 1982, vol. (2003). 62, pp. 185-216. U.S. Patent Aug. 9, 2011 Sheet 1 of 40 US 7,994,135 B2

1200 - 1100 - 1000 - 900 -

800 - -ie- Untreated 700 -

600 - -- cAC10-MC-MMAF4, 500 - 1 mg/kg

400 -

-H cAC10-MC-MMAF4, 300 - 2 mg/kg 200 - 100 -

O 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 DAYS POST TUMOR IMPLANT Fig. 1 U.S. Patent Aug. 9, 2011 Sheet 2 of 40 US 7,994,135 B2

1200 1100

- 1000900 I-umla-K-Untreated ly 800 700 --CAC10-mcMMAF4M D 600 8 mg/kg s 500 Pas 400 - |-8-cAC10-MCMMAF4M2 mg/kg a 300 make 200 100 O O 5 10 15 20 25 30 35 40 45 50 55 DAYS POST TUMOR IMPLANT Fig. 2 U.S. Patent Aug. 9, 2011 Sheet 3 of 40 US 7,994,135 B2

1000 -

900 - -K-Untreated

800 - 700 5c 600 - -H 3cBR96-mc-MMAF4M, mg/kg S 500 - 2 400 - --cBR96-mc-MMAF4M, H 10 mg/kg 300 - c 200 - 100- Schedule q4dx4 O A A A A I T O 20 40 60 80 100 Days Post Tumor implant

Fig. 3a U.S. Patent Aug. 9, 2011 Sheet 4 of 40 US 7,994,135 B2

Efficacy of mAb-mc-MMAF in L2987 Lung Carcinoma

1200 - L2987-AO1 1100 - 1000 - 900 - treatment 800 - -k- Untreated 600 - -H cBR96-mc-MMAF 10 mg/kg

500 - -0-cAC10-mc-MMAF 10 mg/kg 400 - 300 - 200 - 100 -

O -T-I-T- H-I-T-- - 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Schedule q4.dx4 DAYS POST TUMOR IMPLANT Fig. 3b U.S. Patent Aug. 9, 2011 Sheet 5 of 40 US 7,994,135 B2

a CAC 10-mcMMAF

100 O Karpas 299 A L428

75 9 viad C D 50 s S 25

O 0.0001 0.001 0.01 0.1 1 10 100 1000 Concentration (ng/mL) Fig. 4a

b CAC 10-VCMMAF

100 O Karpas 299 A L428

75 S. E D 50 us O st 25

O 0.0001 0.001 0.01 0.1 1 10 100 1000 Concentration (ng/mL) Fig. 4b U.S. Patent Aug. 9, 2011 Sheet 6 of 40 US 7,994,135 B2

a Activity of cBR96-mcMMAF

100 A O H3396 A L2987

O 9 75 (S 9 E D 50 lege O SS 25

O 0.001 0.01 0.1 1 10 100 1000 10000 Concentration (ng/mL) Fig. 5a

b Activity of cBR96-vcMMAF O H3396 100 A L2987 O 9 75

5 50 SS 25

O 0.001 0.01 0.1 1 10 100 1000 10000 Concentration (ng/mL) Fig. 5b U.S. Patent Aug. 9, 2011 Sheet 7 of 40 US 7,994,135 B2

a C1F6-mcMMAF

O Caki-1 100 A 786-O o 9 3. 75 E

O 50 SS 25

O 0.0001 0.001 0.01 0.1 1 10 100 1 000 Concentration (ng/mL) Fig. 6a

b C1F6-VCMMAF

O Caki-1 100 A 786-O C 9 8 75 E

O 50 SS 25

O O.OOO1 O.OO1 O.O1 O.1 1 10 100 1 000 Concentration (ng/mL) Fig. 6b U.S. Patent Aug. 9, 2011 Sheet 8 of 40 US 7,994,135 B2

O H-MC-VC-PAB-MMAF (cys 3.8 drug/Ab O H-MC-MMAF (cys) 4.1 drug/Ab

150OOOO A H-MC-MMAF (cys) 4.8 drug/Ab 5 C SK-BR-3 Cells

o 1 OOOOOO CD O CD d CD 9. E O) 500000 2

CD Y

O O 0.0001 O.OO1 O.O1 0.1 1 lug/ml Fig. 7 U.S. Patent Aug. 9, 2011 Sheet 9 of 40 US 7,994,135 B2

O H-MC-vc-PAB-MMAF (cys) 3.8 drug/Ab O H-MC-MMAF (Cys4.1 drug/Ab a H-MC-MMAF (cys 4.8 drug/Ab 2000000 A

BT-474 Cells 15OOOOO

C - or 1000000

500000

O O O.OOO1 0.001 0.01 O. 1 1 ug/ml Fig. 8 U.S. Patent Aug. 9, 2011 Sheet 10 of 40 US 7,994,135 B2

2000000 MCF-7 CellS

O O O 15OOOOO - a \o-o-o-o- . . . . O. d 1000000 H-MC-vc-PAB-MMAF (cys 3.8 drug/Ab O H-MC-(Me)vc-PAB-MMAF (cys 3.9 drug/Ab A H-MC-MMAF (cys) 4.1 drug Ab 500000

O O 0.0001 0.001 0.01 0.1 1 ug/ml Fig. 9 U.S. Patent Aug. 9, 2011 Sheet 11 of 40 US 7,994,135 B2

2500000 3-9-8 O O O O 2000000 MDA-MB-468 Cells 1500000

BY O H-MC-vc-PAB-MMAE (cys) 4.1 drug/Ab 1 OOOOOO O H-MC-vc-PAB-MMAE (cys) 3.3 drug/Ab A H-MC-vc-PAB-MMAF (cys) 3.7 drug/Ab

500000

O 0.0001 0.001 0.01 0.1 1 g/ml Fig. 10 U.S. Patent Aug. 9, 2011 Sheet 12 of 40 US 7,994,135 B2

H-MC-vc-PAB-MMAF-TEGTotal Ab 100 {X H-MC-vc-PAB-MMAF-TEG Conj A H-MC-VC-PAB-MMAF Total Ab

A H-MC-vc-PAB-MMAF Conj

Time (day) Fig.11 U.S. Patent Aug. 9, 2011 Sheet 13 of 40 US 7,994,135 B2

H-MC-VC-PAB-MMAE in Cynomolgus monkeys

100 -0-0.5 mg/kg,Total Antibody -O-0.5 mg/kg, Conjugated Antibody -h-3 mg/kg, Total Antibody - A-3 mg/kg Conjugated Antibody -0- 1.5 mg/kg,Total Antibody -3-1.5 mg/kg, Conjugated Antibody -- 1.5 mg/kg,Total Antibody -a- 1.5 mg/kg, Conjugated Antibody -0-2.5 mg/kg,Total Antibody -0-2.5 mg/kg. Conjugated Antibody 10 -A-2.5 mg/kg, Total Antibody , Conjugated Antibod

0.01 r-r------H ------O 7 14 21 28 35 42 49 Time (day) Fig. 12 U.S. Patent Aug. 9, 2011 Sheet 14 of 40 US 7,994,135 B2

800

700

600

500 - Vehicle Trastuzumab-MC-vc-PAB-MMAE (1250 ug/m2) 400 -1 300 Trastuzumab-MC-vc-PAB-MMAF (555 ug/m2)

10 20 30 40 50 Day Fig. 13 U.S. Patent Aug. 9, 2011 Sheet 15 of 40 US 7,994,135 B2

1000

800 -

Vehicle

600

Trastuzumab-MC-MMAE 40O 660 ugm

200

Trastuzumab-MC-VC-PAB-MMAE 1250 g/m2

Day Fig. 14 U.S. Patent Aug. 9, 2011 Sheet 16 of 40 US 7,994,135 B2

1000

800 Vehicle

600

400

Trastuzumab-MC-MMAF

200 650 ugm MMAF

Day Fig. 15 U.S. Patent Aug. 9, 2011 Sheet 17 of 40 US 7,994,135 B2

MMAF/H mg/kg Ab Ti PR CR Vehicle (Buffer) 8/8 O/8 O/8 H-MC-VC-PAB-MMAF 5.9 4.1 8/8 2/8 O/8 H-MC-VC-PAB-MMAF 2 12 2/8 2/8 6/8 800 H-MC-vc-PAB-MMAF 4 6 6/7 4/8 28 H-MC-vc-PAB-MMAF 6 -- 4 8/8 3/8 0/8 700

6 O O

5 O O

4 O O Fig. 16 300

2OO

mouse sacrificed 100

O 5 10 15 20 25 30 Day U.S. Patent Aug. 9, 2011 Sheet 18 of 40 US 7,994,135 B2

40.0 -

30.0 - Vehicle 20.0 - Free MMAF / . 5, / ...... scree dg 10.0 - - -

d O.O inition.,,..., 9 s > S-10.0 O Trastuzumab-vc-MMAF / -20.0 (2105 ug/m2) Trastuzumab-VC-MMAE (2105ug/m2)

U.S. Patent Aug. 9, 2011 Sheet 19 of 40 US 7,994,135 B2

40 8 Vehicle Historical Control 30

A 9.94 mg/kg H-MC-val-cit MMAF X 24.90 mg/kg H-MC-val-cit MMAF o 10.69 mg/kg H-MC(Me)- Val-cit-PAB-MMAF o 26.78 mg/kg H-MC(Me)- Wai-Cit-PAB-MMAF a 10.17 mg/kg H-MC-MMAF 8 25.50 mg/kg H-MC-MMAF

1 2 3 4 5 D 21.85 mg/kg H-MC-val-cit PAB-MMAF Study Day Fig. 18 U.S. Patent Aug. 9, 2011 Sheet 20 of 40 US 7,994,135 B2

Change in Body Weight

0 Vehicle Control O 2105ug/m2 H-MC-MMAF O 2105 ug/m2 H-MC-MMAF 3 Wk A 3158 uglim2 H-MC-MMAF

1 2 3 4 5 6 7 8 9 10 Study Day Fig. 19 U.S. Patent Aug. 9, 2011 Sheet 21 of 40 US 7,994,135 B2

Fig. 20 Ab-A-W-Gly-D Ab-A-W-Gly-Gly-D enzymatic enzymatic Cleavage Cleavage Gly-D Gly-Gly-D hydrolysis hydrolysis Drug Drug U.S. Patent Aug. 9, 2011 Sheet 22 of 40 US 7,994,135 B2

Fig. 21

Qm ==

L-A-W-NH-()a V V W \ / ,O--D O p enzymatic Cleavage

NA.(2 - N / -1 O--D O

16-elimination

Drug U.S. Patent Aug. 9, 2011 Sheet 23 of 40 US 7,994,135 B2 Fig. 22

L-A-W-NH-() D p enzymatic cleavage

16-elimination

NH -- Drug U.S. Patent Aug. 9, 2011 Sheet 24 of 40 US 7,994,135 B2

Fig. 23

== /CH2(O(C(O)))-D L-A-W-NH-Q /

enzymatic cleavage 2 drugs U.S. Patent Aug. 9, 2011 Sheet 25 of 40 US 7,994,135 B2

Fig. 24

Luciferase ATP + Luciferin + O-o- Oxyluciferin +. AMP + PPi + CO2 + light Mg"? U.S. Patent Aug. 9, 2011 Sheet 26 of 40 US 7,994,135 B2

Fig. 25

HNPG O -- R7 O PyBrop, DIEA PG P 7 R. R. OH HNRe Re O K-rr Hans-so K A

O R, WhenH 10% PG Pd/C, = Z: EtOH ". - R. d.YT BB -waaaa- R. R. R. R. Ö DEPC, Etan

HC Dioxane R O R7

PSxSrokR O R4 Rs Rs Rs O NSX NY E

U.S. Patent Aug. 9, 2011 Sheet 28 of 40 US 7,994,135 B2

Fig. 27

CH R O DEPC, EtN CH O -- —- N N OH HN O M O Boc Boc R O R O 8 8 Rio Rio G M N

R. R, PG N OH HC/EtOAC CH O y x', 'r HN N k R O R4 R. R. Rs O O F Rs O DEPC, EtN Rio O

R H O R CH R O When PG = Fmoc: PG N N N K DEA/CHCl N N O R. O. R4 R. R. Rs 6 P Rs 0 0

R O R O 3. H CH HCl/dioxane N N N ammermararmarramware HN N O R. O. R4 R. R. R. O Rs. O Rio (Ib) where Z is -O- and R' is t-butyl

R O R CH H 3 Hy N N N N OH R O R. R. R. R. Ó R O R (Ib) where Z is-O- and R is -H U.S. Patent Aug. 9, 2011 Sheet 29 of 40 US 7,994,135 B2

Fig. 28

O O O O o "Hn(xioh - E - N(xioh O O

O O

O & O O ), O O CR Et3N, CH2Cl2 trixto) O O O

O O O Q oc, * H.N. Xh OH "r" O O O C. U.S. Patent Aug. 9, 2011 Sheet 30 of 40 US 7,994,135 B2

Fig. 29

O O 1N1N PPhDAD - HO E OH -tra- NS-EesuoH O O O NO also DEA, DCM n^ero'o O

U.S. Patent Aug. 9, 2011 Sheet 33 of 40 US 7,994,135 B2

Fig. 32

O Ph O Ph H.N.--OH StoH Hin-K)-a Fmoc-N Sos s Fmoc- O OH H O l \ Boc2ODEA (1.5 eq.) AC AD NH AE NH CH2Cl2 Mitr Mt.

Qma OR Qm OH ph o f Ph. O X H S. H S O O l's 1. Diethylamine, DMF (1:2) --n > N H Fmoc-N - H N-(CH2)5 H O s 2.. O H O s O NH C-clascoos AF (m=0) NH AG (R=H, m=0) Mt. O Mitr

p-nitrophenyl-OCOO-p-nitrophenyl (2.0 eq.) DIEA (1.5 eq.), DMF AHR-3-(O O NO2

Ph O O H 1. Diethylamine, DMF (1:2) O 3. OH AE 2. O C-cle H O l Al C-cocoos O NH O U.S. Patent Aug. 9, 2011 Sheet 34 of 40 US 7,994,135 B2

Fig. 33

HATU Drug + Linker-COOH - - Drug-NH-C(O)-Linker (Ib) AJ AK

O O HOBt Drug + Linker-O - C - O NO2 -> Drug-NH-C-O-Linker (Ib) AL AM

Drug + Linker-X base-e- Drug-N-LinkerOO (Ib) AN AO U.S. Patent Aug. 9, 2011 Sheet 35 of 40 US 7,994,135 B2

Fig. 34

O O HaC CH r Z + Drug pyridineHOBt R-3 Nh y O N re's (Ib) O

R = Fmoc AP

Diethylamine

O O l -6PyBrop R = H NH AQ DIEA sO ill- O O AR R = Br1 O ( N-o-o-o: O DEPC, DIEA

O O AS R = ( J-o-o-y O

U.S. Patent Aug. 9, 2011 Sheet 37 of 40 US 7,994,135 B2

Fig. 36

Drug-Linker Antibody - DTT - Partially Reduced Antibody Compound - Drug-Linker-Ligand Conjugatew with Reduced Drug Load U.S. Patent Aug. 9, 2011 Sheet 38 of 40 US 7,994,135 B2

Fig. 37

N Oh

N N N O - -

sch, is OCH irrig

MeVal-Val-Dil-Dap-Phe-OtBu, 1001

O h N N. N N O

&rnO O OCH, O OCH O O K MC-MeVal-Val-Dil-Dap-Phe-OtBu

O h N - i. Pool, I &rnO O OCH O OC. O O

MC-MMAF

U.S. Patent Aug. 9, 2011 Sheet 40 of 40 US 7,994,135 B2

Fig. 39

O O O

1-()- OH . 1".O OCH O N OCH O r

OCH O MB-Me-Val-Dil-Dap-Phe-OtBu

O O O N H

H O Cr'?O OCH O OCH O O MB-MMAF US 7,994,135 B2 1. 2 MONOMETHYLVALINE COMPOUNDS general physiological effects of inappropriate delivery of CAPABLE OF CONUGATION TO LIGANDS Such agents to other cells and tissues, such as uninfected cells. Intracellular targeting may be achieved by methods, com CONTINUITY pounds and formulations which allow accumulation or reten tion of biologically active agents, i.e. active metabolites, This application is a division of U.S. application Ser. No. inside cells. 10/983,340 filed Nov. 5, 2004, now U.S. Pat. No. 7,498,298 Monoclonal antibody therapy has been established for the which claims the benefit of U.S. Provisional Patent Applica targeted treatment of patients with cancer, immunological tion No. 60/622,455, filed Oct. 27, 2004; U.S. Provisional and angiogenic disorders. Patent Application No. 60/598,899, filed Aug. 4, 2004; U.S. 10 The use of antibody-drug conjugates for the local delivery Provisional Patent Application No. 60/557,116, filed Mar. 26, of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit 2004; and U.S. Provisional Patent Application No. 60/518, tumor cells in the treatment of cancer (Syrigos and Epenetos 534, filed Nov. 6, 2003; the disclosures of which are incor (1999) Anticancer Research 19:605-614; Niculescu-Duvaz porated by reference herein. and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. 15 Pat. No. 4,975,278) theoretically allows targeted delivery of JOINT RESEARCH AGREEMENT the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated Some of the Subject matter in this application was made by drug agents may result in unacceptable levels of toxicity to or on behalf of Seattle Genetics, Inc. and Genentech, Inc. as a normal cells as well as the tumor cells sought to be eliminated result of activities undertaken within the scope of a joint (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986):603-05; research agreement effective on or before the date the claimed Thorpe, 1985, Antibody Carriers Of Cytotoxic Agents In invention was made. Cancer Therapy: A Review.” in Monoclonal Antibodies 84: Biological And Clinical Applications, A. Pinchera et al. 1. FIELD OF THE INVENTION (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity 25 is sought thereby. Both polyclonal antibodies and mono The present invention is directed to a Drug Compound and clonal antibodies have been reported as useful in these strat more particularly to Drug-Linker-Ligand Conjugates, Drug egies (Rowland et al., 1986, Cancer Immunol. Immunother. Linker Compounds, and Drug-Ligand Conjugates, to compo 21:183-87). Drugs used in these methods include daunomy sitions including the same, and to methods for using the same cin, doxorubicin, methotrexate, and vindesine (Rowland et to treat cancer, an autoimmune disease or an infectious dis 30 al., 1986, Supra). Toxins used in antibody-toxin conjugates ease. The present invention is also directed to antibody-drug include bacterial toxins such as diphtheria toxin, plant toxins conjugates, to compositions including the same, and to meth such as ricin, small molecule toxins such as geldanamycin ods for using the same to treat cancer, an autoimmune disease (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784; Man or an infectious. disease. The invention also relates to meth dler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19): 1573 ods of using antibody-drug conjugate compounds for in vitro, 35 1581; Mandler et al. (2000) Bioorganic & Med. Chem. Let in situ, and in vivo diagnosis or treatment of mammaliancells, ters 10:1025-1028; Mandler et al. (2002) Bioconjugate or associated pathological conditions. Chem. 13:786-791), maytansinoids (EP 1391213: Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and cali 2. BACKGROUND OF THE INVENTION cheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman 40 et al. (1993) Cancer Res. 53:3336-3342). The toxins may Improving the delivery of drugs and other agents to target affect their cytotoxic and cytostatic effects by mechanisms cells, tissues and tumors to achieve maximal efficacy and including tubulin binding, DNA binding, or topoisomerase minimal toxicity has been the focus. of considerable research inhibition (Meyer, D. L. and Senter, P. D. “Recent Advances for many years. Though many attempts have been made to in Antibody Drug Conjugates for Cancer Therapy” in Annual develop effective methods for importing biologically active 45 Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, molecules into cells, both in vivo and in vitro, none has 229-237). Some cytotoxic drugs tend to be inactive or less proved to be entirely satisfactory. Optimizing the association active when conjugated to largeantibodies or protein receptor of the drug with its intracellular target, while minimizing ligands. intercellular redistribution of the drug, e.g., to neighboring ZEVALINR) (ibritumomab tiuxetan, Biogen/Idec) is an cells, is often difficult or inefficient. 50 antibody-radioisotope conjugate composed of a murine IgG1 Most agents currently administered to a patient parenter kappa monoclonal antibody directed against the CD20 anti ally are not targeted, resulting in systemic delivery of the gen found on the Surface of normal and malignant B lympho agent to cells and tissues of the body where it is unnecessary, cytes and '''In or 'Y radioisotope bound by a thiourea and often undesirable. This may result in adverse drug side linker-chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med. effects, and often limits the dose of a drug (e.g., chemothera 55 27(7):766-77: Wiseman et al. (2002) Blood 99(12):4336-42: peutic (anti-cancer), cytotoxic, enzyme inhibitor agents and Witzigetal. (2002).J. Clin. Oncol. 20(10):2453-63; Witziget antiviral or antimicrobial drugs) that can be administered. By al. (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVA comparison, although oral administration of drugs is consid LIN has activity against B-cell non-Hodgkin’s Lymphoma ered to be a convenient and economical mode of administra (NHL), administration results in severe and prolonged tion, it shares the same concerns of non-specific toxicity to 60 cytopenias in most patients. MYLOTARGTM (gemtuzumab unaffected cells once the drug has been absorbed into the oZogamicin, Wyeth Pharmaceuticals), an antibody drug con systemic circulation. Further complications involve problems jugate composed ofahu CD33 antibody linked to calicheami with oral bioavailability and residence of drug in the gut cin, was approved in 2000 for the treatment of acute myeloid leading to additional exposure of gut to the drug and hence leukemia by injection (Drugs of the Future (2000) 25(7):686; risk of gut toxicities. Accordingly, a major goal has been to 65 U.S. Pat. Nos. 4,970, 198; 5,079,233; 5,585,089; 5,606,040: develop methods for specifically targeting agents to cells and 5,693,762; 5,739,116; 5,767.285; 5,773,001). Cantuzumab tissues. The benefits of such treatment include avoiding the mertansine (Immunogen, Inc.), an antibody drug conjugate US 7,994,135 B2 3 4 composed of the huC242 antibody linked via the disulfide et al., (1989) Mol. Cell. Biol. 9(3): 1165-1172. Maximum linker SPP to the maytansinoid drug moiety, DM1, is advanc inhibition was obtained with the antibody called 4D5 which ing into Phase II trials for the treatment of cancers that express inhibited cellular proliferation by 56%. Other antibodies in CanAg, such as colon, pancreatic, gastric, and others. MLN the panel reduced cellular proliferation to a lesser extent in 2704 (Millennium Pharm. BZL Biologics, Immunogen Inc.), 5 this assay. The antibody 4D5 was further found to sensitize an antibody drug conjugate composed of the anti-prostate ErbB2-overexpressing breast tumor cell lines to the cytotoxic specific membrane antigen (PSMA) monoclonal antibody effects of TNF-C. (U.S. Pat. No. 5,677,171). The anti-ErbB2 linked to the maytansinoid drug moiety, DM1, is under devel antibodies discussed in Hudziak et al. are further character opment for the potential treatment of prostate tumors. The ized in Fendly et al. (1990) Cancer Research 50:1550-1558: same maytansinoid drug moiety, DM1, was linked through a 10 Kotts et al. (1990) In vitro 26(3):59A: Sarup et al. (1991) non-disulfide linker, SMCC, to a mouse murine monoclonal Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin. antibody, TA. 1 (Charietal. (1992) Cancer Research 52:127 Immunol. 11(3):117-127; Kumar et al. (1991) Mol. Cell. 131). This conjugate was reported to be 200-fold less potent Biol. 11(2):979-986; Lewis et al. (1993) Cancer Immunol. than the corresponding disulfidelinker conjugate. The SMCC Immunother. 37:255-263; Pietras et al. (1994) Oncogene linker was considered therein to be "noncleavable.” 15 9:1829-1838; Vitetta et al. (1994) Cancer Research 54:5301 Several short peptidic compounds have been isolated from 5309; Sliwkowski et al. (1994) J. Biol. Chem. 269(20): the marine mollusc Dolabella auricularia and found to have 14661-14665; Scottet al. (1991).J. Biol. Chem. 266:14300-5: biological activity (Pettit et al. (1993) Tetrahedron 49:9151: D’souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206; Nakamura et al. (1995) Tetrahedron Letters 36:5059-5062: Lewis et al. (1996) Cancer Research 56:1457-1465; and Sone et al. (1995) Jour.Org Chem. 60:4474). Analogs of these 20 Schaefer et al. (1997) Oncogene 15:1385-1394. compounds have also been prepared, and some were found to Other anti-ErbB2 antibodies with various properties have have biological activity (for a review, see Pettit et al. (1998) been described in Tagliabue et al. Int. J. Cancer 47:933-937 Anti-Cancer Drug Design 13:243-277). For example, aurista (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier tin E (U.S. Pat. No. 5,635,483) is a synthetic analogue of the et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecu marine natural product Dolastatin 10, an agent that inhibits 25 lar Carcinogenesis 3:350-362 (1990); Stancovski et al. Proc. tubulin polymerization by binding to the same domain on Natl. Acad. Sci. USA 88:8691-8695 (1991); Bacus et al. Can tubulin as the anticancer drug Vincristine (G. R. Pettit, (1997) cer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10, aurista 53:401–408 (1993); WO94/00136; Kasprzyk et al. Cancer tin PE, and auristatin E are linear peptides having four amino Research 52:2771-2776 (1992); Hancock et al. (1991) Can acids, three of which are unique to the dolastatin class of 30 cer Res. 51:4575-4580; Shawver et al. (1994) Cancer Res. compounds, and a C-terminal amide. 54:1367-1373; Arteaga et al. (1994) Cancer Res. 54:3758 The auristatin peptides, auristain E (AE) and monomethy 3765; Harwerth et al. (1992) J. Biol. Chem. 267:15160 lauristatin (MMAE), synthetic analogs of dolastatin, were 15167; U.S. Pat. No. 5,783,186; and Klapper et al. (1997) conjugated to: (i) chimeric monoclonal antibodies chBR96 Oncogene 14:2099-2109. (specific to Lewis Y on carcinomas); (ii) cAC10 which is 35 Homology Screening has resulted in the identification of specific to CD30 on hematological malignancies (Klussman, two other ErbB receptor family members; ErbB3 (U.S. Pat. et al. (2004), Bioconjugate Chemistry 15(4):765-773; Dor Nos. 5,183,884; 5.480,968; Krau.S. etal. (1989) Proc. Natl. onina et al. (2003) Nature Biotechnology 21 (7):778-784: Acad. Sci. USA 86:9193-91.97) and ErbB4 (EP599274; Plow "Monomethylvaline Compounds Capable of Conjugation to man et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750; Ligands'; Francisco et al. (2003) Blood 102(4): 1458-1465; 40 and Plowman et al. (1993) Nature 366:473-475). Both of U.S. Publication 2004/0018194; (iii) anti-CD20 antibodies these receptors display increased expression on at least some such as RITUXANR(WO 04/032828) for the treatment of breast cancer cell lines. CD20-expressing cancers and immune disorders; (iv) anti HERCEPTINR (Trastuzumab) is a recombinant DNA-de EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorec rived humanized monoclonal antibody that selectively binds tal cancer (Mao, et al. (2004) Cancer Research 64(3):781 - 45 with high affinity in a cell-based assay (Kd=5 nM) to the 788); (v) E-selectin antibody (Bhaskar et al. (2003) Cancer extracellular domain of the human epidermal growth factor Res. 63:6387-6394); and (vi) other anti-CD30 antibodies receptor2 protein, HER2 (ErbB2) (U.S. Pat. Nos. 5,821,337; (WO 03/043583). 6,054,297; 6,407,213; 6,639,055; Coussens L, et al. (1985) Auristatin Econjugated to monoclonal antibodies are dis Science 230:1132-9; Slamon DJ, et al (1989) Science 244: closed in Senter et al. Proceedings of the American Associa- 50 707-12). Trastuzumab is an IgG1 kappa antibody that con tion for Cancer Research, Volume 45, Abstract Number 623, tains human framework regions with the complementarity presented Mar. 28, 2004. determining regions of a murine antibody (4D5) that binds to Despite in vitro data for compounds of the dolastatin class HER2. Trastuzumab binds to the HER2 antigen and thus. and its analogs, significant general toxicities at doses required inhibits the growth of cancerous cells. Because Trastuzumab for achieving a therapeutic effect compromise their efficacy 55 is a humanized antibody, it minimizes any HAMA response in clinical studies. Accordingly, there is a clear need in the art in patients. The humanized antibody against HER2 is pro for dolastatin/auristatin derivatives having significantly lower duced by a mammalian cell (Chinese Hamster Ovary, CHO) toxicity, yet useful therapeutic efficiency. These and other suspension culture. The HER2 (or c-erbB2) proto-oncogene limitations and problems of the past are addressed by the encodes a transmembrane receptor protein of 185kDa, which present invention. 60 is structurally related to the epidermal growth factor receptor. The ErbB family of receptor tyrosine kinases are important HER2 protein overexpression is observed in 25%–30% of mediators of cell growth, differentiation and survival. The primary breast cancers and can be determined using an immu receptor family includes four distinct members including epi nohistochemistry based assessment of fixed tumor blocks dermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (Press M F, et al. (1993) Cancer Res 53:4960-70. Trastu (ErbB2 or p185'), HER3 (ErbB3) and HER4 (ErbB4 or 65 Zumab has been shown, in both in vitro assays and in animals, tyro2). A panel of anti-ErbB2 antibodies has been character to inhibit the proliferation of human tumor cells that overex ized using the human breast tumor cell line SKBR3 (Hudziak press HER2 (Hudziak RM, et al. (1989) Mol Cell Biol US 7,994,135 B2 5 9:1165-72; Lewis GD, et al. (1993) Cancer Immunol Immu nother; 37:255-63; Baselga J, et al. (1998) Cancer Res. Ia 58:2825-2831). Trastuzumab is a mediator of antibody-de L-E-A-W-Y-D), pendent cellular cytotoxicity, ADCC (Hotaling T E. et al. (1996) abstract. Proc. Annual Meeting Am Assoc Cancer 5 or a pharmaceutically acceptable salt or Solvate thereof Res; 37:471; Pegram MD, et al. (1997) abstract. Proc Am wherein, Assoc Cancer Res; 38:602). In vitro, Trastuzumab mediated ADCC has been shown to be preferentially exerted on HER2 L- is a Ligand unit; overexpressing cancer cells compared with cancer cells that -A-W, Y, is a Linker unit (LU), wherein the Linker do not overexpress HER2. HERCEPTINR) as a single agent is 10 unit includes: indicated for the treatment of patients with metastatic breast -A- is a Stretcher unit, cancer whose tumors overexpress the HER2 protein and who a is 0 or 1, have received one or more chemotherapy regimens for their each —W - is independently an Amino Acid unit, metastatic disease. HERCEPTINR in combination with 1s w is an integer ranging from 0 to 12, paclitaxel is indicated for treatment of patients with meta static breast cancer whose tumors overexpress the HER2 —Y— is a Spacer unit, and protein and who have not received chemotherapy for their y is 0, 1 or 2: metastatic disease. HERCEPTINR) is clinically active in p ranges from 1 to about 20; and patients with ErbB2-overexpressing metastatic breast can -D is a Drug unit having the Formulas D, and D.

DE R3 O R7 CH R9 H A N s 4 5 R2 O R. R. R8 O R8 O D F R3 O R7 CH3 R9 O

N N N N 71 R11 A N s R2 O R.4 R.5 R8 O R8 O R10 cers that have received extensive prior anti-cancer therapy wherein, independently at each location: (Baselga et al., (1996).J. Clin. Oncol. 14:737-744). R’ is selected from Hand C-C alkyl: The murine monoclonal anti-HER2 antibody inhibits the 40 R is selected from H.C.-Cs alkyl, Cs-Cs carbocycle, aryl, growth of breast cancer cell lines that overexpress HER2 at C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het the 2+ and3+(1-2x10 HER2 receptors percell) level, but has erocycle and C-C alkyl-(C-C heterocycle); no activity on cells that express lower levels of HER2 (Lewis et al., (1993) Cancer Immunol. Immunother. 37:255-263). R" is selected from H, C, -Cs alkyl, C-Cs carbocycle, aryl, Based on this observation, antibody 4D5 was humanized C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337; 45 erocycle and C-C alkyl-(C-Cs heterocycle); Carter et al., (1992) Proc. Natl. Acad. Sci. USA 89: 4285 R is selected from Hand methyl: 4289) and tested in breast cancer patients whose tumors over or R and Rjointly form a carbocyclic ring and have the express HER2 but who had progressed after conventional formula—(CRR), wherein R and Rare independently chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17: selected from H. C-C alkyl and C-C carbocycle and n is 2639-2648). 50 selected from 2, 3, 4, 5 and 6: Although HERCEPTIN is a breakthrough in treating R is selected from Hand C-Cs alkyl: patients with ErbB2-overexpressing breast cancers that have R’ is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, received extensive prior anti-cancer therapy, some patients in C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het this population fail to respond or respond only poorly to erocycle and C-C alkyl-(C-C heterocycle); HERCEPTIN treatment. 55 each R is independently selected from H, OH, C, -Cs Therefore, there is a significant clinical need for develop alkyl, C-C carbocycle and O—(C-C alkyl); ing further HER2-directed cancer therapies for those patients R is selected from Hand C-Cs alkyl: with HER2-overexpressing tumors or other diseases associ R" is selected from aryl or C-Cs heterocycle; ated with HER2 expression that do not respond, or respond Z is O, S, NH, or NR'', wherein R' is C-C alkyl: poorly, to HERCEPTIN treatment. 60 R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero The recitation of any reference in this application is not an cycle, -(RO), R', or -(RO), CH(R'); admission that the reference is prior art to this application. m is an integer ranging from 1-1000; R" is C-C alkyl: 3. SUMMARY OF THE INVENTION R" is H or C-C alkyl: 65 each occurrence of R' is independently H, COOH, In one aspect, the present invention provides Drug-Linker -(CH2), N(R'). -(CH2), SOH, or -(CH2), Ligand compounds having the Formula Ia: SO C-C alkyl, US 7,994,135 B2 7 8 each occurrence of R' is independently H, C-C alkyl, or In still another aspect, the invention provides pharmaceu —(CH), COOH, where; n is an integer ranging from 0 to tical compositions comprising an effective amount of a Drug 6; and Linker Compound and a pharmaceutically acceptable carrier R" is selected from C(R), C(R)-aryl, -C(R) - or vehicle. C(R) (C-Cs heterocycle), and C(R) C(R) (C- 5 In still another aspect, the invention provides compositions Cs carbocycle). comprising an effective amount of a Drug-Ligand Conjugate In another aspect, Drug Compounds having the Formula Ib having a cleavable Drug unit from the Drug-Ligand Conju are provided: gate and a pharmaceutically acceptable carrier or vehicle.

Ib R3 O R7 CH R9 O H N N N Y I N Z R2 y R R5 R8 O R8 O

Ib or pharmaceutically acceptable salts or Solvates thereof, In yet another aspect, the invention provides methods for wherein: killing or inhibiting the multiplication of a tumor cell or R is selected from hydrogen and —C-Cs alkyl; cancer cell including administering to a patient in need R is selected from hydrogen. —C-Cs alkyl, -C-Cs car thereof an effective amount of a Drug-Linker Compound. 25 In another aspect, the invention provides methods for kill bocycle, aryl, -C-Cs alkyl-aryl, —C-C alkyl-(C-Cs car ing or inhibiting the multiplication of a tumor cell or cancer bocycle), —C-Cs heterocycle and —C-Cs alkyl-(C-Cs cell including administering to a patient in need thereof an heterocycle); effective amount of a Drug-Linker-Ligand Conjugate. R" is selected from hydrogen, —C-Cs alkyl, -C-Cs car In another aspect, the invention provides methods for kill bocycle, -aryl. —C-Cs alkyl-aryl, -C-Cs alkyl-(C-C car ing or inhibiting the multiplication of a tumor cell or cancer bocycle), —C-Cs heterocycle and —C-C alkyl-(C-Cs 30 cell including administering to a patient in need thereof an heterocycle) wherein R is selected from-Hand-methyl; or effective amount of a Drug-Ligand Conjugate having a cleav RandR jointly, have the formula—(CRR), wherein R' able Drug unit from the Drug-Ligand Conjugate. In still another aspect, the invention provides methods for and Rare independently selected from -H, -C-C alkyl treating cancer including administering to a patient in need and —C-Cs carbocycle and n is selected from 2, 3, 4, 5 and 35 thereof an effective amount of a Drug-Linker Compound. 6, and form a ring with the carbon atom to which they are In yet another aspect, the invention provides methods for attached; treating cancer including administering to a patient in need R is selected from Hand-C-Cs alkyl: thereof an effective amount of a Drug-Linker-Ligand Conju R’ is selected from H. —C-Cs alkyl, -C-Cs carbocycle, gate. aryl, -C-C alkyl-aryl, -C-C alkyl-(C-C carbocycle), 40 In yet another aspect, the invention provides methods for —C-Cs heterocycle and—C-Cs alkyl-(C-Cs heterocycle); treating cancer including administering to a patient in need each R is independently selected from H, -OH,-C-Cs thereof an effective amount of a Drug-Ligand Conjugatehav alkyl, -C-C carbocycle and —O—(C-C alkyl); ing a cleavable Drug unit from the Drug-Ligand Conjugate. R is selected from Hand-C-Cs alkyl: In still another aspect, the invention provides methods for R" is selected from aryl group or -C-Cs heterocycle; 45 killing or inhibiting the replication of a cell that expresses an Zis-O-, - S - NH-, or - NR' , wherein R' is autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker Com C-Cs alkyl; pound. R'' is selected from H, C, -Coalkyl, aryl, -C-Cs hetero In another aspect, the invention provides methods for kill cycle, -(RO), R', or -(RO), CH(R'); 50 ing or inhibiting the replication of a cell that expresses an m is an integer ranging from 1-1000; autoimmune antibody including administering to a patient in R" is C-C alkyl: need thereof an effective amount of a Drug-Linker-Ligand R'' is Hor—C-Cs alkyl: Conjugate. each occurrence of R' is independently H, -COOH, In another aspect, the invention provides methods for kill —(CH), N(R') —(CH), SOH, or -(CH2), 55 ing or inhibiting the replication of a cell that expresses an SO C-C alkyl; autoimmune antibody including administering to a patient in each occurrence of R' is independently H, —C-Cs alkyl, need thereofan effective amount of a Drug-Ligand Conjugate or —(CH), COOH; and having a cleavable Drug unit from the Drug-Ligand Conju n is an integer ranging from 0 to 6. gate. The compounds of Formula (Ib) are useful for treating 60 In yet another aspect, the invention provides methods for cancer, an autoimmune disease or an infectious disease in a treating an autoimmune disease including administering to a patient or useful as an intermediate for the synthesis of a patient in need thereof an effective amount of a Drug-Linker Drug-Linker, Drug-Linker-Ligand Conjugate, and Drug Compound. Ligand Conjugate having a cleavable Drug unit. In yet another aspect, the invention provides methods for In another aspect, compositions are provided including an 65 treating an autoimmune disease including administering to a effective amount of a Drug-Linker-Ligand Conjugate and a patient in need thereof an effective amount of a Drug-Linker pharmaceutically acceptable carrier or vehicle. Ligand Conjugate. US 7,994,135 B2 10 In yet another aspect, the invention provides methods for thereof an effective amount of a Drug-Ligand Conjugatehav treating an autoimmune disease including administering to a ing a cleavable Drug unit from the Drug-Ligand Conjugate. patient in need thereof an effective amount of a Drug-Ligand In yet another aspect, the invention provides methods for Conjugate having a cleavable Drug unit from the Drug preventing an autoimmune disease including administering Ligand Conjugate. to a patient in need thereof an effective amount of a Drug In still another aspect, the invention provides methods for Linker Compound. treating an infectious. disease including administering to a In yet another aspect, the invention provides methods for patient in need thereof an effective amount of a Drug-Linker preventing an autoimmune disease including administering Compound. to a patient in need thereof an effective amount of a Drug In still another aspect, the invention provides methods for 10 Linker-Ligand Conjugate. treating an infectious disease including administering to a In yet another aspect, the invention provides methods for patient in need thereof an effective amount of a Drug-Linker preventing an autoimmune disease including administering Ligand Conjugate. to a patient in need thereof an effective amount of a Drug In still another aspect, the invention provides methods for Ligand Conjugate having a cleavable Drug unit from the treating an infectious disease including administering to a 15 Drug-Ligand Conjugate. patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug In still another aspect, the invention provides methods for Ligand Conjugate. preventing an infectious disease including administering to a In yet another aspect, the invention provides methods for patient in need thereof an effective amount of a Drug-Linker preventing the multiplication of a tumor cell or cancer cell Compound. including administering to a patient in need thereof an effec In still another aspect, the invention provides methods for tive amount of a Drug-Linker Compound. preventing an infectious disease including administering to a In another aspect, the invention provides methods for pre patient in need thereof an effective amount of a Drug-Linker venting the multiplication of a tumor cell or cancer cell Ligand Conjugate. including administering to a patient in need thereof an effec 25 In still another aspect, the invention provides methods for tive amount of a Drug-Linker-Ligand Conjugate. preventing an infectious disease including administering to a In another aspect, the invention provides methods for pre patient in need thereof an effective amount of a Drug-Ligand venting the multiplication of a tumor cell or cancer cell Conjugate having a cleavable Drug unit from the Drug including administering to a patient in need thereof an effec Ligand Conjugate. tive amount of a Drug-Ligand Conjugate having a cleavable 30 In another aspect, a Drug Compound is provided which can Drug unit from the Drug-Ligand Conjugate. be used as an intermediate for the synthesis of a Drug-Linker In still another aspect, the invention provides methods for Compound having a cleavable Drug unit from the Drug preventing cancer including administering to a patient in need Ligand Conjugate. thereof an effective amount of a Drug-Linker Compound. In another aspect, a Drug-Linker Compound is provided In yet another aspect, the invention provides methods for 35 which can be used as an intermediate for the synthesis of a preventing cancer including administering to a patient in need Drug-Linker-Ligand Conjugate. thereof an effective amount of a Drug-Linker-Ligand Conju In another aspect, compounds having Formula Ia' are pro gate. vided: In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need 40 thereofan effective amount of a Drug-Ligand Conjugate hav Ia ing a cleavable Drug unit from the Drug-Ligand Conjugate. Ab-e-A-Ww-Y-D), In still another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an or a pharmaceutically acceptable salt or Solvate thereof, autoimmune antibody including administering to a patient in 45 wherein: need thereof an effective amount of a Drug-Linker Com Ab includes an antibody including one which binds to pound. CD30, CD40, CD70, and Lewis Yantigen, In another aspect, the invention provides methods for pre A is a Stretcher unit, venting the multiplication of a cell that expresses an autoim a is 0 or 1, mune antibody including administering to a patient in need 50 each W is independently an Amino Acid unit, thereof an effective amount of a Drug-Linker-Ligand Conju w is an integer ranging from 0 to 12, gate. Y is a Spacer unit, and In another aspect, the invention provides methods for pre y is 0, 1 or 2, venting the multiplication of a cell that expresses an autoim p ranges from 1 to about 20, and mune antibody including administering to a patient in need D is a Drug unit selected from Formulas D, and D.

R3 O R7 CH R9 H N N N N N NRIs

R2 O R4 R5 R 6 R 8 O R 8 O US 7,994,135 B2 11 12 -continued DF R3 O R7 CH3 R9 O H N N N 1. R11 N N Z

R 2 O R4 R5 R 6 R 8 O R 8 O R10

10 wherein, independently at each location: membrane domain (TM) and short cytoplasmic domain, R is selected from Hand C-Cs alkyl: (semaphorin) 5B, Genbank accession no. AB040878); R is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, (8) PSCA hlg (2700050C12Rik, C530008O16Rik, C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 erocycle and C-C alkyl-(C-Cs heterocycle); 15 gene, Genbank accession no. AY358628); R is selected from H. C-C alkyl, C-C carbocycle, aryl, (9) ETBR (Endothelin type B receptor, Genbank accession C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het no. AY275463); erocycle and C-C alkyl-(C-Cs heterocycle); (10) MSG783 (RNF124, hypothetical protein FLJ20315, R is selected from Hand methyl; Genbank accession no. NM 017763); or R' and Rjointly form a carbocyclic ring and have the (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, formula—(CRR), wherein RandR are independently STAMP1, STEAP2, STMP prostate cancer associated gene selected from H. C-C alkyl and C-C carbocycle and n is 1, prostate cancer associated protein 1, six transmembrane selected from 2, 3, 4, 5 and 6: epithelial antigen of prostate 2, six transmembrane prostate R is selected from Hand C-Cs alkyl: 25 protein, Genbank accession no. AF455138): R’ is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, (12) TrpM4 (BR224.50, FLJ20041, TRPM4, TRPM4B, C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het transient receptor potential cation channel, Subfamily M. erocycle and C-C alkyl-(C-C heterocycle); member 4, Genbank accession no. NM 017636); each R is independently selected from H, OH, C, -Cs (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, tera alkyl, C-C carbocycle and O—(C-C alkyl); 30 tocarcinoma-derived growth factor, Genbank accession no. R is selected from Hand C-Cs alkyl: NP 003203 or NM 003212); R' is selected from aryl or C-Cs heterocycle: (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/ Z is O, S, NH, or NR", wherein R' is C-C alkyl: Epstein Barr virus receptor) or Hs.73792, Genbank accession R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero no. M26004); cycle, -(R) R', or -(RO), CH(R'); 35 (15) CD79b (IGb (immunoglobulin-associated beta), B29, m is an integer ranging from 1-1000; Genbank accession no. NM 000626); R" is C-Cs alkyl: (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain con R" is Hor C-C alkyl: taining phosphatase anchor protein 1a), SPAP1B, SPAP1C, each occurrence of R' is independently H, COOH, Genbank accession no. NM 030764); —(CH), N(R') —(CH), SOH, or -(CH2), 40 (17) HER2 (Genbank accession no. M11730): SO C-C alkyl; (18) NCA (Genbank accession no. M18728); each occurrence of R' is independently H, C-C alkyl, or (19) MDP (Genbank accession no. BC017023): —(CH), COOH: (20) IL20RC (Genbank accession no. AF 184971); R" is selected from C(R), C(R)-aryl, -C(R) - (21) Brevican (Genbank accession no. AF229053); C(R) (C-Cs heterocycle), and C(R) C(R) (C- 45 (22) Ephb2R (Genbank accession no. NM 004442): Cs carbocycle); and (23) ASLG659 (Genbank accession no. AX092328); n is an integer ranging from 0 to 6. (24) PSCA (Genbank accession no. AJ297436): In one embodiment, Ab is not an antibody which binds to (25) GEDA (Genbank accession no. AY260763); an ErbB receptor or which binds to one or more of receptors (26) BAFF-R (Genbank accession no. NP 443177.1): (1)–(35): 50 (27) CD22 (Genbank accession no. NP-001762. 1); (1) BMPR1B (bone morphogenetic protein receptor-type (28) CD79a (CD79A, CD79C, immunoglobulin-associ IB, Genbank accession no. NM 001203); ated alpha, a B cell-specific protein that covalently interacts (2) E 16 (LAT1, SLC7A5, Genbank accession no. with Ig beta (CD79B) and forms a complex on the surface NM 003.486); with IgM molecules, transduces a signal involved in B-cell (3) STEAP1 (six transmembrane epithelial antigen of pros 55 differentiation, Genbank accession No. NP 001774.1): tate, Genbank accession no. NM 012449); (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein (4) 0772P (CA125, MUC16, Genbank accession no. coupled receptor that is activated by the CXCL13 chemokine, AF361486); functions in lymphocyte migration and humoral defense, (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating plays a role in HIV-2 infection and perhaps development of factor, mesothelin, Genbank accession no. NM 005823); 60 AIDS, lymphoma, myeloma, and leukemia, Genbank acces (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier sion No. NP 001707.1): family 34 (sodium phosphate), member 2, type II sodium (30) HLA-DOB (Beta subunit of MHC class II molecule dependent phosphate transporter 3b, Genbank accession no. (Ia antigen) that binds peptides and presents them to CD4+ T NM 006424); lymphocytes, Genbank accession No. NP 002111.1); (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, 65 (31) P2X5 (Purinergic receptor P2X ligand-gated ion SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, channel 5, an ion channel gated by extracellular ATP may be seven thrombospondin repeats (type 1 and type 1-like), trans involved in synaptic transmission and neurogenesis, defi US 7,994,135 B2 13 14 ciency may contribute to the pathophysiology of idiopathic In still another aspect, the invention provides methods for detrusor instability, Genbank accession No. NP 002552.2): treating an infectious disease including administering to a (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, patient in need thereof an effective amount of a Drug-Linker Genbank accession No. NP 001773.1): Antibody Conjugate. (33) LY64 (Lymphocyte antigen 64 (RP 105), type I mem In still another aspect, the invention provides methods for brane protein of the leucine rich repeat (LRR) family, regu treating an infectious disease including administering to a lates B-cell activation and apoptosis, loss of function is asso patient in need thereof an effective amount of a Drug-Anti ciated with increased disease activity in patients with body Conjugate having a cleavable Drug unit from the Drug systemic lupus erythematosis, Genbank accession No. Antibody Conjugate. NP 005573. 1); 10 In another aspect, the invention provides methods for pre (34) FCRH1 (Fc receptor-like protein 1, a putative receptor venting the multiplication of a tumor cell or cancer cell for the immunoglobulin Fc domain that contains C2 type including administering to a patient in need thereof an effec Ig-like and ITAM domains, may have a role in B-lymphocyte tive amount of a Drug-Linker-Antibody Conjugate. differentiation, Genbank accession No. NP 443170.1); or 15 In another aspect, the invention provides methods for pre (35) IRTA2 (Immunoglobulin superfamily receptor trans venting the multiplication of a tumor cell or cancer cell location associated 2, a putative immunoreceptor with pos including administering to a patient in need thereof an effec sible roles in B cell development and lymphomagenesis; tive amount of a Drug-Antibody Conjugate having a cleav deregulation of the gene by translocation occurs in Some B able Drug unit from the Drug-Antibody Conjugate. cell malignancies, Genbank accession No. NP 112571.1). In yet another aspect, the invention provides methods for In still another aspect, the invention provides pharmaceu preventing cancer including administering to a patient in need tical compositions comprising an effective amount of a Drug thereof an effective amount of a Drug-Linker-Antibody Con Linker-Antibody Conjugate and a pharmaceutically accept jugate. able carrier or vehicle. In yet another aspect, the invention provides methods for In still another aspect, the invention provides compositions 25 preventing cancer including administering to a patient in need comprising an effective amount of a Drug-Antibody Conju thereof an effective amount of a Drug-Antibody Conjugate gate having a cleavable Drug unit (moiety) from the Drug having a cleavable Drug unit from the Drug-Antibody Con Antibody Conjugate and a pharmaceutically acceptable car jugate. rier or vehicle. In another aspect, the invention provides methods for pre In another aspect, the invention provides methods for kill 30 venting the multiplication of a cell that expresses an autoim ing or inhibiting the multiplication of a tumor cell or cancer mune antibody including administering to a patient in need cell including administering to a patient in need thereof an thereof an effective amount of a Drug-Linker-Antibody Con effective amount of a Drug-Linker-Antibody Conjugate. jugate. In another aspect, the invention provides methods for kill In another aspect, the invention provides methods for pre ing or inhibiting the multiplication of a tumor cell or cancer 35 venting the multiplication of a cell that expresses an autoim cell including administering to a patient in need thereof an mune antibody including administering to a patient in need effective amount of a Drug-Antibody Conjugate having a thereof an effective amount of a Drug-Antibody Conjugate cleavable Drug unit from the Drug-Antibody Conjugate. having a cleavable Drug unit from the Drug-Antibody Con In yet another aspect, the invention provides methods for jugate. treating cancer including administering to a patient in need 40 In yet another aspect, the invention provides methods for thereofan effective amount of a Drug-Linker-Antibody Con preventing an autoimmune disease including administering jugate. to a patient in need thereof an effective amount of a Drug In yet another aspect, the invention provides methods for Linker-Antibody Conjugate. treating cancer including administering to a patient in need In yet another aspect, the invention provides methods for thereof an effective amount of a Drug-Antibody Conjugate 45 preventing an autoimmune disease including administering having a cleavable Drug unit from the Drug-Antibody Con to a patient in need thereof an effective amount of a Drug jugate. Antibody Conjugate having a cleavable Drug unit from the In another aspect, the invention provides methods for kill Drug-Antibody Conjugate. ing or inhibiting the replication of a cell that expresses an In still another aspect, the invention provides methods for autoimmune antibody including administering to a patient in 50 preventing an infectious disease including administering to a need thereof an effective amount of a Drug-Linker-Antibody patient in need thereof an effective amount of a Drug-Linker Conjugate. Antibody Conjugate. In another aspect, the invention provides methods for kill In still another aspect, the invention provides methods for ing or inhibiting the replication of a cell that expresses an preventing an infectious disease including administering to a autoimmune antibody including administering to a patient in 55 patient in need thereof an effective amount of a Drug-Anti need thereofan effective amount of a Drug-Antibody Conju body Conjugate having a cleavable Drug unit from the Drug gate having a cleavable Drug unit from the Drug-Antibody Antibody Conjugate. Conjugate. In another aspect, a Drug Compound is provided which can In yet another aspect, the invention provides methods for be used as an intermediate for the synthesis of a Drug-Linker treating an autoimmune disease including administering to a 60 Compound having a cleavable Drug unit from the Drug patient in need thereof an effective amount of a Drug-Linker Antibody Conjugate. Antibody Conjugate. In another aspect, a Drug-Linker Compound is provided In yet another aspect, the invention provides methods for which can be used as an intermediate for the synthesis of a treating an autoimmune disease including administering to a Drug-Linker-Antibody Conjugate. patient in need thereof an effective amount of a Drug-Anti 65 In one aspect, the present invention provides Drug-Linker body Conjugate having a cleavable Drug unit from the Drug Antibody Conjugates (also referred to as antibody-drug con Antibody Conjugate. jugates) having Formula Ic: US 7,994,135 B2 15 16 (18) NCA (Genbank accession no. M18728); Ic (19) MDP (Genbank accession no. BC017023): Ab-e-A-W-Y-D), (20) IL20RC (Genbank accession no. AF 184971); (21) Brevican (Genbank accession no. AF229053); or a pharmaceutically acceptable salt or Solvate thereof, (22) Ephb2R (Genbank accession no. NM 004442): wherein: (23) ASLG659 (Genbank accession no. AX092328); Ab is an antibody which binds to one or more of the (24) PSCA (Genbank accession no. AJ297436): antigens (1)–(35): (25) GEDA (Genbank accession no. AY260763); (1) BMPR1B (bone morphogenetic protein receptor-type 10 (26) BAFF-R (Genbank accession no. NP 443177.1): IB, Genbank accession no. NM 001203); (27) CD22 (Genbank accession no. NP-001762. 1); (2) E 16 (LAT1, SLC7A5, Genbank accession no. (28) CD79a (CD79A, CD79C, immunoglobulin-associ NM 003.486); ated alpha, a B cell-specific protein that covalently interacts (3) STEAP1 (six transmembrane epithelial antigen of pros with Ig beta (CD79B) and forms a complex on the surface tate, Genbank accession no. NM 012449); 15 with IgM molecules, transduces a signal involved in B-cell (4) 0772P (CA125, MUC16, Genbank accession no. differentiation, Genbank accession No. NP 001774.1): AF361486); (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating coupled receptor that is activated by the CXCL13 chemokine, factor, mesothelin, Genbank accession no. NM 005823); functions in lymphocyte migration and humoral defense, (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier plays a role in HIV-2 infection and perhaps development of family 34 (sodium phosphate), member 2, type II sodium AIDS, lymphoma, myeloma, and leukemia, Genbank acces dependent phosphate transporter 3b, Genbank accession no. sion No. NP 001707.1): NM 006424); (30) HLA-DOB (Beta subunit of MHC class II molecule (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, (Ia antigen) that binds peptides and presents them to CD4+ T SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, 25 lymphocytes, Genbank accession No. NP 002111.1); seven thrombospondin repeats (type 1 and type 1-like), trans (31) P2X5 (Purinergic receptor P2X ligand-gated ion membrane domain (TM) and short cytoplasmic domain, channel 5, an ion channel gated by extracellular ATP may be (semaphorin) 5B, Genbank accession no. AB040878); involved in synaptic transmission and neurogenesis, defi (8) PSCA hlg (2700050C12Rik, C530008O16Rik, ciency may contribute to the pathophysiology of idiopathic RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 30 detrusor instability, Genbank accession No. NP 002552.2): gene, Genbank accession no. AY358628); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, (9) ETBR (Endothelin type B receptor, Genbank accession Genbank accession No. NP 001773.1): no. AY275463); (33) LY64 (Lymphocyte antigen 64 (RP 105), type I mem (10) MSG783 (RNF124, hypothetical protein FLJ20315, brane protein of the leucine rich repeat (LRR) family, regu Genbank accession no. NM 017763); 35 lates B-cell activation and apoptosis, loss of function is asso (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, ciated with increased disease activity in patients with STAMP1, STEAP2, STMP prostate cancer associated gene systemic lupus erythematosis, Genbank accession No. 1, prostate cancer associated protein 1, six transmembrane NP 005573.1); epithelial antigen of prostate 2, six transmembrane prostate (34) FCRH1 (Fc receptor-like protein 1, a putative receptor protein, Genbank accession no. AF455138): 40 for the immunoglobulin Fc domain that contains C2 type (12) TrpM4 (BR224.50, FLJ20041, TRPM4, TRPM4B, Ig-like and ITAM domains, may have a role in B-lymphocyte transient receptor potential cation channel, Subfamily M. differentiation, Genbank accession No. NP 443170.1); or member 4, Genbank accession no. NM 017636); (35) IRTA2 (Immunoglobulin superfamily receptor trans (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, tera location associated 2, a putative immunoreceptor with pos tocarcinoma-derived growth factor, Genbank accession no. 45 sible roles in B cell development and lymphomagenesis; NP 003203 or NM 003212); deregulation of the gene by translocation occurs in Some B (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/ cell malignancies, Genbank accession No. NP 112571. 1); Epstein Barr virus receptor) or Hs.73792, Genbank accession A is a Stretcher unit, no. M26004); a is 0 or 1, (15) CD79b (IGb (immunoglobulin-associated beta), B29, 50 each W is independently an Amino Acid unit, Genbank accession no. NM 000626); w is an integer ranging from 0 to 12, (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain con Y is a Spacer unit, and taining phosphatase anchor protein 1a), SPAP1B, SPAP1C, y is 0, 1 or 2, Genbank accession no. NM 030764); p ranges from 1 to about 20, and (17) HER2 (Genbank accession no. M11730): D is a Drug moiety selected from Formulas D, and D. US 7,994,135 B2 17 18 -continued DF R3 O R7 CH3 R9 O H A N N N N N Z - R' R2 R R5 R6 R8 O R8 O R1 O

10 wherein the wavy line of D, and Dindicates the covalent mal a therapeutically effective amount of an antibody-drug attachment site to A. W. or Y, and independently at each conjugate compound of the invention. location: In another aspect, a Substantial amount of the drug moiety R’ is selected from Hand C-Cs alkyl: is not cleaved from the antibody until the antibody-drug con R is selected from H.C.-Cs alkyl, Cs-Cs carbocycle, aryl, 15 jugate compound enters a cell with a cell-surface receptor C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C-C het specific for the antibody of the antibody-drug conjugate, and erocycle and C-C alkyl-(C-Cs heterocycle); the drug moiety is cleaved from the antibody when the anti R" is selected from H, C, -Cs alkyl, C-Cs carbocycle, aryl, body-drug conjugate does enter the cell. C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C-C het In another aspect, the bioavailability of the antibody-drug erocycle and C-C alkyl-(C-Cs heterocycle); conjugate compound or an intracellular metabolite of the R is selected from Hand methyl: compound in a mammal is improved when compared to a or R and Rjointly form a carbocyclic ring and have the drug compound comprising the drug moiety of the antibody formula—(CRR), wherein RandR are independently drug conjugate compound, or when compared to an analog of selected from H. C-C alkyl and C-Cs carbocycle and n is 25 the compound not having the drug moiety. selected from 2, 3, 4, 5 and 6: In another aspect, the drug moiety is intracellularly cleaved R is selected from Hand C-Cs alkyl: in a mammal from the antibody of the compound, or an R’ is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, intracellular metabolite of the compound. C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het In another aspect, the invention includes a pharmaceutical erocycle and C-C alkyl-(C-C heterocycle); 30 composition comprising an effective amount of the antibody each R is independently selected from H, OH, C, -Cs drug conjugate compound of the invention, or a pharmaceu alkyl, C-C carbocycle and O (C-C alkyl); tically acceptable Salt thereof, and a pharmaceutically accept R is selected from Hand C-C alkyl: able diluent, carrier or excipient. The composition may R" is selected from aryl or C-Cs heterocycle; further comprise a therapeutically effective amount of che Z is O, S, NH, or NR", wherein R' is C-C alkyl: 35 R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero motherapeutic agent such as a tubulin-forming inhibitor, a cycle, -(RO), R'', or—(RO), CH(R'); topoisomerase inhibitor, and a DNA binder. m is an integer ranging from 1-1000; In another aspect, the invention includes a method for R" is C-Cs alkyl: killing or inhibiting the proliferation of tumor cells or cancer R" is Hor C-C alkyl: 40 cells comprising treating tumor cells or cancer cells with an each occurrence of R' is independently H, COOH, amount of the antibody-drug conjugate compound of the —(CH), N(R') —(CH), SOH, or -(CH2), invention, or a pharmaceutically acceptable salt or Solvate SO C-C alkyl; thereof, being effective to kill or inhibit the proliferation of each occurrence of R' is independently H, C-C alkyl, or the tumor cells or cancer cells. —(CH), COOH: 45 In another aspect, the invention includes a method of inhib R" is selected from C(R), C(R)-aryl, -C(R) - iting cellular proliferation comprising exposing mammalian C(R), (C-C heterocycle), and C(R), C(R) (C- cells in a cell culture medium to an antibody drug conjugate Cs carbocycle); and compound of the invention, wherein the antibody drug con n is an integer ranging from 0 to 6. jugate compound enters the cells and the drug is cleaved from In another aspect, the antibody of the antibody-drug con 50 the remainder of the antibody drug conjugate compound; jugate (ADC) of the invention specifically binds to a receptor whereby proliferation of the cells is inhibited. encoded by an ErbB2 gene. In another aspect, the invention includes a method of treat In another aspect, the antibody of the antibody-drug con ing cancer comprising administering to a patient a formula jugate is a humanized antibody selected from huMAb4D5-1, tion of an antibody-drug conjugate compound of the inven huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5 55 tion and a pharmaceutically acceptable diluent, carrier or 5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastu excipient. Zumab). In another aspect, the invention includes an article of In another aspect, the invention includes an assay for manufacture comprising an antibody-drug conjugate com detecting cancer cells comprising: pound of the invention; a container, and a package insert or 60 (a) exposing cells to an antibody-drug conjugate com label indicating that the compound can be used to treat cancer pound of the invention; and characterized by the overexpression of an ErbB2 receptor. (b) determining the extent of binding of the antibody-drug In another aspect, the invention includes a method for the conjugate compound to the cells. treatment of cancer in a mammal, wherein the cancer is char The invention will best be understood by reference to the acterized by the overexpression of an ErbB2 receptor and 65 following detailed description of the exemplary embodi does not respond, or responds poorly, to treatment with an ments, taken in conjunction with the accompanying draw anti-ErbB2 antibody, comprising administering to the mam ings, figures, and schemes. The discussion below is descrip US 7,994,135 B2 19 20 tive, illustrative and exemplary and is not to be taken as FIG. 15 shows the meantumor volume change over time in limiting the scope defined by any appended claims. athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with Vehicle and 650 ug/m tras BRIEF DESCRIPTION OF THE DRAWINGS tuZumab-MC-MMAF. FIG.16 shows the meantumor volume change over time in FIG. 1 shows an in Vivo, single dose, efficacy assay of athymic nude mice with MMTV-HER2 Fo5 Mammary tumor cAC10-mcMMAF in subcutaneous Karpas-299 ALCL allografts dosed on Day 0 with Vehicle and 350 ug/m of four Xenografts. trastuzumab-MC-MMAF conjugates where the MMAF/tras FIG. 2 shows an in Vivo, single dose, efficacy assay of tuzumab (H) ratio is 2, 4, 5.9 and 6. cAC10-mcMMAF in subcutaneous L540cy. For this study 10 FIG. 17 shows the Group mean change, with error bars, in there were 4 mice in the untreated group and 10 in each of the animal (rat) body weights (Meant-SD) after administration of treatment groups. Vehicle, trastuzumab-MC-val-cit-MMAF, trastuzumab-MC FIGS. 3a and 3b show in vivo efficacy of cBR96-mcM (Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and MAF in subcutaneous L2987. The filed triangles in FIG.3a trastuzumab-MC-val-cit-PAB-MMAF. 15 FIG. 18 shows the Group mean change in animal (rat) body and arrows in FIG. 3b indicate the days of therapy. weights (Meant-SD) after administration of 9.94 mg/kg FIGS. 4a and 4b show in vitro activity of cAC10-antibody H-MC-vc-MMAF, 24.90 mg/kg H-MC-vc-MMAF, 10.69 drug conjugates against CD30" cell lines. mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg H-MC FIGS.5a and 5b show in vitro activity of cBR96-antibody (Me)-vc-PAB-MMAF, 10.17 mg/kg H-MC-MMAF, 25.50 drug conjugates against Le' cell lines. mg/kg. H-MC-MMAF, and 21.85 mg/kg. H-MC-vc-PAB FIGS. 6a and 6b show in vitro activity of c1F6-antibody MMAF. H=trastuzumab. The MC linker is attached via a drug conjugates against CD70 renal cell carcinoma cell cysteine of trastuzumab for each conjugate. lines. FIG. 19 shows the Group mean change, with error bars, in FIG. 7 shows an in vitro, cell proliferation assay with Sprague Dawley rat body weights (Meant-SD) after admin SK-BR-3 cells treated with antibody drug conjugates (ADC): 25 istration of trastuzumab (H)-MC-MMAF at doses of 2105, -O-Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -o- 3158, and 4210 ug/m. The MC linker is attached via a cys Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -A-Trastu teine of trastuzumab for each conjugate. Zumab-MC-MMAF, 4.8 MMAF/Ab, measured in Relative FIG. 20 shows examples of compounds with a non self Fluorescence Units (RLU) versus Hg/ml concentration of immolative Spacer unit. ADC. H=Trastuzumab where His linked via a cysteine cys. 30 FIG. 21 shows a scheme of a possible mechanism of Drug FIG. 8 shows an in vitro, cell proliferation assay with release from a PAB group which is attached directly to -D via BT-474 cells treated with ADC: -O- Trastuzumab-MC-vc a carbamate or carbonate group. PAB-MMAF, 3.8 MMAF/Ab, -o-Trastuzumab-MC-MMAF, FIG.22 shows a scheme of a possible mechanism of Drug 4.1 MMAF/Ab, and -A- Trastuzumab-MC-MMAF, 4.8 release from a PAB group which is attached directly to -D via MMAF/Ab. 35 an ether or amine linkage. FIG. 9 shows an in vitro, cell proliferation assay with FIG. 23 shows an example of a branched spacer unit, bis MCF-7 cells treated with ADC: -O- Trastuzumab-MC-vc (hydroxymethyl)styrene (BHMS) unit, which can be used to PAB-MMAF, 3.8 MMAF/Ab, -o-Trastuzumab-MC-(N-Me) incorporate and release multiple drug. vc-PAB-MMAF, 3.9 MMAF/Ab, and -A-Trastuzumab-MC FIG. 24 shows a scheme of the CellTiter-Glo R Assay. MMAF, 4.1 MMAF/Ab. 40 FIG. 25 shows the synthesis of an N-terminal tripeptide FIG. 10 shows an in vitro, cell proliferation assay with unit F which is a useful intermediate for the synthesis of the MDA-MB-468 cells treated with ADC: -O- Trastuzumab drug compounds of Formula Ib. MC-vc-PAB-MMAE, 4.1 MMAE/Ab, -o-Trastuzumab-MC FIG. 26 shows the synthesis of an N-terminal tripeptide vc-PAB-MMAE, 3.3 MMAE/Ab, and -A-Trastuzumab-MC unit F which is a useful intermediate for the synthesis of the vc-PAB-MMAF, 3.7 MMAF/Ab. 45 drug compounds of Formula Ib. FIG. 11 shows a plasma concentration clearance study FIG. 27 shows the synthesis of an N-terminal tripeptide after administration of H-MC-vc-PAB-MMAF-TEG and unit F which is a useful intermediate for the synthesis of the H-MC-vc-PAB-MMAF to Sprague-Dawley rats: The admin drug compounds of Formula Ib. istered dose was 2 mg of ADC per kg of rat. Concentrations of FIG. 28 shows the synthesis of useful linkers. total antibody and ADC were measured over time. 50 FIG. 29 shows the synthesis of useful linkers. (H=Trastuzumab). FIG. 30 shows a general synthesis of an illustrative Linker FIG. 12 shows a plasma concentration clearance study unit containing a maleimide Stretcher group and optionally a after administration of H-MC-vc-MMAE to Cynomolgus p-aminobenzyl ether self-immolative Spacer. monkeys at different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg FIG. 31 shows the synthesis of a val-cit dipeptide Linker administered at day 1 and day 21. Concentrations of total 55 having a maleimide Stretcher and optionally a p-aminobenzyl antibody and ADC were measured over time. self-immolative Spacer. (H=Trastuzumab). FIG. 32 shows the synthesis of a phe-lys(Mtr) dipeptide FIG. 13 shows the meantumor volume change overtime in Linker unit having a maleimide Stretcher unit and a p-ami athymic nude mice with MMTV-HER2 Fo5 Mammary tumor nobenzyl self-immolative Spacer unit. allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC 60 FIG.33 shows the synthesis of a Drug-Linker Compound vc-PAB-MMAE (1250 g/m) and Trastuzumab-MC-vc that contains an amide or carbamate group, linking the Drug PAB-MMAF (555 g/m). (H=Trastuzumab). unit to the Linker unit. FIG. 14 shows the meantumor volume change overtime in FIG. 34 shows illustrative methods useful for linking a athymic nude mice with MMTV-HER2 Fo5 Mammary tumor Drug to a Ligand to form a Drug-Linker Compound. allografts dosed on Day 0 with 10 mg/kg (660 ug/m) of 65 FIG. 35 shows the synthesis of a val-cit dipeptide linker Trastuzumab-MC-MMAE and 1250 ug/m Trastuzumab having a maleimide Stretcher unit and a bis(4-hydroxym MC-VC-PAB-MMAE. ethyl)styrene (BHMS) unit. US 7,994,135 B2 21 22 FIG. 36 shows methodology useful for making Drug prising the population are identical except for possible natu Linker-Ligand conjugates having about 2 to about 4 drugs per rally-occurring mutations that may be present in minor antibody. amounts. Monoclonal antibodies are highly specific, being FIG. 37 shows the synthesis of MC-MMAF via t-butyl directed against a single antigenic site. Furthermore, in con ester. 5 trast to polyclonal antibody preparations which include dif FIG. 38 shows the synthesis of MC-MMAF (11) via ferent antibodies directed against different determinants dimethoxybenzyl ester. (epitopes), each monoclonal antibody is directed against a FIG. 39 shows the synthesis of analog of mc-MMAF. single determinant on the antigen. In addition to their speci ficity, the monoclonal antibodies are advantageous in that 4. DETAILED DESCRIPTION OF THE 10 they may be synthesized uncontaminated by other antibodies. EXEMPLARY EMBODIMENTS The modifier "monoclonal indicates the character of the antibody as being obtained from a Substantially homoge 4.1 Definitions and Abbreviations neous population of antibodies, and is not to be construed as Unless stated otherwise, the following terms and phrases as requiring production of the antibody by any particular used herein are intended to have the following meanings: 15 method. For example, the monoclonal antibodies to be used in When trade names are used herein, applicants intend to accordance with the present invention may be made by the independently include the trade name product formulation, hybridoma method first described by Kohler et al. (1975) the generic drug, and the active pharmaceutical ingredient(s) Nature 256:495, or may be made by recombinant DNA meth of the trade name product. ods (see, U.S. Pat. No. 4,816,567). The “monoclonal antibod The term “antibody' herein is used in the broadest sense ies' may also be isolated from phageantibody libraries using and specifically covers intact monoclonal antibodies, poly the techniques described in Clackson et al. (1991) Nature, clonal antibodies, multispecific antibodies (e.g., bispecific 352:624-628 and Marks et al. (1991).J. Mol. Biol., 222:581 antibodies) formed from at least two intact antibodies, and 597, for example. antibody fragments, so long as they exhibit the desired bio The monoclonal antibodies herein specifically include logical activity. An antibody is a protein generated by the 25 "chimeric' antibodies in which a portion of the heavy and/or immune system that is capable of recognizing and binding to light chain is identical with or homologous to corresponding a specific antigen. Described in terms of its structure, an sequences in antibodies derived from a particular species or antibody typically has a Y-shaped protein consisting of four belonging to a particular antibody class or Subclass, while the amino acid chains, two heavy and two light. Each antibody remainder of the chain(s) is identical with or homologous to has primarily two regions: a variable region and a constant 30 corresponding sequences in antibodies derived from another region. The variable region, located on the ends of the arms of species or belonging to another antibody class or Subclass, as the Y, binds to and interacts with the target antigen. This well as fragments of such antibodies, so long as they exhibit variable region includes a complementary determining region the desired biological activity (U.S. Patent mp/4816567; and (CDR) that recognizes and binds to a specific binding site on Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851 a particular antigen. The constant region, located on the tail of 35 6855). the Y, is recognized by and interacts with the immune system Various methods have been employed to produce mono (Janeway, C., Travers, P. Walport, M., Shlomchik (2001) clonal antibodies (MAbs). Hybridoma technology, which Immuno Biology, 5th Ed., Garland Publishing, New York). A refers to a cloned cell line that produces a single type of target antigen generally has numerous binding sites, also antibody, uses the cells of various species, including mice called epitopes, recognized by CDRs on multiple antibodies. 40 (murine), hamsters, rats, and humans. Another method to Each antibody that specifically binds to a different epitope has prepare MAbs uses genetic engineering including recombi a different structure. Thus, one antigen may have more than nant DNA techniques. Monoclonal antibodies made from one corresponding antibody. these techniques include, among others, chimeric antibodies The term “antibody” as used herein, also refers to a full and humanized antibodies. A chimeric antibody combines length immunoglobulin molecule or an immunologically 45 DNA encoding regions from more than one type of species. active portion of a full-length immunoglobulin molecule, i.e., For example, a chimeric antibody may derive the variable a molecule that contains an antigen binding site that immu region from a mouse and the constant region from a human. A nospecifically binds an antigen of a target of interest or part humanized antibody comes predominantly from a human, thereof. Such targets including but not limited to, cancer cell even though it contains nonhuman portions. Like a chimeric or cells that produce autoimmune antibodies associated with 50 antibody, a humanized antibody may contain a completely an autoimmune disease. The immunoglobulin disclosed human constant region. But unlike a chimeric antibody, the herein can be of any type (e.g., IgG, IgE, IgM, Ig), and IgA), variable region may be partially derived from a human. The class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or Sub nonhuman, synthetic portions of a humanized antibody often class of immunoglobulin molecule. The immunoglobulins come from CDRs in murine antibodies. In any event, these can be derived from any species. In one aspect, however, the 55 regions are crucial to allow the antibody to recognize and bind immunoglobulin is of human, murine, or rabbit origin. In to a specific antigen. another aspect, the antibodies are polyclonal, monoclonal, As noted, murine antibodies can be used. While useful for bispecific, human, humanized or chimeric antibodies, single diagnostics and short-term therapies, murine antibodies can chain antibodies, Fv, Fab fragments, F(ab') fragments, F(ab') not be administered to people long-term without increasing fragments, fragments produced by a Fab expression library, 60 the risk of a deleterious immunogenic response. This anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-bind response, called Human Anti-Mouse Antibody (HAMA), ing fragments of any of the above which immunospecifically occurs when a human immune system recognizes the murine bind to cancer cell antigens, viral antigens or microbial anti antibody as foreign and attacks it. A HAMA response can genS. cause toxic shock or even death. The term “monoclonal antibody” as used herein refers to 65 Chimeric and humanized antibodies reduce the likelihood an antibody obtained from a population of Substantially of a HAMA response by minimizing the nonhuman portions homogeneous antibodies, i.e., the individual antibodies com of administered antibodies. Furthermore, chimeric and US 7,994,135 B2 23 24 humanized antibodies have the additional benefit of activat regulin, betacellulin, HB-EGF and epiregulin. ErbB ligands ing secondary human immune responses, such as antibody which bind ErbB3 include heregulins. ErbB ligands capable dependent cellular cytotoxicity. of binding ErbB4 include betacellulin, epiregulin, HB-EGF, Antibody fragments' comprise a portion of an intact anti NRG-2, NRG-3, NRG-4 and heregulins. The ErbB ligand body, preferably comprising the antigen-binding or variable may also be a synthetic ErbB ligand. The synthetic ligand region thereof. Examples of antibody fragments include Fab, may be specific for a particular ErbB receptor, or may recog Fab'. F(ab'), and Fv fragments; diabodies; linear antibodies; nize particular ErbB receptor complexes. An example of a single-chain antibody molecules; and multispecific antibod synthetic ligand is the synthetic heregulin/EGF chimera ies formed from antibody fragment(s). biregulin (see, for example, Jones et al., (1999) FEBS Letters, An “intact' antibody is one which comprises an antigen 10 447:227-231, which is incorporated by reference). binding variable region as well as a light chain constant “Heregulin' (HRG) refers to a polypeptide encoded by the domain (CL) and heavy chain constant domains, CH1, CH2 heregulin gene product as disclosed in U.S. Pat. No. 5,641, and CH3. The constant domains may be native sequence 869 or Marchionni et al., Nature, 362:312-318 (1993). constant domains (e.g., human native sequence constant Examples of heregulins include heregulin-C., heregulin-31, domains) or amino acid sequence variant thereof. 15 heregulin-B2 and heregulin-B3 (Holmes et al., Science, 256: The intact antibody may have one or more “effector func 1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu differ tions” which refer to those biological activities attributable to entiation factor (NDF) (Peles et al., Cell 69: 205-216 (1992)); the Fc region (a native sequence Fc region or amino acid acetylcholine receptor-inducing activity (ARIA) (Falls et al. sequence variant Fc region) of an antibody. Examples of (1993) Cell 72:801-815); glial growth factors (GGFs) (Mar antibody effector functions include C1q binding; comple chionni et al., Nature, 362:312-318 (1993)); sensory and ment dependent cytotoxicity; Fc receptor binding; antibody motor neuron derived factor (SMDF) (Ho et al., J. Biol. dependent cell-mediated cytotoxicity (ADCC), phagocyto Chem., 270:14523-14532 (1995)); Y-heregulin (Schaefer et sis; down regulation of cell Surface receptors (e.g., B cell al., Oncogene, 15:1385-1394 (1997)). The term includes bio receptor, BCR), etc. logically active fragments and/or amino acid sequence vari Depending on the amino acid sequence of the constant 25 ants of a native sequence HRG polypeptide, Such as an EGF domain of their heavy chains, intact antibodies can be like domain fragment thereof (e.g., HRGB1177-244). assigned to different “classes.” There are five major classes of “ErbB hetero-oligomer' is a noncovalently associated oli intact antibodies: IgA, Ig|D, IgE. IgG, and IgM, and several of gomer comprising at least two different ErbB receptors. An these may be further divided into “subclasses” (isotypes), “ErbB dimer' is a noncovalently associated oligomer that e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy 30 comprises two different ErbB receptors. Such complexes chain constant domains that correspond to the different may form when a cell expressing two or more ErbB receptors classes of antibodies are called C, 6, e, Y, and L, respectively. is exposed to an ErbB ligand. ErbB oligomers, such as ErbB The Subunit structures and three-dimensional configurations dimers, can be isolated by immunoprecipitation and analyzed of different classes of immunoglobulins are well known. by SDS-PAGE as described in Sliwkowski et al., J. Biol. The expressions “ErbB2” and “HER2 are used inter 35 Chem., 269(20): 14661-14665 (1994), for example. changeably herein and refer to human HER2 protein Examples of such ErbB hetero-oligomers include EGFR described, for example, in Semba et al., Proc. Natl. Acad. Sci. ErbB2 (also referred to as HER1/HER2), ErbB2-ErbB3 USA, 82:6497-6501 (1985) and Yamamoto et al., (1986) (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes. Nature, 319:230-234 (Genebank accession numberX03363). Moreover, the ErbB hetero-oligomer may comprise two or The term "erbB2 refers to the gene encoding human ErbB2 40 more ErbB2 receptors combined with a different ErbB recep and “neu' refers to the gene encoding rat p185neu. Preferred tor, such as ErbB3, ErbB4 or EGFR (ErbB1). Other proteins, ErbB2 is native sequence human ErbB2. Such as a cytokine receptor Subunit (e.g., gp130) may be Antibodies to ErbB receptors are available commercially included in the hetero-oligomer. from a number of sources, including, for example, Santa Cruz A“native sequence' polypeptide is one which has the same Biotechnology, Inc., California, USA. 45 amino acid sequence as a polypeptide, e.g., tumor-associated By “ErbB ligand” is meant a polypeptide which binds to antigen receptor, derived from nature. Such native sequence and/or activates an ErbB receptor. The ErbB ligand may be a polypeptides can be isolated from nature or can be produced native sequence human ErbB ligand Such as epidermal by recombinant or synthetic means. Thus, a native sequence growth factor (EGF) (Savage et al. (1972) J. Biol. Chem., polypeptide can have the amino acid sequence of naturally 247:7612-7621); transforming growth factor alpha (TGF-C.) 50 occurring human polypeptide, murine polypeptide, or (Marquardt et al. (1984) Science 223: 1079-1082): amphi polypeptide from any other mammalian species. regulin also known as Schwanoma or keratinocyte autocrine The term "amino acid sequence variant” refers to polypep growth factor (Shoyab et al. (1989) Science 243:1074-1076; tides having amino acid sequences that differ to some extent Kimura et al., Nature, 348:257-260 (1990); and Cook et al., from a native sequence polypeptide. Ordinarily, amino acid Mol. Cell. Biol., 11:2547-2557 (1991); betacellulin (Shinget 55 sequence variants will possess at least about 70% homology al., Science, 259:1604-1607 (1993); and Sasada et al., Bio with at least one receptor binding domain of a native ligand, chem. Biophys. Res. Commun., 190:1173 (1993)); heparin or with at least one ligand binding domain of a native receptor, binding epidermal growth factor (HB-EGF) (Higashiyama et Such as a tumor-associated antigen, and preferably, they will al., Science, 251:936-939 (1991)); epiregulin (Toyoda et al., be at least about 80%, more preferably, at least about 90% J. Biol. Chem., 270:7495-7500 (1995); and Komurasaki et al., 60 homologous with Such receptor or ligand binding domains. Oncogene, 15:2841-2848 (1997)); a heregulin (see below); The amino acid sequence variants possess Substitutions, dele neuregulin-2 (NRG-2) (Carraway et al., Nature, 387:512-516 tions, and/or insertions at certain positions within the amino (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. acid sequence of the native amino acid sequence. Acad. Sci., 94:9562–9567 (1997)); neuregulin-4 (NRG-4) “Sequence identity” is defined as the percentage of resi (Hararietal. Oncogene, 18:2681-89 (1999)) or cripto (CR-1) 65 dues in the amino acid sequence variant that are identical after (Kannan et al., J. Biol. Chem., 272(6):3330-3335 (1997)). aligning the sequences and introducing gaps, if necessary, to ErbB ligands which bind EGFR include EGF, TGF-C., amphi achieve the maximum percent sequence identity. Methods US 7,994,135 B2 25 26 and computer programs for the alignment are well known in configuration, connected by three hyperVariable regions, the art. One such computer program is 'Align 2. authored by which form loops connecting, and in Some cases forming part Genentech, Inc., which was filed with user documentation in of the B-sheet structure. The hypervariable regions in each the United States Copyright Office, Washington, D.C. 20559, chain are held together in close proximity by the FRs and, on Dec. 10, 1991. with the hypervariable regions from the other chain, contrib Antibody-dependent cell-mediated cytotoxicity' and ute to the formation of the antigen-binding site of antibodies ADCC refer to a cell-mediated reaction in which nonspe (see Kabat et al. (1991) Sequences of Proteins of Immuno cific cytotoxic cells that express Fc receptors (FcRS) (e.g., logical Interest, 5th Ed. Public Health Service, National Insti Natural Killer (NK) cells, neutrophils, and macrophages) tutes of Health, Bethesda, Md.). The constant domains are not recognize bound antibody on a target cell and Subsequently 10 involved directly in binding an antibody to an antigen, but cause lysis of the target cell. The primary cells for mediating exhibit various effector functions, such as participation of the ADCC, NK cells, express FcyRIII only, whereas monocytes antibody in antibody dependent cellular cytotoxicity express FcyRI, FcyRII and FcyRIII. FcR expression on (ADCC). hematopoietic cells in Summarized is Table 3 on page 464 of The term “hypervariable region' when used herein refers Ravetch and Kinet, (1991) Annu. Rev. Immunol, 9:457-92. To 15 to the amino acid residues of an antibody which are respon assess ADCC activity of a molecule of interest, an in vitro sible for antigen-binding. The hyperVariable region generally ADCC assay, such as that described in U.S. Pat. Nos. 5,500, comprises amino acid residues from a “complementarity 362 or 5,821,337 may be performed. Useful effector cells for determining region' or “CDR (e.g., residues 24-34 (L1), Such assays include peripheral blood mononuclear cells 50-56 (L2) and 89-97 (L3) in the light chain variable domain (PBMC) and Natural Killer (NK) cells. Alternatively, or addi and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy tionally, ADCC activity of the molecule of interest may be chain variable domain; Kabat et al. Supra) and/or those resi assessed in vivo, e.g., in a animal model Such as that disclosed dues from a “hypervariable loop' (e.g., residues 26-32 (L1), in Clynes et al., Preo. Natl. Acad. Sci. USA, 95:652-656 50-52 (L2) and 91-96 (L3) in the light chain variable domain (1998). and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy The terms “Fc receptor” or “FeR” are used to describe a 25 chain variable domain; Chothia and Lesk (1987).J. Mol. Biol. receptor that binds to the Fc region of an antibody. The pre 196:901-917). “Framework Region” or “FR residues are ferred FcR is a native sequence human FcR. Moreover, a those variable domain residues other than the hypervariable preferred FcR is one which binds an IgG antibody (a gamma region residues as herein defined. receptor) and includes receptors of the FcyRI, FcyRII, and Fcy Papain digestion of antibodies produces two identical anti RIII subclasses, including allelic variants and alternatively 30 gen-binding fragments, called “Fab' fragments, each with a spliced forms of these receptors. FeyRII receptors include single antigen-binding site, and a residual "Fo' fragment, FcyRIIA (an "activating receptor) and FcyRIIB (an “inhib whose name reflects its ability to crystallize readily. Pepsin iting receptor), which have similar amino acid sequences treatment yields an F(ab') fragment that has two antigen that differ primarily in the cytoplasmic domains thereof. Acti binding sites and is still capable of cross-linking antigen. Vating receptor FcyRIIA contains an immunoreceptor 35 “Fv' is the minimum antibody fragment which contains a tyrosine-based activation motif (ITAM) in its cytoplasmic complete antigen-recognition and antigen-binding site. This domain. Inhibiting receptor FcyRIIB contains an immunore region consists of a dimer of one heavy chain and one light ceptor tyrosine-based inhibition motif (ITIM) in its cytoplas chain variable domain in tight, non-covalent association. It is mic domain. (See review M. in Daéron, Annu. Rev. Immunol., in this configuration that the three hyperVariable regions of 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, 40 each variable domain interact to define an antigen-binding Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immu site on the surface of the VH-VL dimer. Collectively, the six nomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. hyperVariable regions confer antigen-binding specificity to Med., 126:330-41 (1995). Other FcRs, including those to be the antibody. However, even a single variable domain (or half identified in the future, are encompassed by the term “FcR’ of an Fv comprising only three hyperVariable regions specific herein. The term also includes the neonatal receptor, FcRn, 45 for an antigen) has the ability to recognize and bind antigen, which is responsible for the transfer of maternal IgGs to the although at a lower affinity than the entire binding site. fetus. (Guyer et al., J. Immunol., 117:587 (1976) and Kim et The Fab fragment also contains the constant domain of the al., J. Immunol., 24:249 (1994)). light chain and the first constant domain (CH1) of the heavy “Complement dependent cytotoxicity' or “CDC refers to chain. Fab' fragments differ from Fab fragments by the addi the ability of a molecule to lyse a target in the presence of 50 tion of a few residues at the carboxy terminus of the heavy complement. The complement activation pathway is initiated chain CH1 domain including one or more cysteines from the by the binding of the first component of the complement antibody hinge region. Fab'-SH is the designation herein for system (C1q) to a molecule (e.g., an antibody) complexed Fab' in which the cysteine residue(s) of the constant domains with a cognate antigen. To assess complement activation, a bear at least one free thiol group. F(ab') antibody fragments CDC assay, e.g., as described in Gazzano-Santoro et al., J. 55 originally were produced as pairs of Fab' fragments which Immunol. Methods, 202:163 (1996), may be performed. have hinge cysteines between them. Other chemical cou The term “variable” refers to the fact that certain portions plings of antibody fragments are also known. of the variable domains differ extensively in sequence among The “light chains of antibodies from any vertebrate spe antibodies and are used in the binding and specificity of each cies can be assigned to one of two clearly distinct types, called particular antibody for its particular antigen. However, the 60 kappa (K) and lambda (W), based on the amino acid sequences variability is not evenly distributed throughout the variable of their constant domains. domains of antibodies. It is concentrated in three segments “Single-chain Fv' or “scFv'antibody fragments comprise called hypervariable regions both in the light chain and the the VH and VL domains of antibody, wherein these domains heavy chain variable domains. The more highly conserved are present in a single polypeptide chain. Preferably, the Fv portions of variable domains are called the framework 65 polypeptide further comprises a polypeptide linker between regions (FRs). The variable domains of native heavy and light the VH and VL domains which enables the scEv to form the chains each comprise four FRS, largely adopting a B-sheet desired structure for antigenbinding. For a review of scFv, see US 7,994,135 B2 27 28 Plückthun in The Pharmacology of Monoclonal Antibodies, An antibody which “induces apoptosis” is one which vol. 113, Rosenburg and Moore eds. Springer-Verlag, New induces programmed cell death as determined by binding of York, pp. 269-315 (1994). annexin V, fragmentation of DNA, cell shrinkage, dilation of The term "diabodies' refers to small antibody fragments endoplasmic reticulum, cell fragmentation, and/or formation with two antigen-binding sites, which fragments comprise a of membrane vesicles (called apoptotic bodies). The cell is a variable heavy domain (VH) connected to a variable light tumor cell, e.g., a breast, ovarian, stomach, endometrial, Sali domain (VL) in the same polypeptide chain (VH-VL). By vary gland, lung, kidney, colon, thyroid, pancreatic or bladder using a linker that is too short to allow pairing between the cell. Various methods are available for evaluating the cellular two domains on the same chain, the domains are forced to pair events associated with apoptosis. For example, phosphatidyl with the complementary domains of another chain and create 10 serine (PS) translocation can be measured by annexin bind two antigen-binding sites. Diabodies are described more fully ing; DNA fragmentation can be evaluated through DNA lad in, for example, EP404,097; WO 93/11161; and Hollingeret dering; and nuclear/chromatin condensation along with DNA al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. fragmentation can be evaluated by any increase in hypodip “Humanized forms of non-human (e.g., rodent) antibod loid cells. ies are chimeric antibodies that contain minimal sequence 15 A “disorder is any condition that would benefit from derived from non-human immunoglobulin. For the most part, treatment of the present invention. This includes chronic and humanized antibodies are human immunoglobulins (recipi acute disorders or diseases including those pathological con ent antibody) in which residues from a hypervariable region ditions which predispose the mammal to the disorder in ques of the recipient are replaced by residues from a hypervariable tion. Non-limiting examples of disorders to be treated herein region of a non-human species (donor antibody) Such as include benign and malignant tumors; leukemia and lym mouse, rat, rabbit or nonhuman primate having the desired phoid malignancies, in particular breast, ovarian, Stomach, specificity, affinity, and capacity. In some instances, frame endometrial, salivary gland, lung, kidney, colon, thyroid, pan work region (FR) residues of the human immunoglobulin are creatic, prostate or bladder cancer; neuronal, glial, astrocytal, replaced by corresponding non-human residues. Further hypothalamic and other glandular, macrophagal, epithelial, more, humanized antibodies may comprise residues that are 25 stromal and blastocoelic disorders; and inflammatory, angio not found in the recipient antibody or in the donor antibody. genic and immunologic disorders. These modifications are made to further refine antibody per The term “therapeutically effective amount” refers to an formance. In general, the humanized antibody will comprise amount of a drug effective to treat a disease or disorder in a substantially all of at least one, and typically two, variable mammal. In the case of cancer, the therapeutically effective domains, in which all or substantially all of the hypervariable 30 amount of the drug may reduce the number of cancer cells; loops correspond to those of a non-human immunoglobulin reduce the tumor size; inhibit (i.e., slow to some extent and and all or substantially all of the FRs are those of a human preferably stop) cancer cell infiltration into peripheral organs: immunoglobulin sequence. The humanized antibody option inhibit (i.e., slow to some extent and preferably stop) tumor ally also will comprise at least a portion of an immunoglobu metastasis; inhibit, to some extent, tumor growth; and/or lin constant region (Fc), typically that of a human immuno 35 relieve to Some extent one or more of the symptoms associ globulin. For further details, see Jones et al. (1986) Nature, ated with the cancer. To the extent the drug may prevent 321:522-525; Riechmann et al. (1988) Nature 332:323-329; growth and/or kill existing cancer cells, it may be cytostatic and Presta, (1992) Curr. Op. Struct. Biol. 2:593-596. and/or cytotoxic. For cancer therapy, efficacy can, for Humanized anti-ErbB2 antibodies include huMAb4D5-1, example, be measured by assessing the time to disease pro huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5 40 gression (TTP) and/or determining the response rate (RR). 5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HER The term “substantial amount” refers to a majority, i.e. CEPTINR) as described in Table 3 of U.S. Pat. No. 5,821,337 >50% of a population, of a collection or a sample. expressly incorporated herein by reference; humanized The term “intracellular metabolite' refers to a compound 520C9 (WO 93/21319) and humanized 2C4 antibodies as resulting from a metabolic process or reaction inside a cell on described herein below. 45 an antibody drug conjugate (ADC). The metabolic process or An "isolated antibody is one which has been identified reaction may be an enzymatic process Such as proteolytic and separated and/or recovered from a component of its natu cleavage of a peptide linker of the ADC, or hydrolysis of a ral environment. Contaminant components of its natural envi functional group Such as a hydraZone, ester, or amide. Intra ronment are materials which would interfere with diagnostic cellular metabolites include, but are not limited to, antibodies or therapeutic uses for the antibody, and may include 50 and free drug which have undergone intracellular cleavage enzymes, hormones, and other proteinaceous or nonproteina after entry, diffusion, uptake or transport into a cell. ceous solutes. In preferred embodiments, the antibody will be The terms “intracellularly cleaved' and “intracellular purified (1) to greater than 95% by weight of antibody as cleavage” refer to a metabolic process or reaction inside a cell determined by the Lowry method, and most preferably more on an Drug-Ligand Conjugate, a Drug-Linker-Ligand Con than 99% by weight, (2) to a degree sufficient to obtain at least 55 jugate, an antibody drug conjugate (ADC) or the like whereby 15 residues of N-terminal or internal amino acid sequence by the covalent attachment, e.g., the linker, between the drug use of a spinning cup sequenator, or (3) to homogeneity by moiety (D) and the antibody (Ab) is broken, resulting in the SDS-PAGE under reducing or nonreducing conditions using free drug dissociated from the antibody inside the cell. The Coomassie blue or, preferably, silver stain. Isolated antibody cleaved moieties of the Drug-Ligand Conjugate, a Drug includes the antibody in situ within recombinant cells since at 60 Linker-Ligand Conjugate or ADC are thus intracellular least one component of the antibody's natural environment metabolites. will not be present. Ordinarily, however, isolated antibody The term “bioavailability” refers to the systemic availabil will be prepared by at least one purification step. ity (i.e., blood/plasma levels) of a given amount of drug An antibody “which binds an antigen of interest is one administered to a patient. Bioavailability is an absolute term capable of binding that antigen with sufficient affinity such 65 that indicates measurement of both the time (rate) and total that the antibody is useful in targeting a cell expressing the amount (extent) of drug that reaches the general circulation antigen. from an administered dosage form. US 7,994,135 B2 29 30 The term “cytotoxic activity” refers to a cell-killing, cyto described above. One may also study ErbB2 receptor over static or anti-proliferation effect of an antibody drug conju expression by measuring shed antigen (e.g., ErbB2 extracel gate compound or an intracellular metabolite of an antibody lular domain) in a biological fluid Such as serum (see, e.g., drug conjugate compound. Cytotoxic activity may be U.S. Pat. No. 4,933,294; WO91/05264; U.S. Pat. No. 5,401, expressed as the ICso value which is the concentration (molar 638; and Siaset al., (1990).J. Immunol. Methods, 132: 73-80). or mass) per unit volume at which half the cells survive. Aside from the above assays, various other in Vivo assays are The terms “cancer and "cancerous” refer to or describe the available to the skilled practitioner. For example, one may physiological condition in mammals that is typically charac expose cells within the body of the patient to an antibody terized by unregulated cell growth. A “tumor comprises one which is optionally labeled with a detectable label, e.g., a or more cancerous cells. Examples of cancer include, but are 10 radioactive isotope, and binding of the antibody to cells in the not limited to, carcinoma, lymphoma, blastoma, sarcoma, patient can be evaluated, e.g., by external scanning for radio and leukemia or lymphoid malignancies. More particular activity or by analyzing a biopsy taken from a patient previ examples of such cancers include squamous cell cancer (e.g., ously exposed to the antibody. epithelial squamous cell cancer), lung cancer including The tumors overexpressing HER2 are rated by immuno Small-cell lung cancer, non-Small cell lung cancer 15 histochemical scores corresponding to the number of copies (NSCLC), adenocarcinoma of the lung and squamous car of HER2 molecules expressed per cell, and can been deter cinoma of the lung, cancer of the peritoneum, hepatocellular mined biochemically: 0-0-10,000 copies/cell, 1+=at least cancer, gastric or stomach cancer including gastrointestinal about 200,000 copies/cell, 2+=at least about 500,000 copies/ cancer, pancreatic cancer, glioblastoma, cervical cancer, ova cell, 3+=about 1-2x10 copies/cell. Overexpression of HER2 rian cancer, liver cancer, bladder cancer, hepatoma, breast at the 3+ level, which leads to ligand-independent activation cancer, colon cancer, rectal cancer, colorectal cancer, of the tyrosine kinase (Hudziak et al., (1987) Proc. Natl. endometrial or uterine carcinoma, salivary gland carcinoma, Acad. Sci. USA, 84:7159-71.63), occurs in approximately kidney or renal cancer, prostate cancer, Vulval cancer, thyroid 30% of breast cancers, and in these patients, relapse-free cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, survival and overall survival are diminished (Slamon et al., as well as head and neck cancer. 25 (1989) Science, 244:707-712; Slamon et al., (1987) Science, An “ErbB2-expressing cancer is one which produces suf 235:177-182). ficient levels of ErbB2 at the surface of cells thereof, such that Conversely, a cancer which is “not characterized by over an anti-ErbB2 antibody can bind thereto and have a therapeu expression of the ErbB2 receptor is one which, in a diagnos tic effect with respect to the cancer. tic assay, does not express higher than normal levels of ErbB2 A cancer “characterized by excessive activation of an 30 receptor compared to a noncancerous cell of the same tissue ErbB2 receptor is one in which the extent of ErbB2 receptor type. activation in cancer cells significantly exceeds the level of The term "cytotoxic agent” as used herein refers to a sub activation of that receptor in non-cancerous cells of the same stance that inhibits or prevents the function of cells and/or tissue type. Such excessive activation may result from over causes destruction of cells. The term is intended to include expression of the ErbB2 receptor and/or greater than normal 35 radioactive isotopes (e.g., ''At, I, I, Y, Re, Re, levels of an ErbB2 ligand available for activating the ErbB2 'Sim, ''Bi, P, 'C, and radioactive isotopes of Lu), che receptor in the cancer cells. Such excessive activation may motherapeutic agents, and toxins such as Small molecule cause and/or becaused by the malignant state of a cancer cell. toxins or enzymatically active toxins of bacterial, fungal, In some embodiments, the cancer will be subjected to a diag plant or animal origin, including synthetic analogs and nostic or prognostic assay to determine whether amplification 40 derivatives thereof. In one aspect, the term is not intended to and/or overexpression of an ErbB2 receptor is occurring include radioactive isotopes. which results in such excessive activation of the ErbB2 recep A “chemotherapeutic agent' is a chemical compound use tor. Alternatively, or additionally, the cancer may be subjected ful in the treatment of cancer. Examples of chemotherapeutic to a diagnostic or prognostic assay to determine whether agents include alkylating agents such as thiotepa and amplification and/or overexpression an ErbB2 ligand is 45 CYTOXANR) cyclosphosphamide; alkyl sulfonates such as occurring in the cancer which attributes to excessive activa buSulfan, improsulfan and piposulfan; aziridines Such as ben tion of the receptor. In a Subset of Such cancers, excessive Zodopa, carboquone, meturedopa, and uredopa; ethylen activation of the receptor may result from an autocrine stimu imines and methylamelamines including altretamine, trieth latory pathway. ylenemelamine, trietylenephosphoramide, A cancer which “overexpresses an ErbB2 receptor is one 50 triethiylenethiophosphoramide and trimethylolomelamine; which has significantly higher levels of an ErbB2 receptor at TLK 286 (TELCYTATM); acetogenins (especially bullatacin the cell Surface thereof, compared to a noncancerous cell of and bullatacinone); delta-9-tetrahydrocannabinol (dronab the same tissue type. Such overexpression may be caused by inol, MARINOLR); beta-lapachone; lapachol; colchicines: gene amplification or by increased transcription or transla betulinic acid; a camptothecin (including the synthetic ana tion. ErbB2 receptor overexpression may be determined in a 55 logue topotecan (HYCAMTINR), CPT-11 (irinotecan, diagnostic or prognostic assay by evaluating increased levels CAMPTOSARR), acetylcamptothecin, scopolectin, and of the ErbB2 protein present on the surface of a cell (e.g., via 9-aminocamptothecin); bryostatin: cally statin: CC-1065 (in an immunohistochemistry assay; IHC). Alternatively, or cluding its adoZelesin, carzelesin and bizelesin synthetic ana additionally, one may measure levels of ErbB2-encoding logues); podophyllotoxin: podophyllinic acid; teniposide; nucleic acid in the cell, e.g., via fluorescent in situ hybridiza 60 cryptophycins (particularly cryptophycin 1 and cryptophycin tion (FISH; see WO 98/45479), southern blotting, or poly 8); dolastatin; duocarmycin (including the synthetic ana merase chain reaction (PCR) techniques, such as real time logues, KW-2189 and CB1-TM1); eleutherobin: pancratista quantitative PCR (RT-PCR). Overexpression of the ErbB2 tin; a sarcodictyin; spongistatin: nitrogen mustards Such as ligand, may be determined diagnostically by evaluating levels chlorambucil, chlornaphazine, cholophosphamide, estra of the ligand (or nucleic acid encoding it) in the patient, e.g., 65 mustine, ifosfamide, mechlorethamine, mechlorethamine in a tumor biopsy or by various diagnostic assays such as the oxide hydrochloride, melphalan, novembichin, phenesterine, IHC, FISH, southern blotting, PCR or in vivo assays prednimustine, trofosfamide, uracil mustard; nitroSureas US 7,994,135 B2 31 32 Such as carmustine, chlorozotocin, fotemustine, lomustine, acceptable salts, acids or derivatives of any of the above; as nimustine, and ranimnustine; bisphosphonates, such as clo well as combinations of two or more of the above such as dronate; antibiotics such as the enediyne antibiotics (e.g., CHOP, an abbreviation for a combined therapy of cyclophos calicheamicin, especially calicheamicin gammal I and cali phamide, doxorubicin, Vincristine, and prednisolone, and cheamicin omegal (see, e.g., Agnew, Chem Intl. Ed. Engl., FOLFOX, an abbreviation for a treatment regimen with oxali 33: 183-186 (1994)) and anthracyclines such as annamycin, platin (ELOXATINTM) combined with 5-FU and leucovorin. AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, Also included in this definition are anti-hormonal agents epirubicin, GPX-100, idarubicin, KRN5500, menogaril, that act to regulate or inhibit hormone action on tumors such dynemicin, including dynemicin A, an esperamicin, neo as anti-estrogens and selective estrogen receptor modulators carzinostatin chromophore and related chromoprotein ene 10 (SERMs), including, for example, tamoxifen (including diyne antiobiotic chromophores, aclacinomysins, actinomy NOLVADEXR tamoxifen), raloxifene, droloxifene, 4-hy cin, authramycin, azaserine, bleomycins, cactinomycin, droxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapris carabicin, caminomycin, carzinophilin, chromomycinis, dac tone, and FARESTON toremifene; aromatase inhibitors that tinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, inhibit the enzyme aromatase, which regulates estrogen pro ADRIAMYCINR) doxorubicin (including morpholino-doxo 15 duction in the adrenal glands, Such as, for example, 4(5)- rubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxoru imidazoles, aminoglutethimide, MEGASE(R) megestrol bicin, liposomal doxorubicin, and deoxydoxorubicin), esoru acetate, AROMASINR exemestane, formestanie, fadrozole, bicin, marcellomycin, mitomycins such as mitomycin C, RIVISOR(R) vorozole, FEMARAR) letrozole, and ARIMI mycophenolic acid, nogalamycin, olivomycins, peplomycin, DEX(R) anastroZole; and anti-androgens such as flutamide, potfiromycin, puromycin, quelamycin, rodorubicin, Strep nilutamide, bicalutamide, leuprolide, and goserelin; as well tonigrin, Streptozocin, tubercidin, ubenimex, Zinostatin, and as troxacitabine (a 1,3-dioxolane nucleoside cytosine ana Zorubicin; folic acid analogues such as denopterin, pterop log); antisense oligonucleotides, particularly those that terin, and trimetrexate; purine analogs such as fludarabine, inhibit expression of genes in signaling pathways implicated 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine in abherant cell proliferation, such as, for example, PKC analogs such as ancitabine, azacitidine, 6-azauridine, carmo 25 alpha, Raf, H-Ras, and epidermal growth factor receptor fur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, (EGF-R(R); vaccines such as gene therapy vaccines, for and floXuridine; androgens Such as calusterone, dromo example, ALLOVECTINR vaccine, LEUVECTINR vac stanolone propionate, epitiostanol, mepitioStane, and testo cine, and VAXIDR vaccine: PROLEUKINR rIL-2: LURTO lactone; anti-adrenals such as aminoglutethimide, mitotane, TECANR) topoisomerase 1 inhibitor; ABARELIX(R) rmRH: and triloStane; folic acid replenisher such as folinic acid (leu 30 and pharmaceutically acceptable salts, acids or derivatives of covorin); aceglatone; anti-folate anti-neoplastic agents such any of the above. as ALIMTAR), LY231514 pemetrexed, dihydrofolate reduc As used herein, the term “EGFR-targeted drug” refers to a tase inhibitors such as methotrexate, anti-metabolites such as therapeutic agent that binds to EGFR and, optionally, inhibits 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-1 and EGFR activation. Examples of such agents include antibodies capecitabine, and thymidylate synthase inhibitors and glyci 35 and small molecules that bind to EGFR. Examples of anti namide ribonucleotide formyltransferase inhibitors such as bodies which bind to EGFR include MAb 579 (ATCC CRL raltitrexed (TOMUDEXR, TDX); inhibitors of dihydropyri HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 midine dehydrogenase such as eniluracil; aldophosphamide (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. glycoside; aminolevulinic acid; amsacrine; bestrabucil; Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, bisantrene; ediatraxate, defofamine; demecolcine; diazi 40 such as chimerized 225 (C225 or Cetuximab; ERBITUX(R) quone; elfornithine; elliptinium acetate; an epothilone; eto and reshaped human 225 (H225) (see, WO 96/40210, glucid: gallium nitrate; hydroxyurea; lentinan; lonidainine; Imclone Systems Inc.); antibodies that bind type II mutant maytansinoids Such as maytansine and ansamitocins; EGFR (U.S. Pat. No. 5.212.290); humanized and chimeric mitoguaZone, mitoxantrone; mopidanmol; nitracrine; pen antibodies that bind EGFR as described in U.S. Pat. No. to statin: phenamet, pirarubicin; losoxantrone, 2-ethylhy 45 5,891,996; and human antibodies that bind EGFR, such as drazide; procarbazine: PSKR polysaccharide complex (JHS ABX-EGF (see WO 98/50433, Abgenix). The anti-EGFR Natural Products, Eugene, Oreg.); razoxane; rhizoxin; siZo antibody may be conjugated with a cyotoxic agent, thus gen furan; Spirogermanium; tenuaZonic acid; triaziquone; 2.2.2"- erating an immunoconjugate (see, e.g., EP 659,439A2. trichlorotriethylamine; trichothecenes (especially T-2 toxin, Merck Patent GmbH). Examples of small molecules that bind Verracurin A, roridin A and anguidine); urethan; vindesine 50 to EGFR include ZD1839 or Gefitinib (IRESSATM; Astra (ELDISINE(R), FILDESINR); dacarbazine; mannomustine: Zeneca), Erlotinib HCl (CP-358774, TARCEVATM: Genen mitobronitol; mitolactol; pipobroman, gacytosine; arabino tech/OSI) and AG1478, AG1571 (SU 5271; Sugen). side (Ara-C); cyclophosphamide; thiotepa; taxoids and tax A “tyrosine kinase inhibitor is a molecule which inhibits anes, e.g., TAXOL(R) paclitaxel (Bristol-Myers Squibb to some extent tyrosine kinase activity of a tyrosine kinase Oncology, Princeton, N.J.), ABRAXANETM Cremophor 55 such as an ErbB receptor. Examples of such inhibitors include free, albumin-engineered nanoparticle formulation of pacli the EGFR-targeted drugs noted in the preceding paragraph as taxel (American Pharmaceutical Partners, Schaumberg, Ill.), well as quinazolines such as PD 153035,4-(3-chloroanilino) and TAXOTERE(R) doxetaxel (Rhône-Poulenc Rorer, Ant quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyr ony, France); chloranbucil; gemcitabine (GEMZARR): rolopyrimidines, such as CGP 59326, CGP 60261 and CGP 6-thioguanine; mercaptopurine; platinum; platinum analogs 60 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyr or platinum-based analogs such as cisplatin, oxaliplatin and rolo2,3-dipyrimidines, curcumin (diferuloyl methane, 4.5- carboplatin: vinblastine (VELBANR): etoposide (VP-16); bis(4-fluoroanilino)phthalimide), tyrphostines containing ifosfamide; mitoxantrone; Vincristine (ONCOVINR); Vinca nitrothiophene moieties; PD-0183805 (Warner-Lambert): alkaloid; vinorelbine (NAVELBINE(R); novantrone; ediatrex antisense molecules (e.g., those that bind to ErbB-encoding ate; daunomycin; aminopterin: Xeloda; ibandronate; topoi 65 nucleic acid); quinoxalines (U.S. Pat. No. 5,804.396); try somerase inhibitor RFS 2000; difluoromethylomithine phostins (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); (DMFO); retinoids such as retinoic acid; pharmaceutically PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such US 7,994,135 B2 33 34 as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly): Imatinib into a prodrug form for use in this invention include, but are mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 not limited to, those chemotherapeutic agents described (Glaxo SmithKline): CI-1033 (Pfizer); EKB-569 (Wyeth); above. Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (No A "liposome' is a small vesicle composed of various types vartis/Schering AG); INC-1C11 (Imclone); or as described in of lipids, phospholipids and/or surfactant which is useful for any of the following patent publications: U.S. Pat. No. 5,804, delivery of a drug (such as including the anti-CD30, CD40, 396; WO 99/09016 (American Cyanamid); WO 98/43960 CD70 or Lewis Y antibodies and, optionally, a chemothera (American Cyanamid); WO 97/38983 (Warner Lambert); peutic agent) to a mammal. The components of the liposome WO 99/06378 (Warner Lambert); WO 99/06396 (Warner are commonly arranged in a bilayer formation, similar to the Lambert); WO 96/30347 (Pfizer, Inc): WO 96/33978 (Zen 10 lipid arrangement of biological membranes. eca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca). The term “package insert” is used to refer to instructions An “anti-angiogenic agent” refers to a compound which customarily included in commercial packages of therapeutic blocks, or interferes with to some degree, the development of products, that contain information about the indications, blood vessels. The anti-angiogenic factor may, for instance, usage, dosage, administration, contraindications and/or be a small molecule or antibody that binds to a growth factor 15 warnings concerning the use of Such therapeutic products. or growth factor receptor involved in promoting angiogen An "isolated nucleic acid molecule is a nucleic acid mol esis. In one embodiment, the anti-angiogenic factor is an ecule that is identified and separated from at least one con antibody that binds to Vascular Endothelial Growth Factor taminant nucleic acid molecule with which it is ordinarily (VEGF). associated in the natural source of the antibody nucleic acid. The term “cytokine' is a generic term for proteins released An isolated nucleic acid molecule is other than in the form or by one cell population which act on another cell as intercel setting in which it is found in nature. Isolated nucleic acid lular mediators. Examples of Such cytokines are lymphok molecules therefore are distinguished from the nucleic acid ines, monokines, and traditional polypeptide hormones. molecule as it exists in natural cells. However, an isolated Included among the cytokines are growth hormone Such as nucleic acid molecule includes a nucleic acid molecule con human growth hormone, N-methionyl human growth hor 25 tained in cells that ordinarily express the antibody where, for mone, and bovine growth hormone; parathyroid hormone; example, the nucleic acid molecule is in a chromosomal loca thyroxine; insulin; proinsulin; relaxin; prorelaxin, glycopro tion different from that of natural cells. tein hormones such as follicle stimulating hormone (FSH), The expression “control sequences’ refers to DNA thyroid stimulating hormone (TSH), and luteinizing hormone sequences necessary for the expression of an operably linked (LH); hepatic growth factor; fibroblast growth factor; prolac 30 coding sequence in a particular host organism. The control tin, placental lactogen; tumor necrosis factor-C. and -3; mul sequences that are Suitable for prokaryotes, for example, lerian-inhibiting substance; mouse gonadotropin-associated include a promoter, optionally an operator sequence, and a peptide; inhibin; activin; vascular endothelial growth factor; ribosome binding site. Eukaryotic cells are known to utilize integrin: thrombopoietin (TPO); nerve growth factors such as promoters, polyadenylation signals, and enhancers. NGF-3; platelet-growth factor; transforming growth factors 35 A nucleic acid is “operably linked when it is placed into a (TGFs) such as TGF-C. and TGF-B; insulin-like growth fac functional relationship with another nucleic acid sequence. tor-I and -II; erythropoietin (EPO); osteoinductive factors: For example, DNA for a presequence or secretory leader is interferons such as interferon-C, -f, and -y, colony Stimulat operably linked to DNA for a polypeptide if it is expressed as ing factors (CSFs) such as macrophage-CSF (M-CSF); a preprotein that participates in the secretion of the polypep granulocyte-macrophage-CSF (GM-CSF); and granulocyte 40 tide; a promoter or enhancer is operably linked to a coding CSF (G-CSF); interleukins (ILS) such as IL-1, IL-1C., IL-2, sequence if it affects the transcription of the sequence; or a IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; ribosome binding site is operably linked to a coding sequence a tumor necrosis factor such as TNF-C. or TNF-B; and other if it is positioned so as to facilitate translation. Generally, polypeptide factors including LIF and kit ligand (KL). As “operably linked' means that the DNA sequences being used herein, the term cytokine includes proteins from natural 45 linked are contiguous, and, in the case of a secretory leader, Sources or from recombinant cell culture and biologically contiguous and in reading phase. However, enhancers do not active equivalents of the native sequence cytokines. have to be contiguous. Linking can be accomplished by liga The term “prodrug” as used in this application refers to a tion at convenient restriction sites. If such sites do not exist, precursor or derivative form of a pharmaceutically active the synthetic oligonucleotide adaptors or linkers can be used Substance that is less cytotoxic to tumor cells compared to the 50 in accordance with conventional practice. parent drug and is capable of being enzymatically or hydro As used herein, the expressions “cell. “cell line.” and “cell lytically activated or converted into the more active parent culture' are used interchangeably and all such designations form. See, e.g., Wilman, “Prodrugs in Cancer Chemo include progeny. Thus, the words “transformants' and “trans therapy” Biochemical Society Transactions, 14, pp. 375-382, formed cells' include the primary subject cell and cultures 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A 55 derived therefrom without regard for the number of transfers. Chemical Approach to Targeted Drug Delivery. Directed It is also understood that all progeny may not be precisely Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana identical in DNA content, due to deliberate or inadvertent Press (1985). The prodrugs of this invention include, but are mutations. Mutant progeny that have the same function or not limited to, phosphate-containing prodrugs, thiophos biological activity as screened for in the originally trans phate-containing prodrugs, Sulfate-containing prodrugs, pep 60 formed cell are included. Where distinct designations are tide-containing prodrugs, D-amino acid-modified prodrugs, intended, it will be clear from the context. glycosylated prodrugs, B-lactam-containing prodrugs, An “autoimmune disease herein is a disease or disorder optionally Substituted phenoxyacetamide-containing pro arising from and directed against an individual’s own tissues drugs or optionally Substituted phenylacetamide-containing or a co-segregate or manifestation thereof or resulting condi prodrugs, 5-fluorocytosine and other 5-fluorouridine pro 65 tion therefrom. Examples of autoimmune diseases or disor drugs which can be converted into the more active cytotoxic ders include, but are not limited to arthritis (rheumatoid free drug. Examples of cytotoxic drugs that can be derivatized arthritis, juvenile rheumatoid arthritis, osteoarthritis, psori US 7,994,135 B2 35 36 atic arthritis, and ankylosing spondylitis), psoriasis, dermati monitis (HIV), bronchiolitis obliterans (non-transplant) vs tis including atopic dermatitis; chronic idiopathic urticaria, NSIP. Guillain-Barré Syndrome, Berger's Disease (IgA including chronic autoimmune urticaria, polymyositis/der nephropathy), primary biliary cirrhosis, celiac sprue (gluten matomyositis, toxic epidermal necrolysis, systemic Sclero enteropathy), refractory sprue with co-segregate dermatitis derma and Sclerosis, responses associated with inflammatory herpetiformis, cryoglobulinemia, amylotrophic lateral scle bowel disease (IBD) (Crohn's disease, ulcerative colitis), and rosis (ALS, Lou Gehrig's disease), coronary artery disease, IBD with co-segregate of pyoderma gangrenosum, erythema autoimmune inner ear disease (AIED), autoimmune hearing nodosum, primary Sclerosing cholangitis, and/or episcleritis), loss, opSoclonus myoclonus syndrome (OMS), polychondri respiratory distress syndrome, including adult respiratory tis Such as refractory polychondritis, pulmonary alveolar pro distress syndrome (ARDS), meningitis, IgE-mediated dis 10 teinosis, amyloidosis, giant cell hepatitis, Scleritis, mono eases such as anaphylaxis and allergic rhinitis, encephalitis clonal gammopathy of uncertain/unknown significance Such as Rasmussen's encephalitis, uveitis, colitis such as (MGUS), peripheral neuropathy, paraneoplastic syndrome, microscopic colitis and collagenous colitis, glomerulonephri channelopathies such as epilepsy, migraine, arrhythmia, mus tis (GN) such as membranous GN, idiopathic membranous cular disorders, deafness, blindness, periodic paralysis, and GN, membranous proliferative GN (MPGN), including Type 15 channelopathies of the CNS, autism, inflammatory myopa I and Type II, and rapidly progressive GN, allergic conditions, thy, and focal segmental glomerulosclerosis (FSGS). eczema, asthma, conditions involving infiltration of T cells Alkyl is C-Cs hydrocarbon containing normal, second and chronic inflammatory responses, atherosclerosis, ary, tertiary or cyclic carbon atoms. Examples are methyl autoimmune myocarditis, leukocyte adhesion deficiency, (Me, —CH), ethyl (Et, —CH2CH), 1-propyl (n-Pr, n-pro systemic lupus erythematosus (SLE) such as cutaneous SLE, pyl, —CH2CH2CH), 2-propyl (i-Pr. i-propyl, -CH(CH)), lupus (including nephritis, cerebritis, pediatric, non-renal, 1-butyl (n-Bu, n-butyl, —CHCHCHCH), 2-methyl-1- discoid, alopecia), juvenile onset diabetes, multiple Sclerosis propyl (i-Bu, i-butyl, —CH2CHCCH)), 2-butyl (s-Bu, s-bu (MS) such as spino-optical MS, allergic encephalomyelitis, tyl, CH(CH)CHCH), 2-methyl-2-propyl (t-Bu, t-butyl, immune responses associated with acute and delayed hyper —C(CH)), 1-pentyl (n-pentyl, —CH2CH2CH2CH2CH), sensitivity mediated by cytokines and T-lymphocytes, tuber 25 2-pentyl ( CH(CH)CHCHCH), 3-pentyl ( CH culosis, sarcoidosis, granulomatosis including Wegener's (CHCH)), 2-methyl-2-butyl ( C(CH). CHCH), 3-me granulomatosis, agranulocytosis, vasculitis (including Large thyl-2-butyl ( CH(CH)CH(CH)), 3-methyl-1-butyl Vessel vasculitis (including Polymyalgia Rheumatica and (—CHCH-CH(CH)), 2-methyl-1-butyl ( CHCHCCH) Giant Cell (Takayasu's) Arteritis), Medium Vessel vasculitis CHCH), 1-hexyl ( CHCH2CH2CH2CHCH), 2-hexyl (including Kawasaki's Disease and Polyarteritis Nodosa), 30 (-CH(CH)CHCHCHCH), 3-hexyl (-CH(CHCH.) CNS vasculitis, and ANCA-associated vasculitis, such as (CHCHCH)), 2-methyl-2-pentyl (—C(CH) Churg-Strauss vasculitis or syndrome (CSS), aplastic ane CHCHCH), 3-methyl-2-pentyl ( CH(CH)CH(CH) mia, Coombs positive anemia, Diamond Blackfan anemia, CHCH), 4-methyl-2-pentyl ( CH(CH)CHCH(CH)), immune hemolytic anemia including autoimmune hemolytic 3-methyl-3-pentyl ( C(CH)(CHCH)), 2-methyl-3-pen anemia (AIHA), pernicious anemia, pure red cell aplasia 35 tyl ( CH(CHCH)CH(CH)), 2,3-dimethyl-2-butyl ( C (PRCA), Factor VIII deficiency, hemophilia A, autoimmune (CH)-CH(CH)), 3.3-dimethyl-2-butyl ( CH(CH)C neutropenia, pancytopenia, leukopenia, diseases involving (CH). leukocyte diapedesis, CNS inflammatory disorders, multiple Alkenyl is C-Cs hydrocarbon containing normal, sec organ injury syndrome, myasthenia gravis, antigen-antibody ondary, tertiary or cyclic carbonatoms with at least one site of complex mediated diseases, anti-glomerular basement mem 40 unsaturation, i.e. a carbon-carbon, sp' double bond. brane disease, anti-phospholipid antibody syndrome, allergic Examples include, but are not limited to: ethylene or vinyl neuritis, Bechet disease, Castleman's syndrome, Goodpas (—CH=CH), allyl ( CH-CH=CH-), cyclopentenyl ture's Syndrome, Lambert-Eaton Myasthenic Syndrome, (—CH7), and 5-hexenyl ( CHCHCHCH-CH=CH-). Reynaud's syndrome, Sorgen’s syndrome, Stevens-Johnson Alkynyl is C-Cs hydrocarbon containing normal, sec syndrome, Solid organ transplant rejection (including pre 45 ondary, tertiary or cyclic carbonatoms with at least one site of treatment for high panel reactive antibody titers, IgA deposit unsaturation, i.e. a carbon-carbon, sp triple bond. Examples in tissues, and rejection arising from renal transplantation, include, but are not limited to: acetylenic ( -C=CH) and liver transplantation, intestinal transplantation, cardiac trans propargyl ( CH-C=CH). plantation, etc.), graft versus host disease (GVHD), pemphig Alkylene' refers to a saturated, branched or straight chain oid bullous, pemphigus (including Vulgaris, foliaceus, and 50 or cyclic hydrocarbon radical of 1-18 carbon atoms, and pemphigus mucus-membrane pemphigoid), autoimmune having two monovalent radical centers derived by the polyendocrinopathies, Reiter's disease, stiff-man syndrome, removal of two hydrogen atoms from the same or two differ immune complex nephritis, IgM polyneuropathies or IgM ent carbonatoms of a parent alkane. Typical alkylene radicals mediated neuropathy, idiopathic thrombocytopenic purpura include, but are not limited to: methylene (—CH2—) 1.2- (ITP), thrombotic throbocytopenic purpura (TTP), thromb 55 ethyl (-CH2CH2—), 1,3-propyl (-CH2CH2CH2—), 1,4- ocytopenia (as developed by myocardial infarction patients, butyl ( CHCHCHCH ), and the like. for example), including autoimmune thrombocytopenia, Alkenylene' refers to an unsaturated, branched or straight autoimmune disease of the testis and ovary including autoim chain or cyclic hydrocarbon radical of 2-18 carbonatoms, and mune orchitis and oophoritis, primary hypothyroidism; having two monovalent radical centers derived by the autoimmune endocrine diseases including autoimmune thy 60 removal of two hydrogen atoms from the same or two differ roiditis, chronic thyroiditis (Hashimoto's Thyroiditis), sub ent carbonatoms of a parent alkene. Typical alkenylene radi acute thyroiditis, idiopathic hypothyroidism, Addison's dis cals include, but are not limited to: 1.2-ethylene ease, Grave's disease, autoimmune polyglandular syndromes (-CH=CH-). (or polyglandular endocrinopathy syndromes). Type I diabe Alkynylene' refers to an unsaturated, branched or straight tes also referred to as insulin-dependent diabetes mellitus 65 chain or cyclic hydrocarbon radical of 2-18 carbonatoms, and (IDDM), including pediatric IDDM, and Sheehan’s syn having two monovalent radical centers derived by the drome; autoimmune hepatitis, Lymphoid interstitial pneu removal of two hydrogen atoms from the same or two differ US 7,994,135 B2 37 38 ent carbon atoms of a parent alkyne. Typical alkynylene radi York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The cals include, but are not limited to: acetylene ( -C=C- ), Chemistry of Heterocyclic Compounds. A series of Mono propargyl (—CH2C=C ), and 4-pentynyl graphs” (John Wiley & Sons, New York, 1950 to present), in ( CHCHCH-C=CH-). particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Aryl means a monovalent aromatic hydrocarbon radical Soc. (1960) 82:5566. of6-20 carbonatoms derived by the removal of one hydrogen Examples of heterocycles include by way of example and atom from a single carbon atom of a parent aromatic ring not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl system. Some aryl groups are represented in the exemplary (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized structures as 'Ar. Typical aryl groups include, but are not tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, limited to, radicals derived from benzene, substituted ben 10 pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphtha Zene, naphthalene, anthracene, biphenyl, and the like. lenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimi Arylalkyl refers to an acyclic alkyl radical in which one dazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolido of the hydrogen atoms bonded to a carbon atom, typically a nyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, terminal or sp carbon atom, is replaced with an aryl radical. tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinoli Typical arylalkyl groups include, but are not limited to, ben 15 Zyl. 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylm nyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahy ethyl 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naph droisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, thobenzyl, 2-naphthophenylethan-1-yl and the like. The 2H,6H-1.5.2-dithiazinyl, thienyl, thianthrenyl, pyranyl, arylalkyl group comprises 6 to 20 carbonatoms, e.g., the alkyl isobenzofuranyl, chromenyl, Xanthenyl, phenoxathinyl, moiety, including alkanyl, alkenyl or alkynyl groups, of the 2H-pyrrolyl, isothiazolyl, isoxazolyl pyrazinyl, pyridazinyl, arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 5 to 14 carbon atoms. 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, “Heteroarylalkyl refers to an acyclic alkyl radical in quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carba which one of the hydrogen atoms bonded to a carbon atom, Zolyl, B-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, typically a terminal or sp carbon atom, is replaced with a 25 phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phe heteroaryl radical. Typical heteroarylalkyl groups include, noxazinyl, isochromanyl, chromanyl, imidazolidinyl, imida but are not limited to, 2-benzimidazolylmethyl 2-furylethyl, Zolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, and the like. The heteroarylalkyl group comprises 6 to 20 isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, ben carbon atoms, e.g., the alkyl moiety, including alkanyl, alk Zotriazolyl, benzisoxazolyl, OXindolyl, benzoxazolinyl, and enyl or alkynyl groups, of the heteroarylalkyl group is 1 to 6 30 isatinoyl. carbon atoms and the heteroaryl moiety is 5 to 14 carbon By way of example and not limitation, carbon bonded atoms and 1 to 3 heteroatoms selected from N, O, P, and S. heterocycles are bonded at position 2, 3, 4, 5, or 6 of a The heteroaryl moiety of the heteroarylalkyl group may be a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, monocycle having 3 to 7 ring members (2 to 6 carbon atoms or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, or a bicycle having 7 to 10 ring members (4 to 9 carbonatoms 35 position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, and 1 to 3 heteroatoms selected from N, O, P, and S), for thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of example: a bicyclo 4.5, 5.5, 5.6, or 6.6 system. an oxazole, imidazole or thiazole, position 3, 4, or 5 of an “Substituted alkyl, “substituted aryl', and “substituted isoxazole, pyrazole, or isothiazole, position 2 or 3 of an arylalkyl mean alkyl, aryl, and arylalkyl respectively, in aziridine, position 2, 3, or 4 of anaZetidine, position 2, 3, 4, 5, which one or more hydrogen atoms are each independently 40 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an replaced with a substituent. Typical substituents include, but isoquinoline. Still more typically, carbon bonded hetero are not limited to, —X, —R, —O, —OR, —SR, —S, cycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, —NR - NR =NR, CX - CN, OCN, - SCN, 6-pyridyl, 3-pyridazinyl, 4-pyridaZinyl, 5-pyridazinyl, 6-py -N=C=O, NCS, NO, NO, —N, N NC(=O) ridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-py R, C(=O)R, C(=O)NR, -SO, SOH, 45 rimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, —S(=O), R, OS(=O),OR, S(=O)NR, -S(=O)R, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl. –OP(=O)(OR), -P(=O)(OR), PO, POH, By way of example and not limitation, nitrogen bonded —C(=O)R, —C(=O)x, —C(=S)R, COR, CO., heterocycles are bonded at position 1 of an aziridine, aZeti —C(=S)OR, C(=O)SR, C(=S)SR, C(=O)NR, dine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imida —C(=S)NR, —C(=NR)NR, where each X is indepen 50 Zole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, dently a halogen: F, Cl, Br, or I; and each R is independently pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, pipera —H, C-C alkyl, Co-Co aryl, C-C heterocycle, protect Zine, indole, indoline, 1H-indazole, position 2 of a isoindole, ing group or prodrug moiety. Alkylene, alkenylene, and alky or isoindoline, position 4 of a morpholine, and position 9 of a nylene groups as described above may also be similarly Sub carbazole, or 3-carboline. Still more typically, nitrogen stituted. 55 bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrro “Heteroaryl and “Heterocycle” refer to a ring system in lyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl. which one or more ring atoms is a heteroatom, e.g., nitrogen, “Carbocycle” means a saturated or unsaturated ring having oxygen, and Sulfur. The heterocycle radical comprises 1 to 20 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms carbon atoms and 1 to 3 heteroatoms selected from N, O, P, as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, and S. A heterocycle may be a monocycle having 3 to 7 ring 60 still more typically 5 or 6 ring atoms. Bicyclic carbocycles members (2 to 6 carbonatoms and 1 to 3 heteroatoms selected have 7 to 12 ring atoms, e.g., arranged as a bicyclo4.5, 5.5, from N, O, P, and S) or a bicycle having 7 to 10 ring members 5.6 or 6.6 system, or 9 or 10 ring atoms arranged as a (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N. bicyclo 5.6 or 6.6 system. Examples of monocyclic car O, P, and S), for example: a bicyclo4.5, 5.5, 5.6, or 6.6 bocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cy system. 65 clopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, Heterocycles are described in Paquette, Leo A.; “Principles cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclo of Modern Heterocyclic Chemistry” (W. A. Benjamin, New hex-3-enyl, cycloheptyl, and cyclooctyl. US 7,994,135 B2 39 40 "Linker”, “Linker Unit', or “link' means a chemical moi groups include, but are not limited to, -methyl, -ethyl, -n- ety comprising a covalent bond or a chain of atoms that propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n- covalently attaches an antibody to a drug moiety. In various nonyl and -n-decyl; while branched C-C alkyls include, but embodiments, a linker is specified as LU. Linkers include a are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-bu divalent radical Such as an alkyldiyl, an aryldiyl, a het- 5 tyl, -isopentyl, 2-methylbutyl, unsaturated C-C alkyls eroaryldiyl, moieties such as: —(CR), O(CR), , repeating include, but are not limited to, -vinyl, -allyl, -1-butenyl, units of alkyloxy (e.g., polyethylenoxy, PEG, polymethyl -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-me eneoxy) and alkylamino (e.g., polyethyleneamino, Jeffam thyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-bute ineTM); and diacid ester and amides including Succinate, suc nyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-bu cinamide, diglycolate, malonate, and caproamide. 10 tynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 The term "chiral refers to molecules which have the prop butynyl. methyl, ethyl, propyl, isopropyl. n-butyl, isobutyl, erty of non-Superimposability of the mirror image partner, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, while the term “achiral” refers to molecules which are super imposable on their mirror image partner. isohexyl, 2-methylpentyl, 3-methylpenty1, 2,2-dimethylbu The term “stereoisomers' refers to compounds which have 15 tyl, 2,3-dimethylbutyl, 2,2-dimethylpenty1, 2,3-dimethylpen identical chemical constitution, but differ with regard to the tyl, 3.3-dimethylpenty1, 2,3,4-trimethylpentyl, 3-methyl arrangement of the atoms or groups in space. hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2.5- “Diastereomer' refers to a stereoisomer with two or more dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, centers of chirality and whose molecules are not mirror 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, images of one another. Diastereomers have different physical 20 and isooctyl. A C-C alkyl group can be unsubstituted or properties, e.g., melting points, boiling points, spectral prop Substituted with one or more groups including, but not limited erties, and reactivities. Mixtures of diastereomers may sepa to. —C-Cs alkyl, —O—(C-C alkyl), -aryl, —C(O)R', rate under high resolution analytical procedures such as elec OC(O)R', C(O)OR, C(O)NH, C(O)NHR'. trophoresis and chromatography. C(O)N(R'). NHC(O)R', SOR', S(O).R', S(O)R', "Enantiomers' refer to two stereoisomers of a compound 25 —OH, -halogen, —N, NH, NH(R'), —N(R') and which are non-Superimposable mirror images of one another. —CN; where each R" is independently selected from H. —C- Stereochemical definitions and conventions used herein Cs alkyl and aryl. generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of A "C-C carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered Chemical Terms (1984) McGraw-Hill Book Company, New saturated or unsaturated non-aromatic carbocyclic ring. Rep York; and Eliel, E. and Wilen, S., Stereochemistry of Organic 30 resentative C-Cs carbocycles include, but are not limited to, Compounds (1994) John Wiley & Sons, Inc., New York. -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, Many organic compounds exist in optically active forms, i.e., -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cy they have the ability to rotate the plane of plane-polarized clohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5- light. In describing an optically active compound, the prefixes cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A D and L, or R and S. are used to denote the absolute configu- 35 C-Cs carbocycle group can be unsubstituted or Substituted ration of the molecule about its chiral center(s). The prefixes with one or more groups including, but not limited to. —C- d and 1 or (+) and (-) are employed to designate the sign of Cs alkyl, —O (C-C alkyl), -aryl, —C(O)R', —OC(O)R', rotation of plane-polarized light by the compound, with (-) or C(O)OR, C(O)NH, C(O)NHR', C(O)N(R'), 1 meaning that the compound is levorotatory. A compound NHC(O)R', S(O).R', S(O)R', -OH, -halogen, N. prefixed with (+) or d is dextrorotatory. For a given chemical 40 NH, -NH(R'), N(R'), and –CN; where each R is structure, these stereoisomers are identical except that they independently selected from H. —C-Cs alkyl and aryl. are mirror images of one another. A specific stereoisomer may A "C-Cs carbocyclo” refers to a C-C carbocycle group also be referred to as an enantiomer, and a mixture of Such defined above wherein one of the carbocycle groups hydro isomers is often called an enantiomeric mixture. A 50:50 gen atoms is replaced with a bond. mixture of enantiomers is referred to as a racemic mixture or 45 A "C-Clo alkylene' is a straight chain, saturated hydro a racemate, which may occur where there has been no stereo carbon group of the formula —(CH) . Examples of a selection or stereospecificity in a chemical reaction or pro C-C alkylene include methylene, ethylene, propylene, cess. The terms “racemic mixture' and “racemate” refer to an butylene, pentylene, hexylene, heptylene, ocytylene, non equimolar mixture of two enantiomeric species, devoid of ylene and decalene. optical activity. 50 An “arylene' is an aryl group which has two covalent Examples of a “patient' include, but are not limited to, a bonds and can be in the ortho, meta, or para configurations as human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, shown in the following structures: dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human. Aryl refers to a carbocyclic aromatic group. Examples of 55 aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a hetero cyclic aromatic group can be unsubstituted or Substituted with one or more groups including, but not limited to. —C- Cs alkyl, —O—(C-C alkyl), -aryl, —C(O)R', —OC(O)R', 60 O. C. -C(O)OR, C(O)NH, C(O)NHR', C(O)N(R'), NHC(O)R', S(O).R', S(O)R', -OH, -halogen, N. NH, -NH(R'), N(R'), and CN; wherein each R" is independently selected from H. —C-Cs alkyl and aryl. The term "C-C alkyl, as used herein refers to a straight 65 chain or branched, Saturated or unsaturated hydrocarbon hav ing from 1 to 8 carbon atoms. Representative “C-C alkyl US 7,994,135 B2 41 42 in which the phenyl group can be unsubstituted or substituted plary salts include, but are not limited, to Sulfate, citrate, with up to four groups including, but not limited to. —C-Cs acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, alkyl, —O—(C-C alkyl), -aryl, —C(O)R', —OC(O)R', phosphate, acid phosphate, isonicotinate, lactate, Salicylate, C(O)CR', C(O)NH, C(O)NHR', C(O)N(R') acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, NHC(O)R', S(O).R', S(O)R', -OH, -halogen, N. ascorbate. Succinate, maleate, gentisinate, fumarate, glucon - NH, -NH(R'), N(R'), and CN; wherein each R" is ate, glucuronate, Saccharate, formate, benzoate, glutamate, independently selected from H. —C-C alkyl and aryl. methanesulfonate, ethanesulfonate, benzenesulfonate, A "C-Cs heterocycle” refers to an aromatic or non-aro p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2- matic C-C carbocycle in which one to four of the ring hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable carbon atoms are independently replaced with a heteroatom 10 salt may involve the inclusion of another molecule Such as an from the group consisting of O, S and N. Representative acetate ion, a Succinate ion or other counterion. The counte examples of a C-Cs heterocycle include, but are not limited rion may be any organic or inorganic moiety that stabilizes the to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, charge on the parent compound. Furthermore, a pharmaceu coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thia tically acceptable salt may have more than one charged atom Zolyl, imidazolyl pyrazolyl, triazolyl, quinolinyl, pyrimidi 15 in its structure. Instances where multiple charged atoms are nyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, part of the pharmaceutically acceptable salt can have multiple isoxazolyl and tetrazolyl. A C-Cs heterocycle can be unsub counter ions. Hence, a pharmaceutically acceptable salt can stituted or Substituted with up to seven groups including, but have one or more charged atoms and/or one or more counte not limited to. —C-Cs alkyl, —O—(C-C alkyl), -aryl, 1O. C(O)R', OC(O)R', C(O)OR', C(O)NH, C(O) "Pharmaceutically acceptable solvate” or “solvate” refer to NHR', C(O)N(R'). NHC(O)R', S(O),R, S(O)R', an association of one or more solvent molecules and a com —OH, -halogen, - N - NH, NH(R'), —N(R') and pound of the invention, e.g., an Exemplary Compound or —CN; wherein each R" is independently selected from H. Exemplary Conjugate. Examples of Solvents that form phar —C-Cs alkyl and aryl. maceutically acceptable solvates include, but are not limited "C-Cs heterocyclo” refers to a C-Cs heterocycle group 25 to, water, isopropanol, ethanol, methanol, DMSO, ethyl defined above wherein one of the heterocycle group’s hydro acetate, acetic acid, and ethanolamine. gen atoms is replaced with a bond. A C-Cs heterocyclo can The following abbreviations are used herein and have the be unsubstituted or substituted with up to six groups includ indicated definitions: AE is auristatin E. Boc is N-(t-butoxy ing, but not limited to. —C-Cs alkyl, -O-(C-Cs alkyl). carbonyl), cit is citrulline, dap is dollaproine, DCC is 1,3- -aryl, -C(O)R', OC(O)R', C(O)OR', C(O)NH, 30 dicyclohexylcarbodiimide, DCM is dichloromethane, DEA C(O)NHR', C(O)N(R'). NHC(O)R', S(O).R', is diethylamine, DEAD is diethylazodicarboxylate, DEPC is —S(O)R', -OH, -halogen, - N - NH, -NH(R'), diethylphosphorylcyanidate, DIAD is diisopropylazodicar —N(R'), and —CN; wherein each R" is independently boxylate, DIEA is N,N-diisopropylethylamine, dil is dolai selected from H. —C-Cs alkyl and aryl. soleuine, DMAP is 4-dimethylaminopyridine, DME is ethyl An “Exemplary Compound is a Drug Compound or a 35 eneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is Drug-Linker Compound. N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe An "Exemplary Conjugate' is a Drug-Ligand Conjugate is dollaphenine, dov is N,N-dimethylvaline, DTNB is 5,5'- having a cleavable Drug unit from the Drug-Ligand Conju dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriamine gate or a Drug-Linker-Ligand Conjugate. pentaacetic acid, DTT is dithiothreitol, EDCI is 1-(3-dim In some embodiments, the Exemplary Compounds and 40 ethylaminopropyl)-3-ethylcarbodiimide hydrochloride, Exemplary Conjugates are in isolated or purified form. As EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, used herein, "isolated” means separated from other compo ES-MS is electrospray mass spectrometry, EtOAc is ethyl nents of (a) a natural source. Such as a plant or animal cell or acetate, Fmoc is N-(9-fluorenylmethoxycarbonyl), gly is gly cell culture, or (b) a synthetic organic chemical reaction mix cine, HATU is O-(7-azabenzotriazol-1-yl)-N.N.N',N'-tet ture. As used herein, “purified’ means that when isolated, the 45 ramethyluronium hexafluorophosphate, HOBt is 1-hydroxy isolate contains at least 95%, and in another aspect at least benzotriazole, HPLC is high pressure liquid chromatography, 98%, of Exemplary Compound or Exemplary Conjugate by ile is isoleucine, lys is lysine, MeCN(CHCN) is acetonitrile, weight of the isolate. MeOH is methanol. Mtr is 4-anisyldiphenylmethyl (or Examples of a “hydroxyl protecting group' include, but are 4-methoxytrity1), nor is (1S,2R)-(+)-norephedrine, PAB is not limited to, methoxymethyl ether, 2-methoxyethoxym 50 p-aminobenzyl, PBS is phosphate-buffered saline (pH 7.4), ethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxy PEG is polyethylene glycol, Phis phenyl, Pnp is p-nitrophe benzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopro nyl, MC is 6-maleimidocaproyl phe is L-phenylalanine, pyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl PyBrop is bromo tris-pyrrolidino phosphonium hexafluoro silyl ether, acetate ester, Substituted acetate esters, pivaloate, phosphate, SEC is size-exclusion chromatography, Su is suc benzoate, methanesulfonate and p-toluenesulfonate. 55 cinimide, TBTU is O-benzotriazol-1-yl-N,N.N.N-tetram "Leaving group' refers to a functional group that can be ethyluronium tetrafluoroborate, TFA is trifluoroacetic acid, Substituted by another functional group. Such leaving groups TLC is thin layer chromatography, UV is ultraviolet, and val are well known in the art, and examples include, but are not is valine. limited to, a halide (e.g., chloride, bromide, iodide), methane The following linker abbreviations are used herein and sulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethyl 60 have the indicated definitions: Val Cit is a valine-citrulline, sulfonyl (triflate), and trifluoromethylsulfonate. dipeptide site in protease cleavable linker; PAB is p-ami The phrase “pharmaceutically acceptable salt, as used nobenzylcarbamoyl (Me)vc is N-methyl-valine citrulline, herein, refers to pharmaceutically acceptable organic or inor where the linker peptide bond has been modified to prevent its ganic salts of an Exemplary Compound or Exemplary Con cleavage by cathepsin B; MC(PEG)6-OH is maleimidoca jugate. The Exemplary Compounds and Exemplary Conju 65 proyl-polyethylene glycol; SPP is N-Succinimidyl 4-(2-py gates contain at least one amino group, and accordingly acid ridylthio)pentanoate; and SMCC is N-Succinimidyl 4-(N- addition salts can be formed with this amino group. Exem maleimidomethyl)cyclohexane-1 carboxylate. US 7,994,135 B2 43 44 The terms “treat' or “treatment, unless otherwise indi- 4.2 The Compounds of the Invention cated by context, refer to both therapeutic treatment and pro- 4.2.1 The Compounds of Formula (Ia) phylactic or preventative measures, wherein the object is to In one aspect, the invention provides Drug-Linker-Ligand prevent or slow down (lessen) an undesired physiological Conjugates having Formula Ia: change or disorder, such as the development or spread of 5 Ia cancer. For purposes of this invention, beneficial or desired L-A-Ww-Yy-D), clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease or a pharmaceutically acceptable salt or Solvate thereof 10 wherein, progression, amelioration or palliation of the disease state, L- is a Ligand unit; and remission (whether partial or total), whether detectable or -A-W, Y, is a Linker unit (LU), wherein the Linker undetectable. "Treatment can also mean prolonging Survival unit includes: as compared to expected Survival if not receiving treatment. -A- is a Stretcher unit, Those in need of treatment include those already with the 15 a is 0 or 1, condition O disorder aS well aS those prone to have the each —W - is independently an Amino Acid unit, condition or disorder or those in which the condition or dis- w is an integer ranging from 0 to 12, order is to be prevented. —Y— is a Spacer unit, and In the context of cancer, the term “treating includes any or y is 0, 1 or 2: all of preventing growth of tumor cells, cancer cells, or of a 20 p ranges from 1 to about 20; and tumor, preventing replication of tumor cells or cancer cells, -D is a Drug unit having the Formulas D, and D.

DE R3 O R7 CH R' H N N N N Nin R18

R2 R. R. R6 R8 O R8 O DF R3 O R7 CH3 R9 O A. N 1. N N N 71 R11 R2 R. R. R6 R8 O R8 O R10 lessening of overall tumor burden or decreasing the number wherein, independently at each location: of cancerous cells, and ameliorating one or more symptoms " R’ is selected from Hand C-Cs alkyl: associated with the disease. R is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, In the context of an autoimmune disease, the term “treat- E.l S.SE) C-Cs het ing includes any or all of preventing replication of cells R" is selected from H, C, -Cs alkyl, C-Cs carbocycle, aryl, associated with an autoimmune disease state including, but 45 C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C.-Chet not limited to, cells that produce an autoimmune antibody, erocycle and C-C alkyl-(C-Cs heterocycle); lessening the autoimmune-antibody burden and ameliorating R is selected from Hand methyl: one or more symptoms of an autoimmune disease. or R and Rjointly form a carbocyclic ring and have the formula –(CRR), wherein R* and Rare independently In the context of an infectious disease, the term “treating so selected from H. C-Cs alkyl and C-Cs carbocycle and n is includes any or all of preventing the growth, multiplication selected from 2, 3, 4, 5 and 6: or replication of the pathogen that causes the infectious dis- R is selected from Hand C-Cs alkyl: ease and ameliorating one or more symptoms of an infectious R’ is selected from H.C.-Cs alkyl, Cs-Cs carbocycle, aryl, disease. C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het erocycle and C-C alkyl-(C-Cs heterocycle); The following cytotoxic drug abbreviations are used herein 55 each R is independently selected from H, OH, C, -Cs and have the indicated definitions: MMAE is mono-methyl alkyl, C-Cs carbocycle and O—(C-C alkyl); auristatin E (MW 718); MMAF is N-methylvaline-valine- R is selected from Hand C-Cs alkyl: dolaisoleuine-dollaproine-phenylalanine (MW 731.5); R" is selected from aryl or heterocycle. MMAF-DMAEA is MMAF with DMAEA (dimethylamino- 2 is O.S.Nfor NRT wherein R is CC alkyl, ethylamine) in an amide linkage to the C-terminal phenyla- 60 R'' is selected from H, C-C alkyl, aryl, C-C hetero lanine (MW 801.5); MMAF-TEG is MMAF with tetraethyl- cycle, -(RO), R'', or -(RO), CH(R'); ene glycol esterified to the phenylalanine: MMAF-NtEu is m is an integer ranging from 1-1000; N-t-butyl, attached as an amide to C-terminus of MMAF: R" is C-Cs alkyl: AEVB is auristatin E Valeryl benzylhydrazone, acid labile R" is H or C-C alkyl: linker through the C-terminus of AE (MW 732); and AFP is 65 each occurrence of R' is independently H, COOH, Monoamide of p-phenylene diamine with C-terminal Pheny- -(CH2), N(R'). -(CH2), SOH, or -(CH2), lalanine of Auristatin F (MW 732). SO C-C alkyl, US 7,994,135 B2 45 46 each occurrence of R' is independently H, C-C alkyl, or R'' is Hor—C-Cs alkyl; -(CH), COOH: each occurrence of R' is independently H, -COOH, R" is selected from C(R), C(R)-aryl, -C(R) - -(CH2), N(R'). -(CH2), SOH, or -(CH2), C(R), (C-Cs heterocycle), and C(R), C(R) (C- SO C-C alkyl, Cs carbocycle); and 5 16 n is an integer ranging from 0 to 6. each occurrence of R' is independently H. —C-C alkyl, In another embodiment, the present invention provides or —(CH), COOH; and Drug Compounds having the Formula Ib: n is an integer ranging from 0 to 6.

Ib R3 O R7 CH3 R9 O N N R11 I N 71 R2 R. R. k R8 O R8 O Ib R10 or pharmaceutically acceptable salts or Solvates thereof, * In yet another embodiment, the invention provides Drug wherein: Linker-Ligand Conjugates having the Formula la': R is selected from hydrogen and —C-Cs alkyl; R is selected from hydrogen. —C-Cs alkyl, -C-Cs car bocycle, aryl, -C-Cs alkyl-aryl, —C-C alkyl-(C-Cs car- Formula Ia' bocycle), —C-Cs heterocycle and —C-Cs alkyl-(C-Cs 25 Ab-e-A-W-Y-D), heterocycle); R" is selected from hydrogen, —C-Cs alkyl, -C-Cs car- O preutically acceptable salts or solvates thereof. bocycle, -aryl, -C-C alkyl-aryl. —C-C alkyl-(C-C car- Ab is an antibody, bocycle), C-Cs heterocycle and —C-Cs alkyl-(C-Cs A is a Stretcher unit, heterocycle) wherein R is selected from Hand-methyl; or a is 0 or 1, RandR jointly, have the formula—(CRR), wherein R' each W is independently an Amino Acid unit, and Rare independently selected from -H, -C-Cs alkyl w is an integer ranging from 0 to 12, and —C-Cs carbocycle and n is selected from 2, 3, 4, 5 and Y is a Spacer unit, and 6, and form a ring with the carbon atom to which they are y is 0, 1 or 2, attached; p ranges from 1 to about 20, and R is selected from Hand-C-Cs alkyl: D is a Drug moiety selected from Formulas D, and D.

DE R3 O R7 CH3 R9 A N 1. s N 1Sr. N N NRIs R2 R.4\, R. R6 R8 O R8 O DF R3 O R7 CH3 R9 O A N 1. s N 1Sr. N N 71 R11 R2 R.4\, R. R6 R8 O R8 O R10

R’ is selected from H. —C-Cs alkyl, -C-Cs carbocycle, ss wherein, independently at each location: aryl, -C-C alkyl-aryl, -C-C alkyl-(C-C carbocycle), R’ is selected from Hand C-C alkyl: —C-Cs heterocycle and—C-Cs alkyl-(C-Cs heterocycle); R is selected from H.C.-Cs alkyl, Cs-Cs carbocycle, aryl, each R is independently selected from H. -OH,-C-Cs. C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C-C het alkyl, -C-Cs carbocycle and —O—(C-C alkyl); erocycle and C-C alkyl-(C-Cs heterocycle); R is selected from Hand-C-Cs alkyl: 60 R is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, R" is selected from aryl group or -C-Cs heterocycle; C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C-C het Zis-O-, -S-, -NH , or - NR' , wherein R'' is erocycle and C-C alkyl-(C-Cs heterocycle); C-C alkyl; R is selected from Hand methyl: R'' is selected from H, C, -Coalkyl, aryl, -C-Cs hetero- or R and Rjointly form a carbocyclic ring and have the cycle, -(RO), R'', or—(RO), CH(R): 65 formula—(CRR), wherein RandR are independently m is an integer ranging from 1-1000; selected from H. C-C alkyl and C-Cs carbocycle and n is R" is —C-Cs alkyl: selected from 2, 3, 4, 5 and 6: US 7,994,135 B2 47 48 R is selected from Hand C-Cs alkyl: (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/ R’ is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, Epstein Barr virus receptor) or Hs.73792, Genbank accession C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het no. M26004); erocycle and C-C alkyl-(C-Cs heterocycle); (15) CD79b (IGb (immunoglobulin-associated beta), B29, each R is independently selected from H, OH, C-Cs. Genbank accession no. NM 000626); alkyl, C-Cs carbocycle and O—(C-C alkyl); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain con R is selected from Hand C-Cs alkyl: taining phosphatase anchor protein 1a), SPAP1B, SPAP1C, R' is selected from aryl or C-C heterocycle: Genbank accession no. NM 030764); Z is O, S, NH, or NR, wherein R' is C-C alkyl: (17) HER2 (Genbank accession no. M11730): 10 (18) NCA (Genbank accession no. M18728); R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero (19) MDP (Genbank accession no. BC017023): cycle, -(RO), R', or—(RO), CH(R'); (20) IL20RC (Genbank accession no. AF 184971); m is an integer ranging from 1-1000; (21) Brevican (Genbank accession no. AF229053); R" is C-Cs alkyl: (22) Ephb2R (Genbank accession no. NM 004442): R" is Hor C-C alkyl: 15 (23) ASLG659 (Genbank accession no. AX092328); each occurrence of R' is independently H, COOH, (24) PSCA (Genbank accession no. AJ297436): -(CH2), N(R'). -(CH2), SOH, or -(CH2), (25) GEDA (Genbank accession no. AY260763); SO-C-C alkyl; (26) BAFF-R (Genbank accession no. NP 443177.1): each occurrence of R' is independently H, C-C alkyl, or (27) CD22 (Genbank accession no. NP-001762. 1); —(CH), COOH: (28) CD79a (CD79A, CD79a, immunoglobulin-associated R" is selected from C(R), C(Rs.)-aryl, C(R) - alpha, a B cell-specific protein that covalently interacts with C(R), (C-Cs heterocycle), and C(R), C(R) (C- Igbeta (CD79B) and forms a complex on the surface with Ig Cs carbocycle); and M molecules, transduces a signal involved in B-cell differen n is an integer ranging from 0 to 6. tiation, Genbank accession No. NP 001774.1); Ab is any antibody covalently attached to one or more drug 25 (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein units. Ab includes an antibody which binds to CD30, CD40, coupled receptor that is activated by the CXCL13 chemokine, CD70, Lewis Yantigen. In another embodiment, Ab does not functions in lymphocyte migration and humoral defense, include an antibody which binds to an ErbB receptor or to one plays a role in HIV-2 infection and perhaps development of or more of receptors AIDS, lymphoma, myeloma, and leukemia, Genbank acces (1)–(35): 30 sion No. NP 001707.1): (1) BMPR1B (bone morphogenetic protein receptor-type (30) HLA-DOB (Beta subunit of MHC class II molecule IB, Genbank accession no. NM 001203); (Ia antigen) that binds peptides and presents them to CD4+ T (2) E 16 (LAT1, SLC7A5, Genbank accession no. lymphocytes, Genbank accession No. NP 002111.1); NM 003.486); (31) P2X5 (Purinergic receptor P2X ligand-gated ion (3) STEAP1 (six transmembrane epithelial antigen of pros 35 channel 5, an ion channel gated by extracellular ATP may be tate, Genbank accession no. NM 012449); involved in synaptic transmission and neurogenesis, defi (4) 0772P (CA125, MUC16, Genbank accession no. ciency may contribute to the pathophysiology of idiopathic AF361486); detrusor instability, Genbank accession No. NP 002552.2): (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, factor, mesothelin, Genbank accession no. NM 005823); 40 Genbank accession No. NP 001773.1): (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier (33) LY64 (Lymphocyte antigen 64 (RP 105), type I mem family 34 (sodium phosphate), member 2, type II sodium brane protein of the leucine rich repeat (LRR) family, regu dependent phosphate transporter 3b, Genbank accession no. lates B-cell activation and apoptosis, loss of function is asso NM 006424); ciated with increased disease activity in patients with (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, 45 systemic lupus erythematosis, Genbank accession No. SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, NP 005573.1); seven thrombospondin repeats (type 1 and type 1-like), trans (34) FCRH1 (Fc receptor-like protein 1, a putative receptor membrane domain (TM) and short cytoplasmic domain, for the immunoglobulin Fc domain that contains C2 type (semaphorin) 5B, Genbank accession no. AB040878); Ig-like and ITAM domains, may have a role in B-lymphocyte (8) PSCA hlg (2700050C12Rik, C530008O16Rik, 50 differentiation, Genbank accession No. NP 443170.1); and/ RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 O gene, Genbank accession no. AY358628); (35) IRTA2 (Immunoglobulin superfamily receptor trans (9) ETBR (Endothelin type B receptor, Genbank accession location associated 2, a putative immunoreceptor with pos no. AY275463); sible roles in B cell development and lymphomagenesis; (10) MSG783 (RNF124, hypothetical protein FLJ20315, 55 deregulation of the gene by translocation occurs in Some B Genbank accession no. NM 017763); cell malignancies, Genbank accession No. NP 112571.1). (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, In one embodiment —Ww- is -Val-Cit-. STAMP1, STEAP2, STMP prostate cancer associated gene In another embodiment, R, R and Rare independently 1, prostate cancer associated protein 1, six transmembrane isopropyl or sec-butyl and R is —H. In an exemplary epithelial antigen of prostate 2, six transmembrane prostate 60 embodiment, Rand Rare eachisopropyl, R is -H, and R' protein, Genbank accession no. AF455138): is sec-butyl. In yet another embodiment, Rand Rare each (12) TrpM4 (BR224.50, FLJ20041, TRPM4, TRPM4B, methyl, and R is H. transient receptor potential cation channel, Subfamily M. In still another embodiment, each occurrence of R is member 4, Genbank accession no. NM 017636); –OCH. (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, tera 65 In an exemplary embodiment, Rand Rare each isopro tocarcinoma-derived growth factor, Genbank accession no. pyl, R and Rare each methyl, R is H. R7 is sec-butyl, NP 003203 or NM 003212); each occurrence of R is —OCH, and R is H. US 7,994,135 B2 49 50 In one embodiment, Z is —O— or —NH-. wherein L is an antibody, Val is valine, and Cit is citrulline. In one embodiment, R' is aryl The drug loading is represented by p, the average number In an exemplary embodiment, R' is -phenyl. of drug molecules per antibody in a molecule (e.g., of For mula Ia, Ia' and Ic). Drug loading may range from 1 to 20 In an exemplary embodiment, when Z is O—, R' is drugs (D) per Ligand (e.g., Ab or mAb). Compositions of —H. methyl or t-butyl. Formula Ia and Formula Ia' include collections of antibodies In one embodiment, when Z is NH, R'' is —CH(R'), conjugated with a range of drugs, from 1 to 20. The average wherein R' is —(CH), N(R'), and R' is C-C alkyl number of drugs per antibody in preparation of conjugation or —(CH), COOH. reactions may be characterized by conventional means Such In another embodiment, when Z is NH, R'' is —CH as mass spectroscopy, ELISA assay, and HPLC. The quanti tative distribution of Ligand-Drug-Conjugates in terms of p (R), wherein R' is —(CH), SOH. 10 may also be determined. In some instances, separation, puri In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, fication, and characterization of homogeneous Ligand-Drug hAC10, hBR96, hS2C6, h1F6, and h2F2. conjugates where p is a certain value from Ligand-Drug Exemplary embodiments of Formula Ia have the following Conjugates with other drug loadings may be achieved by Structures: means such as reverse phase HPLC or electrophoresis.

O O H Ab { O O Oul N NA. N Ol NH O On O N Wal-Cit-N On O H O OH p O L-MC-vic-PAB-MMAF

O O OH -- O O o-s, N,7.1N Ol H N Wal-Cit-N | O- O O O H n p O L-MC-vc-PAB-MMAE

-- O O O N N, H ~s NPrio,, N N OH O O O O N O N O 'C p Or L-MC-MMAE

O O H N N, H N N N

O O O O

O OH p L-MC-MMAF US 7,994,135 B2 51 52 4.2.2 The Drug Compounds of Formula (Ib) In another aspect, the present invention provides Drug Compounds having the Formula (Ib):

Ib R3 O R7 CH R9 O H HN N Z

Rl, O R4 R5 R6 R8 O R8 O Ib R10

or a pharmaceutically acceptable salt or Solvate thereof, R'' is Hor—C-Cs alkyl; wherein: each occurrence of R' is independently —H, -COOH, R is selected from -hydrogen and —C-Cs alkyl; -(CH2), N(R'). -(CH2), SOH, or -(CH2), R is selected from -hydrogen, —C-Cs alkyl, -C-Cs SO C-C alkyl, carbocycle, aryl. —C-C alkyl-aryl, -C-C alkyl-(C-Cs each occurrence of R is independently —H. —C-Cs. carbocycle), —C-Cs heterocycle and —C-Cs alkyl-(C-Cs alkyl, or —(CH), COOH; and heterocycle); n is an integer ranging from 0 to 6. R" is selected from -hydrogen, —C-Cs alkyl, -C-Cs In one embodiment, R, R and Rare independently iso carbocycle, -aryl, -C-Cs alkyl-aryl, -C-Cs alkyl-(C-Cs 25 propyl or sec-butyl and R is H. In an exemplary embodi carbocycle), —C-Cs heterocycle and —C-Cs alkyl-(C-Cs ment, R and R are each isopropyl, R is —H, and R7 is heterocycle) wherein R is selected from-Hand-methyl; or sec-butyl. RandR jointly, have the formula—(CRR), wherein R In another embodiment, RandR are each methyl, and R' and Rare independently selected from -H, -C-Cs alkyl is —H. and —C-C carbocycle and n is selected from 2, 3, 4, 5 and 30 In still another embodiment, each occurrence of R is 6, and form a ring with the carbon atom to which they are —OCH. attached; In an exemplary embodiment, Rand Rare each isopro R is selected from Hand-C-Cs alkyl: pyl, R and Rare each methyl, R is H. R7 is sec-butyl, R’ is selected from -H, -C-Cs alkyl, -C-Cs car 35 each occurrence of R is —OCH, and R is H. bocycle, aryl, -C-C alkyl-aryl, -C-C alkyl-(C-C car In one embodiment, Z is —O— or —NH-. bocycle), —C-Cs heterocycle and —C-Cs alkyl-(C-Cs In one embodiment, R' is aryl heterocycle); In an exemplary embodiment, R' is -phenyl. each R is independently selected from -H, -OH, -C- In an exemplary embodiment, when Z is —O , R'' is Cs alkyl, -C-Cs carbocycle and —O—(C-C alkyl); —H. methyl or t-butyl. R is selected from Hand-C-Cs alkyl: 40 In one embodiment, when Z is NH, R'' is —CH(R'), R" is selected from aryl group or —Cs-Cs heterocycle: wherein Ris-(CH2), N(R'), and R' is C-C alkyl Zis-O-, - S - NH-, or - NR' , wherein R' is or —(CH), COOH. C-Cs alkyl; In another embodiment, when Z is NH, R'' is —CH R'' is selected from -H, C-C alkyl, aryl, -C-Cs het (R'), wherein R's is —(CH), SOH. erocycle, -(RO), R'', or -(RO), CH(R'); 45 Illustrative Compounds of Formula (Ib), each of which m is an integer ranging from 1-1000; may be used as drug moieties (D) in ADC, include com R" is C-C alkyl: pounds having the following structures:

1001 US 7,994,135 B2 54 -continued

N N OCH O

2. P OCH O

N N OCH O

N COOH, US 7,994,135 B2

-continued 8 O H No, H HN A. N N N

O O O s N O O N O

SOH 9 O H No, H HN ? N N N

O O O N O O N O NH

r COOH, and 10 O H No, H HN N N N

O O O

N On O O ?oNH

NH2 and pharmaceutically acceptable salts or Solvates thereof. (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating The Compounds of Formula (Ic) 45 factor, mesothelin, Genbank accession no. NM 005823); In another aspect, the invention provides antibody-drug (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier conjugate compounds (ADC) having Formula Ic: family 34 (sodium phosphate), member 2, type II sodium dependent phosphate transporter 3b, Genbank accession no. NM 006424); Ic 50 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, Ab-e-A-Ww-Yy-D), SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), trans comprising an antibody covalently attached to one or more membrane domain (TM) and short cytoplasmic domain, drug units (moieties). The antibody-drug conjugate com (semaphorin) 5B, Genbank accession no. AB040878); pounds include pharmaceutically acceptable salts or Solvates 55 (8) PSCA hlg (2700050C12Rik, C530008O16Rik, thereof. RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 Formula Ic compounds are defined wherein: gene, Genbank accession no. AY358628); Ab is an antibody which binds to one or more tumor (9) ETBR (Endothelin type B receptor, Genbank accession associated antigen receptors (1)–(35): no. AY275463); (1) BMPR1B (bone morphogenetic protein receptor-type 60 IB, Genbank accession no. NM 001203); (10) MSG783 (RNF124, hypothetical protein FLJ20315, (2) E 16 (LAT1, SLC7A5, Genbank accession no. Genbank accession no. NM 017763); NM 003.486); (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, (3) STEAP1 (six transmembrane epithelial antigen of pros STAMP1, STEAP2, STMP prostate cancer associated gene tate, Genbank accession no. NM 012449); 65 1, prostate cancer associated protein 1, six transmembrane (4) 0772P (CA125, MUC16, Genbank accession no. epithelial antigen of prostate 2, six transmembrane prostate AF361486); protein, Genbank accession no. AF455138): US 7,994,135 B2 57 58 (12) TrpM4 (BR224.50, FLJ20041, TRPM4, TRPM4B, lates B-cell activation and apoptosis, loss of function is asso transient receptor potential cation channel, Subfamily M. ciated with increased disease activity in patients with member 4, Genbank accession no. NM 017636); systemic lupus erythematosis, Genbank accession No. (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, tera NP 005573.1); tocarcinoma-derived growth factor, Genbank accession no. (34) FCRH1 (Fc receptor-like protein 1, a putative receptor NP 003203 or NM 003212); for the immunoglobulin Fc domain that contains C2 type (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/ Ig-like and ITAM domains, may have a role in B-lymphocyte Epstein Barr virus receptor) or Hs.73792 Genbank accession differentiation, Genbank accession No. NP 443170.1); and no. M26004); (35) IRTA2 (Immunoglobulin superfamily receptor trans (15) CD79b (CD79B, CD79|B, IGb (immunoglobulin-as 10 location associated 2, a putative immunoreceptor with pos sociated beta), B29, Genbank accession no. NM 000626); sible roles in B cell development and lymphomagenesis; (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain con taining phosphatase anchor protein 1a), SPAP1B, SPAP1C, deregulation of the gene by translocation occurs in Some B Genbank accession no. NM 030764); cell malignancies, Genbank accession No. NP 112571.1). (17) HER2 (Genbank accession no. M11730): 15 A is a Stretcher unit, (18) NCA (Genbank accession no. M18728); a is 0 or 1, (19) MDP (Genbank accession no. BC017023); each W is independently an Amino Acid unit, (20) IL20RC (Genbank accession no. AF184971); w is an integer ranging from 0 to 12, (21) Brevican (Genbank accession no. AF229053); Y is a Spacer unit, and (22) Ephb2R (Genbank accession no. NM 004442): y is 0, 1 or 2, (23) ASLG659 (Genbank accession no. AX092328); p ranges from 1 to about 8, and (24) PSCA (Genbank accession no. AJ297436): D is a Drug moiety selected from Formulas D, and D.

DE R3 O R7 CH3 H N N NN N N R18 R2 R.4\, R. R6 R8 O R8 O

R3 O R7 CH H N N N 1 N N Z R2 R.4\, R. R6 R8 O R8 O

(25) GEDA (Genbank accession no. AY260763: 40 wherein the wavy line of D, and D, indicates the covalent (26) BAFF-R (B cell-activating factor receptor, BLyS attachment site to A. W. or Y, and independently at each receptor 3, BR3, NP 443177.1); location: (27) CD22 (B-cell receptor CD22-B isoform, R’ is selected from Hand C-Cs alkyl: NP 001762. 1); R is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, (28) CD79a (CD79A, CD79C, immunoglobulin-associ 45 ated alpha, a B cell-specific protein that covalently interacts C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het with Ig beta (CD79B) and forms a complex on the surface erocycle and C-C alkyl-(C-Cs heterocycle); with IgM molecules, transduces a signal involved in B-cell R" is selected from H. C-C alkyl, C-C carbocycle, aryl, differentiation, Genbank accession No. NP 001774.1): C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein 50 erocycle and C-C alkyl-(C-Cs heterocycle); coupled receptor that is activated by the CXCL13 chemokine, R is selected from Hand methyl: functions in lymphocyte migration and humoral defense, or R and Rjointly form a carbocyclic ring and have the plays a role in HIV-2 infection and perhaps development of formula—(CRR), wherein RandR are independently AIDS, lymphoma, myeloma, and leukemia, Genbank acces selected from H. C-C alkyl and C-C carbocycle and n is sion No. NP 001707.1): 55 selected from 2, 3, 4, 5 and 6: (30) HLA-DOB (Beta subunit of MHC class II molecule R is selected from Hand C-C alkyl: (Ia antigen) that binds peptides and presents them to CD4+ T R’ is selected from H.C.-Cs alkyl, Cs-Cs carbocycle, aryl, lymphocytes, Genbank accession No. NP 002111.1); C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het (31) P2X5 (Purinergic receptor P2X ligand-gated ion erocycle and C-C alkyl-(C-Cs heterocycle); channel 5, an ion channel gated by extracellular ATP may be 60 each R is independently selected from H, OH, C, -Cs involved in Synaptic transmission and neurogenesis, defi alkyl, C-C carbocycle and O—(C-C alkyl); ciency may contribute to the pathophysiology of idiopathic R is selected from Hand C-Cs alkyl: detrusor instability, Genbank accession No. NP 002552.2): R" is selected from aryl or C-C heterocycle: (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Z is O, S, NH, or NR'', wherein R'' is C-C alkyl: Genbank accession No. NP 001773.1): 65 R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero (33) LY64 (Lymphocyte antigen 64 (RP 105), type I mem cycle, -(RO), R', or -(RO), CH(R'); brane protein of the leucine rich repeat (LRR) family, regu m is an integer ranging from 1-1000; US 7,994,135 B2 59 60 R" is C-Cs alkyl: In an exemplary embodiment, when Z is —O , R'' is R" is Hor C-C alkyl: —H. methyl or t-butyl. each occurrence of R' is independently H, COOH, In one embodiment, when Z is NH, R'' is —CH(R'), -(CH2), N(R'). -(CH2), SOH, or -(CH2), wherein R' is —(CH), N(R'), and R' is C-C alkyl SO-C-C alkyl; 5 each occurrence of R' is independently H, C-C alkyl, or or —(CH), COOH. —(CH), COOH: In another embodiment, when Z is NH, R'' is —CH R" is selected from C(R), C(R)-aryl, C(R) - (R'), wherein R' is —(CH), SOH. C(R), (C-Cs heterocycle), and C(R), C(R) (C- Exemplary embodiments of Formula Ic ADC have the Cs carbocycle); and following structures:

O O

Ab-S O O O l N A. N N NH | | N-a--- Wal-Cit-N n O O H n O OH p O Ab-MC-vic-PAB-MMAF O O H OH Ab { O O -- N NA. N Ol N ~- Wal-Cit- N O O- O O- O p O Ab-MC-vic-PAB-MMAE

Ab S O O O N ~. N, Ol H OH O O O N O On O p

Ab-MC-MMAE

Ab S O O O H N N, H N N N

O O O

O N O-N O O 1 OH O p Ab-MC-MMAF

50 n is an integer ranging from 0 to 6. wherein Ab is an antibody which binds to one or more tumor In one embodiment —Ww- is -Val-Cit-. associated antigen receptors (1)–(35); Val is valine; and Cit is In another embodiment, R, R and R7 are independently citruline. isopropyl or sec-butyl and R is —H. In an exemplary The drug loading is represented by p, the average number 55 of drugs per antibody in a molecule of Formula I. Drug embodiment, Rand Rare eachisopropyl, Ris-H, and R' loading may range from 1 to 20 drugs (D) per antibody (Ab or is sec-butyl. mAb). Compositions of ADC of Formula I include collec In yet another embodiment, RandR are each methyl, and tions of antibodies conjugated with a range of drugs, from 1 to R is H. 20. The average number of drugs per antibody in preparations In still another embodiment, each occurrence of R is 60 of ADC from conjugation reactions may be characterized by –OCH. conventional means such as UV/visible spectroscopy, mass In an exemplary embodiment, RandR are each isopro spectrometry, ELISA assay, and HPLC. The quantitative dis pyl, R and Rare each methyl, R is H. R7 is sec-butyl, tribution of ADC in terms of p may also be determined. In each occurrence of R is —OCH, and R is H. Some instances, separation, purification, and characterization In one embodiment, Z is —O— or —NH-. 65 of homogeneous ADC where p is a certain value from ADC In one embodiment, R' is aryl. with other drug loadings may be achieved by means Such as In an exemplary embodiment, R' is -phenyl. reverse phase HPLC or electrophoresis. US 7,994,135 B2 61 62 For some antibody drug conjugates, p may be limited by —Y— is a Spacer unit; and the number of attachment sites on the antibody. For example, y is 0, 1 or 2. where the attachment is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or sev In the Drug-Linker-Ligand Conjugate, the Linker is eral cysteine thiol groups, or may have only one or several 5 capable of linking the Drug moiety and the Ligand unit. Sufficiently reactive thiol groups through which a linker may 4.3.1 The Stretcher Unit be attached. The Stretcher unit (-A-), when present, is capable of link Typically, fewer than the theoretical maximum of drug ing a Ligand unit to an amino acid unit (—W ). In this moieties are conjugated to an antibody during a conjugation regard a Ligand (L) has a functional group that can form a reaction. An antibody may contain, for example, many lysine 10 residues that do not react with the drug-linker intermediate or bond with a functional group of a Stretcher. Useful functional linker reagent. Only the most reactive lysine groups may react groups that can be present on a ligand, either naturally or via with an amine-reactive linker reagent. Generally, antibodies chemical manipulation include, but are not limited to, Sulf do not contain many, if any, free and reactive cysteine thiol hydryl ( -SH), amino, hydroxyl, carboxy, the anomeric groups which may be linked to a drug moiety. Most cysteine 15 hydroxyl group of a carbohydrate, and carboxyl. In one thiol residues in the antibodies of the compounds of the inven aspect, the Ligand functional groups are sulfhydryl and tion exist as disulfide bridges and must be reduced with a amino. Sulfhydryl groups can be generated by reduction of an reducing agent such as dithiothreitol (DTT). Additionally, the intramolecular disulfide bond of a Ligand. Alternatively, Sulf antibody must be subjected to denaturing conditions to reveal hydryl groups can be generated by reaction of an amino group reactive nucleophilic groups such as lysine or cysteine. The of a lysine moiety of a Ligand using2-iminothiolane (Traut’s loading (drug/antibody ratio) of an ADC may be controlled in reagent) or another sulfhydryl generating reagent. several different manners, including: (i) limiting the molar In one embodiment, the Stretcher unit forms a bond with a excess of drug-linkerintermediate or linker reagent relative to sulfur atom of the Ligand unit. The sulfur atom can be derived antibody, (ii) limiting the conjugation reaction time or tem from a sulfhydryl group of a Ligand. Representative Stretcher perature, and (iii) partial or limiting reductive conditions for 25 cysteine thiol modification. units of this embodiment are depicted within the square It is to be understood that where more than one nucleo brackets of Formulas IIIa and IIIb, wherein L-, —W , philic group reacts with a drug-linker intermediate, or linker - Y -, -D, wandy are as defined above, and R'' is selected reagent followed by drug moiety reagent, then the resulting from —C-Co alkylene-, -C-Cs carbocyclo-, -O-(C- product is a mixture of ADC compounds with a distribution of 30 Cs alkyl)-, -arylene-, -C-Cio alkylene-arylene-, -arylene one or more drug moieties attached to an antibody. The aver C-Co alkylene-, -C-Cio alkylene-(C-Cs carbocyclo)-. age number of drugs per antibody may be calculated from the —(C-C carbocyclo)-C-Cio alkylene-, -C-Cs heterocy mixture by dual ELISA antibody assay, specific for antibody clo-, -C-Cio alkylene-(C-Cs heterocyclo)-, —(C-Cs het and specific for the drug. Individual ADC molecules may be erocyclo)-C-Cio alkylene-, -(CH2CH2O), , and identified in the mixture by mass spectroscopy, and separated 35 —(CH2CH2O), CH ; and r is an integer ranging from by HPLC, e.g., hydrophobic interaction chromatography 1-10. It is to be understood from all the exemplary embodi (“Effect of drug loading on the pharmacology, pharmacoki ments of Formula Ia, such as III-VI, that even where not netics, and toxicity of an anti-CD30 antibody-drug conju denoted expressly, from 1 to 20 drug moieties are linked to a gate'. Hamblett, K. J., et al. Abstract No. 624, American Ligand (p=1-20). Association for Cancer Research; 2004 Annual Meeting, 40 Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; “Controlling the Location of Drug Attachment IIIa in Antibody-Drug Conjugates'. Alley, S. C., et al. Abstract No. 627, American Association for Cancer Research; 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the 45 AACR, Volume 45, March 2004). Thus, a homogeneous ADC with a single loading value may be isolated from the conju gation mixture by electrophoresis or chromatography. 4.3 The Linker Unit A “Linker unit (LU) is a bifunctional compound which 50 can be used to link a Drug unit and an Ligand unit to form Drug-Linker-Ligand Conjugates, or which are useful in the formation of immunoconjugates directed against tumor asso ciated antigens. Such immunoconjugates allow the selective An illustrative Stretcher unit is that of Formula IIIa delivery of toxic drugs to tumor cells. In one embodiment, the 55 wherein R'' is —(CH.) : Linker unit of the Drug-Linker Compound and Drug-Linker Ligand Conjugate has the formula:

-A-W-Y- 60 wherein: -A- is a Stretcher unit; h:-- a is 0 or 1; 65 each —W - is independently an Amino Acid unit; Another illustrative Stretcher unit is that of Formula IIIa w is independently an integer ranging from 0 to 12; wherein R'' is —(CH2CH2O),p CH ; and r is 2: US 7,994,135 B2 63

5 ~~~~\O O

Still another illustrative Stretcher unit is that of Formula 10 IIIb wherein R'' is —(CH.) : 4.3.2 The Amino Acid Unit The Amino Acid unit ( W ), when present, links the 15 Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug moiety if the Spacer unit is absent, and links the Ligand unit to the Drug unit if the /-...--A Stretcher unit and Spacer unit are absent. W – is a dipeptide, tripeptide, tetrapeptide, pentapeptide, In another embodiment, the Stretcher unit is linked to the hexapeptide, heptapeptide, octapeptide, nonapeptide, Ligand unit via a disulfide bond between a sulfur atom of the decapeptide, undecapeptide or dodecapeptide unit. Each Ligand unit and a sulfur atom of the Stretcher unit. A repre —W unit independently has the formula denoted below in sentative Stretcher unit of this embodiment is depicted within the square brackets, and w is an integer ranging from 0 to 12: the square brackets of Formula IV, wherein R'', L-, - W , 25 —Y—, -D, w and y are as defined above.

30 R19 In yet another embodiment, the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of a Ligand. Example of 35 wherein R' is hydrogen, methyl, isopropyl, isobutyl, sec these reactive sites include, but are not limited to, activated butyl, benzyl, p-hydroxybenzyl, —CHOH, -CH(OH)CH, esters such as Succinimide esters, 4-nitrophenyl esters, pen - CHCH-SCH, -CHCONH, -CHCOOH, tafluorophenyl esters, tetrafluorophenyl esters, anhydrides, - CH-CHCONH, CHCHCOOH, -(CH)NHC acid chlorides, Sulfonyl chlorides, isocyanates and isothiocy (—NH)NH, -(CH)NH, -(CH)NHCOCH, anates. Representative Stretcher units of this embodiment are 40 —(CH), NHCHO, -(CH)NHC(=NH)NH, -(CH), depicted within the square brackets of Formulas Va and Vb, NH, -(CH2)NHCOCH, -(CH)NHCHO, -(CH), wherein -R' , L-, -W-, -Y-, -D, w and y are as NHCONH, -(CH.)NHCONH, CHCH-CH(OH) defined above; CH-NH 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylm ethyl-, phenyl, cyclohexyl, 45

In yet another aspect, the reactive group of the Stretcher contains a reactive site that is reactive to a modified carbohy 55 drates (—CHO) group that can be present on a Ligand. For example, a carbohydrate can be mildly oxidized using a reagent Such as sodium periodate and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality Such as a hydrazide, an 60 oXime, a primary or secondary amine, a hydrazine, a thi osemicarbazone, a hydrazine carboxylate, and an arylhy drazide such as those described by Kaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. Representative Stretcher units of this embodiment are depicted within the square brackets of 65 Formulas Vla, VIb, and VIc, wherein - R'7 L-, - W , —Y—, -D, w and y are as defined above. Acco US 7,994,135 B2 65 66 -continued (IX) O R2 O R23 s 5 H H s N N N N H H R20 O R22 O Hi-Q H. O. 10 wherein R. R. R’ and Rare as follows: R20 R21 R22 R23 The Amino Acid unit can be enzymatically cleaved by one 15 or more enzymes, including a tumor-associated protease, to H benzyl isobutyl H; and liberate the Drug unit (-D), which in one embodiment is methyl isobutyl methyl isobutyl. protonated in vivo upon release to provide a Drug (D). Illus trative W units are represented by formulas (VII)-(IX): Exemplary Amino Acid units include, but are not limited to, units of formula (VII) where: R is benzyl and R is (VII) —(CH)NH; R isopropyl and R is -(CH)NH; R' O R21 isopropyl and R is —(CH2)NHCONH2. Another exem H N 25 plary Amino Acid unit is a unit of formula (VIII) wherein R' N is benzyl, R is benzyl, and R’ is —(CH)NH. Useful —W - units can be designed and optimized in R20 O their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease. In one wherein R' and R'' are as follows: 30 embodiment, a - W - unit is that whose cleavage is cata lyzed by cathepsin B, C and D, or a plasmin protease. In one embodiment, —W - is a dipeptide, tripeptide, R20 R21 tetrapeptide or pentapeptide. benzyl (CH2)NH2: methyl (CH2)NH2: 35 When R. R. R. R’ or R’ is other than hydrogen, the isopropyl (CH2)NH2: carbonatom to which R', R. R. R’ or R is attached is isopropyl (CH)NHCONH2: chiral. benzyl (CH)NHCONH2: isobutyl (CH)NHCONH: Each carbon atom to which R, R2, R, R or R is sec-butyl (CH)NHCONH2: attached is independently in the (S) or (R) configuration. 40 (CH)NHCONH2: In one aspect of the Amino Acid unit, the Amino Acid unit is valine-citrulline. In another aspect, the Amino Acid unit is phenylalanine-lysine (i.e. flk). In yet another aspect of the Amino Acid unit, the Amino Acid unit is N-methylvaline " N 'O H 45 citrulline. In yet another aspect, the Amino Acid unit is 5-ami novaleric acid, homo phenylalanine lysine, tetraisoquinolin benzyl methyl; and ecarboxylate lysine, cyclohexylalanine lysine, isonepecotic benzyl (CH)NHC(=NH)NH2: acid lysine, beta-alanine lysine, glycine serine Valine glutamine and isonepecotic acid. 50 In certain embodiments, the Amino Acid unit can comprise (VIII) natural amino acids. In other embodiments, the Amino Acid O R21 O unit can comprise non-natural amino acids. H H 4.3.3 The Spacer Unit N 55 vs. H 1sy The Spacer unit ( Y—), when present, links an Amino R20 O R22 Acid unit to the Drug moiety when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug moiety when the Amino Acid unit is absent. The wherein R, R and Rare as follows: Spacer unit also links the Drug moiety to the Ligand unit 60 when both the Amino Acid unit and Stretcher unit are absent. R20 R21 R22 Spacer units are of two general types: Self-immolative and non self-immolative. A non self-immolative Spacer unit is benzyl benzyl (CH2)NH2: isopropyl benzyl (CH2)NH2; and one in which part orall of the Spacer unit remains bound to the H benzyl (CH2)NH2: 65 Drug moiety after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immo US 7,994,135 B2 67 68 lative Spacer unit include, but are not limited to a (glycine In one aspect, Spacer units ( Y, ) are represented by glycine) Spacer unit and a glycine Spacer unit (both depicted Formulas (X) —(XII): in FIG. 20) (infra). When an Exemplary Compound contain ing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated protease, a cancer-cell-associated protease or a lymphocyte associated protease, a glycine-glycine-Drug moiety or a gly cine-Drug moiety is cleaved from L-A-W-. In one embodiment, an independent hydrolysis reaction takes place within the target cell, cleaving the glycine—Drug moiety 10 bond and liberating the Drug. --- In another embodiment, - Y - is a p-aminobenzyl alco hol (PAB) unit (see FIGS. 21 and 22) whose phenylene por tion is substituted with Q, wherein Q is —C-C alkyl, C-X —O—(C-C alkyl), -halogen, -nitro or -cyano; and m is an 15 integer ranging from 0-4. In one embodiment, a non self-immolative Spacer unit ( Y—) is -Gly-Gly-. In another embodiment, a non self immolative the Spacer unit ( Y—) is -Gly-. In one embodiment, a Drug-Linker Compound or a Drug Linker Ligand Conjugate is provided in which the Spacer unit wherein Q is —C-Cs alkyl, —O—(C-Cs alkyl), -halogen, is absent (y–0), or a pharmaceutically acceptable salt or Sol -nitro or -cyano; and m is an integer ranging from 0-4. vate thereof. Alternatively, an Exemplary Compound containing a self immolative Spacer unit can release-D without the need for a 25 XI separate hydrolysis step. In this embodiment, Y—is a PAB group that is linked to —W - via the amino nitrogen atom HN-CH-CO and of the PAB group, and connected directly to -D via a carbon ate, carbamate or ether group. Without being bound by any particular theory or mechanism, FIG. 21 depicts a possible 30 mechanism of Drug release of a PAB group which is attached directly to -D via a carbamate or carbonate group espoused by Toki et al. (2002) J Org. Chem. 67:1866-1872. In FIG. 21 Q is —C-Cs alkyl, —O—(C-C alkyl), -halo gen, -nitro or -cyano; m is an integer ranging from 0-4; and p 35 XII ranges from 1 to about 20. Without being bound by any particular theory or mecha nism, FIG. 22 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via an ether or amine linkage. 40 In FIG. 22 Q is —C-Cs alkyl, —O—(C-C alkyl), -halo Embodiments of the Formula Ia" and Ic antibody-drug con gen, -nitro or -cyano; m is an integer ranging from 0-4; and p jugate compounds include: ranges from 1 to about 20. Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically 45 O similar to the PAB group Such as 2-aminoimidazol-5-metha nol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. O 9:2237) and ortho or para-aminobenzylacetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, Ab--S N-N-Nu-W-- and, 50 Such as Substituted and unsubstituted 4-aminobutyric acid O amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), p appropriately substituted bicyclo[2.2.1 and bicyclo[2.2.2 ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94. 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine 55 containing drugs that are substituted at the a-position of gly cine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacer useful in Exemplary Compounds. O In one embodiment, the Spacer unit is a branched bis 60 O (hydroxymethyl)styrene (BHMS) unit as depicted in FIG. 23. which can be used to incorporate and release multiple drugs. Ab-S N---- In FIG. 23 Q is C-C alkyl, —O (C-C alkyl), -halo O gen, -nitro or -cyano; m is an integer ranging from 0-4, n is 0 p or 1; and p ranges raging from 1 to about 20. 65 In one embodiment, the -D moieties are the same. In yet another embodiment, the -D moieties are different. wherein w and y are each 0. US 7,994, 135 B2 69 70

Ab-HA-N N su- Y-D H O r p : HN 1. NH2 O O O

Ab-HS N--- su-y- D O H O E ? p and HN 1. NH2 XO O O O O

Ab S N--- Nulls O H O E H ? p HN 1. NH

4.4 The Drug Unit (Moiety) D is a Drug unit (moiety) having a nitrogen atom that can The drug moiety (D) of the antibody drug conjugates form a bond with the Spacer unit when y=1 or 2, with the (ADC) are of the dolastatin/auristatin type (U.S. Pat. Nos. C-terminal carboxyl group of an Amino Acid unit when y=0. 5,635,483; 5,780.588) which have been shown to interfere 45 with microtubule dynamics, GTP hydrolysis, and nuclear and with the carboxyl group of a Stretcher unit when w and y=0, cellular division (Woyke et al. (2001) Antimicrob. Agents and and with the carboxyl group of a Drug unit when a, w, and Chemother. 45(12):3580-3584) and have anticancer (U.S. y–0. It is to be understood that the terms “drug unit” and “drug Pat. No. 5,663,149) and antifungalactivity (Pettitetal. (1998) moiety' are synonymous and used interchangeably herein. Antimicrob. Agents Chemother. 42:2961-2965) In one embodiment. -D is either formula D, or D:

DE R3 O R7 CH R' l N n R 8 R2 ) R' R 6 R8 O R8 O DF R3 O R7 CH R' O H N N N R11 Z 1.

R2 R. R. R.6 R8 O R8 O R10 US 7,994,135 B2 71 72 wherein, independently at each location: each occurrence of R' is independently H, C-Cs alkyl, or R is selected from Hand C-C alkyl: —(CH), COOH: R is selected from H, C, -Cs alkyl, C-Cs carbocycle, aryl, R" is selected from C(R) C(R)-aryl, —C(R) - C-C alkyl-aryl, C-C alkyl-(C-C carbocycle), C-C het erocycle and C-C alkyl-(C-Cs heterocycle); C(R), (C-Cs heterocycle), and C(R), C(R) (C- R is selected from H. C-C alkyl, C-C carbocycle, aryl, Cs carbocycle); and C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het n is an integer ranging from 0 to 6. erocycle and C-C alkyl-(C-C heterocycle); In one embodiment, R, R and Rare independently iso R is selected from Hand methyl: propyl or sec-butyl and R is —H. In an exemplary embodi or R' and Rjointly form a carbocyclic ring and have the ment, Rand Rare each isopropyl, R is H, and R is sec formula—(CRR), wherein RandR are independently 10 butyl. selected from H. C-C alkyl and C-Cs carbocycle and n is In another embodiment, RandR are each methyl, and R' selected from 2, 3, 4, 5 and 6: is H. R is selected from Hand C-Cs alkyl: In still another embodiment, each occurrence of R is R’ is selected from H.C.-Cs alkyl, C-Cs carbocycle, aryl, –OCH. C-Cs alkyl-aryl, C-C alkyl-(C-Cs carbocycle), C-Cs het 15 In an exemplary embodiment, Rand Rare each isopro erocycle and C-C alkyl-(C-Cs heterocycle); pyl, R and Rare each methyl, R is H. R. is sec-butyl, each each R is independently selected from H, OH, C-Cs occurrence of R is —OCH, and R is H. alkyl, C-Cs carbocycle and O—(C-C alkyl); In one embodiment, Z is —O— or —NH-. R is selected from Hand C-C alkyl: In one embodiment, R' is aryl R" is selected from aryl or C-Cs heterocycle; In an exemplary embodiment, R' is -phenyl. Z is O, S, NH, or NR", wherein R' is C-C alkyl: In an exemplary embodiment, when Z is —O R'' is H. R'' is selected from H, C, -Co alkyl, aryl, C-Cs hetero methyl or t-butyl. cycle, -(RO), R'', or—(RO), CH(R'); In one embodiment, when Z is NH, R'' is —CH(R'), m is an integer ranging from 1-1000; 25 wherein R' is —(CH), N(R'), and R' is —C-Cs alkyl R" is C-Cs alkyl: or —(CH), COOH. R" is Hor C-C alkyl: In another embodiment, when Z is NH, R'' is —CH each occurrence of R' is independently H, COOH, (R'), wherein R' is —(CH), SOH. —(CH), N(R') —(CH), SOH, or -(CH2), Illustrative Drug units (-D) include the drug units having SO C-C alkyl; the following structures:

O OH H H N, N N /rO ON O On O 'C MMAE US 7,994,135 B2 73 74 -continued O H N N N

O O O N O N O O 1 NH O

O H N, N w, N N O N N. O OCH O H OCH O O

O H N., H N N N

O O O

N ON O O 1 O

1 N'N US 7,994,135 B2 75 76 -continued

H 2. N, O H A O O O Ol N O N O O 1 NH O

and pharmaceutically acceptable salts or Solvates thereof. In yet another embodiment, the Ligand can have one or In one aspect, hydrophilic groups, such as but not limited to more carbohydrate groups that can be oxidized to provide an triethylene glycol esters (TEG), as shown above, can be aldehyde (-CHO) group (see, for e.g., LaguZZa, et al., J. attached to the Drug Unit at R''. Without being bound by Med. Chem. 1989, 32(3), 548-55). The corresponding alde theory, the hydrophilic groups assist in the internalization and hyde can form a bond with a Reactive Site on a Stretcher. non-agglomeration of the Drug Unit. Reactive sites on a Stretcher that can react with a carbonyl 4.5 The Ligand Unit group on a Ligand include, but are not limited to, hydrazine The Ligand unit (L-) includes within its scope any unit of a 25 and hydroxylamine. Other protocols for the modification of Ligand (L) that binds or reactively associates or complexes proteins for the attachment or association of Drug Units are described in Coligan et al., Current Protocols in Protein with a receptor, antigen or other receptive moiety associated Science, vol. 2, John Wiley & Sons (2002), incorporated with a given target-cell population. A Ligand is a molecule herein by reference. that binds to, complexes with, or reacts with a moiety of a cell 30 Useful non-immunoreactive protein, polypeptide, or pep population sought to be therapeutically or otherwise biologi tide Ligands include, but are not limited to, transferrin, epi cally modified. In one aspect, the Ligand unit acts to deliver dermal growth factors ("EGF), bombesin, gastrin, gastrin the Drug unit to the particular target cell population with releasing peptide, platelet-derived growth factor, IL-2, IL-6, which the Ligand unit reacts. Such Ligands include, but are transforming growth factors (“TGF'), such as TGF-C. and not limited to, large molecular weight proteins such as, for 35 TGF-B, vaccinia growth factor (“VGF'), insulin and insulin example, full-length antibodies, antibody fragments, Smaller like growth factors I and II, lectins and apoprotein from low molecular weight proteins, polypeptide or peptides, lectins, density lipoprotein. glycoproteins, non-peptides, vitamins, nutrient-transport Useful polyclonal antibodies are heterogeneous popula molecules (such as, but not limited to, transferring, or any tions of antibody molecules derived from the sera of immu other cell binding molecule or Substance. 40 nized animals. Various procedures well known in the art may A Ligand unit can formabond to a Stretcher unit, an Amino be used for the production of polyclonal antibodies to an Acid unit, a Spacer Unit, or a Drug Unit. A Ligand unit can antigen-of-interest. For example, for the production of poly form a bond to a Linker unit via a heteroatom of the Ligand. clonal antibodies, various host animals can be immunized by Heteroatoms that may be present on a Ligand unit include injection with an antigen of interest or derivative thereof, Sulfur (in one embodiment, from a sulfhydryl group of a 45 including but not limited to rabbits, mice, rats, and guinea Ligand), oxygen (in one embodiment, from a carbonyl, car pigs. Various adjuvants may be used to increase the immuno boxyl or hydroxyl group of a Ligand) and nitrogen (in one logical response, depending on the host species, and includ embodiment, from a primary or secondary amino group of a ing but not limited to Freunds (complete and incomplete) Ligand). These heteroatoms can be present on the Ligand in adjuvant, mineral gels such as aluminum hydroxide, Surface the Ligand’s natural state, for example a naturally-occurring 50 active Substances Such as lysolecithin, pluronic polyols, antibody, or can be introduced into the Ligand via chemical polyanions, peptides, oil emulsions, keyhole limpet modification. hemocyanins, dinitrophenol, and potentially useful human In one embodiment, a Ligand has a sulfhydryl group and adjuvants such as BCG (bacille Calmette-Guerin) and the Ligand bonds to the Linker unit via the sulfhydryl group's corynebacterium parvum. Such adjuvants are also well Sulfur atom. 55 known in the art. In yet another aspect, the Ligand has one or more lysine Useful monoclonal antibodies are homogeneous popula residues that can be chemically modified to introduce one or tions of antibodies to a particular antigenic determinant (e.g., more Sulfhydryl groups. The Ligand unit bonds to the Linker a cancer cell antigen, a viral antigen, a microbial antigen, a unit via the Sulfhydryl group's Sulfur atom. The reagents that protein, a peptide, a carbohydrate, a chemical, nucleic acid, or can be used to modify lysines include, but are not limited to, 60 fragments thereof). A monoclonal antibody (mAb) to an anti N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothi gen-of-interest can be prepared by using any technique olane hydrochloride (Traut’s Reagent). known in the art which provides for the production of anti In another embodiment, the Ligand can have one or more body molecules by continuous cell lines in culture. These carbohydrate groups that can be chemically modified to have include, but are not limited to, the hybridoma technique origi one or more Sulfhydryl groups. The Ligand unit bonds to the 65 nally described by Köhler and Milstein (1975, Nature 256, Linker Unit, such as the Stretcher Unit, via the sulfhydryl 495-497), the human B cell hybridoma technique (Kozbor et group's Sulfur atom. al., 1983, Immunology Today 4: 72), and the EBV-hybridoma US 7,994,135 B2 77 78 technique (Cole et al., 1985, Monoclonal Antibodies and Bifunctional antibodies are also described, in European Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such anti Patent Publication No. EPA O 105 360. As disclosed in this bodies may be of any immunoglobulin class including IgG, reference, hybrid or bifunctional antibodies can be derived IgM, IgE, IgA, and Ig) and any Subclass thereof. The hybri either biologically, i.e., by cell fusion techniques, or chemi doma producing the mabs of use in this invention may be cally, especially with cross-linking agents or disulfide-bridge cultivated in vitro or in vivo. forming reagents, and may comprise whole antibodies or Useful monoclonal antibodies include, but are not limited fragments thereof. Methods for obtaining such hybrid anti to, human monoclonal antibodies, humanized monoclonal bodies are disclosed for example, in International Publication antibodies, antibody fragments, or chimeric human-mouse WO 83/03679, and European Patent Publication No. EPA 0 (or other species) monoclonal antibodies. Human mono 10 217 577, both of which are incorporated herein by reference. clonal antibodies may be made by any of numerous tech The antibody can be a functionally active fragment, deriva niques known in the art (e.g., Teng et al., 1983, Proc. Natl. tive or analog of an antibody that immunospecifically binds to Acad. Sci. USA. 80, 7308-7312: Kozbor et al., 1983, Immu cancer cell antigens, viral antigens, or microbial antigens or nology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzy other antibodies bound to tumor cells or matrix. In this regard, mol. 92, 3-16). 15 “functionally active” means that the fragment, derivative or The antibody can also be a bispecificantibody. Methods for analog is able to elicit anti-anti-idiotype antibodies that rec making bispecific antibodies are known in the art. Traditional ognize the same antigen that the antibody from which the production of full-length bispecific antibodies is based on the fragment, derivative or analog is derived recognized. Specifi coexpression of two immunoglobulin heavy chain-light chain cally, in an exemplary embodiment the antigenicity of the pairs, where the two chains have different specificities (Mil idiotype of the immunoglobulin molecule can be enhanced by stein et al., 1983, Nature 305:537-539). Because of the ran deletion of framework and CDR sequences that are C-termi dom assortment of immunoglobulin heavy and light chains, nal to the CDR sequence that specifically recognizes the these hybridomas (quadromas) produce a potential mixture of antigen. To determine which CDR sequences bind the anti 10 different antibody molecules, of which only one has the gen, synthetic peptides containing the CDR sequences can be correct bispecific structure. Similar procedures are disclosed 25 used in binding assays with the antigen by any binding assay in International Publication No. WO 93/08829, and in Trau method known in the art (e.g., the BIA core assay) (See, for necker et al., EMBO.J. 10:3655-3659 (1991). e.g., Kabat et al., 1991, Sequences of Proteins of Immunologi According to a different approach, antibody variable cal Interest, Fifth Edition, National Institute of Health, domains with the desired binding specificities (antibody-an Bethesda, Md., Kabat E et al., 1980, J. of Immunology 125 tigen combining sites) are fused to immunoglobulin constant 30 (3):961-969). domain sequences. The fusion preferably is with an immu Other useful antibodies include fragments of antibodies noglobulin heavy chain constant domain, comprising at least such as, but not limited to, F(ab') fragments, which contain part of the hinge, C2, and C3 regions. It is preferred to have the variable region, the light chain constant region and the the first heavy-chain constant region (C1) containing the site CH1 domain of the heavy chain can be produced by pepsin necessary for light chain binding, present in at least one of the 35 digestion of the antibody molecule, and Fab fragments, which fusions. Nucleic acids with sequences encoding the immuno can be generated by reducing the disulfide bridges of the globulin heavy chainfusions and, if desired, the immunoglo F(ab') fragments. Other useful antibodies are heavy chain bulin light chain, are inserted into separate expression vec and light chain dimers of antibodies, or any minimal fragment tors, and are co-transfected into a suitable host organism. This thereof such as FVS or single chain antibodies (SCAS) (e.g., as provides for great flexibility in adjusting the mutual propor 40 described in U.S. Pat. No. 4,946,778; Bird, 1988, Science tions of the three polypeptide fragments in embodiments 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA when unequal ratios of the three polypeptide chains used in 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or the construction provide the optimum yields. It is, however, any other molecule with the same specificity as the antibody. possible to insert the coding sequences for two or all three Additionally, recombinant antibodies, such as chimeric polypeptide chains in one expression vector when the expres 45 and humanized monoclonal antibodies, comprising both sion of at least two polypeptide chains in equal ratios results human and non-human portions, which can be made using in high yields or when the ratios are of no particular signifi standard recombinant DNA techniques, are useful antibodies. CaCC. A chimericantibody is a molecule in which different portions In an embodiment of this approach, the bispecific antibod are derived from different animal species, such as those hav ies have a hybrid immunoglobulin heavy chain with a first 50 ing a variable region derived from a murine monoclonal and binding specificity in one arm, and a hybrid immunoglobulin human immunoglobulin constant regions. (See, e.g., Cabilly heavy chain-light chain pair (providing a second binding et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. specificity) in the other arm. This asymmetric structure facili 4,816,397, which are incorporated herein by reference in their tates the separation of the desired bispecific compound from entirety.) Humanized antibodies are antibody molecules from unwanted immunoglobulin chain combinations, as the pres 55 non-human species having one or more complementarity ence of an immunoglobulin light chain in only one half of the determining regions (CDRS) from the non-human species and bispecific molecule provides for a facile way of separation a framework region from a human immunoglobulin mol (International Publication No. WO94/04690) which is incor ecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is porated herein by reference in its entirety. incorporated herein by reference in its entirety.) Such chi For further details for generating bispecific antibodies see, 60 meric and humanized monoclonal antibodies can be pro for example, Suresh et al., Methods in Enzymology, 1986, duced by recombinant DNA techniques known in the art, for 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954 example using methods described in International Publica 6961; Carteret al., 1992, Bio/Technology 10: 163-167; Carter tion No. WO 87/02671; European Patent Publication No. et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 184, 187: European Patent Publication No. 171496; European 1998, Nature Biotechnology 16:677-681. Using such tech 65 Patent Publication No. 173494; International Publication No. niques, bispecific antibodies can be prepared for use in the WO 86/01533: U.S. Pat. No. 4,816,567; European Patent treatment or prevention of disease as defined herein. Publication No. 12,023; Berteretal, 1988, Science 240: 1041 US 7,994,135 B2 79 80 1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439 chemical modifications can be carried out by known tech 3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., niques, including, but not limited to specific chemical cleav 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et age, acetylation, formylation, metabolic synthesis in the pres al., 1987, Cancer. Res. 47: 999-1005; Wood et al., 1985, ence of tunicamycin, etc. Additionally, the analog or Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer derivative can contain one or more unnatural amino acids. Inst. 80:1553–1559; Morrison, 1985, Science 229:1202 The antibodies include antibodies having modifications 1207: Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. (e.g., Substitutions, deletions or additions) in amino acid resi 5,225,539; Jones et al., 1986, Nature 321:552-525; Verho dues that interact with Fc receptors. In particular, antibodies eyan et al. (1988) Science 239:1534; and Beidler et al., 1988, include antibodies having modifications in amino acid resi J. Immunol. 141:4053-4060; each of which is incorporated 10 herein by reference in its entirety. dues identified as involved in the interaction between the Completely human antibodies are particularly desirable anti-Fc domain and the FcRn receptor (see, e.g., International and can be produced using transgenic mice that are incapable Publication No. WO97/34631, which is incorporated herein of expressing endogenous immunoglobulin heavy and light by reference in its entirety). Antibodies immunospecific for a chains genes, but which can express human heavy and light 15 cancer cell antigen can be obtained commercially, for chain genes. The transgenic mice are immunized in the nor example, from Genentech (San Francisco, Calif.) or produced mal fashion with a selected antigen, e.g., all or a portion of a by any method known to one of skill in the art Such as, e.g., polypeptide of the invention. Monoclonal antibodies directed chemical synthesis or recombinant expression techniques. against the antigen can be obtained using conventional hybri The nucleotide sequence encoding antibodies immunospe doma technology. The human immunoglobulin transgenes cific for a cancer cell antigen can be obtained, e.g., from the harbored by the transgenic mice rearrange during B cell dif GenBank database or a database like it, the literature publi ferentiation, and Subsequently undergo class Switching and cations, or by routine cloning and sequencing. Somatic mutation. Thus, using Such a technique, it is possible In a specific embodiment, known antibodies for the treat to produce therapeutically useful IgG, IgA, IgM and IgE ment or prevention of cancer can be used. Antibodies immu antibodies. For an overview of this technology for producing 25 nospecific for a cancer cell antigen can be obtained commer human antibodies, see Lonberg and Huszar (1995, Int. Rev. cially or produced by any method known to one of skill in the Immunol. 13:65-93). For a detailed discussion of this tech art Such as, e.g., recombinant expression techniques. The nology for producing human antibodies and human mono nucleotide sequence encoding antibodies immunospecific for clonal antibodies and protocols for producing Such antibod a cancer cell antigen can be obtained, e.g., from the GenBank ies. See, e.g., U.S. Pat. Nos. 5,625,126;5,633,425;5,569,825; 30 database or a database like it, the literature publications, or by 5,661.016; 5,545,806; each of which is incorporated herein routine cloning and sequencing. Examples of antibodies by reference in its entirety. Other human antibodies can be available for the treatment of cancer include, but are not obtained commercially from, for example, Abgenix, Inc. limited to, humanized anti-HER2 monoclonal antibody, (Freemont, Calif.) and Genpharm (San Jose, Calif.). HERCEPTINR (trastuzumab; Genentech) for the treatment Completely human antibodies that recognize a selected 35 of patients with metastatic breast cancer; RITUXANR) (rit epitope can be generated using a technique referred to as uximab; Genentech) which is a chimeric anti-CD20 mono 'guided selection.” In this approach a selected non-human clonal antibody for the treatment of patients with non monoclonal antibody, e.g., a mouse antibody, is used to guide Hodgkin’s lymphoma: OvaRex (AltaRex Corporation, the selection of a completely human antibody recognizing the Mass.) which is a murine antibody for the treatment of ova same epitope. (Jespers et al. (1994) Biotechnology 12:899 40 rian cancer; Panorex (Glaxo Wellcome, N.C.) which is a 903). Human antibodies can also be produced using various murine IgG antibody for the treatment of colorectal cancer, techniques known in the art, including phage display libraries Cetuximab Erbitux (Imclone Systems Inc., N.Y.) which is an (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); anti-EGFR IhC chimeric antibody for the treatment of epi Marks et al., J. Mol. Biol., 222:581 (1991); Quan, M. P. and dermal growth factor positive cancers. Such as head and neck Carter, P. 2002. The rise of monoclonal antibodies as thera 45 cancer; Vitaxin (MedImmune, Inc., Md.) which is a human peutics. In Anti-IgE and Allergic Disease, Jardieu, P. M. and ized antibody for the treatment of sarcoma; Campath I/H Fick Jr., R. B., eds., Marcel Dekker, New York, N.Y., Chapter (Leukosite, Mass.) which is a humanized IgG antibody for 20, pp. 427-469). the treatment of chronic lymphocytic leukemia (CLL); Smart In other embodiments, the antibody is a fusion protein of an MI95 (Protein Design Labs, Inc., Calif.) which is a human antibody, or a functionally active fragment thereof, for 50 ized anti-CD33 IgG antibody for the treatment of acute example in which the antibody is fused via a covalent bond myeloid leukemia (AML); LymphoCide (Immunomedics, (e.g., a peptide bond), at either the N-terminus or the C-ter Inc., N.J.) which is a humanized anti-CD22 IgG antibody for minus to an amino acid sequence of another protein (or por the treatment of non-Hodgkin’s lymphoma; Smart ID10 (Pro tion thereof, preferably at least 10, 20 or 50 amino acid tein Design Labs, Inc., Calif.) which is a humanized anti portion of the protein) that is not the antibody. Preferably, the 55 HLA-DR antibody for the treatment of non-Hodgkin’s lym antibody or fragment thereof is covalently linked to the other phoma; Oncolym (Techniclone, Inc., Calif.) which is a protein at the N-terminus of the constant domain. radiolabeled murine anti-HLA-Dr10 antibody for the treat Antibodies include analogs and derivatives that are either ment of non-Hodgkin’s lymphoma; Allomune (BioTrans modified, i.e., by the covalent attachment of any type of plant, Calif.) which is a humanized anti-CD2 mAb for the molecule as long as such covalent attachment permits the 60 treatment of Hodgkin’s Disease or non-Hodgkin’s lym antibody to retain its antigen binding immunospecificity. For phoma; Avastin (Genentech, Inc., Calif.) which is an anti example, but not by way of limitation, the derivatives and VEGF humanized antibody for the treatment of lung and analogs of the antibodies include those that have been further colorectal cancers; EpratuZamab (Immunomedics, Inc., N.J. modified, e.g., by glycosylation, acetylation, pegylation, and Amgen, Calif.) which is an anti-CD22 antibody for the phosphorylation, amidation, derivatization by known protect 65 treatment of non-Hodgkin’s lymphoma; and CEAcide (Im ing/blocking groups, proteolytic cleavage, linkage to a cellu munomedics, N.J.) which is a humanized anti-CEA antibody lar antibody unit or other protein, etc. Any of numerous for the treatment of colorectal cancer. US 7,994,135 B2 81 82 Other antibodies useful in the treatment of cancer include, a company) or produced by any method known to one of skill but are not limited to, antibodies against the following anti in the art Such as, e.g., chemical synthesis or recombinant gens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (car expression techniques. In another embodiment, useful anti cinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X bodies are immunospecific for the treatment of autoimmune (carcinomas), alpha fetoprotein (carcinomas), CA 242 (col diseases include, but are not limited to, Anti-Nuclear Anti orectal), placental alkaline phosphatase (carcinomas), pros body; Anti-ds DNA; Anti-ss DNA, Anti-Cardiolipin Anti tate specific antigen (prostate), prostatic acid phosphatase body IgM, IgG, Anti-Phospholipid Antibody IgM, IgG, Anti (prostate), epidermal growth factor (carcinomas), MAGE-1 SM Antibody; Anti-Mitochondrial Antibody; Thyroid (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcino Antibody; Microsomal Antibody; Thyroglobulin Antibody; mas), MAGE-4 (carcinomas), anti-transferrin receptor (car 10 Anti-SCL-70; Anti-Jo; Anti-URNP; Anti-La/SSB; Anti cinomas), p97 (melanoma), MUC1-KLH (breast cancer), SSA; Anti-SSB; Anti-Perital Cells Antibody; Anti-Histones: CEA (colorectal), gp100 (melanoma), MART 1 (melanoma), Anti-RNP; C-ANCA; P-ANCA; Anti centromere; Anti PSA (prostate), IL-2 receptor (T-cell leukemia and lympho Fibrillarin, and Anti-GBMAntibody. mas), CD20 (non-Hodgkin’s lymphoma), CD52 (leukemia), In certain embodiments, useful antibodies can bind to both CD33 (leukemia), CD22 (lymphoma), human chorionic 15 a receptor or a receptor complex expressed on an activated gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 lymphocyte. The receptor or receptor complex can comprise (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG an immunoglobulin gene Superfamily member, a TNF recep (melanoma), and Neu oncogene product (carcinomas). Some tor Superfamily member, an integrin, a cytokine receptor, a specific, useful antibodies include, but are not limited to, chemokine receptor, a major histocompatibility protein, a BR96 mAb (Trail, P.A., Willner, D., Lasch, S.J., Henderson, lectin, or a complement control protein. Non-limiting A. J., Hofstead, S.J., Casazza, A. M., Firestone, R. A., Hell examples of suitable immunoglobulin Superfamily members ström, I., Hellström, K. E., “Cure of Xenografted Human are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, Carcinomas by BR96-Doxorubicin Immunoconjugates’ Sci CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting ence 1993, 261, 212-215), BR64 (Trail, PA, Willner, D, examples of suitable TNF receptor superfamily members are Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., 25 CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, Trailsmith, M.D., Doyle, T. W. King, H. D., Casazza, A. M., TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Braslawsky, G. R. Brown, J. P. Hofstead, S.J., (Greenfield, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and R.S., Firestone, R.A., Mosure, K., Kadow, D.F., Yang, M.B., APO-3. Non-limiting examples of suitable integrins are Hellstrom, K.E., and Hellstrom, I.'"Effect of LinkerVariation CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, on the Stability, Potency, and Efficacy of Carcinoma-reactive 30 CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and BR64-Doxorubicin Immunoconjugates' Cancer Research CD104. Non-limiting examples of suitable lectins are C-type, 1997, 57, 100-105, mAbs against the CD40 antigen, such as S-type, and I-type lectin. S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., In one embodiment, the Ligand binds to an activated lym Siegall, C. B., and Wahl, A. F. Agonistic properties and in phocyte that is associated with an autoimmune disease. vivo antitumor activity of the anti-CD-40 antibody, SGN-14' 35 In another specific embodiment, useful Ligands immuno Cancer Res. 2000, 60,3225-3231), mAbs against the CD70 specific for a viral or a microbial antigen are monoclonal antigen, Such as 1F6 mAb and 2F2 mAb, and mAbs against antibodies. The antibodies may be chimeric, humanized or the CD30 antigen, such as AC 10 (Bowen, M.A., Olsen, K.J., human monoclonal antibodies. As used herein, the term “viral Cheng, L., Avila, D., and Podack, E. R. “Functional effects of antigen' includes, but is not limited to, any viral peptide, CD30 on a large granular lymphoma cell line YT J. Immu 40 polypeptide protein (e.g., HIV gp120, HIV nef, RSV F gly mol., 151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res. coprotein, influenza virus neuraminidase, influenza virus 62(13):3736–42). Many other internalizing antibodies that hemagglutinin, HTLV tax, herpes simplex virus glycoprotein bind to tumor associated antigens can be used and have been (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D.A., that is capable of eliciting an immune response. As used “Cell surface receptor-targeted therapy of acute myeloid leu 45 herein, the term “microbial antigen' includes, but is not lim kemia: a review''Cancer Biother Radiopharm. 2000, 15, 459 ited to, any microbial peptide, polypeptide, protein, saccha 76; Murray, J. L., “Monoclonal antibody treatment of solid ride, polysaccharide, or lipid molecule (e.g., a bacterial, tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; fungi, pathogenic protozoa, or yeast polypeptide including, Breitling, F., and Dubel, S. Recombinant Antibodies, John e.g., LPS and capsular polysaccharide 5/8) that is capable of Wiley, and Sons, New York, 1998). 50 eliciting an immune response. In certain embodiments, the antibody is not Trastuzumab Antibodies immunospecific for a viral or microbial antigen (full length, humanized anti-HER2 (MW 145167)), Hercept can be obtained commercially, for example, from BD Bio in F(ab') (derived from anti-HER2 enzymatically (MW Sciences (San Francisco, Calif.), Chemicon International, 100000)), 4D5 (full-length, murine antiHER2, from hybri Inc. (Temecula, Calif.), or Vector Laboratories, Inc. (Burlin doma), rhu4D5 (transiently expressed, full-length humanized 55 game, Calif.) or produced by any method known to one of antibody), rhuFab4D5 (recombinant humanized Fab (MW skill in the art such as, e.g., chemical synthesis or recombinant 47738)), 4D5Fc8 (full-length, murine antiHER2, with expression techniques. The nucleotide sequence encoding mutated FcRn binding domain), or Hg (“Hingeless' full antibodies that are immunospecific for a viral or microbial length humanized 4D5, with heavy chain hinge cysteines antigen can be obtained, e.g., from the GenBank database or mutated to serines. Expressed in E. coli (therefore non-gly 60 a database like it, literature publications, or by routine cloning cosylated)). and sequencing. In another specific embodiment, known antibodies for the In a specific embodiment, useful Ligands are those that are treatment or prevention of an autoimmune disease are used in useful for the treatment or prevention of viral or microbial accordance with the compositions and methods of the inven infection in accordance with the methods disclosed herein. tion. Antibodies immunospecific for an antigen of a cell that 65 Examples of antibodies available useful for the treatment of is responsible for producing autoimmune antibodies can be viral infection or microbial infection include, but are not obtained from any organization (e.g., a university Scientist or limited to, SYNAGIS (MedImmune, Inc., Md.) which is a US 7,994,135 B2 83 84 humanized anti-respiratory syncytial virus (RSV) mono Antibodies which comprise Ab in Formula Ic antibody clonal antibody useful for the treatment of patients with RSV drug conjugates (ADC) and which may be useful in the treat infection: PRO542 (Progenics) which is a CD4 fusion anti ment of cancer include, but are not limited to, antibodies body useful for the treatment of HIV infection: OSTAVIR against tumor-associated antigens (TAA). Such tumor-asso (Protein Design Labs, Inc., Calif.) which is a human antibody ciated antigens are known in the art, and can prepared for use useful for the treatment of hepatitis B virus; PROTOVIR in generating antibodies using methods and information (Protein Design Labs, Inc., Calif.) which is a humanized IgG which are well known in the art. Examples of TAA include antibody useful for the treatment of cytomegalovirus (CMV); (1)–(35), but are not limited to TAA (1)–(35) listed below. For and anti-LPS antibodies. convenience, information relating to these antigens, all of Other antibodies useful in the treatment of infectious dis 10 which are known in the art, is listed below and includes eases include, but are not limited to, antibodies against the names, alternative names, Genbank accession numbers and antigens from pathogenic strains of bacteria (Streptococcus primary reference(s). Tumor-associated antigens targeted by pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, antibodies include all amino acid sequence variants and iso Neisseria meningitidis, Corynebacterium diphtheriae, 15 forms possessing at least about 70%, 80%, 85%, 90%, or 95% Clostridium botulinum, Clostridium perfingens, sequence identity relative to the sequences identified in the Clostridium tetani, Hemophilus influenzae, Klebsiella pneu corresponding sequences listed (SEQ ID NOS: 1-35) or the moniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, sequences identified in the cited references. In some embodi Staphylococc aureus, Vibrio Colerae, Escherichia coli, ments, TAA having amino acid sequence variants exhibit Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Substantially the same biological properties or characteristics Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, as a TAA having the sequence found in the corresponding Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuber sequences listed (SEQID NOS: 1-35). For example, a TAA culosis, Shigella dysenteriae, Shigella flexneri, Shigella son having a variant sequence generally is able to bind specifi nei, Salmonella typhimurium, Treponema pallidum, Tre cally to an antibody that binds specifically to the TAA with the ponema pertenue, Treponema carateneum, Borrelia 25 corresponding sequence listed. The sequences and disclosure vincentii, Borrelia burgdorferi, Leptospira icterohemor specifically recited herein are expressly incorporated by ref rhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, CCC. Francisella tularensis, Brucella abortus, Brucella Suis, Bru Tumor-Associated Antigens (1)–(35): cella melitensis, Mycoplasma spp., Rickettsia prowazeki, (1) BMPR1B (bone morphogenetic protein receptor-type IB, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi 30 Genbank accession no. NM 001203, ten Dijke, P., et al. (Coccidioides immitis, Aspergillus fumigatus, Candida albi Science 264 (5155):101-104 (1994), Oncogene 14 (11): cans, Blastomyces dermatitidis, Cryptococcus neoformans, 1377-1382 (1997)); WO2004063362 (Claim 2); Histoplasma capsulatum); protozoa (Entomoeba histolytica, WO2003042661 (Claim 12); US2003.134790-A1 (Page Toxoplasma gondii, Trichomonas tenas, Trichomonas homi 38-39); WO2002102235 (Claim 13: Page 296): nis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypa 35 WO2003055443 (Page 91-92): WO200299122 (Example nosoma rhodesiense, Trypanosoma Cruzi, Leishmania dono 2: Page 528-530); WO2003029421 (Claim 6): vani, Leishmania tropica, Leishmania braziliensis, WO2003024392 (Claim 2: FIG. 112); WO200298358 Pneumocystis pneumonia, Plasmodium vivax, Plasmodium (Claim 1; Page 183); WO200254940 (Page 100-101): falciparum, Plasmodium malaria); or Helminith (Enterobius WO200259377 (Page 349-350); WO200230268 (Claim vermicularis, Trichuris trichiura, Ascaris lumbricoides, Tri 40 27; Page 376): WO200148204 (Example: FIG. 4) chinella spiralis, Strongyloides Stercoralis, Schistosoma NP 001 194 bone morphogenetic protein receptor, type japonicum, Schistosoma mansoni, Schistosoma haemato IB/pid=NP 001 1941– bium, and hookworms). Cross-references: MIM:603248: NP 001194.1; Other antibodies useful in this invention for treatment of NM 001203 1 viral disease include, but are not limited to, antibodies against 45 antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Herpes Simplex sO2 aa (SEQ ID NO: 1) virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviri MLLRSAGKLNWGTKKEDGESTAPTPRPKWLRCKCHHHCPEDSWNNICSTD dae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, GYCFTMIEEDDSGLPVVTSGCLGLEGSDFOCRDTPIPHORRSIECCTERN Retroviridae, influenza viruses, parainfluenza viruses, 50 mumps, measles, respiratory syncytial virus, rubella, Arbo ECNKDLHPTLPPLKNRDFWDGPIHHRALLISWTWCSLLLWLIILFCYFRY viridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, KRQETRPRYSIGLEODETYIPPGESLRDLIEOSQSSGSGSGLPLLVORTI Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, AKOIOMVKOIGKGRYGEVWMGKWRGEKVAVKVFFTTEEASWFRETEIYOT Rotoviridae, and Human Immunodeficiency Virus. 55 In attempts to discover effective cellular targets for cancer VLMRHENILGFIAADIKCTGSWTOLYLITDYHENGSLYDYLKSTTLDAKS diagnosis and therapy, researchers have sought to identify MLKLAYSSVSGLCHLHTEIFSTOGKPAIAHRDLKSKNILWKKNGTCCIAD transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the Surface of one or more LGLAVKFISDTNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFOSYIMADM particular type(s) of cancer cell as compared to on one or 60 YSFGLILWEVARRCVSGGIVEEYOLPYHDLVPSDPSYEDMREIWCIKKLR more normal non-cancerous cell(s). Often, Such tumor-asso ciated polypeptides are more abundantly expressed on the PSFPNRWSSDECLROMGKLMTECWAHNPASRLTALRVKKTLAKMSESODI Surface of the cancer cells as compared to on the Surface of the KL non-cancerous cells. The identification of Such tumor-asso ciated cell Surface antigen polypeptides has given rise to the 65 (2) E 16 (LAT1, SLC7A5, Genbank accession no. ability to specifically target cancer cells for destruction via NM 003486); Biochem. Biophys. Res. Commun. 255(2), antibody-based therapies. 283-288 (1999), Nature 395 (6699):288-291 (1998), US 7,994,135 B2 85 86 Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267 (16): US2003.124140 (Example 16); US2003091580 (Claim 6): 11267-11273); WO2004.048938 (Example 2); WO200206317 (Claim 6: Page 400-408): WO2004032842 (Example IV); WO2003042661 (Claim Cross-references: GI:34501467; AAK74120.3: 12); WO2003016475 (Claim 1); WO200278524 (Example AF361486 1 2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); 6995 a.a. (SEQ ID NO : 4) WO2003003906 (Claim 10: Page 293); WO200264.798 PWTSLLTPGLWITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTWDTEA (Claim 33; Page 93–95); WO200014228 (Claim 5: Page 133-136); US2003224454 (FIG. 3), WO2003025138 MOPSTHTAVTNVRTSISGHESOSSVLSDSETPKATSPMGTTYTMGETSVS (Claim 12: Page 150): NP 003477 solute carrier family 7 10 (cationic amino acid transporter, y+ system), member ISTSDFFETSRIOIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGA 5/pid=NP 003477.3-Homo sapiens TTEVSRTEVISSRGTSMSGPDOFTISPDISTEAITRLSTSPIMTESAESA Cross-references: MIM:600182: NP 0.03477.3; ITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTP NM 015923; NM 003486 1 15 GDWPWPSLPSWEEASSWSSSLSSPAMTSTSFFSTLPESISSSPHPWTALL

5. Of ala (SEQ ID NO: 2) TLGPWKTTDNLRTSSEPETSSPPNLSSTSAEILATSEWTKDREKIHPSSN MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLORNI TPWWNWGTWIYKHLSPSSVLADLWTTKPTSPMATTSTLGNTSVSTSTPAF TLLNGWAIIWGTIIGSGIFWTPTGWLKEAGSPGLALWWWAACGWFSIWGA PETMMTOPTSSLTSGLREISTSOETSSATERSASLSGMPTGATTIVSRTE LCYAELGTTISKSGGDYAYMLEVYGSLPAFLKLWIELLIIRPSSOYIVAL ALSLGRTSTPGPAOSTISPEISTETITRISTPLTTTGSAEMTITPKTGHS VFATYLLKPLFPTCPVPEEAAKLVACLCVLLLTAVNCYSVKAATRVODAF GASSOGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDWSWPSR AAAKLLALALIILLGFWQIGKGVVSNLDPNFSFEGTKLDWGNIWLALYSG 25 PSWEKTSPPSSLWSLSAWTSPSPLYSTPSESSHSSPLRWTSLFTPWMMKT LFAYGGWNYLNFWTEEMINPYRNLPLAIIISLPIWTLWYWLTNLAYFTTL TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIOLSENTAVTOMG STEOMLSSEAVAWDFGNYHLGVMSWIIPVFWGLSCFGSVNGSLFTSSRLF TISAROEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEM FWGSREGHLPSILSMIHPOLLTPWPSLVFTCVMTLLYAFSKDIFSVINFF 30 ESTSTLTPTPRETSTSOEIHSATKPSTVPYKALTSATIEDSMTOVMSSSR SFFNWLCWALAIIGMIWLRHRKPELERPIKWNLALPWFFILACLFLIAWS GPSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATSRGTL FWKTPVECGIGFTIILSGLPVYEFGVWWKNKPKWLLOGIFSTTVLCOKLM TLDTSTTFMSGTHSTASOGFSHSOMTALMSRTPGEVPWLSHPSVEEASSA OVWPOET 35 SFSLSSPWMTSSSPWSSTLPDSIHSSSLPWTSLLTSGLWKTTELLGTSSE (3) STEAP1 (six transmembrane epithelial antigen of pros tate, Genbank accession no. NM 012449 Cancer Res.61 PETSSPPNLSSTSAEILATTEWTTDTEKLSMTNWWTSGYTHESPSSWLAD (15), 5857-5860 (2001), Hubert, R. S., et al. (1999) Proc. SWTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIOTKSKLSLTPGLMETS Natl. Acad. Sci. USA. 96 (25): 14523-14528): WO2004065577 (Claim 6); WO2004027049 (FIG. 1L); 40 ISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAOSTMSSD EP1394274 (Example 11); WO2004016225 (Claim 2); TSMETITRISTPLTRKESTDMAITPKTGPSGATSOGTFTLDSSSTASWPG WO2003042661 (Claim 12); US2003 157089 (Example 5); US2003 185830 (Example 5); US2003064397 (FIG.2): THSATTORFPRSVVTTPMSRGPEDWSWPSPLSVEKNSPPSSLVSSSSVTS WO20028.9747 (Example 5: Page 618-619); PSPLYSTPSGSSHSSPWPWTSLFTSIMMKATDMILDASLEPETTSAPNMNI WO2003022995 (Example 9: FIG. 13A, Example 53; Page 45 173, Example 2: FIG. 2A); NP 036581 six transmem TSDESLAASKATTETEAIHWFENTAASHWETTSATEELYSSSPGFSEPTK brane epithelial antigen of the prostate Cross-references: MIM:604415; NP 036581.1; VISPVVTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSODI NM 012449 1 TSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPGPAOSTMSLDISDEV 50 WTRLSTSPIMTESAEITITTOTGYSLATSOVTLPLGTSMTFLSGTHSTMS 339 a.a. (SEQ ID NO 3) MESRKDITNOEELWKMKPRRNLEEDDYLHKDTGETSMLKRPWLLHLHOTA OGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSS TLLDSSPSSPLPWTSLILPGLWKTTEWLDTSSEPKTSSSPNLSSTSWEIP HADEFDCPSELOHTOELFPOWHLPIKIAAIIASLTFLYTLLREVIHPLAT 55 ATSEIMTDTEKIHPSSNTAWAKWRTSSSWHESHSSWLADSETTITIPSMG SHOOYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVOLHNGTKYKKF ITSAVEDTTWFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITET PHWLDKWMLTRKOFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYOOV

OONKEDAWIEHDVWRMEIYVSLGIWGLAILALLAVTSIPSVSDSLTWREF SAVLFGVPTSATTEVSMTEIMSSNRTHIPDSDOSTMSPDIITEVITRLSS 60 SSMMSESTOMTITTOKSSPGATAOSTLTLATTTAPLARTHSTVPPRFLHS HYIOSKLGIWSLLLGTIHALIFAWNKWIDIKOFWWYTPPTFMIAVFLPIV

WLIFKSILFLPCLRKKILKIRHGWEDWTKINKTEICSOL EMTTLMSRSPENPSWKSSPFWEKTSSSSSLLSLPWTTSPSVSSTLPOSIP (4) 0772P (CA125, MUC16, Genbank accession no. SSSFSWTSLLTPGMWKTTDTSTEPGTSLSPNLSGTSWEILAASEWTTDTE AF361486 J. Biol. Chem. 276 (29):27371-27375 (2001)); 65 KIHPSSSMAWTNWGTTSSGHELYSSWSIHSEPSKATYPWGTPSSMAETSI WO2004.045553 (Claim 14); WO200292836 (Claim 6: FIG. 12); WO200283866 (Claim 15; Page 116-121);

US 7,994,135 B2 91 92 - Continued Genet. 12, 445-447, 1996: Svensson P. J., et al. Hum. Genet. 103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7, DCGRTWYSRWARWCKNDWGGRFLLEDTWTTFMKARLNCSRPGEWPFYYNE 115-124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, LOSAFHLPEODLIYGVFTTNVNSIAASAVCAFNLSAISOAFNGPFRYOEN 198-206; WO2004.045516 (Claim 1); WO2004.048938 (Example 2); WO2004040000 (Claim 151); PRAAWLPIANPIPNFOCGTLPETGPNENLTERSLODAORLFLMSEAVOPV WO2003087768 (Claim 1); WO2003016475 (Claim 1); TPEPCVTODSWRFSHLVVDLVOAKDTLYHVLYIGTESGTILKALSTASRS WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (Claim 12: LHGCYLEELHWLPPGRREPLRSLRILHSARALFWGLRDGWLRWPLERCAA Page 144); WO200198351 (Claim 1; Page 124-125); 10 YRSOGACLGARDPYCGWDGKOORCSTLEDSSNMSLWTONITACPVRNVTR EP522868 (Claim 8: FIG. 2); WO200177172 (Claim 1: Page 297-299); US2003109676; U.S. Pat. No. 6,518.404 DGGFGPWSPWOPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHI (FIG. 3); U.S. Pat. No. 5,773.223 (Claim 1a; Col 31-34): ANCSRNGAWTPWSSWALCSTSCGIGFOVRORSCSNPAPRHGGRICVGKSR WO2004001004; 15 EERFCNENTPCPWPIFWASWGSWSKCSSNCGGGMOSRRRACENGNSCLGC 442 aa (SEO ID NO:9) GVEFKTCNPEGCPEVRRNTPWTPWLPVNVTOGGAROEORFRFTCRAPLAD MOPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLOTAEIMTPPT

PHGLOFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAA KTLWPKGSNASL.ARSLAPAEVPKGDRTAGSPPRTISPPPCOGPIEIKETF

WGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYODCNPOACP KYINTWWSCLWFWLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLH

WRGAWSCWTSWSPCSASCGGGHYORTRSCTSPAPSPGEDICLGLHTEEAL IVIDIPINVYKLLAEDWPFGAEMCKLWPFIOKASVGITVLSLCALSIDRY

CATOACPEGWSPWSEWSKCTDDGAOSRSRHCEELLPGSSACAGNSSOSRP RAWASWSRIKGIGWPKWTAWEIWLIWWWSWWLAWPEAIGFDIITMDYKGS

CPYSEIPWILPASSMEEATGCAGFNLIHLWATGISCFLGSGLLTLAWYLS 25 YLRICLLHPVOKTAFMOFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM

COHCOROSOESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIP LRKKSGMOIALNDHLKORREVAKTVFCLVLVFALCWLPLHLSRILKLTLY

DDRANFYPLOOTNWYTTTYYPSPLNKHSFRPEASPGORCFPNS NONDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSC (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN 30 LCCWCOSFEEKOSLEEKOSCLKFKANDHGYDNFRSSNKYSSS cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. AY358628); US2003129.192 Genbank accession no. NM 017763); WO2003104275 (Claim 2); US2004044180 (Claim 12); US2004044179 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 11); US2003096961 (Claim 11); US2003232056 (Claim 12); WO2003083074 (Claim 14; Page 61): (Example 5); WO2003105758 (Claim 12); 35 WO2003018621 (Claim 1); WO2003024392 (Claim 2: US2003206918 (Example 5); EP1347046 (Claim 1); FIG. 93); WO200166689 (Example 6): WO2003025148 (Claim 20): Cross-references: LocusID:54894; NP 060233.2: Cross-references: GI:37182378; AAQ88991.1; NM 017763. 1 AY358628 1 40 783 a.a. (SEQ ID NO: 10) 141 a.a. (SEQ ID NO:8) MSGGHOLOLAALWPWLLMATLOAGFGRTGLVLAAAVESERSAEOKAIIRV MWWLGIAATFCGLFLLPGFALOIOCYOCEEFOLNNDCSSPEFIVNCTVNV IPLKMDPTGKLNLTLEGVFAGVAETTPAEGKLMOSHPLYLCNASDDDNLE ODMCOKEVMEOSAGIMYRKSCASSAACLIASAGYOSFCSPGKLNSW CISC 45 PGFISIWKLESPRRAPRPCLSLASKARMAGERGASAVLFDITEDRAAAEO CNTPLCNGPRPKKRGSSASALRPGLRTTILFLKLALFSAHC LOOPLGLTWPVVLIWGNDAEKLMEFWYKNOKAHVRIELKEPPAWPDYDVW (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463): Nakamuta M., et al. Biochem. Biophys. ILMTV VGTIFVIILASVLRIRCRPRHSRPDPLOORTAWAISOLATRRYOA Res. Commun. 177, 34-39, 1991; Ogawa Y., et al. Bio 50 SCROARGEWPDSGSSCSSAPVCAICLEEFSEGOELRVISCLHEFHRNCVD chem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992: Arai H., et al. J. PWLHOHRTCPLCVFNITEGDSFSOSLGPSRSYOEPGRRLHLIROHPGHAH Biol. Chem. 268, 3463-3470, 1993: Sakamoto A. Yanag YHLPAAYLLGPSRSAVARPPRPGPFLPSOEPGMGPRHHRFPRAAHPRAPG isawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al. J. Biol. Chem. 55 EOORLAGAQHPYAQGWGMSHLOSTSOHPAACPWPLRRARPPDSSGSGESY 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, S1-S4, 1992; Tsutsumi M., et al. Gene 228, CTERSGYLADGPASDSSSGPCHGSSSDSVVNCTDISLOGVHGSSSTFCSS 43-49, 1999; Strausberg R. L., et al. Proc. Natl. Acad. Sci. LSSDFDPLVYCSPKGDPORVDMOPSVTSRPRSLDSVWPTGETOWSSHWHY USA. 99, 16899-16903, 2002: Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997: Okamoto Y., et 60 HRHRHHHYKKRFOWHGRKPGPETGVPOSRPPIPRTOPOPEPPSPDOOVTG al. Biol. Chem. 272, 21589-21596, 1997; VerheijJ. B., et SNSAAPSGRLSNPQCPRALPEPAPGPWDASSICPSTSSLFNLOKSSLSAR al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffen HPORKRRGGPSEPTPGSRPODATVHPACOIFPHYTPSVAYPWSPEAHPLI berger E. G., et al. Cell 79, 1257-1266, 1994: Attie T. etal, CGPPGLDKRLLPETPGPCYSNSOPVWLCLTPROPLEPHPPGEGPSEWSSD Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A., et al. 65 Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al. Hum. TAEGRPCPYPHCOVLSAOPGSEFELEELCEOAV Mol. Genet. 5,355-357, 1996; Hofstra R. M. W., et al. Nat. US 7,994,135 B2 93 94 (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, - Continued STAMP1, STEAP2, STMP prostate cancer associated gene 1, prostate cancer associated protein 1, six transmem RLEPDAEEAARRKDLAFKFEGMGWDLFGECYRSSEWRAARLLLRRCPLWG brane epithelial antigen of prostate 2, six transmembrane DATCLOLAMOADARAFFAODGWOSLLTOKWWGDMASTTPIWALWLAFFCP prostate protein, Genbank accession no. AF455138, Lab. Invest. 82 (11): 1573-1582 (2002)); WO2003087306; PLIYTRLITFRKSEEEPTREELEFDMDSWINGEGPWGTADPAEKTPLGWP US2003064397 (Claim 1: FIG. 1); WO200272596 (Claim ROSGRPGCCGGRCGGRRCLRRWFHFWGAPVTIFMGNVVSYLLFLLLFSRV 13; Page 54-55); WO200172962 (Claim 1: FIG. 4B); WO2003104270 (Claim 11); WO2003104270 (Claim 16); LLVDFOPAPPGSLELLLYFWAPTLLCEELROGLSGGGGSLASGGPGPGHA US2004.005598 (Claim 22); WO2003042661 (Claim 12); 10 US2003060612 (Claim 12: FIG. 10); WO200226822 SLSORLRLYLADSWNOCDLVALTCFLLGWGCRLTPGLYHLGRTVLCIDFM (Claim 23: FIG. 2); WO200216429 (Claim 12: FIG. 10); WFTVRLLHIFTVNKOLGPKIVIVSKMMKDWFFFLFFLGVWLWAYGVATEG Cross-references: GI:22655488; AANO4080.1; AF455138 1 LLRPRDSDFPSILRRVFYRPYLOIFGOIPOEDMDVALMEHSNCSSEPGFW 15 AHPPGAOAGTCWSOYANWLVWLLLVIFLLVANILLVNLLIAMFSYTFGKV

490 aa (SEQ ID NO: 11) QGNSDLYWKAORYRLIREFHSRPAL.APPFIVISHLRLLLROLCRRPRSPQ MESISMMGSPKSLSETWLPNGINGIKDARKWTWGWIGSGDFAKSLTIRLI PSSPALEHFRWYLSKEAERKLLTWESWHKENFLLARARDKRESDSERLKR RCGYHWWIGSRNPKFASEFFPHWWDWTHHEDALTKTNIIFWAIHREHYTS TSOKVDLALKOLGHIREYEORLKVLEREVOOCSRWLGWVAEALSRSALLP LWDLRHLLVGKILIDWSNNMRINOYPESNAEYLASLFPDSLIVKGFNVVS PGGPPPPDLPGSKD AWALOLGPKDASROVYICSNNIQARQQVIELARQLNFIPIDLGSLSSARE (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, terato IENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYARNOOSDFYKI 25 carcinoma-derived growth factor, Genbank accession no. PIEIWNKTLPIVAITLLSLVYLAGLLAAAYOLYYGTKYRRFPPWLETWLO NP 003203 or NM 003212, Ciccodicola, A., et al. EMBO.J. 8 (7): 1987-1991 (1989), Am. J. Hum. Genet. 49 CRKOLGLLSFFFAMVHVAYSLCLPMRRSERYLFLNMAYOOVHANIENSWN (3):555-565 (1991)); US2003224411 (Claim 1); EEEVWRIEMYISFGIMSLGLLSLLAVTSIPSVSNALNWREFSFIOSTLGY WO2003083041 (Example 1); WO2003034984 (Claim 30 12); WO200288170 (Claim 2: Page 52-53); WALLISTFHWLIYGWKRAFEEEYYRFYTPPNFWLALWLPSIWILGKIILF WO2003024392 (Claim 2: FIG. 58); WO200216413 LPCISOKLKRIKKGWEKSOFLEEGIGGTIPHVSPERVTVM (Claim 1; Page 94-95, 105); WO200222808 (Claim 2: FIG. 1): U.S. Pat. No. 5,854,399 (Example 2: Col 17-18): U.S. (12) TrpM4 (BR224.50, FLJ20041, TRPM4, TRPM4B, tran Pat. No. 5,792,616 (FIG. 2); sient receptor potential cation channel, Subfamily M, mem 35 Cross-references: MIM:187395; NP 003203.1; ber 4, Genbank accession no. NM 017636 Xu, X. Z. etal. NM OO3212 1 Proc. Natl. Acad. Sci. USA. 98 (19):10692-10697 (2001), Cell 109 (3):397–407 (2002), J. Biol. Chem. 278 (33): 30813-30820 (2003)); US2003143557 (Claim 4): 188 a.a. (SEQ ID NO: 13) WO200040614 (Claim 14; Page 100-103): WO200210382 40 MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHOEFARPSRGYLAFRDDS (Claim 1: FIG. 9A); WO2003042661 (Claim 12): WO200230268 (Claim 27; Page 391); US2003219806 IWPOEEPAIRPRSSORVPPMGIOHSKELNRTCCLNGGTCMLGSFCACPPS (Claim 4); WO200162794 (Claim 14: FIG. 1A-D); FYGRNCEHDWRKENCGSVPHDTWLPKKCSLCKCWHGOLRCEPOAFLPGCD Cross-references: MIM:606936; NP 060106.2: GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY NM 017636 1 45 (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/ 1214 aa (SEQ ID NO:12) Epstein Barr virus receptor) or Hs.73792 Genbank acces MVWPEKEOSWISKIFKKKTCTTFIVDSTDPGGTLCOCGRPRTAHPAVAME sion no. M26004, Fujisaku et al. (1989).J. Biol. Chem. 264 (4):21 18-2125); Weis J. J., et al. J. Exp. Med. 167, 1047 DAFGAAWWTWWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPA 50 1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci. USA. 84,9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, AVYSLVTRTWGFRAPNLVVSVLGGSGGPWLOTWLODLLRRGLVRAAOSTG 1025-1031, 1998: Weis J. J., et al. Proc. Natl. Acad. Sci. AWIVTGGLHTGIGRHVGVAVRDHOMASTGGTKVVAMGVAPWGVWRNRDTL USA. 83,5639-5643, 1986: Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320; WO2004.045520 (Example 4): INPKGSFPARYRWRGDPEDGWOFPLDYNYSAFFLVDDGTHGCLGGENRFR 55 US2004005538 (Example 1); WO2003062401 (Claim 9); LRLESYISOOKTGWGGTGIDIPWLLLLIDGDEKMLTRIENATOAOLPCLL WO2004.045520 (Example 4); WO9102536 (FIG. 9.1-9.9); WO2004020595 (Claim 1); WAGSGGAADCLAETLEDTLAPGSGGAROGEARDRIRRFFPKGDLEWLOAQ Accession: P20023; Q13866; Q14212: EMBL: M26004; AAA35786.1. WERIMTRKELLTWYSSEDGSEEFETIWLKALWKACGSSEASAYLDELRLA 60 WAWNRWDIAQSELFRGDIQWRSFHLEASLMDALLNDRPEPVRLLISHGLS 1033 a.a. (SEQ ID NO: 14) LGHPLTPMRLAOLYSAAPSNSLIRNLLDOASHSAGTKAPALKGGAAELRP MGAAGLLGWFLALWAPGWLGISCGSPPPILNGRISYYSTPIAWGTWIRYS

PDVGHVLRMLLGKMCAPRYPSGGAWDPHPGOGFGESMYLLSDKATSPLSL CSGTFRLIGEKSLLCITKDKWDGTWDKPAPKCEYFNKYSSCPEPIWPGGY 65 DAGLGOAPWSDLLLWALLLNRAQMAMYFWEMGSNAVSSALGACLLLRVMA KIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCOANNMWGPTRLPTCVSVF

US 7,994,135 B2 99 100 - Continued - Continued EOCDAGWLSDOTVRYPIOTPREACYGDMDGFPGWRNYGVVDPDDLYDVYC INLPLLDRTIPDYTSFNTVDEWLEAIKMGOYKESFANAGFTSFDVVSOMM YAEDLNGELFLGDPPEKLTLEEARAYCOERGAEIATTGOLYAAWDGGLDH MEDILRVGVTLAGHOKKILNSIOVMRAOMNOIOSVEV CSPGWLADGSVRYPIVTPSORCGGGLPGVKTLELFPNOTGFPNKHSRFNV 5 (23) ASLG659 (B7h, Genbank accession no. AX092328) YCFRDSAOPSAIPEASNPASNPASDGLEAIWTVTETLEELOLPOEATESE US20040101899 (Claim 2); WO2003104399 (Claim 11): SRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETOSMVPPTGFSEEEG WO2004.000221 (FIG. 3); US2003.165504 (Claim 1); US2003.124140 (Example 2); US2003065143 (FIG. 60); KALEEEEKYEDEEEKEEEEEEEEWEDEALWAWPSELSSPGPEASLPTEPA 10 WO2002102235 (Claim 13: Page 299); US2003091580 AOEKSLSOAPARAVLOPGASPLPDGESEASRPPRVHGPPTETLPTPRERN (Example 2); WO200210187 (Claim 6: FIG. 10); WO200194641 (Claim 12: FIG. 7b); WO200202624 LASPSPSTLWEAREWGEATGGPELSGWPRGESEETGSSEGAPSLLPATRA (Claim 13: FIG. 1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2: Page 320-321, claim PEGTRELEAPSEDNSGRTAPAGTSVOAOPVLPTDSASRGGVAVWPASGDC 15 34; Page 321-322); WO200271928 (Page 468-469); WPSPCHNGGTCLEEEEGWRCLCLPGYGGDLCDVGLRFCNPGWDAFOGACY WO200202587 (Example 1: FIG. 1); WO200140269 (Ex ample 3: Pages 190-192); WO200036107 (Example 2: KHFSTRRSWEEAETOCRMYGAHLASISTPEEODFINNRYREYOWIGLNDR Page 205-207); WO2004053079 (Claim 12); TIEGDFLWSDGVPLLYENWNPGOPDSYFLSGENCVWMVWHDOGOWSDVPC WO2003.004989 (Claim 1); WO200271928 (Page 233 234, 452-453): WO 0116318; NYHLSYTCKMGLVSCGPPPELPLAOVFGRPRLRYEVDTVLRYRCREGLAQ

RNLPLIRCOENGRWEAPQISCVPRRPARALHPEEDPEGROGRLLGRWKAL 282 aa (SEQ ID NO:23) MASLGOILFWSIISIIIILAGAIALIIGFGISGRHSITWTTWASAGNIGE LIPPSSPMPGP 25 (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank DGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEODEMPRGR accession no. NM 004442) Chan, J. and Watt, V. M., TAVFADQVIWGNASLRLKNVOLTDAGTYKCYIITSKGKKNANLFYKTGAF Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897 905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. SMPEVNVDYNASSETLRCEAPRWFPOPTVWWASOVDOGANFSEVSNTSFE Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 30 LNSENWTMKWWSWLYNWTINNTYSCMIENDIAKATGDIKVTESEIKRRSH 12); WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9); WO2003004529 LOLLINSKASLCVSSFFAISWALLELSPYLMLK (Page 128-132); WO200053216 (Claim 1; Page 42): (24) PSCA (Prostate stem cell antigen precursor, Genbank Cross-references: MIM:600997; NP 004.433.2: accession no. AJ297436) Reiter R. E., et al. Proc. Natl. NM OO44.42 1. 35 Acad. Sci. USA. 95, 1735-1740, 1998; Gu Z., et al. Onco gene 19, 1288-1296, 2000: Biochem. Biophys. Res. Com 987 a.a. (SEQ ID NO: 22) mun. (2000) 275(3):783-788; WO2004022709; MALRRLGAALLLLPLLAAWEETLMDSTTATAELGWMWHPPSGWEEWSGYD EP1394274 (Example 11); US2004018553 (Claim 17); 40 WO2003008537 (Claim 1); WO200281646 (Claim 1: ENMNTIRTYOVCNWFESSONNWLRTKFIRRRGAHRIHVEMKFSVRDCSSI Page 164); WO2003003906 (Claim 10: Page 288); WO200140309 (Example 1: FIG. 17); US2001055751 PSVPGSCKETFNLYYYEADFDSATKTFPNWMENPWVKVDTIAADESFSOV (Example 1: FIG.1b); WO200032752 (Claim 18: FIG. 1); DLGGRVMKINTEVRSFGPWSRSGFYLAFODYGGCMSLIAVRVFYRKCPRI WO9851805 (Claim 17; Page 97); WO9851824 (Claim 10; Page 94); WO9840403 (Claim 2: FIG. 1B); IONGAIFOETLSGAESTSLVAARGSCIANAEEVDWPIKLYCNGDGEWLVP 45 Accession: O43653; EMBL: AF043498; AAC39607.1. IGRCMCKAGFEAVENGTVCRGCPSGTFKANOGDEACTHCPINSRTTSEGA

TNCVCRNGYYRADLDPLDMPCTTIPSAPOAVISSVNETSLMLEWTPPRDS 123 a.a. (SEQ ID NO:24) MKAVLLALLMAGLALOPGTALLCYSCKAQVSNEDCLOVENCTOLGEOCWT GGREDLWYNIICKSCGSGRGACTRCGDNVOYAPROLGLTEPRIYISDLLA 50 ARIRAVGLLTVISKGCSLNCVDDSODYYVGKKNITCCDTDLCNASGAHAL HTOYTFEIOAVNGVTDOSPFSPOFASVNITTNOAAPSAVSIMHOVSRTVD QPAAAILALLPALGLLLWGPGQL SITLSWSOPDOPNGVILDYELOYYEKELSEYNATAIKSPTNTVTVOGLKA (25) GEDA (Genbank accession No. AY260763); AAP14954 GAIYVFOVRARTVAGYGRYSGKMYFOTMTEAEYOTSIOEKLPLIIGSSAA 55 lipoma HMGIC fusion-partner-like protein/ pid=AAP14954.1—Homo sapiens Species: Homo sapiens GLVFLIAVWWIAIVCNRRRGFERADSEYTDKLOHYTSGHMTPGMKIYIDP (human) WO2003054152 (Claim 20); WO2003000842 FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEWCSGHLKLPGKREIF (Claim 1); WO2003023013 (Example 3, claim 20): US2003194704 (Claim 45); VAIKTLKSGYTEKORRDFLSEASIMGOFDHPNVIHLEGVVTKSTPVMIIT 60 Cross-references: GI:30102449; AAP14954.1; EFMENGSLDSFLRONDGOFTWIOLVGMLRGIAAGMKYLADMNYWHRDLAA AY260763. 1

RNILVNSNLVCKVSDFGLSRFLEDDTSDPTYTSALGGKIPIRWTAPEAIO 236 aa (SEQ ID NO:25) YRKFTSASDVWSYGIVMWEVMSYGERPYWDMTNODVINAIEODYRLPPPM MPGAAAAAAAAAAAMLPAQEAAKLYHTNYWRNSRAIGWLWAIFTICFAIV 65 DCPSALHOLMLDCWOKDRNHRPKFGOIVNTLDKMIRNPNSLKANAPLSSG NVVCFIOPYWIGDGWDTPOAGYEGLFHYCIGNGFSRELTCRGSFTDFSTL US 7,994,135 B2 101 102 - Continued with Igbeta (CD79B) and forms a complex on the surface with IgM molecules, transduces a signal involved in B-cell PSGAFKAASFFIGLSMMLIIACIICFTLFFFCNTATVYKICAWMOLTSAA differentiation) PROTEINSEQUENCE Full mpggpgv ... CLWLGCMIFPDGWDSDEWKRMCGEKTDKYTLGACSWRWAYILAIIGILDA dvdlekp (SEQ ID NO:28) (1 ... 226; 226 aa), p1: 4.84, MW: 25028TM: 2 P Gene Chromosome: 19q13.2, Gen LILSFLAFWLGNRODSLMAEELKAENKVLLSQYSLE bank accession No. NP 001774.1; WO2003088808, US20030228319; WO2OO3O624O1 (claim 9); (26) BAFF-R (B cell-activating factor receptor, BLyS recep US2002150573 (claim 4, pages 13-14); WO9958658 tor 3, BR3, Genbank accession No. NP 443177.1): (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No. NP 443177 BAFF receptor/pid=NP 443177.1 Homo 10 5,644,033: Haetal. (1992) J. Immunol. 148(5):1526-1531; sapiens Thompson, J. S., et al. Science 293 (5537), 2108 Mueller et al. (1992) Eur. J. Biochem. 22:1621-1625; 2111 (2001); WO20040583.09; WO2004011611; Hashimoto et al. (1994) Immunogenetics 40(4):287-295; WO2003045422 (Example; Page 32-33); WO2003.014294 Preud’homme et al. (1992) Clin. Exp. Immunol. 90(1): (Claim 35: FIG. 6B); WO2003035846 (Claim 70; Page 141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637; 615-616); WO200294.852 (Col 136-137); WO200238766 15 Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464; (Claim 3: Page 133); WO200224909 (Example 3: FIG. 3): Cross-references: MIM:606269; NP 443177.1; NM 052945 1 226 aa (SEQ ID NO: 28) MPGGPGVLOALPATIFLLFLL SAVYLGPGCOALWMHKWPASLMWSLGEDA

HFOCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIONVNKSH 184 a.a. (SEQ ID NO: 26) MRRGPRSLRGRDAPAPTPCWPAECFDLLWRHCWACGLLRTPRPKPAGASS GGIYVCRVOEGNESYOOSCGTYLRVROPPPRPFLDMGEGTKNRIITAEGI

PAPRTALOPOESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVS ILLFCAVWPGTLLLFRKRWONEKLGLDAGDEYEDENLYEGLNLDDCSMYE

WRRRORRLRGASSAEAPDGDKDAPEPLDKWIILSPGISDATAPAWPPPGE 25 DISRGLOGTYODWGSLNIGDVOLEKP DPGTTPPGHSVPVPATELGSTELWTTKTAGPEOO (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein coupled receptor that is activated by the CXCL13 chemok (27) CD22 (B-cell receptor CD22-B isoform, Genbank ine, functions in lymphocyte migration and humoral accession No. NP-001762. 1); Stamenkovic, I. and Seed, defense, plays a role in HIV-2 infection and perhaps devel B., Nature 345 (6270), 74-77 (1990); US2003 157113: 30 opment of AIDS, lymphoma, myeloma, and leukemia) US2003118592: WO2OO3O624O1 (Claim 9); PROTEINSEQUENCE Full mnyplt1...atslttf(1... 372; WO2003072036 (Claim 1: FIG. 1); WO200278524 (Ex 372 aa), pI: 8.54 MW. 41959 TM: 7 P. Gene Chromo ample 2); some: 11q23.3, Genbank accession No. NP 001707.1; Cross-references: MIM: 107266; NP 001762.1; WO2004040000; WO2004015426; US2003105292 (Ex NM 001771. 1 35 ample 2); U.S. Pat. No. 6,555,339 (Example 2): WO200261087 (FIG. 1); WO200157188 (claim 20, page 847 a.a. (SEO ID NO: 27) 269); WO200172830 (pages 12-13); WO200022129(Ex MHLLGPWLLLLWLEYLAFSDSSKWWFEHPETLYAWEGACWWIPCTYRALD ample 1, pages 152-153. Example 2, pages 254-256): 40 WO9928468(claim 1, page 38): U.S. Pat. No. 5,440,021 GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEOKRVOFLGDKNK (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, FIG. 5): Dobner et al. (1992) Eur. J. NCTLSIHPVHLNDSGOLGLRMESKTEKWMERIHLNVSERPFPPHIOLPPE Immunol. 22:2795-2799: Barella et al. (1995) Biochem. J. IOESOEVTLTCLLNFSCYGYPIOLOWLLEGVPMROAAVTSTSLTIKSVPT 309:773-779; RSELKFSPOWSHHGKIVTCOLODADGKFLSNDTVOLNWKHTPKLEIKVTP 45 372 a.a. (SEQ ID NO: 29) SDAIVREGDSVTMTCEVSSSNPEYTTWSWLKDGTSLKKONTFTLNLREVT MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLWENHLCPATEGPLMASFK KDOSGKYCCOWSNDWGPGRSEEVFLOVOYAPEPSTVOILHSPAVEGSOVE AVFWPVAYSLIFLLGVIGNVLVLVILERHROTRSSTETFLFHLAVADLLL 50 FLCMSLANPLPTNYTWYHNGKEMOGRTEEKVHIPKILPWHAGTYSCVAEN WFILPFAWAEGSWGWWLGTFLCKTWIALHKWNFYCSSLLLACIAWDRYLA ILGTGORGPGAELDVOYPPKKVTTVIONPMPIREGDTVTLSCNYNSSNPS IVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSOGHHNNSLP VTRYEWKPHGAWEEPSLGVLKIONVGWDNTTIACARCNSWCSWASPVALN RCTFSOENOAETHAWFTSRFLYHVAGFLLPMLVMGWCYWCVVHRLROAOR VOYAPRDVRVRKIKPLSEIHSGNSWSLOCDFSSSHPKEVOFFWEKNGRLL 55 RPOROKAVRVAILVTSIFFLCWSPYHIVIELDTLARLKAVDNTCKLNGSL GKESOLNFDSISPEDAG.SYSCWVNNSIGOTASKAWTLEVLYAPRRLRVSM PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCO

SPGDOVMEGKSATLTCESDANPPVSHYTWFDWNNOSLPHHSOKLRLEPVK LFPSWRRSSLSESENATSLTTF WOHSGAYWCOGTNSVGKGRSPLSTLTWYYSPETIGRRVAVGLGSCLAILI 60 (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia LAICCLKLQRRWKRTOSOOGLOENSSGOSFFWRNKKVRRAPLSEGPHSLG antigen) that binds peptides and presents them to CD4+ T lymphocytes) PROTEINSEQUENCE Full mgsgwVp . . . CYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMORPPRTCDDTWTYSALH villpdisc (SEQID NO:30) (1 ... 273; 273 aa, pI: 6.56 MW: KROVGDYENVIPDFPEDEGIHYSELIOFGVGERPOAOENVDYVILKH 30820 TM: 1 P Gene Chromosome: 6p21.3, Genbank 65 accession No. NP 002111.1: Tonnelle et al. (1985) (28) CD79a (CD79A, CD79C, immunoglobulin-associated EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immu alpha, a B cell-specific protein that covalently interacts nogenetics 29(6):411-413: Becket al. (1992).J. Mol. Biol. US 7,994,135 B2 103 104 228:433-441; Strausberget al. (2002) Proc. Natl. Acad. Sci - Continued USA 99:16899-16903: Servenius et al. (1987) J. Biol. Chem. 262:8759-8766: Beck et al. (1996) J. Mol. Biol. LLTCLLLGWTAICLGWRYLOWSQQLOOTNRWLEWTNSSLRQOLRLKITOL 255:1-13: Naruse et al. (2002) Tissue Antigens 59:512 GOSAEDLQGSRRELAOSOEALOVEORAHOAAEGOLOACQADROKTKETLO 519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No. 6,153, 408(Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); SEEOORRALEOKLSNMENRLKPFFTCGSADTCCPSGWIMHOKSCFYISLT U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et al. SKNWOESOKOCETLSSKLATFSEIYPOSHSYYFLNSLLPNGGSGNSYWTG (1989) Immunogenetics 30(1):66-68; Larhammar et al. LSSNKDWKLTDDTORTRTYAOSSKCNKVHKTWSWWTLESESCRSSLPYIC (1985) J. Biol. Chem. 260(26):14111-141 19: 10 EMTAFRFPD 273 a.a. (SEQ ID NO:30) (33) LY64 (Lymphocyte antigen 64 (RP105), type I mem MGSGWVPWVVALLVNLTRLDSSMTOGTDSPEDFWIOAKADCYPTNGTEKV brane protein of the leucine rich repeat (LRR) family, regu OFVWRFIFNLEEYVRFDSDVGMFVALTKLGOPDAEOWNSRLDLLERSROA lates B-cell activation and apoptosis, loss of function is 15 associated with increased disease activity in patients with WDGVCRHNYRLGAPFTVGRKVOPEWTVYPERTPLLHOHNLLHCSVTGFYP systemic lupus erythematosis) PROTEIN SEQUENCE GDTKIKWFLNGOEERAGVMSTGPIRNGDWTFOTVWMLEMTPELGHWYTCL Full mafdvsc...rwkyghi (SEQ) ID NO:33) (1 ... 661; 661 aa), pI: 6.20, MW: 74.147 TM: 1 P Gene Chromosome: WDHSSLLSPWSWEWRAQSEYSWRKMLSGIAAFLLGLIFLLVGIVIQLRAQ 5q12, Genbank accession No. NP 005573.1; KGYVRTOMSGNEVSRAVLLPOSC US2002 193567; WO9707198 (claim 11, pages 39-42): Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel (1998) Blood 92:2815-2822; WO2003083047; 5, an ion channel gated by extracellular ATP may be WO9744452 (pages 24-26); involved in Synaptic transmission and neurogenesis, defi 25 ciency may contribute to the pathophysiology of idiopathic detrusor instability) PROTEINSEQUENCE Full mgqa 661 a.a. (SEQ ID NO:33) gck ... lephrst (SEQ ID NO:31) (1 . . . 422; 422 aa), pI: MAFDVSCFFWWWLFSAGCKVITSWDOMCIEKEANKTYNCENLGLSEIPDT 7.63, MW: 47206 TM: 1 P Gene Chromosome: 17p13.3, LPNTTEFLEFSFNFLPTIHNRTFSRLMNLTFLDLTRCOINWIHEDTFOSH Genbank accession No. NP 002552.2: Le et al. (1997) 30 FEBS Lett. 418(1-2):195-199: WO2004.047749; HOLSTLVLTGNPLIFMAETSLNGPKSLKHLFLIOTGISNLEFIPVHNLEN WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20): LESLYLGSNHISSIKFPKDFFARNLKVLDFONNAIHYISREDMRSLEOAI WO2003093444 (claim 1); WO2003087768 (claim 1); NLSLNFNGNNVKGIELGAFDSTVFOSLNFGGTPNLSWIFNGLONSTTOSL WO2003029277 (page 82); 35 WLGTFEDIDDEDISSAMLKGLCEMSVESLNLOEHRFSDISSTTFOCFTOL

QELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDOLCQISAANFPSLTHL 422 aa (SEQ ID NO:31) MGOAGCKGLCLSLFDYKTEKYVIAKNKKWGLLYRLLOASILAYLVWWVFL YIRGNVKKLHLGVGCLEKLGNLOTLDLSHNDIEASDCCSLOLKNLSHLOT

IKKGYODVDTSLOSAVITKVKGVAFTNTSDLGORIWDVADYVIPAOGENV 40 LNLSHNEPLGLOSQAFKECPOLELLDLAFTRLHINAPOSPFONLHFLQWL

FFVVTNLIVTPNORONVCAENEGIPDGACSKDSDCHAGEAVTAGNGVKTG NLTYCFLDTSNOHLLAGLPVLRHLNLKGNHFODGTITKTNLLOTWGSLEV

RCLRRENLARGTCEIFAWCPLETSSRPEEPFLKEAEDFTIFIKNHIRFPK LILSSCGLLSIDOOAFHSLGKNSHVDLSHNSLTCDSIDSLSHLKGIYLNL

FNFSKSNWMDWKDRSFLKSCHFGPKNHYCPIERLGSWIRWAGSDFODTAL 45 AANSINIISPRLLPILSOOSTINLSHNPLDCTCSNIHFLTWYKENLHKLE

EGGWIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSWSSGYNFRFARY GSEETTCANPPSLRGWKLSDWKLSCGITAIGIFFLIWFLLLLAILLFFAW

YRDAAGWEFRTLMKAYGIRFDWMWNGKGAFFCDLWLIYLIKKREFYRDKK KYLLRWKYOHI

YEEWRGLEDSSQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKR 50 (34) FCRH1 (Fc receptor-like protein 1, a putative receptor GSSSOKGNGSWCPOLLEPHRST for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lympho (32) CD72 (B-cell differentiation antigen CD72, Lyb-2) cyte differentiation) PROTEINSEQUENCE Full mlprl PROTEINSEQUENCE Full maeaity ... tafrfpd (SEQID ll...vdyedam (SEQIDNO:34) (1... 429:429aa), pI: 5.28, NO:32) (1 ... 359; 359aa), pI: 8.66, MW: 40225TM: 1 IP 55 MW: 46925 TM: 1 P Gene Chromosome: 1921-1q22, Gene Chromosome: 9p13.3, Genbank accession No. Genbank accession No. NP 443170.1; WO2003077836; NP 001773.1; WO2004O42346 (claim 65); WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al. WO2003026493 (pages 51-52, 57-58); WO200075655 (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; (pages 105-106); Von Hoegen et al. (1990) J. Immunol. WO2003089624 (claim 8); EP1347046 (claim 1); 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. 60 WO2003089624 (claim 7); Acad. Sci USA 99:16899-16903;

429 aa (SEQ ID NO:34) 359 a.a. (SEQ ID NO:32) MLPRLLLLICAPLCEPAEL FLIASPSHPTEGSPVTLTCKMPFLOSSDAOF MAEAITYADLRFWKAPLKKSISSRLGODPGADDDGEITYENVOVPAVLGV 65 OFCFFRDTRALGPGWSSSPKLOIAAMWKEDTGSYWCEAOTMASKVLRSRR PSSLASSVLGDKAAVKSEOPTASWRAVTSPAVGRILPCRTTCLRYLLLGL US 7,994,135 B2 105 106 - Continued In an embodiment, the Ligand-Linker-Drug Conjugate has SOINVHRWPVADVSLETOPPGGOVMEGDRLVLICSVAMGTGDITFLWYKG Formula IIIa, where the Ligand is an antibody Ab including one that binds at least one of CD30, CD40, CD70, Lewis Y AVGLNLOSKTORSLTAEYEIPSVRESDAEOYYCVAENGYGPSESGLVSIT antigen, w=0, y=0, and D has Formula Ib. Exemplary Conju WRIPVSRPILMLRAPRAOAAVEDVLELHCEALRGSPPILYWFYHEDITLG gates of Formula IIIa include where R7 is —(CH) . Also included are such Conjugates of Formula IIIa in which D has SRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAORSEAVTLNFTVPTGA the structure of Compound 2 in Example 3 and esters thereof. RSNHLTSG WIEGLLSTLGPATWALLFCYGLKRKIGRRSARDPLRSLPSPL Also included are Such Conjugates of Formula IIIa containing about 3 to about 8, in one aspect, about 3 to about 5 Drug POEFTYLNSPTPGOLOPIYENVNVVSGDEVYSLAYYNOPEOESVAAETLG 10 moieties D. that is, Conjugates of Formula Ia wherein p is a THMEDKWSLDIYSRLRKANITDWDYEDAM value in the range about 3-8, for example about 3-5. Conju gates containing combinations of the structural features noted (35) IRTA2 (Immunoglobulin superfamily receptor translo in this paragraph are also contemplated as within the scope of cation associated 2, a putative immunoreceptor with pos the compounds of the invention. sible roles in B cell development and lymphomagenesis; 15 In another embodiment, the Ligand-Linker-Drug Conju deregulation of the gene by translocation occurs in some B gate has Formula IIIa, where Ligand is an Antibody Ab that cell malignancies) PROTEINSEQUENCE Full millw binds one of CD30, CD40, CD70, Lewis Yantigen, w=1, y=0, vil... assaphr (SEQID NO:35) (1...977;977 aa), pI: 6.88 and D has Formula Ib. Included are such Conjugates of For MW: 106468 TM: 1 P Gene Chromosome: 1 q21, Gen mula IIIain which R'' is —(CH) . Also included are such bank accession No. NP 112571.1; WO2003024392 Conjugates of Formula IIIa in which W is -Val-Cit-, and/or (claim 2, FIG.97); Nakayama et al. (2000) Biochem. Bio where D has the structure of Compound 2 in Example 3 and phys. Res. Commun. 277(1): 124-127; WO2003077836; esters thereof. Also included are such Conjugates of Formula WO200138490 (claim 3, FIG. 18B-1-18B-2); IIIa containing about 3 to about 8, preferably about 3 to about 25 5 Drug moieties D, that is, Conjugates of Formula Ia wherein 977 a.a. (SEO ID NO:35) p is a value in the range of about 3-8, preferably about 3-5. MLLWWILLVLAPVSGOFARTPRPIIFLOPPWTTWEOGERVTLTCKGFRFY Conjugates containing combinations of the structural features noted in this paragraph are also exemplary. SPOKTKWYHRYLGKEILRETPDNILEVOESGEYRCOAOGSPLSSPVHLDF In an embodiment, the Ligand-Linker-Drug Conjugate has SSASLILOAPLSWFEGDSVVLRCRAKAEWTLNNTIYKNDNVLAFLNKRTD 30 Formula IIIa, where the Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Yantigen, w=1, y=1, and FHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIOVOEPFTRPVLRASSEQ D has Formula Ib. Included are Conjugates of Formula IIIa in which R'' is -(CH2)5 -. Also included are such Conjugates PISGNPVTLTCETOLSLERSDWPLRFRFFRDDOTLGLGWSLSPNFOITAM of Formula IIIa where:Wis-Val-Cit-Y has Formula X: Dhas WSKDSGEYWCKAATMPHSVISDSPRSWIOVOIPASHPVLTLSPEKALNFE 35 the structure of Compound 2 in Example3 and esters thereof; p is about 3 to about 8, preferably about 3 to about 5 Drug GTKVTLHCETOEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENS moieties D. Conjugates containing combinations of the struc GNYYCTADNGLGAKPSKAWSLSWTWPWSHPWLNLSSPEDLIFEGAKWTLH tural features noted in this paragraph are also contemplated within the scope of the compounds of the invention. CEAORGSLPILYOFHHEDAALERRSANSAGGVAISFSLTAEHSGNYYCTA 40 A further embodiment is an antibody drug conjugate (ADC), or a pharmaceutically acceptable salt or Solvate DNGFGPORSKAVSLSITWPVSHPVLTLSSASALTFSGATVTLHCEVORGS thereof, wherein Ab is an antibody that binds one of the POILYOFYHEDMPLWSSSTPSWGRVSFSFSLTEGHSGNYYCTADNGFGPO tumor-associated antigens (1)–(35) noted above (the “TAA Compound'). RSEVVSLFVTVPVSRPILTLRVPRAOAVVGDLLELHCEAPRGSPPILYWF 45 Another embodiment is the TAA Compound or pharma YHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAOHSDTISL ceutically acceptable salt or solvate thereof that is in isolated and purified form. SWIVPVSRPILTFRAPRAOAVVGDLLELHCEALRGSSPILYWFYHEDWTL Another embodiment is a method for killing or inhibiting GKISAPSGGGASFNLSLTTEHSGIYSCEADNGPEAORSEMVTLKVAVPVS the multiplication of a tumor cell or cancer cell comprising 50 administering to a patient, for example a human with a hyper RPWLTLRAPGTHAAWGDLLELHCEALRGSPLILYREFHEDWTLGNRSSPS proliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or Solvate thereof, said GGASLNLSLTAEHSGNYSCEADNGLGAORSETVTLYITGLTANRSGPFAT amount being effective to kill or inhibit the multiplication of GWAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDEARSPPDSDSQEPTYH a tumor cell or cancer cell. 55 Another embodiment is a method for treating cancer com NVPAWEELOPVYTNANPRGENVVYSEVRIIOEKKKHAVASDPRHLRNKGS prising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Com PIIYSEWKWASTPWSGSLFLASSAPHR pound or a pharmaceutically acceptable salt or Solvate See also: WOO4/045516 (3 Jun. 2004); WO03/000113 (3 thereof, said amount being effective to treat cancer, alone or Jan. 2003); WO02/016429 (28 Feb. 2002); WO02/16581 (28 60 together with an effective amount of an additional anticancer Feb. 2002); WO03/024392 (27 Mar. 2003); WO04/016225 agent. (26 Feb. 2004); WO01/40309 (7 Jun. 2001), and U.S. Provi Another embodiment is a method for treating an autoim sional patent application Ser. No. 60/520,842 “COMPOSI mune disease, comprising administering to a patient, for TIONS AND METHODS FOR THE TREATMENT OF example a human with a hyperproliferative disorder, an TUMOR OF HEMATOPOIETIC ORIGIN', filed 17 Nov. 65 amount of the TAA Compound or a pharmaceutically accept 2003; all of which are incorporated herein by reference in able salt or solvate thereof, said amount being effective to their entirety. treat an autoimmune disease. US 7,994,135 B2 107 108 The antibodies suitable for use in the invention can be example, but not limited to, chemical mutagenesis and in produced by any method known in the art for the synthesis of vitro site directed mutagenesis (Hutchinson et al., 1978, J. antibodies, in particular, by chemical synthesis or by recom Biol. Chem. 253:6551). binant expression, and are preferably produced by recombi In addition, techniques developed for the production of nant expression techniques. 5 "chimeric antibodies' (Morrison et al., 1984, Proc. Natl. 4.5.1 Production of Recombinant Antibodies Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312: Antibodies of the invention can be produced using any 604-608: Takeda et al., 1985, Nature 314:452-454) by splic method known in the art to be useful for the synthesis of ing genes from a mouse antibody molecule of appropriate antibodies, in particular, by chemical synthesis or by recom antigen specificity together with genes from a human anti 10 body molecule of appropriate biological activity can be used. binant expression. A chimericantibody is a molecule in which different portions Recombinant expression of antibodies, or fragment, are derived from different animal species, such as those hav derivative or analog thereof, requires construction of a ing a variable region derived from a murine monoclonal anti nucleic acid that encodes the antibody. If the nucleotide body and a human immunoglobulin constant region, e.g., sequence of the antibody is known, a nucleic acid encoding 15 humanized antibodies. the antibody may be assembled from chemically synthesized Alternatively, techniques described for the production of oligonucleotides (e.g., as described in Kutmeier et al., 1994, single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, BioTechniques 17:242), which involves the synthesis of over Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. lapping oligonucleotides containing portions of the sequence Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334: encoding the antibody, annealing and ligation of those oligo 544-54) can be adapted to produce single chain antibodies. nucleotides, and then amplification of the ligated oligonucle Single chain antibodies are formed by linking the heavy and otides, e.g., by PCR. light chain fragments of the Fv region via an amino acid Alternatively, a nucleic acid molecule encoding an anti bridge, resulting in a single chain polypeptide. Techniques for body can be generated from a suitable source. If a clone the assembly of functional Fv fragments in E. coli may also be containing the nucleic acid encoding the particular antibody 25 used (Skerra et al., 1988, Science 242:1038-1041). is not available, but the sequence of the antibody is known, a Antibody fragments that recognize specific epitopes can be nucleic acid encoding the antibody can be obtained from a generated by known techniques. For example, such fragments suitable source (e.g., an antibody cINA library, or cDNA include, but are not limited to the F(ab'), fragments that can be library generated from any tissue or cells expressing the produced by pepsin digestion of the antibody molecule and immunoglobulin) by, e.g., PCR amplification using synthetic 30 the Fab fragments that can be generated by reducing the primers hybridizable to the 3' and 5' ends of the sequence or disulfide bridges of the F(ab') fragments. by cloning using an oligonucleotide probe specific for the Once a nucleic acid sequence encoding an antibody has particular gene sequence. been obtained, the vector for the production of the antibody If an antibody that specifically recognizes a particular anti can be produced by recombinant DNA technology using tech gen is not commercially available (or a source for a cDNA 35 niques well known in the art. Methods that are well known to library for cloning a nucleic acid encoding Such an immuno those skilled in the art can be used to construct expression globulin), antibodies specific for a particular antigen can be vectors containing the antibody coding sequences and appro generated by any method known in the art, for example, by priate transcriptional and translational control signals. These immunizing a patient, or Suitable animal model Such as a methods include, for example, in vitro recombinant DNA rabbit or mouse, to generate polyclonal antibodies or, more 40 techniques, synthetic techniques, and in vivo genetic recom preferably, by generating monoclonal antibodies, e.g., as bination. See, for example, the techniques described in Sam described by Kohler and Milstein (1975, Nature 256:495 brooketal. (1990, Molecular Cloning, A Laboratory Manual, 497) or, as described by Kozbor et al. (1983, Immunology 2" Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alterna 45 Molecular Biology, John Wiley & Sons, NY). tively, a clone encoding at least the Fab portion of the anti An expression vector comprising the nucleotide sequence body can be obtained by screening Fab expression libraries of an antibody or the nucleotide sequence of an antibody can (e.g., as described in Huse et al., 1989, Science 246:1275 be transferred to a host cell by conventional techniques (e.g., 1281) for clones of Fab fragments that bind the specific anti electroporation, liposomal transfection, and calcium phos gen or by screening antibody libraries (See, e.g., Clackson et 50 phate precipitation), and the transfected cells are then cul al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. tured by conventional techniques to produce the antibody. In Sci. USA 94:4937). specific embodiments, the expression of the antibody is regu Once a nucleic acid sequence encoding at least the variable lated by a constitutive, an inducible or a tissue, specific pro domain of the antibody is obtained, it can be introduced into moter. a vector containing the nucleotide sequence encoding the 55 The host cells used to express the recombinant antibody constant regions of the antibody (see, e.g., International Pub can be either bacterial cells such as Escherichia coli, or, lication No. WO 86/05807; WO 89/01036; and U.S. Pat. No. preferably, eukaryotic cells, especially for the expression of 5,122,464). Vectors containing the complete light or heavy whole recombinant immunoglobulin molecule. In particular, chain that allow for the expression of a complete antibody mammalian cells such as Chinese hamster ovary cells (CHO), molecule are available. Then, the nucleic acid encoding the 60 in conjunction with a vector Such as the major intermediate antibody can be used to introduce the nucleotide substitutions early gene promoter element from human cytomegalovirus is or deletion necessary to substitute (or delete) the one or more an effective expression system for immunoglobulins (Foeck variable region cysteine residues participating in an intrac ing et al., 198, Gene 45:101; Cockett et al., 1990, BioTech hain disulfide bond with an amino acid residue that does not nology 8:2). contain a Sulfhydyl group. Such modifications can be carried 65 A variety of host-expression vector systems can be utilized out by any method known in the art for the introduction of to express the immunoglobulin antibodies. Such host-expres specific mutations or deletions in a nucleotide sequence, for sion systems represent vehicles by which the coding US 7,994,135 B2 109 110 sequences of the antibody can be produced and Subsequently codon and adjacent sequences. Furthermore, the initiation purified, but also represent cells that can, when transformed codon must be in phase with the reading frame of the desired or transfected with the appropriate nucleotide coding coding sequence to ensure translation of the entire insert. sequences, express an antibody immunoglobulin molecule in These exogenous translational control signals and initiation situ. These include, but are not limited to, microorganisms codons can be of a variety of origins, both natural and Syn Such as bacteria (e.g., E. coli and B. subtilis) transformed with thetic. The efficiency of expression can be enhanced by the recombinant bacteriophage DNA, plasmid DNA or cosmid inclusion of appropriate transcription enhancer elements, DNA expression vectors containing immunoglobulin coding transcription terminators, etc. (see Bittner et al., 1987, Meth sequences; yeast (e.g., Saccharomyces Pichia) transformed ods in Enzymol. 153:51-544). with recombinant yeast expression vectors containing immu 10 In addition, a host cell Strain can be chosen to modulate the noglobulin coding sequences; insect cell systems infected expression of the inserted sequences, or modifies and pro with recombinant virus expression vectors (e.g., baculovirus) cesses the gene product in the specific fashion desired. Such containing the immunoglobulin coding sequences; plant cell modifications (e.g., glycosylation) and processing (e.g., systems infected with recombinant virus expression vectors cleavage) of protein products can be important for the func (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic 15 tion of the protein. Different host cells have characteristic and virus (TMV)) or transformed with recombinant plasmid specific mechanisms for the post-translational processing and expression vectors (e.g., Tiplasmid) containing immunoglo modification of proteins and gene products. Appropriate cell bulin coding sequences; or mammalian cell systems (e.g., lines or host systems can be chosen to ensure the correct COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombi modification and processing of the foreign protein expressed. nant expression constructs containing promoters derived To this end, eukaryotic host cells that possess the cellular from the genome of mammalian cells (e.g., metallothionein machinery for proper processing of the primary transcript, promoter) or from mammalian viruses (e.g., the adenovirus glycosylation, and phosphorylation of the gene product can late promoter; the vaccinia virus 7.5K promoter). be used. Such mammalian host cells include, but are not In bacterial systems, a number of expression vectors can be limited to, CHO, VERY, BH, Hela, COS, MDCK, 293,293T, advantageously selected depending upon the use intended for 25 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, the antibody being expressed. For example, when a large CRL7030 and HS578BSt. quantity of such a protein is to be produced, vectors that direct For long-term, high-yield production of recombinant pro the expression of high levels of fusion protein products that teins, stable expression is preferred. For example, cell lines are readily purified might be desirable. Such vectors include, that stably express an antibody can be engineered. Rather but are not limited, to the E. coli expression vector puR278 30 than using expression vectors that contain viral origins of (Ruther et al., 1983, EMBO.J. 2:1791), in which the antibody replication, host cells can be transformed with DNA con coding sequence may be ligated individually into the vector in trolled by appropriate expression control elements (e.g., pro frame with the lac Z coding region so that a fusion protein is moter, enhancer, sequences, transcription terminators, poly produced; plN vectors (Inouye & Inouye, 1985, Nucleic adenylation sites, etc.), and a selectable marker. Following Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. 35 the introduction of the foreign DNA, engineered cells can be Biol. Chem. 24:5503–5509); and the like. pGEX Vectors can allowed to grow for 1-2 days in an enriched media, and then also be used to express foreign polypeptides as fusion pro are switched to a selective media. The selectable marker in the teins with glutathione S-transferase (GST). In general, such recombinant plasmid confers resistance to the selection and fusion proteins are soluble and can easily be purified from allows cells to stably integrate the plasmid into their chromo lysed cells by adsorption and binding to a matrix glutathione 40 Somes and grow to form foci that in turn can be cloned and agarose beads followed by elution in the presence of free expanded into cell lines. This method can advantageously be glutathione. The pGEX vectors are designed to include used to engineer cell lines which express the antibody. Such thrombin or factor Xa protease cleavage sites so that the engineered cell lines can be particularly useful in Screening cloned target gene product can be released from the GST and evaluation of tumor antigens that interact directly or moiety. 45 indirectly with the antibody. In an insect system, Autographa Californica nuclear poly A number of selection systems can be used, including but hedrosis virus (AcNPV) or the analogous virus from Droso not limited to the herpes simplex virus thymidine kinase phila Melanogaster is used as a vector to express foreign (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine genes. The virus grows in Spodoptera frugiperda cells. The phosphoribosyltransferase (Szybalska & Szybalski, 192, antibody coding sequence can be cloned individually into 50 Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphori non-essential regions (for example the polyhedrin gene) of bosyltransferase (Lowy et al., 1980, Cell 22:817) genes can the virus and placed under control of an AcNPV promoter (for be employed in tk-, hgprt- or aprt- cells, respectively. Also, example the polyhedrin promoter). antimetabolite resistance can be used as the basis of selection In mammalian host cells, a number of viral-based expres for the following genes: DHFR, which confers resistance to sion systems can be utilized. In cases where an adenovirus is 55 methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA used as an expression vector, the antibody coding sequence of 77:357: O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA interest can be ligated to an adenovirus transcription/transla 78: 1527); gpt, which confers resistance to mycophenolic acid tion control complex, e.g., the late promoter and tripartite (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA leader sequence. This chimeric gene can then be inserted in 78:2072); neo, which confers resistance to the aminoglyco the adenovirus genome by in vitro or in Vivo recombination. 60 side G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Insertion in a non-essential region of the viral genome (e.g., 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Phar region E1 or E3) results in a recombinant virus that is viable macol. Toxicol. 32:573-596; Mulligan, 1993, Science 260: and capable of expressing the immunoglobulin molecule in 926-932; and Morgan and Anderson, 1993, Ann. Rev. Bio infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. chem, 62:191-217; May, 1993, TIB TECH 11(5):155-215) Acad. Sci. USA 81:355-359). Specific initiation signals can 65 and hygro, which confers resistance to hygromycin (Santerre also be required for efficient translation of inserted antibody et al., 1984, Gene 30:147). Methods commonly known in the coding sequences. These signals include the ATG initiation art of recombinant DNA technology which can be used are US 7,994,135 B2 111 112 described in Ausubel et al. (eds., 1993, Current Protocols in BR-3 cells, see Stancovski et al. Proc. Natl. Acad. Sci. USA Molecular Biology, John Wiley & Sons, NY: Kriegler, 1990, 88:8691-8695 (1991)) can be used to generate antibodies. Gene Transfer and Expression, A Laboratory Manual, Stock Otherforms of ErbB2 useful for generating antibodies will be ton Press, NY; and in Chapters 12 and 13, Dracopoli et al. apparent to those skilled in the art. (eds), 1994, Current Protocols in Human Genetics, John 5 (i) Polyclonal Antibodies Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Polyclonal antibodies are preferably raised in animals by Biol. 150:1). multiple Subcutaneous (sc) or intraperitoneal (ip) injections The expression levels of an antibody can be increased by of the relevant antigen and an adjuvant. It may be useful to vector amplification (for a review, see Bebbington and conjugate the relevant antigen to a protein that is immuno Hentschel. The use of vectors based on gene amplification for 10 genic in the species to be immunized, e.g., keyhole limpet the expression of cloned genes in mammalian cells in DNA hemocyanin, serum albumin, bovine thyroglobulin, or Soy cloning, Vol. 3. (Academic Press, New York, 1987)). When a bean trypsin inhibitor using a bifunctional or derivatizing marker in the vector System expressing an antibody is ampli agent, for example, maleimidobenzoyl SulfoSuccinimide fiable, an increase in the level of inhibitor present in culture of ester (conjugation through cysteine residues), N-hydroxysuc host cell will increase the number of copies of the marker 15 cinimide (through lysine residues), glutaraldehyde, Succinic gene. Since the amplified region is associated with the nucle anhydride, SOCl, or R'N=C=NR, where R and R' are otide sequence of the antibody, production of the antibody different alkyl groups. will also increase (Crouseet al., 1983, Mol. Cell. Biol. 3:257). Animals are immunized against the antigen, immunogenic The host cell can be co-transfected with two expression conjugates, or derivatives by combining, e.g., 100 g or 5ug vectors, the first vector encoding a heavy chain derived of the protein or conjugate (for rabbits or mice, respectively) polypeptide and the second vector encoding a light chain with 3 Volumes of Freund's complete adjuvant and injecting derived polypeptide. The two vectors can contain identical the solution intradermally at multiple sites. One month later selectable markers that enable equal expression of heavy and the animals are boosted with /s to /10 the original amount of light chain polypeptides. Alternatively, a single vector can be peptide or conjugate in Freund's complete adjuvant by Sub used to encode both heavy and light chain polypeptides. In 25 cutaneous injection at multiple sites. Seven to 14 days later such situations, the light chain should be placed before the the animals are bled and the serum is assayed for antibody heavy chain to avoid an excess of toxic free heavy chain titer. Animals are boosted until the titer plateaus. Preferably, (Proudfoot, 1986, Nature 322:52: Kohler, 1980, Proc. Natl. the animal is boosted with the conjugate of the same antigen, Acad. Sci. USA 77:2197). The coding sequences for the heavy but conjugated to a different protein and/or through a different and light chains can comprise cDNA or genomic DNA. 30 cross-linking reagent. Conjugates also can be made in recom Once the antibody has been recombinantly expressed, it binant cell culture as protein fusions. Also, aggregating can be purified using any method known in the art for purifi agents such as alum are suitably used to enhance the immune cation of an antibody, for example, by chromatography (e.g., response. ion exchange, affinity, particularly by affinity for the specific (ii) Monoclonal Antibodies antigen after Protein A, and sizing column chromatography), 35 Monoclonal antibodies are obtained from a population of centrifugation, differential solubility, or by any other standard Substantially homogeneous antibodies, i.e., the individual technique for the purification of proteins. antibodies comprising the population are identical except for In yet another exemplary embodiment, the antibody is a possible naturally-occurring mutations that may be present in monoclonal antibody. minor amounts. Thus, the modifier "monoclonal indicates In any case, the hybrid antibodies have a dual specificity, 40 the character of the antibody as not being a mixture of discrete preferably with one or more binding sites specific for the antibodies. hapten of choice or one or more binding sites specific for a For example, the monoclonal antibodies may be made target antigen, for example, an antigen associated with a using the hybridoma method first described by Kohler et al., tumor, an autoimmune disease, an infectious organism, or Nature, 256:495 (1975), or may be made by recombinant other disease state. 45 DNA methods (U.S. Pat. No. 4,816,567). 4.5.2 Production of Antibodies In the hybridoma method, a mouse or other appropriate The production of antibodies will be illustrated with refer host animal. Such as a hamster, is immunized as hereinabove ence to anti-CD30 antibodies but it will be apparent for those described to elicitlymphocytes that produce or are capable of skilled in the art that antibodies to other members of the TNF producing antibodies that will specifically bind to the protein receptor family can be produced and modified in a similar 50 used for immunization. Alternatively, lymphocytes may be manner. The use of CD30 for the production of antibodies is immunized in vitro. Lymphocytes then are fused with exemplary only and not intended to be limiting. myeloma cells using a Suitable fusing agent, such as polyeth The CD30 antigen to be used for production of antibodies ylene glycol, to form a hybridoma cell (Goding, Monoclonal may be, e.g., a soluble form of the extracellular domain of Antibodies. Principles and Practice, pp. 59-103 (Academic CD30 or a portion thereof, containing the desired epitope. 55 Press, 1986)). Alternatively, cells expressing CD30 at their cell surface The hybridoma cells thus prepared are seeded and grown in (e.g., L540 (Hodgkin’s lymphoma derived cell line with a T a suitable culture medium that preferably contains one or cell phenotype) and L428 (Hodgkin’s lymphoma derived cell more substances that inhibit the growth or survival of the line with a B cell phenotype)) can be used to generate anti unfused, parental myeloma cells. For example, if the parental bodies. Other forms of CD30 useful for generating antibodies 60 myeloma cells lack the enzyme hypoxanthine guanine phos will be apparent to those skilled in the art. phoribosyl transferase (HGPRT or HPRT), the culture In another exemplary embodiment, the ErbB2 antigen to be medium for the hybridomas typically will include hypoxan used for production of antibodies may be, e.g., a soluble form thine, aminopterin, and thymidine (HAT medium), which of the extracellular domain of ErbB2 or a portion thereof, substances prevent the growth of HGPRT-deficient cells. containing the desired epitope. Alternatively, cells expressing 65 Preferred myeloma cells are those that fuse efficiently, ErbB2 at their cell surface (e.g., NIH-3T3 cells transformed support stable high-level production of antibody by the to overexpress ErbB2; or a carcinoma cell line such as SK selected antibody-producing cells, and are sensitive to a US 7,994,135 B2 113 114 medium such as HAT medium. Among these, preferred The DNA also may be modified, for example, by substi myeloma cell lines are murine myeloma lines, such as those tuting the coding sequence for human heavy chain and light derived from MOPC-21 and MPC-11 mouse tumors available chain constant domains in place of the homologous murine from the Salk Institute Cell Distribution Center, San Diego, sequences (U.S. Pat. No. 4,816,567; and Morrison, et al. Calif. USA, and SP-2 or X63-Ag8-653 cells available from 5 (1984) Proc. Natl. Acad. Sci. USA81:6851), or by covalently the American Type Culture Collection, Rockville, Md. USA. joining to the immunoglobulin coding sequence all or part of Human myeloma and mouse-human heteromyeloma cell the coding sequence for a non-immunoglobulin polypeptide. lines also have been described for the production of human Typically such non-immunoglobulin polypeptides are Sub monoclonal antibodies (Kozbor, J. Immunol. 133:3001 stituted for the constant domains of an antibody, or they are (1984); and Brodeur et al., Monoclonal Antibody Production 10 Substituted for the variable domains of one antigen-combin Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., ing site of an antibody to create a chimeric bivalent antibody New York, 1987)). comprising one antigen-combining site having specificity for Culture medium in which hybridoma cells are growing is an antigen and another antigen-combining site having speci assayed for production of monoclonal antibodies directed ficity for a different antigen. against the antigen. Preferably, the binding specificity of 15 (iii) Humanized Antibodies monoclonal antibodies produced by hybridoma cells is deter A humanized antibody may have one or more amino acid mined by immunoprecipitation or by an in vitro binding residues introduced into it from a source which is non-human. assay, Such as radioimmunoassay (RIA) or enzyme-linked These non-human amino acid residues are often referred to as immunoabsorbent assay (ELISA). The binding affinity of the “import residues, which are typically taken from an monoclonal antibody can, for example, be determined by the “import' variable domain. Humanization can be essentially Scatchard analysis of Munson et al., Anal. Biochem., 107:220 performed following the method of Winter and co-workers (1980). (Jones et al., Nature 321:522-525 (1986); Riechmann et al., After hybridoma cells are identified that produce antibod Nature, 332:323-327 (1988); Verhoeyen et al., Science 239: ies of the desired specificity, affinity, and/or activity, the 1534-1536 (1988)), by substituting hypervariable region clones may be subcloned by limiting dilution procedures and 25 sequences for the corresponding sequences of a human anti grown by standard methods (Goding, Monoclonal Antibod body. Accordingly, such “humanized antibodies are chi ies. Principles and Practice, pp. 59-103 (Academic Press, mericantibodies (U.S. Pat. No. 4,816,567) wherein substan 1986)). Suitable culture media for this purpose include, for tially less than an intact human variable domain has been example, D-MEM or RPMI-1640 medium. In addition, the Substituted by the corresponding sequence from a non-human hybridoma cells may be grown in vivo as ascites tumors in an 30 species. In practice, humanized antibodies are typically animal. human antibodies in which some hyperVariable region resi The monoclonal antibodies secreted by the subclones are dues and possibly some FR residues are substituted by resi Suitably separated from the culture medium, ascites fluid, or dues from analogous sites in rodent antibodies. serum by conventional antibody purification procedures Such The choice of human variable domains, both light and as, for example, protein A-Sepharose, hydroxylapatite chro 35 heavy, to be used in making the humanized antibodies is very matography, gel electrophoresis, dialysis, or affinity chroma important to reduce antigenicity. According to the so-called tography. “best-fit’ method, the sequence of the variable domain of a DNA encoding the monoclonal antibodies is readily iso rodent antibody is screened against the entire library of lated and sequenced using conventional procedures (e.g., by known human variable-domain sequences. The human using oligonucleotide probes that are capable of binding spe 40 sequence which is closest to that of the rodent is then accepted cifically to genes encoding the heavy and light chains of as the human framework region (FR) for the humanized anti murine antibodies). The hybridoma cells serve as a preferred body (Sims et al., J. Immunol., 151:2296 (1993); Chothia et source of such DNA. Once isolated, the DNA may be placed al., J. Mol. Biol., 196:901 (1987)). Another method uses a into expression vectors, which are then transfected into host particular framework region derived from the consensus cells such as E. coli cells, simian COS cells, Chinese Hamster 45 sequence of all human antibodies of a particular subgroup of Ovary (CHO) cells, or myeloma cells that do not otherwise light or heavy chains. The same framework may be used for produce antibody protein, to obtain the synthesis of mono several different humanized antibodies (Carter et al., Proc. clonal antibodies in the recombinant host cells. Review Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immu articles on recombinant expression in bacteria of DNA encod mol., 151:2623 (1993)). ing the antibody include Skerra et al., Curr. Opinion in Immu 50 In another embodiment, the antibodies may be humanized mol., 5:256-262 (1993) and Plückthun, Immunol. Revs., 130: with retention of high affinity for the antigen and other favor 151-188 (1992). able biological properties. Humanized antibodies may be pre In a further embodiment, monoclonal antibodies or anti pared by a process of analysis of the parental sequences and body fragments can be isolated from antibody phage libraries various conceptual humanized products using three-dimen generated using the techniques described in McCafferty et al., 55 sional models of the parental and humanized sequences. Nature, 348:552-554 (1990). Clackson et al., Nature, 352: Three-dimensional immunoglobulin models are commonly 624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 available and are familiar to those skilled in the art. Computer (1991) describe the isolation of murine and human antibod programs are available which illustrate and display probable ies, respectively, using phage libraries. Subsequent publica three-dimensional conformational structures of selected can tions describe the production of high affinity (nM range) 60 didate immunoglobulin sequences. Inspection of these dis human antibodies by chain shuffling (Marks et al., Bio/Tech plays permits analysis of the likely role of the residues in the nology, 10:779-783 (1992)), as well as combinatorial infec functioning of the candidate immunoglobulin sequence, i.e., tion and in vivo recombination as a strategy for constructing the analysis of residues that influence the ability of the can very large phage libraries (Waterhouse et al., Nuc. Acids. Res., didate immunoglobulin to bind its antigen. In this way, FR 21:2265-2266 (1993)). Thus, these techniques are viable residues can be selected and combined from the recipient and alternatives to traditional monoclonal antibody hybridoma import sequences so that the desired antibody characteristic, techniques for isolation of monoclonal antibodies. Such as increased affinity for the target antigen?s), is achieved. US 7,994,135 B2 115 116 In general, the hyperVariable region residues are directly and 610 and 5,229.275). Human anti-CD30 antibodies are most Substantially involved in influencing antigen binding. described in U.S. patent application Ser. No. 10/338,366. Various forms of the humanized antibody are contem (v) Antibody Fragments plated. For example, the humanized antibody may be an Various techniques have been developed for the production antibody fragment, such as a Fab. Alternatively, the human 5 of antibody fragments. Traditionally, these fragments were ized antibody may be an intact antibody, Such as an intact derived via proteolytic digestion of intact antibodies (see, IgG1 antibody. e.g., Morimoto et al., Journal of Biochemical and Biophysical The Examples describe production of an exemplary Methods 24:107-117 (1992); and Brennan et al., Science, humanized anti-ErbB2 antibody. The humanized antibody 229:81 (1985)). However, these fragments can now be pro may, for example, comprise nonhuman hyperVariable region 10 residues incorporated into a human variable heavy domain duced directly by recombinant host cells. For example, the and may further comprise a framework region (FR) substitu antibody fragments can be isolated from the antibody phage tion at a position selected from the group consisting of 69H, libraries discussed above. Alternatively, Fab'-SH fragments 71 Hand 73H utilizing the variable domain numbering system can be directly recovered from E. coli and chemically coupled set forth in Kabat et al., Sequences of Proteins of Immuno 15 to form F(ab'), fragments (Carter et al., Bio/Technology 10: logical Interest, 5th Ed. Public Health Service, National Insti 163-167 (1992)). According to another approach, F(ab') tutes of Health, Bethesda, Md. (1991). In one embodiment, fragments can be isolated directly from recombinant host cell the humanized antibody comprises FR substitutions at two or culture. Other techniques for the production of antibody frag all of positions 69H, 71H and 73H. Another Example ments will be apparent to the skilled practitioner. In other describes preparation of purified trastuzumab antibody from embodiments, the antibody of choice is a single chain FV the HERCEPTINGR) formulation. fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571, (iv) Human Antibodies 894; and 5,587,458. The antibody fragment may also be a As an alternative to humanization, human antibodies can “linear antibody', e.g., as described in U.S. Pat. No. 5,641, be generated. For example, it is now possible to produce 870 for example. Such linear antibody fragments may be transgenic animals (e.g., mice) that are capable, upon immu 25 monospecific or bispecific. nization, of producing a full repertoire of human antibodies in (vi) Bispecific Antibodies the absence of endogenous immunoglobulin production. For Bispecific antibodies are antibodies that have binding example, it has been described that the homozygous deletion specificities for at least two different epitopes. Exemplary of the antibody heavy-chain joining region (J) gene in chi bispecific antibodies may bind to two different epitopes of the meric and germ-line mutant mice results in complete inhibi 30 CD30 protein. Alternatively, an anti-CD30 arm may be com tion of endogenous antibody production. Transfer of the bined with an arm which binds to a Fc receptors for IgG human germ-line immunoglobulin gene array in Such germ (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII line mutant mice will result in the production of human anti (CD16) so as to focus cellular defense mechanisms to the bodies upon antigen challenge. See, e.g., Jakobovits et al., CD30-expressing cell. Bispecificantibodies may also be used Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., 35 to localize cytotoxic agents to cells which express CD30. Nature, 362:255-258 (1993); Bruggermann et al., Year in Traditional production of full length bispecific antibodies Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589, is based on the coexpression of two immunoglobulin heavy 369 and 5,545,807. chain-light chain pairs, where the two chains have different Alternatively, phage display technology (McCafferty et al., specificities (Millstein et al., Nature, 305:537-539 (1983)). Nature 348:552-553 (1990)) can be used to produce human 40 Because of the random assortment of immunoglobulin heavy antibodies and antibody fragments in vitro, from immunoglo and light chains, these hybridomas (quadromas) produce a bulin variable (V) domain gene repertoires from unimmu potential mixture of 10 different antibody molecules, of nized donors. According to this technique, antibody V which only one has the correct bispecific structure. Purifica domain genes are cloned in-frame into eithera major or minor tion of the correct molecule, which is usually done by affinity coat protein gene of a filamentous bacteriophage. Such as 45 chromatography steps, is rather cumbersome, and the product M13 orfa, and displayed as functional antibody fragments on yields are low. Similar procedures are disclosed in WO the Surface of the phage particle. Because the filamentous 93/08829, and in Traunecker et al., EMBO.J., 10:3655-3659 particle contains a single-stranded DNA copy of the phage (1991). According to a different approach, antibody variable genome, selections based on the functional properties of the domains with the desired binding specificities (antibody-an antibody also result in selection of the gene encoding the 50 tigen combining sites) are fused to immunoglobulin constant antibody exhibiting those properties. Thus, the phage mimics domain sequences. The fusion preferably is with an immu some of the properties of the B-cell. Phage display can be noglobulin heavy chain constant domain, comprising at least performed in a variety of formats; for their review see, e.g., part of the hinge, CH2, and CH3 regions. It is preferred to Johnson, Kevin S, and Chiswell, David J. Current Opinion in have the first heavy-chain constant region (CH1) containing Structural Biology 3:564-571 (1993). Several sources of 55 the site necessary for light chain binding, present in at least V-gene segments can be used for phage display. Clackson et one of the fusions. DNAs encoding the immunoglobulin al., Nature, 352:624-628 (1991) isolated a diverse array of heavy chainfusions and, if desired, the immunoglobulin light anti-oxazolone antibodies from a small random combinato chain, are inserted into separate expression vectors, and are rial library of V genes derived from the spleens of immunized co-transfected into a suitable host organism. This provides for mice. A repertoire of V genes from unimmunized human 60 great flexibility in adjusting the mutual proportions of the donors can be constructed and antibodies to a diverse array of three polypeptide fragments in embodiments when unequal antigens (including self-antigens) can be isolated essentially ratios of the three polypeptide chains used in the construction following the techniques described by Marks et al., J. Mol. provide the optimum yields. It is, however, possible to insert Biol. 222:581-597 (1991), or Griffith et al., EMBO.J. 12:725 the coding sequences for two or all three polypeptide chains 734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573, 65 in one expression vector when the expression of at least two 905. As discussed above, human antibodies may also be gen polypeptide chains in equal ratios results in high yields or erated by in vitro activated B cells (see U.S. Pat. Nos. 5,567, when the ratios are of no particular significance.