(12) Unlted States Patent (10) Patent No.: US 7,740,847 B2 Allan Et Al

US007740847B2

(12) Unlted States Patent (10) Patent No.: US 7,740,847 B2 Allan et al. (45) Date of Patent: Jun. 22, 2010

(54) VARIANT FC REGIONS 5,550,362 A 8/1996 Sherman 5,595,721 A 1/1997 Kaminski et a1. (75) Inventors: Barrett Allan, Encinitas, CA (US); 5,624,321 A 4/1997 winter et a1, Weidong Jiallg, Sunnyvale, CA(US); 5,648,260 A 7/1997 Winter etal. Yillg Tang, San Die/g9’ CA (Us); Jeffry 5,677,180 A 10/1997 Robinson et a1. Dean Watkins’ Enclmtas, CA (Us) 5,721,108 A 2/1998 Robinson et a1. (73) Assignee: Applied Molecular Evolution, Inc., San 5’736’l37 A 4/1998 Anderson et al' Diego’ CA (Us) 5,776,456 A 7/1998 Anderson et a1. 5,843,398 A 12/1998 Kaminski et a1. ( * ) Notice: Subject to any disclaimer, the term of this 5,843,439 A 12/1998 Anderson et a1. patent is extended or adjusted under 35 5,985,599 A 11/1999 McKenzie et a1. U-S-C- 154(1)) by 195 days- 6,015,542 A 1/2000 Kaminskietal. 6,090,365 A 7/2000 Kaminski et a1. (21) Appl' NO‘: 11/572’634 6,120,767 A 9/2000 Robinson et a1. (22) PCT FiladZ Jul- 18, 2005 6,121,022 A 9/2000 Presta et a1. 6,165,745 A 12/2000 Ward et a1. (86) PCT No.: PCT/US2005/025276 6,171,586 B1 1/2001 Lam etal. 6,183,744 B1 2/2001 Goldenberg §371 (0X1), 6,187,287 B1 2/2001 Leung etal. (2)’ (4)132“: Jan- 25’ 2007 6,194,551 B1 2/2001 Idusogie et a1. (87) PCT Pub‘ NO‘: W02006/020114 6,204,023 B1 3/2001 Robinson et a1. 6,224,866 B1 5/2001 Barbera-Guillem PCT pub Date; Feb 23, 2006 6,242,195 B1 6/2001 Idusogie et a1. 6,277,375 B1 8/2001 Ward (65) Prior Publication Data 6,287,537 B1 9/2001 Kaminski et a1. Us 2007/0224188 A1 Sep. 27, 2007 6,306,393 B1 10/2001 Goldenberg

Related US. Application Data (60) Provisional application No. 60/ 598,855, ?led on Aug. (Continued) 4, 2004, prov1s1onal appl1cat1on No. 60/602,953, ?led onAug. 19, 2004, provisional application No. 60/604, FOREIGN PATENT DOCUMENTS 339, ?led on Aug. 25, 2004, provisional application No. 60/609,101, ?led on Sep. 10, 2004, provisional W0 WO 88/04936 7/1988 application No. 60/638,442, ?led on Dec. 23, 2004, provisional application No. 60/643,718, ?led on Jan. 13’ 2005' (Continued) (51) Int- 0- OTHER PUBLICATIONS A61K 39/395 (2006.01) C07K 16/28 (2006.01) Carter et al., “HumaniZation of an anti-pl85HER2 Antibody for C07K 16/30 (2006.01) Human CancerTherapy,”Proc.Natl.Acad. Sci.USA, vol. 89,No. l0, C12N 15/13 (2006.01) PP~ 4285-4289 (May 1992) C12N 15/63 (2006.01) . C12N 15/16 (2006.01) (Commued) (52) US. Cl...... 424/1331; 424/1441; 424/1531; Primary ExamineriROn schwadron 424/1551; 424/1561; 424/1731; 424/1741; (74) Anomeyl Agem, 0r FirmiROben L, Sharp; MaryAnn 424/1431; 435/69.6; 435/320.1; 435/325; wiskerchen 435/326; 435/328; 435/343.1; 435/344; 435/344.1; 435/358; 530/387.3; 530/388.22; 530/388.73; (57) ABSTRACT 530/38.8; 530/388.85; 536/2353 (58) Field of Classi?cation Search ...... None See application ?le for complete search history. The present invention provides humanized anti-CD20 anti (56) References Cited bod1es compr1s1ng a human IgG1 Fc reg1on compnsmg an isoleucine at position 247 and a glutamine at position 339 as US. PATENT DOCUMENTS Well as nucleic acids encoding the antibodies and methods of using the antibodies for treating lymphoma. Furthermore, the 4,861,579 A 8/1989 Meyer et a1. invention provides compositions comprising the antibodies 5,348,876 A 9/1994 Michaelsen et a1. and methods of producing them. 5,354,847 A 10/1994 Liu et a1. 5,545,404 A 8/1996 Page 5,545,405 A 8/1996 Page 13 Claims, 8 Drawing Sheets US 7,740,847 B2 Page 2

U.S. PATENT DOCUMENTS W0 WO 2004/029207 A2 4/2004 W0 WO 2004/035607 A2 4/2004 6,399,061 6/2002 Anderson et al. W0 WO 2004/056312 A2 7/2004 6,455,043 9/2002 Grillo-LopeZ W0 WO 2004/063351 A2 7/2004 6,528,624 3/2003 Idusogie et al. W0 WO 2004/074455 A2 9/2004 6,538,124 3/2003 Idusogie et al. W0 W0 2004/ 091657 A2 10/2004 6,565,827 5/2003 Kaminski et al. W0 W0 2004/ 099249 A2 11/2004 6,652,852 11/2003 Robinson et al. W0 WO 2004/103404 A1 12/2004 6,682,734 1/2004 Anderson et al. W0 WO 2005/000901 A2 1/2005 6,737,056 5/2004 Presta W0 WO 2005/016969 A2 2/2005 6,821,505 11/2004 Ward W0 WO 2005/044859 A2 5/2005 6,893,625 5/2005 Robinson et al. W0 WO 2005/056606 A2 6/2005 7,083,784 8/2006 Dall’Acqua et al. W0 WO 2005/070963 A1 8/2005 2002/0164326 11/2002 Young et a1. W0 WO 2005/103081 A2 11/2005 2002/0197256 12/2002 GreWal W0 WO 2006/019447 A1 2/2006 2003/0219433 11/2003 Hansen et al. W0 WO 2006/020114 A2 2/2006 2004/0002587 1/2004 Watkins et al. W0 WO 2006/042240 A2 4/2006 2004/0185045 9/2004 Koenig et a1. W0 WO 2006/053301 A2 5/2006 2005/0054832 3/2005 LaZar et al. W0 WO 2006/064121 A2 6/2006 2005/0202023 9/2005 Ledbetter et al. W0 WO 2006/076651 A2 7/2006 2006/0024300 A1 2/2006 Adams et al. W0 WO 2006/084264 A2 8/2006 2006/0034836 A1 2/2006 Gillies et al. W0 WO 2006/085967 A2 8/2006 2006/0246004 A1 11/2006 Adams et al. W0 WO 2006/105338 A2 10/2006 2006/0251652 A1 11/2006 Watkins et al. OTHER PUBLICATIONS FOREIGN PATENT DOCUMENTS Clynes et al., “Inhibitory Fc Receptors Modulate In Vivo CytoXicity W0 WO 88/07089 9/1988 Against Tumor Targets,” Nature Med., vol. 6, No. 4, pp. 443-446 W0 WO 92/07466 5/1992 (Apr. 2000). W0 WO 92/16562 10/1992 Eisenberg, R, et al., “The therapeutic potential of anti-CD20 What do W0 WO 94/11026 5/1994 B-cells do?,” Clinical Immunology, vol. 117, pp. 207-213, 2005. W0 WO 94/29351 12/1994 Glennie, MJ, et al., “Renaissance of cancer therapeutic antibodies,” W0 WO 95/03770 2/1995 Drug Discovery Today, vol. 8, No. 11, pp. 503-510, (Jun. 1, 2003). W0 WO 95/05468 2/1995 Gopal, AK, et al., “Clinical applications of anti-CD20 antibodies,” W0 WO 97/28267 8/1997 Journal of Laboratory and Clinical Medicine, vol. 134, No. 5, pp. W0 WO 97/34631 9/1997 445-450, 1999. W0 WO 97/44362 11/1997 Grossbard, ML, et al., “Monoclonal Antibody-Based Therapies of W0 WO 98/05787 2/1998 Leukemia and Lymphoma,” Blood, vol. 80, No. 4, pp. 863-878 (Aug. W0 WO 98/23289 6/1998 15, 1992). W0 WO 98/52975 11/1998 Hong, K, et a1 , “Simple quantitative live cell and anti-idiotypic based W0 WO 98/56418 12/1998 ELISA for humanized antibody directed to cell surface protein W0 WO 98/58964 12/1998 CD20,” Journal of Immunological Methods vol. 294, pp. 189-197 W0 WO 99/22764 5/1999 (2004). W0 WO 99/51642 10/1999 Idusogie, EE, et al., “Mapping of the Clq Binding Site on RituXan, A W0 WO 99/58572 11/1999 Chimeric Antibody With a Human IgGl Fc,” Journal of Immunology, W0 WO 00/09160 2/2000 vol. 164, No. 8, pp, 4178-4184 (2000). W0 WO 00/09560 2/2000 Idusogie, EE, et al., “Engineered Antibodies With Increased Activity W0 WO 00/20864 4/2000 to Recruit Complement,” Journal of Immunology, vol. 166, No. 4, pp, W0 WO 00/27428 5/2000 2571-2575 (Feb. 15,2001). W0 WO 00/27433 5/2000 Jones, PT, et al., “Replacing the Complementarity-Determining W0 WO 00/29584 5/2000 Regions in a Human Antibody With Those From a Mouse” Nature W0 WO 00/42072 7/2000 321:522-525 (May 29, 1986). W0 WO 00/44788 8/2000 Liu, AY, et al., “Production of a Mouse-Human Chimeric Monoclonal W0 WO 00/67795 11/2000 Antibody to CD20 With Potent Fc-Dependent Biologic Activity,” W0 WO 00/67796 11/2000 Journal ofImmunology, vol. 139, No. 10, pp. 3521-3526 (1987). W0 WO 00/74718 12/2000 Longo, DL, “Immunotherapy for non-Hodgkin’s lymphoma”, Cur W0 WO 00/76542 A1 12/2000 rent Opinion in Oncology, vol. 8, pp. 353-359, 1996. W0 WO 01/03734 A1 1/2001 Maloney, DG, et al., “Newer Treatments for Non-Hoggkin’s W0 WO 01/10460 A1 2/2001 Lymphoma: Monoclonal Antibodies”, Oncology, vol. 12, No. 10, W0 WO 01/10462 2/2001 Supplement No. 8, pp, 63-76, (Oct. 1998). W0 WO 01/13945 A1 3/2001 Martin, F, et al., “Pathogenic roles of B cells in Human Autoim W0 WO 01/74388 10/2001 munity; Insights from the Clinic,” Immunity, vol. 20, No. 5, pp. W0 WO 01/79299 10/2001 517-527 (May 2004). W0 WO 01/80884 A1 11/2001 Nadler, LM, et al., “A Unique Cell Surface Antigen Identifying W0 WO 01/97858 A2 12/2001 Lymphoid Malignancies of B Cell Origin,” J. Clin. Invest, vol. 67, pp. W0 WO 02/34790 A1 5/2002 134-140, (Jan. 1981). W0 W0 02/060919 A2 8/2002 Paul, WE, “Fv Structure and Diversity in Three Dimensions,” Fun W0 W0 02/060955 A2 8/2002 damental Immunology, 3”’ Edition, pp. 292-295, (1993). W0 W0 02/079255 A1 10/2002 Riechman, L, et al., “Reshaping human antibodies for therapy,” W0 W0 03/002607 A1 1/2003 Nature, vol. 332, pp. 323-327, (Mar. 1988). W0 W0 03/061694 A1 7/2003 Rudikoff, S, et al., “Single Amino Acid Substitution Altering Anti W0 W0 03/068821 A2 8/2003 gen-Binding Speci?city,” Proceedings of the National Academy of W0 W0 03/074679 A2 9/2003 Sciences, vol. 79, pp. 1979-1983 (1982). US 7,740,847 B2 Page 3

Shields, RL, et al., “High Resolution Mapping of the Binding Site on Brown, B.A. et a1 ., “Tumor-Speci?c Genetically Engineered Murine/ Human IgGl for FcyRI, FcryRII, and FcRn and Design of IgGi Human Chimenc Monoclonal Antibody,” Cancer Research, vol. 47: Variants With Improved Binding to the FcyR*,” Journal of Biological pp. 3577-3583 (Jul. 1987). Chemistry, vol. 276, No. 9, pp. 6591-6604, Mar. 2, 2001. Datta-Mannan, et al., “HumaniZed IgGI Variants With Differential Binding Properties to the Neonatal Fc Receptor: Relationship to Tedder, TF, et al., “Isolation and Structure of a cDNA Encoding the Pharmacokinetics in Mice and Primates” (2007) Drug Metabolism Bl (CD20) Cell-Surface Antigen of Human B Lymphocytes,” Proc. and Disposition, 35: 1-9. Natl. Acad. Sci. USA, vol. 85, pp. 208-212 (Jan. 1988). Datta-Mannan, et al.. “Monoclonal Antibody Clearance: Impact of Teeling J L, et al., “Characterization of new human CD20 monoclonal modulating the interaction of I gG With FcRn*” Journal ofBiological antibodies With potent cytolytic activity against non-Hodgkin’s Chemistry, 282(3), pp. 1709-17-17 (2007). lymphomas,” Blood, vol. 104, No. 6, pp. 1793-1800 (Sep. 15, 2004). Datta-Mannan, et al., Vanant Mabs With enhanced FcRn binding: Disposition in mice and cynomolgus monkeys 2006 AAPS National Teeling JL, et al., “The Biological Activity of Human CD20 Biotechnology Meeting. Monoclonal Antibodies Is Linked to Unique Epitopes on CD201” Liang, Y, et al., “CD20 as an Immunotherapy Target,” CD20 Wiley Journal of Immunology, pp. 362-371 (2006). Encyclopedia ofMolecular Medicine, pp. 562-564 (Jan. 15, 2002). Valentine, MA, et al., “Structure and function of the B-cell speci?c Press et al., “Monoclonal AntibodyI F5 (Anti-CD20) Serotherapy of 35-37 kDa CD20 protein,” Leukocyte Typing III, McMichael, Ed., Human B Cell Lymphomas” Blood, vol. 69:2, pp. 584-591 (Feb. Oxford University Press, pp. 440-443 (1987). 1987). US. Patent Jun. 22, 2010 Sheet 1 of8 US 7,740,847 B2

FIG. 1

\\ I’,

US. Patent Jun. 22, 2010 Sheet 5 of8 US 7,740,847 B2

FIG. 5

A. SEQ ID NO:29: AME 133 complete light chain amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

— Constant Region is underlined

B. SEQ ID NO:30: AME 133 complete light chain nucleic acid sequence GAAATTGTGTTGACG'CAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGCTCAAGTGTACCG'I‘ACATCCACTGGTACCAGCAGAAACCTGGC CAGGCTCCCAGGCTCCTCATCTATGCCACATCCGCTCTGGCTTCTGGCATCCCAGACAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAA GATTTTGCAGTGTATTACTGTCAGCAGTGGCTGAGTAACCCACCCACTTTTGGCCAGGGG ACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCT GATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC'C'I‘GC'I‘GAATAACT'I‘CTATCCC AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG US. Patent Jun. 22, 2010 Sheet 6 of8 US 7,740,847 B2

FIG. 6

A. SEQ ID NO:31: AME 133 (247I/339Q variant) complete heavy chain amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSY NQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKWEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKIKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKQKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGWFSCSWHEALHNHYTQKSLSLSPGK

— Constant Region is underlined; variants are bolded

B. SEQ ID NO:32: AME 133 (247I/339Q variant) complete heavy chain nucleic acid sequence GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATC TCC'I‘GTAAGGGTTCTGGCCGTACATTTACCAGTTACAATATGCACTGGGTGCGCCAGATG CCCGGGAAAGGCCTGGAGTGGATGGGGGCTATTTATCCCTTGACGGGTGATACTTCCTAC AATCAGAAGTCGAAACTCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATCGACT TAC‘GTGGGCGGTGACTGGCAGTTCGATGTCTGGGGCAAGGGGACCACGGTCACCGTCTCC TCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC'I‘ACTTCCCCGAACCGGTGACGGTG TCG‘I‘GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTTGAG CCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAAZEQAAGGACACCCTCA'I‘GATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAG‘CCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAEAQAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAC GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG'I'CAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAG'I‘GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGC'I‘GGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG 'I‘GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

— Variants are bolded and underlined US. Patent Jun. 22, 2010 Sheet 7 of8 US 7,740,847 B2

FIG. 6

C. SEQ ID NO:33: AME 133 (247I/339D) variant complete heavy chain amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSY NQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICN'VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKIKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKDKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

— Constant Region is underlined; variants are bolded

D. SEQ ID NO:34: AME 133 (247I/339D) variant complete heavy chain nucleic acid sequence

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATC TCCTGTAAGGGTTCTGGCCGTACATTTACCAGTTACAATATGCACTGGGTGCGCCAGATG CCCGGGAAAGGCCTGGAGTGGATGGGGGCTATTTA'I‘CCCT'I‘GACGGGTGATACTTCCTAC AATCAGAAGTCGAAACTCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATCGACT TACGTGGGCGGTGACTGGCAGTTCGATGTCTGGGGCAAGGGGACCACGGTCACCGTCTCC TCAGCC'I‘CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG TCG'I‘GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG'I‘GCCCTCCAGCAGCTTGGGCACCCAG ACCTACATC'I‘GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTTGAG CCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCC‘CAAAAgEAAGGACACCC'I‘CATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAQPEAAAGGGCAGCCCCGAG'AACCACAG'GTGTACACCCTGCCCCCATCCCGGGAC GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGAC'I'CCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC'I‘AC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

— Variants are bolded and underlined US. Patent Jun. 22, 2010 Sheet 8 of8 US 7,740,847 B2

FIG. 6

E. SEQ ID NO:35: AME 133 (378D) variant complete heavy chain amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSY NQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIDVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMI-IEALHNHYTQKSLSLSPGK

— Constant Region is underlined; variant is bolded

F. SEQ ID NO:36: AME, 133 (378D) variant complete heavy chain nucleic acid sequence

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATC‘ TCCTG'I‘AAGGGTTCTGGCCGTACATTTACCAGTTACAATATGCACTGGGTGCGCCAGATG CCCGGGAAAGGCCTGGAGTGGATGGGGGCTATTTATCCCTTGACGGGTGATACTTCCTAC AATCAGAAGTCGAAACTCCAGGTCACCA'I‘CTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATCGAC'I‘ TACGTGGG‘CGGTGACTGGCAGTTCGATGTCTGGGGCAAGGGGACCACGGTCACCGTCTCC ‘I‘CAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC'I‘CCAAGAGCACCTC'I‘ GGGGGCACAGCGGCCCTGGGCTGCCTGG'I'CAAGGACTACTTCCCCGAACCGGTGACGGTG TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG'I‘GGACAAGAAGGTTGAG CCCAAATC'I‘TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAC' GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCEZEGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

- Variant is bolded and underlined US 7,740,847 B2 1 2 VARIANT FC REGIONS ticles, clearance of immune complexes, release of in?amma tory mediators, placental transfer of the antibody and control This application claims the bene?t of US. ProvisionAppli of immunoglobulin production, (ii) complement-dependent cations With Serial Nos.; and PCT Application Ser. Nos.: cytotoxicity (“CDC”) in Which the Fc region binds the Clq 60/598,855 ?led Aug. 4, 2004; 60/602,953 ?led Aug. 19, component of complement and thereby initiates the classical 2004; 60/604,339 ?ledAug. 25, 2004; 60/609,101 ?led Sep. pathWay of complement activation Which leads to lysis of the 10, 2004; 60/638,442 ?led Dec. 23, 2004; 60/643,718 ?led Jan. 13, 2005; International Application Number: PCT/ target, (iii) antibody dependent cell-mediated cytotoxicity US2005/025276 ?led Jul. 18, 2005; all ofWhich are expressly (“ADCC”) in Which certain human immune system cells, e. g., incorporated herein by reference in their entirely. phagocytes and NK cells, via an Fcy receptor, bind to the Fc region of an antibody via speci?c antibody-binding receptors FIELD OF THE INVENTION on the immune cells and subsequently signal destruction of the entity to Which the antibody is bound, and, (iv) binding to The present invention relates to polypeptides comprising a mast cells, basophils, and eosinophils. The a?inity With novel, variant Fc region. Speci?cally, a novel, variant Fc Which an Fc region can bind a particular FcR (e.g., FcRn), or region of the present invention comprises at least one amino the level With Which an Fc region can mediate CDC orADCC acid substitution described herein that confers an altered activity are important factors for determining the ef?cacy and effector function or altered serum half-life upon an immuno half-life of therapeutic proteins, particularly monoclonal globulin comprising the variant Fc region as compared to the antibodies. parent immunoglobulin lacking that amino acid substitution. 20 ParticulariZed modi?cation of amino acids in the Fc region Furthermore, the invention provides a method for altering an of human IgG is an active area of study yielding structure effector function of a monoclonal antibody or extending the serum half-life of a polypeptide to Which a variant Fc region function relationship information relevant to development of of the invention is operably attached. Therapeutic uses of therapeutic proteins, particularly monoclonal antibodies (see, e.g., US. Pat. No. 6,165,745 and PCT Publication No. polypeptides, proteins, particularly monoclonal antibodies, 25 comprising a variant Fc region of the invention are disclosed. WO2004/035752 regarding alteration of serum half-life of a polypeptide operably linked to an Fc region and US. Pat. No. BACKGROUND OF THE INVENTION 6,737,056 and PCT Publication No. WO2004/ 029207 regard ing alteration of an effector function of a monoclonal anti There are at least seventeen monoclonal antibodies cur 30 body comprising a modi?ed Fc region). rently approved in the United States for use as human thera The development of novel therapeutic proteins, particu peutics. Additionally, there are several hundred monoclonal larly monoclonal antibodies, Would bene?t from the ability to antibodies in clinical trials and thousands in pre-clinical test rationally design an Fc region With particular amino acid ing for treatment of various diseases or disorders including, modi?cations that confer a desired bene?cial property upon e.g., transplant rejection, cancer, in?ammatory diseases, sep 35 the antibody of interest. All monoclonal antibodies Would not sis, nephritis, AlZheimer’s disease, allergies, diabetes, be expected to be improved as a therapeutic due to the same autoimmune diseases, arthritis, multiple sclerosis, and infec particular amino acid modi?cation in the Fc region. A thera tious diseases. The ?eld of therapeutic monoclonal antibodies is positioned for rapid groWth in the coming years. After peutic monoclonal antibody that binds one target antigen may bene?t from an increase in a particular effector function While vaccines, antibodies (or immunoglobulins, “Ig”) constitute 40 the second most common type of biopharrnaceutical agent a different therapeutic monoclonal antibody that binds a dif being tested clinically (StockWin, L. H. et al. Biochemical ferent target antigen may bene?t from an increase in a differ Society Transactions, 311433-436, 2003). ent effector function, or even a decrease. One therapeutic Genetic engineering has contributed substantially to monoclonal antibody may bene?t from the ability to bind a groWth of the ?eld of therapeutic monoclonal antibodies. The 45 particular Fc receptor With greater a?inity While another anti effectiveness of a potential therapeutic monoclonal antibody body may be improved as a therapeutic by binding that Fc Will often vary With modest changes to the protein sequence receptor at a loWer a?inity and therefore being cleared from of the antibody. A single amino acid change in the variable the body at a faster rate. Furthermore, a particular Fc region region of a monoclonal antibody has the potential to alter the amino acid modi?cation or substitution and resulting effect a?inity With Which the antibody binds the antigenic epitope, 50 that Would bene?t a therapeutic antibody may depend upon as Well as antibody properties such as K0” rate or Kof rate. the antigenic target to Which the antibody binds and/or the Such amino acid changes may determine the success or fail disease or disorder to be ameliorated by the antibody. ure of a monoclonal antibody as a therapeutic. Similarly, Methods and compositions that alter particular effector modest changes in the amino acid sequence of the Fc region functions associated With the Fc region of an antibody are of a monoclonal antibody may yield profound changes in the 55 antibody’s effector function properties or the half-life of a necessary to improve the properties of existing therapeutic protein to Which the Fc region is operably linked. antibodies as Well as to generate novel therapeutic antibodies The Fc region of an antibody (i.e., the carboxy-terminal With desired properties. Monoclonal antibodies With variant ends of the heavy chains of an antibody spanning domains Fc regions may be used to treat various diseases or disorders CH2, CH3 and a portion of the hinge region (see FIG. 1)), is 60 including, e.g., in?ammatory disorders, cancer, autoimmune limited in variability and is involved in effecting the physi disorders, cell-signalling disorders and infectious diseases. ological roles played by the antibody. The effector functions Additionally, methods and compositions that alter the serum attributable to the Fc region of an antibody vary With the class half-life of a therapeutic protein, either increasing the half and subclass of antibody and include (i) binding of the anti life and thereby alloWing for feWer doses or decreasing the body via the Fc region to a speci?c Fc receptor (“FcR”) on a 65 half-life and thereby alloWing for more rapid clearance from cell Which triggers various biological responses including, the body, Would bene?t the generation of therapeutic antibod e.g., phagocytosis and destruction of antibody-coated par ies as Well as other therapeutic proteins. US 7,740,847 B2 3 4 What is needed in order to improve the ef?cacy of a thera 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, peutic protein, particularly a monoclonal antibody, are vari 332G, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, ant Fc regions With improved properties. 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341E, 341H, 341I, 341K, SUMMARY OF THE INVENTION 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343G, 343H, 343I, The present invention provides variant Fc regions, i.e., Fc 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, regions comprising an amino acid substitution described 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, herein (e.g., see Table 1), that confer bene?cial properties 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, upon polypeptides comprising said variant Fc regions. 375R, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N, Fc positions of a parent Fc region at Which any amino acid 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, substitution may be made to generate a variant Fc region of 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, the invention include positions 279, 341, 343 and 373 of the 380$, 380T, 382D, 382E, 382H, 382I, 382K, 382L, 382M, Fc region, Wherein the numbering of the residues, i.e., their 382N, 382P, 382Q, 382R, 382$, 382T, 382V, 382W, 382Y, position number, in the Fc region is that of the EU index as in 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, Kabat (see FIG. 2 herein). The present invention provides 427N, 429A, 429F, 429M, 430A, 430D, 430E, 430G, 430H, variant Fc regions comprising an amino acid substitution at 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, position 279, 341, 343 or 373 of a parent Fc region, or any 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, combination thereof. The parent Fc region may optionally 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, have non-native amino acid residues at positions other than 20 440E, 440E, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 279,341,343 and 373. The native amino acid residues at these 440T, 440V or 442K. positions for human IgG are valine (279), glycine (341), In a preferred embodiment, a variant Fc region comprises proline (343) and tyrosine (373). at least 1, 2, 3 or more amino acid substitutions selected from In preferred embodiments throughout the present inven the following: 235G, 236F, 236R, 236Y, 237K, 237N, 237R, tion, the amino acid residue substituted for that present in the 25 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 245R, parent Fc region is a naturally occurring amino acid residue. 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247T, Unless otherWise stated, the parent Fc region may be a native 247W, 247Y, 248E, 248P, 248Q, 248W, 249L, 249M, 249N, or non-native Fc region, preferably of human origin or sub 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, stantially of human origin. The amino acid sequence of the 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R, parent Fc region is preferably that as shoWn in SEQ ID NOs: 30 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, l, 2, 3 or 4. Preferably the parent Fc region has a native amino 256W, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, acid residue present at the position Which is to be substituted 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, to generate a variant Fc region of the invention. Furthermore, 271T, 272H, 272K, 272R, 279A, 279D, 279G, 279H, 279N, throughout, it is understood that a variant Fc region is a parent 279Q, 279S, 279T, 279W, 279Y, 280T, 283E, 283H, 283K, Fc region modi?ed to comprise at least one amino acid sub 35 283M, 283R, 283W, 285N, 286E, 288N, 288P, 292E, 292G, stitution as described herein. Additionally, it is understood 292I, 301W, 304E, 307E, 307M, 312P, 315F, 315L, 315P, that a parent Fc region may be a full-length Fc or a portion 316F, 317P, 317T, 318N, 318P, 318T, 332L, 332M, 332R, thereof comprising the amino acid residue to be substituted to 332S, 332W, 339D, 339F, 339I, 339K, 339M, 339N, 339Q, generate the variant Fc region. 339R, 339S, 339W, 339Y, 341D, 341E, 341E, 341H, 341I, The present invention further provides polypeptides, pref 40 341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, erably monoclonal antibodies, comprising a variant Fc region 341V, 341W, 341Y, 343D, 343E, 343G, 343H, 343K, 343N, (or a functional fragment thereof) comprising at least one 343Q, 343R, 343S, 343T, 343W, 343Y, 373D, 373E, 373G, amino acid substitution at position 279, 341, 343 or 373 as 373H, 373I, 373K, 373L, 373M, 373Q, 373R, 373S, 373T, compared to the parent Fc region. The variant Fc region 373W, 375R, 376G, 376N, 376P, 376Q, 376R, 376S, 376T, comprising at least one amino acid substitution at Fc position 45 376V, 376W, 376Y, 377G, 377K, 377P, 378D, 378N, 379N, 279, 341, 343 or 373 may further comprise at least one addi 379Q, 379T, 380N, 380$, 380T, 382D, 382E, 382I, 382K, tional amino acid substitution in the Fc region as compared to 382L, 382Q, 382R, 382$, 382T, 382V, 382W, 382Y, 385E, the amino acid residue present in the native Fc region of the 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, 429A, same type as the variant Fc region. 429F, 429M, 430A, 430D, 430E, 430G, 430H, 430I, 430K, In one embodiment, a variant Fc region (i.e., a variant of a 50 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, parent Fc region) comprises at least 1, 2, 3 or more amino acid 430W, 430Y, 431H, 431K, 431P, 432R, 4328, 438K, 438L, substitutions selected from the folloWing: 235G, 235R, 236E, 438T, 438W, 439E, 440D, 440I or 440L. 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, The variant Fc regions of the present invention are prefer 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, ably characterized using one of more of the experimental 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 55 methods described herein. Such variant Fc regions confer an 248E, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, altered effector function or altered serum half-life upon a 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, monoclonal antibody that comprises the variant Fc region or 254K, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, an altered serum half-life upon a polypeptide to Which the 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, variant Fc region is operably attached. 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 60 Preferably the parent Fc region of a variant Fc region of the 2648, 265K, 2658, 267H, 267I, 267K, 268K, 269N, 269Q, invention is a native or germline-encoded Fc region of human 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, origin selected from the group consisting of IgG, IgA, IgE, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, IgM and IgD or a polymorphic variant thereof, or a functional 279T, 279W, 279Y, 280T, 283E, 283G, 283H, 283I, 283K, fragment thereof. Preferably the parent Fc region is an I gG Fc 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286E, 65 region, and more preferably, an IgGl, IgG3, or IgG4 Fc 288N, 288P, 292E, 292F, 292G, 292I, 292L, 293S, 293V, region. The parent Fc region may optionally comprise one or 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 3l5P, more additional amino acid substitution(s) as compared to the US 7,740,847 B2 5 6 native Fc region, other than those described herein (i.e., those 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, substitutions listed in Table 1), particularly one or more 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, amino acid substitutions knoWn in the art or as described in 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, Us. Pat. Nos. 6,165,745 or 6,737,056; or PCT Publication 440E, 440G, 440H, 4401, 440K, 440L, 440M, 440Q, 440T, Nos. WO2004/ 035752 or WO2004/029207 (all of Which are 440V or 442K, or (iii) at least tWo amino acid substitutions as incorporated herein in their entirety); such amino acid sub listed in (i) or (ii) above, or (iv) at least 1, 2 or 3 amino acid stitution(s), if present in the parent Fc region, Would then also substitutions as listed in (i) or (ii) above in addition to at least be present in the variant Fc region of the invention and in a one Fc region amino acid substitution not listed in (i) or (ii) polypeptide comprising a variant Fc region of the invention, above. Preferably the altered effector function is an increase unless it Was at a position subsequently substituted to gener in ADCC, a decrease in ADCC, an increase in CDC, a ate the variant Fc region. decrease in CDC, an increase in Clq binding af?nity, a The invention provides a polypeptide, preferably a mono decrease in Clq binding a?inity, an increase in FcR (prefer clonal antibody, comprising a variant Fc region of the inven ably FcRn) binding a?inity or a decrease in FcR (preferably tion, or a functional fragment thereof. In a preferred embodi FcRn) binding af?nity as compared to said polypeptide lack ment, a monoclonal antibody comprising a variant Fc region ing the amino acid substitution in the Fc region (i.e., parent Fc of the invention is a chimeric antibody. In a more preferred region). embodiment, a monoclonal antibody comprising a variant Fc The invention provides a monoclonal antibody comprising region of the invention is a humaniZed antibody or a human a variant Fc region comprising at least one of the folloWing antibody in Which framework sequence and constant region amino acid substitutions in the Fc region: 247A, 247E, 247M, sequence present in the antibody is substantially of human 20 247T, 247V, 247Y, 249E, 249Y, 254P, 254M, 254Y, 256A, origin. The chimeric, humaniZed or human antibody is pref 258D, 279A, 283A, 2831, 283K, 283M, 283R, 288N, 292A, erably a full-length antibody or a single chain antibody. When 311A, 311D, 311N, 311T, 311V, 311Y, 315L, 318N, 318P, a monoclonal antibody comprising a variant Fc region of the 318T, 318V, 332T, 332V, 339D, 339E, 339G, 3391, 339K, invention is to be used as a human therapeutic, the Fc region 339M, 339N, 339Q, 339R, 339S, 339T, 376A, 376V, 377G, is preferably substantially of human origin. 25 377K, 379N, 380N, 380S, 382A, 3821, 385E, 427N, 429M, Preferably a polypeptide comprising, or operably attached 434W, 4361, 440G, 440H, 4401 or 440L, [preferably 247A, to, a variant Fc region of the invention (i.e., “variant polypep 247E, 247M, 247T, 247V, 247Y, 254P, 254Y, 258D, 279A, tide”) has at least one amino acid substitution in the variant Fc 283M, 288N, 292A, 311D, 311N, 311T, 315L, 318N, 318P, region as compared to the parent Fc region, and displays an 318T, 318V, 339D, 3391, 339K, 339M, 339N, 339Q, 339R, altered effector function or altered serum half-life as com 30 339S, 376A, 376V, 377K, 379N, 380N, 382A, 4401 or 440L], pared to that of the polypeptide comprising the parent Fc Wherein the monoclonal antibody comprising the variant Fc region of said variant Fc region, Wherein the “at least one region displays enhanced ADCC as compared to the mono amino acid substitution in the variant Fc region” is (i) any clonal antibody comprising the parent Fc region. amino acid substitution at position 279, 341, 343 or 373 ofthe The invention provides a monoclonal antibody comprising Fc region or (ii) at least one of the folloWing amino acid 35 a variant Fc region comprising at least one of the folloWing substitutions in the Fc region: 235G, 235R, 236E, 236R, amino acid substitutions in the Fc region: 235Q, 235R, 235S, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 2381, 238L, 236E, 236R, 236Y, 237E, 237K, 237N, 237R, 238E, 238G, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247E, 238H, 2381, 238L, 238V, 238W, 238Y, 247G, 247R, 249L, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248E, 249P, 250K, 250M, 250R, 251H, 2511, 251W, 252Y, 254L, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 40 254P, 254Q, 254T, 254V, 256V, 257A, 2571, 257M, 257N, 2511, 251W, 254D, 254E, 254P, 254G, 254H, 2541, 254K, 257S, 257V, 260S, 262L, 264S, 265H, 265K, 2658, 267G, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, 254Y, 267H, 2671, 267K, 269N, 269Q, 270A, 270G, 270K, 270M, 255K, 255N, 256H, 2561, 256K, 256L, 256V, 256W, 256Y, 270N, 271T, 272H, 272K, 272L, 272N, 272R, 279D, 279E, 257A, 2571, 257M, 257N, 257S, 258D, 260S, 262L, 2648, 279K, 279L, 279W, 283D, 283P, 283G, 283H, 283L, 283W, 265K, 2658, 267H, 2671, 267K, 268K, 269N, 269Q, 271T, 45 283Y, 285N, 288P, 292E, 292E, 292G, 2921, 2938, 293V, 272H, 272K, 272L, 272R, 279A, 279D, 279E, 279G, 279H, 301W, 304E, 307A, 307E, 307M, 311F, 3111, 311K, 3118, 2791, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 312P,314F,3141,314V,314W,315E,315P,316F,317P,327T, 279W, 279Y, 280T, 283P, 283G, 283H, 2831, 283K, 283L, 328V, 329Y, 332G, 332K, 332L, 332R, 332W, 341D, 341E, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286E, 288N, 341E, 341H, 3411, 341K, 341L, 341M, 341N, 341P, 341Q, 288P, 292E, 292E, 292G, 2921, 292L, 293S, 293V, 301W, 50 341R, 341S, 341T, 341W, 341Y, 343A, 343D, 343E, 343E, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 343G, 343H, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332E, 332G, 343V, 343W, 343Y, 373A, 373D, 373E, 373F, 373G, 3731, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 339H, 3391, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 373W, 375R, 376A, 376E, 376P, 376G, 376H, 376W, 376Y, 339W, 339Y, 341D, 341E, 341E, 341H, 3411, 341K, 341L, 55 379Q, 382D, 382S, 430H, 430K, 430N, 430Q, 430R, 430W, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 432R, 4328, 4341, 440D, 440T, 440V or 442K, [preferably 341Y, 343A, 343D, 343E, 343E, 343G, 343H, 3431, 343K, 235R, 236E, 236Y, 237E, 237K, 237N, 237R, 238E, 238G, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 238H, 2381, 238L, 238V, 238W, 238Y, 247R, 250K, 251H, 343Y, 373D, 373E, 373F, 373G, 373H, 3731, 373K, 373L, 254T, 2571, 257M, 257N, 257S, 257V, 265H, 265K, 2658, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 60 267G, 267H, 2671, 267K, 269N, 269Q, 270A, 270G, 270K, 376E, 376P, 376G, 376H, 3761, 376L, 376M, 376N, 376P, 270M, 270N, 271T, 272N, 272R, 288P, 292E, 301W, 304E, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 377K, 316F, 317P, 327T, 328V, 329Y, 332K, 332R, 341E, 3411, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 341M, 341P, 341Q, 341R, 341T, 341W, 341Y, 343W, 373A, 380T, 382D, 382P, 382H, 3821, 382K, 382L, 382M, 382N, 373E, 373G, 373S, 376A, 376W, 432R or 4328], Wherein the 382P, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 65 monoclonal antibody comprising the variant Fc region dis 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, plays diminished ADCC activity as compared to the mono 429A, 429E, 429M, 430A, 430D, 430E, 430G, 430H, 4301, clonal antibody comprising the parent Fc region.

US 7,740,847 B2 10 ably the target antigen is selected from the group consisting of ished serum half-life as compared to the parent polypeptide CD3, CD20, CD25, TNFot, Her2/neu, CD33, CD52, EGFR, (i.e., a polypeptide identical to the variant polypeptide but EpCAM, MUC1, GD3, CEA, CA125, HLA-DR, TGFB, lacking the amino acid substitution listed hereinabove). VEGF, GDF8, GDF1 1 , ghrelin, or any precursor or functional In one embodiment the invention provides a method for fragment thereof. increasing the ADCC activity of a monoclonal antibody, pref The invention provides a variant polypeptide comprising a erably a therapeutic monoclonal antibody (or functional frag variant Fc region comprising at least one of the folloWing ment thereof), comprising engineering a nucleic acid com amino acid substitution in the Fc region: 238L, 244L, 245R, prising a nucleic acid encoding a variant Fc region 249P, 252Y, 256P, 257A, 2571, 257M, 257N, 257S, 257V, comprising at least one of the folloWing amino acid substitu 258D, 260S, 262L, 270K, 272L, 272R, 279A, 279D, 279G, tions: 247A, 247E, 247H, 2471, 247L, 247M, 247T, 247V, 279H, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 247Y, 249E, 249Y, 251E, 254E, 254M, 254Y, 256A, 256M, 283A, 283D, 283E, 283G, 283H, 2831, 283K, 283L, 283N, 258D, 268D, 268E, 279A, 280A, 280K, 283A, 2831, 283K, 283P, 283Q, 283R, 283S, 283T, 283W, 283Y, 285N, 286E, 283M, 283R, 288N, 292A, 311A, 311D, 311N, 311T, 311V, 288N, 2881’, 293V, 307E, 307M, 311A, 3111, 311K, 311L, 311Y, 315L, 318N, 318P, 318T, 318V, 330K, 332T, 332V, 311M, 311V, 311W, 3121’, 316K, 3171’, 318N, 318T, 332E, 339D, 339E, 339G, 3391, 339K, 339M, 339N, 339Q, 339R, 332H, 332K, 332L, 332M, 332R, 332S, 332W, 339N, 339T, 339S, 339T, 376A, 376V, 377G, 377K, 379N, 380N, 380S, 339W, 341P, 343E, 343H, 343K, 343Q, 343R, 343T, 343Y, 382A, 3821, 385E, 427N, 429M, 434W, 4361, 440G, 440H, 375R, 376G, 3761, 376M, 376P, 376T, 376V, 377K, 378D, 4401 or 440L [preferably 247A, 247E, 247M, 247T, 247V, 378N, 380N, 380S, 380T, 382E, 382H, 3821, 382K, 382L, 247Y, 254E, 254Y, 258D, 279A, 283M, 288N, 292A, 311D, 382M, 382N, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 20 311N, 311T, 315L, 318N, 318P, 318T, 318V, 339D, 3391, 423N, 427N, 430A, 430E, 430G, 430H, 4301, 430K, 430L, 339K, 339M, 339N, 339Q, 339R, 339S, 376A, 376V, 377K, 430M, 430N, 430Q, 430R, 430S, 430T, 430V, 430Y, 431H, 379N, 380N, 382A, 4401 or 440L]. The nucleic acid molecule 431K, 434E, 434G, 434H, 434W, 434Y, 4361, 436L, 436T, encoding the variant Fc region may be engineered (e. g., from 438K, 438L, 438T, 438W, 440K or 442K, [preferably 245R, a nucleic acid molecule encoding a parent Fc region or a 252Y, 256P, 257A, 2571, 257M, 257N, 257S, 257V, 258D, 25 native Fc region) to comprise at least one amino acid substi 260S, 262L, 279A, 279D, 279G, 279H, 279N, 279Q, 279S, tution as listed above either While the nucleic acid molecule is 279T, 279W, 279Y, 283E, 283H, 283K, 283R, 285N, 286E, operably attached to additional antibody-encoding nucleic 307E, 307M, 3111, 311K, 311L, 311M, 312P, 318N, 318T, acid, (e.g., the nucleic acid sequence encoding the remainder 332S, 339W, 343E, 343H, 343K, 343Q, 343R, 375R, 377K, of the Ig heavy chain), or the method may further comprise 378D, 378N, 380S, 380T, 382E, 382K, 382Q, 382R, 382S, 30 subsequently operably attaching the nucleic acid encoding 382T, 382V, 382W, 382Y, 423N, 427N, 430A, 430E, 430H, the variant Fc region (i.e., after introduction of at least one 4301, 430L, 430M, 430N, 430Q, 430R, 430S, 430V, 430Y, amino acid substitution listed above) to additional antibody 431H, 431K, 434E, 434G, 434H, 434W, 434Y, 4361, 436L, encoding nucleic acid. The method may further comprise 438K, 438L or 438W], Wherein the variant polypeptide dis expression and puri?cation of the monoclonal antibody com plays enhanced serum half-life as compared to the parent 35 prising the variant Fc region. The method may further com polypeptide (i.e., a polypeptide identical to the variant prise expression and puri?cation of the monoclonal antibody polypeptide but lacking the amino acid substitution listed comprising the parent Fc region. The method may further hereinabove). comprise measuring ADCC activity of the monoclonal anti The invention provides a variant polypeptide comprising a body comprising the variant Fc region and of the monoclonal variant Fc region comprising at least one of the folloWing 40 antibody comprising the parent Fc region by any method amino acid substitution in the Fc region: 235Q, 23 6Y, 237K, available in the art or as described herein. The method may 237R, 238E, 238G, 238H, 238W, 247A, 247D, 247E, 247E, further comprise selecting a monoclonal antibody compris 247G, 247H, 2471, 247L, 247M, 247N, 247Q, 247R, 247S, ing a variant Fc region WithADCC activity greater than that of 247W, 247Y, 248A, 248E, 248P, 248Q, 248W, 249E, 249L, the monoclonal antibody comprising the parent Fc region 249M, 249Y, 251E, 251H, 2511, 251W, 254D, 254E, 254E, 45 (i.e., enhanced, preferably by at least 5%, 10%, 12%, 14%, 254G, 254H, 2541, 254K, 254L, 254M, 254N, 254P, 254Q, 16%, 18%, 20% or more). The invention further embodies a 254R, 254T, 254V, 254W, 254Y, 255K, 255N, 256E, 256H, monoclonal antibody, or functional fragment thereof, com 2561, 256K, 256M, 256R, 256W, 256Y, 264$, 265$, 265Y, prising a variant Fc region produced by the method. 267G, 2671, 268D, 268K, 270A, 270M, 2791, 279K, 279L, In one embodiment the invention provides a method for 280T, 292E, 292E, 292G, 2921, 292L, 311D, 311E, 311E, 50 decreasing the ADCC activity of a monoclonal antibody, 311G, 311N 311R, 311Y, 315E, 315K, 3151’, 316E, 317T, preferably a therapeutic monoclonal antibody (or functional 326W, 327T, 339E, 339G, 339L, 339R, 341D, 341E, 341E, fragment thereof), comprising engineering a nucleic acid 3411, 341K, 341L, 341M, 341N, 341Q, 341R, 341S, 341T, comprising a nucleic acid encoding a variant Fc region com 341V, 341W, 341Y, 343M, 343V, 343W, 373A, 373D, 373G, prising at least one of the folloWing amino acid substitutions: 373K, 373L, 373M, 373N, 373Q, 373S, 373T, 373V, 373W, 55 235Q, 235R, 235S, 236E, 236R, 236Y, 237E, 237K, 237N, 376H, 376L, 376W, 376Y, 424M, 424V, 426D, 429A, 429E, 237R, 238E, 238G, 238H, 2381, 238L, 238V, 238W, 238Y, 429M, 430D, 430W, 431P, 432R, 432S, 439Q, 440D, 440E, 247G, 247R, 249L, 2491’, 250K, 250M, 250R, 251H, 2511, 440E or 440M [preferably 237R, 247D, 247E, 247E, 247H, 251W, 252Y, 254L, 254P, S254Q, 254T, 254V, 256V, 257A, 247L, 247M, 247N, 247Q, 247W, 247Y, 248A, 248E, 248P, 2571, 257M, 257N, 257S, 257V, 260S, 262L, 264S, 265H, 248Q, 248W, 249L, 249M, 249Y, 251H, 2511, 251W, 254D, 60 265K, 265S, 267G, S267H, 2671, 267K, 269N, 269Q, 270A, 254E, 254E, 254G, 254H, 2541, 254K, 254M, 254N, 254P, 270G, 270K, 270M, 270N, 271T, 272H, 272K, 272L, 272N, 254Q, 254R, 254T, 254V, 254W, 254Y, 255K, 255N, 256E, 272R, 279D, 279E, 279K, 279L, 279W, 283D, 283E, 283G, 256H, 256K, 256M, 256R, 256W, 265Y, 280T, 292G, 2921, 283H, 283L, 283T, 283W, 283Y, 285N, 2881’, 292E, 292E, 311D, 311E, 311G, 311N, 315E, 315P, 316T, 317T, 327T, 292G, 2921, 293S, 293V, 301W, 304E, 307A, 307E, 307M, 341D, 341E, 341E, 3411, 341L, 341Y, 343W, 373A, 373G, 65 311E, 3111, 311K, 311S, 312P, 314E, 3141, 314V, 314W, 373M, 373Q, 376W, 376Y, 424M, 424V, 430D, 430W, 431P 315E, 315P, 316E, 317P, 327T, 328V, 329Y, 332G, 332K, or 4328], Wherein the variant polypeptide displays dimin 332L, 332R, 332W, 341D, 341E, 341E, 341H, 3411, 341K, US 7,740,847 B2 11 12 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341W, 283K, 283R, 285N, 286E, 307A, 307E, 307M, 3111, 311K, 341Y, 343A, 343D, 343E, 343E, 343G, 343H, 343L, 343M, 311L, 311M, 312P, 318N, 318T, 3328, 339W, 343E, 343H, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 373A, 343K, 343Q, 343R, 375R, 377K, 378D, 378N, 380S, 380T, 373D, 373E, 373F, 373G, 3731, 373K, 373L, 373M, 373N, 382E, 382K, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376A, 376E, 423N, 427N, 430A, 430E, 430H, 4301, 430L, 430M, 430N, 376P, 376G, 376H, 376W, 376Y, 379Q, 382D, 382S, 429A, 430Q, 430R, 430S, 430V, 430Y, 431H, 431K, 434E, 434G, 429E, 430H, 430K, 430N, 430Q, 430R, 430W, 432R, 4328, 434H, 434W, 434Y, 4361, 436L, 438K, 438L or 438W]. The 4341, 440D, 440T, 440V or 442K [preferably 235R, 236E, nucleic acid molecule encoding the variant Fc region may be 236R, 236Y, 237E, 237K, 237N, 237R, 238E, 238G, 238H, engineered (e.g., from a nucleic acid molecule encoding a 2381, 238L, 238V, 238W, 238Y, 247R, 249P, 250K, 251H, parent Fc region or a native Fc region) to comprise at least one 254T, 2571, 257M, 257N, 257S, 257V, 265H, 265K, 2658, amino acid substitution as listed above either While the 267G, 267H, 2671, 267K, 269N, 269Q, 270A, 270G, 270K, nucleic acid molecule is operably attached to additional anti 270M, 270N, 271T, 272R, 288P, 292E, 301W, 304E, 316F, body-encoding nucleic acid, (e.g., the nucleic acid sequence 317P, 327T, 329Y, 332K, 332R, 341E, 3411, 341M, 341P, encoding the remainder of the Ig heavy chain), or the method 341Q, 341R, 341T, 341W, 341Y, 343W, 373A, 373E, 373G, may further comprise subsequently operably attaching the 373S, 376W, 429A, 432R or 4328]. The nucleic acid mol nucleic acid encoding the variant Fc region (i.e., after intro ecule encoding the variant Fc region may be engineered (e. g., duction of at least one amino acid substitution listed above) to from a nucleic acid molecule encoding a parent Fc region or additional antibody-encoding nucleic acid. The method may a native Fc region) to comprise at least one amino acid sub further comprise expression and puri?cation of the mono stitution as listed above either While the nucleic acid molecule 20 clonal antibody comprising the variant Fc region. The method is operably attached to additional antibody-encoding nucleic may further comprise expression and puri?cation of the acid, (e.g., the nucleic acid sequence encoding the remainder monoclonal antibody comprising the parent Fc region. The of the Ig heavy chain), or the method may further comprise method may further comprise measuring FcRn binding a?in subsequently operably attaching the nucleic acid encoding ity of the monoclonal antibody comprising the variant Fc the variant Fc region (i.e., after introduction of at least one 25 region and of the monoclonal antibody comprising the parent amino acid substitution listed above) to additional antibody Fc region by any method available in the art or as described encoding nucleic acid. The method may further comprise herein. The method may further comprise selecting a mono expression and puri?cation of the monoclonal antibody com clonal antibody comprising a variant Fc region With FcRn prising the variant Fc region. The method may further com binding af?nity greater than the FcRn binding af?nity of the prise expression and puri?cation of the monoclonal antibody 30 monoclonal antibody comprising the parent Fc region (i.e., comprising the parent Fc region. The method may further enhanced, preferably by at least 5%, 10%, 12%, 14%, 16%, comprise measuring ADCC activity of the monoclonal anti 18%, 20% or more). The invention further embodies a mono body comprising the variant Fc region and of the monoclonal clonal antibody, or functional fragment thereof, comprising a antibody comprising the parent Fc region by any method variant Fc region produced by the method. available in the art or as described herein. The method may 35 In one embodiment the invention provides a method for further comprise selecting a monoclonal antibody compris increasing the in vivo serum half-life of a polypeptide, pref ing a variant Fc region With ADCC activity less than that of erably a therapeutic polypeptide, comprising engineering a the monoclonal antibody comprising the parent Fc region nucleic acid comprising a nucleic acid encoding a variant Fc (i.e., diminished, preferably by at least 5%, 10%, 12%, 14%, region comprising at least one of the folloWing amino acid 16%, 18%, 20% or more). The invention further embodies a 40 substitutions: 238L, 244L, 245R, 249P, 252Y, 256P, 257A, monoclonal antibody, or functional fragment thereof, com 2571, 257M, 257N, 257S, 257V, 258D, 260S, 262L, 270K, prising a variant Fc region produced by the method. 272L, 272R, 279A, 279D, 279G, 279H, 279M, 279N, 279Q, In one embodiment the invention provides a method for 279R, 279S, 279T, 279W, 279Y, 283A, 283D, 283P, 283G, increasing the FcRn binding af?nity of a monoclonal anti 283H, 2831, 283K, 283L, 283N, 283P, 283Q, 283R, 283S, body, preferably a therapeutic monoclonal antibody (or func 45 283T, 283W, 283Y, 285N, 286E, 288N, 288P, 293V, 307A, tional fragment thereof), comprising engineering a nucleic 307E, 307M, 311A, 3111, 311K, 311L, 311M, 311V, 311W, acid comprising a nucleic acid encoding a variant Fc region 3121’, 316K, 3171’, 318N, 318T, 332E, 332H, 332K, 332L, comprising at least one of the folloWing amino acid substitu 332M, 332R, 332S, 332W, 339N, 339T, 339W, 341P, 343E, tions: 238L, 244L, 245R, 249P, 252Y, 256P, 257A, 2571, 343H, 343K, 343Q, 343R, 343T, 343Y, 375R, 376G, 3761, 257M, 257N, 257S, 257V, 258D, 260S, 262L, 270K, 272L, 50 376M, 376P, 376T, 376V, 377K, 378D, 378N, 380N, 380S, 272R, 279A, 279D, 279G, 279H, 279M, 279N, 279Q, 279R, 380T, 382E, 382H, 3821, 382K, 382L, 382M, 382N, 382Q, 279S, 279T, 279W, 279Y, 283A, 283D, 283P, 283G, 283H, 382R, 382S, 382T, 382V, 382W, 382Y, 423N, 427N, 430A, 2831, 283K, 283L, 283N, 283P, 283Q, 283R, 283S, 283T, 430E, 430G, 430H, 4301, 430K, 430L, 430M, 430N, 430Q, 283W, 283Y, 285N, 286E, 288N, 288P, 293V, 307A, 307E, 430R, 430S, 430T, 430V, 430Y, 431H, 431K, 434E, 434G, 307M, 311A, 3111, 311K, 311L, 311M, 311V, 311W, 312P, 55 434H, 434W, 434Y, 4361, 436L, 436T, 438K, 438L, 438T, 316K, 317P, 318N, 318T, 332E, 332H, 332K, 332L, 332M, 438W, 440K or 442K, [preferably 245R, 252Y, 256P, 257A, 332R, 332S, 332W, 339N, 339T, 339W, 341P, 343E, 343H, 2571, 257M, 257N, 257S, 257V, 258D, 262L, 279A, 279D, 343K, 343Q, 343R, 343T, 343Y, 375R, 376G, 3761, 376M, 279G, 279H, 279N, 279Q, 279S, 279T, 279W, 279Y, 283P, 376P, 376T, 376V, 377K, 378D, 378N, 380N, 380S, 380T, 283H, 283K, 283R, 285N, 286E, 307A, 307E, 307M, 3111, 382E, 382H, 3821, 382K, 382L, 382M, 382N, 382Q, 382R, 60 311K, 311L, 311M, 312P, 318N, 318T, 3328, 339W, 343E, 382S, 382T, 382V, 382W, 382Y, 423N, 427N, 430A, 430E, 343H, 343K, 343Q, 343R, 375R, 377K, 378D, 378N, 380S, 430G, 430H, 4301, 430K, 430L, 430M, 430N, 430Q, 430R, 380T, 382E, 382K, 382Q, 382R, 382S, 382T, 382V, 382W, 430S, 430T, 430V, 430Y, 431H, 431K, 434E, 434G, 434H, 382Y, 423N, 427N, 430A, 430E, 430H, 4301, 430L, 430M, 434W, 434Y, 4361, 436L, 436T, 438K, 438L, 438T, 438W, 430N, 430Q, 430R, 430S, 430V, 430Y, 431H, 431K, 434E, 440K or 442K [preferably 245R, 252Y, 256P, 257A, 2571, 65 434G, 434H, 434W, 434Y, 4361, 436L, 438K, 438L or 257M, 257N, 257S, 257V, 258D, 262L, 279A, 279D, 279G, 438W]. The nucleic acid molecule encoding the variant Fc 279H, 279N, 279Q, 279S, 279T, 279W, 279Y, 283E, 283H, region may be operably linked to a nucleic acid molecule US 7,740,847 B2 13 14 encoding a therapeutic protein. The nucleic acid molecule encoding the variant Fc region after introduction of the at encoding a variant Fc region may be engineered (e. g., from a least one amino acid substitution listed above to additional nucleic acid molecule encoding a parent Fc region or a native antibody-encoding nucleic acid. The method may further Fc region) to comprise at least one amino acid substitution as comprise expression and puri?cation of the monoclonal anti listed above While the nucleic acid molecule is operably body comprising the variant Fc region. The method may attached to additional polypeptide-encoding nucleic acid, further comprise expression and puri?cation of the mono (e. g., the nucleic acid sequence encoding the non-Fc region of clonal antibody comprising the parent Fc region. The method the fusion protein), or the method may further comprise sub may further comprise measuring FcRn binding a?inity of the sequently operably attaching the nucleic acid encoding the monoclonal antibody comprising the variant Fc region and of variant Fc region after introduction of the at least one amino the monoclonal antibody comprising the parent Fc region by acid substitution listed above to nucleic acid encoding a non any method available in the art or as described herein. The Fc fusion partner. The method may further comprise expres method may further comprise selecting a monoclonal anti sion and puri?cation of the polypeptide comprising the vari body comprising a variant Fc region With FcRn binding a?in ant Fc region. The method may further comprise expression ity less than that of the monoclonal antibody comprising the and puri?cation of the polypeptide comprising the parent Fc parent Fc region (i.e., diminished, preferably by at least 5%, region. The method may further comprise measuring in vivo 10%, 12%, 14%, 16%, 18%, 20% or more). The invention serum half-life of the polypeptide comprising the variant Fc further embodies a monoclonal antibody (comprising a vari region and of the polypeptide comprising the parent Fc region ant Fc region) produced by the method. by any method available in the art or as described herein. The In another embodiment the invention provides a method method may further comprise selecting a polypeptide com 20 for decreasing the in vivo serum half-life of a polypeptide, prising a variant Fc region Wherein the polypeptide has preferably a therapeutic polypeptide, comprising engineering increased in vivo serum half life as compared to that of the a nucleic acid encoding a variant Fc region comprising at least polypeptide comprising the parent Fc region (i.e., enhanced, one of the folloWing amino acid substitutions: 235Q, 236Y, preferably by at least 5%, 10%, 12%, 14%, 16%, 18%, 20% or 237K, 237R, 238E, 238G, 238H, 238W, 247A, 247D, 247E, more). The invention further embodies a polypeptide (i.e., a 25 247E, 247G, 247H, 2471, 247L, 247M, 247N, 247Q, 247R, fusion polypeptide) comprising a variant Fc region produced 247S, 247W, 247Y, 248A, 248E, 2481’, 248Q, 248W, 249E, by the method. 249L, 249M, 249Y, 251E, 251H, 2511, 251W, 254D, 254E, In one embodiment the invention provides a method for 254E, 254G, 254H, 2541, 254K, 254L, 254M, 254N, 254P, decreasing the FcRn binding af?nity of a monoclonal anti 254Q, 254R, 254T, 254V, 254W, 254Y, 255K, 255N, 256E, body, preferably a therapeutic monoclonal antibody (or func 30 256H, 2561, 256K, 256M, 256R, 256W, 256Y, 264$, 265$, tional fragment thereof), comprising engineering a nucleic 265Y, S267G, 2671, 268D, 268K, 270A, 270M, 2791, 279K, acid encoding a variant Fc region comprising at least one of 279L, 280T, 292E, 292E, 292G, 2921, 292L, 311D, 311E, the folloWing amino acid substitutions: 235Q, 236Y, 237K, 311E, 311G, 311N, 311R, 311Y, 315E, 315K, 3151’, 316E, 237R, 238E, 238G, 238H, 238W, 247A, 247D, 247E, 247E, 317T, 326W, 327T, 339E, 339G, 339L, 339R, 341D, 341E, 247G, 247H, 2471, 247L, 247M, 247N, 247Q, 247R, 247S, 35 341E, 3411, 341K, 341L, 341M, 341N, 341Q, 341R, 341S, 247W, 247Y, 248A, 248E, 248P, 248Q, 248W, 249E, 249L, 341T, 341V, 341W, 341Y, 343M, 343V, 343W, 373A, 373D, 249M, 249Y, 251E, 251H, 2511, 251W, 254D, 254E, 254P, 373G, 373K, 373L, 373M, 373N, 373Q, 373S, 373T, 373V, 254G, 254H, 2541, 254K, 254L, 254M, 254N, 254P, 254Q, 373W, 376H, 376L, 376W, 376Y, 424M, 424V, 426D, 429A, 254R, 254T, 254V, 254W, 254Y, 255K, 255N, 256E, 256H, 429E, 429M, 430D, 430W, 431P, 432R, 432S, 4341, 439Q, 2561, 256K, 256M, 256R, 256W, 256Y, 264$, 265$, 265Y, 40 440A, 440D, 440E, 440E or 440M [preferably 237R, 247D, S267G, 2671, 268D, 268K, 270A, 270M, 2791, 279K, 279L, 247E, 247E, 247H, 247L, 247M, 247N, 247Q, 247W, 247Y, 280T, 292E, 292E, 292G, 2921, 292L, 311D, 311E, 311E, 248A, 248E, 2481’, 248Q, 248Q, 249L, 249M, 249Y, 251H, 311G, 311N, 311R, 311Y, 315E, 315K, 315P, 316E, 317T, 2511, 251W, 254D, 254E, 254E, 254G, 254H, 2541, 254K, 326W, 327T, 339E, 339G, 339L, 339R, 341D, 341E, 341E, 254M, 254N, 254P, 254R, 254T, 254V, 254W, 254Y, 255K, 3411, 341K, 341L, 341M, 341N, 341Q, 341R, 341S, 341T, 45 255N, 256E, 256H, 256K, 256M, 256R, 256W, 265Y, 280T, 341V, 341W, 341Y, 343M, 343V, 343W, 373A, 373D, 373G, 292G, 2921, 311D, 311E, 311G, 311N, 315E, 315P, 316T, 373K, 373L, 373M, 373N, 373Q, 373S, 373T, 373V, 373W, 317T, 327T, 341D, 341E, 341E, 341L, 341Y, 343W, 373A, 376H, 376L, 376W, 376Y, 424M, 424V, 426D, 429A, 429E, 373G, 373M, 373Q, 376W, 376Y, 424M, 424V, 430D, 430W, 429M, 430D, 430W, 431P, 432R, 4328, 4341, 439Q, 440A, 431P or 4328]. The nucleic acid encoding the Fc variant 440D, 440E, 440E or 440M [preferably 237R, 247D, 247E, 50 region may be operably linked to nucleic acid encoding a 247E, 247H, 247L, 247M, 247N, 247Q, 247W, 247Y, 248A, therapeutic protein. The nucleic acid molecule encoding a 248E, 248P, 248Q, 248Q, 249L, 249M, 249Y, 251H, 2511, variant Fc region may be engineered (e. g., from a nucleic acid 251W, 254D, 254E, 254P, 254G, 254H, 2541, 254K, 254M, molecule encoding a parent Fc region or a native Fc region) to 254N, 254P, 254R, 254T, 254V, 254W, 254Y, 255K, 255N, comprise at least one amino acid substitution as listed above 256E, 256H, 256K, 256M, 256R, 256W, 265Y, 280T, 292G, 55 While the nucleic acid molecule is operably attached to addi 2921, 311D, 311E, 311G, 311N, 315E, 315P, 316T, 317T, tional polypeptide-encoding nucleic acid, (e.g., the nucleic 327T, 341D, 341E, 341E, 341L, 341Y, 343W, 373A, 373G, acid sequence encoding the non-Fc region of the fusion pro 373M, 373Q, 376W, 376Y, 424M, 424V, 430D, 430W, 4311’ tein), or the method may further comprise subsequently oper or 4328]. The nucleic acid molecule encoding a variant Fc ably attaching the nucleic acid encoding the variant Fc region region may be engineered (e.g., from a nucleic acid molecule 60 after introduction of the at least one amino acid substitution encoding a parent Fc region or a native Fc region) to comprise listed above to nucleic acid encoding a non-Fc fusion partner. at least one amino acid substitution as listed above While the The method may further comprise expression and puri?ca nucleic acid encoding the variant Fc region is operably tion of the polypeptide comprising the variant Fc region. The attached to nucleic acid encoding additional antibody method may further comprise expression and puri?cation of sequence, (e.g., the nucleic acid sequence encoding the 65 the polypeptide comprising the parent Fc region. The method remainder of the Ig heavy chain), or the method may further may further comprise measuring in vivo serum half-life of the comprise subsequently operably attaching the nucleic acid polypeptide comprising the variant Fc region and of the US 7,740,847 B2 15 16 polypeptide comprising the parent Fc region by any method method may further comprise expression and puri?cation of available in the art or as described herein. The method may the monoclonal antibody comprising the variant Fc region. further comprise selecting a polypeptide comprising a variant The method may further comprise expression and puri?ca Fc region Wherein the polypeptide has decreased in vivo tion of the monoclonal antibody comprising the parent Fc serum half life as compared to that of the polypeptide com region. The method may further comprise measuring CDC prising the parent Fc region (i.e., diminished, preferably by at activity of the monoclonal antibody comprising the variant Fc least 5%, 10%, 12%, 14%, 16%, 18%, 20% or more). The region and of the monoclonal antibody comprising the parent invention further embodies a polypeptide (i.e., a fusion Fc region by any method available in the art or as described polypeptide) comprising a variant Fc region produced by the herein. The method may further comprise selecting a mono method. clonal antibody comprising a variant Fc region With CDC In another embodiment the invention provides a method activity greater than that of the monoclonal antibody com for increasing the CDC activity of a monoclonal antibody, prising the parent Fc region (i.e., enhanced, preferably by at preferably a therapeutic monoclonal antibody, comprising least 5%, 10%, 12%, 14%, 16%, 18%, 20% or more). The constructing the Fc region of the antibody to comprise at least invention further embodies a monoclonal antibody compris one of the folloWing amino acid substitutions: 236Y, 244L, ing a variant Fc region produced by the method. 247A, 247D, 247E, 247G, 247N, 247Q, 247R, 247S, 247W, In another embodiment the invention provides a method 248E, 248P, 248Q, 248W, 249E, 249L, 249M, 249N, 249P, for decreasing the CDC response of a monoclonal antibody, 249Y, 250K, 250R, 251E, 251H, 2511, 251W, 254A, 254E, preferably a therapeutic monoclonal antibody, comprising 254K, 254L, 254M, 254R, 254Y, 255K, 256A, 256G, 2561, constructing the Fc region of the antibody to comprise at least 256L, 256M, 256P, 256Q, 256W, 256Y, 260S, 268D, 279Q, 20 one of the folloWing amino acid substitutions: 235G, 235S, 279S, 279W, 279Y, 280K, 280T, 283E, 283G, 283H, 2831, 236R, 237E, 237K, 237N, 237R, 238A, 238E, 238G, 238H, 283K, 283L, 283M, 283N, 283P, 283R, 283S, 283W, 292L, 2381, 238L, 238V, 238W, 238Y, 245R, 247H, 2471, 247L, 307A, 307M, 311E, 3111, 311K, 311L, 311M, 311T, 311V, 247T, 247Y, 250M, 252Y, 254D, 254E, 2541, 2541’, 254Q, 311W, 311Y, 312P, 314E, 3141, 314V, 314W, 314Y, 315E, 254T, 254V, 255N, 257A, 2571, 257M, 257N, 257S, 257V, 315K, 315L, 315P, 315R, 316K, 3171’, 317T, 318N, 318T, 25 262L, 264S, 265H, 265Y, 267G, 267H, 2671, 267K, 268K, 332A, 332D, 332E, 332E, 332G, 332H, 332L, 332M, 332N, 269N, 269Q, 270G, 270M, 270N, 271T, 272H, 272L, 272N, 332Q, 332S, 332T, 332V, 332W, 332Y, 339D, 339E, 339G, 292A, 293S, 301W, 307E, 311E, 311S, 316E, 318P, 327T, 339H, 3391, 339K, 339N, 339Q, 339R, 339S, 339T, 339W, 328V, 329Y, 330K, 330R, 332K, 339E, 339M, 3431, 373S, 339Y, 341D, 341E, 341E, 341H, 3411, 341K, 341L, 341M, 378D, 380D, 382D, 382E, 382N, 382P, 382R, 382S, 382W, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 30 382Y, 385E, 385P, 423N, 424H, 424M or 427N [preferably 343A, 343D, 343E, 343G, 343H, 343K, 343L, 343M, 343N, 235G, 235S, 236R, 237E, 237K, 237N, 237R, 238A, 238E, 343Q, 343R, 343S, 343T, 343W, 343Y, 373D, 373E, 373E, 238G, 238H, 2381, 238L, 238V, 238W, 238Y, 245R, 2471, 373H, 3731, 373K, 373L, 373M, 373N, 373Q, 373R, 373T, 247L, 247T, 250M, 257A, 2571, 257M, 262L, 264S, 267G, 373V, 373W, 375R, 376A, 376E, 376G, 376H, 376L, 376N, 267H, 2671, 267K, 268K, 269N, 269Q, 270G, 270M, 270N, 376P, 376Q, 376R, 376S, 376T, 376V, 377P, 379N, 379Q, 35 271T, 272H, 301W, 311S, 327T, 329Y, 330K, 330R, 378D, 379S, 379T, 380A, 380N, 380S, 380T, 3821, 382L, 382Q, 385E, 423N or 424H]. The nucleic acid molecule encoding a 382V, 386K, 426D, 426L, 429A, 429E, 429M, 430A, 430D, variant Fc region may be engineered (e. g., from a nucleic acid 430E, 430G, 430H, 4301, 430K, 430L, 430M, 430N, 430P, molecule encoding a parent Fc region or a native Fc region) to 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431P, 432R, comprise at least one amino acid substitution as listed above 432S, 434W, 434Y, 438L, 438W, 440Q or 440Y [preferably 40 While the nucleic acid molecule is operably attached to addi 236Y, 248E, 248P, 248Q, 248W, 249E, 249L, 249M, 249N, tional antibody-encoding nucleic acid, (e. g., the nucleic acid 249Y, 250K, 250R, 251H, 2511, 251W, 254A, 254E, 254K, sequence encoding the remainder of the Ig heavy chain), or 254L, 254M, 254R, 254Y, 255K, 256A, 256G, 2561, 256L, the method may further comprise subsequently operably 256M, 256P, 256Q, 256W, 260S, 280K, 283W, 307M, 311E, attaching the nucleic acid encoding the variant Fc region after 3111, 311K, 311L, 311M, 311T, 311V, 311W, 311Y, 3141, 45 introduction of the at least one amino acid substitution listed 314V, 314W, 314Y, 3151’, 3171’, 332D, 332L, 332M, 332S, above to additional antibody-encoding nucleic acid. The 332W, 332Y, 339D, 339E, 3391, 339K, 339N, 339S, 339T, method may further comprise expression and puri?cation of 339W, 339Y, 341D, 341E, 341E, 341H, 3411, 341K, 341L, the monoclonal antibody comprising the variant Fc region. 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, The method may further comprise expression and puri?ca 341Y, 343D, 343E, 343G, 343H, 343K, 343N, 343Q, 343R, 50 tion of the monoclonal antibody comprising the parent Fc 343S, 343T, 343W, 343Y, 373D, 373E, 373H, 3731, 373K, region. The method may further comprise measuring CDC 373L, 373Q, 373R, 373T, 373W, 376A, 376G, 376N, 376P, activity of the monoclonal antibody comprising the variant Fc 376Q, 376R, 376S, 376T, 376V, 377P, 379N, 379Q, 379T, region and of the monoclonal antibody comprising the parent 3821, 382L, 386K, 426D, 426L, 429E, 429M, 430A, 430D, Fc region by any method available in the art or as described 430E, 430G, 430H, 4301, 430K, 430L, 430M, 430N, 430P, 55 herein. The method may further comprise selecting a mono 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431P, 432R, clonal antibody comprising a variant Fc region With CDC 434Y, 438L or 440Y]. The nucleic acid molecule encoding a activity less than that of the monoclonal antibody comprising variant Fc region may be engineered (e. g., from a nucleic acid the parent Fc region (i.e., diminished, preferably by at least molecule encoding a parent Fc region or a native Fc region) to 5%, 10%, 12%, 14%, 16%, 18%, 20% or more). The inven comprise at least one amino acid substitution as listed above 60 tion further embodies a monoclonal antibody comprising a While the nucleic acid molecule is operably attached to addi variant Fc region produced by the method. tional antibody-encoding nucleic acid, (e. g., the nucleic acid In another embodiment, the invention provides an isolated sequence encoding the remainder of the Ig heavy chain), or nucleic acid molecule that comprises a nucleic acid molecule the method may further comprise subsequently operably that encodes a variant Fc region of the invention or a func attaching the nucleic acid encoding the variant Fc region after 65 tional fragment thereof. More preferably the isolated nucleic introduction of the at least one amino acid substitution listed acid molecule comprises a nucleic acid that encodes a above to additional antibody-encoding nucleic acid. The polypeptide comprising a variant Fc region of the invention. US 7,740,847 B2 17 18 Preferably the variant Fc region polypeptide encoded by said ies compositions comprising monoclonal antibodies and het nucleic acid has an amino acid substitution as shoWn in Table erologous polypeptides that comprise a variant Fc region l as compared to the parent Fc region of the variant. Prefer described herein, and a physiologically or pharmaceutically ably, the polypeptide is a monoclonal antibody, and even acceptable carrier or diluent. more preferably, the monoclonal antibody is a full length In some embodiments, the present invention provides a antibody or a single-chain antibody. The monoclonal anti polypeptide comprising: i) an unmodi?ed human frameWork body may be a chimeric, humanized, or human monoclonal region (“FR”) (e. g., no alterations have been made to a natu antibody. rally occurring human frameWork), and ii) a variant Fc region. In another embodiment, the invention provides a vector, In certain embodiments, the unmodi?ed human frameWork is preferably (but not limited to) a plasmid, a recombinant a human germline frameWork. In other embodiments, the expression vector, a yeast expression vector, or a retroviral present invention provides compositions comprising a expression vector comprising a polynucleotide encoding a polypeptide, Wherein the polypeptide comprises: i) at least polypeptide comprising a variant Fc region polypeptide of the one randomiZed CDR sequence and ii) a variant Fc region of invention. the invention. In further embodiments, the present invention In another embodiment, the invention provides a host cell provides compositions comprising a polypeptide, Wherein comprising a nucleic acid molecule of the present invention. the polypeptide comprises: i) an unmodi?ed human frame Preferably a host cell of the invention comprises one or more Work (e.g., human germline frameWork), ii) at least one ran vectors or constructs comprising a nucleic acid molecule of domiZed CDR sequence, and iii) a variant Fc region of the the present invention. The host cell of the invention is a cell invention. into Which a vector of the invention has been introduced (e.g., 20 The present invention contemplates therapeutic and diag via transformation, transduction, infection, transfection, nostic uses for monoclonal antibodies heterologous polypep electroporation and the like), said vector comprising a poly tides that comprise a variant Fc region of the invention, dis nucleotide encoding a polypeptide comprising a variant Fc closed herein. region polypeptide of the invention. Optionally, the vector may be stably incorporated into the host cell chromosome. 25 DESCRIPTION OF THE FIGURES The host cell types include mammalian, bacterial, plant and yeast cells. Preferably the host cell is a CHO cell, a COS cell, FIG. 1 shoWs a schematic representation of an IgG mol a SP2/0 cell, a NSO cell, a yeast cell or a derivative or progeny ecule With the various regions and sections labeled. of any preferred cell type. FIG. 2 shoWs an alignment of various parental Fc amino In another embodiment, the invention provides a pharma 30 acid sequences, including human IgG1 ((SEQ ID NO: 1) With ceutical composition comprising a polypeptide comprising a non-a and a allotypes shoWn), human IgG2 (SEQ ID NO:2), variant Fc region of the invention, or functional fragment human IgG3 (SEQ ID NO:3), human IgG4 (SEQ ID NO:4), thereof. Preferably the polypeptide is a monoclonal antibody, murine IgGl (SEQ ID NO:5), murine IgG2A (SEQ ID even more preferably, a therapeutic monoclonal antibody. NO:6), murine IgG2B (SEQ ID NO:7), and murine IgG3 The monoclonal antibody may be a chimeric, humanized, or 35 (SEQ ID NO:8). human monoclonal antibody. Alternatively, the polypeptide FIGS. 3 A-D shoWs various amino acid sequences, includ may be a polypeptide other than an antibody Which bene?ts ing the CH2 region (SEQ ID NO: 9), and CH3 region (SEQ ID from an altered serum half life conferred upon the polypep NO: 10) ofhuman IgGl, as Well as an fallotype (SEQ ID NO: tide by being operably linked to, and coexpressed With, a 11) and a,Z allotype (SEQ ID NO:12) sequences of human variant Fc region of the invention. The pharmaceutical com 40 IgG1 that include the CH1, hinge, CH2 and CH3 regions. position of the invention may further comprise a pharmaceu FIGS. 4 A-D shoWs various amino acid sequences com tically acceptable carrier. In said pharmaceutical composi prised Within: (a) the Light ChainVariable Region (LCVR) of tion, the polypeptide comprising the variant Fc region is the anti-CD20 antibody (I) (SEQ ID NO: 13); (b) Heavy Chain active ingredient. Preferably the pharmaceutical composition Variable Region (HCVR) of anti-CD20 antibody (I) (SEQ ID comprises a homogeneous or substantially homogeneous 45 NO: 14); (c) LCVR of anti-CD20 antibody (II) (SEQ ID NO: population of monoclonal antibody comprising a variant Fc 15); and HCVR of anti-CD20 antibody (II) (SEQ ID NO: 16). region of the invention. The pharmaceutical composition for FIG. 4e shoWs amino acid sequences comprised Within the therapeutic use is preferably sterile and may be lyophiliZed. variable region of an anti-CD20 antibody. (see US. provi The invention provides a method of inhibiting activity of a sional application 60/471,958, ?led May 20, 2003 and US. protein in a mammal, preferably a human, in need thereof 50 Pat. No. 5,843,439, both incorporated herein). comprising administering a therapeutically effective amount, FIG. 5a shoWs the complete light chain amino acid or prophylactically effective amount, of a polypeptide (pref sequence for the anti-CD20 antibody AME 133. FIG. 5b erably a monoclonal antibody) comprising a variant Fc region shoWs the complete light chain nucleic acid sequence for of the invention to said mammal. Preferably, the polypeptide AME 133. comprising the variant Fc region is a binding partner of the 55 FIG. 6 shoWs amino acid and nucleic acid sequences for the protein to be inhibited. The invention further provides a complete heavy chain of three preferred variants of the anti method of treating or preventing a disease or disorder ame CD20 antibody AME 133. Speci?cally, FIG. 6a shoWs the liorated by the inhibition of signal transduction resulting from amino acid sequence of the complete heavy chain of the the binding of a monoclonal antibody comprising a variant Fc 247I/339Q variant. FIG. 6b shoWs the nucleic acid sequence region of the invention to its antigenic epitope that comprises 60 of the complete heavy chain of the 247I/339Q variant. FIG. 6c administering to a patient (e.g., a human) in need of such shows the amino acid sequence of the complete heavy chain treatment or prevention a therapeutically or prophylactically of the 247I/339D variant. FIG. 6d shoWs the nucleic acid effective amount of a monoclonal antibody of the invention. sequence of the complete heavy chain of the 247I/ 339D vari The invention embodies an article of manufacture compris ant. FIG. 6e shoWs the amino acid sequence of the complete ing a packaging material and a polypeptide comprising a 65 heavy chain of the 378D variant. FIG. 6f shoWs the nucleic variant Fc region polypeptide of the invention contained acid sequence of the complete heavy chain of the 378D vari Within said packaging material. The invention further embod ant.