CN116801871A - Combination therapy - Google Patents

Abstract

Provided herein are methods of treating cancer with a combination of an antibody that binds to an immune cell adapter and an antibody-drug conjugate.

Description

Combination therapy

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/111,045 filed on November 8, 2020, U.S. Provisional Application No. 63/172,411 filed on April 8, 2021, and U.S. Provisional Application No. 63/208,179 filed on June 8, 2021, each of which is incorporated herein by reference in its entirety for any purpose.

Technical Field

Provided herein are methods of treating cancer using a combination of antibodies that bind to immune cell engagers and antibody-drug conjugates.

Background Art

Immuno-oncology therapeutics and antibody-drug conjugates (ADCs) have been used to treat cancer in patients. Neither class of therapeutics can treat all patients for the target indication. The present disclosure addresses this and other problems.

Summary of the invention

Embodiment 1. A method of treating cancer, comprising administering to a subject having cancer: (1) an antibody-drug conjugate (ADC) comprising a first antibody that binds to a tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is a tubulin disrupting agent; and (2) a second antibody that binds to an immune cell engager, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs, wherein the activating FcγRs include one or more of FcγRIIIa, FcγRIIa and/or FcγRI.

Embodiment 2. The method of embodiment 1, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa.

Embodiment 3. The method of embodiment 1, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRIIa.

Embodiment 4. The method of embodiment 1, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRI.

Embodiment 5. The method of embodiment 1, wherein the second antibody comprises an Fc that enhances binding to FcγRIIIa, FcγRIIa, and FcγRI.

Embodiment 6. The method of any one of embodiments 1 to 5, wherein the Fc of the second antibody reduces binding to one or more inhibitory FcγRs.

Embodiment 7. The method of embodiment 6, wherein the Fc of the second antibody reduces binding to FcγRIIb.

Embodiment 8. The method of any one of embodiments 1 to 7, wherein the Fc of the second antibody has reduced fucose levels and/or has been engineered to contain one or more mutations such that the Fc enhances binding to one or more activating FcγRs.

Embodiment 9. The method of embodiment 8, wherein the second antibody is non-fucosylated.

Embodiment 10. The method of embodiment 8, wherein the second antibody comprises substitutions S293D, A330L, and I332E in the heavy chain constant region.

Embodiment 11. A method for treating cancer, comprising administering an antibody-drug conjugate to a subject having cancer, wherein the antibody-drug conjugate comprises: a first antibody conjugated to a cytotoxic agent, wherein the cytotoxic agent is a microtubule disrupting agent; and a second antibody that binds to an immune cell engager, wherein the second antibody is non-fucosylated.

Embodiment 12. The method of any one of Embodiments 1 to 11, wherein the first antibody binds to a tumor-associated antigen.

Embodiment 13. A method of treating cancer comprising administering to a subject having cancer: (1) an antibody-drug conjugate (ADC), wherein the ADC comprises a first antibody that binds a tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is a microtubule disrupting agent; and (2) a second antibody that binds an immune cell engager, wherein the second antibody comprises an Fc having enhanced ADCC activity relative to a corresponding wild-type Fc of the same isotype.

Embodiment 14. The method of embodiment 13, wherein the second antibody comprises an Fc having enhanced ADCC and ADCP activities relative to a corresponding wild-type Fc of the same isotype.

Embodiment 15. The method of embodiment 13 or 14, wherein the second antibody is non-fucosylated.

Embodiment 16. The method of any one of embodiments 13 to 15, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs, wherein the activating FcγRs include one or more of FcγRIIIa, FcγRIIa, and/or FcγRI.

Embodiment 17. The method of embodiment 16, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa.

Embodiment 18. The method of embodiment 16, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRIIa.

Embodiment 19. The method of embodiment 16, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRI.

Embodiment 20. The method of embodiment 16, wherein the second antibody comprises an Fc that enhances binding to FcγRIIIa, FcγRIIa, and FcγRI.

Embodiment 21. The method of any one of Embodiments 13 to 20, wherein the Fc of the second antibody reduces binding to one or more inhibitory FcγRs.

Embodiment 22. The method of embodiment 21, wherein the Fc of the second antibody reduces binding to FcγRIIb.

Embodiment 23. A method as described in any one of embodiments 1 to 22, wherein the first antibody binds to an antigen selected from the group consisting of: 5T4 (TPBG), ADAM-9, AG-7, ALK, ALP, AMHRII, APLP2, ASCT2, AVB6, AXL (UFO), B7-H3 (CD276), B7-H4, BCMA, C3a, C3b, C4.4a (LYPD3), C5, C5a, CA6, CA9, CanAg, carbonic anhydrase IX (CAIX), cathepsin D, CCR7, CD1, CD10, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107 a, CD107b, CD108, CD109, CD111, CD112, CD113, CD116, CD117, CD118, CD119, CD11A, CD11b, CD11c, CD120a, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD13, CD13 0. CD131, CD132, CD133, CD135, CD136, CD137, CD138, CD14, CD140a, CD140b, CD141, CD142, CD143, CD144, CD146, CD147, CD148, C D15, CD150, CD151, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158b2, CD158e, CD158f1, CD158h, CD158i, CD159a, CD16, CD160, CD161, CD162, CD163, CD164, CD166, CD16 7b, CD169, CD16a, CD16b, CD170, CD171, CD172a, CD172b, CD172g, CD18, CD180, CD181, CD183, CD184, CD185, CD19, CD194, CD197, CD1 a, CD1b, CD1c, CD1d, CD2, CD20, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD208, CD21, CD213a1, CD213a2, CD217, CD218a, CD22, CD220, CD221, CD222, CD224, CD226, CD 228, CD229, CD23, CD230, CD232, CD239, CD243, CD244, CD248, CD249, CD25, CD26, CD265, CD267, CD269, CD27, CD272, CD273, CD27 4. CD275, CD279, CD28, CD280, CD281, CD282, CD283, CD284, CD289, CD29, CD294, CD295, CD298, CD3, CD3ε, CD30, CD300f, CD302, CD304, CD305, CD307, CD31, CD312, CD315, CD316, CD317, CD318, CD319, CD32, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD32b, CD33, CD331, CD332, CD333, CD334, CD337, C D339, CD34, CD340, CD344, CD35, CD352, CD36, CD37, CD38, CD39, CD3d, CD3g, CD4, CD41, CD42d, CD44, CD44v6, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD5, CD50, CD51, CD51 (integrin alpha-V), CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD6, CD61, CD62L, CD62P, CD63, CD64, CD66a-e, CD67, C D68, CD69, CD7, CD70, CD70L, CD71, CD71 (TfR), CD72, CD73, CD74, CD79a, CD79b, CD8, CD80, CD82, CD83, CD84, CD85f, CD85i, CD85j, CD86, CD87, CD89, CD90, CD91, CD92, CD95, CD96, CD97, CD98, CDH6, CDH6 (cadherin 6), CDw210a, CDw210b, CEA, CEACAM5, CEACAM6, CFC1B, cKIT, CLDN18.2 (clin 18.2), CLDN6, CLDN9, C LL-1, c-MET, complement factor C3, Cripto, CSP-1, CXCR5, DCLK1, DLK-1, DLL3, DPEP3, DR5 (death receptor 5), anti-adhesin (Dysadherin), EFNA4, EGFR, EGFR wild type, EGFRviii, EGP-1 (TROP-2), EGP-2, EMP2, ENPP3, EpCAM, EphA2, EphA3, Ephrin-A4 (EFNA4), ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FOLR, FOLR1, FOLR-α, FSH, GCC, GD2, GD3, globo H, GPC1, GPC-1, GPC3, GPNMB, GPR20, HER2, HER-2, HER3, HER-3, HGFR (c-Met), HLA-DR, HM1.24, HSP90, Ia, IGF-1R, IL-13R, IL-15, IL1RAP, IL-2, IL-3, IL-4, IL7R, integrin alphaVbeta3/integrinαVβ3, integrin beta-6, interleukin-4 receptor (IL4R), KAAG-1, KLK2, LAMP-1, Le(y), Lewis Y antigen (Lewis Y antigen), LGALS3BP, LGR5, LH/hCG, LHRH, lipid rafts, LIV-1 (SLC39A6 or ZIP6), LRP-1, LRRC15, LY6E, macrophage mannose receptor 1, MAGE, mesothelin (MSLN), MET, MHC class I chain-associated proteins A and B (MICA and MICB), MN/CA IX, MRC2, MT1-MMP, MTX3, MTX5, MUC1, MUC16, MUC2, MUC3, MUC4, MUC5, MUC5ac, NaPi2b, NCA-90, NCA-95, connexin-4, Notch3, nucleolin, OAcGD2, OT-MUC1 (tumor tethered MUC1), OX001L, P1GF, PAM4 antigen, p-cadherin (cadherin 3), PD-L1, phosphatidylserine (PS), PRLR, Lactin receptor (PRLR), Pseudomonas, PSMA, PTK4, PTK7, receptor tyrosine kinase (RTK), RNF43, ROR1, ROR2, SAIL, SEZ6, SLAMF7, SLC44A4, SLITRK6, SLMAMF7 (CS1), SLTRK6, Sortilin (SORT1), SSEA-4, SSTR2, Staphylococcus aureus aureus) (antibiotic agent), STEAP-1, STING, STn, T101, TAA, TAC, TDGF1, tenascin, TENB2, TGF-B, Thomson-Friedenreich antigen, Thy1.1, TIM-1, tissue factor (TF; CD142), TM4SF1, Tn antigen, TNF-alpha (TNFα), TRA-1-60, TRAIL receptors (R1 and R2), TROP-2, tumor-associated glycoprotein 72 (TAG-72), uPAR, VEGFR, VEGFR-2, and xCT.

Embodiment 24. The method of any one of embodiments 1 to 23, wherein the first antibody does not bind to connexin-4.

Embodiment 25. The method of any one of Embodiments 1 to 24, wherein the method does not comprise administering an antibody-drug conjugate comprising an antibody that binds connexin-4.

Embodiment 26. The method of any one of embodiments 1 to 25, wherein the first antibody binds to an antigen selected from the group consisting of CD71, Axl, AMHRII and LGR5, Axl, CA9, CD142, CD20, CD22, CD228, CD248, CD30, CD33, CD37, CD48, CD7, CD71, CD79b, CLDN18.2, CLDN6, c-MET, EGFR, EphA2, ETBR, FCRH5, GCC, GloboH, gpNMB, HER-2, IL7R, integrin β-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, mesothelin (MSLN), MET, MRC2, MUC16, NaPi2b, catenin-4, OT-MUC1 (tumor tethered-MUC1), PSMA, ROR1, SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, and tumor-associated glycoprotein 72 (TAG-72).

Embodiment 27. The method of any one of Embodiments 1 to 25, wherein the first antibody binds to an antigen selected from the group consisting of BCMA, GPC1, CD30, cMET, SAIL, HER3, CD70, CD46, CD48, HER2, 5T4, ENPP3, CD19, EGFR, and EphA2.

Embodiment 28. A method as described in any one of embodiments 1 to 25, wherein the first antibody binds to an antigen selected from the group consisting of: Her2, TROP2, BCMA, cMet, integrin αVβ6, CD22, CD79b, CD30, CD19, CD70, CD228, CD47 and CD48.

Embodiment 29. A method as described in any of embodiments 1 to 25, wherein the first antibody binds to an antigen selected from the group consisting of: CD142, integrin beta-6, integrin alpha V beta 6, ENPP3, CD19, Ly6E, cMET, C4.4a, CD37, MUC16, STEAP-1, LRRC15, SLITRK6, ETBR, FCRH5, Axl, EGFR, CD79b, BCMA, CD70, PSMA, CD79b, CD228, CD48, LIV-1, EphA2, SLC44A4, CD30, and sTn.

Embodiment 30. The method of any one of embodiments 1 to 26, wherein the tubulin disrupting agent is an auristatin, tubulysin, colchicine, a vinca alkaloid, a taxane, a cryptophycin, a maytansinoid, or a hemiasterlin.

Embodiment 31. The method of embodiment 30, wherein the tubulin disrupting agent is auristatin.

Embodiment 32. The method of any one of embodiments 1 to 31, wherein the tubulin disrupting agent is dolostatin-10, MMAE (N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), MMAF (N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), auristatin F, AEB, AEVB or AFP (auristatin phenylalanine phenylenediamine).

Embodiment 33. The method of any one of embodiments 1 to 32, wherein the tubulin disrupting agent is MMAE.

Embodiment 33-1. A method as described in Embodiment 33, wherein the antibody-drug conjugate comprises MMAE and is selected from: DP303c, also known as SYSA1501, which targets HER-2 (CSPC Pharmaceutical; Dophen Biomed); SIA01-ADC, also known as ST1, which targets STn (Siamab Therapeutics); Ladiratuzumab vedotin, also known as SGN-LIV1A, which targets LIV-1 (Merck & Co., Inc.; Seagen (Seattle Genetics) Inc.); ABBV-085, also known as Samrotamab vedotin, which targets LRRC15 (Abbvie; Seagen (Seattle Genetics) Inc.); DMOT4039A, also known as RG7600; αMSLN-MMAE, which targets mesothelin (MSLN) (Roche-Genentech); RC68, also known as Remegen EGFR ADC, which targets EGFR (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.)); RC108, also known as RC108-ADC, which targets c-MET (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.)); CMG901, also known as MRG005, which targets CLDN18.2 (Keymed Biosciences; Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)); YBL-001, also known as LCB67, which targets DLK-1 (Lego Chem Biosciences; Pyxis Oncology; Y-Biologics); DCDS0780A, also known as Iladatuzumab vedotin; RG7986, which targets CD79b (Roche-Genentech; Seagen (Seattle Genetics) Inc.); Tisotumab vedotin, also known as Humax-TF-ADC; tf-011-mmae; TIVDAK ^™ , which targets CD142 (GenMab; Seagen (Seattle Genetics) Inc.); GO-3D1-ADC, also known as humAb-3D1-MMAE ADC, which targets MUC1-C (Genus Oncology LLC); ALT-P7, also known as HM2-MMAE, which targets HER-2 (Alteogen, Inc.; Levena Biopharma; 3SBio, Inc.); Vandortuzumab vedotin, also known as DSTP3086S; RG7450, which targets STEAP-1 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); Lifastuzumab vedotin, also known as DSTP3086S; RG7450, which targets STEAP-1 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); Vedotin), also known as DNIB0600A; NaPi2b ADC; RG7599, which targets NaPi2b (Roche-Genentech); Sofituzumab vedotin, also known as DMUC5754A; RG7458, which targets MUC16 (Seagen (Seattle Genetics) Inc.; Roche-Genentech); RG7841, also known as DLYE5953A, which targets Ly6E (Roche-Genentech; Seagen (Seattle Genetics) Inc.); RG7598, also known as DFRF4539A, which targets FCRH5 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); RG7636, also known as DEDN6526A, which targets ETBR (Seagen (Seattle Genetics) Inc.); Genetics) Inc.; Roche-Genentech); Pinatuzumab vedotin, also known as DCDT2980S; RG7593, which targets CD22 (Roche-Genentech); Polatuzumab vedotin, also known as DCDS4501A; POLIVY ^™ ; RG7596; RO-5541077, which targets CD79b (Chugai Pharmaceutical; Roche-Genentech; Seagen (Seattle Genetics) Inc.); DMUC4064A, also known as D-4064a; RG7882, which targets MUC16 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); SYSA1801, also known as CPO102, which targets CLDN18.2 (Conjupro Biotherapeutics Inc.; CSPC ZhongQi Pharmaceutical Technology Co.); RC118, also known as claudin 18.2-ADC; YH005, which targets CLDN18.2 (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Biocytogen); VLS-101, also known as Cirmtuzumab vedotin; MK-2140; UC-961ADC3; Zilovertamab Vedotin, which targets ROR1 (VelosBio. Inc); Glembatumumab vedotin, also known as CDX-011; CR011-vcMMAE, which targets gpNMB (Celldex Therapeutics); BA3021, also known as CAB-ROR2-ADC; Ozuriftamab Vedotin), which targets ROR2 (Bioatla; Himalaya Therapeutics); BA3011, also known as CAB-AXL-ADC; Mecbotamab Vedotin, which targets Axl (Bioatla; Himalaya Therapeutics); CM-09, also known as Bstrongximab-ADC, which targets TRA-1-60 (CureMeta); ABBV-838, also known as Azintuxizumab vedotin, which targets SLAMF7 (Abbvie); Enapotamab vedotin, also known as AXL-107-MMAE; HuMax-AXL-ADC, which targets Axl (GenMab; Seagen (Seattle Genetics) Inc.); ARC-01, also known as anti-CD79b ADC, which targets CD79b (Araris Biotech AG); Disitamab vedotin, also known as RC48, which targets HER-2 (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Seagen (Seattle Genetics) Inc.); ASG-5ME, also known as AGS-5; AGS-5ME, which targets SLC44A4 (Agensys, Inc.; Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); Enfortumab vedotin, also known as AGS-22M6E; ASG-22CE; ASG-22ME; PADCEV ^™ , which targets connexin-4 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); ASG-15ME, also known as AGS-15E; Sirtratumab vedotin, which targets SLITRK6 (Seagen (Seattle Genetics) Inc.; Astellas Pharma Inc.); Brentuximab vedotin, also known as Adcetris; cAC10-vcMMAE; SGN-35, which targets CD30 (Seagen (Seattle Genetics) Inc.; Takeda); Telisotuzumab vedotin, also known as ABBV-399, which targets c-MET (Abbvie); Losatuxizumab vedotin, also known as ABBV-221, which targets EGFR (Abbvie); CX-2029, also known as ABBV-2029, which targets CD71 (Abbvie; CytomX Therapeutics); AB-3A4-ADC, also known as AB-3A4-vcMMAE, which targets KAAG-1 (Alethia Biotherapeutics); Indusatumab vedotin), also known as 5F9-vcMMAE; MLN0264; TAK-264, which targets GCC (Takeda; Millennium Pharmaceuticals, Inc); FOR46, which targets CD46 (Fortis Therapeutics, Inc.); LR004-VC-MMAE, which targets EGFR (Chinese Academy of Medical Sciences Peking Union Medical College Hospital); CD30-ADC, which targets CD30 (NBE Therapeutics; Boehringer Ingelheim); anti-endosialin-MC-VC-PABC-MMAE, which targets CD248 (Genzyme); OBI-998, which targets SSEA-4 (OBI Pharma); MRG002, which targets HER-2 (Lepubiotech; Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd.)); TRS005, which targets CD20 (Teruisi Pharmaceuticals); Oba01, which targets DR5 (death receptor 5) (ObioTechnology (Shanghai) Corp., Ltd.; Yantai Obioadc Biomedical Technology Ltd.); PSMA ADC, which targets PSMA (Progenics Pharmaceuticals, Inc; Seagen (Seattle Genetics) Inc.); SGN-CD48A, which targets CD48 (Seagen (Seattle Genetics) Inc.); IMAB362-vcMMAE, which targets CLDN18.2 (Astellas Pharma Inc.; Ganymed); GB251, which targets HER-2 (Genor Biopharma Co., Ltd.); Innate Pharma BTG-ADC, which targets CD30 (Innate Pharma; Sanofi); ADCendouPARAP ADC, which targets MRC2 (ADCendo); XCN-010, which targets actM (Xiconic Pharmaceuticals, LLC); ANT-043, which targets HER-2 (Antikor Biopharma); OBI-999, which targets Globo H (Abzena; OBIPharma); LY3343544, which targets MET (Eli Lilly and Company); Tagworks anti-TAG72 ADC, which targets TAG-72 (Tagworks Pharmaceuticals); IMAB027-vcMMAE, which targets CLDN6 (Ganymed; AstellasPharma Inc.); LGR5-ADC, which targets LGR5 (Genentech, Inc.); Philochem B12-MMAE ADC, which targets IL-7R (Instituto de Medicina Molecular Biology, Inc.); Lobo Antunes; Philochem AG); TE-1522, which targets CD19 (Immunwork); SGN-STNV, which targets STn (Seagen (Seattle Genetics) Inc.); HTI-1511, which targets EGFR (Abzena; Halozyme Therapeutics); Peptron PAb001-ADC, which targets OT-MUC1 (tumor tethered-MUC1) (Peptron; Qilu Pharmaceutical co. Ltd.); LM-102, which targets CLDN18.2 (LaNova Medicines Limited); Anwita Biosciences MSLN-MMAE, which targets mesothelin (MSLN) (Anwita biosciences); SGN-CD228A, which targets CD228 (Seagen (Seattle Genetics) Inc.); NBT828, which targets HER-2 (NewBio Therapeutics; Genor Biopharma Co., Ltd.); Gamamabs GM103, which targets AMHR2 (GamaMabs Pharma; Exelixis); LCB14-0302, which targets HER-2 (Lego Chem Biosciences); BAY79-4620, which targets carbonic anhydrase IX (CAIX) (Bayer; MorphoSys); NBT508, which targets CD79b (NewBio Therapeutics); PAT-DX3-MMAE, which targets an undisclosed target (Patrys; Yale University); AGS67E, which targets CD37 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); CDX-014, which targets TIM-1 (Celldex Therapeutics); BVX001, which targets CD33; CD7 (Bivictrix therapeutics); SGN-B6A, which targets integrin β-6 (Seagen (Seattle Genetics) Inc.); MRG003, which targets EGFR (Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)), and PYX-202, which targets DLK-1 (Pyxis Oncology; Lego Chem Biosciences).

Embodiment 34. A method as described in embodiment 33, wherein MMAE is bound to the first antibody through a linker comprising valine and citrulline.

Embodiment 35. The method of embodiment 34, wherein the linker-MMAE is vcMMAE.

Embodiment 36. A method as described in embodiment 33, wherein MMAE is bound to the first antibody through a linker comprising leucine, alanine and glutamic acid.

Embodiment 37. The method of embodiment 36, wherein the linker-MMAE is dLAE-MMAE.

Embodiment 38. The method of any one of embodiments 1 to 32, wherein the tubulin disrupting agent is MMAF.

Embodiment 38-1. A method as described in Embodiment 38, wherein the antibody-drug conjugate comprises MMAF and is selected from: CD70-ADC, which targets CD70 (Kochi University; Osaka University); IGN786, which targets SAIL (AstraZeneca; Igenica Biotherapeutics); PF-06263507, which targets 5T4 (Pfizer); GPC1-ADC, which targets GPC-1 (Kochi University); ADC-AVP10, which targets CD30 (Avipep); M290-MC-MMAF, which targets CD103 (The Second Affiliated Hospital of Harbin Medical University); BVX001, which targets CD33; CD7 (Bivictrix therapeutics); Tanabe P3D12-vc-MMAF, which targets c-MET (Tanabe Research Laboratories); LILRB4-targeted ADC, which targets LILRB4 (The University of Texas Health Science Center, Houston); TSD101, also known as ABL201, which targets BCMA (TSD LifeScience; ABL Bio; Lego Chem Biosciences); Depatuxizumab mafodotin, also known as ABT-414, which targets EGFR (Abbvie; Seagen (Seattle Genetics) Inc.); AGS16F, also known as AGS-16C3F; AGS-16M8F, which targets ENPP3 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); AVG-A11 BCMA ADC, also known as AVG-A11-mcMMAF, which targets BCMA (Avantgen); and Belantamab, also known as ABT-414, which targets EGFR (Abbvie; Seagen (Seattle Genetics) Inc.). mafodotin, also known as BLENREP; GSK2857916; J6M0-mcMMAF, which targets BCMA (GlaxoSmithKline; Seagen (Seattle Genetics) Inc.); MP-HER3-ADC, also known as HER3-ADC, which targets HER-3 (MediaPharma); FS-1502, also known as LCB14-0110, which targets HER-2 (Lego Chem Biosciences; Shanghai Fosun Pharmaceutical Development Co, Ltd.); MEDI-547, also known as MI-CP177, which targets EphA2 (AstraZeneca; Seagen (Seattle Genetics) Inc.); Vorsetuzumab mafodotin, also known as SGN-75, which targets CD70 (Seagen (Seattle Genetics) Inc.); Denintuzumab mafodotin, also known as SGN-75, which targets CD70 (Seagen (Seattle Genetics) Inc.); Mafodotin), also known as SGN-CD19A, which targets CD19 (Seagen (Seattle Genetics) Inc.) and HTI-1066, also known as SHR-A1403, which targets c-MET (Jiangsu HengRui Medicine Co., Ltd.).

Embodiment 39. The method of any one of embodiments 1 to 30, wherein the tubulin disrupting agent is tubulysin.

Embodiment 40. The method of embodiment 39, wherein tubulysin is selected from tubulysin D, tubulysin M, tubulysin phenylalanine, and tubulysin tyrosine.

Embodiment 41. A method as described in any one of embodiments 1 to 32, wherein the antibody-drug conjugate is selected from AbGn-107 (Ab1-18Hr1), AGS62P1 (ASP1235), ALT-P7 (HM2-MMAE), BA3011 (CAB-AXL-ADC), martin-belantamab, vebutuximab, visitozumab (VLS-101, UC-961ADC3), pertinentin cofetuzumab (cofetuzumab pelidotin)(PF-06647020, PTK7-ADC, PF-7020, ABBV-647), CX-2029(ABBV-2029), vedicizumab (RC48), veenumab (HuMax-AXL-ADC, AXL-107-MMAE), veenumab (EV), FS-1502(LCB14-0110), gemtuzumab ozogamicin, HTI-1066(SHR-A1403), inotuzumab ozogamicin, PF-06804103(NG-HER2ADC), vepolotuzumab, sacituzumab govitecan), SGN-B6A, SGN-CD228A, SGN-STNV, STI-6129 (CD38 ADC, LNDS1001, CD38-077 ADC), vetlitozumab (ABBV-399), vetizumab (Humax-TF-ADC, tf-011-mmae, TV), trastuzumab deruxtecan, trastuzumab emtansine, and martin-voseluzumab.

Embodiment 42. The method of any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-claudin-18.2 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 61 to 66, respectively.

Embodiment 43. A method as described in Embodiment 42, wherein the anti-claudin-18.2 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:60.

Embodiment 44. The method of embodiment 43, wherein the anti-claudin-18.2 antibody is zolbetuximab (175D10).

Embodiment 45. The method of any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-claudin-18.2 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 69 to 74, respectively.

Embodiment 46. A method as described in Embodiment 45, wherein the anti-claudin-18.2 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:67 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:68.

Embodiment 47. The method of any one of Embodiments 1 to 41, 33 to 1, and 38 to 1, wherein the first antibody is an anti-PD-L1 antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 77 to 82, respectively.

Embodiment 48. A method as described in Embodiment 47, wherein the anti-PD-L1 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:75 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:76.

Embodiment 49. The method of any one of embodiments 1 to 41, 33 to 1, and 38 to 1, wherein the first antibody is an anti-ALP antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 85 to 90, respectively.

Embodiment 50. A method as described in Embodiment 49, wherein the anti-ALP antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:83 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:84.

Embodiment 51. A method as described in any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody comprises an anti-B7H4 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93 to 98, respectively.

Embodiment 52. A method as described in Embodiment 51, wherein the anti-B7H4 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:91 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:92.

Embodiment 53. The method of any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-HER2 antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:100.

Embodiment 54. The method of embodiment 53, wherein the antibody-drug conjugate is vedicizumab.

Embodiment 55. A method as described in any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-NaPi2B antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:102.

Embodiment 56. The method of embodiment 55, wherein the antibody-drug conjugate is velifatuzumab.

Embodiment 57. The method of any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-connexin-4 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 105 to 110, respectively.

Embodiment 58. A method as described in Embodiment 57, wherein the anti-Connexin-4 antibody is an antibody comprising the following: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:103 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:104.

Embodiment 59. The method of embodiment 58, wherein the antibody-drug conjugate is venosumab.

Embodiment 60. The method of any one of embodiments 1 to 41, 33 to 1, and 38 to 1, wherein the first antibody is an anti-AVB6 antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 113 to 118, respectively.

Embodiment 61. A method as described in Embodiment 60, wherein the anti-AVB6 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:111 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:112.

Embodiment 62. The method of any one of embodiments 1 to 41, 33 to 1, and 38 to 1, wherein the first antibody is an anti-AVB6 antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 121 to 126, respectively.

Embodiment 63. A method as described in Embodiment 62, wherein the anti-AVB6 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:119 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:120.

Embodiment 64. A method as described in any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-CD228 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 129 to 134, respectively.

Embodiment 65. A method as described in Embodiment 64, wherein the anti-CD228 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:127 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:128.

Embodiment 66. The method of any one of embodiments 1 to 41, 33 to 1, and 38 to 1, wherein the first antibody is an anti-LIV-1 antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137 to 142, respectively.

Embodiment 67. A method as described in Embodiment 66, wherein the anti-LIV-1 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:135 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:136.

Embodiment 68. The method of any one of embodiments 1 to 41, 33 to 1 and 38 to 1, wherein the first antibody is an anti-tissue factor antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 145 to 150, respectively.

Embodiment 69. A method as described in Embodiment 68, wherein the anti-tissue factor antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:143 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:144.

Embodiment 70. The method of embodiment 69, wherein the antibody-drug conjugate is vesicumab.

Embodiment 71. The method of any one of embodiments 1 to 70, 33 to 1, and 38 to 1, wherein the second antibody binds to an immune cell engager selected from the group consisting of: anti-Mullerian hormone receptor HormoneReceptor)II(AMHR2), B7, B7H1, B7H2, B7H3, B7H4, BAFF-R, BCMA (B cell maturation antigen), Bst1/CD157, C5 complement, CC chemokine receptor 4 (CCR4), CD123, CD137, CD19, CD20, CD25 (IL2RA), CD276, CD278, CD3, CD32, CD33, CD37, CD38, CD4 and HIV-1gp120 binding site, CD40, CD70, CD70 (a member of the TNF receptor ligand family), CD80, CD86, claudin 18.2, c-MET, CSF1R, CTLA-4, EGFR, EGFR MET oncogene, EPHA3, ERBB2, ERBB3, FGFR2b, FLT3, GITR, glucocorticoid-induced TNF receptor (GITR), HER2, HER3, HLA, ICOS, IDO1, IFNAR1, IFNAR2, IGF-1R, IL-3Rα (CD123), IL-5R, IL-5Rα, LAG-3, MET oncogene, OX40 (CD134), PD-1, PD-L1, PD-L2, PVRIG, EBOV glycoprotein The respiratory syncytial virus (RSV) heavily glycosylated mucin-like domain of protein (GP), rhesus macaque (Rh) D, sialic acid immunoglobulin-like lectin 8 (Siglec-8), signaling lymphocyte activation molecule (SLAMF7/CS1), T cell receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA4), TIGIT, TIM3 (HAVCR2), tumor-specific glycoepitope of Muc1 (TA-Muc1), VSIR (VISTA) and VTCN1.

Embodiment 72. The method of any one of Embodiments 1 to 71, 33-1, and 38-1, wherein the second antibody binds TIGIT.

Embodiment 73. A method as described in Embodiment 72, wherein the second antibody comprises: (a) a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 7 to 9; (b) a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 10 to 13; (c) a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 14 to 16; (d) a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 17; (e) a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 18; and (f) a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 19.

Embodiment 74. A method as described in embodiment 72, wherein the second antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the following sequences, respectively: (a) SEQ ID NO:7, 10, 14, 17, 18 and 19; or (b) SEQ ID NO:8, 11, 14, 17, 18 and 19; or (c) SEQ ID NO:9, 12, 15, 17, 18 and 19; or (d) SEQ ID NO:8, 13, 16, 17, 18 and 19; or (e) SEQ ID NO:8, 12, 16, 17, 18 and 19.

Embodiment 75. A method as described in Embodiment 72, wherein the second antibody comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 1 to 5 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 6.

Embodiment 76. A method as described in Embodiment 72, wherein the second antibody comprises: a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20 to 24 and a light chain comprising the amino acid sequence of SEQ ID NO:25.

Embodiment 77. The method of any one of Embodiments 1 to 71, wherein the second antibody binds CD40.

Embodiment 78. A method as described in Embodiment 77, wherein the second antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the following sequences, respectively: (a) SEQ ID NO: 30, 31, 32, 33, 34 and 35; or (b) SEQ ID NO: 30, 36, 32, 33, 34 and 35.

Embodiment 79. A method as described in Embodiment 77, wherein the second antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:29.

Embodiment 80. A method as described in Embodiment 77, wherein the second antibody comprises: a heavy chain comprising an amino acid sequence of SEQ ID NO:26 and a light chain comprising an amino acid sequence of SEQ ID NO:27.

Embodiment 81. The method of any one of Embodiments 1 to 71, 33-1, and 38-1, wherein the second antibody binds CD70.

Embodiment 82. A method as described in Embodiment 81, wherein the second antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 53 to 58, respectively.

Embodiment 83. A method as described in Embodiment 81, wherein the second antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:42.

Embodiment 84. The method of any one of Embodiments 1 to 71, 33-1, and 38-1, wherein the second antibody binds BCMA.

Embodiment 85. A method as described in Embodiment 84, wherein the second antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 47 to 52, respectively.

Embodiment 86. A method as described in Embodiment 84, wherein the second antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:45 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:46.

Embodiment 87. The method of any one of Embodiments 1 to 86, 33-1, and 38-1, wherein the second antibody is an IgG1 or IgG3 antibody.

Embodiment 88. A method as described in any of embodiments 1 to 87, 33-1 and 38-1, wherein the second antibody is contained in an antibody composition, wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the antibodies in the composition are non-fucosylated.

Embodiment 89. A method as described in Embodiment 88, wherein each antibody in the composition comprises the same heavy chain and light chain amino acid sequences as the second antibody.

Embodiment 90. The method of any one of embodiments 1 to 89, 33 to 1, and 38 to 1, wherein the Fc of the second antibody enhances binding to one or more activating FcγRs compared to a corresponding wild-type Fc of the same isotype, wherein the activating FcγRs comprise one or more of FcγRIIIa, FcγRIIa, and/or FcγRI.

Embodiment 91. The method of Embodiment 90, wherein the Fc of the second antibody enhances binding to FcγRIIIa.

Embodiment 92. The method of any one of embodiments 1 to 91, 33-1, and 38-1, wherein the Fc of the second antibody has reduced binding to one or more inhibitory FcγRs compared to a corresponding wild-type Fc of the same isotype.

Embodiment 93. The method of Embodiment 92, wherein the Fc of the second antibody reduces binding to FcγRIIb.

Embodiment 94. The method of any one of embodiments 1 to 93, 33-1, and 38-1, wherein the Fc of the second antibody enhances binding to FcγRIIIa and reduces binding to FcγRIIb.

Embodiment 95. The method of any one of embodiments 1 to 94, 33-1, and 38-1, wherein the second antibody is a monoclonal antibody.

Embodiment 96. The method of any one of embodiments 1 to 95, 33 to 1, and 38 to 1, wherein the second antibody is a humanized antibody or a human antibody.

Embodiment 97. The method of any one of embodiments 1 to 96, 33 to 1, and 38 to 1, wherein the cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, renal clear cell carcinoma, head and neck cancer, lung cancer, lung adenocarcinoma, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, melanoma, central nervous system neoplasm, mesothelioma, lymphoma, leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, or sarcoma.

Embodiment 98. The method of any one of embodiments 1 to 97, 33 to 1, and 38 to 1, wherein the cancer is lymphoma, leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or Hodgkin lymphoma.

Embodiment 99. The method of any one of embodiments 1 to 98, 33-1, and 38-1, wherein the antibody-drug conjugate and the second antibody are administered simultaneously.

Embodiment 100. The method of embodiments 99, 33-1 and 38-1, wherein the antibody-drug conjugate and the second antibody are administered in the form of a single pharmaceutical composition.

Embodiment 101. The method of any one of embodiments 1 to 98, 33-1, and 38-1, wherein the antibody-drug conjugate and the second antibody are administered sequentially.

Embodiment 102. The method of embodiment 101, wherein at least the first dose of the antibody drug conjugate is administered before the first dose of the second antibody; or wherein at least the first dose of the second antibody is administered before the first dose of the antibody drug conjugate.

Embodiment 103. The method of any one of embodiments 1 to 102, 33-1, and 38-1, wherein the second antibody depletes regulatory T cells (Tregs).

Embodiment 104. The method of any one of Embodiments 1 to 103, 33-1, and 38-1, wherein the antibody-drug conjugate induces immune memory against cells expressing an antigen bound by the antibody drug conjugate.

Embodiment 105. A method as described in Embodiment 104, wherein immune memory induction comprises inducing memory T cells.

Embodiment 106. The method of any one of Embodiments 1 to 105, 33-1, and 38-1, wherein the second antibody activates antigen presenting cells (APCs).

Embodiment 107. The method of any one of Embodiments 1 to 106, 33-1, and 38-1, wherein the second antibody enhances the CD8 T cell response.

Embodiment 108. The method of any one of Embodiments 1 to 107, 33-1, and 38-1, wherein the second antibody upregulates a co-stimulatory receptor.

Embodiment 109. The method of any one of Embodiments 1 to 108, 33-1, and 38-1, wherein administration of the ADC and a second antibody promotes the release of immune-activating cytokines.

Embodiment 110. The method of embodiment 109, wherein the immune activating cytokine is CXCL10 or IFNγ.

Embodiment 111. The method of any one of Embodiments 1 to 110, 33-1, and 38-1, wherein the ADC acts synergistically with a second antibody.

Embodiment 112. The method of any one of embodiments 1 to 111, 33-1, and 38-1, wherein administration of the ADC and the second antibody in combination has a toxicity profile comparable to when the ADC or the second antibody is administered as monotherapy.

Embodiment 113. The method of any one of Embodiments 1 to 112, 33-1 and 38-1, wherein the effective dose of the ADC and/or the second antibody when administered in combination is less than the effective dose when administered as monotherapy.

Embodiment 114. The method of any one of Embodiments 1 to 113, 33-1, and 38-1, wherein the cancer has a high tumor mutation burden.

Embodiment 115. The method of any one of Embodiments 1 to 114, 33-1, and 38-1, wherein the cancer has microsatellite instability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that non-targeted chemotherapeutic agents impair T cell responses.

Figure 2 shows BV treatment of CD30+CD8 T cells. Vedotin ADC, which has been targeted to T cells, does not inhibit proliferation.

Fig. 3A to Fig. 3E show that compared with ADC with different payloads, endoplasmic reticulum (ER) stress induction is excellent for auristatin antibody-drug conjugates (ADC), such as ADC based on vedotin. Fig. 3A shows a table of various clinically approved ADC payloads. Fig. 3B shows a diagram of ER stress signaling reactions. Fig. 3C shows a Western blot analysis of MIA-PaCa-2 cells 36 or 48 hours treated with ADC or paclitaxel with different payloads at IC50 concentrations. Fig. 3D to Fig. 3E show MIA-PaCa-2 cells expressing CHOP driven luciferase reporter (Signosis, Inc.) treated with ADC with different payloads within the dosage range (Fig. 3D) or IC50 doses (cytotoxicity) (Fig. 3E). Compared with untreated cells, CHOP induction is expressed by multiple induction.

Figures 4A to 4B show the immunogenic cell death (ICD) potential of clinical ADC payloads. Supernatants were collected from MIA-PaCa-2 pancreatic tumor cells treated with 1 μg/mL ADC with different payloads for 72 hours. Figure 4A shows TP release as measured by Cell Titer Glo. Figure 4B shows HMGB1 secretion as measured by ELISA.

Fig. 5A to Fig. 5C show the immune activation assessment of ADC payload.After 48 hours of co-cultivation (24 hours at IC50 concentration) of L540cy cells of peripheral blood mononuclear cells (PBMC) and administration with ADCs with different payloads, the increase of MHC II class (HLA-DR) on myeloid cells in PBMC was assessed by flow cytometry.24 hours supernatant was assessed by Luminex multiple determination for cytokine levels.Fig. 5A shows the immune activation of ADC.Fig. 5B shows the expression of MHCII on monocytes exposed in response to ADC.Fig. 5C shows the expression of innate cytokine CXCL-10/IP10 in response to ADC exposure as a measure of immune cell activity.

Fig. 6A to Fig. 6E show the effective load evaluation about the main chain of trastuzumab. Fig. 6A shows the table of the trastuzumab ADC evaluated. Fig. 6B shows the figure of ER stress signaling reaction. Fig. 6C shows the protein blot of BT474 cells treated with ADC or drugs for 72 hours. Fig. 6D to Fig. 6E show that the vedotin ADC shows the strong activation of multiple ICD marks. The different ICD reactions of breast cancer cells expressing SKBR3 HER2 are shown with the trastuzumab ADC or free MMAE (100nM) administered with 1 μg/mL. After 48 or 72 hours of treatment, the culture medium is collected and used to measure ATP release (Fig. 6D) and HMGB1 levels (Fig. 6E).

FIG7A further illustrates the immunogenic cell death (ICD) pathway.

FIG. 7B provides information about the payload of certain ADCs used in FIGS. 7B-7E .

7C to 7F show JNK signaling activation in response to treatment with MMAE-ADC compared to maytansine-ADC ( FIG. 7C ), camptothecin-ADC ( FIG. 7D ), anthracycline-ADC ( FIG. 7E ), and calicheamicin-ADC ( FIG. 7F ).

Figures 8A to 8D show CHOP induction in response to treatment with MMAE-ADC compared to maytansine-ADC (Figure 8A), camptothecin-ADC (Figure 8B), anthracycline-ADC (Figure 8C), and other types of ADCs, including ozogamycin-ADC, teserine-ADC, and AT-ADC (Figure 8D).

Figures 9A to 9D show the release of ATP and HMGB1 in response to treatment with MMAE-ADC compared to maytansine-ADC (Figure 9A), camptothecin-ADC (Figure 9B), anthracycline-ADC (Figure 9C), and other types of ADCs, including ozogamicin-ADC, teisline-ADC, and AT-ADC (Figure 9D).

Figures 10A to 10D show MHCII expression and CXCL-10/IP10 release in response to treatment with MMAE-ADC compared to maytansine-ADC (Figure 10A), camptothecin-ADC (Figure 10B), anthracycline-ADC (Figure 10C), and other types of ADCs, including ozogamicin-ADC and teisline-ADC (Figure 10D).

FIG. 10E summarizes the results in FIGS. 7B to 7E , 8A to 8D , 9A to 9D , and 10A to 10D .

Figures 11A to 11D show antibodies binding to FcγRIIa, FcγRIIb and FcγRIIIa in Chinese hamster ovary (CHO cells). Figure 11A shows the antibodies evaluated. Figure 11B shows binding to FcγRIIa. Figure 11C shows binding to FcγRIIIa. Figure 11D shows binding to FcγRIIb.

Figures 12A to 12D show CXCL10 levels (Figure 12A), IFNγ levels (Figure 12B), IL10 levels (Figure 12C), and macrophage-derived cytokine levels (MDC; CCL22) (Figure 12D) in MIA-PaCa2 pancreatic cancer cells treated with CD40 agonists and chemotherapeutic agents.

Figures 13A-13D show CXCL10 levels (Figures 13A-13B) and IL10 levels (Figures 13C-13D) in melanoma cell lines treated with CD40 agonists and chemotherapy.

14A to 14C show CXCL14 levels ( FIG. 14A ), IL14 levels ( FIG. 14B ), and IFNγ levels ( FIG. 14C ) in tumor cells from melanoma, lung, breast, and pancreas.

15 shows in vivo data for the combination of SEA-CD40 vedotin and ADC chemotherapy combinations as evaluated by a human CD40 transgenic model where the tumor target antigen is Thy1.1.

FIG. 16 shows CXCL10 levels in tumor cell lines treated with a combination of ADC-MMAE against tumor-associated antigens and TIGIT-targeting antibodies with various effector function backbones.

FIG. 17 shows IFNγ levels in tumor cell lines treated with a combination of ADC-MMAE against tumor-associated antigens and TIGIT-targeting antibodies with various effector function backbones.

Figures 18A to 18C show in vitro and in vivo data showing the enhanced activity of non-fucosylated TIGIT antibodies and vc-MMAE ADC ("Vedotin ADC"). Figure 18A shows that when these antibodies are co-cultured with tumor cells killed by targeted vc-MMAE ADC, non-fucosylated TIGIT antibodies (SEA-TGT) with enhanced (non-fucosylated) IgG1 Fc main chains are significantly better at driving immune activation via cytokine IP10 induction than corresponding TIGIT antibodies with effector-deficient main chains (LALA) or standard wild-type IgG1 Fc main chains. Combinations with SEA-TGT show synergistic immune cell activation. Figures 18B and 18C show the anti-tumor response when mice implanted with CT26 syngeneic tumor cells (Figure 18B) or Renca syngeneic tumor cells (Figure 18C) were treated with: 1) suboptimal doses of ADC (Thy1.1 vc-MMAE ADC in Figure 18B and EphA2 vc-MMAE ADC in Figure 18C); 2) a series of suboptimal doses of mIgG2a SEA-TGT (reformatted into a non-fucosylated mouse IgG2a form of SEA-TGT antibody corresponding to the non-fucosylated human IgG1 backbone); or 3) a combination of the two agents. As can be seen from these figures, co-administration of these two agents significantly improved the efficacy of the treatment, including the generation of a high percentage of cure responses in these different tumor models.

Figure 19 shows in vivo data demonstrating the enhanced activity of afucosylated TIGIT antibody (SEA-TGT) and SGN-B7H4 vedotin ADC (B7H4V). Figure 19 shows the anti-tumor response when mice implanted with Renca syngeneic tumor cells were treated with subtherapeutic doses of SEA-TGT and subtherapeutic doses of B7H4V or subtherapeutic doses of SEA-TGT and therapeutic doses of oxaliplatin. As shown in Figure 19, co-administration of SEA-TGT and B7H4V significantly improved the efficacy of treatment, even at subtherapeutic doses, including the generation of a high percentage of cure responses.

Figures 20A-20B show the combined effect of SEA-CD70 (afucosylated anti-CD70 antibody) and SGN-35 (anti-CD30 ADC containing MMAE). Figure 20A shows an in vivo tumor growth evaluation of a non-Hodgkin lymphoma (NHL) xenograft model. Figure 20B shows the Kaplan-Meyer survival evaluation of the NHL xenograft model at the 500mm ³ tumor size endpoint. Tx (treatment) and arrows indicate the treatment start day (day 19 after implantation).

Figures 21A-21B show the synergistic effect of SEA-BCMA (afucosylated anti-BCMA antibody) and SGN-CD48A (anti-CD48 ADC containing MMAE). Figure 21A shows the in vivo survival evaluation of the xenograft model, and Figure 21B shows the in vivo luciferase evaluation of the xenograft model.

Figures 22A to 22C show that Vedotin ADC induces in vivo immune cell recruitment and activation. Figure 22A: Tumor xenografts isolated from animals were treated with vc-MMAE ADC or non-binding vc-MMAE isotype ADC for 8 days and subjected to flow cytometry or cytokine analysis. Figure 22B: CD45 positive immune cells were stained for CD11c and activation was observed by staining for MHC class II expression on the cell surface. Figure 22C: Intratumoral cytokines were measured by Luminex.

Figure 23A to Figure 23B show the induction of T cell memory by vc-MMAE ADC. In the Renca syngeneic model, mice were cured by vc-MMAE ADC treatment (Figure 23A). Mice cured by the treatment were attacked again with Renca tumor cells, and those mice rejected the tumor cells implanted subsequently (Figure 23B).

Figure 24 shows protective anti-tumor immunity conferred by cells treated with MMAE or vc-MMAE ADC. A20 cancer cells were treated with brentuximab (BV) or MMAE and dying and dying cells were administered to mice. Figure 24 shows that immunized mice exhibited a strong immune response, rejecting subsequently implanted A20 cells.

Figure 25 shows an exemplary model of receptor aggregation and agonism by non-fucosylated anti-CD40 antibody SEA-CD40; and an exemplary model of receptor agonism and synapse formation by non-fucosylated anti-TIGIT antibody SEA-TGT. As shown, the SEA-CD40 antibody can bind to CD-40 expressed on antigen presenting cells (APCs), wherein the Fc portion of the antibody binds to FcγRIIIa expressed on natural killer (NK) cells or monocytes, which promotes receptor aggregation. In contrast, the SEA-TGT antibody binds to TIGIT expressed on T cells and the Fc region of the antibody binds to FcγRIIIa expressed on APCs.

DETAILED DESCRIPTION

I. Introduction

Cell death by apoptosis is a silent, tolerogenic process. However, some cytotoxic agents, including specific anti-tumor agents, such as anthracyclines, oxaliplatin or radiation induce a characteristic form of cell death called immunogenic cell death (ICD). ICD is a kind of regulation of cell death mode/produces PBMC and T cells for the immune response of apoptotic cancer cells. As shown herein, with some tubulin destroyers, such as auristatin (such as MMAE and MMAF) treatment makes the protein usually found in ER exposed on the cell surface. Tumor antigens increase the absorption and presentation of phagocytes of T cells and then start the acquired immune system. As further shown herein, auristatin such as MMAE and MMAF can obviously drive ICD induction, so that the immune system can recognize and establish cytotoxic activity for tumors. In essence, cells that die from ICD serve as vaccines that stimulate tumor-specific immune responses for any residual disease or in the case of relapse/recurrence.

As shown in the experimental results described herein, tumor cells that undergo ICD in response to auristatins, such as MMAE and MMAF, exhibit a unique set of features that enhance their immunogenicity and apoptosis, including: translocation of calreticulin to the cell surface, secretion of ATP during apoptosis, and release of the nuclear protein HMGB1. ICD induces the release of specific MAMPS and danger-associated molecular patterns (DAMPs), which have the unique ability to establish a pro-inflammatory environment that promotes T cell recognition of tumor antigens. As shown herein, although other chemotherapeutic agents can induce apoptosis, not all chemotherapeutic agents can induce an ICD response as strong as auristatins such as MMAE and MMAF.

The key steps of ICD generate a series of signals that activate the innate immune system to recognize and eliminate tumor cells. First, the drug induces ER stress, which in turn causes DAMPs, including surface exposure of calreticulin, heat shock proteins (HSP70 and HSP90), secretion of ATP, and release of high-mobility group protein B1 (HMGB1). During the ICD process, the exposure of these DAMPs and the secretion of immunomodulators can work together to trigger an immune response, including the activation of dendritic cells and other antigen-presenting cells, leading to phagocytosis and damage of ER-stressed cells.

As mentioned above, the initiation of ICD is related to ER stress. Overloading ER with the ability to fold polypeptides or destroy the protein folding environment triggers ER stress response. ER is tightly connected to a microtubule network that provides structure and elasticity through dynamic assembly and contraction. The destruction of the microtubule network has an impact on the ER network and causes severe ER stress, which triggers the expression of the required features of ICD induction and causes a stress response, known as the unfolded protein response (UPR). The UPR reaction has multiple arms including increased pIRE1, downstream phosphorylation of JNK and ATF4 cutting.

The present invention is based in part on the discovery that certain tubulin disrupting agents such as auristatins (e.g., MMAE and MMAF) are capable of producing a unique ICD response compared to other cytotoxic agents and particularly compared to other payloads used for antibody-drug conjugates. The present invention is further based on the discovery that pairing the unique ability of such agents to drive ICD with agents that enhance immune responses can enhance anti-tumor activity. This discovery was particularly made when antibodies that are capable of binding to certain targets involved in immune signaling and have enhanced Fc binding characteristics and effector functions were used to achieve such immune agonism. Desired Fc binding characteristics include activities such as enhanced binding to activating FcγRs, reduced binding to inhibitory FcγRs, enhanced ADCC activity, and/or enhanced ADCP activity. Certain such antibodies with the desired activity are non-fucosylated.

Based on these common findings, as described in more detail herein, the inventors have demonstrated that induction of ICD using a combination of specific tubulin disrupting agents such as MMAE and MMAF with antibodies to immune cell engagers that also enhance Fc activity synergistically elicits improved anti-tumor responses. The use of ADCs rather than standard chemotherapeutic agents has also been shown to mitigate the attenuation of T cell responses seen with traditional chemotherapy, thus providing another benefit of this specific combination approach.

Thus, some embodiments provided herein are combination therapies comprising administering to a subject having cancer: (1) an antibody-drug conjugate comprising a tubulin disrupting agent conjugated to a first antibody that binds to a tumor-associated antigen; and (2) an antibody that binds to an immune cell adaptor, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs. In some embodiments, the second antibody is non-fucosylated. In certain embodiments, the second antibody has enhanced ADCC and/or ADCP activity.

II. Definitions

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. See, for example, Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th edition, 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to the methods, devices and materials described herein can be used to practice the present invention.

As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "antibody" optionally includes a combination of two or more such molecules, and the like.

As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art.

The term "antibody" includes complete antibodies and antigen-binding fragments thereof, wherein the antigen-binding fragment comprises an antigen-binding region and at least a portion of a heavy chain constant region comprising asparagine (N) 297 located in CH2. Typically, a "variable region" contains the antigen-binding region of an antibody and is related to the specificity and affinity of binding. See Fundamental Immunology 7th edition, Paul, Wolters Kluwer Health/Lippincott Williams & Wilkins (2013). Light chains are generally classified as kappa or lambda. Heavy chains are generally classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. Bivalent and bispecific molecules are described in, for example, Kostelny et al. (1992) J. Immunol. 148: 1547, Pack and Pluckthun (1992) Biochemistry 31: 1579, Hollinger et al. (1993), PNAS. USA 90: 6444, Gruber et al. (1994) J Immunol. 152: 5368, Zhu et al. (1997) Protein Sci. 6: 781, Hu et al. (1996) Cancer Res. 56: 3055, Adams et al. (1993) Cancer Res. 53: 4026 and McCartney, et al. (1995) Protein Eng. 8: 301.

The term "antibody" includes the antibody itself (naked antibody) or the antibody conjugated to a cytotoxic or cytostatic drug.

"Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597, or may be prepared by other methods. The antibodies described herein are monoclonal antibodies.

Specific binding of a monoclonal antibody to its target antigen indicates an affinity of at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ M ^-1 . The detectable magnitude of specific binding is high and can be distinguished from nonspecific binding occurring to at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or a specific spatial fit (e.g., lock and key type), whereas nonspecific binding is typically the result of van der Waals forces.

The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50 to 70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. This variable region is initially expressed connected to a cleavable signal peptide. The variable region without a signal peptide is sometimes referred to as a mature variable region. Therefore, for example, a light chain mature variable region represents a light chain variable region without a light chain signal peptide. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function.

Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and the isotypes of antibodies are defined as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable region and constant region are joined by a "J" region having about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. (See generally Fundamental Immunology (Paul, W., ed., 2nd ed., Raven Press, N.Y., 1989, Chapter 7), which is incorporated by reference in its entirety for all purposes).

The mature variable region of each light chain/heavy chain pair forms an antibody binding site. Therefore, a complete antibody has two binding sites. The difference is that in bifunctional or bispecific antibodies, the two binding sites are identical. The chains all show the same general structure of relatively conservative framework regions (FRs) engaged by three hypervariable regions, also referred to as complementary determining regions or CDRs. The CDRs of the two chains of each pair are aligned by the framework regions so that they can bind to a specific epitope. From the N-terminal to the C-terminal, both the light chain and the heavy chain comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is based on the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989), or Kabat and Chothia, or IMGT (Im Muno GeneTics Information System), AbM or Contact, or other conventional definitions of CDRs. Kabat also provides a widely used numbering convention (Kabat numbering), in which the same numbering is assigned to corresponding residues between different heavy chains or between different light chains. Unless otherwise apparent from the context, Kabat numbering is used to specify the position of an amino acid in a variable region. Unless otherwise apparent from the context, EU numbering is used to specify the position in a constant region.

"Humanized" antibodies are antibodies that retain the reactivity of non-human antibodies while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with the human counterparts. See, for example, Morrison et al., PNAS USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).

As used herein, the term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof is replaced such that the antigen binding site (variable region, CDR or a portion thereof) is linked to a constant region of a different species.

The term "epitope" refers to a site on an antigen that is bound by an antibody. An epitope can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by adjacent amino acids are usually retained after exposure to denaturing solvents, while epitopes formed by tertiary folding usually disappear after treatment with denaturing solvents. An epitope typically includes at least 3 and more typically at least 5 or 8 to 10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, Glenn E. Morris, ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified in a single immunoassay that shows the ability of one antibody to compete with another antibody for binding to a target antigen. Antibody epitopes can also be defined by X-ray crystallography of the antibody in conjunction with its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

Competition between antibodies is determined by an assay in which the test antibody inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). If an excess of the test antibody (e.g., at least 2×, 5×, 10×, 20×, or 100×) inhibits binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%, as measured in a competitive binding assay, then the test antibody competes with the reference antibody. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody to allow steric hindrance to occur.

The phrase "specific binding" refers to binding to a target in a sample with greater affinity, avidity, more easily and/or with longer duration than a molecule (e.g., an antibody or antibody fragment) binding to a non-target compound. In some embodiments, an antibody that specifically binds to a target is an antibody that binds to a target with an affinity at least 2 times greater than that of a non-target compound, such as, for example, at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times, or 100 times greater. For example, an antibody that specifically binds to TIGIT typically binds to TIGIT with an affinity at least 2 times greater than that of a non-TIGIT target. Those of ordinary skill in the art reading this definition should understand that, for example, an antibody (or part or epitope) that specifically or preferably binds to a first target may or may not specifically or preferably bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding.

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or fragment thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

The binding affinity between two molecules, such as an antibody or fragment thereof and an antigen, through a monovalent interaction can be quantified by determining the dissociation constant ( _KD ). Then, as a non-limiting example, _KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore ^™ ). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant _ka (or _kon ) and the dissociation rate constant _kd (or _koff ), respectively. _KD is related to _ka and _kd by the equation _KD = _kd / _ka . The value of the dissociation constant can be determined directly by well-known methods, and can even be calculated for complex mixtures by methods such as those described in, for example, Caceci et al. (1984, Byte 9: 340-362). For example, _K can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong and Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays for evaluating the binding ability of ligands, such as antibodies, to target antigens are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art or as described in the Examples section below, such as surface plasmon resonance (SPR), for example by using a Biacore ^™ system; kinetic exclusion assays, such as and BioLayer interferometry (e.g. using Octet platform). In some embodiments, binding affinity is determined using BioLayer interferometry. See, e.g., Wilson et al., Biochemistry and Molecular Biology Education, 38:400-407 (2010); Dysinger et al., J. Immunol. Methods, 379:30-41 (2012); and Estep et al., Mabs, 2013, 5:270-278.

As used herein, the term "cross-reactivity" refers to the ability of an antibody to bind to an antigen other than the antigen from which the antibody was generated. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen from another species other than the antigen from which the antibody was generated. As a non-limiting example, an anti-TIGIT antibody as described herein generated against a human TIGIT antigen may exhibit cross-reactivity with TIGIT from a different species (e.g., mouse or monkey).

An "isolated" antibody refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment and/or an antibody produced recombinantly. A "purified antibody" is an antibody that is typically at least 50% weight/weight pure from interfering proteins and other impurities produced or purified therefrom, but does not exclude the possibility that the monoclonal antibody is combined with an excess of a pharmaceutically acceptable carrier or other vehicle to facilitate its use. Interfering proteins and other contaminants may include, for example, cellular components of the cells from which the antibody is isolated or recombinantly produced. Sometimes a monoclonal antibody is at least 60%, 70%, 80%, 90%, 95% or 99% weight/weight pure from interfering proteins and contaminants produced or purified therefrom. The antibodies described herein, including rat, chimeric, veneered and humanized antibodies, can be provided in isolated and/or purified form.

The term "LAE" refers to the tripeptide linker Leucine-Alanine-Glutamic acid. The term "dLAE" refers to the tripeptide linker D-Leucine-Alanine-Glutamic acid, wherein the Leucine in the tripeptide linker is in the D-configuration.

"Subject," "patient," "individual," and similar terms are used interchangeably and refer to mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species unless otherwise specified. The terms do not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refer to an individual under medical supervision.

The terms "therapy", "treatment" and "improvement" refer to any alleviation of the severity of symptoms. In the case of treating cancer, treatment may refer to reducing, for example, tumor size, cancer cell number, growth rate, metastatic activity, cell death of non-cancerous cells, etc. As used herein, the terms "treatment" and "prevention" are not intended to be absolute terms. Treatment and prevention may refer to any delayed onset, improved symptoms, improved patient survival, increased survival time or survival rate, etc. Treatment and prevention may be complete (no detectable symptoms remaining) or partial, so that compared with patients without treatment described herein, symptoms become less frequent or less severe. The therapeutic effect of the same patient at different times before or during treatment can be compared with an individual or individual set that has not received treatment. In some aspects, the severity of the disease is reduced by at least 10%, such as compared with an individual before, for example, administration or an individual that has not undergone treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80% or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques.

As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (e.g., an antibody as described herein) is an amount of the agent that prevents, alleviates, ameliorate, improves or reduces the severity of symptoms of a disease (e.g., cancer) in a subject.

The term "administer/administered/administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. Administration techniques that may optionally be employed with the agents and methods described herein include, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, PA, as discussed.

III. Exemplary Antibodies that Bind Immune Cell Engagers

As mentioned above, the inventors have found that inducing ICD by administering an antibody-drug conjugate comprising certain tubulin disruptors (e.g., auristatins, including, for example, MMAE and MMAF) and using an antibody to trigger an immune response in combination can cause improved anti-tumor responses, including synergistic responses, wherein the antibody binds to proteins directly or indirectly involved in immune regulation and has enhanced Fc activity. Therefore, in some embodiments, the method provided herein includes administering to a subject with cancer an antibody that binds to a target involved in regulating an immune response, wherein such binding induces, promotes or enhances an immune response. The target of such an antibody may be referred to as an "immune cell adapter". As used herein, "immune cell adapter" refers to a molecule (e.g., a transmembrane protein) that participates in positive or negative regulation of immune cell responses. In some embodiments, an antibody binds to a receptor on an immune cell or tumor and causes direct immune cell engagement or release of a negative inhibitory signal. In some embodiments, an immune cell adapter is a molecule involved in T cell signaling. In some embodiments, an immune cell adapter regulates (e.g., activates) antigen presenting cells (APCs). In certain embodiments, an immune cell adapter is an immune checkpoint protein. Other examples of potential immune cell engagers are listed below. In some embodiments, the antibody binds to a receptor on an immune cell or tumor. Any of the antibodies that bind to the immune cell engagers described herein can be combined with any of the antibody-drug conjugates described herein.

Antibodies that bind to targets involved in immune regulation (e.g., immune cell engagers) also comprise an Fc having one or more or all of the following characteristics in any combination: 1) enhanced binding to one or more activating FcγRs, 2) reduced binding to inhibitory FcγRs, 3) being non-fucosylated, 4) having enhanced ADCC activity, 5) having enhanced ADCP activity, 6) activating antigen presenting cells (APCs), 7) enhancing CD8 T cell responses, 8) upregulating co-stimulatory receptors, 9) activating innate cellular immune responses and/or 10) engaging NK cells.

Thus, in some embodiments, the antibodies comprise an Fc that enhances binding to one or more activating FcγRs and/or reduces binding to one or more inhibitory FcγRs to obtain the desired enhanced FcγR binding profile. Activating FcγRs include one or more of FcγRIIIa, FcγRIIa, and/or FcγRI. Inhibitory FcγRs include, for example, FcγRIIb.

In certain embodiments, the antibody comprises an Fc that enhances binding to at least FcγRIIIa. In other embodiments, the antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRIIa. In some embodiments, the antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRI. In certain embodiments, the antibody comprises an Fc that enhances binding to FcγRIIIa, FcγRIIa, and FcγRI.

In some embodiments, in addition to enhancing binding to an activating FcγR, the antibody reduces binding to one or more inhibitory FcγRs. Thus, in some embodiments, the antibody reduces binding to FcγRIIa and/or FcγRIIb.

In some embodiments, the antibody is afucosylated.In some embodiments, the antibody further has one of the FcγR binding profiles described above.

In certain embodiments, the Fc of the antibody comprises amino acid changes relative to the wild-type Fc to enhance binding to activating FcγRs and/or reduce binding to one or more inhibitory FcγRs to obtain an FcγR binding profile as described above. For example, in some embodiments, the Fc of the antibody comprises substitutions S293D, A330L, and I332E in the heavy chain constant region.

Additional details regarding methods of obtaining antibodies with a desired FcγR profile are provided below as are methods for obtaining afucosylated antibodies.

A. Exemplary Immune Cell Engagers

Non-limiting exemplary targets or immune cell engagers that the antibodies can target include: Mullerian hormone receptor II (AMHR2), B7, B7H1, B7H2, B7H3, B7H4, BAFF-R, BCMA (B cell maturation antigen), Bst1/CD157, C5 complement, CC chemokine receptor 4 (CCR4), CD123, CD137, CD19, CD20, CD25 (IL2RA), CD276, CD278, CD3, CD32, CD33, CD37, CD38, CD4 and HIV-1 gp120 binding site, CD40, CD70, CD70 (a member of the TNF receptor ligand family), CD80, CD86, Claudin 18.2, c-MET, CSF1R, CTLA-4, EGFR, EGFR MET oncogene, EPHA3, ERBB2, ERBB3, FGFR2b, FLT3, GITR, glucocorticoid-induced TNF receptor (GITR), HER2, HER3, HLA, ICOS, IDO1, IFNAR1, IFNAR2, IGF-1R, IL-3Rα (CD123), IL-5R, IL-5Rα, LAG-3, MET oncogene, OX40 (CD134), PD-1, PD-L1, PD-L2, PVRI G, respiratory syncytial virus (RSV) heavily glycosylated mucin-like domain of EBOV glycoprotein (GP), rhesus macaque (Rh) D, sialic acid immunoglobulin-like lectin 8 (Siglec-8), signaling lymphocyte activation molecule (SLAMF7/CS1), T cell receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA4), TIGIT, TIM3 (HAVCR2), tumor-specific glycotope of Muc1 (TA-Muc1), VSIR (VISTA) and VTCN1.

In some embodiments, the antibody is an agonist of an immune cell engager. In some such embodiments, the antibody is an agonist of an immune cell engager selected from the group consisting of CD80, CD86, OX40 (CD134), GITR, CD137, CD40, VTCN1, CD276, IFNAR2, IFNAR1, CSF1R, VSIR (VISTA), and HLA.

In some embodiments, the antibody is an antagonist of an immune cell engager. In some such embodiments, the antibody is an antagonist of an immune cell engager selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7, TIM3 (HAVCR2), PVRIG, TIGIT, CD25 (IL2RA), and IDO1.

In certain embodiments, the antibody for binding immune cell adapters of the methods provided herein can be an inhibitor for checkpoint proteins. In some embodiments, the antibody for binding immune cell adapters of the methods provided herein can be a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a B7 inhibitor, a TIM3 (HAVCR2) inhibitor, an OX40 (CD134) inhibitor, a GITR agonist, a CD137 agonist, or a CD40 agonist, a VTCN1 inhibitor, an IDO1 inhibitor, a CD276 inhibitor, a PVRIG inhibitor, a TIGIT inhibitor, a CD25 (IL2RA) inhibitor, an IFNAR2 inhibitor, an IFNAR1 inhibitor, a CSF1R inhibitor, a VSIR (VISTA) inhibitor, or a therapeutic agent targeting HLA. Such inhibitors, activators, or therapeutic agents are further provided below. In any embodiment herein, the antibody for binding immune cell adapters can be an antibody comprising an Fc that enhances binding to one or more activating FcγRs. In some embodiments, the antibody that binds the immune cell engager is an afucosylated antibody.

In some embodiments, the antibody that binds to the immune cell adaptor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720, and 7,605,238, all of which are incorporated by reference in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206) or a non-fucosylated version thereof. In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101) or a non-fucosylated version thereof. Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is sold under the trade name Yervoy ^™ .

In certain embodiments, the antibody that binds to the immune cell adaptor is a PD-1/PD-L1 inhibitor. Examples of PD-1/PD-L1 inhibitors include, but are not limited to, U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Patent Application Publication Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699, all of which are incorporated herein by reference in their entirety.

In some embodiments, the antibody that binds to the immune cell adaptor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558 or MDX1106) or pembrolizumab (also known as MK-3475, SCH 900475 or lambrolizumab) or a non-fucosylated version thereof. In one embodiment, the anti-PD-1 antibody is nivolumab or a non-fucosylated version thereof. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is sold under the trade name Opdivo ^TM . In another embodiment, the anti-PD-1 antibody is pembrolizumab or a non-fucosylated version thereof. Pembrolizumab is a humanized monoclonal IgG4 antibody and is sold under the trade name Keytruda ^TM . In yet another embodiment, the anti-PD-1 antibody is CT-011, a humanized antibody, or an afucosylated version thereof. CT-011 alone has failed to show a response in treating acute myeloid leukemia (AML) in the case of relapse. In yet another embodiment, the anti-PD-1 antibody is AMP-224, a fusion protein, or an afucosylated version thereof. In another embodiment, the PD-1 antibody is BGB-A317 or an afucosylated version thereof. BGB-A317 is a monoclonal antibody in which the ability to bind to Fcγ receptor I is specifically engineered, and it has the characteristics of uniquely binding to PD-1 with high affinity and excellent target specificity. In one embodiment, the PD-1 antibody is cemiplimab or an afucosylated version thereof. In another embodiment, the PD-1 antibody is camrelizumab or an afucosylated version thereof. In another embodiment, the PD-1 antibody is sintilimab or a non-fucosylated version thereof. In one embodiment, the PD-1 antibody is tislelizumab or a non-fucosylated version thereof. In certain embodiments, the PD-1 antibody is TSR-042 or a non-fucosylated version thereof. In yet another embodiment, the PD-1 antibody is PDR001 or a non-fucosylated version thereof. In yet another embodiment, the PD-1 antibody is toripalimab or a non-fucosylated version thereof.

In certain embodiments, the antibody that binds to the immune cell engager is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab) or a non-fucosylated version thereof. In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01) or a non-fucosylated version thereof. In yet another embodiment, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A and ) or a non-fucosylated version thereof. In another embodiment, the PD-L1 inhibitor is avelumab or a non-fucosylated version thereof.

In one embodiment, the antibody binding to the immune cell adaptor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHIgM12B7A or its non-fucosylated version.

In one embodiment, the antibody that binds to the immune cell engager is a lymphocyte activation gene-3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor is IMP321 (a soluble Ig fusion protein) (Brignone et al., J. Immunol., 2007, 179, 4202-4211), or a non-fucosylated version thereof. In another embodiment, the LAG-3 inhibitor is BMS-986016 or a non-fucosylated version thereof.

In one embodiment, the antibody that binds to the immune cell adaptor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834), or a non-fucosylated version thereof. In some embodiments, the B7 inhibitor is a B7-H4 inhibitor. A non-limiting exemplary B7-H4 inhibitor is FPA150 (a non-fucosylated antibody for B7-H4). See PCT/US2018/047805.

In one embodiment, the antibody that binds to the immune cell engager is a TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).

In one embodiment, the antibody that binds to an immune cell engager is an OX40 (CD134) agonist antibody.In certain embodiments, the anti-OX40 antibody is MEDI6469 or an afucosylated version thereof.

In one embodiment, the antibody that binds the immune cell engager is a GITR agonist. In one embodiment, the immune cell engager is an anti-GITR antibody or a non-fucosylated version thereof. In one embodiment, the anti-GITR antibody is TRX518 or a non-fucosylated version thereof.

In one embodiment, the antibody that binds the immune cell engager is a CD137 agonist. In one embodiment, the immune cell engager is an anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab or a non-fucosylated version thereof. In another embodiment, the anti-CD137 antibody is PF-05082566 or a non-fucosylated version thereof.

In one embodiment, the antibody that binds to the immune cell adaptor is a CD40 agonist. In one embodiment, the antibody that binds to the immune cell adaptor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870,893 or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is MP0317 (Molecular Partners) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is YH003 (Eucure Biopharma) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is CDX-1140 (Celldex Therapeutics) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is YH003 (Eucure Biopharma) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is mitazalimab (Alligator Bioscience) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is ABBV-927 (AbbVie) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is sotigalimab (Apexigen) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is GEN1042 (Genmab) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is 2141V-11 (Rockefeller University) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is selicrelumab (Roche) or a non-fucosylated version thereof. In one embodiment, the anti-CD40 antibody is SEA-CD40 (Seagen), which is a non-fucosylated humanized version of murine S2C6 and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 30 to 35, respectively. The corresponding VH and VL comprise the amino acid sequences of SEQ ID NOs: 28 and 29, respectively. SEA-CD40 is described in U.S. Patent Publication Nos. 2017/0333556 and 2017/0137528, both of which are incorporated herein by reference.

In some embodiments, the antibody that binds an immune cell engager is an antibody that binds CD70. In some embodiments, the antibody is SEA-CD70. See, e.g., U.S. Patent No. 8,067,546; Sequence Listing herein.

In some embodiments, the antibody binding to the immune cell adaptor is an antibody binding to BCMA. In some embodiments, the antibody is SEA-BCMA. See, for example, U.S. Publication No. 2017/0233484 and WO 2017/143069 (VH and VL of SEQ ID NO: 13 and 19, respectively; CDRs of SEQ ID NO: 60, 61, 62, 90, 91, 92, see U.S. Publication No. 2017/0233484); See also the sequence table herein (VH and VL of SEQ ID NO: 45 and 46, respectively; CDRs of SEQ ID NO: 47 to 52).

In one embodiment, the antibody that binds the immune cell engager is an anti-interleukin-15 antibody.

In one embodiment, the antibody that binds to an immune cell engager is a VTCN inhibitor. In one embodiment, the VTCN inhibitor is FPA150 or a non-fucosylated version thereof.

In one embodiment, the antibody that binds to the immune cell adaptor is an anti-IDO antagonist antibody. In some embodiments, the antibody that binds to the immune cell adaptor is a TIGIT inhibitor. In certain embodiments, the TIGIT inhibitor is an anti-TIGIT antibody. In one embodiment, the TIGIT inhibitor is MTIG7192A or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is BMS-986207 (BMS) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is OMP-313M32 or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is MK-7684. In another embodiment, the TIGIT inhibitor is AB154 or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is CGEN-15137 or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is SEA-TGT. In another embodiment, the TIGIT inhibitor is ASP8374 (Astellas) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is AJUD008 or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is AB308 (Arcus Biosciences) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is AGEN1327 (Agenus) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is AK127 (Akeso Biopharma) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is BAT6005 (Bio-Thera Solutions) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is BAT6021 (Bio-Thera Solutions) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is CASC-674 (Seagen) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is COM902 (Compugen) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is domvanalimab (Arcus Biosciences) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is etigilimab (Mereo BioPharma) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is GSK4428859 (GSK) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is HL186 (HanAll Biopharma) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is MIL-100 (Beijing Mabworks Biotech) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is YH-29143 (Yu Han) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is HLX53 (Shanghai Henlius Biotech) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is IBI939 (Innovent Biologics) or a non-fucosylated version thereof. In yet another embodiment, the TIGIT inhibitor is JS006 (Junshi Biosciences) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is M6223 (Merck KGaA) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is MG1131 (Mogam Institute) or a non-fucosylated version thereof. In yet another embodiment, the TIGIT inhibitor is ociperlimab (BeiGene) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is tiragolumab (Roche; described in U.S. Patent No. 10,047,158) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is TJT6 (I-MabBiopharma) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is vibostolimab (MSD; described in U.S. Pat. No. 10,618,958) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is YBL-012 (Y Biologics) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is IBI-939 (Innovent) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is AZD2936 (AstraZeneca) or a non-fucosylated version thereof. In one embodiment, the TIGIT inhibitor is EOS-448 (iTeos/GSK) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is BAT6005 (Bio-Thera) or a non-fucosylated version thereof. In another embodiment, the TIGIT inhibitor is AGEN1777 (BMS/Agenus) or a non-fucosylated version thereof.

In some embodiments, the antibody that binds to the immune cell adaptor is a VSIR inhibitor. In certain embodiments, the VSIR inhibitor is an anti-VSIR antibody. In one embodiment, the VSIR inhibitor is MTIG7192A or a non-fucosylated version thereof. In another embodiment, the VSIR inhibitor is CA-170 or a non-fucosylated version thereof. In yet another embodiment, the VSIR inhibitor is JNJ 61610588 or a non-fucosylated version thereof. In one embodiment, the VSIR inhibitor is HMBD-002 or a non-fucosylated version thereof.

In some embodiments, the antibody that binds to the immune cell adaptor is a TIM3 inhibitor. In certain embodiments, the TIM3 inhibitor is an anti-TIM3 antibody. In one embodiment, the TIM3 inhibitor is AJUD009 or a non-fucosylated version thereof.

In some embodiments, the antibody that binds to the immune cell engager is a CD25 (IL2RA) inhibitor. In certain embodiments, the CD25 (IL2RA) inhibitor is an anti-CD25 (IL2RA) antibody. In one embodiment, the CD25 (IL2RA) inhibitor is daclizumab or a non-fucosylated version thereof. In another embodiment, the CD25 (IL2RA) inhibitor is basiliximab or a non-fucosylated version thereof.

In some embodiments, the antibody that binds to the immune cell adaptor is an IFNAR1 inhibitor. In certain embodiments, the IFNAR1 inhibitor is an anti-IFNAR1 antibody. In one embodiment, the IFNAR1 inhibitor is anifrolumab or a non-fucosylated version thereof. In another embodiment, the IFNAR1 inhibitor is sifalimumab or a non-fucosylated version thereof.

In some embodiments, the antibody that binds to the immune cell adaptor is a CSF1R inhibitor. In certain embodiments, the CSF1R inhibitor is an anti-CSF1R antibody. In one embodiment, the CSF1R inhibitor is pexidartinib or a non-fucosylated version thereof. In another embodiment, the CSF1R inhibitor is emactuzumab or a non-fucosylated version thereof. In yet another embodiment, the CSF1R inhibitor is cabiralizumab or a non-fucosylated version thereof. In one embodiment, the CSF1R inhibitor is ARRY-382 or a non-fucosylated version thereof. In another embodiment, the CSF1R inhibitor is BLZ945 or a non-fucosylated version thereof. In yet another embodiment, the CSF1R inhibitor is AJUD010 or a non-fucosylated version thereof. In one embodiment, the CSF1R inhibitor is AMG820 or a non-fucosylated version thereof. In another embodiment, the CSF1R inhibitor is IMC-CS4 or a non-fucosylated version thereof. In yet another embodiment, the CSF1R inhibitor is JNJ-40346527 or a non-fucosylated version thereof. In one embodiment, the CSF1R inhibitor is PLX5622 or a non-fucosylated version thereof. In another embodiment, the CSF1R inhibitor is FPA008 or a non-fucosylated version thereof.

In various embodiments, antibodies that bind to immune cell engagers have one or more or all of the following activities in any combination: 1) depletion of regulatory T (Treg) cells, 2) activation of antigen presenting cells (APCs), 3) enhancement of CD8 T cell responses, 4) upregulation of co-stimulatory receptors and/or 5) promotion of release of immune-activating cytokines (such as CXCL10 and/or IFNγ). In some embodiments, antibodies that bind to immune cell engagers promote release of immune-activating cytokines (e.g., CXCL10 and IFNγ) to a greater extent than release of immune-suppressive cytokines (such as IL10 and/or MDC).

B. Exemplary Anti-TIGIT Antibodies

In one aspect, antibodies that bind to human TIGIT (T cell immune receptor with Ig domain and ITIM domain) are provided as antibodies against immune cell adaptors. As described herein, in some embodiments, anti-TIGIT antibodies inhibit the interaction between TIGIT and one or both of the ligands CD155 and CD112. In some embodiments, anti-TIGIT antibodies inhibit the interaction between TIGIT and CD155 in functional bioassays, allowing CD155-CD226 signaling to occur. In some embodiments, anti-TIGIT antibodies exhibit synergistic effects with anti-PD-1 agents (e.g., anti-PD-1 antibodies) or anti-PD-L1 agents (e.g., anti-PD-L1 antibodies). In some embodiments, the anti-TIGIT antibody used in the method of the present invention is SEA-TGT, which is a non-fucosylated IgG1 antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising amino acid sequences SEQ ID NO: 7, 10, 14, 17, 18, and 19, respectively. The corresponding VH and VL comprise the amino acid sequences of SEQ ID NOs: 1 and 6, respectively.

The inventors unexpectedly discovered that anti-TIGIT antibodies with enhanced effector function, such as can be achieved with afucosylated IgG1 antibodies, deplete Treg cells and show improved efficacy in vivo. Accordingly, in various embodiments, afucosylated anti-TIGIT antibodies are provided.

In some embodiments, an anti-TIGIT antibody, such as an afucosylated anti-TIGIT antibody, binds to a human TIGIT protein (SEQ ID NO: 218) or a portion thereof with high affinity. In some embodiments, the antibody has a binding affinity ( _KD ) for human TIGIT of less than 5nM, less than 1nM, less than 500pM, less than 250pM, less than 150pM, less than 100pM, less than 50pM, less than 40pM, less than 30pM, less than 20pM, or less than about 10pM. In some embodiments, the antibody has a binding affinity ( _KD ) for human TIGIT of less than 50pM. In some embodiments, the antibody has a _KD for human TIGIT in the range of about 1pM to about 5nM, for example, about 1pM to about 1nM, about 1pM to about 500pM, about 5pM to about 250pM, or about 10pM to about 100pM.

In some embodiments, in addition to binding to human TIGIT with high affinity, the non-fucosylated anti-TIGIT antibodies exhibit cross-reactivity with cynomolgus monkey ("cyno") TIGIT and/or mouse TIGIT. In some embodiments, the anti-TIGIT antibodies bind to mouse TIGIT with a binding affinity ( _KD ) of 100 nM or less. In some embodiments, the anti-TIGIT antibodies bind to human TIGIT with a _KD of 5 nM or less and cross-react with mouse TIGIT with a _KD of 100 nM or less. In some embodiments, the anti-TIGIT antibodies that bind to human TIGIT also exhibit cross-reactivity with both cynomolgus monkey TIGIT and mouse TIGIT.

In some embodiments, antibody cross-reactivity is determined by detecting specific binding of an anti-TIGIT antibody to TIGIT expressed on a cell (e.g., a cell line expressing human TIGIT, cyno TIGIT, or mouse TIGIT, or a primary cell endogenously expressing TIGIT, such as a primary T cell endogenously expressing human TIGIT, cyno TIGIT, or mouse TIGIT). In some embodiments, antibody binding and antibody cross-reactivity are determined by detecting specific binding of an anti-TIGIT antibody to purified or recombinant TIGIT (e.g., purified or recombinant human TIGIT, purified or recombinant cyno TIGIT, or purified or recombinant mouse TIGIT) or a chimeric protein comprising TIGIT (e.g., an Fc fusion protein comprising human TIGIT, cyno TIGIT, or mouse TIGIT, or a His-tagged protein comprising human TIGIT, cyno TIGIT, or mouse TIGIT).

In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and the ligand CD155. In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and the ligand CD112. In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and both the ligands CD155 and CD112.

In some embodiments, the anti-TIGIT antibody that binds to human TIGIT comprises a light chain variable region sequence or a portion thereof and/or a heavy chain variable region sequence or a portion thereof derived from any of the following antibodies described herein: clone 13, clone 13A, clone 13B, clone 13C, or clone 13D. The amino acid sequences of the CDRs, light chain variable domains (VL), and heavy chain variable domains (VH) of anti-TIGIT antibodies clone 13, clone 13A, clone 13B, clone 13C, and clone 13D are set forth in the following sequence listing.

In some embodiments, the anti-TIGIT antibody comprises one or more (e.g., one, two, three, four, five, or six) of the following:

a heavy chain CDR1 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

a heavy chain CDR2 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13;

a heavy chain CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and 16;

A light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 17;

A light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 18; and/or

The light chain CDR3 sequence comprises the amino acid sequence of SEQ ID NO:19.

In some embodiments, the anti-TIGIT antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13; and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 or 16.

In some embodiments, the anti-TIGIT antibody comprises: a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:17; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:18; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, the anti-TIGIT antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13; a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16; a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:17; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:18; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, the anti-TIGIT antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the following amino acid sequences, respectively:

(a) SEQ ID NO:7, 10, 14, 17, 18 and 19; or

(b) SEQ ID NO: 8, 11, 14, 17, 18 and 19; or

(c) SEQ ID NO: 9, 12, 15, 17, 18 and 19; or

(d) SEQ ID NO: 8, 13, 16, 17, 18 and 19; or

(e) SEQ ID NOs: 8, 12, 16, 17, 18 and 19.

In some embodiments, the anti-TIGIT antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the anti-TIGIT antibody comprises a VH comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, a VH sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human TIGIT and optionally retains the ability to block CD155 and/or CD112 binding to TIGIT.

In some embodiments, the anti-TIGIT antibody comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 6. In some embodiments, the anti-TIGIT antibody comprises a VL comprising the amino acid sequence SEQ ID NO: 6. In some embodiments, the VL sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 6) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human TIGIT and optionally retains the ability to block CD155 and/or CD112 from binding to TIGIT.

In some embodiments, the anti-TIGIT antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO:6. In some embodiments, the anti-TIGIT antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6.

In some embodiments, the anti-TIGIT antibody comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid sequence of SEQ ID NO: 6;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 2 and a VL comprising the amino acid sequence of SEQ ID NO: 6; or

(c) a VH comprising the amino acid sequence of SEQ ID NO: 3 and a VL comprising the amino acid sequence of SEQ ID NO: 6; or

(d) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 6; or

(f) VH comprising the amino acid sequence of SEQ ID NO:5 and VL comprising the amino acid sequence of SEQ ID NO:6.

In some embodiments, the anti-TIGIT antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs:20, 21, 22, 23, and 24 and a light chain comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the anti-TIGIT antibody used in the methods of the invention is a non-fucosylated version of an anti-TIGIT antibody disclosed in US 2009/0258013, US 2016/0176963, US 2016/0376365, or WO 2016/028656.

C. Exemplary Anti-CD40 Antibodies

As mentioned above, in some embodiments, the antibody that binds to the immune cell adaptor is an agonist anti-CD40 antibody. Agonistic CD40 monoclonal antibodies including dacetuzumab have shown encouraging clinical activity in the context of single agent and combination chemotherapy. Dacetuzumab has shown certain clinical activity in a phase 1 study of NHL and a phase 2 study of diffuse large B-cell lymphoma (DLBCL). See, for example, Advani et al., J. Clin. Oncol. 27: 4371-4377 (2009) and De Vos et al., J. Hematol. Oncol. 7: 1-9 (2014). In addition, when combined with paclitaxel or carboplatin or gemcitabine, the humanized IgG2 agonist antibody CP-870,893 for CD40 showed encouraging activity in solid tumor indications. In these studies, activation of antigen presenting cells, cytokine production, and the generation of antigen-specific T cells can be seen. See, e.g., Beatty et al., Clin. Cancer Res. 19:6286-6295 (2013) and Vonderheide et al., Oncoimmunology 2:e23033 (2013).

In some embodiments, a non-fucosylated anti-CD40 antibody is provided for use in the methods of the present invention. In some embodiments, the non-fucosylated anti-CD40 antibody is SEA-CD40, which is a non-fucosylated humanized version of murine S2C6 and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising amino acid sequences SEQ ID NO: 30 to 35, respectively. The corresponding VH and VL comprise amino acid sequences SEQ ID NO: 28 and 29, respectively. SEA-CD40 is described in U.S. Patent Publication Nos. 2017/0333556 and 2017/0137528, both of which are incorporated herein by reference. S2C6 was originally isolated as a murine monoclonal antibody raised against human bladder cancer, referred to herein as mS2C6. See, e.g., Paulie et al., Cancer Immunol. Immunother. 17: 165-179 (1984). The S2C6 antibody is a partial agonist of the CD40 signaling pathway and has the following activities in some embodiments: binding to human CD40 protein, binding to cynomolgus CD40 protein, activating the CD40 signaling pathway, enhancing the interaction of CD40 with its ligand CD40L. See, e.g., U.S. Patent No. 6,946,129.

S2C6 is humanized and this humanized antibody is referred to herein as humanized S2C6, and alternatively as darcibinumab, which is a fucosylated humanized S2C6 (fhS2C6 or SGN-40). See, e.g., WO 2006/128103, which is incorporated herein by reference for any purpose. SEA-CD40 is a non-fucosylated humanized S2C6 antibody. Other versions of humanized S2C6 are disclosed at WO2008/091954; these versions can be non-fucosylated and used in the methods disclosed herein.

In some embodiments, the anti-CD40 antibody comprises one or more (e.g., one, two, three, four, five, or six) of the following:

a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 30;

A heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:36;

a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 32;

A light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 33;

A light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 34; and/or

The light chain CDR3 sequence comprises the amino acid sequence of SEQ ID NO:35.

In some embodiments, the anti-CD40 antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:30; a heavy chain CDR2 sequence comprising any one of the amino acid sequences of SEQ ID NO:31 or SEQ ID NO:36; and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:32.

In some embodiments, the anti-CD40 antibody comprises: a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:33; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:34; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, the anti-CD40 antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:30; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:36; a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:32; a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:33; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:34; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, the anti-CD40 antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the following amino acid sequences, respectively:

(a) SEQ ID NO: 30, 31, 33, 34 and 35; or

(b) SEQ ID NO: 30, 36, 33, 34 and 35.

In some embodiments, the anti-CD40 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 28. In some embodiments, the anti-CD40 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the VH sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 28) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human CD40.

In some embodiments, the anti-CD40 antibody comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 29. In some embodiments, the anti-CD40 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the VL sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 29) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human CD40.

In some embodiments, an anti-CD40 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 28, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 29. In some embodiments, an anti-CD40 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-CD40 antibody comprises the heavy chain variable region and the light chain variable region disclosed as SEQ ID NOs: 28 and 29, respectively. In some embodiments, the anti-CD40 antibody comprises the heavy chain and the light chain disclosed as SEQ ID NOs: 26 and 27, respectively.

D. Exemplary Anti-CD70 Antibodies

In some embodiments, non-fucosylated anti-CD70 antibodies are provided as antibodies that bind immune cell adaptors for use in the methods of the present invention. In some embodiments, the non-fucosylated anti-CD70 antibody is SEA-CD70, as described in U.S. Pat. No. 8,067,546 and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising amino acid sequences SEQ ID NO: 53 to 58, respectively. The corresponding VH and VL comprise amino acid sequences SEQ ID NO: 41 and 42, respectively. The CD70 molecule is a member of the tumor necrosis factor (TNF) ligand superfamily (TNFSF) and binds to the related receptor CD27 (TNFRSF7). The interaction between the two molecules activates intracellular signals from the two receptors. Under normal conditions, CD70 expression is transient and limited to activated T and B cells, mature dendritic cells and natural killer (NK) cells. Similarly, CD27 is expressed on both naive and activated effector T cells and NK and activated B cells. However, CD70 is also abnormally expressed in various blood cancers, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and non-Hodgkin lymphoma (NHL) and carcinomas, and plays a role in both tumor cell survival and/or tumor immune evasion. SEA-CD70 acts by blocking CD70/CD27 axis signaling, inducing antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC), and enhancing antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the anti-CD70 antibody comprises one or more (e.g., one, two, three, four, five, or six) of the following:

a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:53;

a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:54;

a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:55;

a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:56;

A light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 57; and/or

The light chain CDR3 sequence comprises the amino acid sequence of SEQ ID NO:58.

In some embodiments, the anti-CD70 antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:53; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:54; and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:55.

In some embodiments, the anti-CD70 antibody comprises: a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:56; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:57; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:58.

In some embodiments, the anti-CD70 antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:53; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:54; a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:55; a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:56; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:57; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:58.

In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 41. In some embodiments, the anti-CD70 antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 41. In some embodiments, the VH sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 41) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human CD70.

In some embodiments, the anti-CD70 antibody comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 42. In some embodiments, the anti-CD70 antibody comprises a VL comprising the amino acid sequence SEQ ID NO: 42. In some embodiments, the VL sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 42) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human CD70.

In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 42. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the anti-CD70 antibody comprises the heavy chain variable region and light chain variable region disclosed as SEQ ID NOs: 41 and 42, respectively.

E. Exemplary Anti-BCMA Antibodies

In some embodiments, non-fucosylated anti-BCMA antibodies are provided as antibodies that bind to immune cell adaptors for use in the methods of the present invention. In some embodiments, the non-fucosylated anti-BCMA antibody is SEA-BCMA, which is an antibody targeting B cell maturation antigen (BCMA) and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising amino acid sequences SEQ ID NO: 47 to 52, respectively. The corresponding VH and VL comprise amino acid sequences SEQ ID NO: 45 and 46, respectively. BCMA is expressed on multiple myeloma (MM). The antibody works by blocking ligand-mediated BCMA cell signaling, antibody-dependent cellular phagocytosis (ADCP) and enhanced antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the anti-BCMA antibody comprises one or more (e.g., one, two, three, four, five, or six) of the following:

a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:47;

a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:48;

A heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 49;

A light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 50;

A light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO 51; and/or

The light chain CDR3 sequence comprises the amino acid sequence of SEQ ID NO:52.

In some embodiments, the anti-BCMA antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:47; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:48; and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:49.

In some embodiments, the anti-BCMA antibody comprises: a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:50; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:51; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:52.

In some embodiments, the anti-BCMA antibody comprises: a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:47; a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:48; a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:49; a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO:50; a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO:51; and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO:52.

In some embodiments, the anti-BCMA antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 45. In some embodiments, the anti-BCMA antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 45. In some embodiments, the VH sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 45) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human BCMA.

In some embodiments, the anti-BCMA antibody comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 46. In some embodiments, the anti-BCMA antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the VL sequence having at least 90% sequence identity to a reference sequence (e.g., SEQ ID NO: 46) contains one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability to bind to human BCMA.

In some embodiments, an anti-BCMA antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 45, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 46. In some embodiments, an anti-BCMA antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the anti-BCMA antibody comprises the heavy chain variable region and light chain variable region disclosed as SEQ ID NOs: 45 and 46, respectively.

F. Enhanced Fc backbone

As mentioned above, antibodies that bind to immune cell engagers include an Fc with one or more of the following activities: enhancing binding to one or more activating FcγRs; reducing binding to inhibitory FcγRs; enhancing ADCC activity and/or enhancing ADCP activity. Antibodies with Fc with such activities and desired activity profiles can be produced in a variety of ways, including producing non-fucosylated proteins and/or by engineering the Fc to contain certain mutations that produce the desired activity. This section provides additional details on methods for producing non-fucosylated antibodies and exemplary engineering approaches. Additional guidance on the selection of constant regions and the manufacture of antibodies is provided in other sections below.

Antibodies can be glycosylated at conserved positions in their constant regions (Jefferis and Lund, (1997) Chem. Immunol. 65: 111-128; Wright and Morrison, (1997) TibTECH 15: 26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., (1996) Mol. Immunol. 32: 1311-1318; Wittwe and Howard, (1990) Biochem. 29: 4175-4180) as well as intramolecular interactions between parts of the glycoprotein, which can affect the conformation of the glycoprotein and the three-dimensional surface presented (Jefferis and Lund, supra; Wyss and Wagner, (1996) Current Op. Biotech. 7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on specific recognition structures. For example, it has been reported that in agalactosylated IgG, the oligosaccharide moiety is 'flipped' out of the inter-CH2 space and the terminal N-acetylglucosamine residue becomes available for binding to mannose binding protein (Malhotra et al., (1995) Nature Med. 1:237-243). Glycopeptidase removal of oligosaccharides from CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody that recognizes the CDw52 antigen of human lymphocytes) produced in Chinese hamster ovary (CHO) cells resulted in a complete reduction in complement-mediated lysis (CMCL) (Boyd et al., (1996) Mol. Immunol. 32:1311-1318), while selective removal of sialic acid residues using neuraminidase did not result in loss of DMCL. It has also been reported that glycosylation of antibodies can affect antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNAc, were reported to have improved ADCC activity (Umana et al. (1999) Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which the glycosylation pattern of the antibody is altered. Alteration means deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition (glycosylation pattern) of glycosylation, the degree of glycosylation, etc.

Glycosylation sites can be added to an antibody by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding one or more serine or threonine residues to the sequence of the original antibody or replacing the sequence of the original antibody with the residues (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be achieved by amino acid changes within the native glycosylation site of the antibody.

The amino acid sequence is usually altered by changing the underlying nucleic acid sequence. These methods include isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant version of the antibody.

The glycosylation (including glycosylation pattern) of the antibody can also be changed without changing the amino acid sequence or the underlying nucleotide sequence. See, for example, Pereira et al., 2018, MAbs, 10 (5): 693-711. Glycosylation depends largely on the host cell used to express the antibody. Since the cell types used to express recombinant glycoproteins, such as antibodies, as potential therapeutic agents are rarely natural cells, significant changes in the glycosylation pattern of antibodies can be expected. See, for example, Hse et al., (1997) J. Biol. Chem. 272: 9062-9070. In addition to selecting host cells, factors affecting glycosylation during the recombinant production of antibodies include growth mode, medium preparation, culture density, oxygenation, pH, purification schemes, etc. Various methods for changing the glycosylation pattern achieved in a specific host organism have been proposed, including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5047335; 5510261; 5278299). Glycosylation or certain types of glycosylation can be removed from glycoproteins enzymatically, for example using endoglycosidase H (EndoH). In addition, recombinant host cells can be genetically engineered to, for example, be defective in processing certain types of polysaccharides. These and similar techniques are known in the art.

The glycosylation structure of the antibody can be easily analyzed by conventional techniques of carbohydrate analysis, including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, sequential enzymatic digestion and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, but are not limited to, enzymatic treatment (usually performed using peptide-N-glycosidase F/endo-β-galactose), elimination using a harsh alkaline environment to release primarily O-linked structures and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.

A preferred form of glycosylation modification of antibodies is reduced core fucosylation."Core fucosylation" refers to the addition of fucose ("fucosylation") to N-acetylglucosamine ("GlcNAc") at the reducing terminus of N-linked glycans.

A "complex N-glycoside-linked sugar chain" is usually bound to asparagine 297 (according to Kabat's numbering). As used herein, a complex N-glycoside-linked sugar chain has a biantennary complex sugar chain, which essentially has the following structure:

Where ± indicates that sugar molecules may be present or absent, and numbers indicate the position of linkage between sugar molecules. In the above structure, the end of the sugar chain bound to asparagine is called the reducing end (right side), and the other side is called the non-reducing end. Fucose is usually bound to N-acetylglucosamine ("GlcNAc") at the reducing end via an α1,6 bond (position 6 of GlcNAc is connected to position 1 of fucose). "Gal" refers to galactose, and "Man" refers to mannose.

“Complex N-glycoside-linked sugar chains” include 1) a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also called “gal-GlcNAc”) and the non-reducing terminal side of gal-GlcNAc optionally has sialic acid, bisecting N-acetylglucosamine, etc.; and 2) a mixed type in which the non-reducing terminal side of the core structure has a high mannose N-glycoside-linked sugar chain and two branches of a complex N-glycoside-linked sugar chain.

In some methods as provided herein, only a relatively small amount of fucose is incorporated into the complex N-glycoside-linked sugar chains of the antibody. For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibodies in the composition have core fucosylation by fucose. In some embodiments, about 2% of the antibodies in the composition have core fucosylation by fucose. In various embodiments, when less than 60% of the antibodies in the composition have core fucosylation by fucose, the antibodies of the composition can be referred to as "non-fucosylated" or "no fucosylation". In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibodies in the composition are non-fucosylated.

In certain embodiments, only a relatively small amount of the fucose analog (or metabolite or product of the fucose analog) is incorporated into the complex N-glycoside-linked sugar chain. For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibodies have core fucosylation by fucose analogs or metabolites or products of fucose analogs. In some embodiments, about 2% of the antibodies have core fucosylation by fucose analogs or metabolites or products of fucose analogs.

In some embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibodies in the composition have fucose residues on the G0, G1, or G2 glycan structures. (See, e.g., Raju et al., 2012, MAbs 4:385-391, FIG3). In some embodiments, about 2% of the antibodies in the composition have fucose residues on the G0, G1, or G2 glycan structures. In various embodiments, when less than 60% of the antibodies in the composition have fucose residues on the G0, G1, or G2 glycan structures, the antibodies of the composition may be referred to as "non-fucosylated". In some embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibodies in the composition lack fucose on the G0, G1, or G2 glycan structures. It should be noted that G0 glycans include G0-GN glycans. G0-GN glycans are monoantennary glycans with one terminal GlcNAc residue. G1 glycans include G1-GN glycans. G1-GN glycans are monoantennary glycans with one terminal galactose residue. G0-GN and G1-GN glycans can be fucosylated or non-fucosylated.

A variety of methods for producing non-fucosylated antibodies can be used. Exemplary strategies include using cell lines lacking certain biosynthetic enzymes involved in the fucosylation pathway or inhibiting or knocking out certain genes involved in the fucosylation pathway. A review of such pathways is provided by Pereira, et al. (2018) MABS 10: 693-711, which is incorporated herein by reference in its entirety.

For example, a method for preparing non-fucosylated antibodies by incubating antibody-producing cells with fucose analogs is described in, for example, WO2009/135181. In short, cells that have been engineered to express antibodies are incubated in the presence of fucose analogs or intracellular metabolites or products of fucose analogs. The intracellular metabolites may be, for example, GDP-modified analogs or fully or partially deesterified analogs. For example, the product may be a fully or partially deesterified analog. In some embodiments, fucose analogs may inhibit enzymes in the fucose salvage pathway. For example, fucose analogs (or intracellular metabolites or products of fucose analogs) may inhibit the activity of fucokinase or GDP-fucose-pyrophosphorylase. In some embodiments, fucose analogs (or intracellular metabolites or products of fucose analogs) inhibit fucosyltransferases (preferably 1,6-fucosyltransferases, such as FUT8 proteins). In some embodiments, a fucose analog (or an intracellular metabolite or product of a fucose analog) can inhibit the activity of an enzyme in the de novo synthesis pathway of fucose. For example, a fucose analog (or an intracellular metabolite or product of a fucose analog) can inhibit the activity of GDP-mannose 4,6-dehydratase or/and GDP-fucose synthase. In some embodiments, a fucose analog (or an intracellular metabolite or product of a fucose analog) can inhibit a fucose transporter (e.g., a GDP-fucose transporter).

In one embodiment, the fucose analog is 2-fluorofucose.Methods of using fucose analogs and other fucose analogs in growth media are disclosed, for example, in WO 2009/135181, which is incorporated herein by reference.

Other methods for engineering cell lines to reduce core fucosylation include gene knockout, gene knock-in, and RNA interference (RNAi). See, for example, Pereira et al., 2018, MAbs, 10 (5): 693-711. In gene knockout, the gene encoding FUT8 (α1,6-fucosyltransferase) is inactivated. FUT8 catalyzes the transfer of fucosyl residues from GDP-fucose to position 6 of the Asn-linked (N-linked) GlcNac of N-glycans. It is reported that FUT8 is the only enzyme responsible for adding fucose to the biantennary carbohydrates connected to N at Asn297. Gene knock-in adds genes encoding enzymes such as GNTIII or Golgi α-mannosidase II. The increase in the level of such enzymes in the cell diverts the monoclonal antibody from the fucosylation pathway (resulting in a reduction in core fucosylation and an increased amount of bisected N-acetylglucosamine). RNAi also often targets FUT8 gene expression, which results in reduced mRNA transcript levels or complete knockout of gene expression.

Other strategies that can be used include (U.S. Patent No. 6,602,684) and (BioWa).

Any of these methods can be used to generate cell lines that will be capable of producing afucosylated antibodies.

Various engineering approaches can also be used to obtain an Fc region with the desired FcγR activity and effector function. In some embodiments, the Fc is engineered to have a combination of the following mutations: S239D, A330L, and I332E, which increases the affinity of the Fc domain to FcγRIIIA and thus increases ADCC. Additional substitutions that enhance affinity for FcγRIIIa include, for example, T256A, K290A, S298A, E333A, and K334A. Substitutions that enhance binding to activating FcγRI IIa and reduce binding to inhibitory FcγRIIIb include, for example, F243L/R292P/Y300L/V305I/P396L and F243L/R292P/Y300L/L235V/P396L. In some embodiments, the substitution is in the context of IgG1 Fc.

An oligosaccharide covalently linked to the conserved Asn297 is involved in the ability of the Fc region of IgG to bind FcγRs (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, Trends Biotechnol. 15:26-31). Engineering of this glycoform on IgG can significantly enhance IgG-mediated ADCC. Adding a bisecting N-acetylglucosamine modification (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) to this glycoform or removing fucose from this glycoform (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improves the binding between IgG Fc and FcγRs, thereby enhancing Ig-mediated ADCC activity.

Systematic substitution of solvent-exposed amino acids in the Fc region of human IgG1 has generated IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). A subset of these variants involving substitutions of Ala at Thr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 exhibited both increased binding affinity and ADCC activity for FcγRs when compared to the parental IgG1 (Shields et al., 2001, J. Biol. Chem. 276: 6591-604; Okazaki et al., 2004, J. Mol. Biol. 336: 1239-49).

A variety of methods can be used to determine the amount of fucosylation on an antibody. Methods include, for example, LC-MS via PLRP-S chromatography, electrospray ionization quadrupole TOF MS, capillary electrophoresis with laser induced fluorescence (CE-LIF), and hydrophilic interaction chromatography with fluorescence detection (HILIC).

IV. Exemplary Antibody-Drug Conjugates (ADCs)

The previous section describes the relevant aspects of antibodies that are bound to the target (immune cell adaptor) involved in immunomodulation. As mentioned above, some methods provided herein also include administering an antibody-drug conjugate (ADC) comprising a combination of a tubulin disruptor (e.g., auristatin, including, for example, MMAE and MMAF) and an antibody that is bound to an immune cell adaptor. In multiple embodiments, the antibody-drug conjugate comprises an antibody conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a tubulin disruptor. In some embodiments, the antibody binds to an antigen expressed on a tumor cell. Other details about the ADC used in the method provided herein are set forth in this section and in the following embodiments. Any ADC described herein can be combined with any antibody in conjunction with an immune cell adaptor described herein.

A. Exemplary Target Antigens

In some embodiments, the ADC binds to an antigen expressed on a tumor cell.

In some embodiments, the ADC for use in the methods provided herein comprises an antibody conjugated to a cytotoxic agent, wherein the antibody specifically binds to an antigen selected from the group consisting of 5T4 (TPBG), ADAM-9, AG-7, ALK, ALP, AMHRII, APLP2, ASCT2, AVB6, AXL (UFO), B7-H3 (CD276), B7-H4, BCMA, C3a, C3b, C4.4a (LYPD3), C5, C5a, CA6, CA9, CanAg, carbonic anhydrase IX (CAIX), cathepsin D, CCR7, CD1, CD10, CD100, CD101, CD102, CD103, CD104, CD105 , CD106, CD107a, CD107b, CD108, CD109, CD111, CD112, CD113, CD116, CD117, CD118, CD119, CD11A, CD11b, CD11c, CD120a, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD 127. CD13, CD130, CD131, CD132, CD133, CD135, CD136, CD137, CD138, CD14, CD140a, CD140b, CD141, CD142, CD143, CD144, CD146, C D147, CD148, CD15, CD150, CD151, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158b2, CD158e, CD158f1, CD158h, CD158i, CD159a, CD16, CD160, CD161, CD162, CD163, CD16 4. CD166, CD167b, CD169, CD16a, CD16b, CD170, CD171, CD172a, CD172b, CD172g, CD18, CD180, CD181, CD183, CD184, CD185, CD19, CD1 94. CD197, CD1a, CD1b, CD1c, CD1d, CD2, CD20, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD208, CD21, CD213a1, CD213a2, CD217, CD218a, CD22, CD220, CD221, CD222, CD224, CD226, CD228, CD229, CD23, CD230, CD232, CD239, CD243, CD244, CD248, CD249, CD25, CD26, CD265, CD267, CD269, CD27, CD27 2. CD273, CD274, CD275, CD279, CD28, CD280, CD281, CD282, CD283, CD284, CD289, CD29, CD294, CD295, CD298, CD3, CD3ε, CD30, CD300f, CD302, CD304, CD305, CD307, CD31, CD312, CD315, CD316, CD317, CD318, CD319, CD32, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD32b, CD33, CD331, CD332, CD333, C D334, CD337, CD339, CD34, CD340, CD344, CD35, CD352, CD36, CD37, CD38, CD39, CD3d, CD3g, CD4, CD41, CD42d, CD44, CD44v6, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49 e, CD49f, CD5, CD50, CD51, CD51 (integrin α-V), CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD6, CD61, CD62L, CD62P, CD63, CD64, CD 66a-e, CD67, CD68, CD69, CD7, CD70, CD70L, CD71, CD71 (TfR), CD72, CD73, CD74, CD79a, CD79b, CD8, CD80, CD82, CD83, CD84, CD85f, CD85i, CD85j, CD86, CD87, CD89, CD90, CD91, CD92, CD95, CD96, CD97, CD98, CDH6, CDH6 (cadherin 6), CDw210a, CDw210b, CEA, CEACAM5, CEACAM6, CFC1B, cKIT, CLDN18.2 (clin 18.2), C LDN6, CLDN9, CLL-1, c-MET, complement factor C3, Cripto, CSP-1, CXCR5, DCLK1, DLK-1, DLL3, DPEP3, DR5 (death receptor 5), antiadhesin, EFNA4, EGFR, EGFR wild type, EGFRviii, EGP-1 (TROP-2), EGP-2, EMP2, ENPP3, EpCAM, EphA2, EphA3, Ephrin-A4 (EFNA4), ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FOLR, FOLR1, FOLR-α, FSH, GCC, GD2, GD3, globo H, GPC1, GPC-1, GPC3, GPNMB, GPR20, HER2, HER-2, HER3, HER-3, HGFR (c-Met), HLA-DR, HM1.24, HSP90, Ia, IGF-1R, IL-13R, IL-15, IL1RAP, IL-2, IL-3, IL-4, IL7R, integrin αVβ3, integrin β-6, interleukin-4 receptor (IL4R), KAAG-1, KLK2, LAMP-1, Le(y), Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, lipid rafts, LIV-1 (SLC39A6 or ZIP6), LRP-1, LRRC15, LY6E, macrophage mannose receptor 1, MAGE, mesothelin (MSLN), MET, MHC Class I chain-associated proteins A and B (MICA and MICB), MN/CA IX, MRC2, MT1-MMP, MTX3, MTX5, MUC1, MUC16, MUC2, MUC3, MUC4, MUC5, MUC5ac, NaPi2b, NCA-90, NCA-95, connexin-4, Notch3, nucleolin, OAcGD2, OT-MUC1 (tumor tethered MUC1), OX001L, P1GF, PAM4 antigen, p-cadherin (cadherin 3), PD-L1, phosphatidylserine (PS), PRLR, prolactin receptor (PRLR), Pseudomonas, PSMA, PTK4, PTK7, receptor tyrosine kinase (RTK), RNF43, ROR1, ROR2, SAIL, SEZ6, SLAMF 7, SLC44A4, SLITRK6, SLMAMF7 (CS1), SLTRK6, sortin (SORT1), SSEA-4, SSTR2, Staphylococcus aureus (antibiotic agent), STEAP-1, STING, STn, T101, TAA, TAC, TDGF1, tenascin, TENB2, TGF-B, Thomson-Friedrich antigen, Thy1.1, TIM-1, tissue factor (TF; CD142), TM4SF1, Tn antigen, TNF-alpha (TNFα), TRA-1-60, TRAIL receptors (R1 and R2), TROP-2, tumor-associated glycoprotein 72 (TAG-72), uPAR, VEGFR, VEGFR-2, and xCT.

In some embodiments, the ADC binds to an antigen selected from the group consisting of: EGFR, KAAG1, MET, CD30, HER2, CD30, IL7R, CD248, tumor-associated glycoprotein 72 (TAG-72), MRC2, EGFR, CD71, TRA-1-60, STn, CLDN18.2, CLDN6, HER-2, CD33, CD7, OT-MUC1 (tumor tethered MUC1), TRA-1-60, TIM-1, GCC, mesothelin (MSLN), EGFR, gpNMB, CD20, AMHRII, NaPi2b, CD142, ROR1, integrin β6, Ly6E, cMET, CD37, MUC16, STEAP-1, LRRC15, SLITRK6, MUC16, ETBR, FCRH5, Axl, CD79b, Globo H, SLAMF7, PSMA, CD22, CD228, CD48, LIV-1, EphA2, SLC44A4, CA9, Axl, and LGR5.

In some embodiments, the ADC binds an antigen selected from the group consisting of BCMA, GPC1, CD30, cMET, SAIL, HER3, CD70, c-MET, CD46, HER2, 5T4, ENPP3, CD19, EGFR, BCMA, CD70, BCMA, and EphA2.

In some embodiments, the ADC binds an antigen selected from the group consisting of: Her2, TROP2, BCMA, cMet, integrin αVβ6, CD22, CD79b, CD30, CD19, CD70, CD228, and CD47.

In some embodiments, the ADC binds an antigen selected from the group consisting of: CD142, integrin beta-6, ENPP3, CD19, Ly6E, cMET, C4.4a, CD37, MUC16, STEAP-1, LRRC15, SLITRK6, MUC16, ETBR, FCRH5, Axl, EGFR, CD79b, BCMA, CD70, PSMA, CD79b, CD228, CD48, LIV-1, EphA2, SLC44A4, CD30, and sTn.

In some embodiments, the ADC binds to an antigen selected from the group consisting of: 5T4, ADAM-9, AG-7, ALK, AMHRII, APLP2, ASCT2, Axl, B7-H3, B7-H4, BCMA, C4.4a, CA6, CA9, CanAg, carbonic anhydrase IX (CAIX), cathepsin D, CCR7, CD103, CD123, CD133, CD138, CD142, CD147, CD16, CD166, CD184, CD19, CD20, CD205, CD206, CD22, CD228, CD248, CD25, CD3, CD3ε, CD30, CD300f, CD317, CD33, CD352, CD37, CD38, CD44v6, CD45, CD46 、CD47、CD48、CD51、CD56、CD7、CD70、CD71、CD74、CD79b、CDH6、CEA、CEACAM5、CEACAM6、cKIT、CLDN18.2、CLDN6、CLDN9、CLL-1、c-MET、Cripto、CSP-1、CXCR5、DCLK1、DLK-1、DLL3、DPEP3、DR5(death receptor 5), antiadhesin, EFNA4, EGFR, EGFR wild type, EGFRviii, EMP2, ENPP3, EpCAM, EphA2, EphA3, ETBR, FAP, FCRH5, FGFR2, FGFR3, FLT3, FOLR, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, GPR20, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL-15, IL1RAP, IL-2, IL-3, IL-4, IL7R, integrin β-6, interleukin-4 receptor (IL4R), KAAG-1, KLK2, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, lipid rafts, LIV-1, LRP-1, LRRC15, Ly6E, macrophage mannose receptor 1, MAGE, mesothelin (MSLN), MET, MHC Class I chain-associated proteins A and B (MICA and MICB), MRC2, MT1-MMP, MTX3, MTX5, MUC-1, MUC16, NaPi2b, nexin-4, NOTCH3, nucleolin, OAcGD2, OT-MUC1 (tumor tethered MUC1), OX001L, P-cadherin, PD-L1, phosphatidylserine, phosphatidylserine (PS), prolactin receptor (PRLR), Pseudomonas, PSMA, PTK7, receptor tyrosine kinase (RTK), RNF43 , ROR1, ROR2, SAIL, SEZ6, SLAMF7, SLC44A4, SLITRK6, sortin (SORT1), SSEA-4, SSTR2, Staphylococcus aureus (antibiotic agent), STEAP-1, STING, STING (payload target), STn, TAA, TGF-B, TIM-1, TM4SF1, TNF-α, TRA-1-60, TROP-2, tumor-associated glycoprotein 72 (TAG-72), VEGFR-2, xCT.

In some embodiments, the ADC binds to an antigen selected from the group consisting of: AMHRII, Axl, CA9, CD142, CD20, CD22, CD228, CD248, CD30, CD33, CD7, CD48, CD71, CD79b, CLDN18.2, CLDN6, c-MET, EGFR, EphA2, ETBR, FCRH5, GCC, Globo H, gpNMB, HER-2, IL7R, integrin β-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, mesothelin (MSLN), MET, MRC2, MUC16, NaPi2b, catenin-4, OT-MUC1 (tumor tethered MUC1), PSMA, ROR1, SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, tumor-associated glycoprotein 72 (TAG-72).

In some embodiments, the ADC binds an antigen selected from the group consisting of: BCMA, GPC-1, CD30, c-MET, SAIL, HER-3, CD70, CD46, HER-2, 5T4, ENPP3, CD19, EGFR, EphA2.

In some embodiments, the antibody of the ADC does not bind connexin-4.

Typically, the antibody of the ADC and the antibody that binds to the immune cell engager are two separate antibodies. However, in certain embodiments, the antibodies can form bispecific antibodies.

B. Exemplary Cytotoxic Agents

In various embodiments, the methods provided herein comprise administering an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody conjugated to a tubulin disrupting agent.

Various classes of tubulin disrupting agents are known in the art, including but not limited to dolestadin, auristatins, tubulysins, colchicine, vinca alkaloids, taxanes, T67 (Tularik), scutellarin, maytansines, hemipteratin, and other tubulin disrupting agents.

Auristatins are derivatives of the natural product Aplysia caudatum. Exemplary auristatins include Aplysia caudatum-10, Auristatin E, Auristatin T, MMAE (N-methyl valine-valine-aplysia isoleucine-aplysia proline-norephedrine or monomethyl auristatin E) and MMAF (N-methyl valine-valine-aplysia isoleucine-aplysia proline-phenylalanine or polyvaline (dovaline)-valine-aplysia isoleucine-aplysia proline-phenylalanine), AEB (esters produced by reacting Auristatin E with p-acetylbenzoic acid), AEVB (esters produced by reacting Auristatin E with benzoylvaleric acid) and AFP (dimethyl valine-valine-aplysia isoleucine-aplysia proline-phenylalanine-p-phenylenediamine or Auristatin phenylalanine phenylenediamine). WO 2015/057699 describes PEGylated Auristatins, including MMAE. Additional tail Aplysia derivatives contemplated for use are disclosed in U.S. Patent No. 9,345,785, which is incorporated herein by reference for any purpose. Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug units DE and DF, disclosed in "Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, filed March 28, 2004, and described in U.S. Patent Publication No. 2005/0238649, the disclosure of which is expressly incorporated by reference in its entirety.

In certain embodiments, the ADC cytotoxic agent is MMAE.

In other embodiments, the cytotoxic agent conjugated to the ADC is MMAF.

Tubulolysin includes but is not limited to tubulysin D, tubulysin M, tubulolysin A and tubulolysin Tyr. WO2017-096311 and WO 2016-040684 describe non-limiting tubulysin analogs, including tubulysin M.

Colchicine includes, but is not limited to, colchicine and CA-4.

Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR), and vindfesine (VDS).

Taxanes include but are not limited to (paclitaxel) and (docetaxel).

Glomerulin includes, but is not limited to, glomerulin-1 and glomerulin-52.

Maytansinoids include, but are not limited to, maytansine, maytansinol, maytansine analogs, DM1, DM3 and DM4, and ansamatocin-2. Exemplary maytansine-like drug moieties include those maytansine drug moieties having modified aromatic rings, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by reduction of ansamytocin P2 with lithium aluminum hydride); C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides) and those maytansine drug moieties having modifications at other positions.

Maytansine drug moieties also include those with modifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reacting maytansinol with H.sub.25 or P.sub.2S.sub.5); C-14-alkoxymethyl (demethoxy/CH.sub.20R) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH.sub.20H or CH.sub.2OAc) (U.S. Pat. No. 4,450,254) (prepared by Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by converting maytansinol with Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared from Trewia nudlflora) isolation); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylating maytansinol with Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by reducing maytansinol with titanium trichloride/LAH). The cytotoxicity of TA.1-maytansine conjugates binding to HER-2 was tested in vitro on the human breast cancer cell line SK-BR-3 (Chari et al., Cancer Research 52: 127-131 (1992)). The drug conjugates achieved a degree of cytotoxicity similar to that of free maytansine drugs, which can be increased by increasing the number of maytansine molecules per antibody molecule.

Hemitelins include, but are not limited to, hemitelin and HTI-286.

Other tubulin disrupting agents include taccalonolide A, taccalonolide B, taccalonolide AF, taccalonolide AJ, taccalonolide AI-epoxide, discodermolide, baccatin derivatives, taxane analogs (e.g., epothilone A and epothilone B), nocodazole, colchicine, colchicine, estramustine, cemadotin, combretastatins, discodermolide, eleutherobin, eribulin, prolabolin, phomopsin, and laulimalide.

In some embodiments, the ADC used in the methods herein may include a linker unit. For example, the ADC may include a linker region between a cytotoxic agent and an antibody. In some embodiments, the linker is a protease cleavable linker, an acid cleavable linker, a disulfide bond linker, or a self-stabilizing linker. In various embodiments, the linker can be cleaved under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment.

The ADC for the methods herein may include a linker, wherein the therapeutic agent (e.g., tubulin disruptor) can be conjugated to the antibody in a manner that reduces the activity of the antibody unless the linker is stripped from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleavage agent). Such therapeutic agents can be connected to the antibody via a linker. The therapeutic agent conjugated to the linker is also referred to herein as a drug linker. The properties of the linker can be very different. The components constituting the linker are selected based on their characteristics, which can be specified in part according to the conditions at the site where the conjugate is delivered.

The therapeutic agent can be linked to the antibody via a cleavable linker that is sensitive to cleavage in the intracellular environment of the target cell but substantially insensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when it is internalized by the cancer cell (e.g., in an endosome, or, for example, in a lysosomal environment or caveolear environment by virtue of pH sensitivity or protease sensitivity). The therapeutic agent can also be linked to the antibody via a non-cleavable linker.

As indicated, the linker may comprise a cleavable unit. In some such embodiments, the structure and/or sequence of the cleavable unit is selected so that it is cleaved by the action of an enzyme present at the target site (e.g., target cell). In other embodiments, cleavable units that can be cleaved by pH (e.g., acid or base instability), temperature changes, or when irradiated (e.g., light instability) may also be used.

In some embodiments, the cleavable unit may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be a target substrate for an enzyme.

In some aspects, the cleavable unit is a peptidyl unit and is at least two amino acids long. The cleavage agent may include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most typical is a cleavable unit that can be cleaved by an enzyme present in the target cell, i.e., an enzyme-cleavable linker. Thus, the linker may be cleaved, for example, by an intracellular peptidase or protease including a lysosomal or endosomal protease. For example, a linker (e.g., a linker comprising a Phe-Leu or Val-Cit peptide or a Val-Ala peptide) that can be cleaved by the thiol-dependent protease cathepsin B that is highly expressed in cancer tissues may be used.

In some embodiments, the linker will contain a cleavable unit (e.g., a peptidyl unit) and the cleavable unit will be directly conjugated to the therapeutic agent. In other embodiments, the cleavable unit will be conjugated to the therapeutic agent via another functional unit, such as a self-degradable spacer unit or a non-self-degradable spacer unit. A non-self-degradable spacer unit is a spacer unit in which part or all of the spacer unit remains bound to the drug unit after the cleavage of the cleavable unit (e.g., an amino acid) from the antibody-drug conjugate. In order to release the drug, an independent hydrolysis reaction occurs within the target cell to cleave the spacer unit from the drug.

By means of the self-degrading spacer unit, the drug is released without a separate hydrolysis step. In one embodiment, wherein the linker comprises a cleavable unit and a self-degrading group, the cleavable unit can be cleaved by the action of an enzyme and after the cleavage of the cleavable unit, the self-degrading group releases the therapeutic agent. In some embodiments, the cleavable unit of the linker will be directly or indirectly conjugated to the therapeutic agent at one end and directly or indirectly conjugated to the antibody at the other end. In some such embodiments, the cleavable unit will be directly or indirectly conjugated to the therapeutic agent at one end (e.g., via a self-degrading or non-self-degrading spacer unit) and will be conjugated to the antibody via an extension unit at the other end. The extension unit connects the antibody to the remainder of the drug and/or drug linker. In one embodiment, the connection between the antibody and the remainder of the drug or drug linker is via a maleimide group, for example, via a maleimidohexanoyl linker. In some embodiments, the antibody will be connected to the drug via a disulfide bond, such as the disulfide-linked maytansine conjugates SPDB-DM4 and SPP-DM1.

The connection between the antibody and the joint can be via a variety of different approaches, such as through a thioether bond, through a disulfide bond, through an amide bond or through an ester bond. In one embodiment, the connection between the antibody and the joint is formed between the thiol group of the cysteine residue of the antibody and the maleimide group of the joint. In some embodiments, the interchain bond of the antibody is converted into a free thiol group before reacting with the functional group of the joint. In some embodiments, the cysteine residue is introduced into the heavy chain or light chain of the antibody and reacts with the joint. The position of cysteine insertion by substitution in the heavy chain or light chain of the antibody includes the position described in the disclosed U.S. Application No. 2007-0092940 and the International Patent Publication WO2008070593, each of which is incorporated herein by reference in its entirety and for all purposes.

In some embodiments, the antibody-drug conjugate has the following Formula I:

L-(LU-D) _p (I)

Wherein L is an antibody, LU is a linker unit and D is a drug unit (i.e., a therapeutic agent). The subscript p ranges from 1 to 20. Such conjugates comprise an antibody covalently linked to at least one drug via a linker. The linker unit is linked to the antibody at one end and to the drug at the other end.

Drug load is represented by p, which is the number of drug molecules per antibody. Drug load may be within the range of 1 to 20 drug units (D)/antibody. In some aspects, subscript p will be within the range of 1 to 20 (i.e., both integer values and non-integer values of 1 to 20). In some aspects, subscript p will be an integer of 1 to 20, and will represent the number of drug-joints on a single antibody. In other aspects, p represents the average drug-joint molecule number/antibody, such as the average drug-joint number/antibody in a reaction mixture or a composition (such as a pharmaceutical composition), and may be an integer value or a non-integer value. Therefore, in some aspects, for a composition (such as a pharmaceutical composition), p represents the average drug load of an antibody-drug conjugate in the composition, and p is within the range of 1 to 20.

In some embodiments, p is about 1 to about 8 drugs/antibodies. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is about 2 to about 8 drugs/antibodies. In some embodiments, p is about 2 to about 6, 2 to about 5, or 2 to about 4 drugs/antibodies. In some embodiments, p is about 2, about 4, about 6, or about 8 drugs/antibodies.

The average number of drug per antibody unit in the preparation from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, HIC and HPLC. The quantitative distribution of the conjugate can also be determined in terms of p.

Exemplary antibody-drug conjugates include antibody-drug conjugates based on auristatin, i.e., conjugates in which the drug component is an auristatin drug. It has been shown that auristatin binds to tubulin and interferes with microtubule dynamics and nuclear and cell division, and has anticancer activity. Typically, an antibody-drug conjugate based on auristatin comprises a linker between an auristatin drug and an antibody. Auristatin can be connected to an antibody at any position suitable for conjugation with a linker. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Auristatin can be auristatin E or a derivative thereof. Auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E). The synthesis and structure of exemplary auristatins are described in U.S. Patent or Publication Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety and for all purposes.

Exemplary auristatin-based antibody-drug conjugates include vcMMAE, vcMMAF and mcMMAF antibody-drug conjugates as shown below, wherein Ab is an antibody as described herein and val-cit represents a valine-citrulline dipeptide:

Or a pharmaceutically acceptable salt thereof. Drug load is represented by p (number of drug-linker molecules/antibody). Depending on the context, p may represent the average number of drug-linker molecules/antibody, also referred to as average drug load. The variable p is in the range of 1 to 20 and preferably 1 to 8. In some preferred embodiments, when p represents the average drug load, p is in the range of about 2 to about 5. In some embodiments, p is about 2, about 3, about 4, or about 5. In some aspects, the antibody is conjugated to the linker via the sulfur atom of a cysteine residue. In some aspects, the cysteine residue is a residue engineered into the antibody. In other aspects, the cysteine residue is an interchain disulfide bond cysteine residue.

In some other embodiments, the antibody-drug conjugate has a linker unit disclosed in application US20160310612A1 (PCT/US2014/060477), which is incorporated herein by reference in its entirety. In some other embodiments, the antibody-drug conjugate has the following formula (II):

wherein D is a drug unit, PEG is a polyethylene glycol unit that masks the hydrophobicity of the drug-linker, ^Lp is a parallel linker unit that allows the PEG units to be oriented parallel to XD, A is a branching unit that optionally consists of subunits when m is greater than 1 or is absent when m is 1, X is a releasable assembly unit that provides for the release of each D from the LDC and Z is an optional spacer unit through which ^Lp is bound to the antibody L.

In some embodiments, the antibody-drug conjugate has the following Formula III:

wherein AD is a drug linking unit such that the XD moiety indicated by t is additionally linked to the PEG unit in a parallel orientation, and L, ^Lp , Z, A, X, D, m, p and s are as defined in Formula II.

In other main embodiments, the LDC of the present invention is represented by the structure of Formula IV below:

wherein AD, L, ^Lp , PEG, Z, A, X, D, m, p, s and t are as defined in Formula III.

In some embodiments, the antibody-drug conjugate has the following Formula 1:

L-[LU-D'] _p (1)

or a salt thereof, in particular a pharmaceutically acceptable salt, wherein

L is antibody;

LU is a linker unit; and

D' represents 1 to Drug units (D) in each Drug Linker moiety of the formula -LU-D'; and

The subscript p is a number from 1 to 12, from 1 to 10, or from 1 to 8, or is about 4 or about 8,

wherein the antibody is capable of selectively binding to an antigen in tumor tissue to subsequently release the drug unit as a free cytotoxic agent,

Wherein the drug linker moiety of formula -LU-D' in each antibody-drug conjugate of the composition has the structure of Formula 1A:

or a salt thereof, in particular a pharmaceutically acceptable salt,

The wavy line indicates covalent attachment to L;

D is a drug unit of a cytotoxic agent;

_LB is the covalently bound part of the antibody;

A is a first optional Stretcher unit;

The subscript a is 0 or 1, which indicates the absence or presence of A, respectively;

B is an optional branching unit;

The subscript b is 0 or 1, which indicates the absence or presence of B, respectively;

_LO is a secondary linker moiety, wherein the secondary linker has the formula:

wherein the wavy line adjacent to Y indicates the site of covalent attachment of L _O to the Drug unit, and the wavy line adjacent to A' indicates the site of covalent attachment to the remainder of the Drug Linker moiety;

A' is a second optional Stretcher unit which, in the absence of B, becomes a subunit of A,

The subscript a' is 0 or 1, which indicates the absence or presence of A', respectively,

W is a peptide cleavable unit, wherein the peptide cleavable unit is a contiguous sequence of up to 12 (e.g., 3 to 12 or 3 to 10) amino acids, wherein the sequence consists of a selectivity-conferring tripeptide that provides for increased selectivity of tumor tissue relative to normal tissue exposed to free cytotoxic agent released from the antibody-drug conjugate of the composition as compared to the cytotoxic agent released from the antibody-drug conjugate composition of a comparative antibody-drug conjugate composition in which the peptide sequence of its peptide cleavable unit is the dipeptide -valine-citrulline or -valine-alanine-;

wherein the tumor and normal tissue are of a rodent species and wherein the composition of Formula 1 provides said enhanced exposure selectivity as evidenced by:

Maintaining efficacy in a tumor xenograft model of the comparative antibody-drug conjugate composition when administered at the same effective amount and dosage regimen previously determined for the comparative antibody-drug conjugate composition, and

showing that, when administered to non-tumor bearing rodents at an effective amount and at a dosage regimen identical to that in a tumor xenograft model, the plasma concentration of free cytotoxic agent released from the antibody-drug conjugate of the composition is reduced and/or normal cells in the tissue are preserved, compared to an equivalent (e.g., identical) administration of a comparative antibody-drug conjugate composition (the antibody of both conjugate compositions being replaced by a non-binding antibody),

wherein cytotoxicity to cells in human tissue of the same type as normal cells in the tissue of the non-tumor bearing rodent is at least in part responsible for the adverse event in a human subject administered a therapeutically effective amount of the comparative conjugate composition;

Y is a self-immolative spacer unit; and

The subscript y is 0, 1 or 2, which indicates the absence or presence of 1 or 2 Ys, respectively;

The subscript q is an integer ranging from 1 to 4,

The restrictions are that when subscript b is 0, subscript q is 1, and when subscript b is 1, subscript q is 2, 3, or 4; and

The antibody-drug conjugate of the composition has the structure of Formula 1, wherein subscript p is replaced by subscript p', wherein subscript p' is an integer from 1 to 12, 1 to 10 or 1 to 8, or is 4 or 8.

A related embodiment provides a drug linker of Formula V:

LU’-(D’)(V)

or a salt thereof, particularly a pharmaceutically acceptable salt thereof, wherein LU' is capable of providing a covalent bond between L and LU of Formula 1, and is therefore sometimes referred to as a linker unit precursor; and D' represents 1 to 4 drug units, wherein the drug linker is further defined by the structure of Formula VI:

wherein _LB ' is capable of being converted to _LB of Formula VI, thereby forming a covalent bond to L of Formula 1, and is therefore sometimes referred to as an antibody covalently bound promoiety, and the remaining variables of Formula VI are as defined in Formula VI.

In some embodiments, the ADC comprises an antibody (e.g., any antibody as described herein) conjugated to mc-vc-PABC-MMAE (also referred to herein as vcMMAE or 1006), mc-vc-PABC-MMAF, mc-MMAF, or mp-dLAE-PABC-MMAE (also referred to herein as dLAE-MMAE, mp-dLAE-MMAE, or 7092), or a pharmaceutically acceptable salt thereof. mp-dLAE-PABC-MMAE is described in PCT Publication No. WO 2021/055865A1. Such ADCs are as follows, wherein Ab comprises an antigen binding protein (e.g., any antibody as described herein), mc represents maleimidocaproyl, and mp refers to maleimidopropionyl:

val-cit (vc) represents valine-citrulline dipeptide, PABC represents p-aminophenylbenzyloxycarbonyl, and dLAE represents D-leucine-alanine-glutamic acid tripeptide:

mp-dLAE-PABC-MMAE. In some embodiments, drug loading is represented by p (number of drug-linker molecules/antibody). In some embodiments, p can represent the average drug-linker molecule number/antibody in the antibody composition, also referred to as average drug loading. In some embodiments, p is in the range of 1 to 20. In some embodiments, p is in the range of 1 to 8. In some embodiments, when p represents the average drug loading, p is in the range of about 2 to about 5. In some embodiments, p is about 2, about 3, about 4 or about 5. In some embodiments, the average number of drugs/antibody in the preparation can be characterized by conventional means such as mass spectrometry, HIC, ELISA determination and HPLC. In some embodiments, antigen binding proteins (e.g., antibodies) are connected to drug-linkers through the cysteine residues of the antibody. In some embodiments, the cysteine residues are residues engineered into the antibody. In some embodiments, the cysteine residues are interchain disulfide bond cysteine residues.

C. Exemplary ADC

Non-limiting exemplary ADCs for use in the methods of the invention include ADCs comprising an antibody that binds any of the exemplary targets discussed herein conjugated to any of the tubulin disrupting agents described herein.

In some embodiments, the ADC is an anti-sialyl Tn antigen antibody-ADC, which comprises an antibody that binds to a sialyl Tn antigen (sTn) and MMAE. See, e.g., U.S. Patent Publication No. 2018/0327509A1; WO2017083582A1; Sequence Listing herein.

In some embodiments, the ADC is martin-berantuzumab, which comprises antibodies that bind to B-cell maturation antigen (BCMA) and MMAF. See, e.g., U.S. Pat. No. 9,273,141.

In some embodiments, the ADC is an anti-claudin-18.2 ADC comprising an auristatin and the following antibodies:

Zolbetuin (175D10), which is disclosed in U.S. Pat. No. 8,168,427 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 59 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 60; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 61 to 66, respectively;

163E12, which is disclosed in U.S. Pat. No. 8,168,427 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 67 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 68; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 69 to 74, respectively;

Any anti-claudin-18.2 antibody disclosed in PCT Publication No. WO 2020/135674A1; or

Any anti-claudin-18.2 antibody disclosed in PCT Publication No. WO 2021/032157A1.

In some embodiments, the ADC is SGN-PDL1V, which comprises an anti-PD-L1 antibody and MMAE, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 75 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 76; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 77 to 82, respectively.

In some embodiments, the ADC is SGN-ALPV, which comprises an anti-ALP antibody and MMAE, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 83 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 84; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 85 to 90, respectively.

In some embodiments, the ADC is SGN-B7H4V, which comprises an anti-B7H4 antibody and MMAE, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 91 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 92; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93 to 98, respectively.

In some embodiments, the ADC is vedicizumab, which comprises an anti-HER2 antibody and MMAE, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:100.

In some embodiments, the ADC is velifatuzumab, which comprises an anti-NaPi2B antibody and MMAE, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the ADC is venosumab, which comprises an antibody that binds to connexin-4 and MMAE. See, e.g., U.S. Pat. No. 8,637,642; WO 2012/047724. In some embodiments, the antibody of venosumab comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 103 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 104; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 105 to 110, respectively.

In some embodiments, the ADC is SGN-B6A, which comprises an antibody that binds to AVB6 and MMAE. In some embodiments, SGN-B6A comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the ADC comprises an anti-AVB6 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 111 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 112; or comprising heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 113 to 118, respectively. In some embodiments, the ADC comprises an anti-AVB6 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 119 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 120; or comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 121 to 126, respectively.

In some embodiments, the ADC is an anti-CD228 antibody-ADC, which comprises an antibody that binds to CD228 and MMAE. See, for example, U.S. Patent Publication Nos. 2020/0246479A1; WO2020/163225A1. In some embodiments, the ADC is SGN-CD228A, which comprises an anti-CD228 antibody and MMAE, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 127 and a light chain variable region (VL) comprising the amino acid sequence SEQ ID NO: 128; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising amino acid sequences SEQ ID NO: 129 to 134, respectively.

In some embodiments, the ADC is SGN-LIV1A (villadizumab; LV), which comprises an anti-LIV-1 antibody and MMAE, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 135 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 136; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137 to 142, respectively;

Wherein SGN-LIV1A comprises an anti-LIV-1 antibody conjugated to: mc-vc-PAB C-MMAE, mc-vc-PABC-MMAF, mc-MMAF or mp-dLAE-PABC-MMAE.

In some embodiments, the ADC is vetisolomab (TV), which comprises an antibody that binds to tissue factor (TF) and MMAE. See, e.g., U.S. Patent Nos. 9,168,314 and 9,150,658; WO 2011/157741; WO 2010/066803. In some embodiments, the antibody of TV comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 143 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 144; or comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO: 145 to 150, respectively.

In some embodiments, the ADC comprises MMAE and binds to a target selected from the group consisting of: AMHRII, Axl, CA9, CD142, CD20, CD22, CD228, CD248, CD30, CD33, CD7, CD48, CD71, CD79b, CLDN18.2, CLDN6, c-MET, EGFR, EphA2, ETBR, FCRH5, GCC, Globo H, gpNMB, HER-2, IL7R, integrin β-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, mesothelin (MSLN), MET, MRC2, MUC16, NaPi2b, catenin-4, OT-MUC1 (tumor tethered MUC1), PSMA, ROR1, SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, tumor-associated glycoprotein 72 (TAG-72).

In some embodiments, the ADC comprises MMAE and is one of the following: DP303c, also known as SYSA1501, which targets HER-2 (CSPC Pharmaceutical; Doph en Biomed); SIA01-ADC, also known as ST1, which targets STn (Siamab Therapeutics); Ladiratuzumab vedotin, also known as SGN-LIV1A, which targets LIV-1 (Merck & Co., Inc.; Seagen (Seattle Genetics) Inc.); ABBV-085, also known as Samrotamab vedotin, which targets LRRC15 (Abbvie; Seagen (Seattle Genetics) Inc.); DMOT4039A, also known as RG7600; αMSLN-MMAE, which targets mesothelin (MSLN) (Roche-Genentech); RC68, also known as Remegen EGFR ADC, which targets EGFR (Re meGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.)); RC108, also known as RC108-ADC, which targets c-MET (RemeGen (Rongchang Biophar maceutical (Yantai) Co., Ltd.)); CMG901, also known as MRG005, which targets CLDN18.2 (Keymed Biosciences; Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)); YBL-001, also known as LCB67, which targets DLK-1 (Lego ChemBiosciences; Pyxis Oncology; Y-Biologics); DCDS0780A, also known as Iladatuzumab ve dotin); RG7986, which targets CD79b (Roche-Genentech; Seagen (Seattle Genetics) Inc.); Tisotumab vedotin, also known as Humax-TF-ADC; tf-011-mmae; TIVDAK ^™ , which targets CD142 (GenMab; Seagen (Seattle Genetics) Inc.); GO-3D1-ADC, also known as humAb-3D1-MMAE ADC, which targets MUC1-C (Genus Oncology LLC); ALT-P7, also known as HM2-MMAE, which targets HER-2 (Alteogen, Inc.; Levena Biopharma; 3SBio, Inc.); Vandortuzumab vedotin, also known as DSTP3086S; RG7450, which targets STEAP-1 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); Genetics) Inc.); Lifastuzumab vedotin, also known as DNIB0600A; NaPi2b ADC; RG7599, which targets NaPi2b (Roche-Genentech); Sofituzumab vedotin, also known as DMUC5754A; RG7458, which targets MUC16 (Seagen (Seattle Genetics) Inc.; Roche-Genentech); RG7841, also known as DLYE5953A, which targets Ly6E (Roche-Genentech; Seagen (Seattle Genetics) Inc.); RG7598, also known as DFRF4539A, which targets FCRH5 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); RG7636, also known as DEDN6526A, which targets ETBR (Seagen (Seattle Genetics) Inc.; tics) Inc.; Roche-Genentech); vepinatuzumab, also known as DCDT2980S; RG7593, which targets CD22 (Roche-Genentech); vepolotuzumab, also known as DCDS4501A; POLIVY ^™ ; RG7596; RO-5541077, which targets CD79b (Chugai Pharmaceutical; Roche-Genentech; Seagen (Seattle Genetics) Inc.); DMUC4064A, also known as D-4064a; RG7882, which targets MUC16 (Roche-Genentech; Seagen (Seattle Genetics) Inc.); SYSA1801, also known as CPO102, which targets CLDN18.2 (Conjupro Biotherapeutics Inc.; CSPC ZhongQi Pharmaceutical Technology Co.); RC118, also known as claudin 18.2-ADC; YH005, which targets CLDN18.2 (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Biocytogen); VLS-101, also known as visitozumab; MK-2140; UC-961ADC3; Ziloverta mab Vedotin, which targets ROR1 (Velos Bio. Inc); Gle mbatumumab vedotin, also known as CDX-011; CR011-vcMMAE, which targets gpNMB (Celldex Therapeutics); BA3021, also known as CAB-ROR2-ADC; Viocuzumab, which targets ROR2 (Bioatla; Himalaya Therapeutics); BA3011, also known as CAB-AXL-ADC; Vimekumab, which targets Axl (Bioatla; Himalaya Therapeutics); CM-09, also known as Bstrongximab-ADC, which targets TRA-1-60 (CureMeta); ABBV-838, also known as Viagra, which targets SLAMF7 (Abbvie); Vienantuzumab, also known as AXL-107-MMAE; HuMax-AXL-ADC, which targets Axl (GenMab; Seagen (Seattle Genetics) Inc.); ARC-01, also known as anti-CD79b ADC, which targets CD79b (Araris Biotech AG); Vidicizumab, also known as RC48, which targets HER-2 (Reme Gen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Seagen (Seattle Genetics) Inc.); ASG-5ME, also known as AGS-5; AGS-5ME, which targets SLC44A4 (Agensys, Inc.; Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); venosumab, also known as AGS-22M6E; ASG-22CE; ASG-22ME; PADCEV ^™ , which targets connexin-4 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); ASG-15ME, also known as AGS-15E; Visutumab, which targets SLITRK6 (Seagen (Seattle Genetics) Inc.; Astellas Pharma Inc.); velotuximab, also known as Adcetris; cAC10-vcMMAE; SGN-35, which targets CD30 (Seagen (Seattle Genetics) Inc.; Takeda); velotuximab, also known as ABBV-399, which targets c-MET (Abbvie); velotuximab, also known as ABBV-221, which targets EGFR (Abbvie); CX-2029, also known as ABBV-2029, which targets CD71 (Abbvie; CytomX Therapeutics); AB-3A4-ADC, also known as AB-3A4-vcMMAE, which targets KAAG-1 (Alethia Biotherapeutics); velotuximab, also known as 5F9-vcMMAE; MLN0264; TAK-264, which targets GCC (Takeda; Millennium Pharmaceuticals, Inc); FOR46, which targets CD46 (Fortis Therapeutics, Inc.); LR004-VC-MMAE, which targets EGFR (Chinese Academy of Medical Sciences Peking Union Medical College Hospital); CD30-ADC, which targets CD30 (NBE Therapeutics; Boehringer Ingelheim); anti-endosialin-MC-VC-PABC-MM AE, which targets CD248 (Genzyme); OBI-998, which targets SSEA-4 (OBI Pharma); MRG002, which targets HER-2 (Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd.)); TRS005, which targets CD20 (Teruisi Pharmaceuticals); Oba01, which targets DR5 (death receptor 5) (Obio Technology (Shanghai) Corp., Ltd.; Yantai Obioadc Biomedical Technology Ltd.); PSMA ADC, which targets PSMA (Progenics Pharmaceuticals, Inc; Seagen (Seattle Genetics) Inc.); SGN-CD48A, which targets CD48 (Seagen (Seattle Genetics) Inc.); IMAB362-vcMMAE, which targets CLDN18.2 (Astellas Pharma Inc.; Ganymed); GB251, which targets HER-2 (Genor Biopharma Co., Ltd.); Innate Pharma BTG-ADC, which targets CD30 (Innate Pharma; Sanofi); ADCendo uPARAP ADC, which targets MRC2 (ADCendo); XCN-010, which targets actM (Xiconic Pharmaceuticals, LLC); ANT-043, which targets HER-2 (Antikor Biopharma); OBI-999, which targets Globo H (Abzena; OBI Pharma); LY3343544, which targets MET (Eli Lilly and Company); Tagworks anti-TAG72 ADC, which targets TAG-72 (Tagworks Pharmaceuticals); IMAB027-vcMMAE, which targets CLDN6 (Ganymed; Astellas Pharma Inc.); LGR5-ADC, which targets LGR5 (Genent ech, Inc.); Philochem B12-MMAE ADC, which targets IL-7R (Instituto de Medicina Molecular Biology, Inc.); Lobo Antunes; Philochem AG); TE-1522, which targets CD19 (Immunwork); SGN-STNV, which targets STn (Seagen (Seattle Genetics) Inc.); HTI-1511, which targets EGFR (Abzena; Halozyme Therapeutics); Peptron PAb001-ADC, which targets OT-MUC1 (tumor tethered-MUC1) (Peptron; Qilu Pharmaceutical co. Ltd.); LM-102, which targets CL DN18.2 (LaNova Medicines Limited); Anwita Biosciences MSLN-MMAE, which targets mesothelin (MSLN) (Anwitabiosciences); SGN-CD228A, which targets CD228 (Seagen (Seattle Genetics) Inc.); NBT828, which targets HER-2 (NewBio Therapeutics; Genor Biopharma Co., Ltd.); Gamama bs GM103, which targets AMHR2 (GamaMabs Pharma; Exelixis); LCB14-0302, which targets HER-2 (Lego ChemBiosciences); BAY79-4620, which targets carbonic anhydrase IX (CAIX) (Bayer; MorphoSys); NBT508, which targets CD79b (NewBio Therapeutics); PAT-DX3-MMAE, which targets an undisclosed target (Patrys; Yale University); AGS67E, which targets CD37 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); CDX-014, which targets TIM-1 (Celldex Therapeutics); BVX001, which targets CD33; CD7 (Bivictrixtherapeutics); SGN-B6A, which targets integrin β-6 (Seagen (Seattle Genetics) Inc.); MR G003, which targets EGFR (Lepu biotech; Shanghai Miracogen Inc. (Shan ghai Meiya Biotechnology Co., Ltd)) and PYX-202, which targets DLK-1 (Pyxis Oncology; Lego Chem Biosciences).

In some embodiments, the ADC comprises MMAF and binds a target selected from the group consisting of: BCMA, GPC-1, CD30, c-MET, SAIL, HER-3, CD70, CD46, HER-2, 5T4, ENPP3, CD19, EGFR, EphA2.

In some embodiments, the ADC comprises MMAF and is one of the following: CD70-ADC, which targets CD70 (Kochi University; Osaka University); IGN786, which targets SAIL (AstraZeneca; Igenica Biotherapeutics); PF-06263507, which targets 5T4 (Pfizer); GPC1-ADC, which targets GPC-1 (Kochi University); ADC-AVP10, which targets CD30 (Avipep); M290-MC-MMAF, which targets CD103 (The Second Affiliated Hospital of Harbin Medical University); BVX001, which targets CD33; CD7 (Bivictrix therapeutics); Tanabe P3D12-vc-MMAF, which targets c-MET (Tanabe Research Laboratories); LILRB4-targeted ADC, which targets LILRB4 (The University of Texas Health Science Center, Houston); TSD101, also known as ABL201, which targets BCMA (TSD Life Science; ABL Bio; Lego Chem Biosciences); martin-detuximab, also known as ABT-414, which targets EGFR (Abbvie; Seagen (Seattle Genetics) Inc.); AGS16F, also known as AGS-16C3F; AGS-16M8F, which targets ENPP3 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.); AVG-A11 BCMA ADC, also known as AVG-A11-mcMMAF, which targets BCMA (Avantgen); martin-belantamab, also known as BLENREP; GSK2857916; J6M0-mcMMAF, which targets BCMA (GlaxoSmithKline; Seagen (Seattle Genetics) Inc.); Genetics) Inc.); MP-HER3-ADC, also known as HER3-ADC, which targets HER-3 (MediaPharma); FS-1502, also known as LCB14-0110, which targets HER-2 (Lego Chem Biosciences; Shanghai Fosun Pharmaceutical Development Co, Ltd.); MEDI-547, also known as MI-CP177, which targets EphA2 (AstraZeneca; Seagen (Seattle Genetics) Inc.); martin-wortuzumab, also known as SGN-75, which targets CD70 (Seagen (Seattle Genetics) Inc.); martin-dinituzumab, also known as SGN-CD19A, which targets CD19 (Seagen (Seattle Genetics) Inc.) and HTI-1066, also known as SHR-A1403, which targets c-MET (Jiangsu HengRui Medicine Co., Ltd.).

In some embodiments, the ADC is selected from the ADCs in Table A, Table B, or Table C. In Table A, Table B, and Table C, ADCs having sequences provided in the sequence listing are marked with an asterisk (*). In some embodiments, the ADC is not venosumab. In certain embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not venetoclax. In some embodiments, the ADC is not SGN-CD228A.

D. Preparation of Antibodies

To prepare antibodies, many techniques known in the art can be used. See, for example, Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed. 1986).

The genes encoding the heavy and light chains of the antibodies of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas expressing the antibodies and used to produce recombinant monoclonal antibodies. The gene library encoding the heavy and light chains of monoclonal antibodies can also be made from hybridomas or plasma cells. In addition, phage or yeast display technology can be used to identify antibodies and heteropolymeric Fab fragments that specifically bind to selected antigens (see, for example, McCafferty et al., Nature 348: 552-554 (1990); Marks et al., Biotechnology 10: 779-783 (1992); Lou et al. (2010) PEDS 23: 311; and Chao et al., Nature Protocols, 1: 755-768 (2006)). Alternatively, antibodies and antibody sequences can be separated and/or identified using a yeast-based antibody presentation system, such as disclosed in, for example, Xu et al., Protein Eng Des Sel, 2013, 26: 663-670; WO 2009/036379; WO 2010/105256; and the antibody presentation system in WO2012/009568. The random combination of heavy and light chain gene products produces a large number of antibodies with different antigenic specificities (see, for example, Kuby, Immunology (3rd edition, 1997)). The technology for producing single-chain antibodies or recombinant antibodies (U.S. Patent No. 4,946,778, U.S. Patent No. 4,816,567) may also be applicable to producing antibodies. Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or antibodies covalently bound to immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; and WO 92/200373).

Any number of expression systems can be used, including prokaryotic expression systems and eukaryotic expression systems to produce antibodies. In some embodiments, the expression system is a mammalian cell, such as a hybridoma, or a CHO cell. Many such systems are widely available from commercial suppliers. In embodiments in which the antibody comprises both a heavy chain and a light chain, for example, in a bicistronic expression unit or under the control of different promoters, a single vector can be used to express the heavy chain and the heavy chain and the light chain. In other embodiments, a separate vector can be used to express the heavy chain and the light chain region. The heavy chain and the light chain as described herein can optionally include methionine at the N-terminal.

In some embodiments, antibody fragments (such as Fab, Fab', F(ab') ₂ , scFv or diabodies) are produced. Various techniques for producing antibody fragments have been developed. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Meth., 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli (E. coli) cells and chemically coupled to form F(ab') ₂ fragments (see, e.g., Carter et al., BioTechnology, 10: 163-167 (1992)). According to another method, F(ab') ₂ fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See, e.g., PCT Publication No. WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a linear antibody as described, e.g., in U.S. Patent No. 5,641,870.

In some embodiments, the antibody or antibody fragment can be conjugated to another molecule, such as polyethylene glycol (PEGylation) or serum albumin, to provide an extended half-life in vivo. Examples of PEGylation of antibody fragments are provided in Knigh et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br. J. Cancer 70:1126, 1994 (for anti-CEA antibodies); Chapman et al., Nature Biotech. 17:780, 1999; and Humphreys et al., Protein Eng. Des. 20:227, 2007).

In some embodiments, multispecific antibodies, such as bispecific antibodies, are provided.Multispecific antibodies are antibodies that have binding specificity for at least two different antigens or for at least two different epitopes of the same antigen. Methods for making multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of heavy and light chains in a host cell (see, e.g., Zuo et al., Protein Eng Des Sel, 2000, 13:361-367); "knob-and-hole" engineering (see, e.g., Ridgway et al., Protein Eng Des Sel, 1996, 9:617-721); "diabody" technology (see, e.g., Hollinger et al., PNAS (USA), 1993, 90:6444-6448); and intramolecular trimerization (see, e.g., Alvarez-Cienfuegos et al., Scientific Reports, 2016, doi:/10.1038/srep28643); see also Spiess et al., Molecular Immunology, 2015, 67(2), Section A:95-106.

Selection of constant regions

The heavy chain and light chain variable regions of the antibodies described herein may be connected to at least a portion of a human constant region. The selection of constant regions depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and/or complement-dependent cytotoxicity are needed. For example, human isotope IgG1 and IgG3 have stronger complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity and human IgG4 lacks complement-dependent cytotoxicity. In addition, human IgG1 and IgG3 induce cell-mediated effector functions that are stronger than human IgG2 and IgG4. The light chain constant region may be λ or κ. The antibody may be expressed as a tetramer containing two light chains and two heavy chains, expressed as a single heavy chain, a light chain, expressed as Fab, Fab', F(ab') ₂ and Fv, or expressed as a single-chain antibody, wherein the heavy chain and light chain variable domains are connected via a spacer.

Human constant regions show allotypic and xenotypic variation between different individuals, i.e., the constant regions may differ at one or more polymorphic positions in different individuals. Xenotypic is distinguished from allotypic in that serum recognizes an allotype in combination with one or more non-polymorphic regions of the other isotype.

One or more amino acids at the amino or carboxyl terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in a certain proportion or all of the molecules. Substitutions may be made in the constant region to reduce or increase effector functions, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

For the construction of the desired antibody-drug conjugate, in some embodiments, exemplary substitutions including substitutions of a native amino acid to a cysteine residue are introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332, preferably the S239C mutation in the human IgG1 isotype (numbering is according to the EU index (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991); see US Pat. 20100158909, which is incorporated herein by reference). The presence of additional cysteine residues can allow interchain disulfide bond formation. Such interchain disulfide bond formation can lead to steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. One or more cysteine residues introduced into or near the Fc region of the IgG constant region can also serve as a site for conjugating therapeutic agents (i.e., using thiol-specific reagents, such as maleimide derivatives of drugs to couple cytotoxic drugs). The presence of therapeutic agents leads to steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any one of positions 234, 235, 236 and/or 237 reduce the affinity for Fcγ receptors (particularly FcγRI receptors) (see, for example, US 6,624,821, US 5,624,821).

The in vivo half-life of an antibody can also have an impact on its effector function. The half-life of an antibody can be increased or decreased to regulate its therapeutic activity. FcRn is a receptor structurally similar to MHC class I antigens that non-covalently associate with β2-microglobulin. FcRn regulates the catabolism of IgG and its transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). IgG-FcRn interaction occurs at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood); this interaction enables IgG to be recycled back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The region on human IgG1 involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). IgG1 molecules with these substitutions have longer serum half-lives. Therefore, compared with unmodified IgG1, these modified IgG1 molecules may be able to achieve their effector functions over a longer period of time and thus exert their therapeutic efficacy. Other exemplary substitutions for increasing binding to FcRn include Gln at position 250 and/or Leu at position 428. EU numbering is used for all positions in the constant region.

The complement-binding activity (both C1q binding and CDC activity) of antibodies can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166: 2571-2575). The same substitutions on the human IgG2 main chain can convert an antibody isotype that does not bind to C1q sufficiently and severely lacks complement activation activity into an antibody isotype that can bind to C1q and regulate CDC (Idusogie et al., 2001, J. Immunol. 166: 2571-75). Several other methods have also been applied to improve the complement-binding activity of antibodies. For example, transplanting the 18-amino acid carboxyl-terminal tail of IgM to the carboxyl terminus of IgG greatly enhances its CDC activity. This enhancement is observed even in the case of IgG4, which does not normally have detectable CDC activity (Smith et al., 1995, J. Immunol. 154: 2226-36). In addition, substitution of Ser444 located near the carboxyl terminus of the IgG1 heavy chain with Cys induced a 200-fold increase in the CDC activity of tail-to-tail dimerization of IgG1 compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148: 2918-22). In addition, bispecific diabody constructs with specificity for C1q also conferred CDC activity (Kontermann et al., 1997, Nat. Biotech. 15: 629-31).

Complement activity can be reduced by mutating at least one of the amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted nonionic residues, such as Gly, Ile, Leu, or Val, or aromatic nonpolar residues such as Phe, Tyr, Trp, and Pro, replacing any of the three residues, can reduce or eliminate C1q binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322 instead of 318 to reduce or eliminate C1q binding activity. Replacing the 318 (Glu) residue with a polar residue can modulate rather than eliminate C1q binding activity. Replacing residue 297 (Asn) with Ala results in the removal of cleavage activity but only slightly reduces (about three times weaker) the affinity for C1q. This change destroys the glycosylation site and the presence of the carbohydrate required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduced CIq binding: D270A, K322A, P329A and P31IS (see WO 06/036291).

References to human constant regions include constant regions having any natural allotype or any arrangement of residues occupying polymorphic positions in natural allotypes. In addition, there may be up to 1, 2, 5 or 10 mutations relative to the natural human constant region, such as those indicated above to reduce Fcγ receptor binding or increase binding to FcRN.

Nucleic acids, vectors and host cells

In some embodiments, the antibodies described herein are prepared using recombinant methods. Therefore, in some aspects, the present invention provides isolated nucleic acids comprising nucleic acid sequences encoding any of the antibodies described herein (e.g., any one or more of the CDRs described herein); vectors comprising such nucleic acids; and host cells, wherein nucleic acids for replicating antibody encoding nucleic acids and/or expressing antibodies are introduced. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a human cell.

In some embodiments, polynucleotides (e.g., isolated polynucleotides) include nucleotide sequences encoding antibodies described herein. In some embodiments, polynucleotides include nucleotide sequences encoding one or more amino acid sequences disclosed herein (e.g., CDRs, heavy chains, light chains, and/or framework regions). In some embodiments, polynucleotides include nucleotide sequences encoding amino acid sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) with sequences disclosed herein (e.g., CDRs, heavy chains, light chains, or framework regions).

In another aspect, methods for making antibodies as described herein are provided. In some embodiments, the method comprises culturing a host cell as described herein (e.g., a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

Suitable vectors containing polynucleotides encoding antibodies of the present disclosure or fragments thereof include cloning vectors and expression vectors. Although the cloning vector selected may vary depending on the host cell intended for use, useful cloning vectors are generally capable of self-replication, may have a single target for a specific restriction endonuclease and/or may carry genes for markers that can be used to select clones containing the vector. Examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. Cloning vectors are available from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

An expression vector is typically a replicable polynucleotide construct containing a nucleic acid of the present disclosure. The expression vector can replicate in a host cell as an additional gene or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and any other vectors.

Expression of recombinant antibodies

Antibodies are usually produced by recombinant expression. Recombinant polynucleotide constructs usually include expression control sequences operably linked to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. Preferably, the expression control sequence is a eukaryotic promoter system in the vector that can transform or transfect eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences and collection and purification of cross-reactive antibodies.

Mammalian cells are preferred hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Multiple suitable host cell lines capable of secreting complete heterologous proteins have been developed in the art, and the host cell lines include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma, including Sp2/0 and NS0. Preferably, the cells are non-human. Expression vectors for these cells may include expression control sequences, such as replication origins, promoters, enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148:1149 (1992).

Once expressed, the antibodies may be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Antibody characterization

Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art. See, e.g., Ernst et al., Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009). These methods include, but are not limited to, solid phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (SPR, e.g., Biacore ^™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., ), flow cytometry, fluorescence activated cell sorting (FACS), BioLayer interferometry (e.g., Octet ^TM (Forte Bio, Inc., Menlo Park, CA)), and Western blot analysis. SPR technology is reviewed in, for example, Hahnfeld et al., Determination of Kinetic Data Using SPR Biosensors, Molecular Diagnosis of Infectious Diseases (2004). In a typical SPR experiment, one interacting agent (target or targeting agent) is immobilized on an SPR-active gold-coated glass slide in a flow cell, and a sample containing another interacting agent is introduced to flow across the surface. When light of a given wavelength is irradiated on the surface, changes in the optical reflectivity of the gold indicate binding and binding kinetics. In some embodiments, affinity is determined using a kinetic exclusion assay. This technique is described in, for example, Darling et al., Assay and Drug Development Technologies, Vol. 2, No. 6 647-657 (2004). In some embodiments, affinity is determined using a BioLayer interferometry assay. This technique is described, for example, in Wilson et al., Biochemistry and Molecular Biology Education, 38:400-407 (2010); Dysinger et al., J. Immunol. Methods, 379:30-41 (2012).

IV. Treatment Methods

In some embodiments, a method for treating cancer in a subject is provided. In some embodiments, the method comprises administering to the subject: (1) an antibody-drug conjugate (ADC) comprising a first antibody that binds to a tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is a tubulin disruptor; and (2) a second antibody that binds to an immune cell adaptor, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs. In some embodiments, the Fc of the second antibody enhances binding to one or more of FcγRIIIa, FcγRIIa, and/or FcγRI. In some embodiments, the Fc of the second antibody reduces binding to one or more inhibitory FcγRs. In some embodiments, the Fc of the second antibody reduces binding to FcγRIIb.

In some embodiments, a method of treating cancer comprises administering to a subject having cancer: (1) an antibody-drug conjugate (ADC), wherein the ADC comprises a first antibody that binds to a tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is a tubulin disrupting agent; and (2) a second antibody that binds to an immune cell adaptor, wherein the second antibody comprises an Fc that has enhanced ADCC activity relative to a corresponding wild-type Fc of the same isotype. In some embodiments, the second antibody comprises an Fc that has enhanced ADCC and ADCP activity relative to a corresponding wild-type Fc of the same isotype. In some embodiments, the Fc of the second antibody enhances binding to one or more of FcγRIIIa, FcγRIIa, and/or FcγRI. In some embodiments, the Fc of the second antibody reduces binding to one or more inhibitory FcγRs. In some embodiments, the Fc of the second antibody reduces binding to FcγRIIb.

In various embodiments, the second antibody is a non-fucosylated antibody. In various such embodiments, the second antibody is contained in an antibody composition, wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibodies in the composition are non-fucosylated.

In some embodiments, the second antibody binds to TIGIT. In some embodiments, the second antibody binds to CD40. In some embodiments, the second antibody binds to an immune cell engager provided herein.

In various embodiments, the tubulin disruptor conjugated to the first antibody in the ADC is auristatin, tubulysin, colchicine, vinca alkaloids, taxanes, chrysogenin, maytansine or hemiacetylene. In some embodiments, the ADC comprises MMAE or MMAF. In various embodiments, the first antibody binds to a tumor-associated antigen, such as a tumor-associated antigen provided herein.

Any ADC described herein may be combined with any antibody that binds to an immune cell engager described herein. For example, in some embodiments, the ADC is SGN-PDL1V and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-ALPV and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-B7H4V and the second antibody is SEA-BCMA. In some embodiments, the ADC is velifatuzumab and the second antibody is SEA-BCMA. In some embodiments, the ADC is SEA-CD40 and the second antibody is SEA-BCMA. In some embodiments, the ADC is SEA-CD70 and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-B6A and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-CD228A and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-LIV1A and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN-STNV and the second antibody is SEA-BCMA. In some embodiments, the ADC is vedicituzumab (SGN-35) and the second antibody is SEA-BCMA. In some embodiments, the ADC is venitinomab and the second antibody is SEA-BCMA. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-BCMA. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-BCMA. In some embodiments, the ADC is veticituzumab and the second antibody is SEA-BCMA.

In some embodiments, the ADC is SGN-PDL1V and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-ALPV and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-B7H4V and the second antibody is SEA-CD40. In some embodiments, the ADC is velifatuzumab and the second antibody is SEA-CD40. In some embodiments, the ADC is SEA-CD40 and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-CD70 and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-B6A and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-CD228A and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-LIV1A and the second antibody is SEA-CD40. In some embodiments, the ADC is SGN-STNV and the second antibody is SEA-CD40. In some embodiments, the ADC is vedicituzumab (SGN-35) and the second antibody is SEA-CD40. In some embodiments, the ADC is venitinib and the second antibody is SEA-CD40. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-CD40. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-CD40. In some embodiments, the ADC is veticituzumab and the second antibody is SEA-CD40.

In some embodiments, the ADC is SGN-PDL1V and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-ALPV and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-B7H4V and the second antibody is SEA-CD70. In some embodiments, the ADC is velifatuzumab and the second antibody is SEA-CD70. In some embodiments, the ADC is SEA-CD40 and the second antibody is SEA-CD70. In some embodiments, the ADC is SEA-CD70 and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-B6A and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-CD228A and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-LIV1A and the second antibody is SEA-CD70. In some embodiments, the ADC is SGN-STNV and the second antibody is SEA-CD70. In some embodiments, the ADC is vedicituzumab (SGN-35) and the second antibody is SEA-CD70. In some embodiments, the ADC is venitinib and the second antibody is SEA-CD70. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-CD70. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-CD70. In some embodiments, the ADC is veticituzumab and the second antibody is SEA-CD70.

In some embodiments, the ADC is SGN-PDL1V and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-ALPV and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-B7H4V and the second antibody is SEA-TGT. In some embodiments, the ADC is velifatuzumab and the second antibody is SEA-TGT. In some embodiments, the ADC is SEA-CD40 and the second antibody is SEA-TGT. In some embodiments, the ADC is SEA-CD70 and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-B6A and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-CD228A and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-LIV1A and the second antibody is SEA-TGT. In some embodiments, the ADC is SGN-STNV and the second antibody is SEA-TGT. In some embodiments, the ADC is vedicituzumab (SGN-35) and the second antibody is SEA-TGT. In some embodiments, the ADC is venicituzumab and the second antibody is SEA-TGT. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-TGT. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-TGT. In some embodiments, the ADC is vedicituzumab and the second antibody is SEA-TGT.

In some embodiments, the subject is a human.

In some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, renal clear cell carcinoma, head and neck cancer, lung cancer, lung adenocarcinoma, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, melanoma, central nervous system neoplasm, mesothelioma, lymphoma, leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma or sarcoma. In some embodiments, the cancer is selected from gastric cancer, testicular cancer, pancreatic cancer, lung adenocarcinoma, bladder cancer, head and neck cancer, prostate cancer, breast cancer, mesothelioma and renal clear cell carcinoma. In some embodiments, the cancer is a lymphoma or leukemia, including but not limited to acute myeloid, chronic myeloid, acute lymphocytic or chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, primary mediastinal large B-cell lymphoma, splenic marginal zone B-cell lymphoma, or extranodal marginal zone B-cell lymphoma. In some embodiments, the cancer is selected from chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma. In some embodiments, the cancer is a metastatic cancer.

In some embodiments, the cancer is a cancer with a high tumor mutation load, so the cancer has more antigens that drive T cell responses. Thus, in some embodiments, the cancer is a high mutation load cancer, such as lung cancer, melanoma, bladder cancer, or gastric cancer. In some embodiments, the cancer has microsatellite instability.

In various embodiments, the second antibody depletes regulatory T (Treg) cells, activates antigen presenting cells (APCs), enhances CD8 T cell responses, upregulates co-stimulatory receptors and/or promotes the release of immune-activating cytokines (such as CXCL10 and/or IFNγ). In some embodiments, the second antibody promotes the release of immune-activating cytokines to a greater extent than immunosuppressive cytokines (such as IL10 and/or MDC).

The ADC and the second antibody can be administered simultaneously or sequentially. For sequential administration, the first dose of the ADC can be administered before the first dose of the second antibody, or the first dose of the second antibody can be administered before the ADC. For simultaneous administration, in some embodiments, the ADC and the second antibody can be administered in separate pharmaceutical compositions or in the same pharmaceutical composition.

In some embodiments, the therapeutic agent is administered in a therapeutically effective amount or dosage. The daily dosage range of about 0.01mg/kg to about 500mg/kg, or about 0.1mg/kg to about 200mg/kg, or about 1mg/kg to about 100mg/kg, or about 10mg/kg to about 50mg/kg can be used. However, the dosage can vary according to several factors, including the selected route of administration, the formulation of the composition, the patient's response, the severity of the illness, the subject's weight, and the judgment of the prescribing physician. Depending on individual patient needs, the dosage can increase or decrease over time. In some cases, the patient is initially given a low dose, which is then increased to an effective dose that can be tolerated by the patient. The determination of the effective amount is fully within the capabilities of those skilled in the art.

In some embodiments, the enhanced activity observed by the specific combination therapy described herein has certain benefits compared to the corresponding monotherapy treatment. For example, in some embodiments, the combined administration of ADC and the second antibody has a toxicity profile comparable to that when the ADC or the second antibody is administered as a monotherapy. In some embodiments, the effective dose of ADC and/or the second antibody when administered in combination is less than the effective dose when administered as a monotherapy. In some embodiments, compared to the corresponding monotherapy treatment, the combined administration of ADC and the second antibody provides a longer response duration. In some embodiments, compared to the corresponding monotherapy, the combined administration of ADC and the second antibody produces a longer progression-free survival period. In some embodiments, the administration of ADC and the second antibody can be used to treat recurrent cancers that relapse after monotherapy treatment with either agent alone.

The route of administration of the pharmaceutical composition can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other method known in the art. In some embodiments, one or more therapeutic agents are administered orally, intravenously or intraperitoneally.

Co-administered therapeutic agents may be administered together or individually, simultaneously or at different times. When administered, the therapeutic agents may be administered once, twice, three times, four times or more or less times per day as needed. In some embodiments, the administered therapeutic agent is administered once per day. In some embodiments, the administered therapeutic agent is administered, for example, as a mixture, at the same or the same number of times. In some embodiments, one or more of the therapeutic agents are administered in a sustained release formulation.

In some embodiments, the therapeutic agents are administered simultaneously. In some embodiments, the therapeutic agents are administered sequentially. For example, in some embodiments, a first therapeutic agent is administered, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 days or more before the administration of a second therapeutic agent.

In some embodiments, the treatments provided herein are administered to a subject for an extended period of time, e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days, or longer.

V. Compositions and Kits

In another aspect, compositions and kits for treating or preventing cancer in a subject are provided.

Pharmaceutical composition

In some embodiments, pharmaceutical compositions for use in the methods of the invention are provided. In some embodiments, the ADC is administered as a first pharmaceutical composition and the antibody that binds to an immune cell engager is administered as a second pharmaceutical composition. In some embodiments, the ADC and the antibody that binds to an immune cell engager are administered as a single pharmaceutical composition.

Guidance for preparing formulations for use in the present invention can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., 2006, supra; Martindale: The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press; Niazi, Handbook of Pharmaceutical Manufacturing Formulations, 2004, CRC Press; and Gibson, Pharmaceutical Preformulation and Formulation: A Practical Guide from Candidate Drug Selection to Commercial Dosage Form, 2001, Interpharm Press, which are hereby incorporated by reference herein. The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, i.e., by conventional mixing, dissolving, granulating, dragee making, emulsifying, encapsulating, coating or lyophilizing processes. The following methods and excipients are exemplary only and are by no means limiting.

In some embodiments, one or more therapeutic agents are prepared for sustained release, controlled release, extended release, timed release or delayed release preparations, such as delivered in a semi-permeable matrix of a solid hydrophobic polymer containing a therapeutic agent. Various types of sustained release materials have been established and are well known to those skilled in the art. Current extended release preparations include film-coated tablets, multi-granular or pellet systems, matrix technology using hydrophilic or lipophilic materials, and wax-based tablets with pore-forming excipients (see, for example, Huang et al., Drug Dev. Ind. Pharm. 29: 79 (2003); Pearnchob et al., Drug Dev. Ind. Pharm. 29: 925 (2003); Maggi et al. Eur. J. Pharm. Biopharm. 55: 99 (2003); Khanvilkar et al., Drug Dev. Ind. Pharm. 228: 601 (2002); and Schmidt et al., Int. J. Pharm. 216: 9 (2001)). Depending on its design, a sustained release delivery system can release the compound over the course of hours or days, e.g., over 4, 6, 8, 10, 12, 16, 20, 24 hours or longer. Typically, sustained release formulations can be prepared using naturally occurring or synthetic polymers, e.g., polymeric vinyl pyrrolidones, such as polyvinyl pyrrolidone (PVP); carboxyvinyl hydrophilic polymers; hydrophobic and/or hydrophilic hydrocolloids, such as methylcellulose, ethylcellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; and carboxypolymethylene.

For oral administration, the therapeutic agent can be easily formulated by combining with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compound to be formulated into tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. Oral drug preparations can be obtained by mixing the compound with a solid excipient, optionally grinding the resulting mixture, and, if necessary, processing the granular mixture to obtain a tablet or dragee core after adding a suitable auxiliary agent. Suitable excipients include, for example, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, can be added.

The therapeutic agent may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the compound may be formulated into a formulation by dissolving, suspending or emulsifying one or more compounds in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a synthetic aliphatic acid glyceride, an ester of a higher aliphatic acid, or propylene glycol; and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the compound may be formulated in the form of an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or a physiological saline buffer. The formulation for injection may be presented in a unit dosage form, for example, in an ampoule or a multi-dose container, with a preservative added. The composition may be in the form of a suspension, solution, or emulsion, such as in an oily or aqueous vehicle, and may contain a preparatant such as a suspending agent, a stabilizer, and/or a dispersant.

The therapeutic agent can be administered systemically via transmucosal or transdermal means. For transmucosal or transdermal administration, a permeant suitable for permeation of the barrier is used in the formulation. For topical administration, the agent is formulated into an ointment, cream, salves, powders, and gels. In one embodiment, the transdermal delivery agent can be DMSO. The transdermal delivery system may include, for example, a patch. For transmucosal administration, a permeant suitable for permeation of the barrier is used in the formulation. Such permeants are generally known in the art. Exemplary transdermal delivery formulations include those described in U.S. Patent Nos. 6,589,549; 6,544,548; 6,517,864; 6,512,010; 6,465,006; 6,379,696; 6,312,717 and 6,310,177, each of which is hereby incorporated herein by reference.

In some embodiments, the pharmaceutical composition comprises an acceptable carrier and/or excipient. Pharmaceutically acceptable carriers include any solvent, dispersion medium or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical or subcutaneous administration. Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds, which play a role in, for example, stabilizing the composition or increasing or reducing the absorption of the active agent. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; compositions or excipients or other stabilizers and/or buffers that reduce the clearance or hydrolysis of the active agent. Other pharmaceutically acceptable carriers and their preparations are well known and are generally described in, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005. Various pharmaceutically acceptable excipients are well known in the art and can be found, for example, in the Handbook of Pharmaceutical Excipients (5th edition, Ed. Rowe et al., Pharmaceutical Press, Washington, D.C.).

The dosage and desired concentration of the pharmaceutical compositions of the present disclosure may vary depending on the specific use envisioned. Determining the appropriate dosage or route of administration is well within the skill of those skilled in the art. Suitable dosages are also described herein.

Pill Box

In some embodiments, a kit for treating a subject with cancer is provided. In some embodiments, the kit comprises:

an antibody-drug conjugate comprising a first antibody conjugated to a tubulin disrupting agent, as provided herein; and

A second antibody, which binds to an immune cell engager, is provided herein.

In some embodiments, the kit may also include explanatory materials (e.g., instructions for using the kit for treating cancer) containing instructions (i.e., protocols) for practicing the methods of the invention. Although explanatory materials typically include written or printed materials, they are not limited thereto. The present invention encompasses any medium capable of storing these instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CDROMs), etc. Such media may include the address of an Internet site that provides such explanatory materials.

VI. Examples

The examples discussed below are intended only to illustrate the present invention and should not be considered to limit the present invention in any way. The examples are not intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the numbers (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

Example 1: Indirect chemotherapeutic agents impair T cell responses

1.1 Materials and methods

Primary human T cells were induced to proliferate using beads coated with CD3/CD28. Enriched CD3+T cells labeled with 20,000 carboxyfluorescein diacetate succinimidyl ester (CSFE) were incubated with anti-CD3 CD28 beads (1 bead/4 T cells) + 10 ng/mL IL-2 for 4 days. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain (ThermoFisher) and live cells were counted via flow cytometry.

1.2 Results

As shown in Figure 1, all single free-agent chemotherapeutic drugs tested significantly reduced primary human T cell proliferation. These data suggest that systemic exposure to chemotherapeutic agents may limit T cell-mediated activity in patients, including responses from immuno-oncology agents (e.g., antibodies that bind immune cell engagers).

Example 2: Vedotin ADC does not inhibit T cell proliferation even when delivered directly to T cells (BV(SGN-35) treatment of CD30+CD8 T cells)

2.1 Materials and methods

Human primary CD8 T cells were labeled with CSFE and induced with anti-CD3-CD28 beads (1 bead/4 T cells) + 10 ng/mL IL-2 for proliferation for 4 days. During activation, CD30 is upregulated on the surface of T cells. CD30+CD8 T cells were treated with CD30-guided vc-MMAE (vebruximab; BV; SGN-35) or isotype control. Cells were stained with LIVE/DEADFixable Dead Cell Stain (ThermoFisher) and live cells were counted via flow cytometry.

2.2 Results

As shown in Figure 2, cell proliferation of primary human CD30+CD8 T cells was not significantly altered by treatment with BV. These data suggest that systemic exposure to the vedotin ADC, even directly targeting CD8 T cells, does not affect CD8-mediated anti-tumor responses.

Example 3: Endoplasmic reticulum stress induction is superior for vedotin ADC

3.1 Materials and methods

Inducing endoplasmic reticulum (ER) stress is one of the first and necessary steps for eliciting immunogenic cellular responses (Figure 3B). The MIA-PaCa-2 pancreatic cancer cell line was treated with ADCs conjugated with different payloads, including vedotin (MMAE), emtansine (DM1), exotecan DS-8201 (Ex), and free microtubule stabilizer paclitaxel, at IC50 concentrations that induce cell death in this system. After 36 or 48 hours of treatment, cells were harvested for Western blot analysis and the upstream ER stress marker pJNK was evaluated by Western blot (Figure 3B).

3.1.1 Western blotting

Treated cells were centrifuged at 14,000 to 16,000 rpm for 10 min and stored at -20°C. Cell pellets were resuspended in 4X BOLT ^TM LDS sample buffer (Thermo Fisher catalog number B0007) and cut by heating at 95°C for 5 to 10 minutes to produce cell lysates. Sample lysates were run on Bis-Tris4 to 12% gradient gel at 140V in MOPS buffer for 1 hour and 40 minutes. The Bis-Tris gel was then transferred to a nitrocellulose membrane using iBlot2. The membrane was washed once in 1X TBS and incubated overnight at 4°C in Licor blocking buffer. The membrane was washed four times in 1X TBS-T, each time for 5 to 10 min. Images were produced on the Licor Odyssey system using 84 μm resolution and automatic intensity.

3.1.2 CHOP luciferase induction assay

The evaluation of downstream pathways induced by ER stress was performed using MIA-PaCa-2 cells transduced by CHOP-driven luciferase reporter cell lines. CHOP is the last step in the ER stress response cascade and its expression level increases due to ER stress. Several ADC payloads in clinical development (Fig. 3A) were used in the evaluation. CHOP induction was measured using a reporter system for CHOP activity according to manufacturer instructions (Bright-Glo ^™ Luciferase Assay System, Promega). In brief, 100,000 cells/well were plated in 96-well flat-bottomed transparent plates (aliquots, 150 μL/well). 200 μL culture media were aliquoted to the outer holes of the plates to provide a culture medium "coating" around the cell wells. At 24, 48 and 72 hours, the plates were removed from the incubator and brought to room temperature. 100 μL culture media were removed from the holes. 100 μL BrightGlo reagents were added to each well. Before reading, the plates were shaken for at least two minutes. The plates were read using the Envision CTG 96-well standard protocol.

3.2 Results

As shown in Figures 3C to 3E, treatment with auristatin-based ADC (MMAE-ADC or MMAF-ADC) was the only case in which early ER stress response pJNK signaling was found to be induced for the different ADC payloads tested. ER stress induction is associated with microtubule disruption because the ER requires intact microtubules to expand and contract to accommodate the protein translation needs of the cell. The ability of MMAE to induce this ER stress as a microtubule disrupting agent is exemplified in the data shown.

FIG. 3D-3E demonstrate that CHOP, a downstream signal in the ER stress pathway, is significantly induced by MMAE ADC and that downstream pathways of ER stress are also driven differently by MMAE ADC compared to other payloads.

Example 4: ICD Potential of Different Clinical ADC Payloads

4.1 Materials and methods

Typical ICD markers include surface exposure of calreticulin and release of ATP and HMGB1, which occur with the induction of ER stress response. These molecules are considered to be danger signals and activate innate immune cells and increase tumor antigen-specific T cell responses. MIA-PaCa-2 cancer cells are treated with IC50 concentrations of payloads currently in the clinical stage carrying ADC, i.e., MMAE, DM1, and Exitecan (Ex). The treated cells are then analyzed for ICD marker induction. Cisplatin is used as a negative control because it can drive cell death, but is known not to induce ICD.

100,000 to 150,000 cells were seeded per well in a 96-well plate. Cells were made to reach 50% to 60% confluence. Culture medium was removed and fresh culture medium was added to each well of cells. 1 μg/mL of 1 μM drug was added to each cell well. After 24 hours, 250 μL (for ATP release assay) or 200 μL (for HMGB1 assay) culture medium was collected and transferred to a labeled 1.5 mL Eppendorf tube. Each tube sample was centrifuged at 10,000 rpm for 1 min. 50 μL culture medium was transferred to the wells in a 96-well transparent bottom plate. 50 μL CTG was added to each well. Shake the plate for 1 to 2 min. Envision plate reader was used to read the plate.

HMGB1 release levels were monitored by luminescence intensity/well using an Envision plate reader. HMGB1 and ATP release levels were reported as fold changes relative to background values for untreated samples. The acquired values were converted to text files and exported and analyzed using Excel and GraphPad Prism.

4.2 Results

As shown in Figure 4A, vc-MMAE strongly drives ATP release compared to other payloads tested. Although HMGB1 release is associated with the induction of ICD, its release is also seen when cells begin to undergo necrosis and is not directly associated with robust immune cell engagement. In contrast to the topoisomerase inhibitor exotecan (Ex), treatment of MIA-PaCa-2 cells with the tubulin disruptor vc-MMAE and DM1 produced robust HMGB1 release (Figure 4B).

Example 5: Immune Activation Evaluation of ADC Payloads

5.1 Materials and methods

5.1.1 Cells

As shown in Figure 5A, ADC conjugated with MMAE destroys microtubules, causing ER stress response, thereby leading to immunogenic cell death (ICD). The dying cells instead release immune activation molecules-damage associated molecular patterns (DAMPs)-such as HSP70, HSP90, ATP, HMGB1 and calreticulin (CRT). These DAMPs can bind receptors, such as LPR1/CD91, P2RX7, P2RY2, AGER, TLR2 and TLR4, thereby activating the innate immune system. This activation causes, for example, protein upregulation such as CD80, CD86, HLA-DR and CD40, increased MHCII expression on monocytes and cytokine release such as CXCL-10/IP10 and IL-12, thereby initiating anti-tumor T cell response. Such T cell responses can be further enhanced by PD-1/L1 inhibitors. Here, the immunological consequences of ICD were evaluated in human peripheral blood mononuclear cell (PBMC) cultures. Cancer cells exposed to ADCs conjugated with different payloads were added to PBMCs.

L540cy cancer cells exposed to EC50 concentration of ADC or free drug for 18 hours (at 37°C, 5% _CO2 ) were washed and 250ul PBMCs suspended at 10× ¹⁰⁶ cells/mL were added to the cancer cell line killed cells for 48 hours. Tissue culture medium was obtained and cytokines were measured by Luminex evaluation.

Treatments were performed in triplicate on 2 independent PBMC donors.

5.1.2 Surface expression of co-stimulatory molecules

After treatment, the cell pellet was resuspended in 50 mL BD FACs buffer and transferred to a 96-well round-bottom microtiter plate. Fc receptors were blocked with 100 μg/mL Fc fragments on ice for 30 minutes. A master mix consisting of PE-HLA-DR (MHCII) and APC-CD14 diluted 1:100 was prepared in BD FACs buffer containing 100 mg/mL human purified Fc fragments. 10 μl of the master mix was added to each well containing 90 μl of resuspended cells and the samples were incubated on ice for 1 hour. The cells were then centrifuged at 400 x g for 5 minutes in a pre-cooled Eppendorf 5810R centrifuge. The supernatant was removed and the cells were washed with 200 mL BD FACs buffer. Two washes were performed and the cells were resuspended in 200 mL FACs buffer and the samples were analyzed on an Attune flow cytometer. The HLA-DR average fluorescence was determined using FlowJo analysis software.

5.1.3 Cytokine Production

After treatment, the PBMC/cancer cell co-cultures were spun at 800 rpm for 5 minutes in an Eppendorf 5810R using a plate adapter. Serum or tissue culture supernatant was removed and transferred to a 96-tube rack and samples were frozen at -80°C until treatment. Frozen tissue culture supernatant and serum were thawed overnight at 4°C and processed using the Luminex Multiplex Kit from Millipore for cytokine production.

Tissue culture supernatants and serum samples were processed according to the manufacturer's instructions. Briefly, the assay plate was washed with 200 μL wash buffer/well, followed by the addition of 25 μL of standard or buffer, 25 μL of matrix or sample, and 25 μL of multi-analyte beads to each well. Samples were incubated overnight at 4°C with vigorous shaking. The plate was washed twice with wash buffer.

Detection antibody (25 μL) was added to each well and incubated at room temperature for 1 hour. 25 μL of streptavidin-phycoerythrin (SA-PE) was added and the samples were incubated at room temperature for 30 minutes. The plate was washed twice with wash buffer and the beads were resuspended with 150 μL of sheath fluid. The samples were analyzed using a combination of the Luminex MagPix system and the Xponent software system. Cytokine levels were calculated based on the standard curve.

5.2 Results

Innate cell activation was observed, as evidenced by increased release of surface activation markers (MHCII) and inflammatory cytokines (CXCL-10/IP10) (Figures 5B to 5C). Innate immune cells are activated when exposed to vc-MMAE-treated tumor cells. Immune cell activation by vc-MMAE is more robust than activation by other ADC payloads (Figures 5B to 5C).

Vc-MMAE-mediated ICD is a regulated cell death that activates adaptive immune responses against antigens from dead and living tumor cells and allows for robust innate immune cell activation and subsequent cytotoxic T cell responses against specific tumor cell antigens. Here, vc-MMAE-killed cancer cells were demonstrated to elicit an increase in surface MHCII following uptake of dead cells and release of the innate cytokine CXCL10 (a strong chemotactic and inflammatory mediator) from monocytes/macrophages.

Example 6: Evaluation of the Payload on the Trastuzumab Main Chain

6.1 Materials and methods

Trastuzumab ADC conjugates carrying various clinical-stage payloads were evaluated for their ability to induce ER stress and downstream ICD markers ATP and HMGB1. The payloads used were DM1, MMAE, and exotecan (Ex).

6.2 Results

Two observations were made: (1) trastuzumab conjugated to vcMMAE drove the most robust ER stress response, which was associated with the induction of ATP and HMGB1; and (2) the late cell death marker HMGB1 appeared to be elevated in other payload classes, indicating that secondary necrosis may be associated with these payload classes rather than direct ICD (Figures 6C to 6E). The findings here are similar to those described in Example 4 above using Mia-PaCa-2 cells (Figures 4A to 4B).

Example 7: Induction of early ER stress markers (JNK signaling activation) is generally superior for MMAE ADCs

7.1 Materials and methods

As described in Example 5 and shown in Figure 5A, the ICD approach involves various aspects. This approach is further illustrated in Figure 7A. As shown in Figure 7A and mentioned above, microtubule-destroying agents such as MMAE in the initial stage destroy microtubules, thereby causing ER stress and ICD. ICD in turn causes the release of immune activation molecules, such as DAMP, ATP, HMGB1 and CRT. These molecules can then activate innate cells that can trigger anti-tumor T cell responses and can induce T cell memory. Such T cell responses can be further enhanced by combining with other immunomodulators, such as immune cell adapters described herein. The following examples report the results of the study, showing the effectiveness of the aforementioned different aspects of MMAE-induced ICD approaches compared with other ADC payloads.

Induction of endoplasmic reticulum (ER) stress is one of the first steps for eliciting immunogenic cellular responses, and JNK signaling activation is an indicator of ER stress (see FIG3B ). To evaluate this indicator, MIA-PaCa-2 pancreatic cancer cells were treated with 1 μg/mL ADC conjugated with different payloads, as shown in FIG7B . After 24 or 48 hours of treatment, cells were harvested for Western blot analysis and the upstream ER stress marker pJNK was evaluated by simple protein immunoassay (Wes ^TM , Protein Simple).

Use cell scraper to separate treated cells from plate. Suspended cells were centrifuged at 1000rpm, 4°C for 10 minutes. Remove supernatant and resuspend cell pellet in lysis buffer (containing protease and phosphatase inhibitors). After minimum 10 minutes on ice, sample was centrifuged at 13,500g for 10min to precipitate cell debris. Lysis solution was relocated to separate test tubes and stored at -80°C. Bio-Rad DC Protein Assay Kit (Cat. No. 5000112) was used to quantify the amount of lysate protein to allow equal lane loading. Sample lysate and reagent were loaded into assay plate and placed in Wes ^TM . Phosphorylation-JNK was identified using primary antibody (Cell Signaling Technologies Catalog No. 9251S) and immunodetected using secondary antibody (Protein Simple Catalog No. 042-206) conjugated with HRP and chemiluminescent substrate. The resulting chemiluminescent signal was detected, quantified and displayed by integrated Compass software.

7.2 Results

As shown in Figure 7C to Figure 7F, among the different ADC payloads tested, MMAE-ADC (SGD-1006) treatment is one of the strongest inducers of JNK phosphorylation (early ER stress response). In general, compared with the treatments performed with maytansine-ADC (Figure 7C), camptothecin-ADC (Figure 7D), anthracycline-ADC (Figure 7E) and calicheamicin-ADC (Figure 7F), MMAE-ADC treatment produces a stronger pJNK signal. (hIgG in Figure 7C to Figure 7F is a non-targeted conjugate with the same payload as the corresponding ADC). The only exception is the treatment performed with an ADC containing anthracycline mp-EDA-PNU (SGD-8335), which produces a pJNK signal (Figure 7E) comparable to the signal processed with MMAE-ADC. ER stress induction is associated with microtubule destruction, because ER requires complete microtubules to expand and contract to adapt to the protein translation needs of cells. The ability of MMAE to induce this ER stress as a tubulin disruptor is exemplified in the data shown.

Example 8: Induction of late ER stress markers (CHOP induction) is generally superior for MMAE ADCs

8.1 Materials and methods

CHOP is the last step in the ER stress response cascade and its expression level increases due to ER stress (see Figure 3B). MIA-PaCa-2 cells transduced with a CHOP-driven luciferase reporter (Signosis, Inc.) were used to evaluate this downstream pathway of ER stress. Several ADCs containing different payloads were used for evaluation. See Figure 7A.

CHOP induction in MIA-PaCa-2 cells was measured by detecting luciferase signal (Bright-Glo ^™ Luciferase Assay System, Promega). In brief, 10,000 cells/well were plated in 96-well black-walled flat-bottomed transparent plates at 75 μL/well. ADC was administered at 25 μL/well to reach the final IC ₅₀ concentration. At 36, 48 and 72 hours, the plates were removed from the incubator and allowed to reach room temperature. 100 μL Bright-Glo reagent was added to each well. Before reading, the plates were shaken for at least five minutes. The plates were read using the Envision CTG 96-well standard protocol.

8.2 Results

As shown in Figures 8A to 8D, treatment with ADC containing vc-MMAE (SGD-1006) resulted in CHOP induction comparable to that of treatment with ADC containing mertansine (SPP-5351) or ravtansine (SPDB-5352) (Figure 8A) and treatment with ADC containing mp-EDA-PNU (SGD-8335) or mp-Gluc-DXZ (SGD-8248) (Figure 8C). In addition, treatment with ADC containing MMAE (SGD-1006) resulted in CHOP induction stronger than that of treatment with ADC containing camptothecin (Figure 8B), AT (SGD-4830) (Figure 8D) or teisline (SGD-7455) (Figure 8D). In contrast, treatment with ADC containing ozogamicin (SGD-8677) resulted in slightly stronger CHOP induction than treatment with ADC containing vc-MMAE (SGD-1006) ( FIG. 8D ).

Example 9: Induction of immunostimulatory DAMPs is generally superior for MMAE ADCs 9.1 Materials and Methods

ICD causes the release of immune activation molecules - damage associated molecular patterns (DAMPs) - such as ATP, HMGB1 and CRT. To measure ICD, ATP and HMGB1 release were assessed as follows. MIA-PaCa-2 cancer cells were treated with ADCs with various payloads at IC ₅₀ concentrations to assess in vitro ICD marker induction. See Figure 7A.

200,000 cells were plated in each well of a 6-well TC plate and allowed to attach to the plate ON. Cells reached 50% to 60% confluence. IC ₅₀ concentrations of ADC were added to each treatment well. After 72 hours, 500 μL (for ATP release assay) or 750 μL (for HMGB1 assay) of culture supernatant were collected and transferred to labeled 1.5 mL microcentrifuge tubes. Each tube of sample was centrifuged at 13,000 rpm for 1 minute. 50 μL of culture medium was transferred to three replicate wells in a 96-well clear bottom plate. 50 μL of CellTiter- (Promega) was added to each well. The plate was shaken for 1 to 2 minutes. The plate was read using the CTG96 well standard protocol on the Envision plate reader. Then, each test tube sample supernatant was used to measure the HMGB1 release level, which was quantified by ELISA (IBL). HMGB1 and ATP release levels were reported as fold changes relative to the background value of untreated samples. The acquired values were converted to text files and exported and analyzed using Excel and GraphPad Prism.

9.2 Results

As shown in Figures 9A to 9D, the treatment with ADC containing vc-MMAE (SGD-1006) caused ATP release and HMGB1 release, which was stronger than the release of the treatment with ADC containing maytansine (Figure 9A) and the release of the treatment with ADC containing camptothecin (Figure 9B). In addition, the treatment with ADC containing MMAE (SGD-1006) caused ATP release and HMGB1 release, which was stronger than the release of the treatment with ADC containing teisline (SGD-7455) or auristatin AT (SGD-4830) (Figure 9D).

Treatment with ADC containing vc-MMAE (SGD-1006) resulted in ATP and HMGB1 release that was comparable to that of treatment with ADC containing anthracyclines (ATP release was less robust compared to HMGB1) ( FIG. 9C ) and treatment with ADC containing ozogamicin (SGD-8677) ( FIG. 9D ).

Example 10: For MMAE ADCs, innate cell activation (cytokine release) is generally superior 10.1 Materials and Methods

DAMPs activate innate cells that can trigger anti-tumor T cell responses. For example, they can increase the expression of MHCII on monocytes and the release of innate cytokines such as CXCL-10/IP10. MHCII expression and CXCL-10/IP10 were assessed as follows.

L540cy cancer cells exposed to IC ₅₀ concentrations of ADC or paclitaxel for 24 hours (at 37°C, 5% CO ₂ ) were washed, and 0.2×10 ⁶ cells/well of PBMC were added to the killed cancer cells at a 1:10 L540cy:PBMC ratio. The payload of the ADC used for this experiment is described in FIG7A . The co-cultures were incubated for 48 hours. Cell culture supernatants were collected at 24 hours and cytokines, including the innate cytokine CXCL-10/IP10, were measured by Luminex evaluation.

After 48 hours of co-culture incubation, the cell pellet was resuspended in 50 μL BD FACs buffer and transferred to a 96-well round-bottom microtiter plate. Fc receptors were blocked with 100 μg/mL human Fc fragments on ice for 30 minutes. A master mixture of PE-Cy7 anti-HLA-DR (MHCII), PE anti-CD14, PE-Dazzle 594 anti-CD11b, BV605 anti-CD3 and BV421 anti-CD19 diluted 1:100 was prepared in BD FACs buffer containing 100 μg/mL human purified Fc fragments. 10 μL of the master mixture was added to each well containing 90 μL of resuspended cells and the samples were incubated on ice for 1 hour. The cells were then centrifuged at 400 x g for 5 minutes in a pre-cooled Eppendorf 5810R centrifuge. The supernatant was removed and the cells were washed with 200 mL BD FACs buffer. Wash twice and resuspend the cells in 200 mL FACs buffer. Analyze samples on an Attune flow cytometer. Monocytes are defined as CD14+CD11b+CD3-CD19-. HLA-DR mean fluorescence is determined using FlowJo analysis software.

10.2 Results

As shown in Figure 10A to Figure 10D, the treatment carried out by the ADC containing MMAE (SGD-1006) causes monocyte MHCII expression, which is comparable or higher than the expression of the treatment carried out by the ADC containing other payloads, including maytansine (Figure 10A), camptothecin (Figure 10B), anthracyclines (Figure 10C) and calicheamicin ozogamicin (SGD-8677) and PBD teshilin (SGD-7455) (Figure 10D). The treatment carried out by the ADC containing MMAE (SGD-1006) also causes the release of innate cytokines CXCL-10/IP10, which is always higher than the release (Figure 10A to Figure 10D) of the treatment carried out by the ADC containing the same payload.

10.3 Summary of the Superior ICD Potential of MMAE ADC

As shown in these experiments, MMAE-ADC can induce various ICD markers and various immunogenic cellular responses, including induction of early ER stress (e.g., JNK activation), induction of late ER stress (e.g., CHOP induction), induction of immune activation molecules (e.g., ATP and HMGB1 release), and activation of innate immune cells (e.g., macrophage activation). None of the other ADC payloads tested consistently induced these ICD markers. Figure 10E provides a summary of the ICD potential of ADCs with different types of payloads (as measured by the above-mentioned markers) and illustrates the overall superiority of tubulin disruptors, particularly MMAE.

Example 11: Differential FcγR binding based on the Fc backbone to FcγRIIa, FcγRIIb or FcγRIIIa

11.1 Materials and methods

Antibodies SEA-CD40, APX005M, ADC-1013 and Seluzumab (Figure 11A) were evaluated for FcγR binding to CHO cells transfected with human FcγRIIa, FcγRIIb or FcγRIIIa using flow cytometry. CHO cells were incubated with increasing concentrations of antibodies and secondary antibodies for evaluation of binding, as monitored by flow cytometry.

For each cell line, 50 million cells were washed once in 50 mL PBS. Cells were counted again and resuspended in BD staining buffer at 2.2 million cells/mL. Cells were plated at 0.1 mL cells/well in 96-well round-bottom plates.

The antibody solution is diluted to form the following final concentration: 3mg/mL, 1mg/mL, 0.3mg/mL, 0.1mg/mL, 0.03mg/mL, 0.01mg/mL, 0.003mg/mL, 0.001mg/mL, 0.0003mg/mL. Each antibody solution is diluted 10 times (i.e., 11 μL of each antibody solution is added to 89 μL culture medium) to produce the following concentration: 300, 100, 30, 10, 3, 1, 0.03, 0.01, 0.003, 0.001 and 0.0003 μg/ml. Remove the culture medium from the cells and wash the cells with the culture medium. 100 μL of antibody solution is added to each hole. Antibody solution is added in a decreasing concentration in the vertical direction. After incubation at 4°C for 1 hour, the plate is centrifuged and each well cell is washed twice with 200 μL BD staining buffer. The cells of the precipitation are resuspended by vortexing the plate.

Then, PE-conjugated anti-human IgG Fc antibody (1/50 dilution of 1 mg/ml concentration = 33 μg/mL saturated concentration) was prepared in BD staining buffer. The solution was incubated in a dark refrigerator for 30 min. After incubation, the plate was centrifuged, the supernatant was removed, and the cells were washed twice with 200 μL BD staining buffer per well. The cells were resuspended in PBS + 1% paraformaldehyde and kept at 4 ° C until they were analyzed by flow cytometry. Samples were analyzed using an Attune flow cytometer. The data points of the geometric mean (GEO mean fluorescence) of the fluorescence intensity were plotted using GraphpadPrism.

11.2 Results

APX005 S267E exhibited the highest affinity for FcγRIIa and FcγIIb (Figures 11B-11D). SEA-CD40 had the highest affinity for FcγRIIIa and the lowest affinity for FcγRIIb (Figures 11B-11D). The data demonstrate that different Fc backbones affect the potential for binding to different FcγRs. The SEA-CD40 non-fucosylated backbone showed differential binding compared to other CD40 antibodies in development in that it binds to activating rather than inhibitory FcγRs (Figures 11B-11D).

Example 12: Induction of cell death in MIA-PaCa-2 cells

12.1 Materials and Methods

MIA-PaCa-2 pancreatic tumor cells were induced to undergo cell death with the EC50 concentration of the non-ICD inducer Abraxane (which acts similarly to paclitaxel in Example 3 above) or the two ICD inducers oxaliplatin or vc-MMAE. The cells were incubated with each agent for 18 hours. The tumor cells were then added to human PBMCs plus various CD40-directed agonists (1 μg/ml) with different Fc backbones (Figure 11A), and immune activation was assessed 48 hours later.

12.2 Results

The combination of SEA-CD40 and vcMMAE ADC kills tumor cells at least in part by inducing superior release of immune-activating cytokines (CXCL10 and IFNγ; Figures 12A-12B), while other CD40 agonists with different Fc backbones amplify immune-suppressive cytokines (IL-10 and MDC; Figures 12C-12D). This example illustrates the improved immune response observed with an Fc backbone that enhances binding to FcγRIIIa.

Example 13: Differential immune activation against apoptotic melanoma cells varies with the Fc backbone

13.1 Materials and Methods

Two melanoma cell lines (SK-MEL 12 and SK-MEL 28) were treated with EC50 concentrations of abuxaban, oxaloplatin or vc-MMAE for 18 hours. Human PBMCs plus 1 μg/ml of CD40-directed agonists with different Fc backbones ( FIG. 11A ) were added to the treated melanoma/tumor cells. Immune activation was assessed 48 hours later.

13.2 Results

The combination of SEA-CD40 and vc-MMAE ADC induced the release of immune-activating cytokines (CXCL10; FIGS. 13A-13B ), whereas other CD40 agonists amplified immune-suppressive cytokines (IL-10; FIGS. 13C-13D ).

Example 14: Differential immune activation against various apoptotic tumor cell types varies with the Fc backbone

14.1 Materials and Methods

14.1.1 Cells

Cell death was induced in tumor cells from melanoma, lung, breast and pancreas using EC50 concentrations of ICD inducers oxaliplatin or vc-MMAE and incubation for 18 hours. Treated tumor cells were added to human PBMCs and various CD40-directed agonists with different Fc main chains (Figure 11A). Immune activation was assessed after 48 hours. PBMCs and tumor cell lines were treated as described in Example 5 above. Instead of MIA-PaCa-2 cancer cells (which were used in Example 5), the following cell lines were used: melanoma cell lines SK-MEL 12 and SK-MEL 28, lung cancer cell line A549, breast cancer cell line MDA-MB-468 and pancreatic cancer cell line MIA-PaCa-2. Cells were processed in triplicate.

14.1.2 Cytokine Production

Cytokine production was assessed as described in Example 5 above.

14.2 Results

The combination of SEA-CD40 and vc-MMAE ADC drives superior release of immune-activating cytokines (CXCL10 and IFNγ; Figures 14A and 14C), while other CD40 agonists amplify immune-suppressive cytokines (IL-10; Figure 14B). As with Examples 12 and 13, this example demonstrates that improved immune responses are observed with Fc backbones that enhance binding to FcγRIIIa compared to other Fc backbones.

Example 15: Synergistic effect of the combination of non-fucosylated SEA-CD40 antibody and auristatin-based ADC

15.1 Materials and Methods

Human CD40 transgenic mice were implanted with A20 cells engineered to express the antigen Thy 1.1. On day 0, tumor cells were implanted subcutaneously in the flank. When an average tumor size of 100 mm ³ was reached (measured by using the formula: volume (mm ³ ) = 0.5 * length * width ² , where length is the longer dimension), the mice were randomized into treatment groups of 5 mice/group. The animals were then treated with the designated intraperitoneal treatments; each treatment was given once every three days for a total of three treatments. The stock concentration of antibodies was diluted to the appropriate concentration and injected into the animals in a volume of 100 μl. The final dose was 1 mg/kg for SEA-CD40 and 1 mg/kg for vc-MMAE ADC against Thy1.1. Tumor length, tumor width, and mouse weight were measured throughout the study, and tumor volume was calculated using the above formula. Animals were followed until tumor volumes were measured to reach approximately 1,000 mm ³ , at which time the animals were euthanized.

15.2 Results

As shown in Figure 15, treatment of the A20 tumor model with subtherapeutic doses of SEA-CD40 resulted in reduced tumor growth and tumor growth delay. Similarly, the antibody-drug conjugate containing vc-MMAE showed mild tumor growth delay. However, when the two agents were administered to animals in tandem, a curative anti-tumor response was observed. This data demonstrates the synergistic benefits of combining enhanced SEA-CD40 antibodies with immunogenic cell death-inducing chemotherapy delivered via ADC, including the potential to achieve a curative response.

Example 16: Different activities of various tumor cell lines treated with auristatin-based ADCs targeting tumor-associated antigens and TIGIT antibodies with different Fc backbones

16.1 Materials and Methods

Five different human cancer cell lines: SK-MEL 28 (melanoma), MDA-MB-468 (breast), CORL23 (lung), A549 (lung) and HT-26 (colon) were used for this example. Each cell line was treated with 1 μg/ml of a tumor-targeted antibody, vcMMAE ADC, with a drug-antibody ratio (DAR) of 4 for 18 hours. After incubation at 37°C, the tumor cells were washed and human PBMCs were added and treated with 1 μg/ml anti-TIGIT antibodies as indicated in Figure 16. The anti-TIGIT antibodies used had different levels of main chain effector function, with the LALA TIGIT antibody having no FcγR binding and the SEA-TIGIT antibody having enhanced FcγR binding (increasing binding to activating FcγIIIaR and reducing binding to inhibitory FcγRIIbR). The following Table D shows the relative activity of different TGT antibodies. These cultures of immune cells, anti-TIGIT antibody treatments and dead/dying tumor cells were incubated for another 48 hours. Supernatants from each condition were harvested and evaluated for cytokine induction using ELISA according to the manufacturer's instructions.

16.2 Results

As shown in Figure 16, dead/dying tumor cells under PBMC incubation without any other immunomodulatory treatment ("untreated" in Figure 16) have some immune cell stimulation, as seen by the production of the cytokine IP10 associated with innate type I interferon. The level of IP10 activation depends on the tumor cell type, because SK-MEL 28, MDA-MB-468 and CORL23 induce more PBMC activation than A549 or HT-26 cells. However, regardless of the tumor cells, the anti-TIGIT mAb SEA-TGT that enhances effector function is subsequently added to the co-culture to cause a comprehensive further enhancement of immune cell activation. For cell lines that are only co-incubated with dead cells, it is not enough to drive significant activation, and the non-fucosylated TIGIT mAb SEA-TGT shows the most significant increase, which proves its powerful activation ability. In these cells, the IgG1 main chain TIGIT antibody is able to drive immune cell activation that is weakened when compared with the enhanced mAb. The LALA version of anti-TIGIT, which lacks FcγR-engaging ability, is inactive in driving any immune activation.

Example 17: Different activities of various tumor cell lines treated with auristatin-based ADCs targeting tumor-associated antigens and TIGIT antibodies with different Fc backbones

17.1 Materials and Methods

Six different human cancer cell lines HT-26 (colon), A549 (lung), CORL23 (lung), MDA-MB-468 (breast), SK-MEL 28 (melanoma) and Mia-PaCa-2 (pancreas) were used for this example. Each cell line was treated with 1 μg/ml of the tumor-targeting antibody vcMMAE ADC with a drug-antibody ratio (DAR) of 4 for 18 hours. After incubation at 37°C, the tumor cells were washed and human PBMCs were added and treated with 1 μg/ml anti-TIGIT antibodies as indicated in Figure 16. These cultures of immune cells, anti-TIGIT antibody treatments and dead/dying tumor cells were incubated for 48 hours. The supernatant under each condition was harvested and cytokine induction was evaluated using ELISA according to the manufacturer's instructions.

17.2 Results

As shown in Figure 17, dead/dying tumor cells under PBMC incubation without any other immunomodulatory treatment ("untreated" in Figure 16) had some immune cell stimulation, as seen by the production of the adaptive cytokine IFNγ. The level of IFNγ activation depends on the tumor cell type, because SK-MEL 28, MDA-MB-468 and CORL23 induce more PBMC activation than A549 or HT-26 cells. However, regardless of the tumor cells, the subsequent addition of non-fucosylated SEA-TGT mAb that enhances effector function to the co-culture causes a comprehensive further enhancement of immune cell activation. For cell lines that are only co-incubated with dead cells, it is not enough to drive significant activation, and the inclusion of non-fucosylated TIGIT mAb SEA-TGT shows the most significant increase, which indicates its stronger activation ability. In these cells, the IgG1 main chain TIGIT antibody is able to drive some immune cell activation, but it is greatly weakened when compared with the enhanced mAb. The LALA version of anti-TIGIT, which lacks FcγR-engaging ability, is inactive in driving any immune activation.

Example 18: Synergistic effect of the combination of non-fucosylated TIGIT antibodies and auristatin-based ADCs

18.1 Materials and Methods

18.1.1 In vitro evaluation of TIGIT antibodies and auristatin-based ADCs

The conjugation of the ICD inducer vc-MMAE with tumor cell targeting antibodies at EC ₅₀ concentrations induces cell death of A549 non-small cell lung cancer cancer cells. The cells were incubated with the agent for 18 hours and then added to human PBMCs with various concentrations (1, 0.1, 0.01 μg/ml) of anti-TIGIT antibodies with different Fc main chains, including anti-TIGIT LALA, SEA-TGT and antibody 31C6H4/L1 (which is an IgG1 antibody) (US 2018/0066055 A1). Then, immune activation was assessed by measuring cytokine (IP10) levels after 48 hours of co-culture.

18.1.2 In vivo evaluation of TIGIT antibodies and auristatin-based ADCs

Balb/c mice were implanted subcutaneously in the flank with a CT26 syngeneic tumor cell line expressing the tumor antigen Thy1.1 on day 0. When an average tumor size of 100 mm ³ was reached (measured by using the formula: volume (mm ³ ) = 0.5 * length * width ² , where length is the longer dimension), the mice were randomly divided into treatment groups of 5 mice/group. The animals were then treated with the specified intraperitoneal treatment; each treatment was given once every three days, for a total of three treatments. The antibody at the stock concentration was diluted to the appropriate concentration and injected into the animal in a volume of 100 μl. The final dose was 0.1 mg/kg for SEA-TGT mIgG2a and 5 mg/kg for tumor-targeted vc-MMAE Thy1.1 ADC. Both antibodies used were on the mIgG2a backbone, and SEA-TGTmIgG2a was non-fucosylated. Tumor length, tumor width, and mouse weight were measured throughout the study, and tumor volume was calculated using the above formula. Animals were followed until tumor volumes measured to reach approximately 1,000 mm ³ , at which time they were euthanized.

Balb/c mice were implanted subcutaneously in the flank with the Renca syngeneic tumor cell line expressing the tumor antigen EphA2 on day 0. When an average tumor size of 100 mm ³ was reached (measured by using the formula: volume (mm ³ ) = 0.5 * length * width ² , where length is the longer dimension), mice were randomized into treatment groups of 5 mice/group. Animals were then treated with the indicated intraperitoneal treatments; each treatment was given once every three days for a total of three treatments. Antibodies at stock concentrations were diluted to the appropriate concentration and injected into the animals in a volume of 100 μl. The final dose was 0.1 mg/kg for SEA-TGT and 1 mg/kg for the tumor-targeted vc-MMAE EphA2 ADC. Both antibodies used were on the mIgG2a backbone. Tumor length, tumor width, and mouse weight were measured throughout the study, and tumor volume was calculated using the above formula. Animals were followed until tumor volumes were measured to reach approximately 1,000 mm ³ , at which time the animals were euthanized.

18.2 Results

As shown in Figure 18A, incubation of ADC-killed tumor cells with immune cell populations resulted in some immune cell activation due to immunogenic cell death induced by MMAE, as measured by the induction of the cytokine IP10 (see the column marked with "0" on the X-axis). In addition, the addition of increasing concentrations of SEA-TGT to the co-culture resulted in a significant increase in this cytokine induction. This increase was not seen with either the effector-deficient anti-TIGIT antibody (LALA) or the standard anti-TIGIT IgG1 antibody (31C6 H4/L1), indicating that the non-fucosylated backbone of SEA-TGT synergistically drives with the immunogenic cell death-inducing MMAE to provide superior immune cell activation.

As shown in Figure 18B and Figure 18C, the CT26 tumor model and the Renca tumor model were treated with a subtherapeutic dose of 0.1 mg/kg SEA-TGT, respectively, resulting in reduced tumor growth and delayed tumor growth. vc-MMAE (Weidotin) ADC alone showed mild tumor growth delay. When the two agents were administered to animals in series, a significant reduction in tumor growth and a curative response of 40% of the animals were observed. These data demonstrate the synergistic benefits of combining SEA-TGT mIgG2a antibodies (converted to non-fucosylated mouse IgG2a SEA-TGT antibodies corresponding to non-fucosylated human IgG1 main chains) with ADCs that induce immunogenic cell death.

The fact that such observations were obtained in two different tumor models with ADCs targeting two different tumor cell antigens suggests that the antitumor activity of this combination may be broadly applicable to different tumor types.

Example 19: Synergistic effect of the combination of a non-fucosylated TIGIT antibody and another auristatin-based ADC

19.1 Materials and Methods

Renca cells engineered to express murine B7H4 were implanted subcutaneously in Balb/c mice. Tumors were allowed to grow to 100 mm ³ , at which time mice were treated with subtherapeutic doses of SEA-TGT and SGN-B7H4 MMAE ADC (B7H4V) or subtherapeutic doses of SEA-TGT and therapeutic doses of oxaliplatin. Each compound was given on the same day, and mice were treated 7 days apart for a total of 3 doses.

19.2 Results

As shown in Figure 19, when subtherapeutic doses of SEA-TGT were combined with subtherapeutic doses of B7H4V, the SEA-TGT combination activity was extended to increase anti-tumor activity. The combined activity of SEA-TGT (subtherapeutic dose) with B7H4V (subtherapeutic dose) was similar to the combined activity of SEA-TGT (subtherapeutic dose) with oxaliplatin (therapeutic dose), a known ICD inducer. However, oxaliplatin is associated with warnings of allergic reactions and nephrotoxicity, and when given at therapeutic doses, it is not active alone and is often used in combination with a variety of other chemotherapies.

As also shown in Figure 19, the curative effect of SEA-TGT was significantly increased when combined with B7H4V. Although not intending to be bound by theory, it is believed that this effect is due to ICD induction and long-lived memory T cell responses induced with such combinations (see also Example 23 below).

In summary, the results of this experiment are consistent with other results described herein, which show that non-fucosylated antibodies to immune cell engagers (in this experiment, non-fucosylated anti-TIGIT antibodies, such as SEA-TGT) combine well with agents that induce immune cell death, which in this particular example are both MMAE ADC (i.e., B7H4V) and oxaliplatin. However, the combination with MMAE ADC is preferred because of the toxicity associated with oxaliplatin and because comparable therapeutic effects are observed at subtherapeutic doses of ADC compared to therapeutic doses of oxaliplatin.

Example 20: Synergistic effect of the combination of non-fucosylated SEA-CD70 antibody and auristatin-based ADC

SEA-CD70 (SEA-h1F6) is a non-fucosylated antibody targeting the CD70 antigen. The CD70 molecule is a member of the tumor necrosis factor (TNF) ligand superfamily (TNFSF) and binds to the related receptor CD27 (TNFRSF7). The interaction between the two molecules activates intracellular signals from the two receptors. Under normal conditions, CD70 expression is transient and limited to activated T and B cells, mature dendritic cells, and natural killer (NK) cells. Similarly, CD27 is expressed on both initial and activated effector T cells and NK and activated B cells. However, CD70 is also abnormally expressed in various blood cancers, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and non-Hodgkin's lymphoma (NHL) and carcinomas, and plays a role in both tumor cell survival and/or tumor immune escape. SEA-CD70 (which comprises the VH and VL of SEQ ID NOs: 41 and 42, respectively, and the CDRs of SEQ ID NOs: 53 to 58) acts by blocking CD70/CD27 axis signaling, inducing antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC), and enhancing antibody-dependent cellular cytotoxicity (ADCC). As described below, the combination of SEA-CD70 and brentuximab (BV, SGN-35, cAC10-MMAE) was tested in a subcutaneous NHL model. Brentuximab, also known as SGN-35, is a CD30-targeted ADC that contains MMAE conjugated to the monoclonal antibody cAC10. CD30 is expressed in a subset of Hodgkin lymphoma and NHL patients.

20.1 Materials and Methods

In vivo evaluation of subcutaneous tumor growth in NHL xenograft models

Farage cells (2.5×10 ⁶ cells/animal) were resuspended in 0.1 mL 25% Matrigel and injected subcutaneously into SCID mice, which contain active innate immune effector cells to mediate ADCP and ADCC. When an average tumor size of 100 mm ³ was reached (measured by using the formula: volume (mm ³ ) = 0.5*length* ^width2 , where length is the longer dimension), mice were randomized into treatment groups of 6 mice/group. Treatment was given intraperitoneally. Antibodies and chemotherapy at stock concentrations were diluted to appropriate concentrations and injected into animals at 10 μL/g body weight. Tumor length and width and animal weight were measured at least twice a week throughout the study. Nineteen days after implantation, dosing was started at 3 mg/kg SEA-CD70 and/or 1 mg/kg SGN-35. SEA-CD70 was administered intraperitoneally (IP) every 4 days for 5 times and SGN-35 was administered IP once on day 19. Animals were followed until tumor volumes were measured to exceed 500 ^mm3 , at which time the animals were euthanized. 500 ^mm3 was chosen as the tumor size endpoint because tumors tend to ulcerate at larger sizes.

20.2 Results

As shown in Figure 20A, combining SGN-35 with SEA-CD70 delayed tumor growth compared to single-agent SGN-35 or SEA-CD70 treatment. At day 28.5 (day 9.5 after treatment), the volume of all untreated tumors increased by more than 5 times their original size, while tumors treated with a single agent of SEA-CD70 or a single agent of SGN-35 reached an average of 5-fold increase at day 34.5 and day 46 (day 15.5 and day 27 after treatment), respectively. It is worth noting that at the end of the experiment (day 50), tumors treated with a combination of SEA-CD70 and SGN-35 did not reach the determined size endpoint (Figure 20B). No significant toxicity or additional weight loss was observed when SEA-CD70 was combined with SGN-35 compared to single SEA-CD70 or SGN-35 treatment. These data indicate that the combination of an ADC carrying an MMAE payload (SGN-35) and an afucosylated antibody (SEA-CD70) is potent and well tolerated.

Example 21: Synergistic effect of the combination of non-fucosylated SEA-BCMA antibody and auristatin ADC

SEA-BCMA is a non-fucosylated antibody targeting B cell maturation antigen (BCMA), which is expressed on multiple myeloma (MM). SEA-BCMA (which has VH and VL of SEQ ID NO: 45 and 46, respectively, and CDRs of SEQ ID NO: 47 to 52) acts by blocking ligand-mediated BCMA cell signaling, antibody-dependent cellular phagocytosis (ADCP) and enhanced antibody-dependent cellular cytotoxicity (ADCC). As described below, the combination of SEA-BCMA and SGN-CD48A was tested in a disseminated MM tumor xenograft model. SGN-CD48A is a CD48-targeted ADC containing glucuronide-linked MMAE. CD48 is widely expressed in MM.

21.1 Materials and Methods

21.1.1 In vivo survival evaluation of xenograft models

MM1S MM cells were injected intravenously into SCID animals, which contained active innate immune effector cells to mediate ADCP and ADCC. Seven days after implantation, administration was started with 0.1 mg/kg SEA-BCMA and/or 0.01 mg/kg SGN-CD48A, and animal survival was monitored. SEA-BCMA was administered intraperitoneally once a week for 5 weeks and SGN-CD48A was administered intraperitoneally once. The survival of animals was tracked for 160 days (N=8/group). As of the 51st day, all untreated animals were humanely euthanized according to the IACUC protocol.

21.1.2 In vivo luciferase evaluation in xenograft models

L363 luciferase MM cells were injected intravenously into SCID animals and MM cells were homed to the bone marrow. Luciferase signals were monitored over time. Thirty days after implantation, administration was started with 3 mg/kg SEA-BCMA and/or 0.3 mg/kg SGN-CD48A. SEA-BCMA was administered intraperitoneally once a week for 5 weeks and SGN-CD48A was administered intraperitoneally once. Animals were tracked for 175 days (N=5/group). As of day 58, all untreated animals were humanely euthanized according to IACUC protocols.

21.2 Results

As shown in Figures 21A to 21B, the combination of SGN-CD48A and SEA-BCMA induced complete remission and prolonged survival in the tested mouse models. In Figure 21A, as of day 160, five of the eight animals in the group receiving the combination therapy were still alive, compared to zero animals treated with SEA-BCMA alone and one animal treated with SGN-CD8A alone. In Figure 21B, as of day 37, all animals treated with the combination therapy showed no detectable luciferase signal, which was still absent until the end of the study on day 175. This amazing synergy may be due to the unique combination of ICD-induced auristatin ADC and innate immune cell engagement with SEA-BCMA.

Example 22: Vedotin ADC induces immune cell recruitment and activation in vivo

22.1 Materials and Methods

Tumor xenografts were isolated from animals treated with vc-MMAE ADC or non-binding vc-MMAE isotype ADC for 8 days and subjected to flow cytometry or cytokine analysis. CD45 positive immune cells were stained for CD11c and activation was observed by staining for MHC class II expression on the cell surface. Intratumoral cytokines were measured by Luminex.

22.2 Results

As shown in Figure 22, the ADC (vc-MMAE ADC) based on MMAE of targeting common tumor antigens is used to treat tumor-bearing mice so as to promote immune cell recruitment and activation in tumors. Compared with non-binding control (non-binding ADC), when treated with ADC (vcMMAE ADC) based on MMAE of targeted tumors, dendritic cell infiltration and dendritic cell antigen presentation are significantly enhanced (Figure 22 B). When treated with ADC (vc-MMAE ADC) based on MMAE of targeted tumors, intratumoral cytokine levels are also significantly improved (Figure 22 C). These data show that the ADC comprising tubulin disruptors induces ER stress and tumor cell death in a manner that promotes immune cell recruitment and activation in tumors.

These results suggest that MMAE-based ADCs are preferred companions for immune checkpoint blockade.

Example 23: Induction of T cell memory by vedotin ADC

23.1 Materials and Methods

Balb/c mice were implanted subcutaneously in the flank with the Renca syngeneic tumor cell line expressing the tumor antigen Epha2 on day 0. When an average tumor size of 100 mm ³ was reached (measured by using the formula: volume (mm ³ ) = 0.5*length* ^width2 , where length is the longer dimension), mice were randomized into treatment groups of 5 mice/group. Animals were then treated with the designated intraperitoneal treatments ( FIG. 21A ). Each treatment was given once. Antibodies at stock concentrations were diluted to the appropriate concentration and injected into the animals in a volume of 100 μL. The final dose was 5 mg/kg for both the tumor-targeted ADC (ADC-vcMMAE) and the non-binding ADC (isotype vcMMAE, also referred to as h00-vcMMAE). Tumor length, tumor width, and mouse weight were measured throughout the study, and tumor volume was calculated using the above formula. Animals were followed until tumor volume reached approximately 1,000 mm ³ , at which time the animals were euthanized.

Mice were monitored for achieving a curative anti-tumor response. Then, 30 days after achieving a cure, mice were re-challenged with Renca tumor cells (Figure 21B) and assessed for outgrowth and rejection of new tumors.

23.2 Results

As shown in Figure 23A, in the Renca syngeneic model, treatment with a single dose of tumor-targeted MMAE ADC (ADC-vcMMAE) resulted in strong anti-tumor activity and a cure response. As shown in Figure 23B, when mice cured by treatment with MMAE ADC were re-challenged with Renca tumor cells to assess the induction of immune memory, such mice were able to reject subsequently implanted tumor cells. Such results demonstrate that MMAE ADCs are able to elicit specific anti-tumor T cell responses.

Example 24: Brutusimab (BV; SGN-35)-treated cells confer protective anti-tumor immunity

24.1 Materials and Methods

A20 cells expressing human CD30 were treated with CD30-Auristatin ADC (BV; SGN-35) or MMAE for 18 hours. Optionally, an aliquot of the cells was flash frozen. The treated samples were centrifuged on Ficoll to remove viable cells. All samples were analyzed for apoptosis and viability by flow cytometry using Annexin V/7AAD. Dying and dead cells were washed, resuspended in PBS and injected intraperitoneally into mice. Mice were immunized twice, 7 days apart. The immunized mice were left to rest for another 7 days and then challenged with A20 lymphoma cells, and tumor growth or rejection was monitored over time.

24.2 Results

As shown in Figure 24, mice immunized with CD30-expressing A20 cells killed with BV or MMAE exhibited a stronger immune response rejecting the implanted A20 cells compared to mice immunized with CD30-expressing A20 cells killed with flash freezing (a non-ICD method of cell death). These results indicate the induction of a memory T cell response. The induction of immune memory is considered the gold standard for evaluating the ICD activity of a molecule.

In general, the results presented in the aforementioned examples support the unique ability of ADCs based on auristatins (e.g., MMAE and MMAF) to induce immunogenic cell death. As demonstrated by the aforementioned examples, the mechanism of action of auristatins and their ability to destroy microtubule networks appear to be associated with the induction of ER stress responses that cause exposure and secretion of danger signals (DAMPs). The exposure of these DAMPs triggers innate immune cell responses, which can cause antigen-specific T cell responses. Inducing new antigen-specific T cells that can recognize tumor antigens can cause curative anti-tumor activity associated with long-term memory T cell responses that provide long-term immune protection in preclinical settings.

This memory T cell population induced by MMAE ADC can be further increased and/or enhanced by non-fucosylated antibodies. After the establishment of immune memory generated by MMAE ADC that induces ICD (e.g., through the above-mentioned memory T cell response), non-fucosylated antibodies such as SEA-TIGIT can further enhance the immune response through tumor blocking/inhibition mechanisms similar to the checkpoint inhibition mechanism of PD-1/PD-L1.

In addition, pairing the ability of auristatin-based ADCs (e.g., MMAE and MMAF), such as MMAE ADCs, to drive immunogenic cell death with immune cell agonism can amplify anti-tumor activity. Immune agonism can be amplified by using non-fucosylated antibodies or antibodies that have been engineered to enhance binding to activating FcγRs and/or reduce binding to inhibitory FcγRs (e.g., as shown in the above examples for non-fucosylated CD40 and BCMA antibodies). Non-fucosylated antibodies can increase binding to activating FcγRIIIa receptors and reduce or minimize binding to inhibitory FcγRIIb receptors. Depending on the nature of the antibody target, this property is multimodal. In the case where receptors like CD40 have optimal activity when aggregated, non-fucosylated antibodies that bind to FcγRIIA+ cells increase receptor aggregation as well as immune agonism and activation. See Figure 25. As in the case of TIGIT, non-fucosylated antibodies increase the strength of the immune synapse between antigen (+) T cells and antigen presenting cells (Figure 25). Engagement of FcγRIIIa on innate cells increases their activation and the production of factors that can enhance antigen-specific T cell responses. Finally, the afucosylated backbone can bind to innate immune cells or other FcγRIIIa cells such as γδ T cells independently of the target antigen to induce an activation state that can help elicit secondary antigen-specific T cell responses. All of these mechanisms by which afucosylated antibodies work can elicit T cell responses that drive anti-tumor activity and long-lasting immune protection. Reduced or absent binding to FcγRIIb indicates the absence of counter or inhibitory signals that reduce immune activation driven by afucosylated antibodies.

The mechanism of action of auristatin ADCs such as MMAE ADCs results in synergistic and complementary activities through the coupling of non-fucosylated mAbs with immunomodulators, which have been shown to result in enhanced immune activation and curative anti-tumor responses, as demonstrated herein.

All publications, patents, patent applications, and other documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and other document was individually indicated to be incorporated by reference for all purposes.

Sequence Listing

Sequence Listing

<110> SEAGEN INC.

<120> Combination therapy of antibody-drug conjugates and immune cell inhibitors

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<151> 2020-11-08

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<151> 2021-04-08

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<151> 2021-06-08

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100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 4

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Anti-TIGIT antibody clone 13C VH

<400> 4

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Leu Ser Ser

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Leu Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Lys Gly Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Anti-TIGIT antibody clone 13D VH

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Leu Ser Ser

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Tyr Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Lys Gly Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 6

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13, 13A, 13B, 13C and 13D VL proteins

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Arg Arg Ile Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13 VH CDR1

<400> 7

Gly Thr Phe Ser Ser Tyr Ala Ile Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Cloning 13A, 13C and 13D VH CDR1

<400> 8

Gly Thr Phe Leu Ser Ser Ala Ile Ser

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13B VH CDR1

<400> 9

Gly Thr Phe Ser Ala Trp Ala Ile Ser

1 5

<210> 10

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13 VH CDR2

<400> 10

Ser Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 11

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13A VH CDR2

<400> 11

Ser Leu Ile Pro Tyr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 12

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Cloning 13B and 13D VH CDR2

<400> 12

Ser Ile Ile Pro Tyr Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Cloning 13C VH CDR2

<400> 13

Ser Ile Ile Pro Leu Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 14

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13 and 13A VH CDR3

<400> 14

Ala Arg Gly Pro Ser Glu Val Gly Ala Ile Leu Gly Tyr Val Trp Phe

1 5 10 15

Asp Pro

<210> 15

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13B VH CDR3

<400> 15

Ala Arg Gly Pro Ser Glu Val Ser Gly Ile Leu Gly Tyr Val Trp Phe

1 5 10 15

Asp Pro

<210> 16

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Cloning of 13C and 13D VH CDR3

<400> 16

Ala Arg Gly Pro Ser Glu Val Lys Gly Ile Leu Gly Tyr Val Trp Phe

1 5 10 15

Asp Pro

<210> 17

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13, 13A, 13B, 13C and 13D VL CDR1

<400> 17

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 18

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13, 13A, 13B, 13C and 13D VL CDR2

<400> 18

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13, 13A, 13B, 13C and 13D VL CDR3

<400> 19

Met Gln Ala Arg Arg Ile Pro Ile Thr

1 5

<210> 20

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13 heavy chain hIgG1 (and non-fucosylated hIgG1)

Amino acid sequence

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Gly Ala Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 21

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13A heavy chain hIgG1 (and non-fucosylated hIgG1)

Amino acid sequence

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Leu Ser Ser

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Leu Ile Pro Tyr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Gly Ala Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 22

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13B heavy chain hIgG1 (and non-fucosylated hIgG1)

Amino acid sequence

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Trp

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Tyr Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Ser Gly Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 23

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13C heavy chain hIgG1 (and non-fucosylated hIgG1)

Amino acid sequence

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Leu Ser Ser

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Leu Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Lys Gly Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 24

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13D heavy chain hIgG1 (and non-fucosylated hIgG1)

Amino acid sequence

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Leu Ser Ser

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Ile Pro Tyr Phe Gly Lys Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Ser Glu Val Lys Gly Ile Leu Gly Tyr Val Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 25

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Clone 13, 13A, 13B, 13C and 13D light chain hκ

(and non-fucosylated) amino acid sequences

<400> 25

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Arg Arg Ile Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 26

<211> 444

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 (non-fucosylated hS2C6) heavy chain

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Val Ile Pro Asn Ala Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Ile Tyr Trp Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 27

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 (non-fucosylated hS2C6) light chain

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Ser Gln Thr

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 28

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VH

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Val Ile Pro Asn Ala Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Ile Tyr Trp Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 29

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VL

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Ser Gln Thr

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 30

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VH CDR1

<400> 30

Gly Tyr Tyr Ile His

1 5

<210> 31

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VH CDR2

<400> 31

Arg Val Ile Pro Asn Ala Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 32

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VH CDR3

<400> 32

Glu Gly Ile Tyr Trp

1 5

<210> 33

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VL CDR1

<400> 33

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Phe Leu His

1 5 10 15

<210> 34

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VL CDR2

<400> 34

Thr Val Ser Asn Arg Phe Ser

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD40 VL CDR3

<400> 35

Ser Gln Thr Thr His Val Pro Trp Thr

1 5

<210> 36

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Replacement of anti-CD40 antibody CDR2

<400> 36

Arg Val Ile Pro Gln Ala Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 37

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B6A heavy chain variable region

<400> 37

Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 38

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B6A light chain variable region

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-STNV heavy chain variable region

<400> 39

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ile Lys Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Lys Arg Ser Leu Ser Thr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 40

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-STNV heavy chain variable region

<400> 40

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Arg

20 25 30

Gly Asn His Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 41

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 heavy chain variable region

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 42

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD70 light chain variable region

<400> 42

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 43

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Sythetic: SGN-CD228A heavy chain variable region

<400> 43

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 44

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A light chain variable region

<400> 44

Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 45

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA heavy chain variable region

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Asn Thr Ala Tyr

65 70 75 80

Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 46

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA light chain variable region

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Asp Ile Ser Asp Asp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Val

35 40 45

Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Lys Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VH CDR1

<400> 47

Asp Tyr Tyr Ile His

1 5

<210> 48

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VH CDR2

<400> 48

Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 49

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VH CDR3

<400> 49

Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe

1 5 10

<210> 50

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VL CDR1

<400> 50

Leu Ala Ser Glu Asp Ile Ser Asp Asp Leu Ala

1 5 10

<210> 51

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VL CDR2

<400> 51

Thr Thr Ser Ser Leu Gln Ser

1 5

<210> 52

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-BCMA VL CDR3

<400> 52

Gln Gln Thr Tyr Lys Phe Pro Pro Thr

1 5

<210> 53

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VH CDR1

<400> 53

Asn Tyr Gly Met Asn

1 5

<210> 54

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VH CDR2

<400> 54

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe Lys

1 5 10 15

Gly

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VH CDR3

<400> 55

Asp Tyr Gly Asp Tyr Gly Met Asp Tyr

1 5

<210> 56

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VL CDR1

<400> 56

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met His

1 5 10 15

<210> 57

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VL CDR2

<400> 57

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 58

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SEA-CD70 VL CDR3

<400> 58

Gln His Ser Arg Glu Val Pro Trp Thr

1 5

<210> 59

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) heavy chain variable region

<400> 59

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 60

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) light chain variable region

<400> 60

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys

100 105

<210> 61

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) VH CDR1

<400> 61

Ser Tyr Trp Ile Asn

1 5

<210> 62

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) VH CDR2

<400> 62

Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 63

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) VH CDR3

<400> 63

Ser Trp Arg Gly Asn Ser Phe Asp Tyr

1 5

<210> 64

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetocillin (175D10) VL CDR1

<400> 64

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 65

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetuin (175D10) VL CDR2

<400> 65

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Zolbetuin (175D10) VL CDR3

<400> 66

Gln Asn Asp Tyr Ser Tyr Pro Phe Thr

1 5

<210> 67

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 heavy chain variable region

<400> 67

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 68

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 light chain variable region

<400> 68

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 69

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VH CDR1

<400> 69

Asn Tyr Gly Met Asn

1 5

<210> 70

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VH CDR2

<400> 70

Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys

1 5 10 15

Gly

<210> 71

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VH CDR3

<400> 71

Leu Gly Phe Gly Asn Ala Met Asp Tyr

1 5

<210> 72

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VL CDR1

<400> 72

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 73

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VL CDR2

<400> 73

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: 163E12 VL CDR3

<400> 74

Gln Asn Asp Tyr Ser Tyr Pro Leu Thr

1 5

<210> 75

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V heavy chain variable region

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Ser Gly Asp Thr Phe Ser Thr Ala

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 76

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V light chain variable region

<400> 76

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 77

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VH CDR1

<400> 77

Thr Ala Ala Ile Ser

1 5

<210> 78

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VH CDR2

<400> 78

Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 79

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VH CDR3

<400> 79

Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val

1 5 10

<210> 80

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VL CDR1

<400> 80

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 81

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VL CDR2

<400> 81

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 82

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-PDL1V VL CDR3

<400> 82

Gln Gln Arg Ser Asn Trp Pro Thr

1 5

<210> 83

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV heavy chain variable region

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 84

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV light chain variable region

<400> 84

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VH CDR1

<400> 85

Asp Tyr Tyr Met Ser

1 5

<210> 86

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VH CDR2

<400> 86

Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala Ser

1 5 10 15

Val Lys Gly

<210> 87

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VH CDR3

<400> 87

Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

1 5 10

<210> 88

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VL CDR1

<400> 88

Gln Ala Ser Gln Asp Ile Asn Lys Tyr Leu Ala

1 5 10

<210> 89

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VL CDR2

<400> 89

Tyr Thr Ser Ser Leu Gln Ser

1 5

<210> 90

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-ALPV VL CDR3

<400> 90

Leu Gln Tyr Asp Asn Leu Tyr Thr

1 5

<210> 91

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V heavy chain variable region

<400> 91

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Lys Ser Gly

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 92

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V light chain variable region

<400> 92

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 93

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VH CDR1

<400> 93

Gly Ser Ile Lys Ser Gly Ser Tyr Tyr Trp Gly

1 5 10

<210> 94

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VH CDR2

<400> 94

Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser

1 5 10 15

<210> 95

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VH CDR3

<400> 95

Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp Pro

1 5 10

<210> 96

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VL CDR1

<400> 96

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 97

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VL CDR2

<400> 97

Gly Ala Ser Thr Arg Ala Thr

1 5

<210> 98

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-B7H4V VL CDR3

<400> 98

Gln Gln Tyr His Ser Phe Pro Phe Thr

1 5

<210> 99

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Vedicituzumab heavy chain

<400> 99

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asn Pro Asp His Gly Asp Ser Tyr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asn Tyr Leu Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 100

<211> 212

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Vedicizumab light chain

<400> 100

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ile Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Phe Ala Thr Tyr Thr Phe

85 90 95

Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

Arg Gly Glu Cys

210

<210> 101

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Velifatuzumab heavy chain

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Gly Arg Val Ala Phe His Thr Tyr Tyr Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Gly Phe Asp Val Gly His Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 102

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Velifatuzumab light chain

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Glu Thr Leu Val His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Phe Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 103

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) Heavy chain variable

district

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 104

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) light chain variable

district

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 105

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VH CDR1

<400> 105

Ser Tyr Asn Met Asn

1 5

<210> 106

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VH CDR2

<400> 106

Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 107

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VH CDR3

<400> 107

Ala Tyr Tyr Tyr Gly Met Asp Val

1 5

<210> 108

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VL CDR1

<400> 108

Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala

1 5 10

<210> 109

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VL CDR2

<400> 109

Ala Ala Ser Thr Leu Gln Ser

1 5

<210> 110

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: PADCEV (Vienomab) (EV) VL CDR3

<400> 110

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 111

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 heavy chain variable region

<400> 111

Gln Phe Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Leu Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 112

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 light chain variable region

<400> 112

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn Val Leu Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 113

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VH CDR1

<400> 113

Asp Tyr Asn Val Asn

1 5

<210> 114

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VH CDR2

<400> 114

Val Ile Asn Pro Lys Tyr Gly Thr Thr Arg Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 115

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VH CDR3

<400> 115

Gly Leu Asn Ala Trp Asp Tyr

1 5

<210> 116

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VL CDR1

<400> 116

Gly Ala Ser Glu Asn Ile Tyr Gly Ala Leu Asn

1 5 10

<210> 117

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VL CDR2

<400> 117

Gly Ala Thr Asn Leu Glu Asp

1 5

<210> 118

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h2A2 VL CDR3

<400> 118

Gln Asn Val Leu Thr Thr Pro Tyr Thr

1 5

<210> 119

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 heavy chain variable region

<400> 119

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Asp Ser Phe Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Gln Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Leu Arg Arg Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 120

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 light chain variable region

<400> 120

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Glu Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 121

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VH CDR1

<400> 121

Gly Tyr Phe Met Asn

1 5

<210> 122

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VH CDR2

<400> 122

Leu Ile Asn Pro Tyr Asn Gly Asp Ser Phe Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 123

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VH CDR3

<400> 123

Gly Leu Arg Arg Asp Phe Asp Tyr

1 5

<210> 124

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VL CDR1

<400> 124

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn

1 5 10 15

<210> 125

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VL CDR2

<400> 125

Leu Val Ser Glu Leu Asp Ser

1 5

<210> 126

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: h15H3 VL CDR3

<400> 126

Trp Gln Gly Thr His Phe Pro Arg Thr

1 5

<210> 127

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A heavy chain variable region

<400> 127

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 128

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A light chain variable region

<400> 128

Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 129

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VH CDR1

<400> 129

Ser Gly Tyr Trp Asn

1 5

<210> 130

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VH CDR2

<400> 130

Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 131

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VH CDR3

<400> 131

Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 132

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VL CDR1

<400> 132

Arg Ala Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 133

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VL CDR2

<400> 133

Arg Val Ser Asn Arg Phe Ser

1 5

<210> 134

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-CD228A VL CDR3

<400> 134

Ser Gln Ser Thr His Val Pro Pro Thr

1 5

<210> 135

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) heavy chain

Variable region

<400> 135

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Glu Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 136

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) light chain

Variable region

<400> 136

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Pro Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 137

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VH CDR1

<400> 137

Asp Tyr Tyr Met His

1 5

<210> 138

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VH CDR2

<400> 138

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Gly Pro Lys Phe Gln

1 5 10 15

Gly

<210> 139

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VH CDR3

<400> 139

His Asn Ala His Tyr Gly Thr Trp Phe Ala Tyr

1 5 10

<210> 140

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VL CDR1

<400> 140

Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 141

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VL CDR2

<400> 141

Lys Ile Ser Thr Arg Phe Ser

1 5

<210> 142

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: SGN-LIV1A villadizumab (LV) VL CDR3

<400> 142

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 143

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Vectisomab (TV) heavy chain variable region

<400> 143

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Asp Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 144

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: Vectisomab (TV) light chain variable region

<400> 144

Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 145

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VH CDR1 of Vectisomab (TV)

<400> 145

Gly Phe Thr Phe Ser Asn Tyr Ala

1 5

<210> 146

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VH CDR2 of Vectisomab (TV)

<400> 146

Ile Ser Gly Ser Gly Asp Tyr Thr

1 5

<210> 147

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VH CDR3 of Vectisomab (TV)

<400> 147

Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser

1 5 10

<210> 148

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VL CDR1 of Vectisomab (TV)

<400> 148

Gln Gly Ile Ser Ser Arg

1 5

<210> 149

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VL CDR2 of Vectisomab (TV)

<400> 149

Ala Ala Ser

1

<210> 150

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthesis: VL CDR3 of Vectisomab (TV)

<400> 150

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

Claims (115)

1. A method of treating cancer, comprising administering to a subject suffering from cancer: (1) an antibody-drug conjugate (ADC) comprising a first antibody that binds a tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is a tubulin disrupting agent; and (2) a second antibody that binds to an immune cell adapter, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs, The activating FcγR includes one or more of FcγRIIIa, FcγRIIa and/or FcγRI. 2. The method of claim 1, wherein the second antibody comprises an Fc that enhances binding to at least FcyRIIIa. 3. The method of claim 1, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRIIa. 4. The method of claim 1, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRI. 5. The method of claim 1, wherein the second antibody comprises an Fc that enhances binding to FcγRIIIa, FcγRIIa, and FcγRI. 6. The method of any one of claims 1 to 5, wherein the Fc of the second antibody reduces binding to one or more inhibitory FcyRs. 7. The method of claim 6, wherein the Fc of the second antibody reduces binding to FcyRIIb. 8. The method of any one of claims 1 to 7, wherein the Fc of the second antibody has reduced fucose levels and/or has been engineered to contain one or more mutations such that the The Fc enhances binding to the one or more activating FcyRs. 9. The method of claim 8, wherein the second antibody is afucosylated. 10. The method of claim 8, wherein the second antibody comprises substitutions S293D, A330L and I332E in the heavy chain constant region. 11. A method of treating cancer, comprising administering an antibody-drug conjugate to a subject suffering from cancer, wherein the antibody-drug conjugate comprises: a first antibody conjugated to a cytotoxic agent, wherein the cytotoxic agent is a tubulin disrupting agent; and a second antibody that binds an immune cell adapter, wherein the second antibody is afucosylated. 12. The method of any one of claims 1 to 11, wherein the first antibody binds a tumor-associated antigen. 13. A method of treating cancer, comprising administering to a subject suffering from cancer: (1) an antibody-drug conjugate (ADC), wherein the ADC comprises a first antibody that binds a tumor-associated antigen and a cytotoxicity an agent, wherein the cytotoxic agent is a tubulin-disrupting agent; and (2) a second antibody that binds an immune cell adapter, wherein the second antibody comprises an enhanced Fc relative to a corresponding wild-type Fc of the same isotype ADCC active Fc. 14. The method of claim 13, wherein the second antibody comprises an Fc having enhanced ADCC and ADCP activity relative to a corresponding wild-type Fc of the same isotype. 15. The method of claim 13 or 14, wherein the second antibody is afucosylated. 16. The method of any one of claims 13 to 15, wherein the second antibody comprises an Fc that enhances binding to one or more activating FcγRs, wherein the activating FcγRs include FcγRIIIa, FcγRIIa, and /or one or more of FcγRI. 17. The method of claim 16, wherein the second antibody comprises an Fc that enhances binding to at least FcyRIIIa. 18. The method of claim 16, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRIIa. 19. The method of claim 16, wherein the second antibody comprises an Fc that enhances binding to at least FcγRIIIa and FcγRI. 20. The method of claim 16, wherein the second antibody comprises an Fc that enhances binding to FcγRIIIa, FcγRIIa, and FcγRI. 21. The method of any one of claims 13 to 20, wherein the Fc of the second antibody reduces binding to one or more inhibitory FcyRs. 22. The method of claim 21, wherein the Fc of the second antibody reduces binding to FcyRIIb. 23. The method of any one of claims 1 to 22, wherein the first antibody binds an antigen selected from the group consisting of: 5T4 (TPBG), ADAM-9, AG-7, ALK, ALP, AMHRII, APLP2 , ASCT2, AVB6, AXL(UFO), B7-H3(CD276), B7-H4, BCMA, C3a, C3b, C4.4a(LYPD3), C5, C5a, CA6, CA9, CanAg, carbonic anhydrase IX (CAIX ), cathepsin D, CCR7, CD1, CD10, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD111, CD112, CD113, CD116, CD117, CD118, CD119, CD11A, CD11b, CD11c, CD120a, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD13, CD130, CD131, CD132, CD133, CD135, CD136, CD137, CD138, CD14, CD140a, CD140b, CD141 ,CD142, CD143, CD144, CD146, CD147, CD148, CD15, CD150, CD151, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158b2, CD158e, CD158f1, CD158h, CD158i, CD159a, CD16, CD160, CD16 1. CD162, CD163, CD164, CD166, CD167b, CD169, CD16a, CD16b, CD170, CD171, CD172a, CD172b, CD172g, CD18, CD180, CD181, CD183, CD184, CD185, CD19, CD194, CD197, CD1a, CD1b, CD1c, CD1d, CD2, CD20, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD208, CD21, CD213a1, CD213a2, CD217, CD218a, CD22, CD220, CD221, CD222, CD224, CD226, CD228, CD229, CD23, CD230 ,CD232, CD239, CD243, CD244, CD248, CD249, CD25, CD26, CD265, CD267, CD269, CD27, CD272, CD273, CD274, CD275, CD279, CD28, CD280, CD281, CD282, CD283, CD284, CD289, CD29, CD294, CD295, CD298, CD3, CD3ε, CD30, CD300f, CD302, CD304, CD305, CD307, CD31, CD312, CD315, CD316, CD317, CD318, CD319, CD32, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD32b, CD33, CD331, CD332, CD333, CD334, CD337, CD339, CD34, CD340, CD344, CD35, CD352, CD36, CD37, CD38, CD39, CD3d, CD3g, CD4, CD41, CD42d, CD44, CD44v6, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD5, CD50, CD51, CD51 (integrin alpha-V), CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD6, CD61 , CD62L, CD62P, CD63, CD64, CD66a-e, CD67, CD68, CD69, CD7, CD70, CD70L, CD71, CD71(TfR), CD72, CD73, CD74, CD79a, CD79b, CD8, CD80, CD82, CD83, CD84, CD85f, CD85i, CD85j, CD86, CD87, CD89, CD90, CD91, CD92, CD95, CD96, CD97, CD98, CDH6, CDH6 (cadherin 6), CDw210a, CDw210b, CEA, CEACAM5, CEACAM6, CFC1B, cKIT, CLDN18.2 (Claudin 18.2), CLDN6, CLDN9, CLL-1, c-MET, complement factor C3, Cripto, CSP-1, CXCR5, DCLK1, DLK-1, DLL3, DPEP3, DR5 (death receptor body 5), anti-adhesin, EFNA4, EGFR, EGFR wild type, EGFRviii, EGP-1(TROP-2), EGP-2, EMP2, ENPP3, EpCAM, EphA2, EphA3, Ephrin-A4(EFNA4), ETBR , FAP, FcRH5, FGFR2, FGFR3, FLT3, FOLR, FOLR1, FOLR-α, FSH, GCC, GD2, GD3, globo H, GPC1, GPC-1, GPC3, GPNMB, GPR20, HER2, HER-2, HER3, HER-3, HGFR(c-Met), HLA-DR, HM1.24, HSP90, Ia, IGF-1R, IL-13R, IL-15, IL1RAP, IL-2, IL-3, IL-4, IL7R , Integrin αVβ3 (integrin αVβ3), integrin β-6, interleukin-4 receptor (IL4R), KAAG-1, KLK2, LAMP-1, Le(y), Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, lipid rafts, LIV-1 (SLC39A6 or ZIP6), LRP-1, LRRC15, LY6E, macrophage mannose receptor 1, MAGE, mesothelin (MSLN), MET, MHC class I chain-related protein A and B (MICA and MICB), MN/CA IX, MRC2, MT1-MMP, MTX3, MTX5, MUC1, MUC16, MUC2, MUC3, MUC4, MUC5, MUC5ac, NaPi2b, NCA-90, NCA-95, connexin-4 , Notch3, nucleolin, OAcGD2, OT-MUC1 (tumor tethering MUC1), OX001L, P1GF, PAM4 antigen, p-cadherin (cadherin 3), PD-L1, phosphatidylserine (PS), PRLR , prolactin receptor (PRLR), Pseudomonas, PSMA, PTK4, PTK7, receptor tyrosine kinase (RTK), RNF43, ROR1, ROR2, SAIL, SEZ6, SLAMF7, SLC44A4, SLITRK6, SLMAMF7(CS1) , SLTRK6, sortin (SORT1), SSEA-4, SSTR2, Staphylococcus aureus (antibiotic agent), STEAP-1, STING, STn, T101, TAA, TAC, TDGF1, tenascin, TENB2, TGF-B, Thomson-Friedrich antigen, Thy1.1, TIM-1, tissue factor (TF; CD142), TM4SF1, Tn antigen, TNF-alpha (TNFα), TRA-1-60, TRAIL receptors (R1 and R2 ), TROP-2, tumor-associated glycoprotein 72 (TAG-72), uPAR, VEGFR, VEGFR-2 and xCT. 24. The method of any one of claims 1 to 23, wherein the first antibody does not bind connexin-4. 25. The method of any one of claims 1 to 24, wherein the method does not comprise administering an antibody-drug conjugate comprising an antibody that binds Connexin-4. 26. The method of any one of claims 1 to 25, wherein the first antibody binds an antigen selected from the group consisting of: CD71, Axl, AMHRII and LGR5, Axl, CA9, CD142, CD20, CD22, CD228, CD248, CD30, CD33, CD37, CD48, CD7, CD71, CD79b, CLDN18.2, CLDN6, c-MET, EGFR, EphA2, ETBR, FCRH5, GCC, Globo H, gpNMB, HER-2, IL7R, integrin beta -6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, mesothelin (MSLN), MET, MRC2, MUC16, NaPi2b, connexin-4, OT-MUC1 (tumor tethering-MUC1), PSMA, ROR1, SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60 and tumor-associated glycoprotein 72 (TAG-72). 27. The method of any one of claims 1 to 25, wherein the first antibody binds an antigen selected from the group consisting of: BCMA, GPC1, CD30, cMET, SAIL, HER3, CD70, CD46, CD48, HER2, 5T4, ENPP3, CD19, EGFR and EphA2. 28. The method of any one of claims 1 to 25, wherein the first antibody binds an antigen selected from the group consisting of: Her2, TROP2, BCMA, cMet, integrin αVβ6, CD22, CD79b, CD30 , CD19, CD70, CD228, CD47 and CD48. 29. The method of any one of claims 1 to 25, wherein the first antibody binds an antigen selected from the group consisting of: CD142, integrin β-6, integrin αVβ6, ENPP3, CD19, Ly6E, cMET, C4.4a, CD37, MUC16, STEAP-1, LRRC15, SLITRK6, ETBR, FCRH5, Axl, EGFR, CD79b, BCMA, CD70, PSMA, CD79b, CD228, CD48, LIV-1, EphA2, SLC44A4, CD30 and sTn. 30. The method of any one of claims 1 to 29, wherein the tubulin disrupting agent is auristatin, microtubulysin, colchicine, vinca alkaloids, taxanes, nodules steroids, maytansinoids or hamitrine. 31. The method of claim 30, wherein the tubulin disrupting agent is auristatin. 32. The method of any one of claims 1 to 31, wherein the tubulin disrupting agent is Aplysia-10, MMAE (N-methylvaline-valine-Aplysia) Leucine-Aplysia proline-norephedrine), MMAF (N-methylvaline-valine-Aplysia isoleucine-Aplysia proline-phenylalanine), Austrian Restatin F, AEB, AEVB or AFP (aurestatin phenylalanine phenylenediamine). 33. The method of any one of claims 1 to 32, wherein the tubulin disrupting agent is MMAE. 34. The method of claim 33, wherein the MMAE is conjugated to the first antibody through a linker comprising valine and citrulline. 35. The method of claim 34, wherein the linker-MMAE is vcMMAE. 36. The method of claim 33, wherein the MMAE is conjugated to the first antibody through a linker comprising leucine, alanine, and glutamic acid. 37. The method of claim 36, wherein the linker-MMAE is dLAE-MMAE. 38. The method of any one of claims 1 to 32, wherein the tubulin disrupting agent is MMAF. 39. The method of any one of claims 1 to 32, wherein the tubulin disrupting agent is tubulysin. 40. The method of claim 39, wherein said tubulysin is selected from the group consisting of tubulysin D, tubulysin M, microtubule phenylalanine and microtubule tyrosine. 41. The method of any one of claims 1 to 32, wherein the antibody-drug conjugate is selected from the group consisting of AbGn-107 (Ab1-18Hr1), AGS62P1 (ASP1235), ALT-P7 (HM2-MMAE) , BA3011 (CAB-AXL-ADC), Martin-Belantuzumab, Vibutuximab, Vesituzumab (VLS-101, UC-961ADC3), Pertinolizumab (PF-06647020, PTK7-ADC, PF-7020, ABBV-647), CX-2029 (ABBV-2029), vedicitumumab (RC48), veinatumumab (HuMax-AX L-ADC , AXL-107-MMAE), venosumab (EV), FS-1502 (LCB14-0110), gemtuzumab ozogamicin, HTI-1066 (SHR-A1403), intuzumab Ozogamicin, PF-06804103 (NG-HER2ADC), vepolotuzumab, gosatuzumab, SGN-B6A, SGN-CD228A, SGN-STNV, STI-6129 (CD38ADC, LNDS1001, CD38 -077 ADC), Vitilizumab (ABBV-399), Viltuxomab (Humax-TF-ADC, tf-011-mmae, TV), Detrastuzumab, Enmetrastuzumab mAb and martin-versertuzumab. 42. The method of any one of claims 1 to 41, wherein the first antibody is an anti-Claudin-18.2 antibody comprising a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 61 to 66 respectively. , CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. 43. The method of claim 42, wherein the anti-Claudin-18.2 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 59; and the amino acid sequence SEQ ID NO: 60 The light chain variable region (VL). 44. The method of claim 43, wherein the anti-Claudin-18.2 antibody is zobetuximab (175D10). 45. The method of any one of claims 1 to 41, wherein the first antibody is an anti-Claudin-18.2 antibody comprising a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 69 to 74 respectively. , CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. 46. The method of claim 45, wherein the anti-Claudin-18.2 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 67; and comprising the amino acid sequence SEQ ID NO: The light chain variable region (VL) of 68. 47. The method of any one of claims 1 to 41, wherein the first antibody is an anti-PD-L1 antibody comprising heavy chain CDR1, CDR2 respectively comprising the amino acid sequences SEQ ID NO: 77 to 82 and CDR3 as well as light chain CDR1, CDR2 and CDR3. 48. The method of claim 47, wherein the anti-PD-L1 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO:75; and a light chain variable region (VH) comprising the amino acid sequence SEQ ID NO:76. Chain variable region (VL). 49. The method of any one of claims 1 to 41, wherein the first antibody is an anti-ALP antibody comprising heavy chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences SEQ ID NO: 85 to 90 and light chain CDR1, CDR2 and CDR3. 50. The method of claim 49, wherein the ALP antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO:83; and a light chain variable region (VH) comprising the amino acid sequence SEQ ID NO:84. Variable area (VL). 51. The method of any one of claims 1 to 41, wherein the first antibody comprises an anti-B7H4 antibody comprising heavy chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences SEQ ID NO: 93 to 98 and light chain CDR1, CDR2 and CDR3. 52. The method of claim 51, wherein the anti-B7H4 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 91; and a light chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 92. Variable area (VL). 53. The method of any one of claims 1 to 41, wherein the first antibody is an anti-HER2 antibody comprising: a heavy chain comprising the amino acid sequence SEQ ID NO: 99 and a heavy chain comprising the amino acid sequence SEQ ID NO :100 light chain. 54. The method of claim 53, wherein the antibody-drug conjugate is vedesitomab. 55. The method of any one of claims 1 to 41, wherein the first antibody is an anti-NaPi2B antibody comprising: a heavy chain comprising the amino acid sequence SEQ ID NO: 101 and a heavy chain comprising the amino acid sequence SEQ ID NO :102 light chain. 56. The method of claim 55, wherein the antibody-drug conjugate is velifatumumab. 57. The method of any one of claims 1 to 41, wherein the first antibody is an anti-Connexin-4 antibody comprising heavy chain CDR1, respectively comprising the amino acid sequence SEQ ID NO: 105 to 110. CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. 58. The method of claim 57, wherein the anti-Connexin-4 antibody is an antibody comprising: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 103 and a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO :104 light chain variable region (VL). 59. The method of claim 58, wherein the antibody-drug conjugate is venomumab. 60. The method of any one of claims 1 to 41, wherein the first antibody is an anti-AVB6 antibody comprising heavy chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences SEQ ID NO: 113 to 118 and light chain CDR1, CDR2 and CDR3. 61. The method of claim 60, wherein the anti-AVB6 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 38 Area(VL). 62. The method of any one of claims 1 to 41, wherein the first antibody is an anti-AVB6 antibody comprising heavy chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences SEQ ID NO: 121 to 126 and light chain CDR1, CDR2 and CDR3. 63. The method of claim 62, wherein the anti-AVB6 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 119 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 120 Area(VL). 64. The method of any one of claims 1 to 41, wherein the first antibody is an anti-CD228 antibody comprising heavy chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences SEQ ID NO: 129 to 134 and light chain CDR1, CDR2 and CDR3. 65. The method of claim 64, wherein the anti-CD228 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 127 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 128 Area(VL). 66. The method of any one of claims 1 to 41, wherein the first antibody is an anti-LIV-1 antibody comprising heavy chain CDR1, CDR2 respectively comprising the amino acid sequences SEQ ID NO: 137 to 142 and CDR3 as well as light chain CDR1, CDR2 and CDR3. 67. The method of claim 66, wherein the anti-LIV-1 antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 135 and a light chain comprising the amino acid sequence SEQ ID NO: 136 Variable region (VL). 68. The method of any one of claims 1 to 41, wherein the first antibody is an anti-tissue factor antibody comprising heavy chain CDR1, CDR2 and CDR2 respectively comprising the amino acid sequences SEQ ID NO: 145 to 150. CDR3 and light chain CDR1, CDR2 and CDR3. 69. The method of claim 68, wherein the anti-tissue factor antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 143 and a light chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 144. Variable area (VL). 70. The method of claim 69, wherein the antibody-drug conjugate is vitesolumab. 71. The method of any one of claims 1 to 70, wherein the second antibody binds an immune cell adapter selected from the group consisting of anti-Müllerian hormone receptor II (AMHR2), B7, B7H1, B7H2 , B7H3, B7H4, BAFF-R, BCMA (B cell maturation antigen), Bst1/CD157, C5 complement, CC chemokine receptor 4 (CCR4), CD123, CD137, CD19, CD20, CD25 (IL2RA), CD276, CD278, CD3, CD32, CD33, CD37, CD38, CD4 and HIV-1gp120 binding sites, CD40, CD70, CD70 (member of the TNF receptor ligand family), CD80, CD86, claudin 18.2, c-MET , CSF1R, CTLA-4, EGFR, EGFR MET proto-oncogene, EPHA3, ERBB2, ERBB3, FGFR2b, FLT3, GITR, glucocorticoid-induced TNF receptor (GITR), HER2, HER3, HLA, ICOS, IDO1, IFNAR1 , IFNAR2, IGF-1R, IL-3Rα(CD123), IL-5R, IL-5Rα, LAG-3, MET proto-oncogene, OX40(CD134), PD-1, PD-L1, PD-L2, PVRIG, Respiratory syncytial virus (RSV) heavily glycosylated mucin-like domain of EBOV glycoprotein (GP), rhesus (Rh)D, sialic acid immunoglobulin-like lectin 8 (Siglec-8), signaling lymphoid Cell activation molecule (SLAMF7/CS1), T cell receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA4), TIGIT, TIM3 (HAVCR2), tumor-specific glycoepitope of Muc1 (TA-Muc1), VSIR (VISTA) and VTCN1. 72. The method of any one of claims 1 to 71, wherein the second antibody binds TIGIT. 73. The method of claim 72, wherein the second antibody comprises: (a) Heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NO: 7 to 9; (b) Heavy chain CDR2, comprising an amino acid sequence selected from SEQ ID NO: 10 to 13; (c) Heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 14 to 16; (d) light chain CDR1, comprising the amino acid sequence SEQ ID NO: 17; (e) light chain CDR2 comprising the amino acid sequence SEQ ID NO: 18; and (f) Light chain CDR3 comprising the amino acid sequence SEQ ID NO: 19. 74. The method of claim 72, wherein the second antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 respectively comprising the following sequences: (a) SEQ ID NO:7, 10, 14, 17, 18 and 19; or (b) SEQ ID NO:8, 11, 14, 17, 18 and 19; or (c) SEQ ID NO:9, 12, 15, 17, 18 and 19; or (d) SEQ ID NO:8, 13, 16, 17, 18 and 19; or (e) SEQ ID NOs: 8, 12, 16, 17, 18 and 19. 75. The method of claim 72, wherein the second antibody comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 1 to 5 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 6. chain variable region. 76. The method of claim 72, wherein the second antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 20 to 24 and a light chain comprising the amino acid sequence SEQ ID NO: 25. 77. The method of any one of claims 1 to 71, wherein the second antibody binds CD40. 78. The method of claim 77, wherein the second antibody comprises: heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 comprising the following sequences: (a) SEQ ID NO: 30, 31, 32, 33, 34 and 35; or (b) SEQ ID NO: 30, 36, 32, 33, 34 and 35. 79. The method of claim 77, wherein the second antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 29. 80. The method of claim 77, wherein the second antibody comprises: a heavy chain comprising the amino acid sequence SEQ ID NO:26 and a light chain comprising the amino acid sequence SEQ ID NO:27. 81. The method of any one of claims 1 to 71, wherein the second antibody binds CD70. 82. The method of claim 81, wherein the second antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 respectively comprising the sequences of SEQ ID NOs: 53 to 58. 83. The method of claim 81, wherein the second antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 42. 84. The method of any one of claims 1 to 71, wherein the second antibody binds BCMA. 85. The method of claim 84, wherein the second antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 respectively comprising the sequences of SEQ ID NOs: 47 to 52. 86. The method of claim 84, wherein the second antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 46. 87. The method of any one of claims 1 to 86, wherein the second antibody is an IgGl or IgG3 antibody. 88. The method of any one of claims 1 to 87, wherein the second antibody is comprised in an antibody composition, wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibodies are afucosylated. 89. The method of claim 88, wherein each antibody in the composition comprises the same heavy and light chain amino acid sequences as the second antibody. 90. The method of any one of claims 1 to 89, wherein said Fc enhancement of said second antibody is associated with one or more activating FcγRs compared to a corresponding wild-type Fc of the same isotype. The binding, wherein the activating FcγR includes one or more of FcγRIIIa, FcγRIIa and/or FcγRI. 91. The method of claim 90, wherein the Fc of the second antibody enhances binding to FcyRIIIa. 92. The method of any one of claims 1 to 91, wherein said Fc reduction of said second antibody is associated with one or more inhibitory FcγRs compared to a corresponding wild-type Fc of the same isotype. combination. 93. The method of claim 92, wherein the Fc of the second antibody reduces binding to FcyRIIb. 94. The method of any one of claims 1 to 93, wherein the Fc of the second antibody enhances binding to FcγRIIIa and reduces binding to FcγRIIb. 95. The method of any one of claims 1 to 94, wherein the second antibody is a monoclonal antibody. 96. The method of any one of claims 1 to 95, wherein the second antibody is a humanized antibody or a human antibody. 97. The method of any one of claims 1 to 96, wherein the cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, Stomach cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, lung cancer, lung adenocarcinoma, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, melanoma, central nervous system Nervous system tumors, mesothelioma, lymphoma, leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, or sarcoma. 98. The method of any one of claims 1 to 97, wherein the cancer is lymphoma, leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma or Hodgkin lymphoma tumor. 99. The method of any one of claims 1 to 98, wherein the antibody-drug conjugate and the second antibody are administered simultaneously. 100. The method of claim 99, wherein the antibody-drug conjugate and the second antibody are administered in a single pharmaceutical composition. 101. The method of any one of claims 1 to 98, wherein the antibody-drug conjugate and the second antibody are administered sequentially. 102. The method of claim 101, wherein at least the first dose of the antibody-drug conjugate is administered prior to the first dose of the second antibody; or wherein at least the first dose of the second antibody administered prior to the first dose of the antibody-drug conjugate. 103. The method of any one of claims 1 to 102, wherein the second antibody depletes regulatory T cells (Tregs). 104. The method of any one of claims 1 to 103, wherein the antibody-drug conjugate induces immune memory against cells expressing the antigen bound by the antibody-drug conjugate. 105. The method of claim 104, wherein the induction of immune memory includes induction of memory T cells. 106. The method of any one of claims 1 to 105, wherein the second antibody activates an antigen-presenting cell (APC). 107. The method of any one of claims 1 to 106, wherein the second antibody enhances a CD8 T cell response. 108. The method of any one of claims 1 to 107, wherein the second antibody upregulates costimulatory receptors. 109. The method of any one of claims 1 to 108, wherein administration of the ADC and the second antibody promotes the release of immune activating cytokines. 110. The method of claim 109, wherein the immune activating cytokine is CXCL10 or IFNγ. 111. The method of any one of claims 1 to 110, wherein said ADC and said second antibody act synergistically. 112. The method of any one of claims 1 to 111, wherein administration of the ADC and the second antibody in combination has a toxicity comparable to when the ADC or the second antibody is administered as monotherapy Overview. 113. The method of any one of claims 1 to 112, wherein the effective dose of the ADC and/or the second antibody when administered in combination is less than the effective dose when administered as monotherapy. 114. The method of any one of claims 1 to 113, wherein the cancer has a high tumor mutation load. 115. The method of any one of claims 1 to 114, wherein the cancer has microsatellite instability.