USOO9040.042B2

(12) United States Patent (10) Patent No.: US 9,040,042 B2 Bernett et al. (45) Date of Patent: May 26, 2015

(54) OPTIMIZED THAT TARGET (56) References Cited HM124 U.S. PATENT DOCUMENTS (71) Applicants: Matthew J. Bernett, Monrovia, CA 4,816,397 A 3, 1989 BOSS et al. (US); John R. Desjarlais, Pasadena, CA 5,225.348 A 7/1993 Nagata et al. (US); Sher Bahadur Karki, Pomona, 5,266.491 A 11/1993 Nagata et al. 5,328,987 A 7, 1994 Maliszewski CA (US); Gregory Alan Lazar, Arcadia, 5,530,101 A 6/1996 Queen et al. CA (US); Umesh S. Muchhal, 5,541,087 A 7/1996 Lo et al. Monrovia, CA (US); Duc-Hanh Thi 5,576, 184 A 11/1996 Better et al. Nguyen, Sylmar, CA (US); John O. 5,623,053 A 4/1997 Gastinel et al. 5,624,821 A 4/1997 Winter et al. Richards, Duarte, CA (US) 5,633,162 A 5/1997 Keen et al. 5,648,237 A 7, 1997 Carter (72) Inventors: Matthew J. Bernett, Monrovia, CA 5,648,260 A 7, 1997 Winter et al. (US); John R. Desjarlais, Pasadena, CA 5,821,337 A 10, 1998 Carter et al. 5,834,597 A 11/1998 Tso et al. (US); Sher Bahadur Karki, Pomona, 5,885,573 A 3, 1999 Bluestone et al. CA (US); Gregory Alan Lazar, Arcadia, 5,914,252 A 6/1999 Hirano et al. CA (US); Umesh S. Muchhal, 6,030,613 A 2/2000 Blumberg et al. Monrovia, CA (US); Duc-Hanh Thi 6,086,875 A 7/2000 Blumberg et al. 6,121,022 A 9, 2000 Presta et al. Nguyen, Sylmar, CA (US); John O. 6,165,745 A 12/2000 Ward et al. Richards, Duarte, CA (US) 6,188,965 B1 2/2001 Mayo et al. 6,194.551 B1 2/2001 Idusogie et al. (73) Assignee: Xencor, Inc., Monrovia, CA (US) 6.242,195 B1 6/2001 Idusogie et al. 6,269,312 B1 7/2001 Mayo et al. 6,277.375 B1 8, 2001 Ward (*) Notice: Subject to any disclaimer, the term of this 6,331.415 B1 12/2001 Cabilly et al. patent is extended or adjusted under 35 6,358,733 B1 3/2002 Motwani et al. U.S.C. 154(b) by 0 days. 6,365,161 B1 4/2002 Deo et al. 6,403,312 B1 6/2002 Dahiyat et al. 6,444,789 B1 9, 2002 Luo (21) Appl. No.: 13/794,676 6,485,726 B1 1 1/2002 Blumberg et al. 6,503,510 B2 1/2003 Koishihara et al. (22) Filed: Mar 11, 2013 6,528,624 B1 3/2003 Idusogie et al. 6,538,124 B1 3/2003 Idusogie et al. (65) Prior Publication Data 6,613,546 B1 9, 2003 Ohtomo et al. 6,632,927 B2 10/2003 Adair et al. US 2014/02056.05 A1 Jul. 24, 2014 (Continued) FOREIGN PATENT DOCUMENTS Related U.S. Application Data EP O268636 1, 1997 (62) Division of application No. 127441.885, filed as EP O960936 12/1999 application No. PCT/US2007/078800 on Sep. 18, 2007. (Continued) OTHER PUBLICATIONS (60) Provisional application No. 60/885,848, filed on Jan. Aase, A. etal. “The extended hinge region of IgG3 is not required for 19, 2007, provisional application No. 60/828,054, high phagocytic capacity mediated by Fc gamma receptors, but the filed on Sep. 18, 2006. heavy chains must be disulfidebonded.” EurJImmunol. 23(7): 1546 1551 (Jul 1993). (51) Int. C. A 6LX39/395 (2006.01) (Continued) C07K 6/28 (2006.01) A6 IK3 L/454 (2006.01) Primary Examiner — Laura B Goddard A 6LX3/573 (2006.01) Assistant Examiner — Meera Natarajan A6 IK3I/69 (2006.01) (74) Attorney, Agent, or Firm — Polsinelli PC (52) U.S. C. CPC ...... C07K 16/2896 (2013.01); C07K 231 7/24 (57) ABSTRACT (2013.01); C07K 2317/41 (2013.01); C07K The present disclosure describes antibodies that target 231 7/56 (2013.01); C07K 2317/565 (2013.01); HM1.24. In various aspects, the antibodies have specific C07K 2317/72 (2013.01); C07K 2317/92 CDR, variable, or full length sequences, have modifications (2013.01); C07K 231 7/52 (2013.01); A61 K with the parent , or include at least one modification 31/454 (2013.01); A61 K3I/573 (2013.01); relative to a parent antibody that alters affinity to an FcyR or A61 K3I/69 (2013.01); A61 K39/3955 alters effector function as compared to the parent antibody. (2013.01) Nucleic acids encoding the antibodies and methods of using (58) Field of Classification Search the antibodies are also disclosed. CPC ...... C07K 16/00; A61K 39/395 See application file for complete search history. 20 Claims, 19 Drawing Sheets US 9,040,042 B2 Page 2

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U.S. Patent May 26, 2015 Sheet 5 Of 19 US 9,040,042 B2

Figure 4a Position

G1m(3) G1m(f) R E M A G1m(13) G1m(a) R DL A Figure 4b

U.S. Patent May 26, 2015 Sheet 6 of 19 US 9,040,042 B2

Figure 5 Receptor Receptor binding binding Cell activity Therapeutic activity improvement reduction Enhance dendritic cell activity and uptake, and Subsequent Enhance Cell-based Solely presentation of antigens: enhance immune response monocyte and macrophage against target

response to antibOd Enhance ADCC and phagocytosis Increased target cell of broad range of cell types lysis Enhance ADCC and phagocytosis Increased target cell of broad rance of cell types vsis Reduction of activity of all FCR Enhancement of target bearing cell types except NK cells cell lysis selective for and possible activation of NK cells NK cell accessible via Ilc receptor SionalinC tardet Cells Possible NK cell specific activation Enhancement of target and enhancement of NK Cell cell lysis selective for IIb, Illa NK Cell accessible mediated ADCC target cells Enhanced target cell Neutrophil mediated phagocytosis destruction for enhancement neutrophil accessible cells Enhanced target cell Neutrophil mediated phagocytosis destruction for enhancement neutrophil accessible cells Enhance dendritic cell activity and uptake, and Subsequent Enhance Cell-based l, lla, Illa presentation of antigens to T cells; immune response enhance monocyte and against target macrophage response to antibody Reduction in activity of monocytes, Eliminate or reduce macrophages, neutrophils, NK, Cell-mediated Ilala, dendritic and other gamma cytotoxicity against receptor bearind cells tardet bearind cells U.S. Patent May 26, 2015 Sheet 7 Of 19 US 9,040,042 B2

Figure 6

> Murine HM1.24 VL (LO) (SEQ ID NO:9) DVMTOSHKFMSTSVGDRVSTCKASODVNTAVAWYOOKPGGRSPKLLIYSASNRYTGVPDRIT GSGSGTDFTFTISSVOAEDLALYYCOOHYSTPFTFGSGTKLEK > Murine HM1.24 VH (HO) (SEQ ID NO:10) OVOLOOSGAELARPGASVKLSCKASGYTFTPYWMOWVKORPGOGLEWIGSIFPGDGDTRYS OKFKGKATLTADKSSSTAYMOLSLAFEDSAVYYCARGLRRGGYYFDYWGOGTTLTVSS

> Murine HM1.24 VLCDR1 (SEQID NO:11) KASODVNTAVA > Murine HM1.24 VLCDR2 (SEQ ID NO:12) SASNRYT > Murine HM1.24 VLCDR3 (SEQID NO:13) QOHYSTPFT > Murine HM1.24 VHCDR1 (SEQID NO:14) PYWMO > Murine HM1.24 VHCDR2 (SEQID NO:15) SIFPGDGDTRYSOKFKG > Murine HM1.24 VHCDR3 (SEQID NO:16) GLRRGGYYFDY U.S. Patent US 9,040,042 B2

CD3 (T cells)

CD 19 (B cells)

CD56 ********** (NK) U.S. Patent May 26, 2015 Sheet 9 Of 19 US 9,040,042 B2

CD38 (Plasma)

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CD38 (Plasma)

; : : is: 3 : S. & U.S. Patent May 26, 2015 Sheet 10 of 19 US 9,040,042 B2

Figure 9 RPM 18226 (10k) ADCC with PBMC effector cells With HM 1.24 antibodies HHM1.24 HOLO gCG1 WT - HM1.24 HOLO gG1 S239DfI332E aŠas HM1.24 HOLO igg1 G236A \SY HM1.24 HOLO igg1 G236AI332E -0- HM1.24 HOLO gCG1 S239D/H268E/G236A as HM1.24 HOLOlg(G1S239D/332EIG236A -- higG isotype Control

-5 0.01 0.1 1 10 100 1000 10000 Antibody (ng/ml)

U.S. Patent May 26, 2015 Sheet 12 of 19 US 9,040,042 B2

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U.S. Patent May 26, 2015 Sheet 14 of 19 US 9,040,042 B2

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US 9,040,042 B2 1. 2 OPTIMIZED ANTIBODIES THAT TARGET having a CDR1 with amino acid sequence selected from the HM124 group consisting of SEQID NO: 11, 41 or 42, a CDR2 amino acid sequence of SEQ ID NOS:12 and a CDR3 amino acid This application is a divisional application of U.S. patent sequence of SEQ ID NOs: 13; and the heavy chain having a application Ser. No. 12/441,885, filed Dec. 16, 2009, which is CDR1 comprising the amino acid sequence of SEQID NOs: the national stage application of PCT Patent Application No. 114, a CDR2 having an amino acid sequence selected from PCT/US2007/078800, filed Sep. 18, 2007, and which claims SEQID NOS:15, 39 or 40, and a CDR3 having an amino acid benefit under 35 U.S.C. S 119(e) to U.S. Provisional Patent sequence of SEQID NOs: 16, with the proviso that at least one Application Nos. 60/826,054 filed Sep. 18, 2006 and 60/885, of the light chain CDR1 is SEQ ID NO:41, the light chain 848 filed Jan. 19, 2007, each of which are incorporated herein 10 CDR1 is SEQID NO:2, the heavy chain CDR2 is 39, or the by reference in their entirety. heavy chain CDR2 is 40. In a further aspect, the heavy chain sequence is selected SEQUENCE LISTING from SEQ ID NOS: 21-32, and/or the light chain sequence selected from the group consisting of SEQID NOS: 17-20. In A Sequence Listing Submitted in computer readable form 15 an additional aspect, the heavy chain sequence further com (CRF) is hereby incorporated by reference. The CRF file is prises a sequence selected from SEQID NOS: 3-8 and 21-32, named 189572US04. ST25.txt, was created on Feb. 19, and/or the light chain sequence further comprises a sequence 2012, and contains 350 kilobytes selected from the group consisting of SEQ ID NOS:2 and 17-2O. BACKGROUND In an additional aspect, the antibody comprises a heavy chain sequence selected from the group consisting of SEQID B cells are lymphocytes that play a large role in the humoral NOS: 43-114, and/or a variable light chain sequence selected immune response. They are produced in the bone marrow of from the group consisting of SEQID NOS: 115-118. most mammals, and represent 5-15% of the circulating lym In certain aspects, the antibody has an amino acid modifi phoid pool. The principal function of B cells is to make 25 cation. In one variation, the amino acid modification is at a antibodies against various antigens, and are an essential com position selected from among 221, 222, 223, 224, 225, 227, ponent of the adaptive . 228, 230, 231, 232, 233,234, 235, 236, 237,238, 239, 240, Plasma B cells (also known as plasma cells) are a large 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, mature B cells that have been exposed to antigen and produce 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, and secrete large amounts of antibodies, which assist in the 30 276, 278, 280, 281, 282,283, 284, 285, 286, 288, 290, 291, destruction of microbes by binding and facilitating targeting 292, 293,294, 295, 296,297, 298, 299, 300, 301,302,303, by phagocytes, as well as activation of the complement sys 304,305,313,317, 318,320, 322,323,324, 325, 326,327, tem. Plasma cells are sometimes referred to as antibody fac 328,329,330,331,332,333,334,335,336, and 337, wherein tories. numbering is according to the EU index. In a further varia Several plasma cell disorders are known. Multiple 35 tion, the amino acid modification is a Substitution selected myeloma is among the most prominent. Autoimmune disor from among 221K, 221Y. 222E, 222Y, 223E, 223K, 224E, ders resulting from in breakdown of self-tolerance can arise 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, from terminally differentiated plasma cells. Additional 228G, 228K, 228Y. 230A, 230E, 230G, 230Y, 231E, 231G, plasma cell disorders include amyloidosis, which occurs 231 K, 231P 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, when antibodies or protein fragments accumulate in organs of 40 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, the body, Waldenstrom's macroglobulinemia, a malignant 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, blood cancer characterized by a high level of IgM antibodies 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P 234Q, in the blood and the bone marrow, plasmacytoma (which is 234R, 234S, 234T 234V, 234W, 234Y, 235A, 235D, 235E, related to multiple myeloma), POEMS syndrome (character 235F, 23.5G, 235H, 235I, 235K, 235M, 235N, 235P. 235Q, ized by polyneuropathy, organomegaly, endocrinopathy, 45 235R, 235S, 235T, 235V, 235W, 235Y, 236A, 236D, 236E, multiple myeloma, and skin changes), heavy-chain diseases 236F, 236H, 236I, 236K, 23.6L 236M, 236N, 236P, 236Q, (such as gamma heavy-chain disease (Franklin's disease), 23.6R, 236S, 236T, 236V, 236W, 236Y. 237D, 237E, 237F, alpha heavy-chain disease (Seligmann's disease) which 237H, 237I, 237K, 237L, 237M, 237N, 237P. 237O, 237R, occurs together with a small intestinal lymphoma called 237S, 237T, 237V, 237W, 237Y, 238D, 238E, 238F, 238G, immunoproliferative Small intestinal disease), and Mu heavy 50 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, chain disease. 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, To date, there has not been an antibody therapy capable of 239I, 239K, 239L, 23.9M, 239N, 239P, 239Q, 239R, 239T. addressing these indications. This disclosure addresses this 239V, 239W. 239Y, 240A, 24OI, 240M, 240T, 241D, 241E, and other needs. 241L, 241R, 241S, 241 W. 241Y. 243E, 243H, 243L, 243O, 55 243R, 243W, 243Y. 244H, 245A, 246D, 24.6E, 246H, 246Y. SUMMARY 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 26OH, 26OY, 262A, 262E, 262F, 262I, The present application is generally directed to antibodies 262T, 263-A, 263I, 263M, 263T, 264A, 264D, 264E, 264F, that bind HM1.24 (also referred to herein as “anti-HM1.24 264G, 264H, 264-I, 264K, 264L, 264M, 264N, 264P 264Q, antibodies’). In one embodiment, the anti-HM1.24 antibody 60 264R, 264S, 264T, 264W, 264Y. 265F, 265G, 265H, 265I, includes at least one modification in the constant region rela 265K, 265L, 265M, 265N, 265P. 265Q, 265R, 265S, 265T, tive to a parent anti-HM1.24 antibody, and has enhanced 265V, 265W, 265Y. 266A, 266I, 266M, 266T, 267D, 267E, ADCC effector function as compared to the parent antibody. 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P. 267O, In other variations, the antibody binds with increased affinity 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, to the FcyRIIIa receptor as compared to the parent antibody. 65 268I, 268K, 268L, 268M, 268P. 268Q, 268R, 268T, 268V, In other aspects, the present application the antibody 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M, 269N, includes a light chain and a heavy chain, the light chain 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G,

US 9,040,042 B2 7 8 In further aspects, the amino acid modification is 332E. In convention of Kabat (VHCDR1:31-35, VH CDR2: 50–65, additional aspects, the second amino acid modification VHCDR3: 95-102, VLCDR1:24-34, VLCDR2: 50-56, and selected from among: 236A,239D,332E, 268D,268E,330Y, VLCDR3: 89-97). and 330L. FIG. 7. Flow cytometry measuring binding of In a further aspect, the modification is a glycoform modi HM1.24 HOLO IgG1 WT antibody or human IgG control to fication that reduces the level of fucose relative to the parent components of PBMCs. antibody. In a further aspect, the application is directed to a FIG. 8. Flow cytometry measuring binding of composition including a plurality of glycosylated antibodies, HM1.24 HOLO IgG1 WT antibody or human IgG control to wherein about 80-100% of the glycosylated antibody in the RPMI 8226 multiple myeloma cells. composition includes a mature core carbohydrate structure 10 FIG. 9. ADCC assay with WT and variant anti-HM1.24 which lacks fucose. antibodies against RPMI 8226 target cells, using PBMCs as In another aspect, the present disclosure is directed to a effector cells. nucleic acid sequence encoding an antibody disclosed herein. FIGS. 10a-c depict amino acid sequences encoding In a further aspect, the present application is directed to a 15 humanized heavy and light chain variable region variants of method of treating a B-cell related disease, Such as a plasma the anti-HM1.24 antibody. FIG. 10a provides the sequences, cell related disease. In certain variations, the disease is and FIGS. 10b and 10c show alignments of the heavy and selected from multiple myeloma (MM), mantle cell myeloma light humanized chains respectively as compared to WT (HO (MCM), myelodysplastic syndrome with delSq (MDS), and and L0). CDRS are highlighted in gray, and numbering and autoimmune indication. In certain variations, the autoim CDR boundaries are according to the system of Kabat. mune disease is autoimmune disease is rheumatoid arthritis FIG.11. Expression yield of HM1.24 heavy and light chain (RA), systemic erythematosus (SLE or lupus), multiple combinations from a fixed number of plates of 293T cells. All Sclerosis, Sjogren's syndrome, or idiopathic thrombocytope heavy chains comprise the Hybrid S239D/I332E constant nia purpura (ITP). region. In a further aspect, the method of treating a B-cell related 25 FIGS. 12a-b depict binding of humanized anti-HM1.24 disease is further directed to administering a compound antibody variants to HM 1.24+ multiple myeloma RPMI 8226 selected from Velcade(R), Revlimid(R) and dexamethasone. cells measured by flow cytometry. FIG.12a shows the dose A composition comprising an antibody according to claim dependent fluorescence curves; antibody was detected using 1 and an acceptable carrier. R-PE conjugated F(ab')2 fragment Goat anti-human IgG. 30 FIG. 12b shows the EC50's for select antibody binding BRIEF DESCRIPTION OF THE DRAWINGS CUWCS. FIG. 13. ADCC activity of humanized and WT anti The following drawings further illustrate aspects of the HM1.24 antibodies. invention, and are not meant to constrain the invention to any FIG. 14. Library of HM1.24 H1 and HM1.24 H2 variants particular application or theory of operation. 35 to remove potential aspartate isomerization sites from VH FIG. 1: Amino acid sequence of homo sapiens HM1.24, as CDR2. obtained from cDNA clone MGC:45144, IMAGE clone FIG. 15. Amino acid sequences of the HM1.24 H1 and 5217945, GenBank Accession: BC033873. HM1.24 H2 variants to remove potential aspartate isomer FIG.2. Sequences of the natural antibody constant regions, 40 ization sites from VHCDR2. including the kappa constant light chain, and the gamma FIG. 16. Dissociation rate constants of variant and parent constant heavy chains for IgG1, IgG2, IgG3, and IgG4. Also H1 and H2 antibodies for binding to HM1.24 antigen after provided is the sequence of a Hybrid IgG constant chain, and accelerated asp isomerization conditions or no treatment. a Hybrid IgG constant chain comprising the Substitutions FIG. 17. Binding of anti-HM1.24 antibodies to HM1.24 239D and I332E. 45 antigen monitored by Biacore. In this format, HM1.24 anti FIGS. 3a-b depict alignments of the amino acid sequences gen was coupled directly to the chip, and antibodies at mul of the human IgG immunoglobulins IgG1, IgG2, IgG3, and tiple concentrations were used as analyte in this format. The IgG4. FIG. 3a provides the sequences of the CH1 (CY 1) and table provides kinetic rate constants (ka and kd) and equilib hinge domains, and FIG. 3b provides the sequences of the rium dissociation constants (KD) obtained by fitting of these CH2 (Cy2) and CH3(CY3) domains. Positions are numbered 50 data obtained by simultaneous global Langmuir fitting (left according to the EU index of the IgG1 sequence, and differ columns) or by fitting kinetic curves separately (right col ences between IgG1 and the other immunoglobulins IgG2. umns) IgG3, and IgG4 are shown in gray. Allotypic polymorphisms FIG. 18. ADCC activity of anti-HM1.24 antibodies against exist at a number of positions, and thus slight differences the multiple myeloma cell line RPMI 8226. between the presented sequences and sequences in the prior 55 art may exist. The possible beginnings of the Fc region are DETAILED DESCRIPTION labeled, defined herein as either EU position 226 or 230. FIG. 4. The common haplotypes of the gamma chain of The disclosure is directed to modified anti-HM1.24 anti human IgG1 (FIG. 4a) and IgG2 (FIG. 4b) showing the posi bodies and methods of using the same. In various aspects, the tions and the relevantamino acid Substitutions. 60 antibodies can have a having a modified Fc region, specific FIG.5. Preferred embodiments of receptor binding profiles CDR sequences, variable region sequences, and/or constant that include increases to, reductions to, or no effect to the region modifications. In various embodiments, the antibodies binding to various receptors, where such changes may be are humanized. The disclosure is further directed to methods beneficial in certain contexts. ofusing the antibodies in various disease indications, includ FIG. 6. Amino acid sequences of the heavy chain and light 65 ing those of B-cell origin Such as B-cell origin non-Hodgkin’s chain variable regions and CDRs of the murine anti-HM1.24 lymphoma (NHL), acute lymphoblastic leukemia (ALL), and antibody. CDR boundaries are determined according to the autoimmune related diseases. US 9,040,042 B2 9 10 In order that the invention may be more completely under Cgamma2 and Cgamma3 (CY1, Cy2 and Cy3) and the hinge stood, several definitions are set forth below. Such definitions between Cgammal (CY1) and Cgamma2 (CY2). Although the are meant to encompass grammatical equivalents. boundaries of the constant region may vary, the human IgG By ADCC or “antibody dependent cell-mediated cyto heavy chain Fc region is usually defined to comprise residues toxicity’ as used herein is meant the cell-mediated reaction 5 to its carboxyl-terminus, wherein the numbering is according wherein nonspecific cytotoxic cells that express FcyRs rec to the EU indexas in Kabat. The constant light chain typically ognize bound antibody on a target cell and Subsequently comprises a single domain, and as defined herein refers to cause lysis of the target cell. In various aspects, the enhanced positions 108-214 of CK or CW, wherein numbering is accord ADCC effector function can mean enhanced potency or ing to the EU index. For full length IgG antibodies, the con enhanced efficacy. By “potency” as used in the experimental 10 stant heavy chain, as defined herein, refers to the N-terminus context is meant the concentration of antibody when a par of the CH1 domain to the C-terminus of the CH3 domain, or ticular therapeutic effect is observed EC50 (half maximal positions 118-447, wherein numbering is according to the EU effective concentration). By “efficacy” as used in the experi index. "Constant region' may refer to this region in isolation, mental context is meant the maximal possible effector func or a truncation or fusion include antibodies, Fc fusions, iso tion at Saturating levels of antibody. 15 lated Fcs, and Fc fragments. In various embodiments, the By ADCP or antibody dependent cell-mediated phago constant region may be the region of the antibody that is cytosis as used herein is meant the cell-mediated reaction encoded by one of the light or heavy chain immunoglobulin wherein nonspecific cytotoxic cells that express FcyRs rec constant region genes, i.e. the region of an antibody encoded ognize bound antibody on a target cell and Subsequently by the kappa (CK) or lambda (CW) light chains. In various cause phagocytosis of the target cell. embodiments, the constant heavy chain or heavy chain con By “amino acid and “amino acid identity” as used herein stant region can be the region of an antibody encoded by the is meant one of the 20 naturally occurring amino acids or any mu, delta, gamma, alpha, or epsilon genes to define the anti non-natural analogues that may be present at a specific, body's isotype as IgM, Ig), IgG, IgA, or IgE, respectively. defined position. Thus "amino acid as used herein means By “effector function” as used herein is meant a biochemi both naturally occurring and synthetic amino acids. For 25 cal event that results from the interaction of an antibody Fc example, homophenylalanine, citrulline and norleucine are region with an Fc receptor or ligand. Effector functions considered amino acids for the purposes of the invention. include Fcy R-mediated effector functions such as ADCC and “Amino acid also includes imino acid residues such as pro ADCP, and complement-mediated effector functions such as line and hydroxyproline. The side chain may be in either the CDC. By “effector cell as used herein is meant a cell of the (R) or the (S) configuration. In the preferred embodiment, the 30 immune system that expresses one or more Fc receptors and amino acids are in the (S) or L-configuration. If non-naturally mediates one or more effector functions. Effector cells occurring side chains are used, non-amino acid substituents include but are not limited to monocytes, macrophages, neu may be used, for example to prevent or retard in vivo degra trophils, dendritic cells, eosinophils, mast cells, platelets, B dation. cells, large granular lymphocytes, Langerhans cells, natural By “B cell' or “B lymphocyte' as used herein is meant a 35 killer (NK) cells, and Yö T cells, and may be from any organ type of lymphocyte developed in bone marrow that circulates ism including but not limited to humans, mice, rats, rabbits, in the blood and lymph, and provides humoral immunity. B and monkeys. cells recognize free antigen molecules and differentiate or By “Fab’ or "Fab region” as used herein is meant the mature into plasma cells that secrete immunoglobulin (anti polypeptides that comprise the V, CH1, V, and C, immu bodies) that inactivate the antigens. Memory cells are also 40 noglobulin domains. Fab may refer to this region in isolation, generated that make the specific Immunoglobulin (antibody) or this region in the context of a full length antibody or on Subsequent encounters with Such antigen. B cells are also antibody fragment. known as “Beta cells” in the islet of Langerhans. By “Fe' or “Fe region', as used herein is meant the By “B-cell antigen' or “B-cell marker” as used herein is polypeptide comprising the constant region of an antibody meant any protein that is expressed on B cells. 45 excluding the first constant region immunoglobulin domain. By “HM1.24 as used herein is meant the protein encoded Thus Fc refers to the last two constant region immunoglobu by the gene designated HM1.24. HM1.24 is also referred to as lin domains of IgA, Ig|D, and IgG, and the last three constant bone marrow stromal cell antigen 2 (BST2 or BST-2), region immunoglobulin domains of IgE and IgM, and the EMP24, and CD317. Human HM1.24 is designated GeneID: flexible hinge N-terminal to these domains. For IgA and IgM, 684 by Entrez Gene, and HGNC:1119 by HGNC. The use of 50 Fc may include the J chain. For IgG, Fc comprises immuno HM1.24 herein is meant to encompass all known or as yet globulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) undiscovered alleles and polymorphic forms of HM1.24. The and the hinge between Cgammal (CY1) and Cgamma2 (CY2). sequence of human HM1.24 antigen used in the present study Although the boundaries of the Fc region may vary, the is provided in FIG. 1, SEQ ID:1. human IgG heavy chain Fc region is usually defined to com By “CDC or “complement dependent cytotoxicity’ as 55 prise residues C226 or P230 to its carboxyl-terminus, wherein used herein is meant the reaction wherein one or more the numbering is according to the EU index as in Kabat. Fc complement protein components recognize bound antibody may refer to this region in isolation, or this region in the on a target cell and Subsequently cause lysis of the target cell. context of an Fc polypeptide, for example an antibody. By “Fe By "constant region' as used herein is meant the polypep polypeptide' as used herein is meant a polypeptide that com tide including at least a portion of the first three constant 60 prises all or part of an Fc region. Fc polypeptides include regions of an antibody, having at least one effector function. antibodies, Fc fusions, isolated Fcs, and Fc fragments. Thus constant region thus refers to the last three constant By “Fe gamma receptor or “FeyRas used herein is meant region immunoglobulin domains of IgA, Ig|D, and IgG, and any member of the family of proteins that bind the IgG anti the last four constant region immunoglobulin domains of IgE body Fc region. In various embodiments, FcyR are substan and IgM, and the flexible hinge N-terminal to these domains. 65 tially encoded by the FcyR genes. In humans this family For IgA and IgM, Fc may include the J chain. For IgG, the includes but is not limited to FcyRI (CD64), including iso constant region include immunoglobulin domains Cgammal, forms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), includ US 9,040,042 B2 11 12 ing isoforms FcyRIIa (including allotypes H131 and R131), By “glycoform modification' or “modified glycoform” or FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc: “engineered glycoform' as used herein is meant a carbohy and FcyRIII (CD16), including isoforms FcyRIIIa (including drate composition that is covalently attached to a protein, for allotypes V158 and F158) and FcyRIIIb (including allotypes example an antibody, wherein said carbohydrate composition FcyRIIIb-NA1 and FcyRIIIb-NA2) (Jefferis et al., 2002, differs chemically from that of a parent protein. Modified Immunol Lett 82:57-65, incorporated entirely by reference), glycoform typically refers to the different carbohydrate or as well as any undiscovered human FcyRs or FcyR isoforms oligosaccharide; thus for example an antibody may comprise or allotypes. Mouse FcyRs include but are not limited to a modified glycoform. Alternatively, modified glycoform FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and may refer to the antibody that comprises the different carbo FcyRIII-2 (CD16-2), as well as any undiscovered mouse 10 hydrate or oligosaccharide. FcyRs or FcyR isoforms or allotypes. An FcyR may be from By “parent polypeptide', 'parent protein”, “precursor any organism, including but not limited to humans, mice, rats, polypeptide', or “precursor protein’ as used herein is meant rabbits, and monkeys. an unmodified polypeptide that is Subsequently modified to By “Fe ligand’ or “Fc receptor as used herein is meanta generate a variant. Said parent polypeptide may be a naturally molecule, preferably a polypeptide, from any organism that 15 occurring polypeptide, or a variant or engineered version of a binds to the Fc region of an antibody to form an Fc-ligand naturally occurring polypeptide. Parent polypeptide may complex. Fc ligands include but are not limited to FcyRs, refer to the polypeptide itself, compositions that comprise the FcRn, C1q, C3, mannan binding lectin, mannose receptor, parent polypeptide, or the amino acid sequence that encodes staphylococcal protein A, Streptococcal protein G, and viral it. Accordingly, by "parent antibody' or “parent immunoglo FcyR. Fc ligands also include Fc receptor homologs (FcRH), bulin' as used herein is meant an antibody or immunoglobu which are a family of Fc receptors that are homologous to the lin that is modified to generate a variant. By "parent anti FcyRs (Davis et al., 2002, Immunological Reviews 190: 123 HM124 antibody' O “parent anti-HM1.24 136, incorporated entirely by reference). Fc ligands may immunoglobulin' as used herein is meant an antibody or include undiscovered molecules that bind Fc. immunoglobulin that binds HM1.24 and is modified to gen By "IgG” as used herein is meant a polypeptide belonging 25 erate a variant. to the class of antibodies that are substantially encoded by a By plasma cell (also called plasma B cells or plas recognized immunoglobulin gamma gene. In humans this mocytes) antibody secreting cells. class comprises IgG1, IgG2, IgG3, and IgG4. In mice this By “protein’ or “polypeptide' as used herein is meant at class comprises IgG1, IgG2a, IgG2b, IgG3. By “immunoglo least two covalently attached amino acids, which includes bulin (Ig) herein is meant a protein consisting of one or more 30 proteins, polypeptides, oligopeptides and peptides. The pro polypeptides Substantially encoded by immunoglobulin tein may be made up of naturally occurring amino acids and genes. Immunoglobulins include but are not limited to anti peptide bonds, or synthetic peptidomimetic structures, i.e. bodies. Immunoglobulins may have a number of structural “analogs'. Such as peptoids. forms, including but not limited to full length antibodies, By “position' as used herein is meant a location in the antibody fragments, and individual immunoglobulin 35 sequence of a protein. Positions may be numbered sequen domains. By “immunoglobulin (Ig) domain herein is meant tially, or according to an established format, for example the a region of an immunoglobulin that exists as a distinct struc EU index as in Kabat. Corresponding positions are deter tural entity as ascertained by one skilled in the art of protein mined as outlined herein, generally through alignment with structure. Ig domains typically have a characteristic -sand other parent sequences. wich folding topology. The known Ig domains in the IgG 40 By “residue' as used herein is meant a position in a protein class of antibodies are V, Cyl, Cy2, Cy3, V, and C. and its associated amino acid identity. For example, Aspar By "modification herein is meant an alteration in the agine 297 (also referred to as Asn297 and N297) is a residue physical, chemical, or sequence properties of a protein, at position 297 in the human antibody IgG1. polypeptide, antibody, or immunoglobulin. Preferred modi By “target antigen' or “target' or “antigen' as used herein fications of the invention are amino acid modifications and 45 is meant the molecule that is bound specifically by the vari glycoform modifications. able region of a given antibody. A target antigen may be a By “amino acid modification' herein is meant an amino protein, carbohydrate, lipid, or other chemical compound. By acid Substitution, insertion, and/or deletion in a polypeptide “target cell as used herein is meant a cell that expresses a sequence. By “amino acid substitution” or “substitution” target antigen. herein is meant the replacement of an amino acid at a particu 50 By "variable region' is meant the variable region of an lar position in a parent polypeptide sequence with another antibody heavy chain or light chain. The heavy chain variable amino acid. For example, the substitution I332E refers to a region (VH), as defined herein, refers to the N-terminus to the variant polypeptide, in this case a constant heavy chain vari C-terminus of the VH domain, defined by residues 1-113 ant, in which the isoleucine at position 332 is replaced with according to the numbering convention of Kabat. The light glutamic acid. The WT residue may or may not be designated. 55 chain variable region (VL), as defined herein, refers to the For the preceding example, 332E indicates the substitution of N-terminus to the C-terminus of the VL domain, defined by position 332 with a glutamic acid. For the purposes herein, residues 1-107 according to the numbering convention of multiple Substitutions are typically separated by a slash. For Kabat. Those skilled in the art will recognize that the Kabat example, 239D/332E refers to a double variant comprising variable region numbering convention employs letters to the substitutions 239D and 332E. By “amino acid insertion 60 account for the variable length of CDRs. Thus that a VH is or “insertion' as used herein is meant the addition of an amino defined by Kabat residues 1-113, and that a VL is defined by acid at a particular position in a parent polypeptide sequence. Kabat 1-107, does not necessarily mean that the VH domain For example, insert -236 designates an insertion of glycine at contains exactly 113 residues, nor that VL contains exactly position 236. By “amino acid deletion” or “deletion” as used 107 residues. Rather, residues 1-113 of VH and 1-107 of VL herein is meant the removal of an amino acid at a particular 65 according to Kabat are meant to encompass the structural position in a parent polypeptide sequence. For example, domains that were determined by sequence alignments of a G236-designates the deletion of glycine at position 236. large set of variable length antibody variable regions of vary US 9,040,042 B2 13 14 ing length ((Kabat et al., 1991, Sequences of Proteins of mals, including humans and mice, the full length antibody of Immunological Interest, 5th Ed., United States Public Health the IgG class is a tetramer and consists of two identical pairs Service, National Institutes of Health, Bethesda, incorporated of two immunoglobulin chains, each pair having one light and entirely by reference). In certain embodiments, the variable one heavy chain, each light chain comprising immunoglobu region can comprises one or more Ig domains substantially lin domains V, and C, and each heavy chain comprising encoded by any of the VK, VW, and/or V genes that make up immunoglobulin domains V, CH1 (CY1), CH2 (Cy2), and the kappa, lambda, and heavy chain immunoglobulin genetic CH3 (Cy3). In some mammals, for example in camels and loci respectively. llamas, IgG antibodies may consist of only two heavy chains, By "variant protein”, “protein variant”, “variant polypep each heavy chain comprising a variable domain attached to tide', or “polypeptide variant as used herein is meant a 10 the Fc region. polypeptide sequence that differs from that of a parent The term “antibody” also includes antibody fragments. polypeptide sequence by virtue of at least one amino acid Specific antibody fragments include, but are not limited to, (i) modification. Variant polypeptide may refer to the polypep the Fab fragment consisting of VL, VH, CL and CH1 tide itself, a composition comprising the polypeptide, or the domains, (ii) the Fd fragment consisting of the VH and CH1 amino sequence that encodes it. Preferably, the variant 15 domains, (iii) the Fv fragment consisting of the VL and VH polypeptide has at least one amino acid modification com domains of a single antibody; (iv) the dAb fragment (Ward et pared to the parent polypeptide, e.g. from about one to about al., 1989, Nature 341:544-546) which consists of a single ten amino acid modifications, and preferably from about one variable, (v) isolated CDR regions, (vi) F(ab')2 fragments, a to about five amino acid modifications compared to the par bivalent fragment comprising two linked Fab fragments (vii) ent. The variant polypeptide sequence herein will preferably single chain Fv molecules (sclv), wherein a VH domain and possess at least about 80% homology with a parent polypep a VL domain are linked by a peptide linker which allows the tide sequence, and most preferably at least about 90% homol two domains to associate to forman antigenbinding site (Bird ogy, more preferably at least about 95% homology. Accord et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. ingly, by “variant antibody' or “antibody variant as used Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii) bispecific herein is meant an antibody sequence that differs from that of 25 single chain Fv dimers (PCT/US92/09965) and (ix)"diabod a parent antibody sequence by virtue of at least one amino ies' or “triabodies', multivalent or multispecific fragments acid modification. Antibody variant may refer to the antibody constructed by gene fusion (Tomlinson et. al., 2000, Methods polypeptide itself, compositions comprising the antibody Enzymol. 326:461–479; WO94/13804; Holliger et al., 1993, variant polypeptide, or the amino acid sequence that encodes Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448). In certain it. Accordingly, by “variant antibody' or “antibody variant” 30 embodiments, antibodies are produced by recombinant DNA as used herein is meant an antibody, as defined above, that techniques. Other examples of antibody formats and archi differs in sequence from that of a parent antibody sequence by tectures are described in Holliger & Hudson, 2006, Nature virtue of at least one amino acid modification. Variant anti Biotechnology 23(9): 1126-1136, and Carter 2006, Nature body may refer to the protein itself, compositions comprising Reviews Immunology 6:343-357 and references cited the protein, or the amino acid sequence that encodes it. 35 therein, all expressly incorporated by reference. In additional Accordingly, by “constant heavy chain variant' or “constant embodiments, antibodies are produced by enzymatic or light chain variant' or “Fe variant as used herein is meant a chemical cleavage of naturally occurring antibodies. constant heavy chain, constant light chain, or Fc region Natural antibody structural units typically comprise a tet polypeptide or sequence, respectively, that differs in ramer. Each tetramer is typically composed of two identical sequence from that of a parent sequence by virtue of at least 40 pairs of polypeptide chains, each pair having one “light one amino acid modification. (typically having a molecular weight of about 25 kDa) and By “wild type or WT" herein is meant an amino acid one "heavy chain (typically having a molecular weight of sequence or a nucleotide sequence that is found in nature, about 50-70 kDa). Each of the light and heavy chains are including allelic variations. A WT protein, polypeptide, anti made up of two distinct regions, referred to as the variable and body, immunoglobulin, IgG, etc., has an amino acid sequence 45 constant regions. For the IgG class of immunoglobulins, the or a nucleotide sequence that has not been intentionally modi heavy chain is composed of four immunoglobulin domains fied. linked from N- to C-terminus in the order V-CH1-CH2 For all immunoglobulin heavy chain constant region posi CH3, referring to the heavy chain variable domain, heavy tions discussed in the present disclosure, numbering is chain constant domain 1, heavy chain constant domain 2, and according to the EU index as in Kabat (Kabat et al., 1991, 50 heavy chain constant domain 3 respectively (also referred to Sequences of Proteins of Immunological Interest, 5th Ed., as V-CY1-Cy2-Cy3, referring to the heavy chain variable United States Public Health Service, National Institutes of domain, constant gamma 1 domain, constant gamma 2 Health, Bethesda, incorporated entirely by reference). The domain, and constant gamma 3 domain respectively). The “EU index as in Kabat’ refers to the residue numbering of the IgG light chain is composed of two immunoglobulin domains human IgG1 EU antibody, as described in Edelman et al., 55 linked from N- to C-terminus in the order V-C, referring to 1969, Biochemistry 63:78-85, incorporated entirely by refer the light chain variable domain and the light chain constant CCC. domain respectively. The constant regions show less Antibodies sequence diversity, and are responsible for binding a number As used herein, the term “antibody' refers to a monomeric of natural proteins to elicit important biochemical events. or multimeric protein comprising one or more polypeptide 60 The variable region of an antibody contains the antigen chains. An antibody binds specifically to an antigen (e.g. binding determinants of the molecule, and thus determines HM1.24) and may be able to modulate the biological activity the specificity of an antibody for its target antigen. The vari of the antigen. As used herein, the term “antibody’ can able region is so named because it is the most distinct in include “full length antibody” and “Fc polypeptide.” sequence from other antibodies within the same class. In the By "full length antibody' herein is meant the structure that 65 variable region, three loops are gathered for each of the V constitutes the natural biological form of an antibody, includ domains of the heavy chain and light chain to form an anti ing variable and constant regions. For example, in most mam gen-binding site. Each of the loops is referred to as a comple US 9,040,042 B2 15 16 mentarity-determining region (hereinafter referred to as a CDR3 having an amino acid sequence of SEQID NOs: 16. In “CDR), in which the variation in the amino acid sequence is this embodiment, the at least one of the light chain CDR1 is most significant. There are 6 CDRs total, three each per heavy SEQ ID NO:41, the light chain CDR1 is SEQID NO:2, the and light chain, designated V, CDR1, VCDR2. V. CDR3, heavy chain CDR2 is 39, or the heavy chain CDR2 is 40. V, CDR1, V, CDR2, and V, CDR3. The variable region These CDRs can be in any combination. outside of the CDRs is referred to as the framework (FR) Alternatively, the antibody can have a heavy chain region. Although not as diverse as the CDRs, sequence Vari sequence is selected from SEQ ID NOS: 21-32, and/or the ability does occur in the FR region between different antibod light chain sequence selected from the group consisting of ies. Overall, this characteristic architecture of antibodies pro SEQID NOS: 17-20. The heavy chain can also include a wild vides a stable scaffold (the FR region) upon which substantial 10 type or modified Fc region, such as an Fc region having a antigen binding diversity (the CDRs) can be explored by the sequence selected from SEQID NOS: 3-8 and 21-32. Simi immune system to obtain specificity for a broad array of larly, the light chain sequence can further include a wild type antigens. A number of high-resolution structures are available or modified Fc region, such as an Fc region having a sequence for a variety of variable region fragments from different selected from SEQID NOS:2 and 17-20. It will be recognized organisms, some unbound and some in complex with antigen. 15 that the wild type or modified Fc region can be added in Sequence and structural features of antibody variable regions combination with the sequences identified by CDRs, or the are disclosed, for example, in Morea et al., 1997, Biophys variable chain, with or without any intervening sequences. Chem 68:9-16; Morea et al., 2000, Methods 20:267-279, and Alternatively, the antibody can include a heavy chain the conserved features of antibodies are disclosed, for sequence selected from among SEQID NOS: 43-114, and/or example, in Maynard et al., 2000, Annu Rev Biomed Eng a variable light chain sequence selected from among SEQID 2:339-376, both incorporated entirely by reference. NOS: 115-118. Antibodies are grouped into classes, also referred to as As described in U.S. Ser. No. 1 1/256,060, filed Oct. 21, isotypes, as determined genetically by the constant region. 2005, entitled “IgG Immunoglobulin Variants with Opti Human constant light chains are classified as kappa (CK) and mized Effector Function’, herein expressly incorporated by lambda (CW) light chains. Heavy chains are classified as mu, 25 reference, it is possible to engineer amino acid modifications delta, gamma, alpha, or epsilon, and define the antibody’s in an antibody that comprise constant regions from other isotype as IgM, Ig), IgG, IgA, and IgE, respectively. The IgG immunoglobulin classes, for example as those illustrated in class is the most commonly used for therapeutic purposes. In the alignments in FIG. 3. Such engineered hybrid IgG com humans this class comprises Subclasses IgG1, IgG2, IgG3. positions may provide improved effector function properties, and IgG4. In mice this class comprises Subclasses IgG1. 30 including improved ADCC, phagocytosis, CDC, and serum IgG2a, IgG2b, IgG3. IgM has subclasses, including, but not half-life. For example, as illustrated by FIG.3, an IgG1/IgG3 limited to, IgM1 and IgM2. IgA has several subclasses, hybrid variant may be constructed by substituting IgG1 posi including but not limited to IgA1 and IgA2. Thus, “isotype' tions in the CH2 and CH3 region with the amino acids from as used herein is meant any of the classes or Subclasses of IgG3 at positions where the two isotypes differ. Thus a hybrid immunoglobulins defined by the chemical and antigenic 35 variant IgG antibody may be constructed that comprises one characteristics of their constant regions. The known human or more Substitutions selected from the group consisting of immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, 274Q, 276K,300F, 339T., 356E, 358M, 384S, 392N, 397M, IgA2, IgM1, IgM2, Igl), and IgE. FIG. 2 provides the 422I, 435R, and 436F, wherein numbering is according to the sequences of the human light chain kappa and heavy chain EU index. Such variant may provide alternate and/or gamma constant chains. FIG. 3 shows an alignment of the 40 improved effector function properties. human IgG constant heavy chains. As another example, relatively poor effector function of Also useful for the invention may be IgGs that are hybrid IgG2 may be improved by replacing key FcyR binding resi compositions of the natural human IgG isotypes. Effector dues with the corresponding amino acids in an IgG with better functions such as ADCC, ADCP, CDC, and serum half-life effector function. For example, key residue differences differ significantly between the different classes of antibod 45 between IgG2 and IgG1 with respect to FcyR binding may ies, including for example human IgG1, IgG2, IgG3, IgG4. include P233, V234, A235, -236 (referring to a deletion in IgA1, IgA2, Igl), IgE, IgG, and IgM (Michaelsen et al., 1992, IgG2 relative to IgG1), and G327. Thus one or more amino Molecular Immunology, 29(3): 319-326, entirely incorpo acid modifications in the parent IgG2 wherein one or more of rated by reference). A number of studies have explored IgG1. these residues is replaced with the corresponding IgG1 amino IgG2, IgG3, and IgG4 variants in order to investigate the 50 acids, P233E, V234L, A235L, -236G (referring to an inser determinants of the effector function differences between tion of a glycine at position 236), and G327A, may provide them. See for example Canfield & Morrison, 1991, J. Exp. enhanced effector function. The sequence of Such an IgG, Med. 173: 1483-1491; Chappelet al., 1991, Proc. Natl. Acad. comprising a hybrid of residues from IgG1 and IgG2, referred Sci. USA 88(20):9036-9040; Chappelet al., 1993, Journal of to herein as “Hybrid” in the Examples and Figures, is pro Biological Chemistry 268:25124-25131; Tao et al., 1991, J. 55 vided in FIG. 2. Exp. Med. 173: 1025-1028; Tao et al., 1993, J. Exp. Med. 178: As is well known in the art, immunoglobulin polymor 661-667: Redpath et al., 1998, Human Immunology, 59, 720 phisms exist in the human population. Gm polymorphism is 727, all entirely incorporated by reference. determined by the IGHG1, IGHG2 and IGHG3 genes which The anti-HM1.24 antibodies can be sequence based. For have alleles encoding allotypic antigenic determinants example, the antibody includes a light chain and a heavy 60 referred to as G1 m, G2m, and G3m allotypes for markers of chain. The light chain can have a CDR1 with amino acid the human IgG1, IgG2 and IgG3 molecules (no Gmallotypes sequence selected from the group consisting of SEQID NO: have been found on the gamma 4 chain). Markers may be 11, 41 or 42, a CDR2 amino acid sequence of SEQID NOS:12 classified into allotypes and isoallotypes. These are distin and a CDR3 amino acid sequence of SEQ ID NOs: 13. The guished on different serological bases dependent upon the heavy chain can have a CDR1 comprising the amino acid 65 strong sequence homologies between isotypes. Allotypes are sequence of SEQID NOs: 114, a CDR2 having an amino acid antigenic determinants specified by allelic forms of the Ig sequence selected from SEQ ID NOS:15, 39 or 40, and a genes. Allotypes represent slight differences in the amino US 9,040,042 B2 17 18 acid sequences of heavy or light chains of different individu which other sequences are compared, including but not lim als. Even a single amino acid difference can give rise to an ited to sequences from other organisms, for example rodent allotypic determinant, although in many cases there are sev and primate sequences, as well as sequences from other eral amino acid Substitutions that have occurred. Allotypes immunoglobulin classes such as IgA, IgE, IgGD, IgGM, and are sequence differences between alleles of a subclass the like. It is contemplated that, although the antibodies of the whereby the antisera recognize only the allelic differences. present disclosure are engineered in the context of one parent An isoallotype is an allele in one isotype which produces an antibody, the variants may be engineered in or “transferred epitope which is shared with a non-polymorphic homologous to the context of another, second parent antibody. This is done region of one or more other isotypes and because of this the by determining the “equivalent” or “corresponding residues antisera will react with both the relevant allotypes and the 10 and substitutions between the first and second antibodies, relevant homologous isotypes (Clark, 1997, IgG effector typically based on sequence or structural homology between mechanisms, Chem. Immunol. 65:88-110; Gorman & Clark, the sequences of the two antibodies. In order to establish 1990, Semin Immunol 2(6):457-66, both incorporated homology, the amino acid sequence of a first antibody out entirely by reference). lined herein is directly compared to the sequence of a second Allelic forms of human immunoglobulins have been well 15 antibody. After aligning the sequences, using one or more of characterized (WHO Review of the notation for the allotypic the homology alignment programs known in the art (for and related markers of human immunoglobulins. J Immuno example using conserved residues as between species), gen 1976, 3: 357-362; WHO Review of the notation for the allowing for necessary insertions and deletions in order to allotypic and related markers of human immunoglobulins. maintain alignment (i.e., avoiding the elimination of con 1976, Eur. J. Immunol. 6, 599-601: E. van Loghem, 1986, served residues through arbitrary deletion and insertion), the Allotypic markers, Monogr 19: 40-51, all incorpo residues equivalent to particular amino acids in the primary rated entirely by reference). Additionally, other polymor sequence of the first antibody are defined. Alignment of con phisms have been characterized (Kim et al., 2001, J. Mol. served residues preferably should conserve 100% of such Evol. 54:1-9, incorporated entirely by reference). At present, residues. However, alignment of greater than 75% or as little 18 Gm allotypes are known: G1 m (1, 2, 3, 17) or Glm (a, X, 25 as 50% of conserved residues is also adequate to define f, Z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, equivalent residues. Equivalent residues may also be defined 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, by determining structural homology between a first and sec u, V, g5) (Lefranc, et al., The human IgG Subclasses: molecu ond antibody that is at the level of tertiary structure for anti lar analysis of structure, function and regulation. Pergamon, bodies whose structures have been determined. In this case, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. 30 equivalent residues are defined as those for which the atomic Genet.: 50, 199-211, both incorporated entirely by reference). coordinates of two or more of the main chain atoms of a Allotypes that are inherited in fixed combinations are called particular amino acid residue of the parent or precursor (Non Gm haplotypes. FIG. 4 shows common haplotypes of the N, CA on CA, C on C and O on O) are within 0.13 nm and gamma chain of human IgG1 (FIG. 4a) and IgG2 (FIG. 4b) preferably 0.1 nm after alignment. Alignment is achieved showing the positions and the relevantamino acid substitu 35 after the best model has been oriented and positioned to give tions. Amino acid sequences of these allotypic versions of the maximum overlap of atomic coordinates of non-hydrogen IgG1 and IgG2 are provided as SEQ IDs: 33-38). The anti protein atoms of the proteins. Regardless of how equivalentor bodies of the present disclosure may be substantially encoded corresponding residues are determined, and regardless of the by any allotype, isoallotype, or haplotype of any immunoglo identity of the parent antibody in which the antibodies are bulin gene. 40 made, what is meant to be conveyed is that the antibodies Antibodies of the present disclosure may be substantially discovered by the present disclosure may be engineered into encoded by genes from any organism, preferably mammals, any second parent antibody that has significant sequence or including but not limited to humans, rodents including but not structural homology with said antibody. Thus for example, if limited to mice and rats, lagomorpha including but not limited a variant antibody is generated wherein the parent antibody is to rabbits and hares, camelidae including but not limited to 45 human IgG1, by using the methods described above or other camels, llamas, and dromedaries, and non-human primates, methods for determining equivalent residues, said variant including but not limited to Prosimians, Platyrrhini (New antibody may be engineered in a human IgG2 parent anti World monkeys), Cercopithecoidea (Old World monkeys), body, a human IgA parent antibody, a mouse IgG2a or IgG2b and Hominoidea including the Gibbons and Lesser and Great parent antibody, and the like. Again, as described above, the Apes. In a most preferred embodiment, the antibodies of the 50 context of the parent antibody does not affect the ability to present disclosure are substantially human. The antibodies of transfer the antibodies of the present disclosure to other par the present disclosure may be substantially encoded by ent antibodies. For example, the variant antibodies that are immunoglobulin genes belonging to any of the antibody engineered in a human IgG1 antibody that targets one antigen classes. In a most preferred embodiment, the antibodies of the epitope may be transferred into a human IgG2 antibody that present disclosure comprise sequences belonging to the IgG 55 targets a different antigen epitope, and so forth. class of antibodies, including human Subclasses IgG1, IgG2. In the IgG class of immunoglobulins, there are several IgG3, and IgG4. In an alternate embodiment, the antibodies immunoglobulin domains in the heavy chain. By “immuno of the present disclosure comprise sequences belonging to the globulin (Ig) domain herein is meant a region of an immu IgA (including human Subclasses IgA1 and IgA2), Ig|D, IgE, noglobulin having a distinct tertiary structure. Of interest in IgG, or IgM classes of antibodies. The antibodies of the 60 the present disclosure are the domains of the constant heavy present disclosure may comprise more than one protein chain. chain, including, the constant heavy (CH) domains and the That is, the present disclosure may find use in an antibody that hinge. In the context of IgG antibodies, the IgG isotypes each is a monomer or an oligomer, including a homo- or hetero have three CH regions: “CH1 refers to positions 118-220, oligomer. “CH2 refers to positions 237-340, and “CH3” refers to posi In the most preferred embodiment, the antibodies of the 65 tions 341-447 according to the EU index as in Kabat. By invention are based on human IgG sequences, and thus human “hinge' or “hinge region' or “antibody hinge region” or IgG sequences are used as the “base' sequences against “immunoglobulin hinge region' herein is meant the flexible US 9,040,042 B2 19 20 polypeptide comprising the amino acids between the first and “Chimeric antibodies' traditionally comprise variable second constant domains of an antibody. Structurally, the IgG region(s) from a mouse (or rat, in some cases) and the con CH1 domain ends at EU position 220, and the IgG CH2 stant region(s) from a human (Morrison et al., 1984, Proc Natl domain begins at residue EU position 237. Thus for IgG the AcadSci USA 81: 6851-6855, incorporated entirely by refer hinge is herein defined to include positions 221 (D221 in 5 ence). IgG1) to 236 (G236 in IgG1), wherein the numbering is By “humanized antibody as used herein is meant an anti according to the EU index as in Kabat. In some embodiments, body comprising a human framework region (FR) and one or for example in the context of an Fc region, the lower hinge is more complementarity determining regions (CDR's) from a included, with the “lower hinge’ generally referring to posi non-human (usually mouse or rat) antibody. The non-human tions 226 or 230. The constant heavy chain, as defined herein, 10 antibody providing the CDR's is called the “donor” and the refers to the N-terminus of the CH1 domainto the C-terminus human immunoglobulin providing the framework is called of the CH3 domain, thus comprising positions 118-447, the “acceptor. In certain embodiments, humanization relies wherein numbering is according to the EU index. The con principally on the grafting of donor CDRS onto acceptor stant light chain comprises a single domain, and as defined (human)VL and VH frameworks (Winter U.S. Pat. No. 5.225, herein refers to positions 108-214 of CK or CW, wherein 15 539, incorporated entirely by reference). This strategy is numbering is according to the EU index. referred to as “CDR grafting”. “Backmutation of selected Antibodies of the invention may include multispecificanti acceptor framework residues to the corresponding donor resi bodies, notably bispecific antibodies, also sometimes dues is often required to regain affinity that is lost in the initial referred to as "diabodies'. These are antibodies that bind to grafted construct (U.S. Pat. No. 5,693.762, incorporated two (or more) different antigens. Diabodies can be manufac- 20 entirely by reference). The humanized antibody optimally tured in a variety of ways known in the art, e.g., prepared also will comprise at least a portion of an immunoglobulin chemically or from hybrid hybridomas. In one embodiment, constant region, typically that of a human immunoglobulin, the antibody is a minibody. Minibodies are minimized anti and thus will typically comprise a human Fc region. A variety body-like proteins comprising a schv joined to a CH3 of techniques and methods for humanizing and reshaping domain. In some cases, the Sclv can be joined to the Fc 25 non-human antibodies are well known in the art (See Tsu region, and may include some or all of the hinge region. For rushita & Vasquez, 2004, Humanization of Monoclonal Anti a description of multispecific antibodies see Holliger & Hud bodies, Molecular Biology of B Cells, 533-545, Elsevier Sci son, 2006, Nature Biotechnology 23(9): 1126-1136 and ref ence (USA), and references cited therein, all incorporated erences cited therein, all expressly incorporated by reference. entirely by reference). Humanization or other methods of In one embodiment, the antibody of the invention is an 30 reducing the immunogenicity of nonhuman antibody variable antibody fragment. Of particular interest are antibodies that regions may include resurfacing methods, as described for comprise Fc regions, Fc fusions, and the constant region of example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA the heavy chain (CH1-hinge-CH2-CH3). Antibodies of the 91:969-973, incorporated entirely by reference. In one present disclosure may comprise Fc fragments. An Fc frag embodiment, selection based methods may be employed to ment of the present disclosure may comprise from 1-90% of 35 humanize and/or affinity mature antibody variable regions, the Fc region, with 10-90% being preferred, and 30-90% that is, to increase the affinity of the variable region for its being most preferred. Thus for example, an Fc fragment of the target antigen. Other humanization methods may involve the present disclosure may comprise an IgG1 Cy2 domain, an grafting of only parts of the CDRs, including but not limited IgG1 Cy2 domain and hinge region, an IgG1 CY3 domain, and to methods described in U.S. Ser. No. 09/810,502; Tan et al., So forth. In one embodiment, an Fc fragment of the present 40 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. disclosure additionally comprises a fusion partner, effectively Immunol. 169:3076-3084, incorporated entirely by refer making it an Fc fragment fusion. Fc fragments may or may ence. Structure-based methods may be employed for human not contain extra polypeptide sequence. ization and affinity maturation, for example as described in Chimeric, Humanized, and Fully Human Antibodies U.S. Ser. No. 10/153,159 and related applications, all incor Immunogenicity is the result of a complex series of 45 porated entirely by reference. responses to a substance that is perceived as foreign, and may In certain variations, the immunogenicity of the antibody is include production of neutralizing and non-neutralizing anti reduced using a method described in U.S. Ser. No. 11/004, bodies, formation of immune complexes, complement acti 590, entitled “Methods of Generating Variant Proteins with Vation, activation, inflammation, hypersensitivity Increased Host String Content and Compositions Thereof, responses, and . Several factors can contribute to 50 filed on Dec. 3, 2004, incorporated entirely by reference. protein immunogenicity, including but not limited to protein Modifications to reduce immunogenicity may include sequence, route and frequency of administration, and patient modifications that reduce binding of processed peptides population. Immunogenicity may limit the efficacy and safety derived from the parent sequence to MHC proteins. For of a protein therapeutic in multiple ways. Efficacy can be example, amino acid modifications would be engineered Such reduced directly by the formation of neutralizing antibodies. 55 that there are no or a minimal number of immune that Efficacy may also be reduced indirectly, as binding to either are predicted to bind, with high affinity, to any prevalent MHC neutralizing or non-neutralizing antibodies typically leads to alleles. Several methods of identifying MHC-binding rapid clearance from serum. Severe side effects and even epitopes in protein sequences are known in the art and may be death may occur when an immune reaction is raised. Thus in used to score epitopes in an antibody of the present disclo a preferred embodiment, protein engineering is used to 60 sure. See for example U.S. Ser. No. 09/903,378, U.S. Ser. No. reduce the immunogenicity of the antibodies of the present 10/754,296, U.S. Ser. No. 1 1/249,692, and references cited disclosure. therein, all expressly incorporated by reference. In some embodiments, the scaffold components can be a In an alternate embodiment, the antibodies of the present mixture from different species. Such antibody may be a chi disclosure may be fully human, that is the sequences of the mericantibody and/or a humanized antibody. In general, both 65 antibodies are completely or substantially human. “Fully "chimeric antibodies' and "humanized antibodies' refer to human antibody' or “complete human antibody” refers to a antibodies that combine regions from more than one species. human antibody having the gene sequence of an antibody US 9,040,042 B2 21 22 derived from a human chromosome with the modifications part of anti-HM1.24 mechanism of action in MM therapy, and outlined herein. A number of methods are known in the art for further that an anti-HM1.24 antibody may benefit from tech generating fully human antibodies, including the use of trans nologies that enhance effector function. genic mice (Bruggemann et al., 1997, Curr Opin Biotechnol The antibodies of the present disclosure may be virtually 8:455-458) or human antibody libraries coupled with selec 5 any antibody that binds to HM1.24. The variable regions of tion methods (Griffiths et al., 1998, Curr Opin Biotechnol any known or undiscovered anti-HM1.24 antibodies may find 9:102-108) both incorporated entirely by reference. use in the present disclosure. Antibodies of the invention may The antibodies of the present disclosure may find use in a display selectivity for HM1.24 versus alternative targets, or wide range of products. In one embodiment the antibody of selectivity for a specific form of the target versus alternative the invention is a therapeutic, a diagnostic, or a research 10 forms. Examples include full-length versus splice variants, reagent, preferably a therapeutic. Alternatively, the antibody cell-surface vs. soluble forms, selectivity for various poly of the present disclosure may be used for agricultural or morphic variants, or selectivity for specific conformational industrial uses. An antibody of the present disclosure may forms of a target. An antibody of the present disclosure may find use in an antibody composition that is monoclonal or bind any epitope or region on HM1.24, and may be specific polyclonal. The antibodies of the present disclosure may be 15 for fragments, mutant forms, splice forms, or aberrant forms agonists, antagonists, neutralizing, inhibitory, or stimulatory. of said antigens. In a preferred embodiment, the antibodies of the present Fc Optimization of Anti-HM1.24 Antibodies disclosure are used to kill target cells that bear the target There are a number of characterized mechanisms by which antigen, for example cancer cells. In an alternate embodi antibodies mediate cellular effects, including anti-prolifera ment, the antibodies of the present disclosure are used to tion via blockage of needed growth pathways, intracellular block, antagonize, or agonize the target antigen. In an alter signaling leading to apoptosis, enhanced down regulation nately preferred embodiment, the antibodies of the present and/or turnover of receptors, complement-dependent cyto disclosure are used to block, antagonize, or agonize the target toxicity (CDC), antibody-dependent cell-mediated cytotox antigen and kill the target cells that bear the target antigen. icity (ADCC), antibody-dependent cell-mediated phagocyto Anti-HM1.24 Antibodies as Therapeutics to Treat Plasma 25 sis (ADCP) and promotion of an adaptive immune response Cell Disorders (Cragget al., 1999, Curr Opin Immunol 11:541-547: Glennie A number of favorable properties of antibodies, including et al., 2000, Immunol Today 21:403-410, both incorporated but not limited to specificity for target, ability to mediate entirely by reference). Antibody efficacy may be due to a immune effector mechanisms, and long half-life in serum, combination of these mechanisms, and their relative impor make antibodies powerful therapeutics. The present disclo 30 tance in clinical therapy for oncology appears to be cancer sure describes antibodies against the B-cell antigen HM1.24. dependent. HM1.24/BST-2, also referred to as CD317, or EMP24 is a The importance of Fcy R-mediated effector functions for type II transmembrane protein found in the endoplasmic the activity of some antibodies has been demonstrated in mice reticulum and cell surface; it has a molecular weight of 29 to (Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656: 33 kD and is expressed selectively on terminally differenti 35 Clynes et al., 2000, Nat Med 6:443-446, both incorporated ated normal and neoplastic B cells. The protein has a unique entirely by reference), and from observed correlations topology that is present in lipid rafts via a glycosylphosphati between clinical efficacy in humans and their allotype of high dylinositol anchor at the C-terminus. The HM1.24 gene is one (V158) or low (F158) affinity polymorphic forms of FcyRIIIa of the important activators of the nuclear factor KB pathway (Cartronet al., 2002, Blood 99:754-758; Weng & Levy, 2003, that is involved in the pathogenesis of Multiple Myeloma 40 Journal of Clinical Oncology, 21:3940-3947, both incorpo (MM) (Matsuda et al., 2003, Oncogene 22:3307-3318, incor rated entirely by reference). Together these data Suggest that porated entirely by reference), Suggesting that HM 1.24 pro an antibody that is optimized for binding to certain FcyRs vides a platform for a signaling complex at the cell. HM1.24 may better mediate effector functions, and thereby destroy was originally identified as an antigen preferentially overex target cells more effectively in patients. Thus a promising pressed on Multiple Myeloma cells (Goto et al., 1994, Blood: 45 means for enhancing the anti-tumor potency of antibodies is 84:1922-1930) and later was found to be identical to bone via enhancement of their ability to mediate cytotoxic effector marrow stromal cell antigen 2 (BST-2) (Ohtomo et al., 1999, functions such as ADCC, ADCP, and CDC. Additionally, Biochem Biophys Res Commun 258:583-591; Ishikawa et antibodies can mediate anti-tumor mechanism via growth al., 1995, Genomics 26:527-534, all incorporated entirely by inhibitory or apoptotic signaling that may occur when an reference. HM1.24 is also specifically up-regulated in meta 50 antibody binds to its target on tumor cells. Such signaling static tumor cells and chemoresistant cancer cells (Walter may be potentiated when antibodies are presented to tumor Yohrling et al., 2003, Cancer Res 63:8939-8947; Becker et cells bound to immune cells via FcyR. Therefore increased al., 2005, Mol Cancer Ther 4:151-168), both incorporated affinity of antibodies to FcyRs may result in enhanced anti entirely by reference. proliferative effects. The HM1.24 antigen is an attractive target for the antibody 55 Antibody engineering for optimized effector function has based immunotherapy of multiple myeloma. The relevance of been achieved using amino acid modifications (see for the immune system in MM treatment (Gahrton et al., 2001, Br example U.S. Ser. No. 10/672,280 and U.S. Ser. No. 11/124, J Haematol 113:209-216: Lokhorst et al., 2000, J. Clin Oncol 620 and references cited therein, all incorporated entirely by 18:3031-3037: Kroger et al., 2004, Blood 104:3361-3363, all reference), and engineered glycoforms (see for example incorporated entirely by reference), as well as the observation 60 Umafia et al., 1999, Nat Biotechnol 17:176-180; Shinkawa et of an increased number and activity of natural killer (NK) al., 2003, JBiol Chem 278:3466-3473, Yamane-Ohnuki et al., cells in the peripheral blood and bone marrow of myeloma 2004, Biotechnology and Bioengineering 87(5):614-621, all patients (Saito et al., 1986, Jpn.JClin Immunol 9:441; Uchida incorporated entirely by reference). et al., 1984, IntJ Cancer 34:375; Osterborg et al., 1990, Eur J Modifications for Optimizing Effector Function Haematol 45:153; Gonzalez et al., 1992, Am J Hematol 65 The present disclosure is directed to antibodies comprising 39:84, all incorporated entirely by reference) suggest that modifications, wherein said modifications alter affinity to one antibody effector function has the potential to be an important or more Fc receptors, and/or alter the ability of the antibody to

US 9,040,042 B2 27 28 tions where the two isotypes differ. Thus a hybrid variant IgG For the purposes herein, a “mature core carbohydrate struc antibody may be constructed that comprises one or more ture” refers to a processed core carbohydrate structure Substitutions selected from the group consisting of 274Q. attached to an Fc region which generally consists of the 276K, 300F, 339T., 356E, 358M, 384S, 392N, 397M, 422I, following carbohydrate structure GlcNAc(Fucose)-GlcNAc 435R, and 436F. In other embodiments of the invention, an 5 Man-(Man-GlcNAc) typical of biantennary oligosaccha IgG1/IgG2 hybrid variant may be constructed by substituting rides. The mature core carbohydrate structure is attached to IgG2 positions in the CH2 and/or CH3 region with amino the Fc region of the glycoprotein, generally via N-linkage to acids from IgG1 at positions where the two isotypes differ. Asn297 of a CH2 domain of the Fc region. A “bisecting Thus a hybrid variant IgG antibody may be constructed that GlcNAc is a GlcNAc residue attached to the B1.4 mannose 10 of the mature core carbohydrate structure. The bisecting comprises one or more modifications selected from the group GlcNAc can be enzymatically attached to the mature core consisting of 233E, 234L, 235L, -236G (referring to an inser carbohydrate structure by a B(1,4)-N-acetylglucosaminyl tion of a glycine at position 236), and 327A. transferase III (GnTIII). CHO cells do not normally Glycoform Modifications express GnTIII (Stanley et al., 1984, J. Biol. Chem. 261: Many polypeptides, including antibodies, are subjected to 15 13370-13378), but may be engineered to do so (Umana et al., a variety of post-translational modifications involving carbo 1999, Nature Biotech. 17:176-180). hydrate moieties, such as glycosylation with oligosaccha The present disclosure contemplates antibodies that com rides. There are several factors that can influence glycosyla prise modified glycoforms or engineered glycoforms. By tion. The species, tissue and cell type have all been shown to “modified glycoform” or “engineered glycoform as used be important in the way that glycosylation occurs. In addition, herein is meant a carbohydrate composition that is covalently the extracellular environment, through altered culture condi attached to a protein, for example an antibody, wherein said tions such as serum concentration, may have a direct effect on carbohydrate composition differs chemically from that of a glycosylation. (Lifely et al., 1995, Glycobiology 5(8): 813 parent protein. Engineered glycoforms may be useful for a 822), incorporated entirely by reference. variety of purposes, including but not limited to enhancing or Allantibodies contain carbohydrate at conserved positions 25 reducing Fcy R-mediated effector function. In a preferred in the constant regions of the heavy chain. Each antibody embodiment, the antibodies of the present disclosure are isotype has a distinct variety of N-linked carbohydrate struc modified to control the level of fucosylated and/or bisecting tures. Aside from the carbohydrate attached to the heavy oligosaccharides that are covalently attached to the Fc region. chain, up to 30% of human IgGs have a glycosylated Fab Historically, antibodies produced in Chinese Hamster region. IgG has a single N-linked biantennary carbohydrate at 30 Ovary Cells (CHO), one of the most commonly used indus Asn297 of the CH2 domain. For IgG from either serum or trial hosts, contain about 2 to 6% in the population that are produced ex vivo in hybridomas or engineered cells, the IgG nonfucosylated. YB2/0 (rat myeloma) and Lec13 cell line (a are heterogeneous with respect to the ASn297 linked carbo lectin mutant of CHO line which has a deficient GDP-man hydrate (Jefferis et al., 1998, Immunol. Rev. 163:59-76: nose 4.6 dehydratase leading to the deficiency of GDP-fucose Wright et al., 1997, Trends Biotech 15:26-32, both incorpo 35 or GDP-sugar intermediates that are the substrate of C.1.6- rated entirely by reference). For human IgG, the core oli fucosyltransferase (Ripka et al., 1986), however, can produce gosaccharide normally consists of GlcNAc-Man-GlcNAc, antibodies with 78% to 98% nonfucosylated species. Unfor with differing numbers of outer residues. tunately, the yield of antibody from these cells is extremely The carbohydrate moieties of the present disclosure will be poor and therefore these cell lines are not useful to make described with reference to commonly used nomenclature for 40 therapeutic antibody products commercially. The FUT8 gene the description of oligosaccharides. A review of carbohydrate encodes the C.1.6-fucosyltransferase enzyme that catalyzes chemistry which uses this nomenclature is found in Hubbard the transfer of a fucosyl residue from GDP-fucose to position et al. 1981, Ann. Rev. Biochem. 50:555-583, incorporated 6 of Asn-linked (N-linked) GlcNac of an N-glycan entirely by reference. This nomenclature includes, for (Yanagidani et al., 1997, J Biochem 121:626-632). It is instance, Man, which represents mannose; GlcNAc, which 45 known that the C.1.6 fucosyltransferase is the only enzyme represents 2-N-acetylglucosamine; Gal which represents responsible for adding fucose to the N-linked biantennary galactose; Fuc for fucose, and Glc, which represents glucose. carbohydrate at Asn297 in the CH2 domain of the IgG anti Sialic acids are described by the shorthand notation NeuNAc, body. for 5-N-acetylneuraminic acid, and NeuNGc for 5-glycolyl A variety of methods are well known in the art for gener neuraminic. 50 ating modified glycoforms (Umafia et al., 1999, Nat Biotech The term “glycosylation” means the attachment of oli nol 17:176-180; Davies et al., 2001, Biotechnol Bioeng gosaccharides (carbohydrates containing two or more simple 74:288-294; Shields et al., 2002, J Biol Chem 277:26733 Sugars linked together e.g. from two to about twelve simple 26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473); Sugars linked together) to a glycoprotein. The oligosaccha (U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. ride side chains are typically linked to the backbone of the 55 No. 10/113,929; PCT WO 00/61739A1; PCT WO glycoprotein through either N- or O-linkages. The oligosac 01/29246A1; PCTWO 02/31140A1; PCT WOO2/30954A1); charides of the present disclosure occur generally are Yamane-Ohnuki et al., 2004, Biotechnology and Bioengi attached to a CH2 domain of an Fc region as N-linked oli neering 87(5):614-621; (PotelligentTM technology Biowa, gosaccharides. “N-linked glycosylation” refers to the attach Inc., Princeton, N.J.; GlycoMAbTM glycosylation engineer ment of the carbohydrate moiety to an asparagine residue in a 60 ing technology (GLYCART biotechnology AG, Zürich, Swit glycoprotein chain. The skilled artisan will recognize that, for zerland; all of which are expressly incorporated by refer example, each of murine IgG1, IgG2a, IgG2b and IgG3 as ence). These techniques control the level of fucosylated and/ well as human IgG1, IgG2, IgG3, IgG4, IgA and Ig|D CH2 or bisecting oligosaccharides that are covalently attached to domains have a single site for N-linked glycosylation at the Fc region, for example by expressing an IgG in various amino acid residue 297 (Kabat et al. Sequences of Proteins of 65 organisms or cell lines, engineered or otherwise (for example Immunological Interest, 1991, incorporated entirely by ref Lec-13 CHO cells or rathybridomaYB2/0 cells), by regulat erence). ing involved in the glycosylation pathway (for US 9,040,042 B2 29 30 example FUT8 C.1.6-fucosyltranserase and/or B1-4-N- the variant antibodies and those of the parent antibodies are acetylglucosaminyltransferase III GnTIII), or by modifying Substantially homologous. For example, the variant antibody carbohydrate(s) after the IgG has been expressed. sequences herein will possess about 80% homology with the Other methods for modifying glycoforms of the antibodies parent antibody sequence, preferably at least about 90% of the invention include using glycoengineered strains of 5 homology, and most preferably at least about 95% homology. yeast (Liet al., 2006, Nature Biotechnology 24(2):210-215), In a most preferred embodiment, the antibodies of the moss (Nechansky et al., 2007, Mol Immunjol 44(7): 1826-8), present disclosure comprise amino acid modifications that and plants (Cox et al., 2006, Nat Biotechnol 24(12): 1591-7). provide optimized effector function properties relative to the Methods for modifying glycoforms include but are not lim parent. Most preferred substitutions and optimized effector ited to using a glycoengineered strain of yeast Pichia pastoris 10 function properties are described in U.S. Ser. No. 10/672.280, (Liet al., 2006, Nature Biotechnology 24(2):210-215), a gly PCT USO3/30249, and U.S. Ser. No. 10/822,231, and U.S. coengineered Strain of the moss Physcomitrella patens Ser. No. 60/627,774, filed Nov. 12, 2004 and entitled “Opti wherein the enzymes B12-xylosyltransferase and/or C. 1,3- mized Fc Variants'. Properties that may be optimized include fucosyltransferase are knocked out in (Nechansky et al., but are not limited to enhanced or reduced affinity for an 2007, Mol Immunjol 44(7):1826-8), and the use of RNA 15 FcyR. In a preferred embodiment, the antibodies of the interference to inhibit endogenous alpha-1,3-fucosyltrans present disclosure are optimized to possess enhanced affinity ferase and/or beta-1,2-Xylosyltransferase in the aquatic plant for a human activating FcyR, preferably FcyRI, FcyRIIa, Lemna minor (Cox et al., 2006, Nat Biotechnol 24(12): 1591 FcyRIIc, FcyRIIIa, and FcyRIIIb, most preferably FcyRIIIa. 7). In an alternately preferred embodiment, the antibodies are Modified or engineered glycoform typically refers to the optimized to possess reduced affinity for the human inhibi different carbohydrate or oligosaccharide; thus for example tory receptor FcyRIIb. These preferred embodiments are an antibody may comprise an engineered glycoform. Alter anticipated to provide antibodies with enhanced therapeutic natively, engineered glycoform may refer to the antibody that properties in humans, for example enhanced effector function comprises the different carbohydrate or oligosaccharide. For and greater anti-cancer potency. In an alternate embodiment, the purposes of modified glycoforms described herein, apar 25 the antibodies of the present disclosure are optimized to have ent antibody' is a glycosylated antibody having the same reduced or ablated affinity for a human FcyR, including but amino acid sequence and mature core carbohydrate structure not limited to FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, as an engineered glycoform of the present disclosure, except and FcyRIIIb. These embodiments are anticipated to provide that fucose is attached to the mature core carbohydrate struc antibodies with enhanced therapeutic properties in humans, ture of the parent antibody. For instance, in a composition 30 for example reduced effector function and reduced toxicity. comprising the parent glycoprotein about 50-100% or about In other embodiments, antibodies of the present disclosure 70-100% of the parent glycoprotein comprises a mature core provide enhanced affinity for one or more FcyRs, yet reduced carbohydrate structure having fucose attached thereto. affinity for one or more other FcyRs. For example, an anti The present disclosure provides a composition comprising body of the present disclosure may have enhanced binding to a glycosylated antibody having an Fc region, wherein about 35 FcyRIIIa, yet reduced binding to FcyRIIb. Alternately, an 51-100% of the glycosylated antibody in the composition antibody of the present disclosure may have enhanced bind comprises a mature core carbohydrate structure which lacks ing to FcyRIIa and FcyRI, yet reduced binding to FcyRIIb. In fucose, attached to the Fc region of the antibody. More pref yet another embodiment, an antibody of the present disclo erably, about 80-100% of the antibody in the composition sure may have enhanced affinity for FcyRIIb, yet reduced comprises a mature core carbohydrate structure which lacks 40 affinity to one or more activating FcyRs. fucose and most preferably about 90–99% of the antibody in The modification of the invention preferably enhance bind the composition lacks fucose attached to the mature core ing affinity for one or more FcyRs. By “greater affinity” or carbohydrate structure. In a most preferred embodiment, the “improved affinity” or "enhanced affinity” or “better affinity” antibody in the composition both comprises a mature core than a parent immunoglobulin, as used herein is meant that an carbohydrate structure that lacks fucose and additionally 45 Fc variant binds to an Fc receptor with a significantly higher comprises at least one amino acid modification in the Fc equilibrium constant of association (KA) or lower equilib region. In the most preferred embodiment, the combination of rium constant of dissociation (KD) than the parent polypep engineered glycoform and amino acid modification provides tide when the amounts of variant and parent polypeptide in optimal Fc receptor binding properties to the antibody. the binding assay are essentially the same. For example, the Optimized Properties of Antibodies 50 Fc variant with improved FcyR binding affinity may display The present disclosure provides variant antibodies that are from about 5 fold to about 1000 fold, e.g. from about 10 fold optimized for a number of therapeutically relevant properties. to about 500 fold improvement in Fc receptor binding affinity A variant antibody comprises one or more amino acid modi compared to the parent polypeptide, where Fc receptor bind fications relative to a parent antibody, wherein said amino ing affinity is determined, for example, as disclosed in the acid modification(s) provide one or more optimized proper 55 Examples herein. Accordingly, by “reduced affinity” as com ties. Thus the antibodies of the present disclosure are variants pared to a parent Fc polypeptide as used herein is meant that antibodies. An antibody of the present disclosure differs in an Fc variant binds an Fc receptor with significantly lower KA amino acid sequence from its parent antibody by virtue of at or higher KD than the parent polypeptide. least one amino acid modification. Thus variant antibodies of Data in the present study indicate that human WT IgG1 the present disclosure have at least one amino acid modifica 60 binds to human V158 FcyRIIIa with an affinity of approxi tion compared to the parent. Alternatively, the variant anti mately 240 nM (Example 1). This is consistent with the bodies of the present disclosure may have more than one literature which indicate that binding is approximately 200 amino acid modification as compared to the parent, for 500 nM, as determined by Biacore (210 nM as shown in example from about one to fifty amino acid modifications, Okazaki et al., 2004, J Mol Bio 336:1239-49; 250 nM as preferably from about one to ten amino acid modifications, 65 shown in Lazaretal, Proc Natl AcadSci USA 103(11):4005 and most preferably from about one to about five amino acid 4010) and calorimetry (530 nM, Okazaki et al., 2004, J Mol modifications compared to the parent. Thus the sequences of Bio 336:1239-49). However affinity as low as 750 nM was US 9,040,042 B2 31 32 measured in one study (Ferrara et al., 2006, J Biol Chem antibodies that provide enhanced binding to the inhibitory 281 (8):5032-5036). Although binding to F 158 FcyRIIIa was FcyRIIb, yet WT level, reduced, or ablated binding to activat lower than the 5 uM cutoff applied in the present study, the ing FcyRs. This may be particularly useful, for example, literature indicates that human WT IgG1 binds to human when the goal of an antibody is to inhibit inflammation or F158 FcyRIIIa with an affinity of approximately 3-5 uM, as 5 auto-immune disease, or modulate the immune system in indicated by calorimetry (2.7 uM, in Okazaki et al., 2004, J Some way. Mol Bio 336:1239–49) and Biacore (5.0 uM, Ferrara et al., Clearly an important parameter that determines the most 2006, J Biol Chem 281 (8):5032-5036). beneficial selectivity of a given antibody to treat a given Preferred embodiments comprise optimization of Fc bind disease is the context of the antibody, that is what type of ing to a human FcyR, however in alternate embodiments the 10 antibody is being used. Thus the Fc ligand selectivity or antibodies of the present disclosure possess enhanced or specificity of a given antibody will provide different proper reduced affinity for FcyRs from nonhuman organisms, ties depending on whether it composes an antibody or an including but not limited to rodents and non-human primates. antibodies with a coupled fusion or conjugate partner. For antibodies that are optimized for binding to a nonhuman FcyR example, toxin, radionucleotide, or other conjugates may be may find use in experimentation. For example, mouse models 15 less toxic to normal cells if the antibody that comprises them are available for a variety of diseases that enable testing of has reduced or ablated binding to one or more Fc ligands. As properties such as efficacy, toxicity, and pharmacokinetics for another example, in order to inhibit inflammation or auto a given drug candidate. As is known in the art, cancer cells can immune disease, it may be preferable to utilize an antibody be grafted or injected into mice to mimic a human cancer, a with enhanced affinity for activating FcyRs, such as to bind process referred to as Xenografting. Testing of antibodies that these FcyRs and prevent their activation. Conversely, an anti comprise antibodies that are optimized for one or more mouse body that comprises two or more Fc regions with enhanced FcyRs, may provide valuable information with regard to the FcyRIIb affinity may co-engage this receptor on the surface of efficacy of the protein, its mechanism of action, and the like. immune cells, thereby inhibiting proliferation of these cells. The antibodies of the present disclosure may also be opti Whereas in some cases an antibodies may engage its target mized for enhanced functionality and/or solution properties 25 antigen on one cell type yet engage FcyRs on separate cells in aglycosylated form. In a preferred embodiment, the agly from the target antigen, in other cases it may be advantageous cosylated antibodies of the present disclosure bind an Fc to engage FcyRs on the Surface of the same cells as the target ligand with greater affinity than the aglycosylated form of the antigen. For example, if an antibody targets an antigen on a parent antibody. Said Fc ligands include but are not limited to cell that also expresses one or more FcyRs, it may be benefi FcyRs, C1q, FcRn, and proteins A and G, and may be from 30 cial to utilize an antibody that enhances or reduces binding to any source including but not limited to human, mouse, rat, the FcyRs on the surface of that cell. This may be the case, for rabbit, or monkey, preferably human. In an alternately pre example when the antibody is being used as an anti-cancer ferred embodiment, the antibodies are optimized to be more agent, and co-engagement of target antigen and FcyR on the stable and/or more soluble than the aglycosylated form of the Surface of the same cell promote signaling events within the parent antibody. 35 cell that result in growth inhibition, apoptosis, or other anti Antibodies of the invention may comprise modifications proliferative effect. Alternatively, antigen and FcyR co-en that modulate interaction with Fc ligands other than FcyRs, gagement on the same cell may be advantageous when the including but not limited to complement proteins, FcRn, and antibody is being used to modulate the immune system in Fc receptor homologs (FcRHs). FcRHs include but are not Some way, wherein co-engagement of target antigen and limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and 40 FcyR provides some proliferative or anti-proliferative effect. FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, Likewise, antibodies that comprise two or more Fc regions incorporated entirely by reference). may benefit from antibodies that modulate FcyR selectivity or Preferably, the Fc ligand specificity of the antibody of the specificity to co-engage FcyRs on the Surface of the same cell. present disclosure will determine its therapeutic utility. The The Fc ligand specificity of the antibodies of the present utility of a given antibody for therapeutic purposes will 45 disclosure can be modulated to create different effector func depend on the epitope or form of the target antigen and the tion profiles that may be Suited for particular antigen epitopes, disease or indication being treated. For some targets and indications or patient populations. FIG. 5 describes several indications, enhanced Fcy R-mediated effector functions may preferred embodiments of receptor binding profiles that be preferable. This may be particularly favorable for anti include improvements to, reductions to or no effect to the cancer antibodies. Thus antibodies may be used that comprise 50 binding to various receptors, where such changes may be antibodies that provide enhanced affinity for activating FcyRs beneficial in certain contexts. The receptor binding profiles in and/or reduced affinity for inhibitory FcyRs. For some targets FIG. 5 could be varied by degree of increase or decrease to the and indications, it may be further beneficial to utilize anti specified receptors. Additionally, the binding changes speci bodies that provide differential selectivity for different acti fied could be in the context of additional binding changes to Vating FcyRs; for example, in Some cases enhanced binding to 55 other receptors such as C1q or FcRn, for example by com FcyRIIa and FcyRIIIa may be desired, but not FcyRI, whereas bining with ablation of binding to C1q to shutoff complement in other cases, enhanced binding only to FcyRIIa may be activation, or by combining with enhanced binding to C1q to preferred. For certain targets and indications, it may be pref increase complement activation. Other embodiments with erable to utilize antibodies that enhance both Fcy R-mediated other receptor binding profiles are possible, the listed receptor and complement-mediated effector functions, whereas for 60 binding profiles are exemplary. other cases it may be advantageous to utilize antibodies that The presence of different polymorphic forms of FcyRs enhance either Fcy R-mediated or complement-mediated provides yet another parameter that impacts the therapeutic effector functions. For some targets or cancer indications, it utility of the antibodies of the present disclosure. Whereas the may be advantageous to reduce or ablate one or more effector specificity and selectivity of a given antibody for the different functions, for example by knocking out binding to C1q, one 65 classes of FcyRS significantly affects the capacity of an anti or more FcyRs, FcRn, or one or more other Fc ligands. For body to target a given antigen for treatment of a given disease, other targets and indications, it may be preferable to utilize the specificity or selectivity of an antibody for different poly US 9,040,042 B2 33 34 morphic forms of these receptors may in part determine system. These strategies may be particularly advantageous which research or pre-clinical experiments may be appropri when the targeted antigenis shed from the cellular Surface. An ate for testing, and ultimately which patient populations may additional application of these concepts arises with idiotype or may not respond to treatment. Thus the specificity or selec vaccine immunotherapies, in which clone-specific antibodies tivity of antibodies of the present disclosure to Fc ligand produced by a patient’s lymphoma cells are used to vaccinate polymorphisms, including but not limited to FcyR, C1q, the patient. FcRn, and FcRH polymorphisms, may be used to guide the In a preferred embodiment, modifications are made to selection of valid research and pre-clinical experiments, clini improve biophysical properties of the antibodies of the cal trial design, patient selection, dosing dependence, and/or present disclosure, including but not limited to stability, Solu other aspects concerning clinical trials. 10 bility, and oligomeric state. Modifications can include, for Other Modifications example, Substitutions that provide more favorable intramo Antibodies of the present disclosure may comprise one or lecular interactions in the antibody Such as to provide greater more modifications that provide optimized properties that are stability, or Substitution of exposed nonpolar amino acids not specifically related to effector function perse. Said modi with polar amino acids for higher solubility. A number of fications may be amino acid modifications, or may be modi 15 optimization goals and methods are described in U.S. Ser. No. fications that are made enzymatically or chemically. Such 10/379,392, incorporated entirely by reference, that may find modification(s) likely provide Some improvement in the anti use for engineering additional modifications to further opti body, for example an enhancement in its stability, Solubility, mize the antibodies of the present disclosure. The antibodies function, or clinical use. The present disclosure contemplates of the present disclosure can also be combined with addi a variety of improvements that made be made by coupling the tional modifications that reduce oligomeric State or size. Such antibodies of the present disclosure with additional modifi that tumor penetration is enhanced, or in vivo clearance rates cations. are increased as desired. In one embodiment, the variable region of an antibody of Other modifications to the antibodies of the present disclo the present disclosure may be affinity matured, that is to say sure include those that enable the specific formation or that amino acid modifications have been made in the VH 25 homodimeric or homomultimeric molecules. Such modifica and/or VL domains of the antibody to enhance binding of the tions include but are not limited to engineered disulfides, as antibody to its target antigen. Such types of modifications well as chemical modifications or aggregation methods may improve the association and/or the dissociation kinetics which may provide a mechanism for generating covalent for binding to the target antigen. Other modifications include homodimeric or homomultimers. For example, methods of those that improve selectivity for target antigen vs. alternative 30 engineering and compositions of Such molecules are targets. These include modifications that improve selectivity described in Kan et al., 2001, J. Immunol., 2001, 166: 1320 for antigen expressed on target vs. non-target cells. Other 1326; Stevenson et al., 2002, Recent Results Cancer Res. 159: improvements to the target recognition properties may be 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. provided by additional modifications. Such properties may Med. 176:1191-1195, and Shopes, 1992, J. Immunol. 148(9): include, but are not limited to, specific kinetic properties (i.e. 35 2918-22, all incorporated entirely by reference. Additional association and dissociation kinetics), selectivity for the par modifications to the variants of the present disclosure include ticular target versus alternative targets, and selectivity for a those that enable the specific formation or heterodimeric, specific form of target versus alternative forms. Examples heteromultimeric, bifunctional, and/or multifunctional mol include full-length versus splice variants, cell-Surface Vs. ecules. Such modifications include, but are not limited to, one soluble forms, selectivity for various polymorphic variants, 40 or more amino acid substitutions in the CH3 domain, in which or selectivity for specific conformational forms of the target the Substitutions reduce homodimer formation and increase antigen. heterodimer formation. For example, methods of engineering Antibodies of the invention may comprise one or more and compositions of Such molecules are described in Atwell modifications that provide reduced or enhanced internaliza et al., 1997, J. Mol. Biol. 270(1):26-35, and Carter et al., tion of an antibody. In one embodiment, antibodies of the 45 2001, J. Immunol. Methods 248:7-15, both incorporated present disclosure can be utilized or combined with addi entirely by reference. Additional modifications include modi tional modifications in order to reduce the cellular internal fications in the hinge and CH3 domains, in which the modi ization of an antibody that occurs via interaction with one or fications reduce the propensity to form dimers. more Fc ligands. This property might be expected to enhance In further embodiments, the antibodies of the present dis effector function, and potentially reduce immunogenicity of 50 closure comprise modifications that remove proteolytic deg the antibodies of the invention. Alternatively, antibodies of radation sites. These may include, for example, protease sites the presentantibodies of the present disclosure can be utilized that reduce production yields, as well as protease sites that directly or combined with additional modifications in order to degrade the administered protein in vivo. In a preferred enhance the cellularinternalization of an antibody that occurs embodiment, additional modifications are made to remove via interaction with one or more Fc ligands. For example, in 55 covalent degradation sites Such as deamidation (i.e. deami a preferred embodiment, an antibody is used that provides dation of glutaminyl and asparaginyl residues to the corre enhanced binding to FcyRI, which is expressed on dendritic sponding glutamyland aspartyl residues), oxidation, and pro cells and active early in immune response. This strategy could teolytic degradation sites. Deamidation sites that are be further enhanced by combination with additional modifi particular useful to remove are those that have enhance pro cations, either within the antibody or in an attached fusion or 60 pensity for deamidation, including, but not limited to aspar conjugate partner, that promote recognition and presentation aginyl and glituamyl residues followed by glycines (NG and of Fc peptide fragments by MHC molecules. These strategies QG motifs, respectively). In such cases, substitution of either are expected to enhance target antigen processing and thereby residue can significantly reduce the tendency for deamida improve antigenicity of the target antigen (Bonnerot and tion. Common oxidation sites include methionine and cys Amigorena, 1999, Immunol Rev. 172:279-84, incorporated 65 teine residues. Other covalent modifications, that can either entirely by reference), promoting an adaptive immune be introduced or removed, include hydroxylation of proline response and greater target cell killing by the human immune and lysine, phosphorylation of hydroxyl groups of Seryl or US 9,040,042 B2 35 36 threonyl residues, methylation of the "-amino groups of c) redox active moieties; d) optical dyes; enzymatic groups lysine, arginine, and histidine side chains (T. E. Creighton, (e.g. horseradish peroxidase, 6-galactosidase, luciferase, Proteins: Structure and Molecular Properties, W.H. Free alkaline phosphatase); e) biotinylated groups; and f) prede man & Co., San Francisco, pp. 79-86 (1983), incorporated termined polypeptide epitopes recognized by a secondary entirely by reference), acetylation of the N-terminal amine, 5 reporter (e.g., leucine Zipper pair sequences, binding sites for and amidation of any C-terminal carboxyl group. Additional secondary antibodies, metal binding domains, epitope tags, modifications also may include but are not limited to post etc.). In some embodiments, the labeling group is coupled to translational modifications such as N-linked or O-linked gly the antibody via spacer arms of various lengths to reduce cosylation and phosphorylation. potential steric hindrance. Various methods for labeling pro Modifications may include those that improve expression 10 teins are known in the art and may be used in performing the and/or purification yields from hosts or host cells commonly present disclosure. Specific labels include optical dyes, used for production of biologics. These include, but are not including, but not limited to, chromophores, phosphors and limited to various mammaliancell lines (e.g. CHO), yeast cell fluorophores, with the latter being specific in many instances. lines, bacterial cell lines, and plants. Additional modifications Fluorophores can be either “small molecule' fluores, or pro include modifications that remove or reduce the ability of 15 teinaceous fluores. By “fluorescent label' is meant any mol heavy chains to form inter-chain disulfide linkages. Addi ecule that may be detected via its inherent fluorescent prop tional modifications include modifications that remove or erties. reduce the ability of heavy chains to form intra-chain disul Antibody Conjugates and Fusions fide linkages. In one embodiment, the antibodies of the invention are The antibodies of the present disclosure may comprise antibody “fusion proteins, sometimes referred to herein as modifications that include the use of unnatural amino acids 'antibody conjugates'. The fusion partner or conjugate part incorporated using, for example, the technologies developed ner can be proteinaceous or non-proteinaceous; the latter by Schultz and colleagues, including but not limited to meth generally being generated using functional groups on the ods described by Cropp & Shultz, 2004, Trends Genet. antibody and on the conjugate partner. Conjugate and fusion 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. 25 partners may be any molecule, including Small molecule U.S.A. 101(2):7566-71, Zhanget al., 2003,303(5656):371-3, chemical compounds and polypeptides. For example, a vari and Chin et al., 2003, Science 301 (5635):964-7, all incorpo ety of antibody conjugates and methods are described in Trail rated entirely by reference. In some embodiments, these et al., 1999, Curr. Opin. Immunol. 11:584-588, incorporated modifications enable manipulation of various functional, bio entirely by reference. Possible conjugate partners include but physical, immunological, or manufacturing properties dis 30 are not limited to , cytotoxic agents, toxins, radio cussed above. In additional embodiments, these modifica isotopes, chemotherapeutic agent, anti-angiogenic agents, a tions enable additional chemical modification for other tyrosine kinase inhibitors, and other therapeutically active purposes. Other modifications are contemplated herein. For agents. In some embodiments, conjugate partners may be example, the antibody may be linked to one of a variety of thought of more as payloads, that is to say that the goal of a nonproteinaceous polymers, e.g., polyethylene glycol (PEG), 35 conjugate is targeted delivery of the conjugate partner to a polypropylene glycol, polyoxyalkylenes, or copolymers of targeted cell, for example a cancer cellor immune cell, by the polyethylene glycol and polypropylene glycol. Additional antibody. Thus, for example, the conjugation of a toxin to an amino acid modifications may be made to enable specific or antibody targets the delivery of said toxin to cells expressing non-specific chemical or posttranslational modification of the the target antigen. As will be appreciated by one skilled in the antibodies. Such modifications, include, but are not limited to 40 art, in reality the concepts and definitions of fusion and con PEGylation and glycosylation. Specific substitutions that can jugate are overlapping. The designation of an antibody as a be utilized to enable PEGylation include, but are not limited fusion or conjugate is not meant to constrain it to any particu to, introduction of novel cysteine residues or unnatural amino lar embodiment of the present disclosure. Rather, these terms acids Such that efficient and specific coupling chemistries can are used loosely to convey the broad concept that any anti be used to attach a PEG or otherwise polymeric moiety. 45 body of the present disclosure may be linked genetically, Introduction of specific glycosylation sites can be achieved chemically, or otherwise, to one or more polypeptides or by introducing novel N-X-T/S sequences into the antibodies molecules to provide Some desirable property. of the present disclosure. Suitable conjugates include, but are not limited to, labels as Covalent modifications of antibodies are included within described below, drugs and cytotoxic agents including, but the scope of this invention, and are generally, but not always, 50 not limited to, cytotoxic drugs (e.g., chemotherapeutic done post-translationally. For example, several types of cova agents) or toxins or active fragments of such toxins. Suitable lent modifications of the antibody are introduced into the toxins and their corresponding fragments include diphtheria molecule by reacting specific amino acid residues of the A chain, exotoxin A chain, ricin A chain, abrin A chain, antibody with an organic derivatizing agent that is capable of curcin, crotin, phenomycin, enomycin and the like. Cytotoxic reacting with selected side chains or the N- or C-terminal 55 agents also include radiochemicals made by conjugating residues. radioisotopes to antibodies, or binding of a radionuclide to a In some embodiments, the covalent modification of the chelating agent that has been covalently attached to the anti antibodies of the invention comprises the addition of one or body. Additional embodiments utilize calicheamicin, aurist more labels. The term “labeling group” means any detectable atins, geldanamycin, maytansine, and duocarmycins and ana label. In some embodiments, the labeling group is coupled to 60 logs; for the latter, see U.S. 2003/0050331, incorporated the antibody via spacer arms of various lengths to reduce entirely by reference. potential steric hindrance. Various methods for labeling pro In one embodiment, the antibodies of the present disclo teins are known in the art and may be used in performing the Sure are fused or conjugated to a cytokine. By “cytokine’ as present disclosure. In general, labels fall into a variety of used herein is meant a generic term for proteins released by classes, depending on the assay in which they are to be 65 one cell population that act on another cell as intercellular detected: a) isotopic labels, which may be radioactive or mediators. For example, as described in Penichet et al., 2001, heavy isotopes; b) magnetic labels (e.g., magnetic particles); J. Immunol. Methods 248:91-101, incorporated entirely by US 9,040,042 B2 37 38 reference, cytokines may be fused to antibody to provide an PAPII, and PAP-S), momordica charantia inhibitor, curcin, array of desirable properties. Examples of Such cytokines are crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, lymphokines, monokines, and traditional polypeptide hor restrictocin, phenomycin, enomycin and the tricothecenes. mones. Included among the cytokines are growth hormone See, for example, PCT WO93/21232, incorporated entirely Such as human growth hormone, N-methionyl human growth 5 by reference. The present disclosure further contemplates a hormone, and bovine growth hormone; parathyroid hormone; conjugate between an antibody of the present disclosure and thyroxine; insulin; proinsulin; relaxin; prorelaxin, glycopro a compound with nucleolytic activity, for example a ribonu tein hormones such as follicle stimulating hormone (FSH), clease or DNA endonuclease such as a deoxyribonuclease thyroid stimulating hormone (TSH), and luteinizing hormone (Dnase). (LH); hepatic growth factor; fibroblast growth factor; prolac 10 In an alternate embodiment, an antibody of the present tin, placental lactogen; tumor necrosis factor-alpha and -beta; disclosure may be fused, conjugated, or operably linked to a mullerian-inhibiting Substance; mouse gonadotropin-associ radioisotope to form a radioconjugate. A variety of radioac ated peptide; inhibin; activin; vascular endothelial growth tive isotopes are available for the production of radioconju factor; integrin: thrombopoietin (TPO); nerve growth factors gate antibodies. Examples include, but are not limited to, such as NGF-beta; platelet-growth factor; transforming 15 At 211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, growth factors (TGFs) such as TGF-alpha and TGF-beta; and radioactive isotopes of Lu. insulin-like growth factor-I and -II; erythropoietin (EPO); In yet another embodiment, an antibody of the present osteoinductive factors; interferons such as interferon-alpha, disclosure may be conjugated to a “receptor’ (Such strepta beta, and -gamma; colony stimulating factors (CSFs) such as vidin) for utilization in tumor pretargeting wherein the anti macrophage-CSF (M-CSF); granulocyte-macrophage-CSF body-receptor conjugate is administered to the patient, fol (GM-CSF); and granulocyte-CSF (G-CSF); lowed by removal of unbound conjugate from the circulation (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, using a clearing agent and then administration of a “ligand IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a factor such as TNF-alpha or TNF-beta: C5a, and other radionucleotide). In an alternate embodiment, the antibody is polypeptide factors including LIF and kit ligand (KL). As 25 conjugated or operably linked to an enzyme in order to used herein, the term cytokine includes proteins from natural employ Antibody Dependent Enzyme Mediated Prodrug Sources or from recombinant cell culture, and biologically Therapy (ADEPT). ADEPT may be used by conjugating or active equivalents of the native sequence cytokines. operably linking the antibody to a prodrug-activating enzyme In an alternate embodiment, the antibodies of the present that converts a prodrug (e.g. a peptidyl chemotherapeutic disclosure are fused, conjugated, or operably linked to a 30 agent, see PCT WO 81/01145, incorporated entirely by ref toxin, including but not limited to Small molecule toxins and erence) to an active anti-cancer drug. See, for example, PCT enzymatically active toxins of bacterial, fungal, plant or ani WO 88/07378 and U.S. Pat. No. 4,975,278, both incorporated mal origin, including fragments and/or variants thereof. For entirely by reference. The enzyme component of the immu example, a variety of immunotoxins and immunotoxin meth noconjugate useful for ADEPT includes any enzyme capable ods are described in Thrush et al., 1996, Ann. Rev. Immunol. 35 of acting on a prodrug in Such a way so as to covert it into its 14:49-71, incorporated entirely by reference. Small molecule more active, cytotoxic form. Enzymes that are useful in the toxins include but are not limited to calicheamicin, may method of this invention includebutare not limited to alkaline tansine (U.S. Pat. No. 5,208,020, incorporated entirely by phosphatase useful for converting phosphate-containing pro reference), trichothene, and CC1065. In one embodiment of drugs into free drugs: arylsulfatase useful for converting Sul the invention, the antibody is conjugated to one or more 40 fate-containing prodrugs into free drugs; cytosine deaminase maytansine molecules (e.g. about 1 to about 10 maytansine useful for converting non-toxic 5-fluorocytosine into the anti molecules per antibody molecule). Maytansine may, for cancer drug. 5-fluorouracil; proteases, such as serratia pro example, be converted to May-SS-Me which may be reduced tease, thermolysin, Subtilisin, carboxypeptidases and cathep to May-SH3 and reacted with modified antibody (Chariet al., sins (such as cathepsins B and L), that are useful for 1992, Cancer Research 52: 127-131, incorporated entirely by 45 converting peptide-containing prodrugs into free drugs; reference) to generate a maytansinoid-antibody conjugate. D-alanylcarboxypeptidases, useful for converting prodrugs Another conjugate of interest comprises an antibody conju that contain D-amino acid Substituents; carbohydrate-cleav gated to one or more calicheamicin molecules. The cali ing enzymes such as beta.-galactosidase and neuramimidase cheamicin family of antibiotics are capable of producing useful for converting glycosylated prodrugs into free drugs; double-stranded DNA breaks at sub-picomolar concentra 50 beta-lactamase useful for converting drugs derivatized with tions. Structural analogues of calicheamicin that may be used ..alpha.-lactams into free drugs; and penicillin amidases, such include but are not limited to Y', C.", Cls, N-acetyl-Y', as penicillin V amidase or penicillin G. amidase, useful for PSAG, and 0', (Hinman et al., 1993, Cancer Research converting drugs derivatized at their amine nitrogens with 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925 phenoxyacetyl or phenylacetyl groups, respectively, into free 2928) (U.S. Pat. No. 5,714,586: U.S. Pat. No. 5,712.374; U.S. 55 drugs. Alternatively, antibodies with enzymatic activity, also Pat. No. 5.264,586: U.S. Pat. No. 5,773,001, all incorporated known in the art as “abzymes', can be used to convert the entirely by reference). Dolastatin 10 analogs such as aurista prodrugs of the invention into free active drugs (see, for tin E (AE) and monomethylauristatin E (MMAE) may find example, Massey, 1987, Nature 328: 457-458, incorporated use as conjugates for the antibodies of the present disclosure entirely by reference). Antibody-abzyme conjugates can be (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Fran 60 prepared for delivery of the abzyme to a tumor cell popula cisco et al., 2003 Blood 102(4): 1458-65, both incorporated tion. A variety of additional conjugates are contemplated for entirely by reference). Useful enzymatically active toxins the antibodies of the present disclosure. A variety of chemo include but are not limited to diphtheria A chain, nonbinding therapeutic agents, anti-angiogenic agents, tyrosine kinase active fragments of diphtheria toxin, eXotoxin A chain (from inhibitors, and other therapeutic agents are described below, Pseudomonas aeruginosa), ricin A chain, abrin A chain, 65 which may find use as antibody conjugates. modeccin. A chain, alpha-sarcin, Aleurites fordii proteins, Also contemplated as fusion and conjugate partners are Fc dianthin proteins, Phytolaca americana proteins (PAPI, polypeptides. Thus an antibody may be a multimeric Fc US 9,040,042 B2 39 40 polypeptide, comprising two or more Fc regions. The advan ering sequence' or grammatical equivalents thereof, herein is tage of Such a molecule is that it provides multiple binding meant a molecule or group of molecules (such as a monomer sites for Fc receptors with a single protein molecule. In one or polymer) that connects two molecules and often serves to embodiment, Fc regions may be linked using a chemical place the two molecules in a preferred configuration. Linkers engineering approach. For example, Fab's and Fc's may be 5 are known in the art; for example, homo- or hetero-bifunc linked by thioether bonds originating at cysteine residues in tional linkers as are well known (see, 1994 Pierce Chemical the hinges, generating molecules such as FabFc. Fc regions Company catalog, technical section on cross-linkers, pages may be linked using disulfide engineering and/or chemical 155-200, incorporated entirely by reference). A number of cross-linking. In a preferred embodiment, Fc regions may be strategies may be used to covalently link molecules together. linked genetically. In a preferred embodiment, Fc regions in 10 These include, but are not limited to polypeptide linkages an antibody are linked genetically to generated tandemly between N- and C-termini of proteins or protein domains, linked Fc regions as described in U.S. Ser. No. 1 1/022,289. linkage via disulfide bonds, and linkage via chemical cross filed Dec. 21, 2004, entitled “Fc polypeptides with novel Fc linking reagents. In one aspect of this embodiment, the linker ligand binding sites, incorporated entirely by reference. Tan is a peptide bond, generated by recombinant techniques or demly linked Fc polypeptides may comprise two or more Fc 15 peptide synthesis. The linker may contain amino acid resi regions, preferably one to three, most preferably two Fc dues that provide flexibility. Thus, the linker peptide may regions. It may be advantageous to explore a number of predominantly include the following amino acid residues: engineering constructs in order to obtain homo- or hetero Gly, Ser, Ala, or Thr. The linker peptide should have a length tandemly linked antibodies with the most favorable structural that is adequate to link two molecules in Such a way that they and functional properties. Tandemly linked antibodies may assume the correct conformation relative to one another so be homo-tandemly linked antibodies, that is an antibody of that they retain the desired activity. Suitable lengths for this one isotype is fused genetically to another antibody of the purpose include at least one and not more than 50 amino acid same isotype. It is anticipated that because there are multiple residues. Preferably, the linker is from about 1 to 30 amino Fc R, C1q, and/or FcRn binding sites on tandemly linked Fc acids in length, with linkers of 1 to 20 amino acids in length polypeptides, effector functions and/or pharmacokinetics 25 being most preferred. Useful linkers include glycine-serine may be enhanced. In an alternate embodiment, antibodies polymers (including, for example, (GS)n, (GSGGS)n from different isotypes may be tandemly linked, referred to as (GGGGS)n and (GGGS)n, where n is an integer of at least hetero-tandemly linked antibodies. For example, because of one), glycine-alanine polymers, alanine-serine polymers, and the capacity to target Fcy Rand FcCRI receptors, an antibody other flexible linkers, as will be appreciated by those in the that binds both FcyRs and FcCRI may provide a significant 30 art. Alternatively, a variety of nonproteinaceous polymers, clinical improvement. including but not limited to polyethylene glycol (PEG), In addition to antibodies, an antibody-like protein that is polypropylene glycol, polyoxyalkylenes, or copolymers of finding an expanding role in research and therapy is the Fc polyethylene glycol and polypropylene glycol, may find use fusion (Chamow et al., 1996, Trends Biotechnol 14:52-60; as linkers, that is may find use to link the antibodies of the Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both 35 present disclosure to a fusion or conjugate partner, or to link incorporated entirely by reference). “Fe fusion' is herein the antibodies of the present disclosure to a conjugate. meant to be synonymous with the terms “immunoadhesin'. Production of Antibodies “Ig fusion”, “Ig chimera', and “receptor globulin' (some The present disclosure provides methods for producing times with dashes) as used in the prior art (Chamow et al., and experimentally testing antibodies. The described meth 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, 40 ods are not meant to constrain the present disclosure to any Curr Opin Immunol 9:195-200). An Fc fusion is a protein particular application or theory of operation. Rather, the pro wherein one or more polypeptides is operably linked to Fc. vided methods are meant to illustrate generally that one or An Fc fusion combines the Fc region of an antibody, and thus more antibodies may be produced and experimentally tested its favorable effector functions and pharmacokinetics, with to obtain variant antibodies. General methods for antibody the target-binding region of a receptor, ligand, or some other 45 molecular biology, expression, purification, and screening protein or protein domain. The role of the latter is to mediate are described in Antibody Engineering, edited by Duebel & target recognition, and thus it is functionally analogous to the Kontermann, Springer-Verlag, Heidelberg, 2001; and Hay antibody variable region. Because of the structural and func hurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689: tional overlap of Fc fusions with antibodies, the discussion on Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76: antibodies in the present disclosure extends also to Fc. 50 Antibodies: A Laboratory Manual by Harlow & Lane, New Virtually any protein or small molecule may be linked to Fc York: Cold Spring Harbor Laboratory Press, 1988, all incor to generate an Fc fusion. Protein fusion partners may include, porated entirely by reference. but are not limited to, the variable region of any antibody, the In one embodiment of the present disclosure, nucleic acids target-binding region of a receptor, an adhesion molecule, a are created that encode the antibodies, and that may then be ligand, an enzyme, a cytokine, a chemokine, or some other 55 cloned into host cells, expressed and assayed, if desired. protein or protein domain. Small molecule fusion partners Thus, nucleic acids, and particularly DNA, may be made that may include any therapeutic agent that directs the Fc fusion to encode each protein sequence. These practices are carried out a therapeutic target. Such targets may be any molecule, pref using well-known procedures. For example, a variety of erably an extracellular receptor, that is implicated in disease. methods that may find use in the present disclosure are Fusion and conjugate partners may be linked to any region 60 described in Molecular Cloning A Laboratory Manual, 3" of an antibody of the present disclosure, including at the N- or Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New C-termini, or at some residue in-between the termini. In a York, 2001), and Current Protocols in Molecular Biology preferred embodiment, a fusion or conjugate partner is linked (John Wiley & Sons), both incorporated entirely by reference. at the N- or C-terminus of the antibody, most preferably the As will be appreciated by those skilled in the art, the genera N-terminus. A variety of linkers may find use in the present 65 tion of exact sequences for a library comprising a large num disclosure to covalently link antibodies to a fusion or conju ber of sequences is potentially expensive and time consum gate partner. By “linker”, “linker sequence”, “spacer”, “teth ing. By “library' herein is meanta set of variants in any form, US 9,040,042 B2 41 42 including but not limited to a list of nucleic acid oramino acid The nucleic acids that encode the antibodies of the present sequences, a list of nucleic acid oramino acid substitutions at disclosure may be incorporated into an expression vector in variable positions, a physical library comprising nucleic acids order to express the protein. A variety of expression vectors that encode the library sequences, or a physical library com may be utilized for protein expression. Expression vectors prising the variant proteins, either in purified or unpurified may comprise self-replicating extra-chromosomal vectors or form. Accordingly, there are a variety of techniques that may vectors which integrate into a host genome. Expression vec be used to efficiently generate libraries of the present disclo tors are constructed to be compatible with the host cell type. Sure. Such methods that may find use in the present disclosure Thus expression vectors which find use in the present disclo are described or referenced in U.S. Pat. No. 6,403,312; U.S. sure include but are not limited to those which enable protein Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No. 10 expression in mammalian cells, bacteria, insect cells, yeast, 10/218,102: PCTWO 01/40091; and PCT WO 02/25588, all and in in vitro systems. As is known in the art, a variety of incorporated entirely by reference. Such methods include but expression vectors are available, commercially or otherwise, are not limited to gene assembly methods, PCR-based that may find use in the present disclosure for expressing method and methods which use variations of PCR, ligase antibodies. chain reaction-based methods, pooled oligo methods such as 15 Expression vectors typically comprise a protein operably those used in synthetic shuffling, error-prone amplification linked with control or regulatory sequences, selectable mark methods and methods which use oligos with random muta ers, any fusion partners, and/or additional elements. By tions, classical site-directed mutagenesis methods, cassette “operably linked herein is meant that the nucleic acid is mutagenesis, and other amplification and gene synthesis placed into a functional relationship with another nucleic acid methods. AS is known in the art, there are a variety of com sequence. Generally, these expression vectors include tran mercially available kits and methods for gene assembly, Scriptional and translational regulatory nucleic acid operably mutagenesis, vector Subcloning, and the like, and Such com linked to the nucleic acid encoding the antibody, and are mercial products find use in the present disclosure for gener typically appropriate to the host cell used to express the ating nucleic acids that encode antibodies. protein. In general, the transcriptional and translational regu The antibodies of the present disclosure may be produced 25 latory sequences may include promoter sequences, ribosomal by culturing a host cell transformed with nucleic acid, pref binding sites, transcriptional start and stop sequences, trans erably an expression vector, containing nucleic acid encoding lational start and stop sequences, and enhancer or activator the antibodies, under the appropriate conditions to induce or sequences. AS is also known in the art, expression vectors cause expression of the protein. The conditions appropriate typically contain a selection gene or marker to allow the for expression will vary with the choice of the expression 30 selection of transformed host cells containing the expression vector and the host cell, and will be easily ascertained by one vector. Selection genes are well known in the art and will vary skilled in the art through routine experimentation. A wide with the host cell used. variety of appropriate host cells may be used, including but Antibodies may be operably linked to a fusion partner to not limited to mammalian cells, bacteria, insect cells, and enable targeting of the expressed protein, purification, yeast. For example, a variety of cell lines that may find use in 35 screening, display, and the like. Fusion partners may be the present disclosure are described in the ATCC(R) cell line linked to the antibody sequence via a linker sequences. The catalog, available from the American Type Culture Collec linker sequence will generally comprise a small number of tion. amino acids, typically less than ten, although longer linkers In a preferred embodiment, the antibodies are expressed in may also be used. Typically, linker sequences are selected to mammalian expression systems, including systems in which 40 beflexible and resistant to degradation. As will be appreciated the expression constructs are introduced into the mammalian by those skilled in the art, any of a wide variety of sequences cells using virus Such as retrovirus or adenovirus. Any mam may be used as linkers. For example, a common linker malian cells may be used, with human, mouse, rat, hamster, sequence comprises the amino acid sequence GGGGS. A and primate cells being particularly preferred. Suitable cells fusion partner may be a targeting or signal sequence that also include known research cells, including but not limited to 45 directs antibody and any associated fusion partners to a Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PER desired cellular location or to the extracellular media. As is C.6, HeLa, Sp2/0, NS0 cells and variants thereof. In an alter known in the art, certain signaling sequences may target a nately preferred embodiment, library proteins are expressed protein to be either secreted into the growth media, or into the in bacterial cells. Bacterial expression systems are well periplasmic space, located between the inner and outer mem known in the art, and include Escherichia coli (E. coli), Bacil 50 brane of the cell. A fusion partner may also be a sequence that lus subtilis, Streptococcus Cremoris, and Streptococcus liv encodes a peptide or protein that enables purification and/or idans. In alternate embodiments, antibodies are produced in screening. Such fusion partners include but are not limited to insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b 1-4) or polyhistidine tags (His-tags) (for example H and Ho or other yeast cells (e.g. S. cerevisiae, Pichia, etc). In an alternate tags for use with Immobilized Metal Affinity Chromatogra embodiment, antibodies are expressed in vitro using cell free 55 phy (IMAC) systems (e.g. Ni" affinity columns)), GST translation systems. In vitro translation systems derived from fusions, MBP fusions, Strep-tag, the BSP biotinylation target both prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheat sequence of the bacterial enzyme BirA, and epitope tags germ, rabbit reticulocytes) cells are available and may be which are targeted by antibodies (for example c-myc tags, chosen based on the expression levels and functional proper flag-tags, and the like). As will be appreciated by those skilled ties of the protein of interest. For example, as appreciated by 60 in the art, Such tags may be useful for purification, for screen those skilled in the art, in vitro translation is required for some ing, or both. For example, an antibody may be purified using display technologies, for example ribosome display. In addi a His-tag by immobilizing it to a Ni" affinity column, and tion, the antibodies may be produced by chemical synthesis then after purification the same His-tag may be used to immo methods. Also transgenic expression systems both animal bilize the antibody to a Ni" coated plate to performan ELISA (e.g. cow, sheep or goat milk, embryonated hen's eggs, whole 65 or other binding assay (as described below). A fusion partner insect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, may enable the use of a selection method to screen antibodies etc.) (see below). Fusion partners that enable a variety of selection US 9,040,042 B2 43 44 methods are well-known in the art, and all of these find use in a) immune labels, which may be an epitope incorporated as a the present disclosure. For example, by fusing the members of fusion partner that is recognized by an antibody, b) isotopic an antibody library to the gene III protein, phage display can labels, which may be radioactive or heavy isotopes, and c) be employed (Kay et al., Phage display of peptides and pro small molecule labels, which may include fluorescent and teins: a laboratory manual, Academic Press, San Diego, 5 colorimetric dyes, or molecules Such as biotin that enable Calif., 1996: Lowman et al., 1991, Biochemistry 30:10832 other labeling methods. Labels may be incorporated into the 10838; Smith, 1985, Science 228:1315-1317, incorporated compound at any position and may be incorporated in vitro or entirely by reference). Fusion partners may enable antibodies in vivo during protein expression. to be labeled. Alternatively, a fusion partner may bind to a In a preferred embodiment, the functional and/or biophysi specific sequence on the expression vector, enabling the 10 cal properties of antibodies are screened in an in vitro assay. fusion partner and associated antibody to be linked covalently In vitro assays may allow a broad dynamic range for screen or noncovalently with the nucleic acid that encodes them. ing properties of interest. Properties of antibodies that may be The methods of introducing exogenous nucleic acid into screened includebut are not limited to stability, solubility, and host cells are well known in the art, and will vary with the host affinity for Fc ligands, for example FcyRs. Multiple proper cell used. Techniques include but are not limited to dextran 15 ties may be screened simultaneously or individually. Proteins mediated transfection, calcium phosphate precipitation, cal may be purified or unpurified, depending on the requirements cium chloride treatment, polybrene mediated transfection, of the assay. In one embodiment, the screen is a qualitative or protoplast fusion, electroporation, viral or phage infection, quantitative binding assay for binding of antibodies to a pro encapsulation of the polynucleotide(s) in liposomes, and tein or nonprotein molecule that is known or thought to bind direct microinjection of the DNA into nuclei. In the case of the antibody. In a preferred embodiment, the screen is a mammalian cells, transfection may be either transient or binding assay for measuring binding to the target antigen. In stable. an alternately preferred embodiment, the screen is an assay In a preferred embodiment, antibodies are purified or iso for binding of antibodies to an Fc ligand, including but are not lated after expression. Proteins may be isolated or purified in limited to the family of FcyRs, the neonatal receptor FcRn, a variety of ways known to those skilled in the art. Standard 25 the complement protein C1q, and the bacterial proteins A and purification methods include chromatographic techniques, G. Said Fc ligands may be from any organism, with humans, including ion exchange, hydrophobic interaction, affinity, mice, rats, rabbits, and monkeys preferred. Binding assays sizing or gel filtration, and reversed-phase, carried out at can be carried out using a variety of methods known in the art, atmospheric pressure or at high pressure using systems such including but not limited to FRET (Fluorescence Resonance as FPLC and HPLC. Purification methods also include elec 30 Energy Transfer) and BRET (Bioluminescence Resonance trophoretic, immunological, precipitation, dialysis, and chro Energy Transfer)-based assays, AlphaScreenTM (Amplified matofocusing techniques. Ultrafiltration and diafiltration Luminescent Proximity Homogeneous Assay), Scintilation techniques, in conjunction with protein concentration, are Proximity Assay, ELISA (Enzyme-Linked Immunosorbent also useful. As is well known in the art, a variety of natural Assay), SPR (Surface Plasmon Resonance, also known as proteins bind Fc and antibodies, and these proteins can find 35 BiacoreTM), isothermal titration calorimetry, differential use in the present disclosure for purification of antibodies. For scanning calorimetry, gel electrophoresis, and chromatogra example, the bacterial proteins A and G bind to the Fc region. phy including gel filtration. These and other methods may Likewise, the bacterial protein L binds to the Fab region of take advantage of some fusion partner or label of the antibody. Some antibodies, as of course does the antibody's target anti Assays may employ a variety of detection methods including gen. Purification can often be enabled by a particular fusion 40 but not limited to chromogenic, fluorescent, luminescent, or partner. For example, antibodies may be purified using glu isotopic labels. tathione resin if a GST fusion is employed, Ni' affinity The biophysical properties of antibodies, for example sta chromatography if a His-tag is employed, or immobilized bility and solubility, may be screened using a variety of meth anti-flag antibody if a flag-tag is used. For general guidance in ods known in the art. Protein stability may be determined by Suitable purification techniques, see, e.g. incorporated 45 measuring the thermodynamic equilibrium between folded entirely by reference Protein Purification: Principles and and unfolded states. For example, antibodies of the present Practice, 3" Ed., Scopes, Springer-Verlag, NY, 1994, incor disclosure may be unfolded using chemical denaturant, heat, porated entirely by reference. The degree of purification nec or pH, and this transition may be monitored using methods essary will vary depending on the screen or use of the anti including but not limited to circular dichroism spectroscopy, bodies. In some instances no purification is necessary. For 50 fluorescence spectroscopy, absorbance spectroscopy, NMR example in one embodiment, if the antibodies are secreted, spectroscopy, calorimetry, and proteolysis. As will be appre screening may take place directly from the media. As is well ciated by those skilled in the art, the kinetic parameters of the known in the art, some methods of selection do not involve folding and unfolding transitions may also be monitored purification of proteins. Thus, for example, if a library of using these and other techniques. The solubility and overall antibodies is made into a phage display library, protein puri 55 structural integrity of an antibody may be quantitatively or fication may not be performed. qualitatively determined using a wide range of methods that In Vitro Experimentation are known in the art. Methods which may find use in the Antibodies may be screened using a variety of methods, present disclosure for characterizing the biophysical proper including but not limited to those that use in vitro assays, in ties of antibodies include gel electrophoresis, isoelectric Vivo and cell-based assays, and selection technologies. Auto 60 focusing, capillary electrophoresis, chromatography Such as mation and high-throughput Screening technologies may be size exclusion chromatography, ion-exchange chromatogra utilized in the screening procedures. Screening may employ phy, and reversed-phase high performance liquid chromatog the use of a fusion partner or label. The use of fusion partners raphy, peptide mapping, oligosaccharide mapping, mass has been discussed above. By “labeled herein is meant that spectrometry, ultraviolet absorbance spectroscopy, fluores the antibodies of the invention have one or more elements, 65 cence spectroscopy, circular dichroism spectroscopy, isother isotopes, or chemical compounds attached to enable the mal titration calorimetry, differential scanning calorimetry, detection in a screen. In general, labels fall into three classes: analytical ultra-centrifugation, dynamic light scattering, pro US 9,040,042 B2 45 46 teolysis, and cross-linking, turbidity measurement, filter morphological changes of cells as a response to the presence retardation assays, immunological assays, fluorescent dye of an antibody. Cell types for Such assays may be prokaryotic binding assays, protein-staining assays, microscopy, and or eukaryotic, and a variety of cell lines that are known in the detection of aggregates via ELISA or other binding assay. art may be employed. Alternatively, cell-based screens are Structural analysis employing X-ray crystallographic tech performed using cells that have been transformed or trans niques and NMR spectroscopy may also find use. In one fected with nucleic acids encoding the antibodies. embodiment, stability and/or solubility may be measured by In vitro assays includebut are not limited to binding assays, determining the amount of protein solution after some ADCC, CDC, phagocytosis, cytotoxicity, proliferation, apo defined period of time. In this assay, the protein may or may ptosis, necrosis, cell cycle arrest, peroxide/oZone release, not be exposed to Some extreme condition, for example 10 elevated temperature, low pH, or the presence of denaturant. chemotaxis of effector cells, inhibition of such assays by Because function typically requires a stable, soluble, and/or reduced effector function antibodies; ranges of activities Such well-folded/structured protein, the aforementioned func as >100x improvement or >100x reduction, blends of recep tional and binding assays also provide ways to perform Such tor activation and the assay outcomes that are expected from a measurement. For example, a solution comprising an anti 15 Such receptor profiles. body could be assayed for its ability to bind target antigen, In Vivo Experimentation then exposed to elevated temperature for one or more defined The biological properties of the antibodies of the present periods of time, then assayed for antigen binding again. disclosure may be characterized in cell, tissue, and whole Because unfolded and aggregated protein is not expected to organism experiments. AS is know in the art, drugs are often be capable of binding antigen, the amount of activity remain tested in animals, including but not limited to mice, rats, ing provides a measure of the antibody's stability and solu rabbits, dogs, cats, pigs, and monkeys, in order to measure a bility. drug's efficacy for treatment against a disease or disease In a preferred embodiment, the library is screened using model, or to measure a drug's pharmacokinetics, toxicity, and one or more cell-based or in vitro assays. For such assays, other properties. Said animals may be referred to as disease antibodies, purified or unpurified, are typically added exog 25 models. With respect to the antibodies of the present disclo enously such that cells are exposed to individual variants or Sure, a particular challenge arises when using animal models groups of variants belonging to a library. These assays are to evaluate the potential for in-human efficacy of candidate typically, but not always, based on the biology of the ability of polypeptides—this is due, at least in part, to the fact that the antibody to bind to antigen and mediate Some biochemical antibodies that have a specific effect on the affinity for a event, for example effector functions like cellular lysis, 30 human Fc receptor may not have a similar affinity effect with phagocytosis, ligand/receptor binding inhibition, inhibition the orthologous animal receptor. These problems can be fur of growth and/or proliferation, apoptosis and the like. Such ther exacerbated by the inevitable ambiguities associated assays often involve monitoring the response of cells to anti with correct assignment of true orthologues (Mechetina et al., body, for example cell Survival, cell death, cellular phagocy Immunogenetics, 2002 54:463-468, incorporated entirely by tosis, cell lysis, change in cellular morphology, or transcrip 35 reference), and the fact that Some orthologues simply do not tional activation Such as cellular expression of a natural gene exist in the animal (e.g. humans possess an FcyRIIa whereas or reporter gene. For example, Such assays may measure the mice do not). Therapeutics are often tested in mice, including ability of antibodies to elicit ADCC, ADCP, or CDC. For but not limited to nude mice, SCID mice, xenograft mice, and Some assays additional cells or components, that is in addition transgenic mice (including knockins and knockouts). For to the target cells, may need to be added, for example serum 40 example, an antibody of the present disclosure that is complement, or effector cells Such as peripheral blood mono intended as an anti-cancer therapeutic may be tested in a cytes (PBMCs), NK cells, macrophages, and the like. Such mouse cancer model, for example a Xenograft mouse. In this additional cells may be from any organism, preferably method, a tumor or tumor cell line is grafted onto or injected humans, mice, rat, rabbit, and monkey. Crosslinked or mono into a mouse, and Subsequently the mouse is treated with the meric antibodies may cause apoptosis of certain cell lines 45 therapeutic to determine the ability of the antibody to reduce expressing the antibody's target antigen, or they may mediate or inhibit cancer growth and metastasis. An alternative attack on target cells by immune cells which have been added approach is the use of a SCID murine model in which to the assay. Methods for monitoring cell death or viability are immune-deficient mice are injected with human Periferal known in the art, and include the use of dyes, fluorophores, Blood Lymphocytes (PBLs), conferring a semi-functional immunochemical, cytochemical, and radioactive reagents. 50 and human immune system—with an appropriate array of For example, caspase assays or annexin-flourconjugates may human FcRs—to the mice that have subsequently been enable apoptosis to be measured, and uptake or release of injected with antibodies or Fc-polypeptides that target radioactive Substrates (e.g. Chromium-51 release assays) or injected human tumor cells. In Such a model, the Fc-polypep the metabolic reduction of fluorescent dyes such as alamar tides that target the desired antigen (Such as her2/neu on blue may enable cell growth, proliferation or activation to be 55 SkOV3 ovarian cancer cells) interact with human PBLs monitored. In a preferred embodiment, the DELFIAR) within the mice to engage tumoricidal effector functions. EuTDA-based cytotoxicity assay (Perkin Elmer, Mass.) is Such experimentation may provide meaningful data for deter used. Alternatively, dead or damaged target cells may be mination of the potential of said antibody to be used as a monitored by measuring the release of one or more natural therapeutic. Any organism, preferably mammals, may be intracellular proteins, for example lactate dehydrogenase. 60 used for testing. For example because of their genetic simi Transcriptional activation may also serve as a method for larity to humans, monkeys can be suitable therapeutic mod assaying function in cell-based assays. In this case, response els, and thus may be used to test the efficacy, toxicity, phar may be monitored by assaying for natural genes or proteins macokinetics, or other property of the antibodies of the which may be upregulated or down-regulated, for example present disclosure. Tests of the antibodies of the present dis the release of certain interleukins may be measured, or alter 65 closure in humans are ultimately required for approval as natively readout may be via a luciferase or GFP-reporter drugs, and thus of course these experiments are contem construct. Cell-based assays may also involve the measure of plated. Thus the antibodies of the present disclosure may be US 9,040,042 B2 47 48 tested in humans to determine their therapeutic efficacy, tox candidate therapeutic antibodies in a human patient, some icity, pharmacokinetics, and/or other clinical properties. variant polypeptides of the present disclosure may find utility The antibodies of the present disclosure may confer supe as proxies for assessing potential in-human efficacy. Such rior performance on Fc-containing therapeutics in animal proxy molecules would preferably mimic—in the animal sys models or in humans. The receptor binding profiles of Such tem—the FcRand/or complement biology of a corresponding antibodies, as described in this specification, may, for candidate human antibody. This mimicry is most likely to be example, be selected to increase the potency of cytotoxic manifested by relative association affinities between specific drugs or to target specific effector functions or effector cells to antibodies and animal vs. human receptors. For example, if improve the selectivity of the drug's action. Further, receptor one were using a mouse model to assess the potential in binding profiles can be selected that may reduce Some or all 10 human efficacy of an antibody that has enhanced affinity for effector functions thereby reducing the side-effects or toxic human FcyRIIIa, an appropriate proxy variant would have ity of such Fc-containing drug. For example, an antibody with enhanced affinity for mouse FcyRIII-2 (mouse CD16-2). reduced binding to FcyRIIIa, FcyRI and FcyRIIa can be Alternatively if one were using a mouse model to assess the selected to eliminate most cell-mediated effector function, or potential in-human efficacy of an antibody that has reduced an antibody with reduced binding to C1q may be selected to 15 affinity for the inhibitory human FcyRIIb, an appropriate limit complement-mediated effector functions. In some con proxy variant would have reduced affinity for mouse FcyRII. texts, such effector functions are known to have potential It should also be noted that the proxy antibodies could be toxic effects, therefore eliminating them may increase the created in the context of a human antibody, an animal anti safety of the Fc-bearing drug and Such improved safety may body, or both. be characterized in animal models. In some contexts, such In a preferred embodiment, the testing of antibodies may effector functions are known to mediate the desirable thera include study of efficacy in primates (e.g. cynomolgus mon peutic activity, therefore enhancing them may increase the key model) to facilitate the evaluation of depletion of specific activity or potency of the Fc-bearing drug and Such improved target cells harboring target antigen. Additional primate mod activity or potency may be characterized in animal models. els include but not limited to that of the rhesus monkey and Fc Optimized antibodies can be tested in a variety of orthoto 25 polypetides in therapeutic studies of autoimmune, transplan pic tumor models. These clinically relevant animal models tation and cancer. are important in the study of pathophysiology and therapy of Toxicity studies are performed to determine the antibody or aggressive cancers like pancreatic, prostate and breast cancer. Fc-fusion related-effects that cannot be evaluated in standard Immune deprived mice including, but not limited to athymic pharmacology profile or occur only after repeated adminis nude or SCID mice are frequently used in scoring of local and 30 tration of the agent. Most toxicity tests are performed in two systemic tumor spread from the site of intraorgan (e.g. pan species—a rodentanda non-rodent—to ensure that any unex creas, prostate or mammary gland) injection of human tumor pected adverse effects are not overlooked before new thera cells or fragments of donor patients. peutic entities are introduced into man. In general, these In preferred embodiments, antibodies of the present dis models may measure a variety of toxicities including geno closure may be assessed for efficacy in clinically relevant 35 toxicity, chronic toxicity, immunogenicity, reproductive/de animal models of various human diseases. In many cases, velopmental toxicity and carcinogenicity. Included within the relevant models include various transgenic animals for spe aforementioned parameters are standard measurement of cific tumor antigens. food consumption, bodyweight, antibody formation, clinical Relevant transgenic models such as those that express chemistry, and macro- and microscopic examination of stan human Fc receptors (e.g., CD16 including the gamma chain, 40 dard organs/tissues (e.g. cardiotoxicity). Additional param FcyRI, RIIa/b, and others) could be used to evaluate and test eters of measurement are injection site trauma and the mea antibodies and Fc-fusions in their efficacy. The evaluation of Surement of neutralizing antibodies, if any. Traditionally, antibodies by the introduction of human genes that directly or monoclonal antibody therepeutics, naked or conjugated are indirectly mediate effector function in mice or other rodents evaluated for cross-reactivity with normal tissues, immuno that may enable physiological studies of efficacy in tumor 45 genicity/antibody production, conjugate or linker toxicity toxicity or other diseases Such as autoimmune disorders and and “bystander toxicity of radiolabeled species. Nonethe RA. Human Fc receptors such as FcyRIIIa may possess poly less, such studies may have to be individualized to address morphisms such as that in position 158 V or F which would specific concerns and following the guidance set by ICH S6 further enable the introduction of specific and combinations (Safety studies for biotechnological products also noted of human polymorphisms into rodents. The various studies 50 above). As such, the general principles are that the products involving polymorphism-specific FcRS are not limited to this are sufficiently well characterized and for which impurities/ section, however, and encompasses all discussions and appli contaminants have been removed, that the test material is cations of FcRS in general as specified in throughout this comparable throughout development, and GLP compliance. application. antibodies of the present disclosure may confer The pharmacokinetics (PK) of the antibodies of the inven Superior activity on Fc-containing drugs in Such transgenic 55 tion can be studied in a variety of animal systems, with the models, in particular variants with binding profiles optimized most relevant being non-human primates Such as the cyno for human FcyRIIIa mediated activity may show superior molgus, rhesus monkeys. Single or repeated i.v./s.c. admin activity in transgenic CD16 mice. Similar improvements in istrations over a dose range of 6000-fold (0.05-300 mg/kg) efficacy in mice transgenic for the other human Fc receptors, can be evaluated for the half-life (days to weeks) using plasma e.g. FcyRIIa, FcyRI, etc., may be observed for antibodies with 60 concentration and clearance as well as Volume of distribution binding profiles optimized for the respective receptors. Mice at a steady State and level of systemic absorbance can be transgenic for multiple human receptors would show measured. Examples of Such parameters of measurement improved activity for antibodies with binding profiles opti generally include maximum observed plasma concentration mized for the corresponding multiple receptors, for example (Cmax), the time to reach Cmax (Tmax), the area under the as outlined in FIG. 5. 65 plasma concentration-time curve from time 0 to infinity Because of the difficulties and ambiguities associated with AUC(0-inf and apparent elimination half-life (T/2). Addi using animal models to characterize the potential efficacy of tional measured parameters could include compartmental US 9,040,042 B2 49 50 analysis of concentration-time data obtained following i.v. comprises treatment of the disease. Those “in need of treat administration and bioavailability. Examples of pharmaco ment' include mammals already having the disease or disor logical/toxicological studies using cynomolgus have been der, as well as those prone to having the disease or disorder, established for Rituxan and Zevalin in which monoclonal including those in which the disease or disorder is to be antibodies to CD20 are cross-reactive. Biodistribution, prevented. dosimetry (for radiolabled antibodies), and PK studies can In one embodiment, an antibody of the present disclosure is also be done in rodent models. Such studies would evaluate administered to a patient having a disease involving inappro tolerance at all doses administered, toxicity to local tissues, priate expression of a protein or other molecule. Within the preferential localization to rodent Xenograft animal models, Scope of the present disclosure this is meant to include dis depletion of target cells (e.g. CD20 positive cells). 10 eases and disorders characterized by aberrant proteins, due The antibodies of the present disclosure may confer supe for example to alterations in the amount of a protein present, rior pharmacokinetics on Fc-containing therapeutics in ani protein localization, posttranslational modification, confor mal systems or in humans. For example, increased binding to mational state, the presence of a mutant or pathogen protein, FcRn may increase the half-life and exposure of the Fc etc. Similarly, the disease or disorder may be characterized by containing drug. Alternatively, decreased binding to FcRn 15 alterations molecules including but not limited to polysaccha may decrease the half-life and exposure of the Fc-containing rides and gangliosides. An overabundance may be due to any drug in cases where reduced exposure is favorable Such as cause, including but not limited to overexpression at the when such drug has side-effects. molecular level, prolonged or accumulated appearance at the It is known in the art that the array of Fc receptors is site of action, or increased activity of a protein relative to differentially expressed on various immune cell types, as well normal. Included within this definition are diseases and dis as in different tissues. Differential tissue distribution of Fc orders characterized by a reduction of a protein. This reduc receptors may ultimately have an impact on the pharmacody tion may be due to any cause, including but not limited to namic (PD) and pharmacokinetic (PK) properties of antibod reduced expression at the molecular level, shortened or ies of the present disclosure. Because antibodies of the pre reduced appearance at the site of action, mutant forms of a sentation have varying affinities for the array of Fc receptors, 25 protein, or decreased activity of a protein relative to normal. further screening of the polypeptides for PD and/or PK prop Such an overabundance or reduction of a protein can be erties may be extremely useful for defining the optimal bal measured relative to normal expression, appearance, or activ ance of PD. PK, and therapeutic efficacy conferred by each ity of a protein, and said measurement may play an important candidate polypeptide. role in the development and/or clinical testing of the antibod Pharmacodynamic studies may include, but are not limited 30 ies of the present disclosure. to, targeting specific tumor cells or blocking signaling mecha By "cancer and "cancerous' herein refer to or describe the nisms, measuring depletion of target antigen expressing cells physiological condition in mammals that is typically charac or signals, etc. The antibodies of the present disclosure may terized by unregulated cell growth. Examples of cancer target particular effector cell populations and thereby direct include but are not limited to carcinoma, lymphoma, blas Fc-containing drugs to recruit certain activities to improve 35 toma, sarcoma (including liposarcoma), neuroendocrine potency or to increase penetration into a particularly favor tumors, mesothelioma, Schwanoma, meningioma, adenocar able physiological compartment. For example, neutrophil cinoma, melanoma, and leukemia or lymphoid malignancies. activity and localization can be targeted by an antibody that More particular examples of Such cancers include hema preferentially targets FcyRIIIb. Such pharmacodynamic tologic malignancies, such as non-Hodgkin’s lymphomas effects may be demonstrated in animal models or in humans. 40 (NHL). NHL cancers include but are not limited to Burkitt's Clinical Use lymphoma (BL), Small lymphocytic lymphoma/chronic lym The antibodies of the present disclosure may be used for phocytic leukemia (SLL/CLL), mantle cell lymphoma various therapeutic purposes. As will be appreciated by those (MCL), follicular lymphoma (FL), diffuse large B-cell lym in the art, the antibodies of the present disclosure may be used phoma (DLCL), marginal Zone lymphoma (MZL), hairy cell for any therapeutic purpose that uses antibodies and the like. 45 leukemia (HCL) and lymphoplasmacytic leukemia (LPL), In a preferred embodiment, the antibodies are administered to extranodal marginal Zone B-cell lymphoma of mucosa-asso a patient to treat disorders including but not limited to cancer, ciated lymphoid tissue (MALT), nodal marginal Zone B cell autoimmune and inflammatory diseases, and infectious dis lymphoma, mediastinal large cell lymphoma, intravascular CaSCS. large cell lymphoma, primary effusion lymphoma, precursor A “patient' for the purposes of the present disclosure 50 B-lymphoblastic leukemia/lymphoma, precursor T- and NK includes both humans and other animals, preferably mam cells lymphoma (precursor T lymphoblastic lymphoma, blas mals and most preferably humans. Thus the antibodies of the tic NK cell lymphoma), tumors of the mature T and NK cells, present disclosure have both human therapy and Veterinary including peripheral T-cell lymphoma and leukemia (PTL). applications. The term “treatment” or “treating in the present adult T-cell leukemia/T-cell lymphomas and large granular disclosure is meant to include therapeutic treatment, as well 55 lymphocytic leukemia, T-cell chronic lymphocytic leukemia/ as prophylactic, or Suppressive measures for a disease or prolymphocytic leukemia, T-cell large granular lymphocytic disorder. Thus, for example, successful administration of an leukemia, aggressive NK-cell leukemia, extranodal T/NK antibody prior to onset of the disease results in treatment of cell lymphoma, enteropathy-type T-cell lymphoma, the disease. As another example, Successful administration of hepatosplenic T-cell lymphoma, anaplastic large cell lym an optimized antibody after clinical manifestation of the dis 60 phoma (ALCL), angiocetric and angioimmunoblastic T-cell ease to combat the symptoms of the disease comprises treat lymphoma, mycosis fungoides/Sezary syndrome, and cuta ment of the disease. “Treatment” and “treating also encom neous T-cell lymphoma (CTCL). Other cancers that may be passes administration of an optimized antibody after the treatable by the antibodies disclosed herein include but are appearance of the disease in order to eradicate the disease. not limited to Hodgkin’s lymphoma, tumors of lymphocyte Successful administration of an agent after onset and after 65 precursor cells, including B-cell acute lymphoblastic leuke clinical symptoms have developed, with possible abatement mia/lymphoma (B-ALL), and T-cell acute lymphoblastic leu of clinical symptoms and perhaps amelioration of the disease, kemia/lymphoma (T-ALL), thymoma, histo US 9,040,042 B2 51 52 cytosis, multiple myeloma, myeloid neoplasias Such as acute nia purpura (ITP), IgA neuropathy, IgM polyneuropathies, myelogenous leukemias (AML), including AML with matu immune mediated thrombocytopenia, juvenile arthritis, ration, AML without differentiation, acute promyelocytic Kawasaki's disease, lichen plantus, lupus erthematosis, leukemia, acute myelomonocytic leukemia, and acute mono Meniere's disease, mixed connective tissue disease, multiple cytic leukemias, myelodysplastic syndromes, and chronic Sclerosis (MS), type 1 diabetes mellitus, myasthenia gravis, myeloproliferative disorders (MDS), including chronic pemphigus Vulgaris, pernicious anemia, polyarteritis nodosa, myelogenous leukemia (CML). Other cancers that may be polychrondritis, polyglandular syndromes, polymyalgia treatable by the antibodies of the invention include but are not rheumatica, polymyositis and dermatomyositis, primary limited to tumors of the central nervous system Such as agammaglobinulinemia, primary biliary cirrhosis, psoriasis, glioma, glioblastoma, neuroblastoma, astrocytoma, medullo 10 psoriatic arthritis, Reynauld's phenomenon, Reiter's Syn blastoma, ependymoma, and retinoblastoma; Solid tumors of drome, rheumatoid arthritis (RA), sarcoidosis, Scleroderma, the head and neck (eg. nasopharyngeal cancer, salivary gland Sjogren's syndrome, Solid organ transplant rejection, stiff carcinoma, and esophageal cancer), lung (eg. Small-cell lung man syndrome, systemic lupus erythematosus (SLE), taka cancer, non-Small cell lung cancer, adenocarcinoma of the yasu arteritis, temporal arteristis/giant cell arteritis, throm lung and squamous carcinoma of the lung), digestive system 15 botic thrombocytopenia purpura, ulcerative colitis, uveitis, (eg. gastric or stomach cancer including gastrointestinal can vasculitides Such as dermatitis herpetiformis vasculitis, viti cer, cancer of the bile duct or biliary tract, colon cancer, rectal ligo, and Wegner's granulomatosis. cancer, colorectal cancer, and anal carcinoma), reproductive By “inflammatory disorders' herein include acute respira system (eg. testicular, penile, or prostate cancer, uterine, vagi tory distress syndrome (ARDS), acute septic arthritis, adju nal, Vulval, cervical, ovarian, and endometrial cancer), skin vant arthritis, juvenile idiopathic arthritis, allergic encepha (eg. melanoma, basal cell carcinoma, squamous cell cancer, lomyelitis, allergic rhinitis, allergic vasculitis, allergy, actinic keratosis), liver (eg. liver cancer, hepatic carcinoma, asthma, atherosclerosis, chronic inflammation due to chronic hepatocellular cancer, and hepatoma), bone (eg. osteoclas bacterial or viral infectionis, chronic obstructive pulmonary toma, and osteolytic bone cancers) additional tissues and disease (COPD), coronary artery disease, encephalitis, organs (eg. pancreatic cancer, bladder cancer, kidney or renal 25 inflammatory bowel disease, inflammatory osteolysis, cancer, thyroid cancer, breast cancer, cancer of the perito inflammation associated with acute and delayed hypersensi neum, and Kaposi's sarcoma), and tumors of the vascular tivity reactions, inflammation associated with tumors, periph System (eg. angiosarcoma and hemangiopericytoma). eral nerve injury or demyelinating diseases, inflammation Preferred indications that can be treated by the anti associated with tissue trauma Such as burns and ischemia, HM1.24 antibodies include, but are not limited to, multiple 30 inflammation due to meningitis, multiple organ injury Syn myeloma (MM), mantle cell myeloma (MCM), myelodys drome, pulmonary fibrosis, sepsis and septic shock, Stevens plastic syndrome with del5q (MDS), and autoimmune indi Johnson syndrome, undifferentiated arthropy, and undiffer cations. Preferred autoimmune indications that can be treated entiated spondyloarthropathy. by anti-HM1.24 antibodies include but are not limited to By “infectious diseases herein include diseases caused by rheumatoid arthritis (RA), systemic lupus erythematosus 35 pathogens such as , bacteria, fungi, protozoa, and (SLE or lupus), multiple Sclerosis, Sjogren's syndrome, and parasites. Infectious diseases may be caused by viruses idiopathic thrombocytopenia purpura (ITP). including adenovirus, cytomegalovirus, dengue, Epstein Autoimmunity results from a breakdown of self-tolerance Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes sim involving humoral and/or cell-mediated immune mecha plex type I, herpes simplex type II, human immunodeficiency nisms in. Among of the consequences of failure in central 40 virus, (HIV), human papilloma virus (HPV), influenza, and/or peripheral tolerance, are Survival and activation of measles, mumps, papova virus, polio, respiratory syncytial self-reactive B cells and T cells. Several autoimmune diseases virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, are defined by excessive activation of both B and/or T lym Smallpox, viral meningitis, and the like. Infections diseases phocytes. Activation of these cells requires in cooperation, may also be caused by bacteria including Bacillus antracis, antigen engagement and co-stimulatory signals from inter 45 Borrelia burgdorferi, Campylobacter jejuni, Chlamydia acting lymphocytes. Antibody-mediated depletion, inhibi chomatis, Clostridium botulinum, Clostridium tetani, tion, anti-proliferation, and/or blockade of B cells are thera Diptheria, E. coli, Legionella, Helicobacterpylori, Mycobac peutic approaches for the treatment of autoimmune disease. terium rickettsia, Mycoplasma mesisseria, Pertussis, By “autoimmune diseases herein include allogenic islet Pseudomonas aeruginosa, S. pneumonia, Streptococcus, Sta graft rejection, alopecia greata, ankylosing spondylitis, 50 phylococcus, Vibria cholerae, Yersinia pestis, and the like. antiphospholipid syndrome, autoimmune Addison's disease, Infectious diseases may also be caused by fungi such as antineutrophil cytoplasmic (ANCA), autoim Aspergillus filmigatus, Blastomyces dermatitidis, Candida mune diseases of the adrenal gland, autoimmune hemolytic albicans, Coccidioides immitis, Cryptococcus neoformans, anemia, autoimmune hepatitis, autoimmune myocarditis, Histoplasma capsulatum, Penicillium marneffei, and the like. autoimmune neutropenia, autoimmune oophoritis and orchi 55 Infectious diseases may also be caused by protozoa and para tis, autoimmune thrombocytopenia, autoimmune urticaria, sites Such as chlamydia, kokzidioa, leishmania, malaria, rick Behcet’s disease, bullous pemphigoid, cardiomyopathy, ettsia, trypanosoma, and the like. Castleman’s syndrome, celiac spruce-dermatitis, chronic Furthermore, antibodies of the present disclosure may be fatigue immune disfunction syndrome, chronic inflammatory used to prevent or treat additional conditions including but demyelinating polyneuropathy, Churg-Strauss syndrome, 60 not limited to heart conditions such as congestive heart failure cicatrical pemphigoid, CREST syndrome, cold agglutinin (CHF), myocarditis and other conditions of the myocardium; disease, Crohn's disease, dermatomyositis, discoid lupus, skin conditions such as rosacea, acne, and eczema, bone and essential mixed cryoglobulinemia, factor VIII deficiency, tooth conditions such as bone loss, osteoporosis, Paget’s dis fibromyalgia-fibromyositis, glomerulonephritis, Grave's dis ease, Langerhans cell histiocytosis, periodontal disease, dis ease, Guillain-Barre, Goodpasture's syndrome, graft-Versus 65 use osteopenia, osteomalacia, monostotic fibrous dysplasia, host disease (GVHD), Hashimoto's thyroiditis, hemophilia polyostotic fibrous dysplasia, bone metastasis, bone pain A, idiopathic pulmonary fibrosis, idiopathic thrombocytope management, humoral malignant hypercalcemia, periodontal US 9,040,042 B2 53 54 reconstruction, spinal cord injury, and bone fractures; meta patients are selected for inclusion in a clinical trial or for bolic conditions such as Gaucher's disease; endocrine condi receipt of therapy post-approval based on their polymorphism tions such as Cushing's syndrome; and neurological condi genotype, where such therapy contains an antibody engi tions. neered to be specifically efficacious for Such population, or A number of the receptors that may interact with the anti alternatively where Such therapy contains an antibody that bodies of the present disclosure are polymorphic in the does not show differential activity to the different forms of the human population. For a given patient or population of polymorphism. patients, the efficacy of the antibodies of the present disclo Included in the present disclosure are diagnostic tests to sure may be affected by the presence or absence of specific identify patients who are likely to show a favorable clinical polymorphisms in proteins. For example, FcyRIIIA is poly 10 response to an antibody of the present disclosure, or who are morphic at position 158, which is commonly either V (high likely to exhibit a significantly better response when treated affinity) or F (low affinity). Patients with the V/V homozy with an antibody of the present disclosure versus one or more gous genotype are observed to have a better clinical response currently used antibody therapeutics. Any of a number of to treatment with the anti-CD20 antibody Rituxan R (ritux methods for determining FcyR polymorphisms in humans imab), likely because these patients mount a stronger NK 15 known in the art may be used. response (Dall'Ozzo et al. (2004) Cancer Res. 64:4664-9, Furthermore, the present disclosure comprises prognostic incorporated entirely by reference). Additional polymor tests performed on clinical samples Such as blood and tissue phisms include but are not limited to FcyRIIAR131 or H131, samples. Such tests may assay for effector function activity, and Such polymorphisms are known to either increase or including but not limited to ADCC, CDC, phagocytosis, and decrease Fc binding and Subsequent biological activity, opSonization, or for killing, regardless of mechanism, of can depending on the polymorphism. antibodies of the present cerous or otherwise pathogenic cells. In a preferred embodi disclosure may bind preferentially to a particular polymor ment, ADCC assays, such as those described previously, are phic form of a receptor, for example FcyRIIIA 158 V, or to used to predict, for a specific patient, the efficacy of a given bind with equivalent affinity to all of the polymorphisms at a antibody of the present disclosure. Such information may be particular position in the receptor, for example both the 158V 25 used to identify patients for inclusion or exclusion in clinical and 158F polymorphisms of FcyRIIIA. In a preferred trials, or to inform decisions regarding appropriate dosages embodiment, antibodies of the present disclosure may have and treatment regemins. Such information may also be used equivalent binding to polymorphisms may be used in an anti to select a drug that contains a particular antibody that shows body to eliminate the differential efficacy seen in patients Superior activity in Such assay. with different polymorphisms. Such a property may give 30 Formulation greater consistency in therapeutic response and reduce non Pharmaceutical compositions are contemplated wherein responding patient populations. Such variant Fc with identi an antibody of the present disclosure and one or more thera cal binding to receptor polymorphisms may have increased peutically active agents are formulated. Formulations of the biological activity, such as ADCC, CDC or circulating half antibodies of the present disclosure are prepared for storage life, or alternatively decreased activity, via modulation of the 35 by mixing said antibody having the desired degree of purity binding to the relevant Fc receptors. In a preferred embodi with optional pharmaceutically acceptable carriers, excipi ment, antibodies of the present disclosure may bind with ents or stabilizers (Remington’s Pharmaceutical Sciences higher or lower affinity to one of the polymorphisms of a 16th edition, Osol, A. Ed., 1980, incorporated entirely by receptor, either accentuating the existing difference in bind reference), in the form of lyophilized formulations oraqueous ing or reversing the difference. Such a property may allow 40 Solutions. Acceptable carriers, excipients, or stabilizers are creation of therapeutics particularly tailored for efficacy with nontoxic to recipients at the dosages and concentrations a patient population possessing such polymorphism. For employed, and include buffers such as phosphate, citrate, example, a patient population possessing a polymorphism acetate, and other organic acids; antioxidants including ascor with a higher affinity for an inhibitory receptor such as bic acid and methionine; preservatives (such as octade FcyRIIB could receive a drug containing an antibody with 45 cyldimethylbenzyl ammonium chloride; hexamethonium reduced binding to Such polymorphic form of the receptor, chloride; benzalkonium chloride, benzethonium chloride; creating a more efficacious drug. phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl In a preferred embodiment, patients are screened for one or or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pen more polymorphisms in order to predict the efficacy of the tanol; and m-cresol); low molecular weight (less than about antibodies of the present disclosure. This information may be 50 10 residues) polypeptides; proteins, such as serum albumin, used, for example, to select patients to include or exclude gelatin, or immunoglobulins; hydrophilic polymers such as from clinical trials or, post-approval, to provide guidance to polyvinylpyrrolidone; amino acids such as glycine, physicians and patients regarding appropriate dosages and glutamine, asparagine, histidine, arginine, or lysine; treatment options. For example, in patients that are homozy monosaccharides, disaccharides, and other carbohydrates gous or heterozygous for FcyRIIIA 158F antibody drugs such 55 including glucose, mannose, or dextrins; chelating agents as the anti-CD20 mAb, Rituximab are minimally effective Such as EDTA; Sugars such as Sucrose, mannitol, trehalose or (Carton 2002 Blood 99: 754-758; Weng 2003 J. Clin. Oncol. Sorbitol; Sweeteners and other flavoring agents; fillers such as 21:3940-3947, both incorporated entirely by reference); such microcrystalline cellulose, lactose, corn and other starches; patients may show a much better clinical response to the binding agents; additives; coloring agents; salt-forming antibodies of the present disclosure. In one embodiment, 60 counter-ions such as Sodium; metal complexes (e.g. Zn-pro patients are selected for inclusion in clinical trials for an tein complexes); and/or non-ionic Surfactants such as antibody of the present disclosure if their genotype indicates TWEENTM, PLURONICSTM or polyethylene glycol (PEG). that they are likely to respond significantly better to an anti In a preferred embodiment, the pharmaceutical composition body of the present disclosure as compared to one or more that comprises the antibody of the present disclosure may be currently used antibody therapeutics. In another embodiment, 65 in a water-soluble form, such as being present as pharmaceu appropriate dosages and treatment regimens are determined tically acceptable salts, which is meant to include both acid using Such genotype information. In another embodiment, and base addition salts. “Pharmaceutically acceptable acid US 9,040,042 B2 55 56 addition salt” refers to those salts that retain the biological acetate, degradable lactic acid-glycolic acid copolymers such effectiveness of the free bases and that are not biologically or as the Lupron DepotR) (which are injectable microspheres otherwise undesirable, formed with inorganic acids such as composed of lactic acid-glycolic acid copolymer and leupro hydrochloric acid, hydrobromic acid, Sulfuric acid, nitric lide acetate), poly-D-(-)-3-hydroxybutyric acid, and Pro acid, phosphoric acid and the like, and organic acids such as Lease R (commercially available from Alkermes), which is a acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic microsphere-based delivery system composed of the desired acid, maleic acid, malonic acid, Succinic acid, fumaric acid, bioactive molecule incorporated into a matrix of poly-DL tartaric acid, citric acid, benzoic acid, cinnamic acid, man lactide-co-glycolide (PLG). delic acid, methanesulfonic acid, ethanesulfonic acid, p-tolu Administration enesulfonic acid, salicylic acid and the like. “Pharmaceuti 10 Administration of the pharmaceutical composition com cally acceptable base addition salts' include those derived prising an antibody of the present disclosure, preferably in the from inorganic bases Such as Sodium, potassium, lithium, form of a sterile aqueous solution, may be done in a variety of ammonium, calcium, magnesium, iron, Zinc, copper, manga ways, including, but not limited to orally, Subcutaneously, nese, aluminum salts and the like. Particularly preferred are intravenously, intranasally, intraotically, transdermally, topi the ammonium, potassium, Sodium, calcium, and magnesium 15 salts. Salts derived from pharmaceutically acceptable organic cally (e.g., gels, salves, lotions, creams, etc.), intraperito non-toxic bases include salts of primary, secondary, and ter neally, intramuscularly, intrapulmonary, vaginally, parenter tiary amines, Substituted amines including naturally occur ally, rectally, or intraocularly. In some instances, for example ring Substituted amines, cyclic amines and basic ion exchange for the treatment of wounds, inflammation, etc., the antibody resins, such as isopropylamine, trimethylamine, diethy may be directly applied as a solution or spray. As is known in lamine, triethylamine, tripropylamine, and ethanolamine. the art, the pharmaceutical composition may be formulated The formulations to be used for in vivo administration are accordingly depending upon the manner of introduction. preferably sterile. This is readily accomplished by filtration Subcutaneous administration may be preferable in some through sterile filtration membranes or other methods. circumstances because the patient may self-administer the The antibodies disclosed herein may also be formulated as 25 pharmaceutical composition. Many protein therapeutics are immunoliposomes. A liposome is a small vesicle comprising not sufficiently potent to allow for formulation of a therapeu various types of lipids, phospholipids and/or Surfactant that is tically effective dose in the maximum acceptable volume for useful for delivery of a therapeutic agent to a mammal. Lipo Subcutaneous administration. This problem may be Somes containing the antibody are prepared by methods addressed in part by the use of protein formulations compris known in the art, such as described in Epstein et al., 1985, 30 ing arginine-HCl, histidine, and polysorbate (see WO Proc Natl AcadSci USA, 82:3688; Hwang et al., 1980, Proc Natl AcadSci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. 04.091658, incorporated entirely by reference). Antibodies of Pat. No. 4,544,545; and PCT WO97/38731, all incorporated the present disclosure may be more amenable to Subcutane entirely by reference. Liposomes with enhanced circulation ous administration due to, for example, increased potency, time are disclosed in U.S. Pat. No. 5,013,556, incorporated 35 improved serum half-life, or enhanced solubility. entirely by reference. The components of the liposome are As is known in the art, protein therapeutics are often deliv commonly arranged inabilayerformation, similar to the lipid ered by IV infusion or bolus. The antibodies of the present arrangement of biological membranes. Particularly useful disclosure may also be delivered using Such methods. For liposomes can be generated by the reverse phase evaporation example, administration may Venious be by intravenous infu method with a lipid composition comprising phosphatidyl 40 sion with 0.9% sodium chloride as an infusion vehicle. choline, cholesterol and PEG-derivatized phosphatidyletha Pulmonary delivery may be accomplished using an inhaler nolamine (PEG-PE). Liposomes are extruded through filters or nebulizer and a formulation comprising an aerosolizing of defined pore size to yield liposomes with the desired diam agent. For example, AERX(R) inhalable technology commer eter. A chemotherapeutic agent or other therapeutically active cially available from Aradigm, or InhanceTM pulmonary agentis optionally contained within the liposome (Gabizon et 45 delivery system commercially available from Nektar Thera al., 1989, J National Cancer Inst 81: 1484, incorporated peutics may be used. Antibodies of the present disclosure may entirely by reference). be more amenable to intrapulmonary delivery. FcRn is The antibody and other therapeutically active agents may present in the lung, and may promote transport from the lung also be entrapped in microcapsules prepared by methods to the bloodstream (e.g. Syntonix WO 04004798, Bitontiet including but not limited to coacervation techniques, interfa 50 al. (2004) Proc. Nat. Acad. Sci. 101:9763-8, both incorpo cial polymerization (for example using hydroxymethylcellu rated entirely by reference). Accordingly, antibodies that bind lose or gelatin-microcapsules, or poly-(methylmethacylate) FcRn more effectively in the lung or that are released more microcapsules), colloidal drug delivery systems (for efficiently in the bloodstream may have improved bioavail example, liposomes, albumin microspheres, microemul ability following intrapulmonary administration. Antibodies sions, nano-particles and nanocapsules), and macroemul 55 of the present disclosure may also be more amenable to intra sions. Such techniques are disclosed in Remington’s Pharma pulmonary administration due to, for example, improved ceutical Sciences 16th edition, Osol, A. Ed., 1980, solubility or altered isoelectric point. incorporated entirely by reference. Sustained-release prepa Furthermore, antibodies of the present disclosure may be rations may be prepared. Suitable examples of Sustained more amenable to oral delivery due to, for example, improved release preparations include semipermeable matrices of solid 60 stability at gastric pH and increased resistance to proteolysis. hydrophobic polymer, which matrices are in the form of Furthermore, FcRn appears to be expressed in the intestinal shaped articles, e.g. films, or microcapsules. Examples of epithelia of adults (Dickinson et al. (1999) J. Clin. Invest. Sustained-release matrices include polyesters, hydrogels (for 104:903-11, incorporated entirely by reference), so antibod example poly(2-hydroxyethyl-methacrylate), or poly(vinyla ies of the present disclosure with improved FcRn interaction lcohol)), polylactides (U.S. Pat. No. 3,773.919, incorporated 65 profiles may show enhanced bioavailability following oral entirely by reference), copolymers of L-glutamic acid and administration. FcRn mediated transport of antibodies may gamma ethyl-L-glutamate, non-degradable ethylene-vinyl also occur at other mucus membranes such as those in the US 9,040,042 B2 57 58 gastrointestinal, respiratory, and genital tracts (Yoshida et al. sequence of administration one or more times. The number of (2004) Immunity 20:769-83, incorporated entirely by refer cycles is typically from 2-10. Cycling therapy may reduce the ence). development of resistance to one or more agents, may mini In addition, any of a number of delivery systems are known mize side effects, or may improve treatment efficacy. in the art and may be used to administer the antibodies of the Combination Therapies present disclosure. Examples include, but are not limited to, The antibodies disclosed herein may be administered con encapsulation in liposomes, microparticles, microspheres comitantly with one or more other therapeutic regimens or (eg. PLA/PGA microspheres), and the like. Alternatively, an agents. The additional therapeutic regimes or agents may be implant of a porous, non-porous, or gelatinous material, used to improve the efficacy or safety of the antibody. Also, including membranes or fibers, may be used. Sustained 10 release systems may comprise a polymeric material or matrix the additional therapeutic regimes or agents may be used to Such as polyesters, hydrogels, poly(vinylalcohol), polylac treat the same disease or a comorbidity rather than to alter the tides, copolymers of L-glutamic acid and ethyl-L-glutamate, action of the antibody. For example, an antibody of the ethylene-Vinyl acetate, lactic acid-glycolic acid copolymers present disclosure may be administered to the patient along such as the Lupron Depot(R), and poly-D-(-)-3-hydroxybu 15 with chemotherapy, radiation therapy, or both chemotherapy tyric acid. It is also possible to administer a nucleic acid and radiation therapy. The antibody of the present disclosure encoding the antibodies, for example by retroviral infection, may be administered in combination with one or more other direct injection, or coating with lipids, cell Surface receptors, prophylactic or therapeutic agents, including but not limited or other transfection agents. In all cases, controlled release to cytotoxic agents, chemotherapeutic agents, cytokines, systems may be used to release the antibody at or close to the growth inhibitory agents, anti-hormonal agents, kinase desired location of action. inhibitors, anti-angiogenic agents, cardioprotectants, immu Dosing nostimulatory agents, immunosuppressive agents, agents that The dosing amounts and frequencies of administration are, promote proliferation of hematological cells, angiogenesis in a preferred embodiment, selected to be therapeutically or inhibitors, protein tyrosine kinase (PTK) inhibitors, addi prophylactically effective. As is known in the art, adjustments 25 tional antibodies, FcyRIIb or other Fc receptor inhibitors, or for protein degradation, Systemic versus localized delivery, other therapeutic agents. and rate of new protease synthesis, as well as the age, body The terms “in combination with and “co-administration' weight, general health, sex, diet, time of administration, drug are not limited to the administration of said prophylactic or interaction and the severity of the condition may be necessary, therapeutic agents at exactly the same time. Instead, it is and will be ascertainable with routine experimentation by 30 meant that the antibody of the present disclosure and the other those skilled in the art. agent or agents are administered in a sequence and within a The concentration of the therapeutically active antibody in time interval such that they may act together to provide a the formulation may vary from about 0.1 to 100 weight%. In benefit that is increased versus treatment with only either the a preferred embodiment, the concentration of the antibody is antibody of the present disclosure or the other agentoragents. in the range of 0.003 to 1.0 molar. In order to treat a patient, 35 It is preferred that the antibody and the other agent or agents a therapeutically effective dose of the antibody of the present act additively, and especially preferred that they act synergis disclosure may be administered. By “therapeutically effec tically. Such molecules are Suitably present in combination in tive dose' herein is meant a dose that produces the effects for amounts that are effective for the purpose intended. The which it is administered. The exact dose will depend on the skilled medical practitioner can determine empirically, or by purpose of the treatment, and will be ascertainable by one 40 considering the pharmacokinetics and modes of action of the skilled in the art using known techniques. Dosages may range agents, the appropriate dose or doses of each therapeutic from 0.0001 to 100 mg/kg of body weight or greater, for agent, as well as the appropriate timings and methods of example 0.1. 1, 10, or 50 mg/kg of body weight, with 1 to 10 administration. mg/kg being preferred. In one embodiment, the anti-HM1.24 antibody is admin In some embodiments, only a single dose of the antibody is 45 istered with one or more additional molecules comprising used. In other embodiments, multiple doses of the antibody antibodies or Fc. The antibodies may be co-administered with are administered. The elapsed time between administrations one or more other antibodies that have efficacy in treating the may be less than 1 hour, about 1 hour, about 1-2 hours, about same disease or an additional comorbidity; for example two 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, antibodies may be administered that recognize two antigens about 24 hours, about 48 hours, about 2-4 days, about 4-6 50 that are overexpressed in a given type of cancer, or two anti days, about 1 week, about 2 weeks, or more than 2 weeks. gens that mediate pathogenesis of an autoimmune or infec In other embodiments the antibodies of the present disclo tious disease. An example of Such an antibody is an anti Sure are administered in metronomic dosing regimes, either CD40 antibody. by continuous infusion or frequent administration without In certain variations, an anti-HM1.24 antibody is adminis extended rest periods. Such metronomic administration may 55 tered in combination with one or more compounds selected involve dosing at constant intervals without rest periods. from VelcadeR (bortezomib) (Millenium Pharmaceuticals), Typically such regimens encompass chronic low-dose or con Revlimid(R) (lenalidomide) (Chelgene Corporation), dexam tinuous infusion for an extended period of time, for example ethasone and/or other multiple myeloma treatments. 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more. Velcade(R) is a proteasome inhibitor used to treat multiple The use of lower doses may minimize side effects and the 60 myeloma. It is believed that velcade acts on myeloma cells to need for rest periods. cause cell death, and or acts indirectly to inhibit myeloma cell In certain embodiments the antibody of the present disclo growth and Survival by acting on the bone microenvironment. Sure and one or more other prophylactic ortherapeutic agents Without being limited to a specific theory or mode of action, are cyclically administered to the patient. Cycling therapy Velcade thus disrupts normal cellular processes, resulting in involves administration of a first agent at one time, a second 65 proteasome inhibition that promotes apoptosis. In various agent at a second time, optionally additional agents at addi circumstances, the anti-HM1.24 antibodies and velcade may tional times, optionally a rest period, and then repeating this act synergistically. US 9,040,042 B2 59 60 Revlimid R is an immunomodulatory agent. It is thought and RencareXR); anti-GD2 ganglioside antibodies such as that Revlimid(R) affects multiple pathways in myeloma path EMD-273063 and TriGem; anti-GD3 ganglioside antibodies ways, thereby inducing apoptosis, inhibiting myeloma cell such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIa growth, inhibiting vasculare entdothelial growth factor antibodies Such as ReoPro; anti-heparinase antibodies; anti (VEGF) thereby inhibiting angiogenesis, and reducing adhe Her2/ErbB2 antibodies such as Herceptin R (trastuzumab), sion of myeloma cells to bone marrow to stromal cells. MDX-210, and pertuzumab; anti-HLA antibodies such as Anti-HM1.24 antibodies can be used in combination with OncolymR), Smart 1D10; anti-HM1.24 antibodies; anti dexamethasone. Dexamethasone is a synthetic glucocorti ICAM antibodies such as ICM3; anti-IgA receptor antibod coid steroid hormone that acts as an anti-inflammatory and ies; anti-IGF-1 antibodies such as CP-751871 and EM-164: immunosuppressant. When administered to cancer patients, 10 anti-IGF-1 Rantibodies such as IMC-A12; anti-IL-6 antibod dexamethasone can counteract side effects of cancer therapy. ies such as CNTO-328 and elsilimomab; anti-IL-15 antibod Dexamethasone can also be given alone or together with other ies such as HuMaxTM-IL15; anti-KDR antibodies; anti-lami anticancer agents, including thalidomide, adriamycin or Vin nin 5 antibodies; anti-Lewis Y antigen antibodies such as cristine. Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1 Additional combination therapies can include antibodies 15 antibodies such as BravaRex and TriAb; anti-NCAM anti used to treat multiple myeloma, including anti-CD40 mono bodies such as ERIC-1 and ICRT anti-PEM antigen antibod clonal antibodies as disclosed in Tai et al. Cancer Res. 2005; ies such as Theragyn and Therex; anti-PSA antibodies; anti 65(24): 11712-20. PSCA antibodies such as IG8; anti-Ptkantibodies; anti-PTN Other anti-cancer antibodies can be co-administered with antibodies; anti-RANKL antibodies such as AMG-162; anti the anti-HM1.24 antibodies disclosed herein, including but RLIP76 antibodies; anti-SK-1 antigen antibodies such as not limited to anti-17-1A cell surface antigen antibodies such Monopharm C: anti-STEAPantibodies; anti-TAG72 antibod as PanorexTM (edrecolomab); anti-4-1BB antibodies; anti ies such as CC49-SCA and MDX-220; anti-TGF-B antibod 4Dc antibodies; anti-A33 antibodies such as A33 and CDP ies such as CAT152; anti-TNF-C. antibodies such as 833; anti-C4B1 integrin antibodies such as natalizumab; anti CDP571, CDP870, D2E7, Humira(R) (adalimumab), and C.437 integrin antibodies such as LDP-02; anti-CVB1 integrin 25 Remicade(R) (infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies such as F-200, M-200, and SJ-749; anti-CVB3 antibodies; anti-VE-cadherin-2 antibodies; and anti-VLA-4 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, antibodies such as AntegrenTM. Furthermore, anti-idiotype and VitaxinTM; anti-complement factor 5 (C5) antibodies antibodies including but not limited to the GD3 epitope anti such as 5G 1.1; anti-CA125 antibodies such as OvaRexR body BEC2 and the gp72 epitope antibody 105AD7, may be (oregovomab); anti-CD3 antibodies such as Nuvion(R) (visili 30 used. In addition, bispecific antibodies including but not lim Zumab) and Rexomab; anti-CD4 antibodies such as IDEC ited to the anti-CD3/CD20 antibody Bi20 may be used. 151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Examples of antibodies that may be co-administered to Oncolysin B and Oncolysin CD6; anti-CD7 antibodies such treat autoimmune or inflammatory disease, transplant rejec as HB2; anti-HM1.24 antibodies such as B43, MT-103, and tion, GVHD, and the like include, but are not limited to, Oncolysin B; anti-CD20 antibodies such as 2H7, 2H7.V16, 35 anti-C4f7 integrin antibodies such as LDP-02, anti-beta2 2H7.V114, 2H7.v115, Bexxar R (tositumomab, I-131 labeled integrin antibodies such as LDP-01, anti-complement (C5) anti-CD20), Rituxan R (rituximab), and Zevalin R. (Ibritumo antibodies such as 5G1.1, anti-CD2 antibodies such as BTI mabtiuxetan, Y-90 labeled anti-CD20); anti-CD22 antibodies 322, MEDI-507, anti-CD3 antibodies such as OKT3, such as Lymphocide.M (epratuzumab, Y-90 labeled anti SMART anti-CD3, anti-CD4 antibodies such as IDEC-151, CD22); anti-CD23 antibodies such as IDEC-152; anti-CD25 40 MDX-CD4, OKT4A, anti-CD11a antibodies, anti-CD14 antibodies such as basiliximab and Zenapax(R) (daclizumab); antibodies such as IC 14, anti-CD18 antibodies, anti-CD23 anti-CD30 antibodies such as AC10, MDX-060, and SGN antibodies such as IDEC 152, anti-CD25 antibodies such as 30; anti-CD33 antibodies such as Mylotarg(R) (gemtuzumab Zenapax, anti-CD40L antibodies such as 5c8, Antova, IDEC ozogamicin). Oncolysin M, and Smart M195; anti-CD38 131, anti-CD64 antibodies such as MDX-33, anti-CD80 anti antibodies; anti-CD40 antibodies such as SGN-40 and torali 45 bodies such as IDEC-114, anti-CD147 antibodies such as Zumab; anti-CD40L antibodies such as 5c8, AntovaTM, and ABX-CBL, anti-E-selectin antibodies such as CDP850, anti IDEC-131; anti-CD44 antibodies such as bivatuzumab; anti gplb/IIIa antibodies such as ReoPro/Abcixima, anti CD46 antibodies; anti-CD52 antibodies such as Campath(R) ICAM-3 antibodies such as ICM3, anti-ICE antibodies such (alemtuzumab); anti-CD55 antibodies such as SC-1; anti as VX-740, anti-FcyR1 antibodies such as MDX-33, anti-IgE CD56 antibodies such as huN901-DM1; anti-CD64 antibod 50 antibodies such as rhuMab-E25, anti-IL-4 antibodies such as ies such as MDX-33; anti-CD66eantibodies such as XR-303; SB-240683, anti-IL-5 antibodies such as SB-240563, anti-CD74 antibodies such as IMMU-110; anti-CD80 anti SCH55700, anti-IL-8 antibodies such as ABX-IL8, anti-in bodies such as galiximab and IDEC-114; anti-CD89 antibod terferon gamma antibodies, and anti-TNFa antibodies such as ies such as MDX-214; anti-CD123 antibodies; anti-CD138 CDP571, CDP870, D2E7, Infliximab, MAK-195F, anti antibodies such as B-B4-DM1; anti-CD146 antibodies such 55 VLA-4 antibodies such as Antegren. Examples of other Fc as AA-98; anti-CD148 antibodies; anti-CEA antibodies such containing molecules that may be co-administered to treat as cT84.66, labetuzumab, and PentaceaTM; anti-CTLA-4 autoimmune or inflammatory disease, transplant rejection, antibodies such as MDX-101; anti-CXCR4 antibodies; anti GVHD, and the like include, but are not limited to, the p75 EGFR antibodies such as ABX-EGF, Erbitux(R) (cetuximab), TNF receptor/Fc fusion Enbrel(R) (etanercept) and Regener IMC-C225, and Merck Mab 425; anti-EpCAM antibodies 60 on's IL-1 trap. such as Crucells anti-EpCAM, ING-1, and IS-IL-2; anti Examples of antibodies that may be co-administered to ephrin B2/EphB4 antibodies; anti-Her2 antibodies such as treat infectious diseases include, but are not limited to, anti Herceptin R, MDX-210; anti-FAP (fibroblast activation pro anthrax antibodies such as ABthrax, anti-CMV antibodies tein) antibodies such as sibrotuzumab; anti-ferritin antibodies Such as CytoGam and Sevirumab, anti-cryptosporidium anti such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3 anti 65 bodies such as CryptoGAM, Sporidin-G, anti-helicobacter bodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti antibodies such as Pyloran, anti-hepatitis Bantibodies such as fibrin antibodies; anti-G250 antibodies such as WX-G250 HepeX-B, Nabi-HB, anti-HIV antibodies such as HRG-214, US 9,040,042 B2 61 62 anti-RSV antibodies such as felvizumab, HNK-20, palivi 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxiflu Zumab, RespiGam, and anti-staphylococcus antibodies Such ridine, enocitabine, floXuridine, 5-FU; taxanes, e.g. paclitaxel as Aurexis, Aurograb. BSYX-A110, and SE-Mab. (TAXOL(R), Bristol-Myers Squibb Oncology, Princeton, N.J.) Alternatively, the antibodies of the present disclosure may and docetaxel (TAXOTERE(R), Rhine-Poulenc Rorer, Antony, be co-administered or with one or more other molecules that France); topoisomerase inhibitor RFS 2000; thymidylate syn compete for binding to one or more Fc receptors. For thase inhibitor (such as Tomudex); additional chemothera example, co-administering inhibitors of the inhibitory recep peutics including aceglatone; aldophosphamide glycoside; tor FcyRIIb may result in increased effector function. Simi aminolevulinic acid; amsacrine; bestrabucil; bisantrene; larly, co-administering inhibitors of the activating receptors ediatraxate; defofamine; demecolcine; diaziquone; difluo such as FcyRIIIa may minimize unwanted effector function. 10 romethylornithine (DMFO): elformithine; elliptinium Fc receptor inhibitors include, but are not limited to, Fc mol acetate; etoglucid: gallium nitrate; hydroxyurea; lentinan; ecules that are engineered to act as competitive inhibitors for lonidamine; mitoguaZone; mitoxantrone; mopidamol; nitra binding to FcyRIIb FcyRIIIa, or other Fc receptors, as well as crine; pentostatin: phenamet, pirarubicin; podophyllinic other immunoglobulins and specifically the treatment called acid; 2-ethylhydrazide; procarbazine: PSKR); razoxane: sizo IVIg (intravenous immunoglobulin). In one embodiment, the 15 furan; spirogermanium; tenuaZonic acid; triaziquone; 2.2.2"- inhibitor is administered and allowed to act before the anti trichlorotriethylamine, urethan; vindesine; dacarbazine; body is administered. An alternative way of achieving the mannomustine, mitobronitol; mitolactol; pipobroman, gacy effect of sequential dosing would be to provide an immediate tosine; arabinoside ("Ara-C); cyclophosphamide; thiotepa; release dosage form of the Fc receptor inhibitor and then a chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; sustained release formulation of the antibody of the invention. methotrexate: etoposide (VP-16); ifosfamide: mitomycin C: The immediate release and controlled release formulations mitoxantrone; Vincristine; vinorelbine; navelbine; could be administered separately or be combined into one novantrone; teniposide; daunomycin; aminopterin: Xeloda; unit dosage form. Administration of an FcyRIIb inhibitor may ibandronate; CPT-11; retinoic acid; esperamicins; capecitab also be used to limit unwanted immune responses, for ine. Pharmaceutically acceptable salts, acids or derivatives of example anti-Factor VIII antibody response following Factor 25 any of the above may also be used. VIII administration to hemophiliacs. A chemotherapeutic or other cytotoxic agent may be In one embodiment, the antibodies of the present disclo administered as a prodrug. By "prodrug” as used herein is Sure are administered with a chemotherapeutic agent. By meant a precursor or derivative form of a pharmaceutically “chemotherapeutic agent” as used herein is meant a chemical active Substance that is less cytotoxic to tumor cells compared compound useful in the treatment of cancer. Examples of 30 to the parent drug and is capable of being enzymatically chemotherapeutic agents include but are not limited to alky activated or converted into the more active parent form. See, lating agents such as thiotepa and cyclosphosphamide (CY for example Wilman, 1986, Biochemical Society Transac TOXANTM); alkyl sulfonates such as busulfan, improsulfan tions, 615th Meeting Belfast, 14:375-382; Stella et al., “Pro and piposulfan; androgens such as calusterone, dromo drugs: A Chemical Approach to Targeted Drug Delivery.” stanolone propionate, epitioStanol, mepitioStane, testolac 35 Directed Drug Delivery; and Borchardt et al., (ed.): 247-267, tone; anti-adrenals such as aminoglutethimide, mitotane, Humana Press, 1985, all incorporated entirely by reference. triloStane; anti-androgens Such as flutamide, nilutamide, The prodrugs that may find use with the present disclosure bicalutamide, leuprolide, and goserelin; antibiotics Such as include but are not limited to phosphate-containing prodrugs, aclacinomysins, actinomycin, authramycin, aZaserine, bleo thiophosphate-containing prodrugs, Sulfate-containing pro mycins, cactinomycin, calicheamicin, carabicin, caminomy 40 drugs, peptide-containing prodrugs, D-amino acid-modified cin, carzinophilin, chromomycins, dactinomycin, daunorubi prodrugs, glycosylated prodrugs, beta-lactam-containing cin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, prodrugs, optionally Substituted phenoxyacetamide-contain epirubicin, esorubicin, idarubicin, marcellomycin, mitomy ing prodrugs or optionally Substituted phenylacetamide-con cins, mycophenolic acid, nogalamycin, olivomycins, peplo taining prodrugs, 5-fluorocytosine and other 5-fluorouridine mycin, potfiromycin, puromycin, quelamycin, rodorubicin, 45 prodrugs which can be converted into the more active cyto streptonigrin, Streptozocin, tubercidin, ubenimex, Zinostatin, toxic free drug. Examples of cytotoxic drugs that can be Zorubicin; anti estrogens including for example tamoxifen, derivatized into a prodrug form for use with the antibodies of raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydrox the present disclosure include but are not limited to any of the ytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, aforementioned chemotherapeutic agents. and toremifene (Fareston); anti-metabolites such as methotr 50 A variety of other therapeutic agents may find use for exate and 5-fluorouracil (5-FU); folic acid analogues such as administration with the antibodies of the present disclosure. denopterin, methotrexate, pteropterin, trimetrexate, aziri In one embodiment, the antibody is administered with an dines such as benzodopa, carboquone, meturedopa, and ure anti-angiogenic agent. By “anti-angiogenic agent as used dopa; ethylenimines and methylamelamines including altre herein is meant a compound that blocks, or interferes to some tamine, triethylenemelamine, trietylenephosphoramide, 55 degree, the development of blood vessels. The anti-angio triethylenethiophosphaoramide and trimethylolomelamine; genic factor may, for instance, be a small molecule or a folic acid replenisher such as frolinic acid; nitrogen mustards protein, for example an antibody, Fc fusion, or cytokine, that Such as chlorambucil, chlornaphazine, cholophosphamide, binds to a growth factor or growth factor receptor involved in estramustine, ifosfamide, mechlorethamine, mechlore promoting angiogenesis. The preferred anti-angiogenic fac thamine oxide hydrochloride, melphalan, novembichin, 60 tor herein is an antibody that binds to Vascular Endothelial phenesterine, prednimustine, trofosfamide, uracil mustard; Growth Factor (VEGF). Other agents that inhibit signaling nitroSureas such as carmustine, chlorozotocin, fotemustine, through VEGF may also be used, for example RNA-based lomustine, nimustine, ranimustine; platinum analogs such as therapeutics that reduce levels of VEGF or VEGF-R expres cisplatin and carboplatin: vinblastine; platinum; proteins sion, VEGF-toxin fusions, Regeneron's VEGF-trap, and anti Such as arginine deiminase and asparaginase; purine analogs 65 bodies that bind VEGF-R. In an alternate embodiment, the Such as fludarabine, 6-mercaptopurine, thiamiprine, thiogua antibody is administered with atherapeutic agent that induces nine; pyrimidine analogs such as ancitabine, azacitidine, or enhances adaptive immune response, for example an anti US 9,040,042 B2 63 64 body that targets CTLA-4. Additional anti-angiogenesis , thromboxanes, and ; as well as agents include, but are not limited to, angiostatin (plasmino topical Steroids such as anthralin, calcipotriene, clobetasol, gen fragment), antithrombin III, angiozyme, ABT-627, Bay and tazaroteine); cytokines such as TGFb. IFNa, IFNb, IFNg, 12-9566, benefin, bevacizumab, bisphosphonates, BMS IL-2, IL-4, IL-10; cytokine, chemokine, or receptor antago 275291, cartilage-derived inhibitor (CM), CAI, CD59 nists including antibodies, Soluble receptors, and receptor-Fc complement fragment, CEP-7055, Col3, combretastatin A-4, fusions against BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, endostatin (collagen XVIII fragment), farnesyl transferase CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, inhibitors, fibronectin fragment, gro-beta, halofluginone, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, heparinases, heparin hexasaccharide fragment, HMV833, CD80, CD86, CD147, CD152, complement factors (C5, D) human chorionic gonadotropin (hCG), IM-862, interferon 10 CTLA4, eotaxin, Fas, ICAM, ICOS, IFNC, IFNB, IFNY, alpha, interferon beta, interferon gamma, interferon inducible IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 protein 10 (IP-10), -12, kringle 5 (plasminogen IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA fragment), marimastat, metalloproteinase inhibitors (eg. 1, LFA-3, MHC, selectins, TGFB, TNFC, TNFB, TNF-R1, TIMPs), 2-methodyestradiol, MMI 270 (CGS 27023A), plas T-cell receptor, including Enbrel(R) (etanercept), Humira R. minogen activator inhibitor (PAI), platelet factor-4 (PF4), 15 (adalimumab), and Remicade(R) (infliximab); heterologous prinomastat, prolactin 16 kDa fragment, proliferin-related anti-lymphocyte globulin; other immunomodulatory mol protein (PRP), PTK787/ZK 222594, retinoids, solimastat, ecules Such as 2-amino-6-aryl-5 Substituted pyrimidines, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahy anti-idiotypic antibodies for MHC binding peptides and drocortisol-S, tetrathiomolybdate, thalidomide, thrombo MHC fragments, azathioprine, brequinar, bromocryptine, spondin-1 (TSP-1), TNP-470, transforming growth factor cyclophosphamide, cyclosporine A, D-penicillamine, deox beta (TGF-B), vasculostatin, vasostatin (calreticulin frag yspergualin, FK506, glutaraldehyde, gold, hydroxychloro ment), ZS6126, and ZD6474. quine, leflunomide, malononitriloamides (eg. leflunomide), In a preferred embodiment, the antibody is administered methotrexate, minocycline, mizoribine, mycophenolate with a tyrosine kinase inhibitor. By “tyrosine kinase inhibi mofetil, rapamycin, and Sulfasasazine. tor” as used herein is meant a molecule that inhibits to some 25 In an alternate embodiment, antibody of the present dis extent tyrosine kinase activity of a tyrosine kinase. Examples closure are administered with a cytokine. By “cytokine' as of such inhibitors include but are not limited to quinazolines, used herein is meant a generic term for proteins released by such as PD 153035, 4-(3-chloroanilino) quinazoline; pyri one cell population that act on another cell as intercellular dopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, mediators. Examples of Such cytokines are lymphokines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazol 30 monokines, and traditional polypeptide hormones. Included opyrimidines, 4-(phenylamino)-7H-pyrrolo2,3-dipyrim among the cytokines are growth hormone Such as human idines; curcumin (diferuloyl methane, 4,5-bis(4-fluoroa growth hormone, N-methionyl human growth hormone, and nilino)phthalimide); tyrphostines containing nitrothiophene bovine growth hormone; parathyroid hormone; thyroxine; moieties; PD-0183805 (Warner-Lambert); antisense mol insulin; proinsulin; relaxin; prorelaxin, glycoprotein hor ecules (e.g. those that bind to ErbB-encoding nucleic acid); 35 mones such as follicle stimulating hormone (FSH), thyroid quinoxalines (U.S. Pat. No. 5,804.396); tryphostins (U.S. Pat. stimulating hormone (TSH), and luteinizing hormone (LH); No. 5,804.396); ZD6474 (Astra Zeneca); PTK-787 (Novar hepatic growth factor; fibroblast growth factor; prolactin; tis/Schering A G); pan-ErbB inhibitors such as CI-1033 placental lactogen; tumor necrosis factor-alpha and -beta; (Pfizer); Affinitac (ISIS 3521; Isis/Lilly): Imatinib mesylate mullerian-inhibiting Substance; mouse gonadotropin-associ (STI571, Gleevec(R); Novartis); PKI 166 (Novartis); GW2016 40 ated peptide; inhibin; activin; vascular endothelial growth (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); factor; integrin: thrombopoietin (TPO); nerve growth factors Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (No such as NGF-beta; platelet-growth factor; transforming vartis/Schering AG); INC-1C11 (Imclone); or as described in growth factors (TGFs) such as TGF-alpha and TGF-beta; any of the following patent publications: U.S. Pat. No. 5,804, insulin-like growth factor-I and -II; erythropoietin (EPO); 396; PCT WO 99/09016 (American Cyanimid); PCT WO 45 osteoinductive factors; interferons such as interferon-alpha, 98/43960 (American Cyanamid); PCT WO 97/38983 beta, and -gamma; colony stimulating factors (CSFs) such as (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); macrophage-CSF (M-CSF); granulocyte-macrophage-CSF PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (Pfizer, Inc); PCTWO 96/33978 (AstraZeneca); PCT WO96/ (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, 3397 (AstraZeneca); PCTWO 96/33980 (AstraZeneca), gefi 50 IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis tinib (IRESSATM, ZD1839, AstraZeneca), and OSI-774 factor such as TNF-alpha or TNF-beta; and other polypeptide (TarcevaTM, OSI Pharmaceuticals/Genentech), all patent pub factors including LIF and kit ligand (KL). As used herein, the lications incorporated entirely by reference. term cytokine includes proteins from natural sources or from In another embodiment, the antibody is administered with recombinant cell culture, and biologically active equivalents one or more immunomodulatory agents. Such agents may 55 of the native sequence cytokines. increase or decrease production of one or more cytokines, up In a preferred embodiment, cytokines or other agents that or down-regulate self-antigen presentation, mask MHC anti stimulate cells of the immune system are co-administered gens, or promote the proliferation, differentiation, migration, with the antibody of the present disclosure. Such a mode of or activation state of one or more types of immune cells. treatment may enhance desired effector function. For Immunomodulatory agents include but not limited to: non 60 example, agents that stimulate NK cells, including but not steroidal anti-inflammatory drugs (NSAIDs) such as asprin, limited to IL-2 may be co-administered. In another embodi ibuprofed, celecoxib, diclofenac, etodolac, fenoprofen, ment, agents that stimulate macrophages, including but not indomethacin, ketoralac, oxaprozin, nabumentone, Sulindac, limited to C5a, formyl peptides such as N-formyl-methionyl tolmentin, rofecoxib, naproxen, ketoprofen, and nabume leucyl-phenylalanine (Beigier-Bompadre et al. (2003) Scand. tone; steroids (eg. glucocorticoids, dexamethasone, corti 65 J. Immunol. 57: 221-8, incorporated entirely by reference), Sone, hydroxycortisone, methylprednisolone, prednisone, may be co-administered. Also, agents that stimulate neutro prednisolone, trimcinolone, aZulfidineicosanoids such as phils, including but not limited to G-CSF, GM-CSF, and the US 9,040,042 B2 65 66 like may be administered. Furthermore, agents that promote whole body irradiation, or may be directed locally to a spe migration of Such immunostimulatory cytokines may be cific site or tissue in or on the body, such as the lung, bladder, used. Also additional agents including but not limited to inter or prostate. Typically, radiation therapy is administered in feron gamma, IL-3 and IL-7 may promote one or more effec pulses over a period of time from about 1 to 2 weeks. The tor functions. radiation therapy may, however, be administered over longer In an alternate embodiment, cytokines or other agents that periods of time. For instance, radiation therapy may be inhibit effector cell function are co-administered with the administered to patients having head and neck cancer for antibody of the present disclosure. Such a mode of treatment about 6 to about 7 weeks. Optionally, the radiation therapy may limit unwanted effector function. may be administered as a single dose or as multiple, sequen In an additional embodiment, the antibody is administered 10 tial doses. The skilled medical practitioner can determine with one or more antibiotics, including but not limited to: empirically the appropriate dose or doses of radiation therapy aminoglycoside antibiotics (eg. apramycin, arbekacin, bam useful herein. In accordance with another embodiment of the bermycins, butirosin, dibekacin, gentamicin, kanamycin, invention, the antibody of the present disclosure and one or neomycin, netilmicin, paromomycin, ribostamycin, sisomy more other anti-cancer therapies are employed to treat cancer cin, spectrinomycin), aminocyclitols (eg. Spretinomycin), 15 cells ex vivo. It is contemplated that such ex vivo treatment amphenicol antibiotics (eg. azidamfenicol, chloramphenicol, may be useful in bone marrow transplantation and particu florfrnicol, and thiamphemicol), ansamycin antibiotics (eg. larly, autologous bone marrow transplantation. For instance, rifamide and rifampin), carbapenems (eg. imipenem, mero treatment of cells or tissue(s) containing cancer cells with penem, panipenem); cephalosporins (eg. cefaclor, cefadroxil, antibody and one or more other anti-cancer therapies, such as cefamandole, cefatrizine, cefazedone, cefoZopran, ce?pimi described above, can be employed to deplete or substantially Zole, cefpiramide, cefpirome, cefprozil, cefuroxine, deplete the cancer cells prior to transplantation in a recipient cefixime, cephalexin, cephradine), cephamycins (cefbupera patient. Zone, cefoxitin, cefiminox, cefinetazole, and cefotetan); lin It is of course contemplated that the antibodies of the cosamides (eg. clindamycin, lincomycin); macrollide (eg. invention may employ in combination with still other thera azithromycin, brefeldin A, clarithromycin, erythromycin, 25 peutic techniques such as Surgery or phototherapy. roXithromycin, tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam); mupirocin, oxacephems (eg. EXAMPLES flomoxeflatamoxef, and moxalactam); penicillins (eg. amdi nocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, Examples are provided below to illustrate the present dis beXZylpenicillinic acid, benzylpenicillin Sodium, epicillin, 30 closure. These examples are not meant to constrain the fenbenicillin, floxacillin, penamecillin, penethamate hydrio present disclosure to any particular application or theory of dide, penicillin o-benethamine, penicillin O, penicillin V. operation. penicillin V benzoate, penicillin V hydrabamine, penimepi For reference to immunoglobulin variable regions, posi cycline, and phencihicillin potassium); polypeptides (eg. tions are numbered according to the Kabat numbering bacitracin, colistin, polymixin B, teicoplanin, Vancomycin); 35 scheme. For reference to immunoglobulin constant regions, quinolones (amifloxacin, cinoxacin, ciprofloxacin, enoxacin, positions are numbered according to the EU index as in Kabat enrofloxacin, feroxacin, flumequine, gatifloxacin, gemifloxa (Kabat et al., 1991, Sequences of Proteins of Immunological cin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic Interest, 5th Ed., United States Public Health Service, acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipe National Institutes of Health, Bethesda). midic acid, roSoxacin, rufloxacin, sparfloxacin, temafloxacin, 40 to Sufloxacin, trovafloxacin); rifampin; Streptogramins (eg. Example 1 quinupristin, dalfopristin); Sulfonamides (sulfanilamide, Sul famethoxazole); tetracyclenes (chlortetracycline, demeclo Antibodies that Bind the Plasma Cell Antigen cycline hydrochloride, demethylchlortetracycline, doxycy HM124 cline, duramycin, minocycline, neomycin, oxytetracycline, 45 streptomycin, tetracycline, Vancomycin). Sequences of the heavy and light chain variable regions of Anti-fungal agents such as amphotericin B, ciclopirox, the anti-HM1.24 antibody HM1.24, as disclosed in clotrimazole, econazole, fluconazole, flucytosine, itracona US23103970A1, herein expressly incorporated by reference, Zole, ketoconazole, niconazole, nystatin, terbinafine, tercona are provided in FIG. 6. The anti-HM1.24 antibody was gen Zole, and tioconazole may also be used. 50 erated by immunization of BALB/c mice with the plasma cell Antiviral agents including protease inhibitors, reverse tran line KPC-32 (Goto et al., 1994, Blood:84:1922-1930, incor Scriptase inhibitors, and others, including type I interferons, porated entirely by reference). The genes for murine WT viral fusion inhibitors, and neuramidase inhibitors, may also HM1.24 VH and VL, designated H0 and L0 respectively, be used. Examples of antiviral agents include, but are not were constructed using gene synthesis techniques and Sub limited to, acyclovir, adefovir, amantadine, , cle 55 cloned into the mammalian expression vector pcDNA3.1Zeo Vadine, , entecavir, , gangcyclovir, comprising the full length light kappa (CK) and heavy chain idoxuridine, , , pleconaril, ribavirin, riman IgG1 constant regions. tadine, , , trifluridine, Vidarabine, and Plasmids containing heavy chain gene (VH-CH1-CH2 , may be used. CH3) were co-transfected with plasmid containing light The antibodies of the present disclosure may be combined 60 chain gene (VL-CLK) into 293T cells. grown in 10% ultra low with other therapeutic regimens. For example, in one embodi IgG fetal bovine serum with 1 mM sodium pyruvate and 1 x ment, the patient to be treated with an antibody of the present non-essential amino acids (Gibco R, Invitrogen, Hayward disclosure may also receive radiation therapy. Radiation Calif.). Five days after transfection, the culture media was therapy can be administered according to protocols com removed and ran through a protein A column. The antibody monly employed in the art and known to the skilled artisan. 65 produced is referred to as HM1.24 HOL0 IgG1 WT. Such therapy includes but is not limited to cesium, iridium, Flow cytometry was used to confirm the binding of anti iodine, or cobalt radiation. The radiation therapy may be HM1.24 antibody to HM1.24 antigen and plasma cells, as US 9,040,042 B2 67 68 well as to evaluate the expression of HM1.24 on various cell engineering Fc variants with enhanced FcyR affinity enables types in PBMCs. 1x10 PBMCs were first mixed with either a significant amount of ADCC activity. unlabelled HM1.24 HOL0 IgG1 WT or isotype control IgG1 (hIgG Kappa, from Sigma) antibody. A mixture of cell Example 3 type specific labeled antibodies (all mouse anti-human CD, BD Biosciences) was then added to tag the specific cell types Anti-HM1.24 Antibodies with Reduced Potential for in the PBMCs. The following antibodies were used: anti Immunogenicity CD15-FITC (BD 555401) for labeling PMNs, anti-CD19 PerCP-Cy5.5 (BD3409510) for labeling B-cells, anti-CD14 In order to reduce the potential for immunogenicity of the PE-Cy7 (BD 557742) for labeling monocytes, ant-CD56 10 H0 and L0 HM1.24 variable regions, the immunogenicity was reduced using a method described in U.S. Ser. No. APC (BD 555518) for labeling NK cells, and anti-CD3-APC 11/004,590, entitled “Methods of Generating Variant Pro Cy3 (BD557832) for labeling T cells. After incubation on ice teins with Increased Host String Content and Compositions for 30 min. in the dark, the cells were washed and mixed with Thereof, filed on Dec. 6, 2004. The method reduces the R-PE conjugated F(ab')2 fragment Goat anti-human IgG 15 potential for immunogenicity by increasing the human string (against fe-gamma fragment) to specifically label the primary content of the antibody through mutations. The anti-CD5 testantibody (anti-HM1.24 or isotype control). After incuba variable region variants with reduced potential for immuno tion, cells were washed again and analyzed by 6-color flow genicity are named H1, H2,H3, and H4 for the heavy chain, cytometry using a FACS Canto II flow cytometer to determine and L1, L2, L3, and L4 for the light chain. The sequences for the expression of HM 1.24 on any of these specific cell types these variants are provided in FIG. 10. Light and heavy chains in PBMCs. The results, shown in FIG. 7, indicate that were constructed by gene synthesis. Light chains were Sub HM1.24 is not expressed to a significant degree on any of the cloned into the pcDNA3.1Zeo comprising the full length light tested PBMC cell types. In contrast, a similar experiment kappa (CK) constant region, and heavy chains were subcloned carried out against a multiple myeloma cell line RPMI 8226 into pcNDA3.1Zeo vectors comprising both the heavy chain (1 million cells), using FITC-labeled anti-CD38 to label 25 Hybrid IgG constant region with mutations S239D/I332E plasma cells, indicates that the anti-HM1.24 antibodies bind (designated Hybrid S239D/I332E). All sequences were well to plasma cells (FIG. 8). These results suggest that the sequenced to confirm the fidelity of the sequence. Combina anti-HM1.24 antibodies of the invention may be useful for tions of the different heavy and light chains were expressed in selectively targeting multiple myeloma cells relative to other 293T cells and purified as described above. For example, an blood cell types. 30 anti-HM1.24 antibody comprising the H3 heavy chain and L1 light chain would be designated as HM1.24 H3L1. Expres Example 2 sion levels of the HM1.24 heavy and light chain combinations from a fixed number of plates of 293T cells are shown in FIG. Modifications to Anti-HM1.24 Antibodies Provide 11. Enhanced Effector Function 35 Humanized variants were screened for their binding to HM1.24+ multiple myeloma RPMI 8226 cells by flow cytometry using R-PE conjugated F(ab')2 fragment Goat In order to evaluate the effector function capacity of an anti-human IgG, similar to as described above. The dose anti-HM1.24 antibody, as well as the possibility of improving dependent binding curves for this experiment are shown in the effector function of any such activity, variants were con 40 FIG.12a, and the EC50's for selectantibodies, obtained from structed in Fc region of the HM1.24 HOLO antibody. Variants the fits of these data, are provided in FIG. 12b. The results S239D/I332E, G236A, G236A/I332E, S239D/H268E/ show that H1 and H2 and L2 and L4 combinations bind with G236A, and S239D/I332E/G236A were constructed in the Fc comparable affinity to the target antigen as the parent region of the IgG1 heavy chain in the pcDNA3.1Zeo vector HM1.24 HOL0 antibody. using quick-change mutagenesis techniques. All sequences 45 The humanized variants were also screened for their capac were sequenced to confirm the fidelity of the sequence. Plas ity to mediate effector function against HM 1.24+ cells in an mids containing heavy and light chain genes were co-trans ADCC assay, as described above. FIG. 13 shows that human fected, and antibody was expressed and purified as described ized variants, comprising Fc modifications, mediate a com above. parable level of ADCC activity as the parent HM.1.24 HOLO The capacity of the variant antibodies to mediate effector 50 comprising the same Fc modifications. Indeed it would be function against HM 1.24 expressing cells was tested in a difficult to screen humanized variants for effector function cell-based ADCC assay (FIG. 9). Human peripheral blood with a WT IgG1 Fc region because activity in this context is monocytes (PBMCs) were isolated from leukopaks and used So low. as effector cells, and HM1.24+ RPMI8226 cells were used as target cells. 10,0000 RPMI8226 target cells were incubated 55 Example 4 with 450,000 effector cells (non-adherent PBMC cells from a healthy donor) in 100 ul of 1% FBS/RPMI1640 ADCC assay Engineering of Humanized Anti-HM1.24 Antibodies media in the presence of antibodies. After 4 hours at 37° C. to Remove Potential Deamidation Sites relative ADCC activity was detected using fluorescent LDH (lactate dehydrogenase release) detection system from 60 Two potential aspartate (Asp, D) isomerization sites were Promega as per manufacturer's protocol. The ADCC activity observed in CDR2 of the anti-HM1.24 heavy chain variable was determined by the following: ((Experimental Value-(Ef region. Asp isomerization can lead to the formation of isoas fector and Target))/(Total Target-Target Alone))* 100 where partate and cyclic imide variants, and if modification occurs at Total Target value is RPMI8226 cells lysed with 1% Tri a site involved in antigen binding can reduce the affinity and tonX100. The results show that the WT anti-HM1.24 anti 65 impact therapeutic potency. Asp isomerization is accelerated body does not mediate ADCC against RPMI8225 multiple when glycine is at the C-terminal side of the reacting Asp myeloma cells. However, optimization of the antibody by (Asp-Gly), and when conditions are relatively acidic. A set of US 9,040,042 B2 69 70 variants, shown in FIG. 14, were engineered in the H1 and H2 ated, wherein the His-tagged HM1.24 antigen was coupled heavy chain variable regions to remove the Asp-Gly site. The directly to the chip using standard NHS chemistry. Antibodies variants, designated H1.1-H1.4 and H2.1-H2.4, were con at multiple concentrations were used as analyte in this format. structed as described above. FIG. 15 lists the amino acid sequences of these variants. Antibodies comprising these Kinetic rate constants (ka and kd) and equilibrium dissocia heavy chain variants and the HM1.24 L4 light chains were tion constants (KD) obtained by fitting of these data were expressed and purified as described above. consistent whether obtained by simultaneous global Lang Susceptibility of WT and variant antibodies to Asp isomer muir fitting or by fitting kinetic curves separately (FIG. 17). ization was evaluated in an accelerated isomerization assay. The agreement suggests that this format provides accurate Antibodies were incubated at pH9, 37 C for 4 days, and then binding parameters for the antibody/HM1.24 interaction. The tested for binding to antigen using BiacoreTM. DNA encoding 10 results indicate that the anti-HM1.24 antibodies of the inven the HM1.24 antigen was obtained from the ATCC (IMAGE tion bindantigen with an affinity of 3-8x10' M(30-80pM). clone 5217945, MGC:45144, Genbank accession number The ability of the variant anti-HM1.24 antibodies to medi BC033873). Residues N49-A163 of the extracellular domain ate effector function was evaluated in an ADCC assay, as of HM1.24 (Rew et al., 2005, Clin Cancer Res 11 (9):3377 described above. FIG. 18 shows that the optimized anti 3384) were subcloned with an N-terminal His-tag and Factor 15 Xa cleavage site into pcDNA3.1Zeo. Protein was expressed HM1.24 antibodies of the invention have significant cyto in 293T cells and purified using Ni-NTA chromatography. toxic capacity against the multiple myeloma cell line RPMI Antibody, untreated and post incubation, was immobilized on 8226, substantially greater than the parent a protein A chip and HM1.24 antigen analytes was flowed HM1.24 HOL0 IgG1 WT antibody. over at increasing concentrations. Although this particular The use of particular modifications, for example the sub BiacoreTM format did not provide consistent fitting affinities stitutions 239D and 332E, to enhance effector function are between global Langmuir fits and individual fitting of on (ka) not meant to constrain the anti-HM1.24 antibodies to these and off (kd) rates, a comparison between variants was still particular modifications. As described above in the section possible by comparing relative off rates. FIG. 16 shows that entitled “Modifications for optimizing effector function', a no significant difference in binding was observed between 25 incubated and nonincubated Samples, indicating either that large number of modifications, including amino acid modifi Asp isomerization does not occur, or that it does occur but cations and modified glycoforms, are contemplated for anti does not affect binding to antigen. HM1.24 antibodies to improve their effector function prop erties. Example 5 All cited references are herein expressly incorporated by 30 reference in their entirety. Antigen Binding and Effector Function of Whereas particular embodiments of the invention have Anti-HM1.24 Antibodies been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous varia In order to obtain valid binding affinities of the anti tions of the details may be made without departing from the HM1.24 antibodies, an alternate BiacoreTM format was evalu invention as described in the appended claims.

is Human HM124 (SEQ ID NO: MASTSYDYCRWPMEDGDKRCKLLL.GIGILWLLIIWILGWPLIIFTIKANSEACRDGLRAWMECRNWT

HLLOOELTEAOKGFQDWEAQAATCNHTVMALMASLDAEKAOGOKKWEELEGEITTLNHKLODA

SAEVERLRRENOWLSWRIADKKYYPSSODSSSAAAPOLLIWLLGLSALLO > Kappa constant light chain (C) (SEQ ID NO: RTVAAPSVFIFPPSDEOLKSGTASVWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKD

STYSLSSTLTLSKADYEKHKWYACEVTHOGLSSPVTKSFNRGEC > IgG1 constant heavy chain (CH) (SEQ ID NO: ASTKGPSWFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLY

SLSSWWTWPSSS LGTOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLT

WLHODWLNGKEY KCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOWSLTCLVK

GFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEAL

HNHYTOKSLSLS > IgG2 constant heavy chain (CH) (SEQ ID NO: ASTKGPSWFPLA PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLY

SLSSWWTWPSSN FGTOTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSHEDPEVOFNWYWDGVEVHNAKTKPREEOFNSTFRVVSWLTV VH

US 9,040,042 B2 111 112 - Continued > HM1.24 L2 Kappa constant light chain (C) (SEQ ID NO: 116) DIVMTOSPSSLSASVGDRVTITCOASODVNTAVAWYOOKPDOSPKLLIYSASNRYTGVPDRITG

SGSGTDFTLTISSLOAEDVAVYYCOOHYSTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEOLKSGT

ASVWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKWYH

ACEVTHOGLSSPVTKSFNRGEC > HM1.24 L3 Kappa constant light chain (C) (SEO ID NO : 117) DIVMTOSPPTLSLSPGERVTLSCRASODVNTAVAWYOOKPDOSPKLLIYSASNRYTGVPDRIT

GSGSGTDFTLTISSLOPEDFAVYYCOOHYSTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEOLKSG

TASVWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHOGLSSPVTKSFNRGEC > HM1.24 L4 Kappa constant light chain (C) (SEQ ID NO: 118) DIVMTOSPLSLPVTLGOPASISCKASODVNTAVAWYLOKPGKSPKLLIYSASNRYTGVPDRITG

SGSGTDFTLKISRVEAEDVGVYYCOOHYSTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEOLKSG

TASVWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHOGLSSPVTKSFNRGEC

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 119

<210s, SEQ ID NO 1 &211s LENGTH: 18O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 1 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly 1. 5 1O 15 Asp Lys Arg Cys Llys Lieu Lleu Lieu. Gly Ile Gly Ile Lieu Val Lieu. Lieu 2O 25 3O Ile Ile Val Ile Leu Gly Val Pro Leu. Ile Ile Phe Thr Ile Lys Ala 35 4 O 45 Asn Ser Glu Ala Cys Arg Asp Gly Lieu. Arg Ala Wal Met Glu. Cys Arg SO 55 6 O Asn Val Thir His Lieu. Lieu. Glin Glin Glu Lieu. Thr Glu Ala Glin Lys Gly 65 70 7s 8O Phe Glin Asp Val Glu Ala Glin Ala Ala Thr Cys Asn His Thr Val Met 85 90 95 Ala Lieu Met Ala Ser Lieu. Asp Ala Glu Lys Ala Glin Gly Glin Llys Llys 1OO 105 11 O Val Glu Glu Lieu. Glu Gly Glu Ile Thir Thr Lieu. Asn His Llys Lieu. Glin 115 12 O 125 Asp Ala Ser Ala Glu Val Glu Arg Lieu. Arg Arg Glu Asn. Glin Val Lieu 13 O 135 14 O Ser Val Arg Ile Ala Asp Llys Llys Tyr Tyr Pro Ser Ser Glin Asp Ser 145 150 155 160 Ser Ser Ala Ala Ala Pro Gln Lieu. Lieu. Ile Val Lieu. Lieu. Gly Lieu. Ser 1.65 17O 17s

Ala Lieu. Lieu. Glin 18O US 9,040,042 B2 113 114 - Continued

<210s, SEQ ID NO 2 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2

Arg Thir Wall Ala Ala Pro Ser Wall Phe Ile Phe Pro Pro Ser Asp Glu 1. 5 1O 15

Glin Lieu Lys Ser Gly Thr Ala Ser Wall Wall Cys Lell Lell Asn Asn Phe 2O 25 3O

Pro Arg Glu Ala Lys Val Glin Trp Wall Asp Asn Ala Luell Glin 35 4 O 45

Ser Gly Asn Ser Glin Glu Ser Val Thir Glu Glin Asp Ser Asp Ser SO 55 6 O

Thir Tyr Ser Leu Ser Ser Thr Lieu. Thir Luell Ser Ala Asp Glu 65 70

His Llys Val Tyr Ala Cys Glu Wall Thir His Glin Gly Luell Ser Ser 85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 1OO 105

<210s, SEQ ID NO 3 &211s LENGTH: 330 212. TYPE: PRT <213> ORGANISM: Homo sapiens < 4 OO SEQUENCE: 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thir Ser Gly Gly Thr Ala Ala Luell Gly Cys Lell Wall Lys Asp 2O 25

Phe Pro Glu Pro Wall. Thir Wal Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wal Wall. Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Cys Asn Val Asn His Llys Pro Ser ASn Thir Wall Asp 85 90 95

Val Glu Pro Llys Ser Cys Asp Lys Thir His Thir Pro Pro 1OO 105 11 O

Pro Ala Pro Glu Lieu. Lieu. Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Lys Asp Thr Lieu Met Ile Ser Arg Thir Pro Glu Wall Thir 13 O 135 14 O

Wall Val Val Asp Val Ser His Glu Asp Pro Glu Wall Phe Asn Trp 145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Gln Tyr Asn Ser Thr Tyr Arg Wall Wall Ser Wall Lell Thir Wall Luell 18O 185 19 O

His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195 2OO 2O5

Ala Lieu Pro Ala Pro Ile Glu Thir Ile Ser Ala Gly 21 O 215 22O US 9,040,042 B2 115 116 - Continued

Glin Pro Arg Glu Pro Glin Val Tyr Thir Luell Pro Pro Ser Arg Glu Glu 225 23 O 235 24 O

Met Thir Lys Asn Glin Val Ser Lieu. Thir Cys Luell Wall Gly Phe 245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Tyr Lys Thr Thr Pro Pro Val Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285

Lell Tyr Ser Lys Lieu. Thr Val Asp Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Cys Ser Val Met His Glu Ala Luell His Asn His Thir 3. OS 310 315 32O

Glin Llys Ser Lieu. Ser Lieu. Ser Pro Gly Lys 3.25 330

<210s, SEQ ID NO 4 &211s LENGTH: 326 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 4

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Luell Ala Pro Ser Arg 1. 5 15

Ser Thir Ser Glu Ser Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp 2O 25

Phe Pro G. Pro Wall. Thir Wal Ser Trp Asn Ser Gly Ala Luell Thr Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wal Wall. Thir Wall Pro Ser Ser ASn Phe Gly Thir Glin Thir 65 70 8O

Thir Cys Asn. Wall Asp His Lys Pro Ser ASn Thir Wall Asp 85 90 95

Thir Val Glu Arg Lys Cys Cys Val Glu Pro Pro Pro Ala Pro 1OO 105 11 O

Pro Val Ala Gly Pro Ser Val Phe Luell Phe Pro Pro Lys Pro Asp 115 12 O 125

Thir Leu Met Ile Ser Arg Thr Pro Glu Wall Thir Cys Wall Wall Wall Asp 13 O 135 14 O

Wall Ser His Glu Asp Pro Glu Val Glin Phe ASn Trp Wall Asp Gly 145 150 155 160

Wall Glu Val His Asn Ala Lys Thr Pro Arg Glu Glu Glin Phe Asn 1.65 17O 17s

Ser Thr Phe Arg Val Val Ser Val Luell Thir Wall Wall His Glin Asp Trp 18O 185 19 O

Lell Asin Gly Lys Glu Tyr Lys Cys Wall Ser Asn Lys Gly Luell Pro 195 2OO

Ala Pro Ile Glu Lys Thr Ile Ser Thir Gly Glin Pro Arg Glu 21 O 215 22O

Pro Glin Val Tyr Thr Lieu Pro Pro Ser Arg Glu Glu Met Thir Asn 225 23 O 235 24 O

Glin Val Ser Lieu. Thr Cys Lieu Val Gly Phe Pro Ser Asp Ile 245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Thir US 9,040,042 B2 117 118 - Continued

26 O 265 27 O

Thir Pro Pro Met Lieu. Asp Ser Asp Gly Ser Phe Phe Lell Tyr Ser 28O 285

Lell Thir Wall Asp Lys Ser Arg Trp Glin Glin Gly Asn Wall Phe Ser 29 O 295 3 OO

Ser Wall Met His Glu Ala Lell His Asn His Tyr Thir Glin Llys Ser Luell 3. OS 310 315 32O

Ser Luell Ser Pro Gly Lys 3.25

<210s, SEQ ID NO 5 &211s LENGTH: 377 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5

Ala Ser Thr Lys Gly Pro Ser Wall Phe Pro Luell Ala Pro Cys Ser Arg 1. 15

Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp 25 3O

Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn Ser Gly Ala Lieu. Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Lieu. Tyr Ser SO 55 6 O

Lell Ser Ser Wall Wall Thir Wall Pro Ser Ser Ser Lell Gly Thr Gin Thir 65 70 8O

Thir Asn Wall Asn His Pro Ser ASn Thir Val Asp 85 90 95

Arg Wall Glu Luell Thir Pro Luell Gly Asp Thir Thir His Thr Cys Pro 105 11 O

Arg Pro Glu Pro Ser Cys Asp Thir Pro Pro Pro Cys Pro Arg 115 12 O 125

Pro Glu Pro Ser Cys Asp Thir Pro Pro Pro Pro Arg 13 O 135 14 O

Pro Glu Pro Ser Cys Asp Thir Pro Pro Pro Pro Arg Cys Pro 145 150 155 160

Ala Pro Glu Luell Lell Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 1.65 17O 17s

Pro Asp Thir Lell Met Ile Ser Arg Thir Pro Glu Wall Thr Cys Wall 18O 185 19 O

Wall Wall Asp Wall Ser His Glu Asp Pro Glu Wall Glin Phe 195

Wall Asp Gly Wall Glu Wall His Asn Ala Thir Lys Pro Arg Glu Glu 21 O 215 22O

Glin Tyr Asn Ser Thir Phe Arg Wall Wall Ser Wall Lell Thir Wall Lieu. His 225 23 O 235 24 O

Glin Asp Trp Luell Asn Gly Glu Tyr Lys Wall Ser Asn 245 250 255

Ala Luell Pro Ala Pro Ile Glu Thir Ile Ser Thir Lys Gly Glin 26 O 265 27 O

Pro Arg Glu Pro Glin Wall Thir Luell Pro Pro Ser Arg Glu Glu Met 28O 285

Thir Lys Asn Glin Wall Ser Lell Thir Luell Wall Lys Gly Phe Tyr Pro 29 O 295 3 OO US 9,040,042 B2 119 120 - Continued

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Glin Pro Glu Asn Asn 3. OS 310 315 32O

Asn Thir Thir Pro Pro Met Leu. Asp Ser Asp Gly Ser Phe Phe Luell 3.25 330 335

Ser Luell Thr Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Ile 34 O 345 35. O

Phe Ser Cys Ser Wal Met His Glu Ala Luell His Asn Arg Phe Thir Glin 355 360 365

Ser Luell Ser Lieu. Ser Pro Gly 37 O 375

<210s, SEQ ID NO 6 &211s LENGTH: 327 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Luell Ala Pro Ser Arg 1. 15

Ser Thir Ser Glu Ser Thir Ala Ala Luell Gly Lell Wall Lys Asp 25

Phe Pro Glu Pro Wall. Thir Wal Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Ser SO 55 6 O

Lell Ser Ser Wall Wall. Thir Wall Pro Ser Ser Ser Lell Gly Thir Thir 65 70 75

Thir Asn Val Asp His Lys Pro Ser ASn Thir Wall Asp 85 90 95

Arg Wall Glu Ser Lys Tyr Gly Pro Pro Pro Ser Pro Ala Pro 105 11 O

Glu Phe Luell Gly Gly Pro Ser Val Phe Luell Phe Pro Pro Pro 115 12 O 125

Asp Thir Luell Met Ile Ser Arg Thr Pro Glu Wall Thir Wall Wall Wall 13 O 135 14 O

Asp Wall Ser Glin Glu Asp Pro Glu Wall Glin Phe Asn Trp Wall Asp 145 150 155 160

Gly Wall Glu Wall His Asn Ala Lys Thir Lys Pro Arg Glu Glu Glin Phe 1.65 17s

Asn Ser Thir Tyr Arg Val Val Ser Wall Luell Thir Wall Lell His Glin Asp 18O 185 19 O

Trp Luell Asn Gly Lys Glu Tyr Lys Wall Ser Asn Gly Luell 195 2OO

Pro Ser Ser Ile Glu Lys Thir Ile Ser Ala Lys Gly Glin Pro Arg 21 O 215 22O

Glu Pro Glin Wall Tyr Thr Lieu Pro Pro Ser Glin Glu Glu Met Thir Lys 225 23 O 235 24 O

Asn Glin Wall Ser Lieu. Thir Cys Lieu. Wall Lys Gly Phe Pro Ser Asp 245 250 255

Ile Ala Wall Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Lys 26 O 265 27 O

Thir Thir Pro Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Luell Ser 27s 28O 285

Arg Luell Thir Wall Asp Llys Ser Arg Trp Glin Glu Gly Asn Wall Phe Ser 29 O 295 3 OO US 9,040,042 B2 121 122 - Continued

Cys Ser Val Met His Glu Ala Lieu. His Asn His Tyr Thr Glin Lys Ser 3. OS 310 315 Lieu. Ser Lieu. Ser Lieu. Gly Lys 3.25

SEO ID NO 7 LENGTH: 330 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Hybrid constant heavy chain (CH)

<4 OOs, SEQUENCE: 7

Ala Ser Thr Lys Gly Pro Ser Wall Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp Tyr 25

Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wall Wall Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Asn Wall Asn His Pro Ser ASn Thir Wall Asp 85 90 95

Wall Glu Pro Ser Asp Lys Thir His Thir Pro Pro 105 11 O

Pro Ala Pro Glu Lell Lell Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Asp Thir Lell Met Ile Ser Arg Thir Pro Glu Wall Thir 13 O 135 14 O

Wall Wall Wall Asp Wall Ser His Glu Asp Pro Glu Wall Glin Phe Asn Trp 145 150 155 160

Wall Asp Gly Wall Glu Wall His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Glin Phe Asn Ser Thir Phe Arg Wall Wall Ser Wall Lell Thir Wall Wall 18O 185 19 O

His Glin Asp Trp Lell Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195 2O5

Ala Luell Pro Ala Pro Ile Glu Thir Ile Ser Thir Gly 21 O 215 22O

Glin Pro Arg Glu Pro Glin Wall Thir Luell Pro Pro Ser Arg Glu Glu 225 23 O 235 24 O

Met Thir Asn Glin Wall Ser Luell Thir Cys Luell Wall Gly Phe Tyr 245 250 255

Pro Ser Asp Ile Ala Wall Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Lys Thir Thir Pro Pro Met Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285

Lell Tyr Ser Lell Thir Wall Asp Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Ser Wall Met His Glu Ala Luell His Asn His Thir 3. OS 310 315 32O

Glin Ser Luell Ser Lell Ser Pro Gly Lys 3.25 330 US 9,040,042 B2 123 124 - Continued

SEQ ID NO 8 LENGTH: 330 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Hybrid constant heavy chain (CH) With 239D and 332E substitutions

<4 OOs, SEQUENCE: 8

Ala Ser Thr Lys Gly Pro Ser Wall Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp Tyr 25

Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wall Wall Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Asn Wall Asn His Pro Ser ASn Thir Wall Asp 85 90 95

Wall Glu Pro Ser Asp Lys Thir His Thir Pro Pro 105 11 O

Pro Ala Pro Glu Lell Lell Gly Gly Pro Asp Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Asp Thr Lieu Met Ile Ser Arg Thr Pro Glu Wall Thr Cys 13 O 135 14 O

Wall Wall Wall Asp Wall Ser His Glu Asp Pro Glu Wall Glin Phe Asn Trp 145 150 155 160

Wall Asp Gly Wall Glu Wall His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Glin Phe Asn Ser Thir Phe Arg Wall Wall Ser Wall Lell Thir Wall Wall 18O 185 19 O

His Glin Asp Trp Lell Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195

Ala Luell Pro Ala Pro Glu Glu Thir Ile Ser Thir Gly 21 O 215 22O

Glin Pro Arg Glu Pro Glin Wall Thir Luell Pro Pro Ser Arg Glu Glu 225 23 O 235 24 O

Met Thir Asn Glin Wall Ser Luell Thir Cys Luell Wall Gly Phe Tyr 245 250 255

Pro Ser Asp Ile Ala Wall Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Lys Thir Thir Pro Pro Met Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285

Lell Tyr Ser Lell Thir Wall Asp Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Ser Wall Met His Glu Ala Luell His Asn His Thir 3. OS 310 315 32O

Glin Ser Luell Ser Lell Ser Pro Gly Lys 3.25 330

<210s, SEQ ID NO 9 &211s LENGTH: 1. Of 212. TYPE : PRT US 9,040,042 B2 125 126 - Continued

<213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 9 Asp Ile Val Met Thr Glin Ser His Llys Phe Met Ser Thr Ser Val Gly 1. 5 1O 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Ile Thr Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Glin Ala 65 70 7s 8O Glu Asp Leu Ala Lieu. Tyr Tyr Cys Glin Glin His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Lieu. Glu Ile Llys 1OO 105

<210s, SEQ ID NO 10 &211s LENGTH: 120 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 10 Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Ala Arg Pro Gly Ala 1. 5 1O 15 Ser Val Llys Lieu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Pro Tyr 2O 25 3O Trp Met Gln Trp Val Lys Glin Arg Pro Gly Glin Gly Lieu. Glu Trp Ile 35 4 O 45 Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser Glin Llys Phe SO 55 6 O Lys Gly Lys Ala Thr Lieu. Thir Ala Asp Llys Ser Ser Ser Thir Ala Tyr 65 70 7s 8O Met Glin Leu Ser Ile Leu Ala Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Thr Lieu. Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 11 &211s LENGTH: 11 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 11 Lys Ala Ser Glin Asp Val Asn. Thir Ala Val Ala 1. 5 1O

<210s, SEQ ID NO 12 &211s LENGTH: 7 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 12 Ser Ala Ser Asn Arg Tyr Thr 1. 5 US 9,040,042 B2 127 128 - Continued

<210s, SEQ ID NO 13 &211s LENGTH: 9 212. TYPE : PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 13 Gln Gln His Tyr Ser Thr Pro Phe Thr 1. 5

<210s, SEQ ID NO 14 &211s LENGTH: 5 212. TYPE : PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 14

Pro Tyr Trp Met Glin 1. 5

SEO ID NO 15 LENGTH: 17 TYPE : PRT ORGANISM: Mus musculus

< 4 OOs SEQUENCE: 15 Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Llys Phe Lys 1. 5 15 Gly

<210 SEQ ID NO 16 &211s LENGTH: 11 212. TYPE : PRT &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 16 Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 1. 5

SEO ID NO 17 LENGTH: 1. Of TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM124 L1

<4 OOs, SEQUENCE: 17

Asp Ile Val Met Thir Glin Ser Pro Ser Ser Luell Ser Ala Ser Wall Gly 1. 5 15

Asp Arg Wall Thir Ile Thir Glin Ala Ser Glin Asp Wall Asn Thir Ala 25

Wall Ala Trp Tyr Glin Glin Pro Gly Lys Ser Pro Lys Luell Luell Ile 35 4 O 45

Ser Ala Ser Asn Arg Tyr Thir Gly Wall Pro Asp Arg Ile Thir Gly SO 55 6 O

Ser Gly Ser Gly Thir Asp Phe Thir Phe Thir Ile Ser Ser Luell Glin Pro 65 70 8O

Glu Asp Ile Ala Thir Glin Glin His Tyr Ser Thir Pro Phe 85 90 95

Thir Phe Gly Ser Gly Thir Luell Glu Ile Lys 1OO 105

<210s, SEQ ID NO 18 US 9,040,042 B2 129 130 - Continued

&211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: HM124 L2

<4 OOs, SEQUENCE: 18 Asp Ile Val Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Glin Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Asp Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Ile Thr Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Glin Ala 65 70 7s 8O Glu Asp Val Ala Val Tyr Tyr Cys Glin Glin His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Lieu. Glu Ile Llys 1OO 105

<210s, SEQ ID NO 19 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: HM124 L3

<4 OOs, SEQUENCE: 19 Asp Ile Val Met Thr Glin Ser Pro Pro Thr Lieu Ser Leu Ser Pro Gly 1. 5 1O 15 Glu Arg Val Thir Lieu. Ser Cys Arg Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3O Val Ala Trp Tyr Glin Gln Llys Pro Asp Glin Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Ile Thr Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Lieu. Glu Ile Llys 1OO 105

<210s, SEQ ID NO 2 O &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: HM124 L4

<4 OOs, SEQUENCE: 2O Asp Ile Val Met Thr Glin Ser Pro Leu Ser Leu Pro Val Thr Lieu. Gly 1. 5 1O 15

Glin Pro Ala Ser Ile Ser Cys Lys Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3O

Val Ala Trp Tyr Lieu. Glin Llys Pro Gly Lys Ser Pro Llys Lieu. Lieu. Ile 35 4 O 45 US 9,040,042 B2 131 132 - Continued

Ser Ala Ser Asn Arg Tyr Thir Gly Wall Pro Asp Arg Ile Thr Gly SO 55 6 O

Ser Gly Ser Gly Thr Asp Phe Thir Luell Ile Ser Arg Val Glu Ala 65 70 7s

Glu Asp Wall Gly Val Tyr Glin Glin His Tyr Ser Thr Pro Phe 85 90 95

Thir Phe Gly Ser Gly Thir Luell Glu Ile 1OO 105

SEQ ID NO 21 LENGTH: 120 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM124 H1

<4 OOs, SEQUENCE: 21

Glin Wall Glin Lieu Wall Glin Ser Gly Ala Glu Wall Pro Gly Ala 1. 5 1O 15

Ser Wall Lys Wall Ser Ala Ser Gly Thir Phe Thir Pro Tyr 2O 25

Trp Met Glin Trp Val Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Gly Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Wall. Thir Ile Ser Ala Asp Ser Ile Ser Thir Ala Tyr 65 70

Met Glu Luell Ser Arg Lell Arg Ser Asp Thir Ala Wall Tyr Cys 85 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Thir Luell Wall. Thir Wall Ser Ser 115 12 O

SEQ ID NO 22 LENGTH: 120 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM124 H2

<4 OOs, SEQUENCE: 22

Glu Wall Glin Lieu Wall Glu Ser Gly Gly Gly Luell Wall Pro Gly Gly 1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Thir Phe Thir Pro Tyr 2O 25 3O

Trp Met Glin Trp Val Arg Glin Ala Pro Gly Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Gly Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Wall. Thir Ile Ser Ala Asp Ser Ile Ser Thir Ala 65 70

Met Glu Luell Ser Ser Lell Arg Ser Glu Thir Ala Wall Tyr Cys 85 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Thir Luell Wall. Thir Wall Ser Ser 115 12 O US 9,040,042 B2 133 134 - Continued

SEQ ID NO 23 LENGTH: 12O TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM124 H3

<4 OOs, SEQUENCE: 23

Glin Wall Glin Lieu. Wall Glu Ser Gly Gly Luell Wall Glin Pro Gly Arg 1. 5 1O 15

Ser Luell Arg Luell Ser Thir Ser Gly Tyr Thir Phe Thir Pro Tyr 25

Trp Met Trp Wall Arg Glin Pro Gly Glin Gly Lell Glu Trp Met 45

Gly Ser Phe Pro Gly Asp Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Wall Thir Ile Ser Asp Ser Ile Ser Thir Ala Tyr 65 70

Lell Glin Ser Ser Lell Glu Met Ala Met Tyr Cys 85 95

Ala Arg Luell Arg Arg Gly Tyr Phe Asp Trp Gly Glin 105 11 O

Gly Thir Wall Thir Wall Ser Ser 12 O

SEQ ID NO 24 LENGTH: 12O TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM124 H4

<4 OOs, SEQUENCE: 24

Glin Wall Glin Lieu. Glin Glu Ser Gly Ser Gly Luell Wall Pro Pro Gly 1. 5 1O 15

Thir Luell Ser Luell Thir Ala Ala Ser Gly Tyr Thir Phe Thir Pro Tyr 25

Trp Met Glin Trp Wall Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Gly Asp Thir Arg Tyr Ser Glin Phe SO 55 6 O

Glin Gly Arg Wall Thir Ile Ser Ala Asp Ser Ile Ser Thir Ala Tyr 65 70

Met Glu Luell Ser Ser Lell Arg Ser Glu Thir Ala Met Tyr Cys 85 95

Ala Arg Gly Luell Arg Arg Gly Gly Tyr Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Thir Luell Wall Thir Wall Ser Ser 115 12 O

SEO ID NO 25 LENGTH: 12O TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM1.24 H1.1

SEQUENCE: 25 US 9,040,042 B2 135 136 - Continued

Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Wall Lys Lys Pro Gly Ala 15

Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Pro Tyr 2O 25

Trp Met Gln Trp Val Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Glu Gly Asp Thr Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gl y Glin Val Thir Ile Ser Ala Asp Llys Ser Ile Ser Thir Ala Tyr 65 70

Met Gl u Lieu. Ser Arg Lieu. Arg Ser Asp Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

SEQ ID NO 26 LENGTH: 120 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM1.24 H1.2

SEQUENCE: 26

Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Wall Pro Gly Ala 1. 15

Ser Val Llys Val Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Pro Tyr 2O 25

Trp Met Gln Trp Val Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Ser Gly Asp Thr Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gl y Glin Val Thir Ile Ser Ala Asp Llys Ser Ile Ser Thir Ala Tyr 65 70

Met Gl u Lieu. Ser Arg Lieu. Arg Ser Asp Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

SEO ID NO 27 LENGTH: 120 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM1.24 H1.3

SEQUENCE: 27

Glin Wa l Glin Lieu Val Glin Ser Gly Ala Glu Wall Pro Gly Ala 1. 5 1O 15

Ser Wa l Llys Val Ser Cys Lys Ala Ser Gly Thir Phe Thir Pro Tyr 2O 25

Trp Me t Gln Trp Val Arg Glin Ala Pro Gly Glin Gly Lell Glu Trp Met 35 4 O 45

Gly Se r Ile Phe Pro Gly Asp Ser Asp Thr Arg Tyr Ser Glin Ser Phe SO 55 6 O US 9,040,042 B2 137 138 - Continued

Glin Gly Glin Wall. Thir Ile Ser Ala Asp Llys Ser Ile Ser Thr Ala Tyr 65 70 8O

Met Glu Lieu. Ser Arg Lell Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Glin 105 11 O

Gly Thir Lieu Val Thir Wall Ser Ser 115 12 O

SEQ ID NO 28 LENGTH: 12O TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM1.24 H1.4

<4 OOs, SEQUENCE: 28

Glin Wall Glin Lieu. Wall Glin Ser Gly Ala Glu Wall Llys Pro Gly Ala 1. 5 1O 15

Ser Wall Lys Wall Ser Ala Ser Gly Tyr Thir Phe Thr Pro Tyr 25 3O

Trp Met Glin Trp Wall Arg Glin Ala Pro Gly Glin Gly Lieu. Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Ala Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Wall Thir Ile Ser Ala Asp Ser Ile Ser Thr Ala Tyr 65 70 75

Met Glu Luell Ser Arg Lell Arg Ser Asp Thir Ala Val Tyr Tyr Cys 85 95

Ala Arg Gly Luell Arg Arg Gly Gly Tyr Phe Asp Tyr Trp Gly Glin 105 11 O

Gly Thir Luell Wall Thir Wall Ser Ser 115 12 O

SEQ ID NO 29 LENGTH: 12O TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: HM1.24 H2.1

<4 OOs, SEQUENCE: 29

Glu Wall Glin Lieu. Wall Glu Ser Gly Gly Gly Luell Wall Llys Pro Gly Gly 1. 5 1O 15

Ser Luell Arg Luell Ser Ala Ala Ser Gly Tyr Thir Phe Thr Pro Tyr 25 3O

Trp Met Glin Trp Wall Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Glu Gly Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Wall Thir Ile Ser Ala Asp Ser Ile Ser Thr Ala 65 70 7s

Met Glu Luell Ser Ser Lell Arg Ser Glu Thir Ala Val Tyr Tyr Cys 85 95

Ala Arg Gly Luell Arg Arg Gly Gly Tyr Phe Asp Tyr Trp Gly Glin 1OO 105 11 O

Gly Thir Luell Wall Thir Wall Ser Ser US 9,040,042 B2 139 140 - Continued

115 12 O

<210s, SEQ ID NO 3 O &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: HM1.24 H2.2

<4 OOs, SEQUENCE: 30

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Pro Gly Gly 1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Tyr Thir Phe Thir Pro Tyr 2O 25

Trp Met Glin Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Ser Asp Thr Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Val Thir Ile Ser Ala Asp Llys Ser Ile Ser Thir Ala Tyr 65 70

Met Glu Lieu. Ser Ser Lieu. Arg Ser Glu Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Thir Lieu Val Thir Wal Ser Ser 115 12 O

<210s, SEQ ID NO 31 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: HM1.24 H2.3

<4 OOs, SEQUENCE: 31

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Luell Wall Pro Gly Gly 1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Tyr Thir Phe Thir Pro Tyr 2O 25

Trp Met Glin Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Ser Asp Thr Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Val Thir Ile Ser Ala Asp Llys Ser Ile Ser Thir Ala Tyr 65 70

Met Glu Lieu. Ser Ser Lieu. Arg Ser Glu Asp Thir Ala Wall Tyr Cys 85 90 95

Ala Arg Gly Lieu. Arg Arg Gly Gly Tyr Tyr Phe Asp Trp Gly Glin 1OO 105 11 O

Gly Thir Lieu Val Thir Wal Ser Ser 115 12 O

<210s, SEQ ID NO 32 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: HM1.24 H2.4

<4 OOs, SEQUENCE: 32 US 9,040,042 B2 141 142 - Continued

Glu Wall Glin Luell Val Glu Ser Gly Gly Gly Luell Wall Lys Pro Gly Gly 1O 15

Ser Luell Arg Luell Ser Cys Ala Ala Ser Gly Thir Phe Thir Pro 25

Trp Met Glin Trp Val Arg Glin Ala Pro Gly Gly Lell Glu Trp Met 35 4 O 45

Gly Ser Ile Phe Pro Gly Asp Ala Asp Thir Arg Tyr Ser Glin Ser Phe SO 55 6 O

Glin Gly Glin Wall Thir Ile Ser Ala Asp Ser Ile Ser Thir Ala Tyr 65 70 7s

Met Glu Luell Ser Ser Lieu. Arg Ser Glu Thir Ala Wall Tyr 85 95

Ala Arg Gly Luell Arg Arg Gly Gly Tyr Phe Asp Trp Gly Glin 105 11 O

Gly Thir Luell Wall Thir Wal Ser Ser 115 12 O

<210s, SEQ ID NO 33 &211s LENGTH: 330 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 33

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thr Ser Gly Gly Thr Ala Ala Lieu Gly Lieu Wall Lys Asp 25

Phe Pro Glu Pro Wall. Thir Wal Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Ser SO 55 6 O

Lell Ser Ser Wall Wall. Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Asn Val Asn His Lys Pro Ser ASn Thir Wall Asp 85 90 95

Wall Glu Pro Llys Ser Cys Asp Lys Thir His Thir Pro Pro 105 11 O

Pro Ala Pro Glu Lieu. Lieu. Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Asp Thir Lieu Met Ile Ser Arg Thir Pro Glu Wall Thir 13 O 135 14 O

Wall Wall Wall Asp Wal Ser His Glu Asp Pro Glu Wall Phe Asn Trp 145 150 155 160

Wall Asp Gly Wall Glu Wal His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Glin Asn Ser Thr Tyr Arg Wall Wall Ser Wall Lell Thir Wall Luell 18O 185 19 O

His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195 2OO 2O5

Ala Luell Pro Ala Pro Ile Glu Thir Ile Ser Ala Gly 21 O 215 22O

Glin Pro Arg Glu Pro Glin Val Tyr Thir Luell Pro Pro Ser Arg Asp Glu 225 23 O 235 24 O

Lell Thir Asn Glin Wal Ser Lieu Thir Cys Luell Wall Gly Phe Tyr US 9,040,042 B2 143 144 - Continued

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Lys Thir Thir Pro Pro Wall Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285

Lell Tyr Ser Lieu. Thr Val Asp Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Ser Wal Met His Glu Ala Luell His Asn His Thir 3. OS 310 315 32O

Glin Ser Luell Ser Leu Ser Pro Gly Lys 3.25 330

<210s, SEQ ID NO 34 &211s LENGTH: 330 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 34

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thir Ser Gly Gly. Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp 25

Phe Pro Glu Pro Wall. Thir Wal Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wall Wall. Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Asn Val Asn His Lys Pro Ser ASn Thir Wall Asp 85 90 95

Wall Glu Pro Llys Ser Cys Asp Lys Thir His Thir Pro Pro 105 11 O

Pro Ala Pro Glu Lieu. Lieu. Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Asp Thir Lieu Met Ile Ser Arg Thir Pro Glu Wall Thir 13 O 135 14 O

Wall Wall Wall Asp Wal Ser His Glu Asp Pro Glu Wall Phe Asn Trp 145 150 155 160

Wall Asp Gly Wall Glu Wal His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Glin Tyr Asn Ser Thr Tyr Arg Wall Wall Ser Wall Lell Thir Wall Luell 18O 185 19 O

His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195 2OO 2O5

Ala Luell Pro Ala Pro Ile Glu Thir Ile Ser Ala Gly 21 O 215 22O

Glin Pro Arg Glu Pro Glin Val Tyr Thir Luell Pro Pro Ser Arg Asp Glu 225 23 O 235 24 O

Lell Thir Asn Glin Wal Ser Lieu Thir Cys Luell Wall Gly Phe Tyr 245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Lys Thir Thir Pro Pro Wall Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285 US 9,040,042 B2 145 146 - Continued

Lell Tyr Ser Lys Lieu. Thr Wall Asp Llys Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Cys Ser Wall Met His Glu Gly Lieu. His Asn His Tyr Thr 3. OS 310 315

Glin Lys Ser Lieu. Ser Lell Ser Pro Gly Lys 3.25 330

<210s, SEQ ID NO 35 &211s LENGTH: 330 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 35

Ala Ser Thr Lys Gly Pro Ser Wall Phe Pro Luell Ala Pro Ser Ser 1. 5 15

Ser Thir Ser Gly Gly Thir Ala Ala Luell Gly Cys Lell Wall Lys Asp Tyr 25

Phe Pro Glu Pro Wall Thir Wall Ser Trp Asn Ser Gly Ala Luell Thir Ser 35 4 O 45

Gly Wall His Thir Phe Pro Ala Wall Luell Glin Ser Ser Gly Luell Tyr Ser SO 55 6 O

Lell Ser Ser Wall Wall Thir Wall Pro Ser Ser Ser Lell Gly Thir Glin Thir 65 70 8O

Ile Asn Wall Asn His Pro Ser ASn Thir Wall Asp 85 90 95

Arg Wall Glu Pro Ser Asp Lys Thir His Thir Pro Pro 105 11 O

Pro Ala Pro Glu Lell Lell Gly Gly Pro Ser Wall Phe Lell Phe Pro Pro 115 12 O 125

Pro Asp Thir Lell Met Ile Ser Arg Thir Pro Glu Wall Thir 13 O 135 14 O

Wall Wall Wall Asp Wall Ser His Glu Asp Pro Glu Wall Phe Asn Trp 145 150 155 160

Wall Asp Gly Wall Glu Wall His Asn Ala Thir Pro Arg Glu 1.65 17O

Glu Glin Tyr Asn Ser Thir Arg Wall Wall Ser Wall Lell Thir Wall Luell 18O 185 19 O

His Glin Asp Trp Lell Asn Gly Lys Glu Tyr Lys Wall Ser Asn 195 2O5

Ala Luell Pro Ala Pro Ile Glu Thir Ile Ser Ala Gly 21 O 215 22O

Glin Pro Arg Glu Pro Glin Wall Thir Luell Pro Pro Ser Arg Glu Glu 225 23 O 235 24 O

Met Thir Asn Glin Wall Ser Luell Thir Cys Luell Wall Gly Phe Tyr 245 250 255

Pro Ser Asp Ile Ala Wall Glu Trp Glu Ser ASn Gly Glin Pro Glu Asn 26 O 265 27 O

Asn Lys Thir Thir Pro Pro Wall Luell Asp Ser Asp Gly Ser Phe Phe 27s 28O 285

Lell Tyr Ser Lell Thir Wall Asp Ser Arg Trp Glin Glin Gly Asn 29 O 295 3 OO

Wall Phe Ser Ser Wall Met His Glu Ala Luell His Asn His Thir 3. OS 310 315 32O

Glin Ser Luell Ser Lell Ser Pro Gly Lys 3.25 330