Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION THERAPY ANTIBODY DRUG CONJUGATE WITH IMMUNE CELL INHIBITOR
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of US Provisional
Application No.
63/111,045, filed November 8, 2020, US Provisional Application No. 63/172,411,
filed April 8,
2021, and US Provisional Application No. 63/208,179, filed June 8, 2021, each
of which is
incorporated by reference herein in its entirety for any purpose.
FIELD
[0002] Provided herein are methods of treating cancer with an antibody that
binds an
immune cell engager in combination with an antibody-drug conjugate.
BACKGROUND
[0003] Immuno-oncology therapeutics and antibody-drug conjugates (ADCs)
have been
used to treat cancer in patients. Neither class of therapeutics has been able
to treat the full
complement of patients in targeted indications. This disclosure solves this
and other problems.
BRIEF SUMMARY
[0004] Embodiment 1. A method of treating cancer, comprising administering
to a subject
with cancer (1) an antibody-drug conjugate (ADC) that comprises a first
antibody that binds a
tumor-associated antigen and a cytotoxic agent, wherein the cytotoxic agent is
a tubulin
disrupter; and (2) a second antibody that binds to an immune cell engager,
wherein the second
antibody comprises an Fc with enhanced binding to one or more activating
FcyRs, wherein the
activating FcyRs include one or more of FcyRIIIa, FcyRIIa, and/or FcyRI.
[0005] Embodiment 2. The method of embodiment 1, wherein the second
antibody
comprises an Fc with enhanced binding to at least FcyRIIIa.
[0006] Embodiment 3. The method of embodiment 1, wherein second antibody
comprises
an Fc with enhanced binding to at least FcyRIIIa and FcyRIIa.
[0007] Embodiment 4. The method of embodiment 1, wherein the second
antibody
comprises an Fc with enhanced binding to at least FcyRIIIa and FcyRI.
[0008] Embodiment 5. The method of embodiment 1, wherein the second
antibody
comprises an Fc with enhanced binding to FcyRIIIa, FcyRIIa, and FcyRI.
[0009] Embodiment 6. The method of any one of embodiments 1-5, wherein the
Fc of the
second antibody has reduced binding to one or more inhibitory FcyRs.
1
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[0010] Embodiment 7. The method of embodiment 6, wherein the Fe of the
second
antibody has reduced binding to FcyRIIb.
[0011] Embodiment 8. The method of any one of embodiments 1-7, wherein the
Fe of the
second antibody has reduced fucose levels and/or has been engineered to
comprise one or more
mutations such that the Fe has enhanced binding to the one or more activating
FcyRs.
[0012] Embodiment 9. The method of embodiment 8, wherein the second
antibody is
nonfucosylated.
[0013] Embodiment 10. The method of embodiment 8, wherein the second
antibody
comprises substitutions S293D, A330L, and I332E in the heavy chain constant
region.
[0014] Embodiment 11. A method of treating cancer, comprising administering
to a subject
with cancer an antibody-drug conjugate, wherein the antibody-drug conjugate
comprises a first
antibody conjugated to a cytotoxic agent, wherein the cytotoxic agent is a
tubulin disrupter; and
a second antibody that binds an immune cell engager, wherein the second
antibody is
nonfucosylated.
[0015] Embodiment 12. The method of any one of embodiments 1-11, wherein
the first
antibody binds a tumor-associated antigen.
[0016] Embodiment 13. A method of treating cancer, comprising administering
to a subject
with 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
tubulin disrupter, and (2) a second antibody that binds an immune cell
engager, wherein the
second antibody comprises an Fe with enhanced ADCC activity relative to a
corresponding
wild-type Fe of the same isotype.
[0017] Embodiment 14. The method of embodiment 13, wherein the second
antibody
comprises an Fe with enhanced ADCC and ADCP activity relative to a
corresponding wild-type
Fe of the same isotype.
[0018] Embodiment 15. The method of embodiment 13 or 14, wherein the second
antibody
is nonfucosylated.
[0019] Embodiment 16. The method of any one of embodiments 13-15, wherein
the second
antibody comprises an Fe with enhanced binding to one or more activating
FcyRs, wherein the
activating FcyRs include one or more of FcyRIIIa, FcyRIIa, and/or FcyRI.
[0020] Embodiment 17. The method of embodiment 16, wherein the second
antibody
comprise an Fe with enhanced binding to at least FcyRIIIa.
[0021] Embodiment 18. The method of embodiment 16, wherein second antibody
comprises an Fe with enhanced binding to at least FcyRIIIa and FcyRIIa.
2
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[0022] Embodiment 19. The method of embodiment 16, wherein the second
antibody
comprises an Fc with enhanced binding to at least FcyRIIIa and FcyRI.
[0023] Embodiment 20. The method of embodiment 16, wherein the second
antibody
comprises an Fc with enhanced binding to FcyRIIIa, FcyRIIa, and FcyRI.
[0024] Embodiment 21. The method of any one of embodiments 13-20, wherein
the Fc of
the second antibody has reduced binding to one or more inhibitory FcyRs.
[0025] Embodiment 22. The method of embodiment 21, wherein the Fc of the
second
antibody has reduced binding to FcyRIIb.
[0026] Embodiment 23. The method of any one of embodiments 1-22, wherein
the first
antibody binds an antigen selected from 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, CD161, CD162, CD163, CD164, CD166, CD167b, CD169, CD16a,
CD16b, CD170, CD171, CD172a, CD172b, CD172g, CD18, CD180, CD181, CD183, CD184,
CD185, CD19, CD194, CD197, CD1a, CD1b, CD lc, 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, CD3 epsilon, 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, CD7OL, 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,
3
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CEA, CEACAM5, CEACAM6, CFC1B, cKIT, CLDN18.2 (claudin 18.2), CLDN6, CLDN9,
CLL-1, c-MET, complement factors C3, Cripto, CSP-1, CXCR5, DCLK1, DLK-1, DLL3,
DPEP3, DR5 (Death receptor 5), 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-alpha, 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 (integrin aVf33), integrin beta-6, Interleukin-4
Receptor (IL4R),
KAAG-1, KLK2, LAMP-1, Le(y), Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH,
Lipid raft, 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, Nectin-4, Notch3, Nucleolin,
OAcGD2,
OT-MUC1 (onco-tethered-MUC1), OX001L, P1GF, PAM4 antigen, p-cadherin (cadherin
3),
PD-L1, Phosphatidyl Serine(PS), PRLR, Prolactin Receptor (PRLR), Pseudomonas,
PSMA,
PTK4, PTK7, Receptor tyrosine kinase (RTK), RNF43, ROR1, ROR2, SAIL, SEZ6,
SLAMF7,
5LC44A4, SLITRK6, SLMAMF7 (CS1), SLTRK6, Sortilin (SORT1), SSEA-4, SSTR2,
Staphylococcus aureus (antibiotic agent), STEAP-1, STING, STn, T101, TAA, TAC,
TDGF1,
tenascin, TENB2, TGF-B, Thomson-Friedenreich antigens, Thy1.1, TIM-1, tissue
factor (TF;
CD142), TM4SF1, Tn antigen, TNF-alpha (TNFa), TRA-1-60, TRAIL receptor (R1 and
R2),
TROP-2, Tumor-associated glycoprotein 72 (TAG-72), uPAR, VEGFR, VEGFR-2, and
xCT.
[0027] Embodiment 24. The method of any one of embodiments 1-23, wherein
the first
antibody does not bind Nectin-4.
[0028] Embodiment 25. The method of any one of embodiments 1-24, wherein
the method
does not comprise administering an antibody-drug conjugate comprising an
antibody that binds
Nectin-4.
[0029] Embodiment 26. The method of any one of embodiments 1-25, wherein
the first
antibody binds an antigen selected from CD71, Axl, AMUR'', 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, Nectin-4, OT-MUC1 (onco-tethered-MUC1), PSMA, ROR1, SLAMF7,
5LC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, and Tumor-associated
glycoprotein
72 (TAG-72).
4
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[0030] Embodiment 27. The method of any one of embodiments 1-25, wherein
the first
antibody binds an antigen selected from BCMA, GPC1, CD30, cMET, SAIL, HER3,
CD70,
CD46, CD48, HER2, 5T4, ENPP3, CD19, EGFR, and EphA2.
[0031] Embodiment 28. The method of any one of embodiments 1-25, wherein
the first
antibody binds an antigen selected from Her2, TROP2, BCMA, cMet, integrin
alphVbeta6
(integrin aVf36), CD22, CD79b, CD30, CD19, CD70, CD228, CD47, and CD48.
[0032] Embodiment 29. The method of any one of embodiments 1-25, wherein
the first
antibody binds an antigen selected from CD142, Integrin beta-6, integrin
alphaVbeta6, 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, 5LC44A4,
CD30, and sTn.
[0033] Embodiment 30. The method of any one of embodiments 1-26, wherein
the tubulin
disrupter is an auristatins, a tubulysin, a colchicine, a vinca alkaloid, a
taxane, a cryptophycin, a
maytansinoid, or a hemiasterlin.
[0034] Embodiment 31. The method of embodiment 30, wherein the tubulin
disrupter is an
auristatin.
[0035] Embodiment 32. The method of any one of embodiments 1-31, wherein
tubulin
disrupter 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).
[0036] Embodiment 33. The method of any one of embodiments 1-32, wherein
the tubulin
disrupter is MMAE.
[0037] Embodiment 33-1. The method of embodiment 33, wherein the antibody-
drug
conjugate comprises MMAE and is selected from: DP303c, also known as SYSA1501,
targeting HER-2 (CSPC Pharmaceutical; Dophen Biomed), SIA01-ADC, also known as
ST1,
targeting STn (Siamab Therapeutics), Ladiratuzumab vedotin, also known as SGN-
LIV1A,
targeting LIV-1 (Merck & Co., Inc.; Seagen (Seattle Genetics) Inc.), ABBV-085,
also known as
Samrotamab vedotin, targeting LRRC15 (Abbvie; Seagen (Seattle Genetics) Inc.),
DMOT4039A, also known as RG7600; aMSLN-MMAE, targeting Mesothelin (MSLN)
(Roche-Genentech), RC68, also known as Remegen EGFR ADC, targeting EGFR
(RemeGen
(Rongchang Biopharmaceutical (Yantai) Co., Ltd.)), RC108, also known as RC108-
ADC,
targeting c-MET (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.)),
CMG901, also
known as MRG005, targeting CLDN18.2 (Keymed Biosciences; Lepu biotech;
Shanghai
Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)), YBL-001, also known
as LCB67,
targeting DLK-1 (Lego Chem Biosciences; Pyxis Oncology; Y-Biologics),
DCDS0780A, also
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known as Iladatuzumab vedotin; RG7986, targeting CD79b (Roche-Genentech;
Seagen (Seattle
Genetics) Inc.), Tisotumab vedotin, also known as Humax-TF-ADC; tf-011-mmae;
TIVDAKTm,
targeting CD142 (GenMab; Seagen (Seattle Genetics) Inc.), GO-3D1-ADC, also
known as
humAb-3D1-MMAE ADC, targeting MUC1-C (Genus Oncology LLC), ALT-P7, also known
as
HM2-MMAE, targeting HER-2 (Alteogen, Inc.; Levena Biopharma; 3SBio, Inc.),
Vandortuzumab vedotin, also known as D5TP30865; RG7450, targeting STEAP-1
(Roche-
Genentech; Seagen (Seattle Genetics) Inc.), Lifastuzumab Vedotin, also known
as DNIB0600A;
NaPi2b ADC; RG7599, targeting NaPi2b (Roche-Genentech), Sofituzumab vedotin,
also known
as DMUC5754A; RG7458, targeting MUC16 (Seagen (Seattle Genetics) Inc.; Roche-
Genentech), RG7841, also known as DLYE5953A, targeting Ly6E (Roche-Genentech;
Seagen
(Seattle Genetics) Inc.), RG7598, also known as DFRF4539A, targeting FCRH5
(Roche-
Genentech; Seagen (Seattle Genetics) Inc.), RG7636, also known as DEDN6526A,
targeting
ETBR (Seagen (Seattle Genetics) Inc.; Roche-Genentech), Pinatuzumab vedotin,
also known as
DCDT2980S; RG7593, targeting CD22 (Roche-Genentech), Polatuzumab vedotin, also
known
as DCDS4501A; POLIVYTm; RG7596; RO-5541077, targeting CD79b (Chugai
Pharmaceutical;
Roche-Genentech; Seagen (Seattle Genetics) Inc.), DMUC4064A, also known as D-
4064a;
RG7882, targeting MUC16 (Roche-Genentech; Seagen (Seattle Genetics) Inc.),
SYSA1801, also
known as CP0102, targeting CLDN18.2 (Conjupro Biotherapeutics Inc.; CSPC
ZhongQi
Pharmaceutical Technology Co.), RC118, also known as Claudin18.2- ADC; YHO05,
targeting
CLDN18.2 (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.);
Biocytogen), VLS-
101, also known as Cirmtuzumab vedotin; MK-2140; UC-961ADC3; Zilovertamab
Vedotin,
targeting ROR1 (VelosBio. Inc), Glembatumumab vedotin, also known as CDX-011;
CR011-
vcMMAE, targeting gpNMB (Celldex Therapeutics), BA3021, also known as CAB-ROR2-
ADC; Ozuriftamab Vedotin, targeting ROR2 (Bioatla; Himalaya Therapeutics),
BA3011, also
known as CAB-AXL-ADC; Mecbotamab Vedotin, targeting Axl (Bioatla; Himalaya
Therapeutics), CM-09, also known as Bstrongximab-ADC, targeting TRA-1-60
(CureMeta),
ABBV-838, also known as Azintuxizumab vedotin, targeting SLAMF7 (Abbvie),
Enapotamab
vedotin, also known as AXL-107-MMAE; HuMax-AXL-ADC, targeting Axl (GenMab;
Seagen
(Seattle Genetics) Inc.), ARC-01, also known as anti-CD79b ADC, targeting
CD79b (Araris
Biotech AG), Disitamab vedotin, also known as Aidexig; RC48, targeting HER-2
(RemeGen
(Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Seagen (Seattle Genetics)
Inc.), ASG-5ME,
also known as AGS-5; AGS-5ME, targeting 5LC44A4 (Agensys, Inc.; Astellas
Pharma Inc.;
Seagen (Seattle Genetics) Inc.), Enfortumab vedotin, also known as AGS-22M6E;
ASG-22CE;
ASG-22ME; PADCEVTM, targeting Nectin-4 (Astellas Pharma Inc.; Seagen (Seattle
Genetics)
Inc.), ASG-15ME, also known as AGS-15E; Sirtratumab vedotin, targeting SLITRK6
(Seagen
6
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(Seattle Genetics) Inc.; Astellas Pharma Inc.), Brentuximab vedotin, also
known as Adcetris;
cAC10-vcMMAE; SGN-35, targeting CD30 (Seagen (Seattle Genetics) Inc.; Takeda),
Telisotuzumab vedotin, also known as ABBV-399, targeting c-MET (Abbvie),
Losatuxizumab
vedotin, also known as ABBV-221, targeting EGFR (Abbvie), CX-2029, also known
as ABBV-
2029, targeting CD71 (Abbvie; CytomX Therapeutics), AB-3A4-ADC, also known as
AB-3A4-
vcMMAE, targeting KAAG-1 (Alethia Biotherapeutics), Indusatumab vedotin, also
known as
5F9-vcMMAE; 1V11LN0264; TAK-264, targeting GCC (Takeda; Millennium
Pharmaceuticals,
Inc), F0R46 targeting CD46 (Fortis Therapeutics, Inc.), LR004-VC-MMAE
targeting EGFR
(Chinese Academy of Medical Sciences Peking Union Medical College Hospital),
CD30-ADCs
targeting CD30 (NBE Therapeutics; Boehringer Ingelheim), Anti-endosialin-MC-VC-
PABC-
MMAE targeting CD248 (Genzyme), OBI-998 targeting SSEA-4 (OBI Pharma), MRG002
targeting HER-2 (Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya
Biotechnology Co.,
Ltd)), TRS005 targeting CD20 (Teruisi Pharmaceuticals), Oba01 targeting DR5
(Death receptor
5) (Obio Technology (Shanghai) Corp.,Ltd.; Yantai Obioadc Biomedical
Technology Ltd.),
PSMA ADC targeting PSMA (Progenics Pharmaceuticals, Inc; Seagen (Seattle
Genetics) Inc.),
SGN-CD48A targeting CD48 (Seagen (Seattle Genetics) Inc.), IIVIAB362-vcMMAE
targeting
CLDN18.2 (Astellas Pharma Inc.; Ganymed), GB251 targeting HER-2 (Genor
Biopharma Co.,
Ltd.), Innate Pharma BTG-ADCs targeting CD30 (Innate Pharma; Sanofi), ADCendo
uPARAP
ADC targeting MRC2 (ADCendo), XCN-010 targeting actM (Xiconic Pharmaceuticals,
LLC),
ANT-043 targeting HER-2 (Antikor Biopharma), OBI-999 targeting Globo H
(Abzena; OBI
Pharma), LY3343544 targeting MET (Eli Lilly and Company), Tagworks anti-TAG72
ADC
targeting TAG-72 (Tagworks Pharmaceuticals), IIVIAB027-vcMMAE targeting CLDN6
(Ganymed; Astellas Pharma Inc.), LGR5-ADC targeting LGR5 (Genentech, Inc.),
Philochem
B12-MMAE ADC targeting IL-7R (Institut de Medicina Molecular Joao Lobo
Antunes;
Philochem AG), TE-1522 targeting CD19 (Immunwork), SGN-STNV targeting STn
(Seagen
(Seattle Genetics) Inc.), HTI-1511 targeting EGFR (Abzena; Halozyme
Therapeutics), Peptron
PAb001-ADC targeting OT-MUC1 (onco-tethered-MUC1) (Peptron; Qilu
Pharmaceutical co.
Ltd.), LM-102 targeting CLDN18.2 (Lallova Medicines Limited), Anwita
Biosciences MSLN-
MMAE targeting Mesothelin (MSLN) (Anwita biosciences), SGN-CD228A targeting
CD228
(Seagen (Seattle Genetics) Inc.), NBT828 targeting HER-2 (NewBio Therapeutics;
Genor
Biopharma Co., Ltd.), Gamamabs GM103 targeting AMHR2 (GamaMabs Pharma;
Exelixis),
LCB14-0302 targeting HER-2 (Lego Chem Biosciences), BAY79-4620 targeting
carbonic
anhydrase IX (CAIX) (Bayer; MorphoSys), NBT508 targeting CD79b (NewBio
Therapeutics),
PAT-DX3-MMAE targeting Undisclosed (Patrys; Yale University), AGS67E targeting
CD37
(Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.), CDX-014 targeting TIM-
1 (Celldex
7
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Therapeutics), BVX001 targeting CD33; CD7 (Bivictrix therapeutics), SGN-B6A
targeting
Integrin beta-6 (Seagen (Seattle Genetics) Inc.), MRG003 targeting EGFR (Lepu
biotech;
Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)), and PYX-202
targeting
DLK-1 (Pyxis Oncology; Lego Chem Biosciences).
[0038] Embodiment 34. The method of embodiment 33, wherein the MMAE is
conjugated
to the first antibody through a linker that comprises valine and citrulline.
[0039] Embodiment 35. The method of embodiment 34, wherein the linker-MMAE
is
vcMMAE.
[0040] Embodiment 36. The method of embodiment 33, wherein the MMAE is
conjugated
to the first antibody through a linker that comprises leucine, alanine, and
glutamic acid.
[0041] Embodiment 37. The method of embodiment 36, wherein the linker-MMAE
is
dLAE-MMAE.
[0042] Embodiment 38. The method of any one of embodiments 1-32, wherein
the tubulin
disrupter is MMAF.
[0043] Embodiment 38-1. The method of embodiment 38, wherein the antibody-
drug
conjugate comprises MMAF and is selected from: CD70-ADC targeting CD70 (Kochi
University; Osaka University), IGN786 targeting SAIL (AstraZeneca; Igenica
Biotherapeutics),
PF-06263507 targeting 5T4 (Pfizer), GPC1-ADC targeting GPC-1 (Kochi
University), ADC-
AVP10 targeting CD30 (Avipep), M290-MC-MMAF targeting CD103 (The Second
Affiliated
Hospital of Harbin Medical University), BVX001 targeting CD33; CD7 (Bivictrix
therapeutics),
Tanabe P3D12-vc-MMAF targeting c-MET (Tanabe Research Laboratories), LILRB4-
Targeting
ADC targeting LILRB4 (The University of Texas Health Science Center, Houston),
TSD101,
also known as ABL201, targeting BCMA (TSD Life Science; ABL Bio; Lego Chem
Biosciences), Depatuxizumab mafodotin, also known as ABT-414, targeting EGFR
(Abbvie;
Seagen (Seattle Genetics) Inc.), AGS16F, also known as AGS-16C3F; AGS-16M8F,
targeting
ENPP3 (Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.), AVG-All BCMA
ADC, also
known as AVG-All-mcMMAF, targeting BCMA (Avantgen), Belantamab mafodotin, also
known as BLENREP; G5K2857916; J6M0-mcMMAF, targeting BCMA (GlaxoSmithKline;
Seagen (Seattle Genetics) Inc.), MP-HER3-ADC, also known as HER3-ADC,
targeting HER-3
(MediaPharma), FS-1502, also known as LCB14-0110, targeting HER-2 (Lego Chem
Biosciences; Shanghai Fosun Pharmaceutical Development Co, Ltd.), MEDI-547,
also known as
MI-CP177, targeting EphA2 (AstraZeneca; Seagen (Seattle Genetics) Inc.),
Vorsetuzumab
mafodotin, also known as SGN-75, targeting CD70 (Seagen (Seattle Genetics)
Inc.),
Denintuzumab mafodotin, also known as SGN-CD19A, targeting CD19 (Seagen
(Seattle
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Genetics) Inc.), and HTI-1066, also known as SHR-A1403, targeting c-MET
(Jiangsu HengRui
Medicine Co., Ltd).
[0044] Embodiment 39. The method of embodiment any one of embodiments 1-30,
wherein
the tubulin disrupter is a tubulysin.
[0045] Embodiment 40. The method of embodiment 39, wherein the tubulysin is
selected
from tubulysin D, tubulysin M, tubuphenylalanine, and tubutyrosine.
[0046] Embodiment 41. The method of any one of embodiments 1-32, wherein
the
antibody-drug conjugate is selected from AbGn-107 (Ab1-18Hr1), AGS62P1
(ASP1235), ALT-
P7 (HM2-MMAE), BA3011 (CAB-AXL-ADC), belantamab mafodotin, brentuximab
vedotin,
cirmtuzumab vedotin (VLS-101, UC-961ADC3), cofetuzumab pelidotin (PF-06647020,
PTK7-
ADC, PF-7020, ABBV-647), CX-2029 (ABBV-2029), disitamab vedotin (RC48),
enapotamab
vedotin (HuMax-AXL-ADC, AXL-107-MMAE), enfortumab vedotin (EV), FS-1502 (LCB14-
0110), gemtuzumab ozogamicin, HTI-1066 (SHR-A1403), inotuzumab ozogamicin, PF-
06804103 (NG-HER2 ADC), polatuzumab vedotin, sacituzumab govitecan, SGN-B6A,
SGN-
CD228A , SGN-STNV, STI-6129 (CD38 ADC, LNDS1001, CD38-077 ADC), telisotuzumab
vedotin (ABBV-399), tisotumab vedotin (Humax-TF-ADC, tf-011-mmae, TV),
trastuzumab
deruxtecan, trastuzumab emtansine, and vorsetuzumab mafodotin.
[0047] Embodiment 42. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-claudin-18.2 antibody that comprises a
heavy chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:61-66.
[0048] Embodiment 43. The method of 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.
[0049] Embodiment 44. The method of embodiment 43, wherein the anti-claudin-
18.2
antibody is zolbetuximab (175D10).
[0050] Embodiment 45. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-claudin-18.2 antibody that comprises a
heavy chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs: 69-74.
[0051] Embodiment 46. The method of 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.
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[0052] Embodiment 47. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-PD-Li antibody that comprises a heavy
chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:77-82.
[0053] Embodiment 48. The method of embodiment 47, wherein the anti-PD-Li
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.
[0054] Embodiment 49. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-ALP antibody that comprises a heavy
chain CDR1, CDR2,
and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising the
amino acid
sequences of SEQ ID NOs:85-90.
[0055] Embodiment 50. The method of 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.
[0056] Embodiment 51. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody comprises an anti-B7H4 antibody that comprises a
heavy chain
CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively
comprising
the amino acid sequences of SEQ ID NOs:93-98.
[0057] Embodiment 52. The method of embodiment Si, 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.
[0058] Embodiment 53. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-HER2 antibody that 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.
[0059] Embodiment 54. The method of embodiment 53, wherein the antibody-
drug
conjugate is disitamab vedotin.
[0060] Embodiment 55. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-NaPi2B antibody that 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.
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[0061] Embodiment 56. The method of embodiment 55, wherein the antibody-
drug
conjugate is lifastuzumab vedotin.
[0062] Embodiment 57. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-nectin-4 antibody that comprises a heavy
chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:105-110.
[0063] Embodiment 58. The method of embodiment 57, wherein the anti-nectin-
4 antibody
is an antibody that 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.
[0064] Embodiment 59. The method of embodiment 58, wherein the antibody-
drug
conjugate is enfortumab vedotin.
[0065] Embodiment 60. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-AVB6 antibody that comprises a heavy
chain CDR1, CDR2,
and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising the
amino acid
sequences of SEQ ID NOs:113-118.
[0066] Embodiment 61. The method of 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.
[0067] Embodiment 62. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-AVB6 antibody that comprises a heavy
chain CDR1, CDR2,
and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising the
amino acid
sequences of SEQ ID NOs:121-126.
[0068] Embodiment 63. The method of 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.
[0069] Embodiment 64. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-CD228 antibody that comprises a heavy
chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:129-134.
[0070] Embodiment 65. The method of embodiment 64, wherein the anti-CD228
antibody
comprises a heavy chain variable region (VH) comprising the amino acid
sequence of SEQ ID
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NO:127 and a light chain variable region (VL) comprising the amino acid
sequence of SEQ ID
NO:128.
[0071] Embodiment 66. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-LIV-1 antibody that comprises a heavy
chain CDR1, CDR2,
and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising the
amino acid
sequences of SEQ ID NOs:137-142.
[0072] Embodiment 67. The method of 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.
[0073] Embodiment 68. The method of any one of embodiments 1-41, 33-1, and
38-1,
wherein the first antibody is an anti-tissue factor antibody that comprises
heavy chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:145-150.
[0074] Embodiment 69. The method of 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.
[0075] Embodiment 70. The method of embodiment 69, wherein the antibody-
drug
conjugate is tisotumab vedotin.
[0076] Embodiment 71. The method of any one of embodiments 1-70, 33-1, and
38-1,
wherein the second antibody binds an immune cell engager selected from anti-
Mullerian
Hormone Receptor II (AMHR2), B7, B7H1, B7H2, B7H3, B7H4, BAFF-R, BCMA (B-cell
maturation antigen), Bstl/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 sites, CD40, CD70, CD70 (a 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, IDO 1, IFNAR1, IFNAR2, IGF-1R, IL-
3Ralpha
(CD123), IL-5R, IL-5Ralpha, LAG-3, MET proto-oncogene , 0X40 (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 lectins
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 Mucl (TA-Mud), VSIR (VISTA), and VTCN1.
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[0077] Embodiment 72. The method of any one of embodiments 1-71, 33-1, and
38-1,
wherein the second antibody binds TIGIT.
[0078] Embodiment 73. The method of embodiment 72, wherein the second
antibody
comprises: (a) a heavy chain CDR1 comprising an amino acid sequence selected
from SEQ ID
NOs: 7-9; (b) a heavy chain CDR2 comprising an amino acid sequence selected
from SEQ ID
NOs: 10-13; (c) a heavy chain CDR3 comprising an amino acid sequence selected
from SEQ ID
NOs: 14-16; (d) a light chain CDR1 comprising the amino acid sequence of SEQ
ID NO: 17; (e)
a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18; and
(f) a light chain
CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
[0079] Embodiment 74. The method of embodiment 72, wherein the second
antibody
comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR, and
CDR3
comprising the sequences of: (a) SEQ ID NOs: 7, 10, 14, 17, 18, and 19,
respectively; or (b)
SEQ ID NOs: 8, 11, 14, 17, 18, and 19, respectively; or (c) SEQ ID NOs: 9, 12,
15, 17, 18, and
19, respectively; or (d) SEQ ID NOs: 8, 13, 16, 17, 18, and 19, respectively;
or (e) SEQ ID NOs:
8, 12, 16, 17, 18, and 19, respectively.
[0080] Embodiment 75. The method of embodiment 72, wherein the second
antibody
comprises a heavy chain variable region comprising an amino acid sequence
selected from SEQ
ID NOs: 1-5 and a light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 6.
[0081] Embodiment 76. The method of embodiment 72, wherein the second
antibody
comprises a heavy chain comprising an amino acid sequence selected from SEQ ID
NOs: 20-24
and a light chain comprising the amino acid sequence of SEQ ID NO: 25.
[0082] Embodiment 77. The method of any one of embodiments 1-71, wherein
the second
antibody binds CD40.
[0083] Embodiment 78. The method of embodiment 77, wherein the second
antibody
comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR, and
CDR3
comprising the sequences of: (a) SEQ ID NOs: 30, 31, 32, 33, 34, and 35,
respectively; or (b)
SEQ ID NOs: 30, 36, 32, 33, 34, and 35, respectively.
[0084] Embodiment 79. The method of 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.
[0085] Embodiment 80. The method of embodiment 77, wherein the second
antibody
comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26
and a light
chain comprising the amino acid sequence of SEQ ID NO: 27.
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[0086] Embodiment 81. The method of any one of embodiments 1-71, 33-1, and
38-1,
wherein the second antibody binds CD70.
[0087] Embodiment 82. The method of embodiment 81, wherein the second
antibody
comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR, and
CDR3
comprising the sequences of SEQ ID NOs: 53-58, respectively.
[0088] Embodiment 83. The method of 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.
[0089] Embodiment 84. The method of any one of embodiments 1-71, 33-1, and
38-1,
wherein the second antibody binds BCMA.
[0090] Embodiment 85. The method of embodiment 84, wherein the second
antibody
comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR, and
CDR3
comprising the sequences of SEQ ID NOs: 47-52, respectively.
[0091] Embodiment 86. The method of 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.
[0092] Embodiment 87. The method of any one of embodiments 1-86, 33-1, and
38-1,
wherein the second antibody is an IgG1 or IgG3 antibody.
[0093] Embodiment 88. The method of any one of embodiments 1-87, 33-1, and
38-1,
wherein the second antibody is comprised in a composition of antibodies,
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
nonfucosylated.
[0094] Embodiment 89. The method of embodiment 88, wherein each antibody in
the
composition comprises the same heavy chain and light chain amino acid
sequences as the
second antibody.
[0095] Embodiment 90. The method of any one of embodiments 1-89, 33-1, and
38-1,
wherein the Fc of the second antibody has enhanced binding to one or more
activating FcyRs as
compared to a corresponding wild-type Fc of the same isotype, wherein the
activating FcyRs
include one or more of FcyRIIIa, FcyRIIa, and/or FcyRI.
[0096] Embodiment 91. The method of embodiment 90, wherein the Fc of the
second
antibody has enhanced binding to FcyRIIIa.
[0097] Embodiment 92. The method of any one of embodiments 1-91, 33-1, and
38-1,
wherein the Fc of the second antibody has reduced binding to one or more
inhibitory FcyRs as
compared to a corresponding wild-type Fc of the same isotype.
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[0098] Embodiment 93. The method of embodiment 92, wherein the Fe of the
second
antibody has reduced binding to FcyRIIb.
[0099] Embodiment 94. The method of any one of embodiments 1-93, 33-1, and
38-1,
wherein the Fe of the second antibody has enhanced binding to FcyRIIIa and
reduced binding to
FcyRIIb.
[00100] Embodiment 95. The method of any one of embodiments 1-94, 33-1, and 38-
1,
wherein the second antibody is a monoclonal antibody.
[00101] Embodiment 96. The method of any one of embodiments 1-95, 33-1, and 38-
1,
wherein the second antibody is a humanized antibody or a human antibody.
[00102] Embodiment 97. The method of any one of embodiments 1-96, 33-1, and 38-
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,
clear cell renal
carcinoma, head and neck cancer, lung cancer, lung adenocarcinoma, stomach
cancer, germ cell
cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, melanoma,
neoplasm of the
central nervous system, mesothelioma, lymphoma, leukemia, chronic lymphocytic
leukemia,
diffuse large B cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma,
or sarcoma.
[00103] Embodiment 98. The method of any one of embodiments 1-97, 33-1, and 38-
1,
wherein the cancer is lymphoma, leukemia, chronic lymphocytic leukemia,
diffuse large B cell
lymphoma, follicular lymphoma, or Hodgkin lymphoma.
[00104] Embodiment 99. The method of any one of embodiments 1-98, 33-1, and 38-
1,
wherein the antibody-drug conjugate and the second antibody are administered
concurrently.
[00105] Embodiment 100. The method of embodiment 99, 33-1, and 38-1, wherein
the
antibody-drug conjugate and the second antibody are administered in a single
pharmaceutical
composition.
[00106] Embodiment 101. The method of any one of embodiments 1-98, 33-1, and
38-1,
wherein the antibody-drug conjugate and the second antibody are administered
sequentially.
[00107] Embodiment 102. The method of embodiment 101, wherein at least a first
dose of
the antibody-drug conjugate is administered prior to a first dose of the
second antibody; or
wherein at least a first dose of the second antibody is administered prior to
a first dose of the
antibody-drug conjugate.
[00108] Embodiment 103. The method of any one of embodiments 1-102, 33-1, and
38-1,
wherein the second antibody depletes T regulatory cells (Tregs).
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[00109] Embodiment 104. The method of any one of embodiments 1-103, 33-1, and
38-1,
wherein the antibody-drug conjugate induces immune memory against cells
expressing the
antigen bound by the antibody-drug conjugate.
[00110] Embodiment 105. The method of embodiment 104, wherein the induction of
immune memory comprises induction of memory T cells.
[00111] Embodiment 106. The method of any one of embodiments 1-105, 33-1, and
38-1,
wherein the second antibody activates antigen presenting cells (APCs).
[00112] Embodiment 107. The method of any one of embodiments 1-106, 33-1, and
38-1,
wherein the second antibody enhances CD8 T cell responses.
[00113] Embodiment 108. The method of any one of embodiments 1-107, 33-1, and
38-1,
wherein the second antibody upregulates co-stimulatory receptors.
[00114] Embodiment 109. The method of any one of embodiments 1-108, 33-1, and
38-1,
wherein administration of the ADC and the second antibody promotes release of
an immune
activating cytokine.
[00115] Embodiment 110. The method of embodiment 109, wherein the immune
activating
cytokine is CXCL10 or IFNy.
[00116] Embodiment 111. The method of any one of embodiments 1-110, 33-1, and
38-1,
wherein the ADC and the second antibody act synergistically.
[00117] Embodiment 112. The method of any one of embodiments 1-111, 33-1, and
38-1,
wherein administration of the ADC and the second antibody in combination has a
toxicity
profile comparable to that of the ADC or the second antibody when either is
administered as
monotherapy.
[00118] Embodiment 113. The method of any one of embodiments 1-112, 33-1, and
38-1,
wherein the effective dose of the ADC and/or the second antibody when dosed in
combination is
less than when administered as monotherapy.
[00119] Embodiment 114. The method of any one of embodiments 1-113, 33-1, and
38-1,
wherein the cancer has high tumor mutation burden.
[00120] Embodiment 115. The method of any one of embodiments 1-114, 33-1, and
38-1,
wherein the cancer has microsatellite instability.
BRIEF DESCRIPTION OF THE DRAWINGS
[00121] FIG. 1 shows that non-directed chemotherapeutic agents impair T cell
responses.
[00122] FIG. 2 shows brentuximab vedotin (BV) treatment of CD30+ CD8 T cells.
Vedotin
ADCs that have directed delivery to T cells do not inhibit proliferation.
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[00123] FIG. 3A-E show that endoplasmic reticulum (ER) stress induction is
superior for
auristatin antibody-drug conjugates (ADCs), such as vedotin-based ADCs, as
compared to
ADCs with different payloads. FIG. 3A shows a table of various clinically
approved ADC
payloads. FIG. 3B shows a graphic of ER stress signaling response. FIG. 3C
shows a Western
blot analysis of MIA-PaCa-2 cells treated with ADCs with differing payloads or
paclitaxel at
IC50 concentrations for 36 or 48 hours. FIG. 3D-E show MIA-PaCa-2 cells
expressing CHOP-
driven luciferase reporter (Signosis, Inc.) that were treated with ADCs with
different payloads
over a dose range (FIG. 3D) or at IC50 dose (cytotoxicity) (FIG. 3E). CHOP
induction is
expressed by fold induction compared to untreated cells.
[00124] FIG. 4A-B show immunogenic cell death (ICD) potential of clinical ADC
payloads.
Supernatants were collected from MIA-PaCa-2 pancreatic tumor cells that were
treated with 1
pg/mL ADCs with different payloads for 72 hours. FIG. 4A shows TP released as
determined by
Cell Titer Glo. FIG. 4B shows HMGB1 secretion as determined by ELISA.
[00125] FIG. 5A-C show immune activation assessment of ADC payloads.
Upregulation of
MHC-Class II (HLA-DR) on myeloid cells within peripheral blood mononuclear
cells (PBMC)
was assessed by flow cytometry following a 48-hour co-incubation of PBMC with
L540cy cells
dosed with ADCs with different payloads (24 hours at IC50 concentration). 24-
hour
supernatants were assessed by Luminex multiplex assay for cytokine levels.
FIG. 5A shows
immune activation by ADC. FIG. 5B shows MHCII expression on monocytes in
response to
ADC exposure. FIG. 5C shows innate cytokine CXCL-10/IP10 expression in
response to ADC
exposure as a measure of immune cell activity.
[00126] FIG. 6A-E show payload evaluation on trastuzumab backbone. FIG. 6A
shows a
table of trastuzumab ADCs that were evaluated. FIG. 6B shows a graphic of ER
stress signaling
response. FIG. 6C shows a Western blot of BT474 cells treated with ADCs or
drug for 72 hours.
FIG. 6D-E show that Vedotin ADC demonstrate strong activation of multiple ICD
hallmarks.
Trastuzumab ADCs dosed at 1 pg/mL or free MMAE (100 nM) demonstrate different
ICD
responses in SKBR3 HER2 expressing breast cancer cells. After 48 or 72 hours
of treatment,
media was collected and used to measure ATP release (FIG. 6D) and HMGB1 levels
(FIG. 6E).
[00127] FIG. 7A further illustrates the immunogenic cell death (ICD) pathway.
[00128] FIG. 7B provides information regarding the payloads of certain ADCs
used in FIG.
7B-E.
[00129] FIG. 7C-F show JNK signaling activation generated in response to
treatment with
MMAE-ADCs compared to maytansine-ADCs (FIG. 7C), to camptothecin-ADCs (FIG.
7D), to
anthracycline-ADCs (FIG. 7E), and calicheamicin-ADCs (FIG. 7F).
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[00130] FIG. 8A-D show CHOP induction generated in response to treatment with
MMAE-
ADCs compared to maytansine-ADCs (FIG. 8A), to camptothecin-ADCs (FIG. 8B), to
anthracycline-ADCs (FIG. 8C), and to other types of ADCs, including ozogamycin-
ADCs,
teserine-ADCs, and AT-ADCs (FIG. 8D).
[00131] FIG. 9A-D show release of ATP and HMGB1 generated in response to
treatment
with MMAE-ADCs compared to maytansine-ADCs (FIG. 9A), to camptothecin-ADCs
(FIG.
9B), to anthracycline-ADCs (FIG. 9C), and to other types of ADCs, including
ozogamycin-
ADCs, teserine-ADCs, and AT-ADCs (FIG. 9D).
[00132] FIG. 10A-D show MHCII expression and CXCL-10/IP10 release generated in
response to treatment with MMAE-ADCs compared to maytansine-ADCs (FIG. 10A),
to
camptothecin-ADCs (FIG. 10B), to anthracycline-ADCs (FIG. 10C), and to other
types of
ADCs, including ozogamycin-ADCs and teserine-ADCs (FIG. 10D).
[00133] FIG. 10E summarizes the results in FIG. 7B-E, FIG. 8A-D, FIG. 9A-D,
and FIG.
10A-D.
[00134] FIG. 11A-D show antibody binding to FcyRIIa, FcyRIIb, and FcyRIIIa in
Chinese
hamster ovary (CHO cells). FIG. 11A shows antibodies assessed. FIG. 11B shows
binding to
FcyRIIa. FIG. 11C shows binding to FcyRIIIa. FIG. 11D shows binding to
FcyRIIb.
[00135] FIG. 12A-D show levels of CXCL10 (FIG. 12A), IFNy (FIG. 12B), IL10
(FIG.
12C), and macrophage derived cytokine (MDC; CCL22) (FIG. 12D) in MIA-PaCa 2
pancreatic
cancer cells treated with CD40 agonists and chemotherapy agents.
[00136] FIG. 13A-D show levels of CXCL10 (FIG. 13A-B) and IL10 (FIG. 13C-D) in
melanoma cell lines treated with CD40 agonists and chemotherapy.
[00137] FIG. 14A-C show levels of CXCL14 (FIG. 14A), IL14 (FIG. 14B), and IFNy
(FIG.
14C) in tumor cells from melanoma, lung, breast, and pancreas.
[00138] FIG. 15 shows in vivo data for SEA-CD40 Vedotin in combination with
ADC
chemotherapy combination as assessed by a human CD40 transgenic model where
the tumor
target antigen was Thy1.1.
[00139] FIG. 16 shows CXCL10 levels in tumor cell lines that were treated with
an ADC-
MMAE directed to a tumor-associated antigen in combination with TIGIT targeted
antibodies
with various effector function backbones.
[00140] FIG. 17 shows IFNy levels in tumor cell lines that were treated with
an ADC-
MMAE directed to a tumor-associated antigen in combination with TIGIT targeted
antibodies
with various effector function backbones.
[00141] FIG. 18A-C show in vitro and in vivo data demonstrating the enhanced
activity of a
nonfucosylated TIGIT antibody and a vc-MMAE ADC ("Vedotin ADC"). FIG. 18A
shows that
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a nonfucosylated TIGIT antibody having an enhanced (nonfucosylated) IgG1 Fc
backbone
(SEA-TGT) was significantly better at driving immune activation via cytokine
IP10 induction as
compared to either a corresponding TIGIT antibody with an effector null
backbone (LALA) or a
standard wildtype IgG1 Fc backbone when these antibodies were co-cultured with
tumor cells
killed by a targeting ye-WI:MAE ADC. The combination with SEA-TGT exhibited
synergistic
immune cell activation. FIG. 18B and FIG. 18C show the anti-tumor response
when mice
implanted with either CT26 syngeneic tumor cells (FIG. 18B) or Renca syngeneic
tumor cells
(FIG. 18C) were treated with: 1) a sub-optimal dose of an ADC (Thy1.1 vc-MMAE
ADC in
FIG. 18B and EphA2 ye-WI:MAE ADC in FIG. 18C); 2) a series of sub-optimal
doses of mIgG2a
SEA-TGT (the SEA-TGT antibody reformatted as a nonfucosylated mouse IgG2a that
corresponds to a nonfucosylated human IgG1 backbone); or 3) a combination of
both agents. As
can be seen from these figures, co-administration of these two agents
significantly increased
treatment efficacy, including generating a high percentage of curative
responses in these distinct
tumor models.
[00142] FIG. 19 shows in vivo data demonstrating the enhanced activity of a
nonfucosylated
TIGIT antibody (SEA-TGT) and SGN-B7H4 vedotin ADC (B7H4V). FIG. 19 shows the
anti-
tumor response when mice implanted with Renca syngeneic tumor cells were
treated with a
subtherapeutic dose of SEA-TGT and a subtherapeutic dose of B7H4V, or a
subtherapeutic dose
of SEA-TGT and a therapeutic dose of oxaliplatin. As shown in FIG. 19, co-
administration of
SEA-TGT and B7H4V significantly increased treatment efficacy, even at
subtherapeutic doses
of each, including generating a high percentage of curative responses.
[00143] FIG. 20A-B show combinatorial effects of SEA-CD70, a nonfucosylated
anti-CD70
antibody, and SGN-35, an anti-CD30 ADC containing MMAE. FIG. 20A shows in vivo
tumor
growth evaluation of a non-Hodgkin lymphoma (NHL) xenograft model. FIG. 20B
shows
Kaplan-Meyer survival evaluation of an NHL xenograft model at a 500 mm3 tumor
size
endpoint. Tx (treatment) and arrow indicate the treatment starting day (19
days post
implantation).
[00144] FIG. 21A-B show synergistic effects of SEA-BCMA, a nonfucosylated anti-
BCMA
antibody, and SGN-CD48A, an anti-CD/1.8 ADC containing IVIMAE. FIG. 21A shows
in vivo
survival evaluation of a xenograft model, and FIG. 21B shows in vivo hid
ferase evaluation of a
xenograft model.
[00145] FIG. 22A-C show vedotin ADC induces immune cell recruitment and
activation in
vivo. FIG. 22A: Tumor xenografts isolated from animals treated with a vc-MMAE
ADC or non-
binding vc-MMAE isotype ADC for 8 days, and subject to flow cytometry or
cytokine profiling.
FIG. 22B: CD45 positive immune cells were stained for CD11 c and activation
observed by
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staining for the expression of MHC-Class II on the cell surface. FIG. 22C:
Intratumoral
cytokines were measured by Luminex.
[00146] FIG. 23A-B show induction of T cell memory by vc-MMAE ADC. In a Renca
syngeneic model, mice were cured with a vc-MMAE ADC treatment (FIG. 23A). The
mice
cured with the treatment were rechallenged with Renca tumor cells, and those
mice rejected the
subsequently implanted tumor cells (FIG. 23B).
[00147] FIG. 24 shows protective anti-tumor immunity conferred by MMAE or vc-
MMAE
ADC-treated cells. A20 cancer cells were treated with brentuximab vedotin (BV)
or MMAE,
and the dying and dead cells were administered to mice. FIG. 24 shows that
immunized mice
displayed stronger immune responses rejecting subsequently implanted A20
cells.
[00148] FIG. 25 shows an exemplary model of receptor clustering and agonism by
a
nonfucosylated anti-CD40 antibody, SEA-CD40; and an exemplary model of
receptor agonist
and synapse formation by a nonfucosylated anti-TIGIT antibody, SEA-TGT. As
shown, the
SEA-CD40 antibody can bind CD-40 expressed on antigen presenting cells (APCs)
with the Fc
portion of the antibody binding to FcyRIIIa expressed on natural killer (NK)
cells or on
monocytes, which promotes receptor clustering. The SEA-TGT antibody, in
contrast, binds to
TIGIT expressed on T-cells and the Fc region of the antibody binds to FcyRIIIa
expressed on
APCs.
DETAILED DESCRIPTION
I. Introduction
[00149] Cell death through apoptosis is a silent, tolerogenic process.
However, certain
cytotoxic agents, including specific antitumor agents such as anthracyclines,
oxaliplatin, or
radiation, induce a characteristic form of cell death termed Immunogenic Cell
Death (ICD). ICD
is a mode of regulated cell death/ that generates immune responses of PBMCs
and T-cells
against the apoptotic cancerous cells. As demonstrated herein, treatment with
certain tubulin
disrupting agents such as auristatins (e.g., MMAE and MMAF) cause proteins
normally found
within the ER to become exposed on the cell surface. Increased phagocytic
uptake and
presentation of tumor antigens to T cells subsequently prime the adaptive
immune system. As
further shown herein, auristatins such as MMAE and MMAF are distinctly capable
of driving
ICD induction, thereby enabling the immune system to recognize and mount
cytotoxic activity
against tumors. In essence, cells dying from ICD serve as a vaccine to
stimulate tumor-specific
immune responses against any residual disease, or in the event of
relapse/recurrence.
[00150] As demonstrated by the experimental results described herein, tumor
cells
undergoing ICD in response to auristatins such as MMAE and MMAF display a
unique set of
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characteristics that potentiate 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 release of specific MAMPS and danger-associated
molecular
patterns (DAMPS) which have the unique capability to establish a pro-
inflammatory
environment that promotes T cell recognition of tumor antigens. As shown
herein, while other
chemotherapeutic agents can induce apoptosis, not all can induce as robust of
an ICD response
as auristatins such as MMAE and MMAF.
[00151] Key steps of ICD generate a series of signals that activate the innate
immune system
to recognize tumor cells and clear them. First, a drug induces ER stress. and
this in turn results
in surface exposure of DAMPs including calreticulin, heat shock proteins
(HSP70 and HSP90),
secretion of ATP, and release of high mobility group protein B1 (HMGB1).
Exposure of these
DAMPs and secretion of the immune modulatory agents during the process of ICD
can act in
concert to initiate immune responses including activation of dendritic cells
and other antigen
presenting cells, leading to phagocytosis and destruction of the ER-stressed
cell.
[00152] As noted above, initiation of ICD is linked to ER stress. Overloading
the ER's
capacity for unfolded polypeptides or disruption of the protein-folding
environment initiates ER
stress responses. The ER is intimately connected to the microtubule network
which provides
structure and elasticity through dynamic assembly and contraction. Disruption
of microtubule
network impinges on the ER network and results in severe ER stress, which
triggers expression
of the characteristics required for ICD induction and results in a stress
response, referred to as
the unfolded protein response (UPR). The UPR response has multiple arms which
include
increased pIRE1, downstream phosphorylation of JNK, and ATF4 cleavage.
[00153] The present invention is based in part on the finding that certain
tubulin disrupting
agents such as auristatins (e.g., MMAE and MMAF) are capable of generating an
unique ICD
response compared with other cytotoxic agents and, in particular, as compared
to other payloads
that are used on antibody-drug conjugates. The invention is further based on
the discovery that
pairing the unique ability of such agents to drive ICD with agents that
enhance an immune
response can amplify anti-tumor activity. This was particularly found to be
the case when such
immune agonism was achieved using antibodies having the ability to bind
certain targets
involved in immune signaling and having enhanced Fc binding characteristics
and effector
function. The desired Fc binding characteristics included activities such as
enhanced binding to
activating FcyRs, decreased binding to inhibitory FcyRs, enhanced ADCC
activity, and/or
enhanced ADCP activity. Certain such antibodies with the desired activities
were
nonfucosylated.
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[00154] Based upon these collective findings, the present inventors have
demonstrated as
described in greater detail herein that inducing ICD using particular tubulin
disrupters such as
MMAE and MMAF in combination with antibodies directed to immune cell engagers
that also
have enhanced Fc activity results in synergistically improved anti-tumor
responses. The use of
ADCs rather than standard chemotherapy agents was also shown to mitigate the
impairment of T
cell responses seen with traditional chemotherapy, thus providing a further
advantage to this
particular combination approach.
[00155] Accordingly, some embodiments provided herein are combination
therapies which
comprise administering to a subject with cancer: (1) an antibody-drug
conjugate comprising a
tubulin disrupter conjugated to a first antibody that binds a tumor-associated
antigen; and (2)
antibody that binds to an immune cell engager, wherein the second antibody
comprises an Fc
with enhanced binding to one or more activating FcyRs. In some embodiments,
the second
antibody is nonfucosylated. In certain embodiments, the second antibody has
enhanced ADCC
and/or ADCP activity.
DEFINITIONS
[00156] Unless defined otherwise, technical and scientific terms used herein
have the same
meaning as commonly understood by a person of ordinary skill in the art. See,
e.g., Lackie,
DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et
at.,
MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold
Springs
Harbor, NY 1989). Any methods, devices and materials similar or equivalent to
those described
herein can be used in the practice of this invention.
[00157] As used herein, the singular forms "a", "an" and "the" include plural
referents unless
the content clearly dictates otherwise. Thus, for example, reference to "an
antibody" optionally
includes a combination of two or more such molecules, and the like.
[00158] The term "about," as used herein, refers to the usual error range for
the respective
value readily known to the skilled person in this technical field.
[00159] The term "antibody" includes intact antibodies and antigen-binding
fragments
thereof, wherein the antigen-binding fragments comprise the antigen-binding
region and at least
a portion of the heavy chain constant region comprising asparagine (N) 297,
located in CH2.
Typically, the "variable region" contains the antigen-binding region of the
antibody and is
involved in specificity and affinity of binding. See, Fundamental Immunology
7th Edition, Paul,
ed., Wolters Kluwer Health/Lippincott Williams & Wilkins (2013). Light chains
are typically
classified as either kappa or lambda. Heavy chains are typically classified as
gamma, mu, alpha,
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delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM,
IgA, IgD and IgE,
respectively.
[00160] The term "antibody" also includes bivalent or bispecific molecules,
diabodies,
triabodies, and tetrabodies. Bivalent and bispecific molecules are described
in, e.g., Kostelny et
at. (1992)1 Immunol. 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579,
Hollinger et
at. (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.
[00161] The term "antibody" includes an antibody by itself (naked antibody) or
an antibody
conjugated to a cytotoxic or cytostatic drug.
[00162] A "monoclonal antibody" refers to an antibody obtained from a
population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population
are identical except for possible naturally occurring mutations that may be
present in minor
amounts. The modifier "monoclonal" indicates the character of the antibody as
being obtained
from a substantially homogeneous population of antibodies, and is not to be
construed as
requiring production of the antibody by any particular method. For example,
the monoclonal
antibodies to be used in accordance with the present invention may be made by
the hybridoma
method first described by Kohler et al. (1975) Nature 256:495, or may be made
by recombinant
DNA methods (see, for example, U.S. Patent No. 4816567). The "monoclonal
antibodies" may
also be isolated from phage antibody libraries using the techniques described
in Clackson et al.
(1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597,
for example or
may be made by other methods. The antibodies described herein are monoclonal
antibodies.
[00163] Specific binding of a monoclonal antibody to its target antigen
means an affinity of at
least 106, 107, 108, 109, or 1010 M1. Specific binding is detectably higher in
magnitude and
distinguishable from non-specific binding occurring to at least one unrelated
target. Specific
binding can be the result of formation of bonds between particular functional
groups or
particular spatial fit (e.g., lock and key type) whereas nonspecific binding
is usually the result of
van der Waals forces.
[00164] The basic antibody structural unit is a tetramer of subunits. Each
tetramer includes
two identical pairs of polypeptide chains, each pair having one "light" (about
25 kDa) and one
"heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable
region of about 100 to 110 or more amino acids primarily responsible for
antigen recognition.
This variable region is initially expressed linked to a cleavable signal
peptide. The variable
region without the signal peptide is sometimes referred to as a mature
variable region. Thus, for
example, a light chain mature variable region, means a light chain variable
region without the
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light chain signal peptide. The carboxy-terminal portion of each chain defines
a constant region
primarily responsible for effector function.
[00165] Light chains are classified as either kappa or lambda. Heavy chains
are classified as
gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG,
IgM, IgA, IgD
and IgE, respectively. Within light and heavy chains, the variable and
constant regions are
joined by a "J" region of about 12 or more amino acids, with the heavy chain
also including a
"D" region of about 10 or more amino acids. (See generally, Fundamental
Immunology (Paul,
W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7, incorporated by reference in
its entirety for all
purposes).
[00166] The mature variable regions of each light/heavy chain pair form the
antibody binding
site. Thus, an intact antibody has two binding sites. Except in bifunctional
or bispecific
antibodies, the two binding sites are the same. The chains all exhibit the
same general structure
of relatively conserved framework regions (FR) joined by three hypervariable
regions, also
called complementarity determining regions or CDRs. The CDRs from the two
chains of each
pair are aligned by the framework regions, enabling binding to a specific
epitope. From N-
terminal to C-terminal, both light and heavy chains comprise the domains FR1,
CDR1, FR2,
CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in
accordance
with 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 a composite of Kabat
and Chothia, or
IMGT (ImMunoGeneTics information system), AbM or Contact or other conventional
definition
of CDRs. Kabat also provides a widely used numbering convention (Kabat
numbering) in
which corresponding residues between different heavy chains or between
different light chains
are assigned the same number. Unless otherwise apparent from the context,
Kabat numbering is
used to designate the position of amino acids in the variable regions. Unless
otherwise apparent
from the context EU numbering is used to designated positions in constant
regions.
[00167] A "humanized" antibody is an antibody that retains the reactivity of a
non-human
antibody while being less immunogenic in humans. This can be achieved, for
instance, by
retaining the non-human CDR regions and replacing the remaining parts of the
antibody with
their human counterparts. See, e.g., Morrison et al., PNAS USA, 81:6851-6855
(1984); Morrison
and 0i, 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).
[00168] As used herein, the term "chimeric antibody" refers to an antibody
molecule in which
(a) the constant region, or a portion thereof, is replaced so that the antigen
binding site (variable
region, CDR, or portion thereof) is linked to a constant region of a different
species.
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[00169] The term "epitope" refers to a site on an antigen to which an antibody
binds. An
epitope can be formed from contiguous amino acids or noncontiguous amino acids
juxtaposed
by tertiary folding of one or more proteins. Epitopes formed from contiguous
amino acids are
typically retained on exposure to denaturing solvents whereas epitopes formed
by tertiary
folding are typically lost on treatment with denaturing solvents. An epitope
typically includes at
least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial
conformation.
Methods of determining spatial conformation of epitopes include, for example,
x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,
Epitope Mapping
Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed.
(1996).
[00170] Antibodies that recognize the same or overlapping epitopes can be
identified in a
simple immunoassay showing the ability of one antibody to compete with the
binding of another
antibody to a target antigen. The epitope of an antibody can also be defined
by X-ray
crystallography of the antibody bound to 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 binding of one antibody reduce or eliminate binding of the other.
Two antibodies have
overlapping epitopes if some amino acid mutations that reduce or eliminate
binding of one
antibody reduce or eliminate binding of the other.
[00171] Competition between antibodies is determined by an assay in which an
antibody
under test inhibits specific binding of a reference antibody to a common
antigen (see, e.g.,
Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a
reference
antibody if an excess of a test antibody (e.g., at least 2x, 5x, 10x, 20x or
100x) inhibits binding
of the reference antibody by at least 50% but preferably 75%, 90% or 99% as
measured in a
competitive binding assay. Antibodies identified by competition assay
(competing antibodies)
include antibodies binding to the same epitope as the reference antibody and
antibodies binding
to an adjacent epitope sufficiently proximal to the epitope bound by the
reference antibody for
steric hindrance to occur.
[00172] The phrase "specifically binds" refers to a molecule (e.g.,
antibody or antibody
fragment) that binds to a target with greater affinity, avidity, more readily,
and/or with greater
duration to that target in a sample than it binds to a non-target compound. In
some embodiments,
an antibody that specifically binds a target is an antibody that binds to the
target with at least 2-
fold greater affinity than non-target compounds, such as, for example, at
least 4-fold, 5-fold, 6-
fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold
greater affinity. For
example, an antibody that specifically binds TIGIT will typically bind to
TIGIT with at least a 2-
fold greater affinity than to a non-TIGIT target. It will be understood by a
person of ordinary
skill in the art reading this definition, for example, that an antibody (or
moiety or epitope) that
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specifically or preferentially binds to a first target may or may not
specifically or preferentially
bind to a second target. As such, "specific binding" does not necessarily
require (although it can
include) exclusive binding.
[00173] The term "binding affinity" is herein used as a measure of the
strength of a non-
covalent interaction between two molecules, e.g., an antibody, or fragment
thereof, and an
antigen. The term "binding affinity" is used to describe monovalent
interactions (intrinsic
activity).
[00174] Binding affinity between two molecules, e.g. an antibody, or fragment
thereof, and
an antigen, through a monovalent interaction may be quantified by
determination of the
dissociation constant (K6). In turn, KD can be determined by measurement of
the kinetics of
complex formation and dissociation using, as a nonlimiting example, the
surface plasmon
resonance (SPR) method (BiacoreTm). The rate constants corresponding to the
association and
the dissociation of a monovalent complex are referred to as the association
rate constants ka (or
Icon) and dissociation rate constant ka (or /coif), respectively. KD is
related to ka and ka through the
equation KD = ka I ka. The value of the dissociation constant can be
determined directly by well-
known methods, and can be computed even for complex mixtures by methods such
as those, for
example, set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the
KD may be
established using a double-filter nitrocellulose filter binding assay such as
that disclosed by
Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard
assays to
evaluate the binding ability of ligands such as antibodies towards target
antigens are known in
the art, including for example, ELISAs, Western blots, RIAs, and flow
cytometry analysis, and
other assays exemplified elsewhere herein. The binding kinetics and binding
affinity of the
antibody also can be assessed by standard assays known in the art or as
described in the
Examples section below, such as Surface Plasmon Resonance (SPR), e.g. by using
a BiacoreTM
system; kinetic exclusion assays such as KinExAg; and BioLayer interferometry
(e.g., using the
ForteBio Octet platform). In some embodiments, binding affinity is determined
using a
BioLayer interferometry assay. See, e.g., Wilson et al., Biochemistry and
Molecular Biology
Education, 38:400-407 (2010); Dysinger et al., I Immunol. Methods, 379:30-41
(2012); and
Estep et al., Mabs, 2013, 5:270-278.
[00175] The term "cross-reacts," as used herein, refers to the ability of
an antibody to bind to
an antigen other than the antigen against which the antibody was raised. In
some embodiments,
cross-reactivity refers to the ability of an antibody to bind to an antigen
from another species
than the antigen against which the antibody was raised. As a non-limiting
example, an anti-
TIGIT antibody as described herein that is raised against a human TIGIT
antigen can exhibit
cross-reactivity with TIGIT from a different species (e.g., mouse or monkey).
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[00176] An "isolated" antibody refers to an antibody that has been
identified and
separated and/or recovered from components of its natural environment and/or
an antibody that
is recombinantly produced. A "purified antibody" is an antibody that is
typically at least 50%
w/w pure of interfering proteins and other contaminants arising from its
production or
purification but does not exclude the possibility that the monoclonal antibody
is combined with
an excess of pharmaceutical acceptable carrier(s) or other vehicle intended to
facilitate its use.
Interfering proteins and other contaminants can include, for example, cellular
components of the
cells from which an antibody is isolated or recombinantly produced. Sometimes
monoclonal
antibodies are at least 60%, 70%, 80%, 90%, 95 or 99% w/w pure of interfering
proteins and
contaminants from production or purification. The antibodies described herein,
including rat,
chimeric, veneered and humanized antibodies can be provided in isolated and/or
purified form.
[00177] The term "LAB" 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.
[00178] "Subject," "patient," "individual" and like terms are used
interchangeably and refer
to, except where indicated, mammals such as humans and non-human primates, as
well as
rabbits, rats, mice, goats, pigs, and other mammalian species. The term does
not necessarily
indicate that the subject has been diagnosed with a particular disease, but
typically refers to an
individual under medical supervision.
[00179] The terms "therapy," "treatment," and "amelioration" refer to any
reduction in the
severity of symptoms. In the case of treating cancer, treatment can refer to
reducing, e.g., tumor
size, number of cancer cells, growth rate, metastatic activity, cell death of
non-cancer cells, etc.
As used herein, the terms "treat" and "prevent" are not intended to be
absolute terms. Treatment
and prevention can refer to any delay in onset, amelioration of symptoms,
improvement in
patient survival, increase in survival time or rate, etc. Treatment and
prevention can be complete
(no detectable symptoms remaining) or partial, such that symptoms are less
frequent or severe
than in a patient without the treatment described herein. The effect of
treatment can be
compared to an individual or pool of individuals not receiving the treatment,
or to the same
patient prior to treatment or at a different time during treatment. In some
aspects, the severity of
disease is reduced by at least 10%, as compared, e.g., to the individual
before administration or
to a control individual not undergoing treatment. In some aspects, the
severity of disease is
reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer
detectable using
standard diagnostic techniques.
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[00180] 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,
abates, ameliorates, or reduces the severity of symptoms of a disease (e.g., a
cancer) in a subject.
[00181] The terms "administer," "administered," or "administering" refer to
methods of
delivering agents, compounds, or compositions to the 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 are optionally
employed with the agents
and methods described herein, include e.g., as discussed in Goodman and
Gilman, The
Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's,
Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, PA.
III. Exemplary Antibodies that Bind an Immune Cell Engager
[00182] As noted above, the present inventors have found that inducing ICD by
administering
an antibody-drug conjugate comprising certain tubulin disrupters (e.g.,
auristatins, including for
instance MMAE and MMAF) in combination with triggering an immune response
using an
antibody that binds a protein directly or indirectly involved in immune
regulation and that has
enhanced Fc activity can result in improved anti-tumor responses, including
synergistic
responses. As such, in some embodiments, the methods provided herein comprise
administering
to a subject with cancer an antibody that binds a target involved in
regulating an immune
response, wherein such binding induces, promotes, or enhances an immune
response. The target
of such an antibody can be referred to as an "immune cell engager." An "immune
cell engager"
as used herein refers to a molecule (e.g., a transmembrane protein) that is
involved in
modulating an immune cell response, either positively or negatively. In some
embodiments, the
antibody binds to a receptor on immune cells or tumors and results in direct
immune cell
engagement or releases a negative inhibitory signal. In some embodiments, the
immune cell
engager is a molecule involved in T-cell signaling. In some embodiments, the
immune cell
engager modulates (e.g. activates) antigen-present cells (APCs). The immune
cell engager in
certain embodiments 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
immune cells or tumors. Any of the antibodies that bind an immune cell engager
described
herein may be combined with any of the antibody-drug conjugates described
herein.
[00183] The antibody that binds the target involved in immune regulation
(e.g, the immune
cell engager) also comprises an Fc that has one or more or all of the
following features in any
combination: 1) enhanced binding to one or more activating FcyRs, 2) reduced
binding to
28
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inhibitory FcyRs, 3) is nonfucosylated, 4) has enhanced ADCC activity, 5) has
enhanced ADCP
activity, 6) activates antigen presenting cells (APCs), 7) enhances CD8 T cell
responses, 8)
upregulates co-stimulatory receptors, 9) activates an innate cell immune
response, and/or 10)
engages NK cells. .
[00184] Thus, in some embodiments, the antibody comprises an Fc with enhanced
binding to
one or more activating FcyRs and/or reduced binding to one or more inhibitory
FcyRs to obtain
the desired enhanced FcyR binding profile. Activating FcyRs include one or
more of FcyRIIIa,
FcyRIIa, and/or FcyRI. Inhibitory FcyRs include, for example, FcyRIIb.
[00185] In certain embodiments, the antibody comprises an Fc with enhanced
binding to at
least FcyRIIIa. In other embodiments, the antibody comprises an Fc with
enhanced binding to at
least FcyRIIIa and FcyRIIa. In some embodiments, the antibody comprises an Fc
with enhanced
binding to at least FcyRIIIa and FcyRI. In certain embodiments, the antibody
comprises an Fc
with enhanced binding to FcyRIIIa, FcyRIIa, and FcyRI.
[00186] In some embodiments, the antibody, in addition to or separately from
enhanced
binding to an activating FcyR, has reduced binding to one or more inhibitory
FcyRs. Thus, in
some embodiments, the antibody has reduced binding to FcyRIIa and/or FcyRIIb.
[00187] In some embodiments, the antibody is nonfucosylated. In some
embodiments, the
antibody further has one of the FcyR binding profiles described above.
[00188] In certain embodiments, the Fc of the antibody comprises amino acid
changes
relative to a wild-type Fc to enhance binding to an activating FcyR, and/or
reduce binding to one
or more inhibitory FcyRs to obtain an FcyR binding profile such as described
above. For
example, in some embodiments the Fc of the antibody comprises the
substitutions S293D,
A330L, and I332E in the heavy chain constant region.
[00189] Additional details on methods for obtaining antibodies with the
desired FcyR profile
are provided below, as are methods for obtaining nonfucosylated antibodies.
A. Exemplary Immune Cell Engagers
[00190] Nonlimiting exemplary targets or immune cell engagers to which an
antibody can be
targeted include: Mullerian Hormone Receptor II (AMHR2), B7, B7H1, B7H2, B7H3,
B7H4,
BAFF-R, BCMA (B-cell maturation antigen), Bstl/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 sites, CD40, CD70, CD70 (a
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, IDO
1,
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IFNAR1, IFNAR2, IGF-1R, IL-3Ralpha (CD123), IL-5R, IL-5Ralpha, LAG-3, MET
proto-
oncogene , 0X40 (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 lectins 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 Mucl (TA-Mud), VSIR (VISTA), and
VTCN1.
[00191] In some embodiments, an antibody is an agonist of an immune cell
engager. In some
such embodiments, an antibody is an agonist of an immune cell engager selected
from CD80,
CD86, 0X40 (CD134), GITR, CD137, CD40, VTCN1, CD276, IFNAR2, IFNARE CSF1R,
VSIR (VISTA), and HLA.
[00192] In some embodiments, an antibody is an antagonist of an immune cell
engager. In
some such embodiments, an antibody is an antagonist of an immune cell engager
selected from
CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7, TIM3 (HAVCR2), PVRIG, TIGIT, CD25
(IL2RA),
and ID01.
[00193] In certain embodiments, the antibody that binds an immune cell engager
for the
methods provided herein can be an inhibitor against a checkpoint protein. In
some
embodiments, the antibody that binds an immune cell engager for the methods
provided herein
can be a PD-1 inhibitor, a PD-Li inhibitor, a PD-L2 inhibitor, a CTLA-4
inhibitor, a LAG-3
inhibitor, a B7 inhibitor, a TIM3 (HAVCR2) inhibitor, an 0X40 (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 of the embodiments herein, the antibody that binds an immune
cell engager may
be an antibody comprising an Fc with enhanced binding to one or more
activating FcyRs. In
some embodiments, the antibody that binds an immune cell engager is a
nonfucosylated
antibody.
[00194] In some embodiments, the antibody that binds an immune cell engager 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 US
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 herein in their entireties. In one embodiment,
the anti-CTLA-4
antibody is tremelimumab (also known as ticilimumab or CP-675,206) or a
nonfucosylated
version thereof In another embodiment, the anti-CTLA-4 antibody is ipilimumab
(also known
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as MDX-010 or MDX-101) or a nonfucosylated version thereof. Ipilimumab is a
fully human
monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the
trade name
YervoyTM.
[00195] In certain embodiments, the antibody that binds an immune cell engager
is a PD-
1/PD-L1 inhibitor. Examples of PD-1/PD-L1 inhibitors include, but are not
limited to, those
described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757;
8,217,149, and PCT
Patent Application Publication Nos. W02003042402, W02008156712, W02010089411,
W02010036959, W02011066342, W02011159877, W02011082400, and W02011161699, all
of which are incorporated herein in their entireties.
[00196] In some embodiments, the antibody that binds an immune cell engager 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), pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab),
or a
nonfucosylated version thereof In one embodiment, the anti-PD-1 antibody is
nivolumab or a
nonfucosylated version thereof. Nivolumab is a human IgG4 anti-PD-1 monoclonal
antibody,
and is marketed under the trade name OpdivoTM. In another embodiment, the anti-
PD-1
antibody is pembrolizumab or a nonfucosylated version thereof. Pembrolizumab
is a humanized
monoclonal IgG4 antibody and is marketed under the trade name KeytrudaTM. In
yet another
embodiment, the anti-PD-1 antibody is CT-011, a humanized antibody, or a
nonfucosylated
version thereof CT-011 administered alone has failed to show response in
treating acute
myeloid leukemia (AML) at relapse. In yet another embodiment, the anti-PD-1
antibody is
AMP-224, a fusion protein, or a nonfucosylated version thereof In another
embodiment, the
PD-1 antibody is BGB-A317, or a nonfucosylated version thereof. BGB-A317 is a
monoclonal
antibody in which the ability to bind Fc gamma receptor I is specifically
engineered out, and
which has a unique binding signature to PD-1 with high affinity and superior
target specificity.
In one embodiment, the PD-1 antibody is cemiplimab or a nonfucosylated version
thereof. In
another embodiment, the PD-1 antibody is camrelizumab or a nonfucosylated
version thereof
In a further embodiment, the PD-1 antibody is sintilimab or a nonfucosylated
version thereof. In
some embodiments, the PD-1 antibody is tislelizumab or a nonfucosylated
version thereof. In
certain embodiments, the PD-1 antibody is TSR-042 or a nonfucosylated version
thereof. In yet
another embodiment, the PD-1 antibody is PDR001 or a nonfucosylated version
thereof In yet
another embodiment, the PD-1 antibody is toripalimab or a nonfucosylated
version thereof
[00197] In certain embodiments, the antibody that binds an immune cell engager
is a PD-Li
inhibitor. In one embodiment, the PD-Li inhibitor is an anti-PD-Li antibody.
In one
embodiment, the anti-PD-Li antibody is 1V1EDI4736 (durvalumab) or a
nonfucosylated version
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thereof. In another embodiment, the anti-PD-Li antibody is BMS-936559 (also
known as
MDX-1105-01) or a nonfucosylated version thereof In yet another embodiment,
the PD-Li
inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriqg) or a
nonfucosylated
version thereof. In a further embodiment, the PD-Li inhibitor is avelumab or a
nonfucosylated
version thereof
[00198] In one embodiment, the antibody that binds an immune cell engager 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 rHIgMl2B7A or a nonfucosylated version
thereof.
[00199] In one embodiment, the antibody that binds an 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 at., I Immunol., 2007, 179, 4202-4211),
or a
nonfucosylated version thereof. In another embodiment, the LAG-3 inhibitor is
BMS-986016 or
a nonfucosylated version thereof.
[00200] In one embodiment, the antibody that binds an immune cell engager 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 at.,
Cl/n. Cancer
Res., 2012, 3834), or a nonfucosylated version thereof. In some embodiments,
the B7 inhibitor
is a B7-H4 inhibitor. A nonlimiting exemplary B7-H4 inhibitor is FPA150, a
nonfucosylated
antibody against B7-H4. See PCT/US2018/047805.
[00201] In one embodiment, the antibody that binds an immune cell engager is a
TIM3 (T-
cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et at., I
Exp. Med.,
2010, 207, 2175-86; Sakuishi et al.,1 Exp. Med., 2010, 207, 2187-94).
[00202] In one embodiment, the antibody that binds an immune cell engager is
an 0X40
(CD134) agonist antibody. In certain embodiments, the anti-0X40 antibody is
MEDI6469 or a
nonfucosylated version thereof.
[00203] In one embodiment, the antibody that binds an immune cell engager is a
GITR
agonist. In one embodiment, the immune cell engager is an anti-GITR antibody
or a
nonfucosylated version thereof In one embodiment, the anti-GITR antibody is
TRX518 or a
nonfucosylated version thereof.
[00204] In one embodiment, the antibody that binds an immune cell engager is a
CD137
agonist. In one embodiment, the immune cell engager is an anti-CD i37
antibody. In one
embodiment, the anti-CD137 antibody is urelumab or a nonfucosylated version
thereof. In
another embodiment, the anti-CD i37 antibody is PF-05082566 or a
nonfucosylated version
thereof.
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[00205] In one embodiment, the antibody that binds an immune cell engager is a
CD40
agonist. In one embodiment, the antibody that binds an immune cell engager is
an anti-CD40
antibody. In one embodiment, the anti-CD40 antibody is CF-870,893 or a
nonfucosylated
version thereof In one embodiment, the anti-CD40 antibody is MP0317 (Molecular
Partners) or
a nonfucosylated version thereof. In one embodiment, the anti-CD40 antibody is
YH003
(Eucure Biopharma) or a nonfucosylated version thereof. In one embodiment, the
anti-CD40
antibody is CDX-1140 (Celldex Therapeutics) or a nonfucosylated version
thereof. In one
embodiment, the anti-CD40 antibody is YH003 (Eucure Biopharma) or a
nonfucosylated
version thereof In one embodiment, the anti-CD40 antibody is mitazalimab
(Alligator
Bioscience) or a nonfucosylated version thereof. In one embodiment, the anti-
CD40 antibody is
ABBV-927 (AbbVie) or a nonfucosylated version thereof In one embodiment, the
anti-CD40
antibody is sotigalimab (Apexigen) or a nonfucosylated version thereof In one
embodiment, the
anti-CD40 antibody is GEN1042 (Genmab) or a nonfucosylated version thereof In
one
embodiment, the anti-CD40 antibody is 2141 V-11 (Rockefeller University) or a
nonfucosylated
version thereof In one embodiment, the anti-CD40 antibody is selicrelumab
(Roche) or a
nonfucosylated version thereof In one embodiment, the anti-CD40 antibody is
SEA-CD40
(Seagen), which is a nonfucosylated, humanized version of murine S2C6 and
which comprises
heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3
comprising the
amino acid sequences of SEQ ID NOs: 30-35, respectively. The corresponding VH
and VL
comprise the amino acid sequences of SEQ ID NOs: 28 and 29, respectively. SEA-
CD40 is
described in US Patent Publication Nos. 2017/0333556 and 2017/0137528, both of
which are
herein incorporated by reference.
[00206] 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., US
Patent No.
8,067,546; Table of Sequences herein.
[00207] In some embodiments, the antibody that binds an immune cell engager is
an antibody
that binds BCMA. In some embodiments, the antibody is SEA-BCMA. See, e.g., US
Publication No. 2017/0233484 and WO 2017/143069 (VH and VL of SEQ ID NOs: 13
and 19,
respectively; CDRs of SEQ ID NOs: 60, 61, 62, 90, 91, 92, see US Publication
No.
2017/0233484); see also Table of Sequences herein (VH and VL of SEQ ID NOs: 45
and 46,
respectively; CDRs of SEQ ID NOs: 47-52).
[00208] In one embodiment, the antibody that binds an immune cell engager is
an anti-
interleukin-15 antibody.
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[00209] In one embodiment, the antibody that binds an immune cell engager is a
VTCN
inhibitor. In one embodiment, the VTCN inhibitor is FPA150 or a nonfucosylated
version
thereof.
[00210] In one embodiment, the antibody that binds an immune cell engager is
an anti-DO
antagonist antibody. In some embodiments, the antibody that binds an immune
cell engager 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 nonfucosylated version
thereof. In
another embodiment, the TIGIT inhibitor is BMS-986207 (BMS) or a
nonfucosylated version
thereof. In yet another embodiment, the TIGIT inhibitor is OMP-313M32 or a
nonfucosylated
version thereof In one embodiment, the TIGIT inhibitor is MK-7684. In another
embodiment,
the TIGIT inhibitor is AB154 or a nonfucosylated version thereof In yet
another embodiment,
the TIGIT inhibitor is CGEN-15137 or a nonfucosylated version thereof. In one
embodiment,
the TIGIT inhibitor is SEA-TGT. In another embodiment, the TIGIT inhibitor is
ASP8374
(Astellas) or a nonfucosylated version thereof. In yet another embodiment, the
TIGIT inhibitor
is AJUD008 or a nonfucosylated version thereof In one embodiment, the TIGIT
inhibitor is
AB308 (Arcus Biosciences) or a nonfucosylated version thereof In another
embodiment, the
TIGIT inhibitor is AGEN1327 (Agenus) or a nonfucosylated version thereof. In
yet another
embodiment, the TIGIT inhibitor is AK127 (Akeso Biopharma) or a nonfucosylated
version
thereof. In another embodiment, the TIGIT inhibitor is BAT6005 (Bio-Thera
Solutions) or a
nonfucosylated version thereof. In another embodiment, the TIGIT inhibitor is
BAT6021 (Bio-
Thera Solutions) or a nonfucosylated version thereof In one embodiment, the
TIGIT inhibitor
is CASC-674 (Seagen) or a nonfucosylated version thereof. In another
embodiment, the TIGIT
inhibitor is C0M902 (Compugen) or a nonfucosylated version thereof In yet
another
embodiment, the TIGIT inhibitor is domvanalimab (Arcus Biosciences) or a
nonfucosylated
version thereof In one embodiment, the TIGIT inhibitor is etigilimab (Mereo
BioPharma) or a
nonfucosylated version thereof In another embodiment, the TIGIT inhibitor is
G5K4428859
(GSK) or a nonfucosylated version thereof In yet another embodiment, the TIGIT
inhibitor is
HL186 (HanAll Biopharma) or a nonfucosylated version thereof. In one
embodiment, the
TIGIT inhibitor is MIL-100 (Beijing Mabworks Biotech) or a nonfucosylated
version thereof.
In another embodiment, the TIGIT inhibitor is YH-29143 (Yu Han) or a
nonfucosylated version
thereof. In one embodiment, the TIGIT inhibitor is HLX53 (Shanghai Henlius
Biotech) or a
nonfucosylated version thereof. In another embodiment, the TIGIT inhibitor is
IBI939
(Innovent Biologics) or a nonfucosylated version thereof. In yet another
embodiment, the
TIGIT inhibitor is J5006 (Junshi Biosciences) or a nonfucosylated version
thereof In one
embodiment, the TIGIT inhibitor is M6223 (Merck KGaA) or a nonfucosylated
version thereof.
34
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In another embodiment, the TIGIT inhibitor is MG1131 (Mogam Institute) or a
nonfucosylated
version thereof In yet another embodiment, the TIGIT inhibitor is ociperlimab
(BeiGene) or a
nonfucosylated version thereof In one embodiment, the TIGIT inhibitor is
tiragolumab (Roche;
described in US Patent No. 10,047,158) or a nonfucosylated version thereof. In
another
embodiment, the TIGIT inhibitor is TJT6 (I-Mab Biopharma) or a nonfucosylated
version
thereof. In yet another embodiment, the TIGIT inhibitor is vibostolimab (MSD;
described in US
Patent No. 10,618,958) or a nonfucosylated version thereof. In one embodiment,
the TIGIT
inhibitor is YBL-012 (Y Biologics) or a nonfucosylated version thereof. In
another
embodiment, the TIGIT inhibitor is IBI-939 (Innovent) or a nonfucosylated
version thereof. In
yet another embodiment, the TIGIT inhibitor is AZD2936 (AstraZeneca) or a
nonfucosylated
version thereof In one embodiment, the TIGIT inhibitor is EOS-448 (iTeos/GSK)
or a
nonfucosylated version thereof. In another embodiment, the TIGIT inhibitor is
BAT6005 (Bio
Thera) or a nonfucosylated version thereof. In yet another embodiment, the
TIGIT inhibitor is
AGEN1777 (BMS/Agenus) or a nonfucosylated version thereof
[00211] In some embodiments, the antibody that binds an immune cell engager 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 nonfucosylated version
thereof In another
embodiment, the VSIR inhibitor is CA-170 or a nonfucosylated version thereof
In yet another
embodiment, the VSIR inhibitor is JNJ 61610588 or a nonfucosylated version
thereof. In one
embodiment, the VSIR inhibitor is HMBD-002 or a nonfucosylated version
thereof.
[00212] In some embodiments, the antibody that binds an immune cell engager 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 nonfucosylated version thereof.
[00213] In some embodiments, the antibody that binds an 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
nonfucosylated version thereof In another embodiment, the CD25 (IL2RA)
inhibitor is
basiliximab or a nonfucosylated version thereof.
[00214] In some embodiments, the antibody that binds an immune cell engager 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 nonfucosylated version
thereof. In
another embodiment, the IFNAR1 inhibitor is sifalimumab or a nonfucosylated
version thereof.
[00215] In some embodiments, the antibody that binds an immune cell engager 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 nonfucosylated version
thereof In
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another embodiment, the CSF1R inhibitor is emactuzumab or a nonfucosylated
version thereof.
In yet another embodiment, the CSF1R inhibitor is cabiralizumab or a
nonfucosylated version
thereof. In one embodiment, the CSF1R inhibitor is ARRY-382 or a
nonfucosylated version
thereof. In another embodiment, the CSF1R inhibitor is BLZ945 or a
nonfucosylated version
thereof. In yet another embodiment, the CSF1R inhibitor is AJUD010 or a
nonfucosylated
version thereof. In one embodiment, the CSF1R inhibitor is AMG820 or a
nonfucosylated
version thereof In another embodiment, the CSF1R inhibitor is IMC-CS4 or a
nonfucosylated
version thereof. In yet another embodiment, the CSF1R inhibitor is JNJ-
40346527 or a
nonfucosylated version thereof In one embodiment, the CSF1R inhibitor is
PLX5622 or a
nonfucosylated version thereof. In another embodiment, the CSF1R inhibitor is
FPA008 or a
nonfucosylated version thereof.
[00216] In various embodiments, an antibody that binds an immune cell engager
has one or
more or all of the following activities in any combination: 1) depletes T
regulatory (Treg) cells,
2) activates antigen presenting cells (APCs), 3) enhances CD8 T cell
responses, 4) upregulates
co-stimulatory receptors, and/or 5) promotes release of immune activating
cytokines (such as
CXCL10 and/or IFNy). In some embodiments, the antibody that binds an immune
cell engager
promotes release of immune-activating cytokines (e.g., CXCL10 and IFNy) to a
greater extent
than immune suppressive cytokines (such as IL10 and/or MDC).
B. Exemplary Anti-TIGIT Antibodies
[00217] In one aspect, antibodies that bind to human TIGIT (T-cell
immunoreceptor with Ig
and ITIM domains) are provided as the antibodies against the immune cell
engager. As
described herein, in some embodiments, the anti-TIGIT antibody inhibits
interaction between
TIGIT and one or both of the ligands CD155 and CD112. In some embodiments, the
anti-TIGIT
antibody inhibits the interaction between TIGIT and CD155 in a functional
bioassay, allowing
CD155-CD226 signaling to occur. In some embodiments, the anti-TIGIT antibody
exhibits
synergy with an anti-PD-1 agent (e.g., an anti-PD-1 antibody) or an anti-PD-Li
agent (e.g., an
anti-PD-Li antibody). In some embodiments, an anti-TIGIT antibody for use in
the present
methods is SEA-TGT, which is a nonfucosylated IgG1 antibody comprising heavy
chain CDR1,
CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 comprising the amino acid
sequences of SEQ ID NOs: 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.
[00218] The present inventors found that, surprisingly, anti-TIGIT antibodies
with enhanced
effector function, such as may be achieved with nonfucosylated IgG1
antibodies, deplete Treg
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cells and show improved efficacy in vivo. Accordingly, in various embodiments,
nonfucosylated anti-TIGIT antibodies are provided.
[00219] In some embodiments, an anti-TIGIT antibody, such as a nonfucosylated
anti-TIGIT
antibody, binds to 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 5 nM, less than 1 nM, less than 500 pM, less than 250 pM, less than 150
pM, less than 100
pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, or
less than about 10
pM. In some embodiments, the antibody has a binding affinity (KD) for human
TIGIT of less
than 50 pM. In some embodiments, the antibody has a KD for human TIGIT in the
range of
about 1 pM to about 5 nM, e.g., about 1 pM to about 1 nM, about 1 pM to about
500 pM, about
pM to about 250 pM, or about 10 pM to about 100 pM.
[00220] In some embodiments, in addition to binding to human TIGIT with high
affinity, a
nonfucosylated anti-TIGIT antibody exhibits cross-reactivity with cynomolgus
monkey ("cyno")
TIGIT and/or mouse TIGIT . In some embodiments, the anti-TIGIT antibody binds
to mouse
TIGIT with a binding affinity (KD) of 100 nM or less. In some embodiments, the
anti-TIGIT
antibody binds to human TIGIT with a KD of 5 nM or less, and cross-reacts with
mouse TIGIT
with a KD of 100 nM or less. In some embodiments, an anti-TIGIT antibody that
binds to a
human TIGIT also exhibits cross-reactivity with both cynomolgus monkey TIGIT
and mouse
TIGIT.
[00221] In some embodiments, antibody cross-reactivity is determined by
detecting specific
binding of the anti-TIGIT antibody to TIGIT that is expressed on a cell (e.g.,
a cell line that
expresses human TIGIT, cynomolgus monkey TIGIT, or mouse TIGIT, or a primary
cell that
endogenously expresses TIGIT, e.g., primary T cells that endogenously express
human TIGIT,
cyno TIGIT, or mouse TIGIT). In some embodiments, antibody binding and
antibody cross-
reactivity is determined by detecting specific binding of the 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, cynomolgus monkey TIGIT, or mouse
TIGIT, or
a His-tagged protein comprising human TIGIT, cyno TIGIT, or mouse TIGIT).
[00222] In some embodiments, the anti-TIGIT antibodies provided herein inhibit
interaction
between TIGIT and the ligand CD155. In some embodiments, the anti-TIGIT
antibodies
provided herein inhibit interaction between TIGIT and the ligand CD112. In
some embodiments,
the anti-TIGIT antibodies provided herein inhibit interaction between TIGIT
and both of the
ligands CD155 and CD112.
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[00223] In some embodiments, an 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
CDR, light chain variable domain (VL), and heavy chain variable domain (VH) of
the anti-
TIGIT antibodies Clone 13, Clone 13A, Clone 13B, Clone 13C, and Clone 13D are
set forth in
the Table of Sequences below.
[00224] In some embodiments, an anti-TIGIT antibody comprises one or more
(e.g., one,
two, three, four, five, or six) of:
a heavy chain CDR1 sequence comprising an amino acid sequence selected from
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
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
SEQ ID NO:14, SEQ ID NO:15 and 16;
a light chain CDR1 sequence comprising an amino acid sequence of SEQ ID
NO:17;
a light chain CDR2 sequence comprising an amino acid sequence of SEQ ID
NO:18; and/or
a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID
NO:19.
[00225] In some embodiments, an 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.
[00226] In some embodiments, an 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.
[00227] In some embodiments, an 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
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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.
[00228] In some embodiments, an anti-TIGIT antibody comprises a heavy chain
CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 comprising the amino
acid
sequences of:
(a) SEQ ID NOs: 7, 10, 14, 17, 18, and 19, respectively; or
(b) SEQ ID NOs: 8, 11, 14, 17, 18, and 19, respectively; or
(c) SEQ ID NOs: 9, 12, 15, 17, 18, and 19, respectively; or
(d) SEQ ID NOs: 8, 13, 16, 17, 18, and 19, respectively; or
(e) SEQ ID NOs: 8, 12, 16, 17, 18, and 19, respectively.
[00229] In some embodiments, an anti-TIGIT antibody comprises a heavy chain
variable
region (VH) comprising an amino acid sequence that has 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, an anti-TIGIT antibody
comprises a
VH 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. 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 binding of CD155 and/or CD112 to TIGIT.
[00230] In some embodiments, an anti-TIGIT antibody comprises a light chain
variable
region (VL) comprising an amino acid sequence that has 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, an anti-
TIGIT antibody comprises a VL comprising the amino acid sequence of SEQ ID
NO:6. In some
embodiments, a 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 binding of CD155 and/or CD112 to TIGIT.
[00231] In some embodiments, an anti-TIGIT antibody comprises a heavy chain
variable
region comprising an amino acid sequence that has at least 90% sequence
identity (e.g., at least
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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 comprises a light chain variable region
comprising an amino
acid sequence that has 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, an 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 comprises a light chain
variable
region comprising the amino acid sequence of SEQ ID NO:6.
[00232] In some embodiments, an 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) a VH comprising the amino acid sequence of SEQ ID NO:5 and a
VL
comprising the amino acid sequence of SEQ ID NO:6.
[00233] In some embodiments, an 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.
[00234] In some embodiments, an anti-TIGIT antibody for use in the present
methods is a
nonfucosylated 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
[00235] As noted above, in some embodiments, the antibody that binds an immune
cell
engager is an agonist anti-CD40 antibody. Agonistic CD40 monoclonal antibodies
including
dacetuzumab have shown encouraging clinical activity in single-agent and
combination
chemotherapy settings. Dacetuzumab demonstrated some clinical activity in a
phase 1 study in
NHL and a phase 2 study in diffuse large B-cell lymphoma (DLBCL). See, e.g.,
Advani et al.,
Cl/n. Oncol. 27:4371-4377 (2009) and De Vos et al., I Hematol. Oncol. 7:1-9
(2014).
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Additionally, CP-870,893, a humanized IgG2 agonist antibody to CD40, showed
encouraging
activity in solid tumor indications when combined with paclitaxel or
carboplatin or gemcitabine.
In these studies, activation of antigen presenting cells, cytokine production,
and generation of
antigen- specific T cells were seen. See, e.g., Beatty et al., Cl/n. Cancer
Res. 19:6286-6295
(2013) and Vonderheide et al., Oncoimmunology 2:e23033 (2013).
[00236] In some embodiments, a nonfucosylated anti-CD40 antibody is provided
for use in
the present methods. In some embodiments, the nonfucosylated anti-CD40
antibody is SEA-
CD40, which is a nonfucosylated, humanized version of murine S2C6 and which
comprises
heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3
comprising the
amino acid sequences of SEQ ID NOs: 30-35, respectively. The corresponding VH
and VL
comprise the amino acid sequences of SEQ ID NOs: 28 and 29, respectively. SEA-
CD40 is
described in US Patent Publication Nos. 2017/0333556 and 2017/0137528, both of
which are
herein incorporated by reference. 52C6 was originally isolated as a murine
monoclonal
antibody raised against a human bladder carcinoma, referred to herein as
mS2C6. See, e.g.,
Paulie et al., Cancer Immunol. Immunother. 17:165-179 (1984). The 52C6
antibody is a partial
agonist of the CD40 signaling pathway and, in some embodiments, has the
following activities:
binding to human CD40 protein, binding to cynomolgus CD40 protein, activation
of the CD40
signaling pathway, potentiation of the interaction of CD40 with its ligand,
CD4OL. See, e.g., US
Patent No. 6,946,129.
[00237] 52C6 was humanized and this humanized antibody is referred to as
humanized 52C6,
herein, and alternatively as dacetuzumab, which is fucosylated humanized 52C6
(fhS2C6, or
SGN-40). See, e.g., WO 2006/128103, which is incorporated herein by reference
for any
purpose. SEA-CD40 is a nonfucosylated humanized 52C6 antibody. Other versions
of
humanized 52C6 are disclosed at W02008/091954; these can be nonfucosylated and
used in the
methods disclosed herein.
[00238] In some embodiments, an anti-CD40 antibody comprises one or more
(e.g., one, two,
three, four, five, or six) of:
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 is SEQ ID
NO :33;
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a light chain CDR2 sequence comprising the amino acid is SEQ ID NO:34;
and/or
a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID
NO:35.
[00239] In some embodiments, an 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 any 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.
[00240] In some embodiments, an 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 of SEQ ID NO:34; and a light chain CDR3 sequence
comprising the
amino acid sequence of SEQ ID NO:35.
[00241] In some embodiments, an 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.
[00242] In some embodiments, an anti-CD40 antibody comprises a heavy chain
CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 comprising the amino
acid
sequences of:
(a) SEQ ID NOs: 30, 31, 33, 34, and 35, respectively; or
(b) SEQ ID NOs: 30, 36, 33, 34, and 35, respectively.
[00243] In some embodiments, an anti-CD40 antibody comprises a heavy chain
variable
region (VH) comprising an amino acid sequence that has 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, an anti-
CD40 antibody comprises a VH comprising the amino acid sequence of NO:28. In
some
embodiments, a 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.
[00244] In some embodiments, an anti-CD40 antibody comprises a light chain
variable region
(VL) comprising an amino acid sequence that has at least 90% sequence identity
(e.g., at least
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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 VL comprising the amino acid sequence of SEQ ID NO:29. In
some
embodiments, a 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.
[00245] In some embodiments, an anti-CD40 antibody comprises a heavy chain
variable
region comprising an amino acid sequence that has 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 comprises a light
chain variable
region comprising an amino acid sequence that has 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 comprises a light chain variable region comprising the amino
acid sequence of
SEQ ID NO:29.
[00246] In some embodiments, the anti-CD40 antibody comprises the heavy chain
variable
region and light chain variable region disclosed as SEQ ID NO:28 and 29,
respectively. In some
embodiments, the anti-CD40 antibody comprises the heavy chain and light chain
disclosed as
SEQ ID NO:26 and 27, respectively.
D. Exemplary Anti-CD70 Antibodies
[00247] In some embodiments, a nonfucosylated anti-CD70 antibody is provided
for use in
the present methods as the antibody that binds an immune cell engager. In some
embodiments,
the nonfucosylated anti-CD70 antibody is SEA-CD70, as described in US Patent
No. 8,067,546
and which comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1,
CDR2, and
CDR3 comprising the amino acid sequences of SEQ ID NOs: 53-58, respectively.
The
corresponding VH and VL comprise the amino acid sequences of SEQ ID NOs: 41
and 42,
respectively. The CD70 molecule is a member of the tumor necrosis factor (TNF)
ligand
superfamily (TNFSF) and it binds to the related receptor, CD27 (TNFRSF7). The
interaction
between the two molecules activates intracellular signals from both receptors.
In normal
conditions, CD70 expression is transient and limited to activated T and B
cells, mature dendritic,
and natural killer (NK) cells. Similarly, CD27 is expressed on both naïve and
activated effector
T cells, as well as NK and activated B cells. However, CD70 is also aberrantly
expressed in
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various hematologic cancers, including acute myeloid leukemia (AML),
myelodysplastic
syndrome (MDS), and non-Hodgkin lymphoma (NHL), as well as carcinomas, and
plays a role
in both tumor cell survival and/or tumor immune evasion. SEA-CD70 acts through
blocking
CD70/CD27 axis signaling, eliciting antibody dependent cellular phagocytosis
(ADCP) and
complement dependent cytotoxicity (CDC), and enhancing antibody dependent
cellular
cytotoxicity (ADCC).
[00248] In some embodiments, an anti-CD70 antibody comprises one or more
(e.g., one, two,
three, four, five, or six) of:
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 is SEQ ID
NO:56;
a light chain CDR2 sequence comprising the amino acid is SEQ ID NO:57;
and/or
a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID
NO:58.
[00249] In some embodiments, an 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 any of SEQ ID NO:54; and a heavy chain
CDR3
sequence comprising the amino acid sequence of SEQ ID NO:55.
[00250] In some embodiments, an 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 of SEQ ID NO:57; and a light chain CDR3 sequence
comprising the
amino acid sequence of SEQ ID NO:58.
[00251] In some embodiments, an 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.
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[00252] In some embodiments, an anti-CD70 antibody comprises a heavy chain
variable
region (VH) comprising an amino acid sequence that has 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, an anti-
CD70 antibody comprises a VH comprising the amino acid sequence of NO:41. In
some
embodiments, a 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.
[00253] In some embodiments, an anti-CD70 antibody comprises a light chain
variable region
(VL) comprising an amino acid sequence that has 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,
an anti-CD70
antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:42. In
some
embodiments, a 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.
[00254] In some embodiments, an anti-CD70 antibody comprises a heavy chain
variable
region comprising an amino acid sequence that has 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 comprises a light
chain variable
region comprising an amino acid sequence that has 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,
an anti-CD70
antibody comprises a heavy chain variable region comprising the amino acid
sequence of SEQ
ID NO:41, and comprises a light chain variable region comprising the amino
acid sequence of
SEQ ID NO:42.
[00255] In some embodiments, the anti-CD70 antibody comprises the heavy chain
variable
region and light chain variable region disclosed as SEQ ID NO:41 and 42,
respectively.
E. Exemplary Anti-BCMA Antibodies
[00256] In some embodiments, a nonfucosylated anti-BCMA antibody is provided
for use in
the present methods as the antibody that binds an immune cell engager. In some
embodiments,
the nonfucosylated anti-BCMA antibody is SEA-BCMA, which is an antibody
targeting B-cell
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maturation antigen (BCMA) and which comprises heavy chain CDR1, CDR2, and
CDR3, and
light chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID
NOs: 47-
52, respectively. The corresponding VH and VL comprise the amino acid
sequences of SEQ ID
NOs: 45 and 46, respectively. BCMA is expressed on multiple myeloma (MM). The
antibody
acts through blocking ligand mediated BCMA cell signaling, antibody dependent
cellular
phagocytosis (ADCP), and enhanced antibody dependent cellular cytotoxicity
(ADCC).
[00257] In some embodiments, an anti-BCMA antibody comprises one or more
(e.g., one,
two, three, four, five, or six) of:
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 is SEQ ID
NO:50;
a light chain CDR2 sequence comprising the amino acid is SEQ ID NO:51;
and/or
a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID
NO:52.
[00258] In some embodiments, an 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 any of SEQ ID NO:48; and a heavy chain
CDR3
sequence comprising the amino acid sequence of SEQ ID NO:49.
[00259] In some embodiments, an 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 of SEQ ID NO:51; and a light chain CDR3 sequence
comprising the
amino acid sequence of SEQ ID NO:52.
[00260] In some embodiments, an 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.
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[00261] In some embodiments, an anti-BCMA antibody comprises a heavy chain
variable
region (VH) comprising an amino acid sequence that has 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, an anti-
BCMA antibody comprises a VH comprising the amino acid sequence of NO:45. In
some
embodiments, a 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.
[00262] In some embodiments, an anti-BCMA antibody comprises a light chain
variable
region (VL) comprising an amino acid sequence that has 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 VL comprising the amino acid sequence of SEQ ID
NO:46. In
some embodiments, a 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.
[00263] In some embodiments, an anti-BCMA antibody comprises a heavy chain
variable
region comprising an amino acid sequence that has 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 comprises a light
chain variable
region comprising an amino acid sequence that has 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 comprises a light chain variable region comprising the
amino acid
sequence of SEQ ID NO:46.
[00264] In some embodiments, the anti-BCMA antibody comprises the heavy chain
variable
region and light chain variable region disclosed as SEQ ID NO:45 and 46,
respectively.
F. Enhanced Fc Backbone
[00265] As noted above, the antibodies that bind the immune cell engager
comprise an Fc that
has one or more of the following activities: enhanced binding to one or more
activating FcyRs;
reduced binding to inhibitory FcyRs; enhanced ADCC activity; and/or enhanced
ADCP activity.
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Antibodies having Fe with such activities and the desired activity profile can
be generated in a
variety of ways, including producing a nonfucosylated protein and/or by
engineering the Fe to
contain certain mutations that yield the desired activity. This section
provides additional details
on methods for generating nonfucosylated antibodies and exemplary engineering
approaches.
Additional guidance on selection of constant regions and manufacturing of
antibodies is
provided in other sections below.
[00266] Antibodies may 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 the immunoglobulins
affect the
protein's function (Boyd et at., (1996) Mol. Immunol. 32:1311-1318; Wittwe and
Howard,
(1990) Biochem. 29:4175-4180), and the intramolecular interaction between
portions of the
glycoprotein which can affect the conformation and presented three-dimensional
surface of the
glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin.
Biotech. 7:409-
416). Oligosaccharides may also serve to target a given glycoprotein to
certain molecules based
upon specific recognition structures. For example, it has been reported that
in agalactosylated
IgG, the oligosaccharide moiety 'flips' out of the inter-CH2 space and
terminal N-
acetylglucosamine residues become available to bind mannose binding protein
(Malhotra et at.,
(1995) Nature Med. 1:237-243). Removal by glycopeptidase of the
oligosaccharides from
CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody which
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
at., (1996)
Mol. Immunol. 32:1311-1318), while selective removal of sialic acid residues
using
neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has
also been reported
to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with
tetracycline-regulated expression of13(1,4)-N-acetylglucosaminyltransferase
III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to
have improved
ADCC activity (Umana et at. (1999) Mature Biotech. 17:176-180).
[00267] Glycosylation of antibodies is typically either N-linked or 0-
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 the recognition sequences for enzymatic attachment of
the carbohydrate
moiety to the asparagine side chain. Thus, the presence of either of these
tripeptide sequences in
a polypeptide creates a potential glycosylation site. 0-linked glycosylation
refers to the
attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to
a hydroxyamino
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acid, most commonly serine or threonine, although 5-hydroxyproline or 5-
hydroxylysine may
also be used.
[00268] Glycosylation variants of antibodies are variants in which the
glycosylation pattern of
an antibody is altered. By altering is meant deleting one or more carbohydrate
moieties found in
the antibody, adding one or more carbohydrate moieties to the antibody,
changing the
composition of glycosylation (glycosylation pattern), the extent of
glycosylation, etc.
[00269] Addition of glycosylation sites to the antibody can be accomplished by
altering the
amino acid sequence such that it contains one or more of the above-described
tripeptide
sequences (for N-linked glycosylation sites). The alteration may also be made
by the addition
of, or substitution by, one or more serine or threonine residues to the
sequence of the original
antibody (for 0-linked glycosylation sites). Similarly, removal of
glycosylation sites can be
accomplished by amino acid alteration within the native glycosylation sites of
the antibody.
[00270] The amino acid sequence is usually altered by altering the underlying
nucleic acid
sequence. These methods include isolation from a natural source (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 a non-variant version of the antibody.
[00271] The glycosylation (including glycosylation pattern) of antibodies may
also be altered
without altering the amino acid sequence or the underlying nucleotide
sequence. See, e.g.,
Pereira et al., 2018, MAbs, 10(5): 693-711. Glycosylation largely depends on
the host cell used
to express the antibody. Since the cell type used for expression of
recombinant glycoproteins,
e.g., antibodies, as potential therapeutics is rarely the native cell,
significant variations in the
glycosylation pattern of the antibodies can be expected. See, e.g., Hse et
at., (1997) J. Biol.
Chem. 272:9062-9070. In addition to the choice of host cells, factors which
affect glycosylation
during recombinant production of antibodies include growth mode, media
formulation, culture
density, oxygenation, pH, purification schemes and the like. Various methods
have been
proposed to alter the glycosylation pattern achieved in a particular host
organism including
introducing or overexpressing certain enzymes involved in oligosaccharide
production (U.S.
Patent Nos. 5047335; 5510261; 5278299). Glycosylation, or certain types of
glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo
H). In addition, the recombinant host cell can be genetically engineered,
e.g., make defective in
processing certain types of polysaccharides. These and similar techniques are
known in the art.
[00272] The glycosylation structure of antibodies can be readily analyzed by
conventional
techniques of carbohydrate analysis, including lectin chromatography, NMR,
Mass
spectrometry, HPLC, GPC, monosaccharide compositional analysis, sequential
enzymatic
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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, without limitation, enzymatic treatment
(commonly
performed using peptide-N-glycosidase Fiendo-13-galactosidase), elimination
using harsh
alkaline environment to release mainly 0-linked structures, and chemical
methods using
anhydrous hydrazine to release both N- and 0-linked oligosaccharides
[00273] A preferred form of modification of glycosylation of antibodies is
reduced core
fucosylation. "Core fucosylation" refers to addition of fucose
("fucosylation") to N-
acetylglucosamine ("GlcNAc") at the reducing terminal of an N-linked glycan.
[00274] A "complex N-glycoside-linked sugar chain" is typically bound to
asparagine 297
(according to the number of Kabat). As used herein, the complex N-glycoside-
linked sugar
chain has a biantennary composite sugar chain, mainly having the following
structure:
+/-Fucal
+/-Ga1131¨ 4GIcNAc131 ¨2Mana1
6 6
+/- GIcNAc131 4Man1:31-4G1cNAc131 p 4GIcNAc
3
+/-Ga1131¨ 4GIcNAc131¨ 2Mana1
where + indicates the sugar molecule can be present or absent, and the numbers
indicate the
position of linkages between the sugar molecules. In the above structure, the
sugar chain
terminal which binds to asparagine is called a reducing terminal (at right),
and the opposite side
is called a non-reducing terminal. Fucose is usually bound to N-
acetylglucosamine ("GlcNAc")
of the reducing terminal, typically by an a1,6 bond (the 6-position of GlcNAc
is linked to the 1-
position of fucose). "Gal" refers to galactose, and "Man" refers to mannose.
[00275] A "complex N-glycoside-linked sugar chain" includes 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 referred to as "gal-GlcNAc") and the non-reducing
terminal side of
gal-GlcNAc optionally has a sialic acid, bisecting N-acetylglucosamine or the
like; and 2) a
hybrid type, in which the non-reducing terminal side of the core structure has
both branches of a
high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked
sugar chain.
[00276] In some methods as provided herein, only a minor amount of fucose is
incorporated
into the complex N-glycoside-linked sugar chain(s) of the antibodies. 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
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than about 3% of the antibodies in a 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 that 60% of the antibodies in a composition
have core
fucosylation by fucose, the antibodies of the composition may be referred to
as
"nonfucosylated" or "afucosylated." 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 nonfucosylated.
[00277] In certain embodiments, only a minor amount of a fucose analog (or a
metabolite or
product of the fucose analog) is incorporated into the complex N-glycoside-
linked sugar
chain(s). 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 a
fucose analog or a metabolite or product of the fucose analog. In some
embodiments, about 2%
of the antibodies have core fucosylation by a fucose analog or a metabolite or
product of the
fucose analog.
[00278] In some embodiments, less that 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 a composition have a
fucose residue on
a GO, Gl, or G2 glycan structure. (See, e.g., Raju et al., 2012, MAbs 4: 385-
391, Figure 3.) In
some embodiments, about 2% of the antibodies in the composition have a fucose
residue on a
GO, Gl, or G2 glycan structure. In various embodiments, when less than 60% of
the antibodies
in a composition have a fucose residue on a GO, Gl, or G2 glycan structure,
the antibodies of the
composition may be referred to as "nonfucosylated." 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
a GO, Gl, or G2
glycan structure. It should be noted that GO glycans include GO-GN glycans. GO-
GN glycans are
monoantenary glycans with one terminal GlcNAc residue. G1 glycans include G1-
GN glycans.
G1-GN glycans are monoantenary glycans with one terminal galactose residue. GO-
GN and Gl-
GN glycans can be fucosylated or nonfucosylated.
[00279] A variety of methods for generating nonfucosylated antibodies can be
utilized.
Exemplary strategies include the use of cell lines lacking certain
biosynthetic enzymes involved
in fucosylation pathways or the inhibition or the knockout of certain genes
involved in the
fucosylation pathway. A review of such approaches is provided by Pereira, et
al. (2018) MABS
10:693-711, which is incorporated herein by reference in its entirety.
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[00280] For example, methods of making nonfucosylated antibodies by incubating
antibody-
producing cells with a fucose analogue are described, e.g., in W02009/135181.
Briefly, cells
that have been engineered to express the antibodies are incubated in the
presence of a fucose
analogue or an intracellular metabolite or product of the fucose analog. An
intracellular
metabolite can be, for example, a GDP-modified analog or a fully or partially
de-esterified
analog. A product can be, for example, a fully or partially de-esterified
analog. In some
embodiments, a fucose analogue can inhibit an enzyme(s) in the fucose salvage
pathway. For
example, a fucose analog (or an intracellular metabolite or product of the
fucose analog) can
inhibit the activity of fucokinase, or GDP-fucose-pyrophosphorylase. In some
embodiments, a
fucose analog (or an intracellular metabolite or product of the fucose analog)
inhibits
fucosyltransferase (preferably a 1,6-fucosyltransferase, e.g., the FUT8
protein). In some
embodiments, a fucose analog (or an intracellular metabolite or product of the
fucose analog)
can inhibit the activity of an enzyme in the de novo synthetic pathway for
fucose. For example,
a fucose analog (or an intracellular metabolite or product of the fucose
analog) can inhibit the
activity of GDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In some
embodiments,
the fucose analog (or an intracellular metabolite or product of the fucose
analog) can inhibit a
fucose transporter (e.g., GDP-fucose transporter).
[00281] In one embodiment, the fucose analogue is 2-flurofucose. Methods of
using fucose
analogues in growth medium and other fucose analogues are disclosed, e.g., in
WO
2009/135181, which is herein incorporated by reference.
[00282] Other methods for engineering cell lines to reduce core fucosylation
included gene
knock-outs, gene knock-ins and RNA interference (RNAi). See, e.g., Pereira et
al., 2018, MAbs,
10(5): 693-711. In gene knock-outs, the gene encoding FUT8 (alpha 1,6-
fucosyltransferase
enzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residue from
GDP-fucose to
position 6 of Asn-linked (N-linked) GlcNac of an N-glycan. FUT8 is reported to
be the only
enzyme responsible for adding fucose to the N-linked biantennary carbohydrate
at
Asn297. Gene knock-ins add genes encoding enzymes such as GNTIII or a golgi
alpha
mannosidase II. An increase in the levels of such enzymes in cells diverts
monoclonal
antibodies from the fucosylation pathway (leading to decreased core
fucosylation), and having
increased amount of bisecting N-acetylglucosamines. RNAi typically also
targets FUT8 gene
expression, leading to decreased mRNA transcript levels or knocking out gene
expression
entirely.
[00283] Other strategies that may be used include GlycoMAb (US Patent No.
6,602,684)
and Potelligent (BioWa).
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[00284] Any of these methods can be used to generate a cell line that would be
able to
produce a nonfucosylated antibody.
[00285] Various engineering approaches can also be utilized to obtain Fc
regions with the
desired FcyR activity and effector function. In some embodiments, the Fc is
engineered to have
the following combination of mutations: S239D, A330L and 1332E, which
increases the affinity
of the Fc domain for FcyRIIIA and consequently increases ADCC. Additional
substitutions that
enhance affinity for FcyRIIIa include, for example, T256A, K290A, S298A,
E333A, and
K334A. Substitutions that enhance binding to activating FcyRIIIa and reduced
binding to
inhibitory FcyRIIIb include, for example, F243L/R292P/Y300LN3051/P396L and
F243L/R292P/Y300L/L235V/P396L. In some embodiments, the substitutions are in
an IgG1 Fc
background.
[00286] Oligosaccharides covalently attached to the conserved Asn297 are
involved in the
ability of the Fc region of an IgG to bind FcyR (Lund et al., 1996,1 Immunol.
157:4963-69;
Wright and Morrison, 1997, Trends Biotechnol. 15:26-31). Engineering of this
glycoform on
IgG can significantly improve IgG-mediated ADCC. Addition of bisecting N-
acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol. 17:176-
180; Davies et
at., 2001, Biotech. Bioeng. 74:288-94) to this glycoform or removal of fucose
(Shields et al.,
2002,1 Biol. Chem. 277:26733-40; Shinkawa et al., 2003,1 Biol. Chem. 278:6591-
604; Niwa
et at., 2004, Cancer Res. 64:2127-33) from this glycoform are two examples of
IgG Fc
engineering that improves the binding between IgG Fc and FcyR, thereby
enhancing Ig-
mediated ADCC activity.
[00287] A systemic substitution of solvent-exposed amino acids of human IgG1
Fc region has
generated IgG variants with altered FcyR binding affinities (Shields et at.,
2001, 1 Biol. Chem.
276:6591-604). When compared to parental IgGl, a subset of these variants
involving
substitutions at Thr256/5er298, 5er298/G1u333, 5er298/Lys334, or
5er298/G1u333/Lys334 to
Ala demonstrate increased in both binding affinity toward FcyR and ADCC
activity (Shields et
at., 2001,1 Biol. Chem. 276:6591-604; Okazaki et al., 2004,1 Mot. Biol.
336:1239-49).
[00288] Many methods are available to determine the amount of fucosylation on
an
antibody. Methods include, e.g., 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).
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IV. Exemplary Antibody-Drug Conjugates (ADCs)
[00289] The preceding section described the relevant aspects of the antibody
that binds to a
target that is involved in immune regulation (an immune cell engager). As
noted above, some of
the methods that are provided herein also comprise administering an antibody-
drug conjugate
(ADC) comprising a tubulin disrupter (e.g., auristatins, including for
instance MMAE and
MMAF) in combination with the antibody that binds to the immune cell engager.
In various
embodiments, the antibody-drug conjugate comprises an antibody conjugated to a
cytotoxic
agent. In some embodiments, the cytotoxic agent is a tubulin disrupter. In
some embodiments,
the antibody binds an antigen expressed on a tumor cell. Further details
regarding the ADC that
is utilized in the methods provided herein are set forth in this section and
in the Examples below.
Any of the ADCs described herein may be combined with any of the antibodies
that bind an
immune cell engager described herein.
A. Exemplary Target Antigens
[00290] In some embodiments, the ADC binds an antigen expressed on a tumor
cell.
[00291] In some embodiments, an ADC used in the methods provided herein
comprises an
antibody conjugated to a cytotoxic agent, wherein the antibody specifically
binds an antigen
selected from 5T4 (TPBG), ADAM-9 , AG-7, ALK, ALP, AMUR'', 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, CD161,
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, CD3 epsilon, 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,
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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,
CD7OL, 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
factors C3, Cripto, CSP-1, CXCR5, DCLK1, DLK-1, DLL3, DPEP3, DR5 (Death
receptor 5),
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-alpha, 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 (integrin
aVf33), integrin beta-6, Interleukin-4 Receptor (IL4R), KAAG-1, KLK2, LAMP-1,
Le(y), Lewis
Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, Lipid raft, 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, Nectin-4, Notch3, Nucleolin, OAcGD2, OT-MUC1 (onco-tethered-MUC1),
OX001L,
P1GF, PAM4 antigen, p-cadherin (cadherin 3), PD-L1, Phosphatidyl Serine(PS),
PRLR,
Prolactin 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
(antibiotic agent),
STEAP-1, STING, STn, T101, TAA, TAC, TDGF1, tenascin, TENB2, TGF-B, Thomson-
Friedenreich antigens, Thy1.1, TIM-1, tissue factor (TF; CD142), TM45F1, Tn
antigen, TNF-
alpha (TNFa), TRA-1-60, TRAIL receptor (R1 and R2), TROP-2, Tumor-associated
glycoprotein 72 (TAG-72), uPAR, VEGFR, VEGFR-2, and xCT
[00292] In some embodiments, the ADC binds an antigen selected from 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
(onco-tethered-MUC1), TRA-1-60, TIM-1, GCC, Mesothelin (MSLN), EGFR, gpNMB,
CD20,
AMUR'', NaPi2b, CD142, ROR1, Integrin beta-6, Ly6E, cMET, CD37, MUC16, STEAP-
1,
LRRC15, SLITRK6, MUC16, ETBR, FCRH5, Axl, CD79b, Globo H, SLAMF7, PSMA, CD22,
CD228, CD48, LIV-1, EphA2, 5LC44A4, CA9, Axl, and LGR5.
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[00293] In some embodiments, the ADC binds an antigen selected from BCMA,
GPC1,
CD30, cMET, SAIL, HER3, CD70, c-MET, CD46, HER2, 5T4, ENPP3, CD19, EGFR, BCMA,
CD70, BCMA, and EphA2.
[00294] In some embodiments, the ADC binds an antigen selected from Her2,
TROP2,
BCMA, cMet, integrin alphVbeta6 (integrin aVf36), CD22, CD79b, CD30, CD19,
CD70,
CD228, and CD47.
[00295] In some embodiments, the ADC binds an antigen selected from 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, 5LC44A4, CD30, and sTn.
[00296] In some embodiments, the ADC binds an antigen selected from 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, CD3 epsilon, 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), Dysadherin, EFNA4 ,
EGFR, EGFR wild type, EGFRviii, EMP2, ENPP3, EpCAM, EphA2, EphA3, ETBR, FAP,
FCRH5, FGFR2, FGFR3, FLT3, FOLR, FOLR-alpha, 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 beta-6, Interleukin-4 Receptor (IL4R), KAAG-
1, KLK2,
LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, Lipid raft, LIV-1, LRP-
1,
LRRC15, Ly6E, Macrophage mannose receptor 1, MAGE, Mesothelin (MSLN), MET, MHC
class I chain-related protein A and B (MICA and MICB), MRC2, MT1-MMP, MTX3,
MTX5,
MUC-1, MUC16, NaPi2b, Nectin-4, NOTCH3, Nucleolin, OAcGD2, OT-MUC1 (onco-
tethered-MUC1), OX001L, P-Cadherin, PD-L1, Phosphatidyl Serine,
Phosphatidylserine (PS),
Prolactin Receptor (PRLR), Pseudomonas, PSMA, PTK7, Receptor tyrosine kinase
(RTK),
RNF43, ROR1, ROR2, SAIL, SEZ6, SLAMF7, 5LC44A4, SLITRK6, Sortilin (SORT1),
SSEA-
4, SSTR2, Staphylococcus aureus (antibiotic agent), STEAP-1, STING, STING
(payload target),
STn, TAA, TGF-B, TIM-1, TM45F1, TNF-alpha, TRA-1-60, TROP-2, Tumor-associated
glycoprotein 72 (TAG-72), VEGFR-2, xCT.
[00297] In some embodiments, the ADC binds an antigen selected from 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,
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Integrin beta-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, Mesothelin (MSLN), MET,
MRC2,
MUC16, NaPi2b, Nectin-4, OT-MUC1 (onco-tethered-MUC1), PSMA, ROR1, SLAMF7,
SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, Tumor-associated glycoprotein
72
(TAG-72).
[00298] In some embodiments, the ADC binds an antigen selected from BCMA, GPC-
1,
CD30, c-MET, SAIL, HER-3, CD70, CD46, HER-2, 5T4, ENPP3, CD19, EGFR, EphA2.
[00299] In some embodiments, the antibody of the ADC does not bind Nectin-4.
[00300] Typically, the antibody of the ADC and the antibody that binds the
immune cell
engager are two separate antibodies. In certain embodiments, however, the
antibodies may form
a bispecific antibody.
B. Exemplary Cytotoxic Agents
[00301] 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.
[00302] Various categories of tubulin disrupting agent are known in the field,
including, but
not limited to, dolastatins, auristatins, tubulysins, colchicine, vinca
alkaloids, taxanes, T67
(Tularik), cryptophycins, maytansinoids, hemiasterlins, and other tubulin
disrupting agents.
[00303] Auristatins are derivatives of the natural product dolastatin.
Exemplary auristatins
include dolostatin-10, auristatin E, auristatin T, MMAE (N-methylvaline-valine-
dolaisoleuine-
dolaproine-norephedrine or monomethyl auristatin E) and MMAF (N-methylvaline-
valine-
dolaisoleuine-dolaproine-phenylalanine or dovaline-valine-dolaisoleunine-
dolaproine-
phenylalanine), AEB (ester produced by reacting auristatin E with paraacetyl
benzoic acid),
AEVB (ester produced by reacting auristatin E with benzoylvaleric acid), and
AFP
(dimethylvaline-valine-dolaisoleuine- dolaproine-phenylalanine-p-
phenylenediamine or
auristatin phenylalanine phenylenediamine). WO 2015/057699 describes PEGylated
auristatins
including MMAE. Additional dolostatin derivatives contemplated for use are
disclosed in U.S.
Pat. No. 9,345,785, incorporated herein by reference for any purpose.
Exemplary auristatin
embodiments include the N-terminus linked monomethylauristatin drug units DE
and DF,
disclosed in "Senter et al, Proceedings of the American Association for Cancer
Research,
Volume 45, Abstract Number 623, presented 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.
[00304] In certain embodiments, the ADC cytotoxic agent is MMAE.
[00305] In other embodiments, the cytotoxic agent conjugated to the ADC is
MMAF.
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[00306] Tubulysins include, but are not limited to, tubulysin D, tubulysin M,
tubuphenylalanine and tubutyrosine. WO 2017-096311 and WO 2016-040684 describe
nonlimiting tubulysin analogs including tubulysin M.
[00307] Colchicines include, but are not limited to, colchicine and CA-4.
[00308] Vinca alkaloids include, but are not limited to, Vinblastine (VBL),
vinorelbine
(VRL), vincristine (VCR) and vindfesine (VDS).
[00309] Taxanes include, but are not limited to, Taxol (paclitaxel) and
Taxotere
(docetaxel).
[00310] Cryptophycins include but are not limited to cryptophycin-1 and
cryptophycin-52.
[00311] Maytansinoids include, but are not limited to, maytansine,
maytansinol, maytansine
analogs, DM1, DM3 and DM4, and ansamatocin-2. Exemplary maytansinoid drug
moieties
include those having a modified aromatic ring, such as: C-19-dechloro (U.S.
Pat. No. 4,256,746)
(prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-
hydroxy (or C-20-
demethy1)+/¨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 (-000R), +/¨dechloro (U.S. Pat. No. 4,294,757)
(prepared by
acylation using acyl chlorides), and those having modifications at other
positions.
[00312] Maytansinoid drug moieties also include those having modifications
such as: C-9-SH
(U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with
H25 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 (CH20H or CH20Ac) (U.S. Pat. No.
4,450,254)
(prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866)
(prepared by the
conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos.
4,313,946 and
4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos.
4,362,663 and
4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and
4,5-deoxy
(U.S. Pat. No. 4,371,533) (prepared by the titanium trichloride/LAH reduction
of maytansinol).
The cytotoxicity of the TA.1-maytansonoid conjugate that binds HER-2 (Chari et
al., Cancer
Research 52:127-131 (1992) was tested in vitro on the human breast cancer cell
line SK-BR-3.
The drug conjugate achieved a degree of cytotoxicity similar to the free
maytansinoid drug,
which could be increased by increasing the number of maytansinoid molecules
per antibody
molecule.
[00313] Hemiasterlins include but are not limited to, hemiasterlin and HTI-
286.
[00314] 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,
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colcimid, estramustine, cemadotin, combretastatins, discodermolide,
eleutherobin, eribulin,
prolabolin, phomopsin, and laulimalide.
[00315] The ADC for use in the methods herein may, in some embodiments,
comprise linker
units. For example, the ADC may comprise a linker region between the cytotoxic
agent and the
antibody. In some embodiments, the linker is a protease cleavable linker, an
acid-cleavable
linker, a disulfide linker, or a self-stabilizing linker. In various
embodiments, the linker is
cleavable under intracellular conditions, such that cleavage of the linker
releases the therapeutic
agent from the antibody in the intracellular environment.
[00316] The ADC for use in the methods herein may comprise a linker
wherein the
therapeutic agent (e.g., tubulin disrupter) can be conjugated to the antibody
in a manner that
reduces its activity unless it is detached from the antibody (e.g., by
hydrolysis, by antibody
degradation, or by a cleaving agent). Such therapeutic agent can be attached
to the antibody
via a linker. A therapeutic agent conjugated to a linker is also referred to
herein as a drug linker.
The nature of the linker can vary widely. The components that make up the
linker are chosen on
the basis of their characteristics, which may be dictated in part, by the
conditions at the site to
which the conjugate is delivered.
[00317] The therapeutic agent can be attached to the antibody with a
cleavable
linker that is sensitive to cleavage in the intracellular environment of a
target cell but is not
substantially sensitive to the extracellular environment, such that the
conjugate is cleaved from
the antibody when it is internalized by the cancer cell (e.g., in the
endosomal or, for example by
virtue of pH sensitivity or protease sensitivity, in the lysosomal environment
or in the caveolear
environment). The therapeutic agent can also be attached to the antibody with
a non-cleavable
linker.
[00318] As indicated, the linker may comprise a cleavable unit. In
some such
embodiments, the structure and/or sequence of the cleavable unit is selected
such that it is
cleaved by the action of enzymes present at the target site (e.g., the target
cell). In other
embodiments, cleavable units that are cleavable by changes in pH (e.g. acid or
base labile),
temperature or upon irradiation (e.g. photolabile) may also be used.
[00319] In some embodiments, the cleavable unit may comprise one
amino acid or
a contiguous sequence of amino acids. The amino acid sequence may be the
target substrate for
an enzyme.
[00320] In some aspects, the cleavable unit is a peptidyl unit and
is at least two
amino acids long. Cleaving agents can include cathepsins B and D and plasmin
(see, e.g.,
Dubowchik and Walker, 1999, Pharm . Therapeutics 83:67-123). Most typical are
cleavable unit
that are cleavable by enzymes that are present in the target cells, i.e., an
enzyme cleavable
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linker. Accordingly, the linker can be cleaved by an intracellular peptidase
or protease enzyme,
including a lysosomal or endosomal protease. For example, a linker that is
cleavable by the
thiol-dependent protease cathepsin-B, which is highly expressed in cancerous
tissue, can be used
(e.g., a linker comprising a Phe-Leu or a Val-Cit peptide or a Val-Ala
peptide).
[00321] In some embodiments, the linker will comprise 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 an
additional functional unit, e.g., a self-immolative spacer unit or a non-self-
immolative spacer
unit. A non-self-immolative spacer unit is one in which part or all of the
spacer unit remains
bound to the drug unit after cleavage of a cleavable unit (e.g., amino acid)
from the antibody-
drug conjugate. To liberate the drug, an independent hydrolysis reaction takes
place within the
target cell to cleave the spacer unit from the drug.
[00322] With a self-immolative spacer unit, the drug is released
without the need
for drug for a separate hydrolysis step. In one embodiment, wherein the linker
comprises a
cleavable unit and a self immolative group, the cleavable unit is cleavable by
the action of an
enzyme and after cleavage of the cleavable unit, the self-immolative group(s)
release the
therapeutic agent. In some embodiments, the cleavable unit of the linker will
be directly or
indirectly conjugated to the therapeutic agent on one end and on the other end
will be directly or
indirectly conjugated to the antibody. In some such embodiments, the cleavable
unit will be
directly or indirectly (e.g., via a self-immolative or non-self-immolative
spacer unit) conjugated
to the therapeutic agent on one end and on the other end will be conjugated to
the antibody via a
stretcher unit. A stretcher unit links the antibody to the rest of the drug
and/or drug linker. In
one embodiment, the connection between the antibody and the rest of the drug
or drug linker is
via a maleimide group, e.g., via a maleimidocaproyl linker. In some
embodiments, the antibody
will be linked to the drug via a disulfide, for example the disulfide linked
maytansinoid
conjugates SPDB-DM4 and SPP-DM1.
[00323] The connection between the antibody and the linker can be
via a number
of different routes, e.g., 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 linker is formed between a thiol group of a cysteine residue of the
antibody and a maleimide
group of the linker. In some embodiments, the interchain bonds of the antibody
are converted
to free thiol groups prior to reaction with the functional group of the
linker. In some
embodiments, a cysteine residue is an introduced into the heavy or light chain
of an antibody
and reacted with the linker. Positions for cysteine insertion by substitution
in antibody heavy or
light chains include those described in Published U.S. Application No. 2007-
0092940 and
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International Patent Publication W02008070593, each of which are incorporated
by reference
herein in its entirety and for all purposes.
[00324] In some embodiments, the antibody-drug conjugates have 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., the
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
connected at one end
to the antibody and at the other end to the drug.
[00325] The drug loading is represented by p, the number of drug
molecules per
antibody. Drug loading may range from 1 to 20 Drug units (D) per antibody. In
some aspects,
the subscript p will range from 1 to 20 (i.e., both integer and non-integer
values from 1 to 20).
In some aspects, the subscript p will be an integer from 1 to 20, and will
represent the number of
drug-linkers on a singular antibody. In other aspects, p represents the
average number of drug-
linker molecules per antibody, e.g., the average number of drug-linkers per
antibody in a
reaction mixture or composition (e.g., pharmaceutical composition), and can be
an integer or
non-integer value. Accordingly, in some aspects, for compositions (e.g.,
pharmaceutical
compositions), p represents the average drug loading of the antibody-drug
conjugates in the
composition, and p ranges from 1 to 20.
[00326] In some embodiments, p is from about 1 to about 8 drugs per
antibody. In
some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p
is from
about 2 to about 8 drugs per antibody. In some embodiments, p is from about 2
to about 6, 2 to
about 5, or 2 to about 4 drugs per antibody. In some embodiments, p is about
2, about 4, about 6
or about 8 drugs per antibody.
[00327] The average number of drugs per antibody unit in a
preparation from a
conjugation reaction may be characterized by conventional means such as mass
spectroscopy,
ELISA assay, HIC, and HPLC. The quantitative distribution of conjugates in
terms of p may
also be determined.
[00328] Exemplary antibody-drug conjugates include auristatin based
antibody-
drug conjugates, i.e., conjugates wherein the drug component is an auristatin
drug. Auristatins
bind tubulin, have been shown to interfere with microtubule dynamics and
nuclear and cellular
division, and have anticancer activity. Typically, the auristatin based
antibody-drug conjugate
comprises a linker between the auristatin drug and the antibody. The
auristatins can be linked to
the antibody at any position suitable for conjugation to a linker. The linker
can be, for example,
a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g.,
linker released by
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degradation of the antibody). The auristatin can be auristatin E or a
derivative thereof. The
auristatin can be, for example, an ester formed between auristatin E and a
keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid or
benzoylvaleric acid to
produce AEB and AEVB, respectively. Other typical auristatins include MMAF
(monomethyl
auristatin F), and M_MAE (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.
[00329] 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 the valine-citrulline dipeptide:
H2Ny0
( ;NH
CH3
Ab __ a FNLA: N FNI
0 0
. ,3- - ,3el H3C CH3 H3C46...)
0 N 0
0 (HA
,_, ,,,_,
eH HO Ph
3 X
)7Y rrnr-N N CH
OCHH
H3C CH3 OCH3`-'
/3 P
Ab-vcMMAE
Ab
o H 0
0
NVal-Cit¨N I 0 ....7õõ.., I 0,, 0
0, 0
p
Ab-vcMMAF
Ab 0
f 0 H 0
0, 0
0 OH
\ / P
Ab-mcMMAF
or a pharmaceutically acceptable salt thereof. The drug loading is represented
by p, the number
of drug-linker molecules per antibody. Depending on the context, p can
represent the average
number of drug-linker molecules per antibody, also referred to the average
drug loading. The
variable p ranges from 1 to 20 and is preferably from 1 to 8. In some
preferred embodiments,
when p represents the average drug loading, p ranges from about 2 to about 5.
In some
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embodiments, p is about 2, about 3, about 4, or about 5. In some aspects, the
antibody is
conjugated to the linker via a sulfur atom of a cysteine residue. In some
aspects, the cysteine
residue is one that is engineered into the antibody. In other aspects, the
cysteine residue is an
interchain disulfide cysteine residue.
[00330] In some other embodiments, the antibody-drug conjugates have
the linker
units disclosed in the application US20160310612A1 (PCT/US2014/060477) herein
incorporated in its entirety by reference. In some other embodiments, the
antibody- drug
conjugates have following formula (II):
PEG
L Z ___
drue-link er
(II)
wherein D is a drug unit, PEG is the polyethylene glycol unit that masks the
hydrophobicity of
the drug-linker, LP is the parallel connector unit that allows for a PEG Unit
to be in a parallel
orientation with respect to X-D, A is a branching unit when m is greater than
1, optionally
comprised of subunits, or A is absent when m is 1, X is a Releasable Assembly
unit that
provides for release of each D from the LDC and Z is an optional spacer unit
through which LP
is bonded to L, which is the antibody.
[00331] In some embodiments, the antibody-drug conjugates have the
following
formula III:
( PEG
L (Z-A,
X
D t m
-
\
drug-linker (III)
wherein AD is a drug attachment unit that allows for additional attachment of
X-D moieties
indicated by tin parallel orientation to the PEG Unit and L, LP, Z, A, X, D,
m, p and s are as
defined for Formula II
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[00332] In yet other principle embodiments an LDC of the present
invention is
represented by the structure of Formula IV below:
EG
L ______________________ Z¨A, Li P--AD--AD\
I I
X X
1 I
D D /
_ _t mi P
drug-linker (IV)
wherein AD, L, LP, PEG, Z, A, X, D, m, p, s and t are as defined for Formula
III.
[00333] In some embodiments, the antibody-drug conjugates have the
following
formula 1:
L-[LU-Dlp (1)
or a salt thereof, in particular a pharmaceutically acceptable salt, wherein
L is an antibody;
LU is a Linker Unit; and
D' represents from 1 to Drug Units (D) in each drug linker moiety of formula -
LU-D';
and
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 selective binding to an antigen of tumor
tissue for
subsequent release of the Drug Unit as free cytotoxic agent,
wherein the drug linker moiety of formula -LU-D' in each of the antibody-drug
conjugate of the composition has the structure of Formula IA:
¨1¨I_B¨Aa Bb ( LO¨D )
q (IA)
or a salt thereof, in particular a pharmaceutically acceptable salt,
wherein the wavy line indicates covalent attachment to L;
D is the Drug Unit of the cytotoxic agent;
LB is an antibody covalent binding moiety;
A is a first optional Stretcher Unit;
subscript a is 0 or 1 indicating the absence of presence of A, respectively;
B is an optional Branching Unit;
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subscript b is 0 or 1, indicating the absence of presence of B, respectively;
Lo is a secondary linker moiety, wherein the secondary linker has the formula
of;
_FA,a,_w_yy
wherein the wavy line adjacent to Y indicates the site of covalent attachment
of
Lo to the Drug Unit and the wavy line adjacent A' to 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,
subscript a' is 0 or 1, indicating 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., 3-12 or 3-10) amino acids, wherein the
sequence is
comprised of a selectivity conferring tripeptide that provides improved
selectivity for
exposure of tumor tissue over normal tissue to free cytotoxic agent released
from the
antibody-drug conjugates of the composition in comparison to the cytotoxic
agent released
from antibody-drug conjugate composition of a comparator 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 tissues are of rodent species and wherein the
Formula 1
composition provides said improved exposure selectivity demonstrated by:
retaining efficacy in a tumor xenograft model of the comparator antibody-drug
conjugate
composition when administered at the same effective amount and dose schedule
previously
determined for the comparator antibody-drug conjugate composition, and
showing a reduction in plasma concentration of the free cytotoxic agent
released from
the antibody-drug conjugates of the composition, and/or preservation of normal
cells in
tissue when administered at the same effective amount and dose schedule as in
the tumor
xenograft model to a non-tumor bearing rodent in comparison to the equivalent
(e.g., same)
administration of the comparator antibody-drug conjugate composition in which
the
antibody of both conjugate compositions are replaced by a non-binding
antibody,
wherein cytotoxicity to cells in human tissue of the same type as the normal
cells in the
tissue of the non-tumor bearing rodent is responsible at least in part to an
adverse event in a
human subject to whom is administered a therapeutically effective amount of
the comparator
conjugate composition;
Y is a self-immolative Spacer Unit; and
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subscript y is 0, 1 or 2 indicating the absence or presence of 1 or 2 of Y,
respectively;
subscript q is an integer ranging from 1 to 4,
provided that subscript q is 1 when subscript b is 0 and subscript q is 2, 3
or 4 when
subscript b is 1; and
wherein the antibody-drug conjugates of the composition have the structure of
Formula 1
in which 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.
[00334] A related embodiment provides for a Drug Linker of Formula
V:
LU'-(D') (V)
or a salt thereof, in particular a pharmaceutically acceptable salt thereof,
wherein LU' is
capable of providing a covalent bond between L and LU of Formula 1, and
therefore is
sometimes referred to as a Linker Unit precursor; and D' represents from 1 to
4 Drug Units,
wherein the Drug Linker is further defined by the structure of Formula VI:
LB' ¨Aa Bb _________ LO¨D
(VI)
wherein LB' is capable of transformation to LB of Formula VI thereby forming a
covalent
bond to L of Formula 1, and therefore is sometimes referred to an antibody
covalent binding
precursor moiety, and the remaining variable groups of Formula VI are as
defined for
Formula VI.
[00335] 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 2021/055865 Al. Such
ADCs are shown below, wherein Ab comprises an antigen-binding protein (e.g.,
any antibody as
described herein), mc represents a maleimidocaproyl group, mp refers to
maleimidopropionyl:
=-=õ, .
, val-cit (vc) represents a valine-citrulline dipeptide, PABC represents ap-
aminobenzyloxycarbonyl group, and dLAE represents a D-leucine-alanine-glutamic
acid
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tripeptide:
\
/
144:
,..,, r ss., ,......... õ..,. 0,
,
.z.,
t
\ L .,
p
i
1.
e -w.
mc-vc-PABC-MMAE
$ \
el 0 '"Nr."'" 0 **===,-,-"==
c"...'"4 0 \
,e*-1--;k
l'1::' ..,
===" ,....," \ .,..,1L ektet. , ..A. :....k ...---- -,N-4, ..,-\....
õA',....,",,,, . 1
...., ., ...... , .: .
,K....sx, .... .... 1 .; ,...... , ......õ.??.. \ N....,, -
-... ,..... , .1 ...r. 4 a n ,........., ,µ 1 \ \e
µk.'N = k
\ v ,:.,
i
,---,& mc-vc-PABC-MMAF
e
/
/ \
, \ y..0 k
0 ....,... -,.
\r- ,is..,
0 1
1 <1 il 1
-ctr, NI .....õ...---,.....,.....,-\,,,,....-11, N.0,¨......,.. NI :,..õ,,11.,
2,4 .õ.õ..õ....,K,..14 ,...t., ,....14,,,,,....õ,õ., I
\ d i tl i 1 i n
OCH, 0 i 11 j$1 I
OCHa 0
....."' ...\\,Nef.''''''=\:,. = /
\ i
i
\
mc-MIVIAF
\
; o ):" **e'e.- \ \ 3")
=04.-Z:' il 11 ''.
:-.., <,.. ........,,,.õ...õ,,,,,,,,
1 ,.., õ 44 3
.1.;. ,i t...=:*1 ti''' ..4:::X.. <=:' .==;.=Z 1
ii \,,, \ .... ..."4, , ''''s: = J., .......,,, ,==== \......."- \
.,....0 \ ...=:',Z.
4 N.."
..µ= ,,,. t.µ! '''
. / 1
i
\
N sp
mp-dLAE-PABC-MIVIAE. In some embodiments, the drug loading is represented by
p, the
number of drug-linker molecules per antibody. In some embodiments, p can
represent the
average number of drug-linker molecules per antibody in a composition of
antibodies, also
referred to the average drug loading. In some embodiments, p ranges from 1 to
20. In some
embodiments, p ranges from 1 to 8. In some embodiments, when p represents the
average drug
loading, p ranges from 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 per antibody in a
preparation
may be characterized by conventional means such as mass spectroscopy, HIC,
ELISA assay, and
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HPLC. In some embodiments, the antigen-binding protein (e.g,. an antibody) is
attached to the
drug-linker through a cysteine residue of the antibody. In some embodiments,
the cysteine
residue is one that is engineered into the antibody. In some embodiments, the
cysteine residue is
an interchain disulfide cysteine residue.
C. Exemplary ADCs
[00336] Nonlimiting exemplary ADCs for use in the present methods include ADCs
comprising an antibody that binds any of the exemplary targets discussed
herein conjugated to
any of the tubulin disrupters described herein.
[00337] In some embodiments, the ADC is an anti-sialyl Tn antigen antibody-
ADC, which
comprises an antibody that binds to sialyl Tn antigen (sTn) and MMAE. See,
e.g., US Patent
Publication No. 2018/0327509A1; W02017083582A1; Table of Sequences herein.
[00338] In some embodiments, the ADC is belantamab mafodotin, which comprises
an
antibody that binds to B-cell maturation antigen (BCMA) and MMAF. See, e.g.,
US Patent No.
9,273,141.
[00339] In some embodiments, the ADC is an anti-claudin-18.2 ADC, comprising
an
auristatin and an antibody as follows:
zolbetuximab (175D10), disclosed in US Patent No. 8,168,427, and comprising 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
comprising a
heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3
respectively
comprising the amino acid sequences of SEQ ID NOs:61-66;
163E12, disclosed in US Patent No. 8,168,427, and comprising 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 comprising a heavy
chain
CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively
comprising
the amino acid sequences of SEQ ID NOs:69-74;
any anti-claudin-18.2 antibody disclosed in PCT Publication No. WO 2020/135674
Al; or
any anti-claudin-18.2 antibody disclosed in PCT Publication No. WO 2021/032157
Al.
[00340] In some embodiments, the ADC is SGN-PDL1V, comprising an anti-PD-Lb
antibody
and MMAE, the antibody comprising 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 comprising a heavy chain CDR1, CDR2, and
CDR3, and a
light chain CDR1, CDR2, and CDR3 respectively comprising the amino acid
sequences of SEQ
ID NOs:77-82.
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[00341] In some embodiments, the ADC is SGN-ALPV, comprising an anti-ALP
antibody
and MIVIAE, the antibody comprising 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 comprising a heavy chain CDR1, CDR2, and
CDR3, and a
light chain CDR1, CDR2, and CDR3 respectively comprising the amino acid
sequences of SEQ
ID NOs:85-90.
[00342] In some embodiments, the ADC is SGN-B7H4V, comprising an anti-B7H4
antibody
and MIVIAE, the antibody comprising 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 comprising a heavy chain CDR1, CDR2, and
CDR3, and a
light chain CDR1, CDR2, and CDR3 respectively comprising the amino acid
sequences of SEQ
ID NOs:93-98.
[00343] In some embodiments, the ADC is disitamab vedotin, comprising an anti-
HER2
antibody and MMAE, the 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.
[00344] In some embodiments, the ADC is lifastuzumab vedotin, comprising an
anti-NaPi2B
antibody and MMAE, the 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.
[00345] In some embodiments, the ADC is enfortumab vedotin, which comprises an
antibody
that binds nectin-4 and MMAE. See, e.g., US Patent No. 8,637,642; WO
2012/047724. In some
embodiments, the antibody of enfortumab vedotin 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 comprising a heavy
chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:105-110.
[00346] 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
a heavy
chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3
respectively
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comprising the amino acid sequences of SEQ ID NOs:113-118. 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 a heavy
chain CDR1,
CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3 respectively comprising
the
amino acid sequences of SEQ ID NOs:121-126.
[00347] In some embodiments, the ADC is an anti-CD228 antibody-ADC, which
comprises
an antibody that binds CD228 and MMAE. See, e.g., US Patent Publication No.
2020/0246479A1; W02020/163225A1. In some embodiments, the ADC is SGN-CD228A,
comprising an anti-CD228 antibody and MMAE, the antibody comprising 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 or
comprising a
heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3
respectively
comprising the amino acid sequences of SEQ ID NOs:129-134.
[00348] In some embodiments, the ADC is SGN-LIV1A (ladiratuzumab vedotin; LV),
comprising an anti-LIV-1 antibody and MMAE, the antibody comprising 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 comprising
a heavy
chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3
respectively
comprising the amino acid sequences of SEQ ID NOs:137-142;
wherein SGN-LIV1A comprises the anti-LIV-1 antibody conjugated to mc-vc-PABC-
MMAE,
mc-vc-PABC-MMAF, mc-MMAF, or mp-dLAE-PABC-MMAE.
[00349] In some embodiments, the ADC is tisotumab vedotin (TV), which
comprises an
antibody that binds tissue factor (TF) and MMAE. See, e.g., US 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 comprising a heavy chain CDR1, CDR2, and CDR3, and a light chain
CDR1, CDR2,
and CDR3 respectively comprising the amino acid sequences of SEQ ID NOs:145-
150.
[00350] In some embodiments, an ADC comprises MMAE and binds a target selected
from
AMUR'', 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 beta-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, Mesothelin
(MSLN),
MET, MRC2, MUC16, NaPi2b, Nectin-4, OT-MUC1 (onco-tethered-MUC1), PSMA, ROR1,
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SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, Tumor-associated
glycoprotein 72 (TAG-72).
[00351] In some embodiments, an ADC comprises MMAE and is one of: DP303c, also
known as SYSA1501, targeting HER-2 (CSPC Pharmaceutical; Dophen Biomed), SIA01-
ADC,
also known as ST1, targeting STn (Siamab Therapeutics), Ladiratuzumab vedotin,
also known
as SGN-LIV1A, targeting LIV-1 (Merck & Co., Inc.; Seagen (Seattle Genetics)
Inc.), ABBV-
085, also known as Samrotamab vedotin, targeting LRRC15 (Abbvie; Seagen
(Seattle Genetics)
Inc.), DMOT4039A, also known as RG7600; aMSLN-MMAE, targeting Mesothelin
(MSLN)
(Roche-Genentech), RC68, also known as Remegen EGFR ADC, targeting EGFR
(RemeGen
(Rongchang Biopharmaceutical (Yantai) Co., Ltd.)), RC108, also known as RC108-
ADC,
targeting c-MET (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.)),
CMG901, also
known as MRG005, targeting CLDN18.2 (Keymed Biosciences; Lepu biotech;
Shanghai
Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)), YBL-001, also known
as LCB67,
targeting DLK-1 (Lego Chem Biosciences; Pyxis Oncology; Y-Biologics),
DCDS0780A, also
known as Iladatuzumab vedotin; RG7986, targeting CD79b (Roche-Genentech;
Seagen (Seattle
Genetics) Inc.), Tisotumab vedotin, also known as Humax-TF-ADC; tf-011-mmae;
TIVDAKTm,
targeting CD142 (GenMab; Seagen (Seattle Genetics) Inc.), GO-3D1-ADC, also
known as
humAb-3D1-MMAE ADC, targeting MUC1-C (Genus Oncology LLC), ALT-P7, also known
as
HM2-MMAE, targeting HER-2 (Alteogen, Inc.; Levena Biopharma; 3SBio, Inc.),
Vandortuzumab vedotin, also known as D5TP30865; RG7450, targeting STEAP-1
(Roche-
Genentech; Seagen (Seattle Genetics) Inc.), Lifastuzumab Vedotin, also known
as DNIB0600A;
NaPi2b ADC; RG7599, targeting NaPi2b (Roche-Genentech), Sofituzumab vedotin,
also known
as DMUC5754A; RG7458, targeting MUC16 (Seagen (Seattle Genetics) Inc.; Roche-
Genentech), RG7841, also known as DLYE5953A, targeting Ly6E (Roche-Genentech;
Seagen
(Seattle Genetics) Inc.), RG7598, also known as DFRF4539A, targeting FCRH5
(Roche-
Genentech; Seagen (Seattle Genetics) Inc.), RG7636, also known as DEDN6526A,
targeting
ETBR (Seagen (Seattle Genetics) Inc.; Roche-Genentech), Pinatuzumab vedotin,
also known as
DCDT2980S; RG7593, targeting CD22 (Roche-Genentech), Polatuzumab vedotin, also
known
as DCDS4501A; POLIVYTm; RG7596; RO-5541077, targeting CD79b (Chugai
Pharmaceutical;
Roche-Genentech; Seagen (Seattle Genetics) Inc.), DMUC4064A, also known as D-
4064a;
RG7882, targeting MUC16 (Roche-Genentech; Seagen (Seattle Genetics) Inc.),
SYSA1801, also
known as CP0102, targeting CLDN18.2 (Conjupro Biotherapeutics Inc.; CSPC
ZhongQi
Pharmaceutical Technology Co.), RC118, also known as Claudin18.2- ADC; YHO05,
targeting
CLDN18.2 (RemeGen (Rongchang Biopharmaceutical (Yantai) Co., Ltd.);
Biocytogen), VLS-
101, also known as Cirmtuzumab vedotin; MK-2140; UC-961ADC3; Zilovertamab
Vedotin,
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targeting ROR1 (VelosBio. Inc), Glembatumumab vedotin, also known as CDX-011;
CR011-
vcMMAE, targeting gpNMB (Celldex Therapeutics), BA3021, also known as CAB-ROR2-
ADC; Ozuriftamab Vedotin, targeting ROR2 (Bioatla; Himalaya Therapeutics),
BA3011, also
known as CAB-AXL-ADC; Mecbotamab Vedotin, targeting Axl (Bioatla; Himalaya
Therapeutics), CM-09, also known as Bstrongximab-ADC, targeting TRA-1-60
(CureMeta),
ABBV-838, also known as Azintuxizumab vedotin, targeting SLAMF7 (Abbvie),
Enapotamab
vedotin, also known as AXL-107-MMAE; HuMax-AXL-ADC, targeting Axl (GenMab;
Seagen
(Seattle Genetics) Inc.), ARC-01, also known as anti-CD79b ADC, targeting
CD79b (Araris
Biotech AG), Disitamab vedotin, also known as Aidexig; RC48, targeting HER-2
(RemeGen
(Rongchang Biopharmaceutical (Yantai) Co., Ltd.); Seagen (Seattle Genetics)
Inc.), ASG-5ME,
also known as AGS-5; AGS-5ME, targeting 5LC44A4 (Agensys, Inc.; Astellas
Pharma Inc.;
Seagen (Seattle Genetics) Inc.), Enfortumab vedotin, also known as AGS-22M6E;
ASG-22CE;
ASG-22ME; PADCEVTM, targeting Nectin-4 (Astellas Pharma Inc.; Seagen (Seattle
Genetics)
Inc.), ASG-15ME, also known as AGS-15E; Sirtratumab vedotin, targeting SLITRK6
(Seagen
(Seattle Genetics) Inc.; Astellas Pharma Inc.), Brentuximab vedotin, also
known as Adcetris;
cAC10-vcMMAE; SGN-35, targeting CD30 (Seagen (Seattle Genetics) Inc.; Takeda),
Telisotuzumab vedotin, also known as ABBV-399, targeting c-MET (Abbvie),
Losatuxizumab
vedotin, also known as ABBV-221, targeting EGFR (Abbvie), CX-2029, also known
as ABBV-
2029, targeting CD71 (Abbvie; CytomX Therapeutics), AB-3A4-ADC, also known as
AB-3A4-
vcMMAE, targeting KAAG-1 (Alethia Biotherapeutics), Indusatumab vedotin, also
known as
5F9-vcMMAE; 1V11LN0264; TAK-264, targeting GCC (Takeda; Millennium
Pharmaceuticals,
Inc), F0R46 targeting CD46 (Fortis Therapeutics, Inc.), LR004-VC-MMAE
targeting EGFR
(Chinese Academy of Medical Sciences Peking Union Medical College Hospital),
CD30-ADCs
targeting CD30 (NBE Therapeutics; Boehringer Ingelheim), Anti-endosialin-MC-VC-
PABC-
MMAE targeting CD248 (Genzyme), OBI-998 targeting SSEA-4 (OBI Pharma), MRG002
targeting HER-2 (Lepu biotech; Shanghai Miracogen Inc. (Shanghai Meiya
Biotechnology Co.,
Ltd)), TRS005 targeting CD20 (Teruisi Pharmaceuticals), Oba01 targeting DR5
(Death receptor
5) (Obio Technology (Shanghai) Corp.,Ltd.; Yantai Obioadc Biomedical
Technology Ltd.),
PSMA ADC targeting PSMA (Progenics Pharmaceuticals, Inc; Seagen (Seattle
Genetics) Inc.),
SGN-CD48A targeting CD48 (Seagen (Seattle Genetics) Inc.), IIIVIAB362-vcMMAE
targeting
CLDN18.2 (Astellas Pharma Inc.; Ganymed), GB251 targeting HER-2 (Genor
Biopharma Co.,
Ltd.), Innate Pharma BTG-ADCs targeting CD30 (Innate Pharma; Sanofi), ADCendo
uPARAP
ADC targeting MRC2 (ADCendo), XCN-010 targeting actM (Xiconic Pharmaceuticals,
LLC),
ANT-043 targeting HER-2 (Antikor Biopharma), OBI-999 targeting Globo H
(Abzena; OBI
Pharma), LY3343544 targeting MET (Eli Lilly and Company), Tagworks anti-TAG72
ADC
72
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WO 2022/098972 PCT/US2021/058208
targeting TAG-72 (Tagworks Pharmaceuticals), IIVIAB027-vcMMAE targeting CLDN6
(Ganymed; Astellas Pharma Inc.), LGR5-ADC targeting LGR5 (Genentech, Inc.),
Philochem
B12-MMAE ADC targeting IL-7R (Institut de Medicina Molecular Joao Lobo
Antunes;
Philochem AG), TE-1522 targeting CD19 (Immunwork), SGN-STNV targeting STn
(Seagen
(Seattle Genetics) Inc.), HTI-1511 targeting EGFR (Abzena; Halozyme
Therapeutics), Peptron
PAb001-ADC targeting OT-MUC1 (onco-tethered-MUC1) (Peptron; Qilu
Pharmaceutical co.
Ltd.), LM-102 targeting CLDN18.2 (Lallova Medicines Limited), Anwita
Biosciences MSLN-
MMAE targeting Mesothelin (MSLN) (Anwita biosciences), SGN-CD228A targeting
CD228
(Seagen (Seattle Genetics) Inc.), NBT828 targeting HER-2 (NewBio Therapeutics;
Genor
Biopharma Co., Ltd.), Gamamabs GM103 targeting AMHR2 (GamaMabs Pharma;
Exelixis),
LCB14-0302 targeting HER-2 (Lego Chem Biosciences), BAY79-4620 targeting
carbonic
anhydrase IX (CAIX) (Bayer; MorphoSys), NBT508 targeting CD79b (NewBio
Therapeutics),
PAT-DX3-MMAE targeting Undisclosed (Patrys; Yale University), AGS67E targeting
CD37
(Astellas Pharma Inc.; Seagen (Seattle Genetics) Inc.), CDX-014 targeting TIM-
1 (Celldex
Therapeutics), BVX001 targeting CD33; CD7 (Bivictrix therapeutics), SGN-B6A
targeting
Integrin beta-6 (Seagen (Seattle Genetics) Inc.), MRG003 targeting EGFR (Lepu
biotech;
Shanghai Miracogen Inc. (Shanghai Meiya Biotechnology Co., Ltd)), and PYX-202
targeting
DLK-1 (Pyxis Oncology; Lego Chem Biosciences).
[00352] In some embodiments an ADC comprises MMAF and binds a target selected
from
BCMA, GPC-1, CD30, c-MET, SAIL, HER-3, CD70, CD46, HER-2, 5T4, ENPP3, CD19,
EGFR, EphA2.
[00353] In some embodiments, an ADC comprises MMAF and is one of: CD70-ADC
targeting CD70 (Kochi University; Osaka University), IGN786 targeting SAIL
(AstraZeneca;
Igenica Biotherapeutics), PF-06263507 targeting 5T4 (Pfizer), GPC1-ADC
targeting GPC-1
(Kochi University), ADC-AVP10 targeting CD30 (Avipep), M290-MC-MMAF targeting
CD103 (The Second Affiliated Hospital of Harbin Medical University), BVX001
targeting
CD33; CD7 (Bivictrix therapeutics), Tanabe P3D12-vc-MMAF targeting c-MET
(Tanabe
Research Laboratories), LILRB4-Targeting ADC targeting LILRB4 (The University
of Texas
Health Science Center, Houston), TSD101, also known as ABL201, targeting BCMA
(TSD Life
Science; ABL Bio; Lego Chem Biosciences), Depatuxizumab mafodotin, also known
as ABT-
414, targeting EGFR (Abbvie; Seagen (Seattle Genetics) Inc.), AG516F, also
known as AGS-
16C3F; AGS-16M8F, targeting ENPP3 (Astellas Pharma Inc.; Seagen (Seattle
Genetics) Inc.),
AVG-All BCMA ADC, also known as AVG-All-mcMMAF, targeting BCMA (Avantgen),
Belantamab mafodotin, also known as BLENREP; G5K2857916; J6M0-mcMMAF,
targeting
BCMA (GlaxoSmithKline; Seagen (Seattle Genetics) Inc.), MP-HER3-ADC, also
known as
73
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WO 2022/098972 PCT/US2021/058208
HER3-ADC, targeting HER-3 (MediaPharma), FS-1502, also known as LCB14-0110,
targeting
HER-2 (Lego Chem Biosciences; Shanghai Fosun Pharmaceutical Development Co,
Ltd.),
MEDI-547, also known as MI-CP177, targeting EphA2 (AstraZeneca; Seagen
(Seattle Genetics)
Inc.), Vorsetuzumab mafodotin, also known as SGN-75, targeting CD70 (Seagen
(Seattle
Genetics) Inc.), Denintuzumab mafodotin, also known as SGN-CD19A, targeting
CD19 (Seagen
(Seattle Genetics) Inc.), and HTI-1066, also known as SHR-A1403, targeting c-
MET (Jiangsu
HengRui Medicine Co., Ltd).
[00354] In some embodiments, an ADC is selected from the ADCs in Table A,
Table B, or
Table C. In Table A, Table B, and Table C, the ADCs with sequences provided in
the Table of
Sequences are marked with an asterisk (*). In some embodiments the ADC is not
enfortumab
vedotin. In certain embodiments, the ADC is not brentuximab vedotin. In some
embodiments,
the ADC is not tisotumab vedotin. In some embodiments, the ADC is not
ladiratuzumab
vedotin. In some embodiments, the ADC is not SGN-CD228A.
74
Table A
_______________________________________________________________________________
___________________________________________ 0
Patent Drug Names Target Moiety
Linker Payload t..)
o
Polatuzumab Isotype: IgG1
t..)
t..)
US 8,545,850 Polatuzumab vedotin CD79b Origin: Humanized
Valine-Citrulline MIVIAE O-
,o
cio
Gemtuzumab Gemtuzumab Isotype: IgG4
,o
-4
t..)
ozogamicin CD33 Origin: Humanized
AcBut acyl hydrazone-disulfide Calicheamicin
Belantamab (J6M0) Isotype:
IgG1 Origin: Humanized
US 9,273,141 Belantamab mafodotin BCMA Format: mAB
mc MMAF
Trastuzumab Trastuzumab Isotype: IgG1
GGFG (Glycine-Glycine- DXd/DX8951
US 9,808,537 deruxtecan HER-2 Origin: Humanized
Phenylalanine-Glycine) (MAAA-1181a)
US 8,637,642 (WO Enfortumab Isotype: IgGlk
2012/047724) Enfortumab vedotin* Nectin-4 Origin: Human
Valine-Citrulline MIVIAE P
Inotuzumab Inotuzumab Isotype: IgG4
g
US 8,153,768 ozogamicin CD22 Origin: Humanized
AcBut acyl hydrazone-disulfide Calicheamicin
Brentuximab vedotin Brentuximab Isotype: IgG1
,9
---1
,õ
(a, WO 2004/010957 (5GN-35)* CD30 Origin: Chimeric
Valine-Citrulline MMAE
,
Sacituzumab
(hR57) Isotype: IgG1
US 7,517,964; US Origin: Humanized Format:
8,877,901 Sacituzumab govitecan TROP-2 mAB
CL2A SN-38
Trastuzumab Trastuzumab Isotype: IgG1
US 8,337,856 emtansine HER-2 Origin: Humanized
SMCC DM1
1-d
n
1-i
cp
t..)
=
t..)
,-,
'a
u,
oe
t..)
=
oe
Table B
Other Drug
0
t..)
Drug Names Names Target Moiety
Linker Payload =
t..)
t..)
CX-2029 ABBV-2029 CD71 Probody
Valine-Citrulline MMAE O-
,o
DP303c HER-2
Unknown MMAE cio
,o
-4
t..)
anti-C-MET Isotype: IgG2
Origin: Humanized Format:
HTI-1066 SHR-A1403 c-MET mAB
3-proplythio-mc MMAF
23AG2 Isotype: IgG1 Origin:
Human Format: mAB Other:
FOR46 CD46 anti-CD46 humanized
Valine-Citrulline MMAF
Anti-FLT3 monoclonal
antibody Isotype: IgG1
P
AGS62P1 ASP1235 FLT3 Origin: Human
Oxime AGD-0182
BT8009 Nectin-4
Valine-Citrulline MMAE
ARX788 HER-2
Oxime Amberstatin269
---1
"
XMT-1535 Isotype: IgG1
,
Origin: Humanized Format:
XMT-1536 NaPi2b mAB
Fleximer Polymer Auristatin F-HPA
Trastuzumab Isotype: IgG1
Origin: Humanized Format:
mAB Other: NexMab variant
ALT-P7 HM2-MMAE HER-2 of trastuzumab
Valine-Citrulline MMAE
P-glucuronidase (BG)
1-d
FS-1502 LCB14-0110 HER-2 Trastuzumab
linker MMAF n
1-i
PF-06647020,
cp
Cofetuzumab PTK7-ADC, PF- h6M24 Isotype: IgG1
Origin: t..)
o
t..)
pelidotin 7020, ABBV-647 PTK7 Humanized Format: mAB
Valine-Citrulline PF-06380101
O-
Rituximab Isotype: IgG1
u,
oo
t..)
Origin: Chimeric Format:
TRS005 CD20 mAb
Valine-Citrulline MMAE
CD38 ADC,
LNDS1001, STI-5171 Isotype: IgG1
0
STI-6129 CD38-077 ADC CD38 Origin: Human Format:
mAB Unknown Duostatin 5.2 t..)
o
HuMab (HuMax
t..)
t..)
O-
Tisotumab Humax-TF-ADC, antibody) Isotype: IgG1
,.tD
cio
vedotin* tf-011-mmae CD142 Origin: Human
Valine-Citrulline MMAE ,.tD
-.1
t..)
Cirmtuzumab (UC-
Cirmtuzumab VLS-101, UC- 961) Isotype: IgG1
Origin:
vedotin 961ADC3 ROR1 Humanized Format: mAB
Protease Cleavable MMAE
AbGn-7 Isotype: IgG1 Origin:
AbGn-107 Ab1-18Hr1 AG-7 Humanized Format: mAB
Valine-Citrulline MIVIAD
Undisclosed Format: scFvFc
ASN-004 5T4 antibody
Fleximer Polymer Dolastatin
P
SGN-B6A* Integrin beta-6 h2A2 Origin: Humanized
Valine-Citrulline MMAE .
Hertuzumab Isotype: IgG1 o Disitamab
Origin: Humanized Format:
vedotin* RC48 HER-2 mAB
Valine-Citrulline MMAE 2
---1
w
,
--A Telisotuzumab
u2
,
vedotin ABBV-399 c-MET ABT-700
Valine-Citrulline MMAE u2
HuMax-AXL-
Enapotamab ADC, AXL-107- HuMax Antibody Origin:
vedotin MMAE Axl Human Format: Full
length Valine-Citrulline MMAE
OBI-888 Isotype: IgG1
Origin: Humanized Format:
OBI-999 Globo H mAb
Disulphide MMAE 1-d
n
XMT-1592 NaPi2b Unknown
Unknown Auristatin F-HPA
hL49 (anti-CD228A
cp
t..)
monoclonal antibody) Origin: P-glucuronidase (BG)
=
t..)
SGN-CD228A CD228 Humanized Format: mAB
linker MMAE
O-
u,
Ladiratuzumab (hLIV-
cao
t..)
o
Ladiratuzumab 22) Isotype: IgG1
Origin: cee
vedotin* SGN-LIV1A LIV-1 Humanized Format: mAB
Valine-Citrulline MMAE
BT5528 EphA2 Unknown
Valine-Citrulline MMAE
Unknown Isotype: IgG1
0
PF-06804103 NG-HER2 ADC HER-2 Origin: Human
Valine-Citrulline PF-06380101 (Aur 101) a)
hz208F2-4 (anti-IGF1R
antibody) Origin: Humanized
W0101 IGF-1R Format: mAB
mc Auristatin
CAB-Ax! Other:
Conditionally Active
Biologics (CAB) anti-Ax!
BA3011 CAB-AXL-ADC Ax! antibody
Unknown MMAE
anti-HER2 Isotype: IgG1
Origin: Humanized Format:
MRG002 HER-2 mAB
Unknown MMAE
ZW25 Isotype: IgG1 Format:
Bispecific, ZW25 Isotype:
ZW49 HER-2,HER-2 IgG1 Format:
Bispecific Valine-Citrulline Auristatin
00
Table C
Patent ADC names
targets:
CD142 (tissue
US 9,168,314 (WO 2011/157741) Tisotumab vedotin (TV)*
factor)
US 9,273,141 Belantamab mafodotin BCMA
1-d
US 8,637,642 (WO 2012/047724) Enfortumab vedotin (EV)* Nectin-
4
US 11,028,181 (W02017083582A1); see Table
of Sequences herein SGN-STNV STN
Enapotamab vedotin Ax!
US 2020/0246479 (WO 2020/163225; VH/VL
of SEQ ID NO:7 and 8, respectively (CDRs SEQ
ID NOs: 1-6)); see Table of Sequences herein SGN-CD228A* CD228
US 2021/0198367 (claiming priority to USSN
62/943,959 and USSN 62/012,584); See Table of
0
Sequences herein SGN-B6A* Integrin
beta-6
Ladiratuzumab vedotin
W02012/078688 (LV)* LIV-1
cio
Brentuximab vedotin (SGN-
WO 2004/010957 35)* CD30
US 8,329,173 Telisotuzumab vedotin c-MET
US 8,545,850 Polatuzumab vedotin CD79b
US 7,662,387 Vorsetuzumab mafodotin CD70
1-d
cio
cio
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D. Preparation of Antibodies
[00355] For preparing an antibody, many techniques known in the art can be
used. See, e.g.,
Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et at., Immunology Today
4: 72 (1983);
Cole et at., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 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)).
[00356] The genes encoding the heavy and light chains of an antibody of
interest can be
cloned from a cell, e.g., the genes encoding a monoclonal antibody can be
cloned from a
hybridoma that expresses the antibody and used to produce a recombinant
monoclonal antibody.
Gene libraries encoding heavy and light chains of monoclonal antibodies can
also be made from
hybridoma or plasma cells. Additionally, phage or yeast display technology can
be used to
identify antibodies and heteromeric Fab fragments that specifically bind to
selected antigens
(see, e.g., 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,
1:7 55-7 68
(2006)). Alternatively, antibodies and antibody sequences may be isolated
and/or identified
using a yeast-based antibody presentation system, such as that disclosed in,
e.g., Xu et al.,
Protein Eng Des Set, 2013, 26:663-670; WO 2009/036379; WO 2010/105256; and WO
2012/009568. Random combinations of the heavy and light chain gene products
generate a large
pool of antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3rd ed.
1997)). Techniques for the production of single chain antibodies or
recombinant antibodies
(U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can also be adapted to
produce antibodies.
Antibodies can also be made bispecific, i.e., able to recognize two different
antigens (see, e.g.,
WO 93/08829, Traunecker et at., EMBO 1 10:3655-3659 (1991); and Suresh et at.,
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. Patent No.
4,676,980, WO 91/00360; and WO 92/200373).
[00357] Antibodies can be produced using any number of expression systems,
including
prokaryotic and eukaryotic expression systems. 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 an antibody comprises both
a heavy chain
and light chain, the heavy chain and heavy chain and light chain may be
expressed using a single
vector, e.g., in a di-cistronic expression unit, or be under the control of
different promoters. In
other embodiments, the heavy chain and light chain region may be expressed
using separate
vectors. Heavy chains and light chains as described herein may optionally
comprise a
methionine at the N-terminus.
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[00358] In some embodiments, antibody fragments (such as a Fab, a Fab', a
F(ab')2, a scFv,
or a diabody) are generated. Various techniques have been developed for the
production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of
intact antibodies (see, e.g., Morimoto et al., I Biochem. Biophys. Meth.,
24:107-117 (1992); and
Brennan et at., 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 E.
coil cells and chemically coupled to form F(ab')2 fragments (see, e.g., Carter
et at.,
BioTechnology, 10:163-167 (1992)). According to another approach, F(ab')2
fragments can be
isolated directly from recombinant host cell culture. Other techniques for the
production of
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.
[00359] In some embodiments, the antibody or antibody fragment can be
conjugated to
another molecule, e.g., polyethylene glycol (PEGylation) or serum albumin, to
provide an
extended half-life in vivo. Examples of PEGylation of antibody fragments are
provided in
Knight et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br. I
Cancer 70:1126, 1994
(for an anti-CEA antibody); Chapman et at., Nature Biotech. 17:780, 1999; and
Humphreys, et
at., Protein Eng. Des. 20: 227, 2007).
[00360] In some embodiments, multispecific antibodies are provided, e.g., a
bispecific
antibody. Multispecific antibodies are antibodies that have binding
specificities 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 chain and light chain in a host cell (see, e.g., Zuo et al., Protein
Eng Des Set, 2000,
13:361-367); "knobs-into-holes" engineering (see, e.g., Ridgway et al.,
Protein Eng Des Set,
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), Part A:95-106.
Selection of Constant Region
[00361] Heavy and light chain variable regions of the antibodies described
herein can be
linked to at least a portion of a human constant region. The choice of
constant region depends, in
part, whether antibody-dependent cell-mediated cytotoxicity, antibody
dependent cellular
phagocytosis and/or complement dependent cytotoxicity are desired. For
example, human
81
CA 03200974 2023-05-05
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isotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human
isotype IgG2
weak complement-dependent cytotoxicity and human IgG4 lacks complement-
dependent
cytotoxicity. Human IgG1 and IgG3 also induce stronger cell mediated effector
functions than
human IgG2 and IgG4. Light chain constant regions can be lambda or kappa.
Antibodies can
be expressed as tetramers containing two light and two heavy chains, as
separate heavy chains,
light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in
which heavy and light
chain variable domains are linked through a spacer.
[00362] Human constant regions show allotypic variation and isoallotypic
variation between
different individuals, that is, the constant regions can differ in different
individuals at one or
more polymorphic positions. Isoallotypes differ from allotypes in that sera
recognizing an
isoallotype binds to a non-polymorphic region of one or more other isotypes.
[00363] One or several amino acids at the amino or carboxy terminus of the
light and/or
heavy chain, such as the C-terminal lysine of the heavy chain, may be missing
or derivatized in a
proportion or all of the molecules. Substitutions can be made in the constant
regions to reduce
or increase effector function such as complement-mediated cytotoxicity or ADCC
(see, e.g.,
Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597;
and Lazar et al.,
Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans
(see, e.g., Hinton
et al., J. Biol. Chem. 279:6213, 2004).
[00364] For constructing desired antibody-drug conjugates, in some
embodiments, exemplary
substitution include the amino acid substitution of the native amino acid to a
cysteine residue is
introduced at amino acid position 234, 235, 237, 239, 267, 298, 299, 326, 330,
or 332,
preferably an 5239C mutation in a 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 20100158909, which is herein incorporated
reference).
The presence of an additional cysteine residue may allow interchain disulfide
bond formation.
Such interchain disulfide bond formation can cause steric hindrance, thereby
reducing the
affinity of the Fc region-FcyR binding interaction. The cysteine residue(s)
introduced in or in
proximity to the Fc region of an IgG constant region can also serve as sites
for conjugation to
therapeutic agents (i.e., coupling cytotoxic drugs using thiol specific
reagents such as maleimide
derivatives of drugs. The presence of a therapeutic agent causes steric
hindrance, thereby further
reducing the affinity of the Fc region-FcyR binding interaction. Other
substitutions at any of
positions 234, 235, 236 and/or 237 reduce affinity for Fcy receptors,
particularly FcyRI receptor
(see, e.g., US 6,624,821, US 5,624,821.)
[00365] The in vivo half-life of an antibody can also impact its effector
functions. The half-
life of an antibody can be increased or decreased to modify its therapeutic
activities. FcRn is a
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receptor that is structurally similar to MHC Class I antigen that non-
covalently associates with
f32-microglobulin. FcRn regulates the catabolism of IgGs and their
transcytosis across tissues
(Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002,
Immunol.
Res. 25:97-113). The IgG-FcRn interaction takes place at pH 6.0 (pH of
intracellular vesicles)
but not at pH 7.4 (pH of blood); this interaction enables IgGs to be recycled
back to 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 at., 2001, 1 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., 2001,1 Biol. Chem. 276:6591-604). IgG1 molecules
harboring
these substitutions have longer serum half-lives. Consequently, these modified
IgG1 molecules
may be able to carry out their effector functions, and hence exert their
therapeutic efficacies,
over a longer period of time compared to unmodified IgGl. Other exemplary
substitutions for
increasing binding to FcRn include a Gln at position 250 and/or a Leu at
position 428. EU
numbering is used for all positions in the constant region.
[00366] Complement fixation activity of antibodies (both Clq binding and CDC
activity) can
be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001,1
Immunol.
166:2571-2575). The same substitutions on a human IgG2 backbone can convert an
antibody
isotype that binds poorly to Clq and is severely deficient in complement
activation activity to
one that can both bind Clq and mediate CDC (Idusogie et at., 2001, 1 Immunol.
166:2571-75).
Several other methods have also been applied to improve complement fixation
activity of
antibodies. For example, the grafting of an 18-amino acid carboxyl-terminal
tail piece of IgM to
the carboxyl-termini of IgG greatly enhances their CDC activity. This is
observed even with
IgG4, which normally has no detectable CDC activity (Smith et at., 1995, 1
Immunol.
154:2226-36). Also, substituting 5er444 located close to the carboxy-terminal
of IgG1 heavy
chain with Cys induced tail-to-tail dimerization of IgG1 with a 200-fold
increase of CDC
activity over monomeric IgG1 (Shopes et at., 1992, 1 Immunol. 148:2918-22). In
addition, a
bispecific diabody construct with specificity for Clq also confers CDC
activity (Kontermann et
at., 1997, Nat. Biotech. 15:629-31).
[00367] 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 having a different
side chain, such as
Ala. Other alkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or
Val, or such aromatic
non-polar residues as Phe, Tyr, Trp and Pro in place of any one of the three
residues also reduce
or abolish Clq binding. Ser, Thr, Cys, and Met can be used at residues 320 and
322, but not 318,
to reduce or abolish Clq binding activity. Replacement of the 318 (Glu)
residue by a polar
83
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residue may modify but not abolish Clq binding activity. Replacing residue 297
(Asn) with Ala
results in removal of lytic activity but only slightly reduces (about three
fold weaker) affinity for
Clq. This alteration destroys the glycosylation site and the presence of
carbohydrate that is
required for complement activation. Any other substitution at this site also
destroys the
glycosylation site. The following mutations and any combination thereof also
reduce Clq
binding: D270A, K322A, P329A, and P311S (see WO 06/036291).
[00368] Reference to a human constant region includes a constant region with
any natural
allotype or any permutation of residues occupying polymorphic positions in
natural allotypes.
Also, up to 1, 2, 5, or 10 mutations may be present relative to a natural
human constant region,
such as those indicated above to reduce Fcy receptor binding or increase
binding to FcRN.
Nucleic Acids, Vectors, and Host Cells
[00369] In some embodiments, the antibodies described herein are prepared
using
recombinant methods. Accordingly, in some aspects, the invention provides
isolated nucleic
acids comprising a nucleic acid sequence 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 into which the nucleic acids are introduced that are used to replicate
the antibody-encoding
nucleic acids and/or to express the antibodies. In some embodiments, the host
cell is eukaryotic,
e.g., a Chinese Hamster Ovary (CHO) cell; or a human cell.
[00370] In some embodiments, a polynucleotide (e.g., an isolated
polynucleotide) comprises
a nucleotide sequence encoding an antibody described herein. In some
embodiments, the
polynucleotide comprises a nucleotide sequence encoding one or more amino acid
sequences
(e.g., CDR, heavy chain, light chain, and/or framework regions) disclosed
herein. In some
embodiments, the polynucleotide comprises a nucleotide sequence encoding an
amino acid
sequence 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) to a sequence (e.g., a CDR, heavy chain, light
chain, or framework
region sequence) disclosed herein.
[00371] In a further aspect, methods of making an antibody described herein
are provided. In
some embodiments, the method includes culturing a host cell as described
herein (e.g., a host
cell expressing a polynucleotide or vector as described herein) under
conditions suitable for
expression of the antibody. In some embodiments, the antibody is subsequently
recovered from
the host cell (or host cell culture medium).
[00372] Suitable vectors containing polynucleotides encoding antibodies of
the present
disclosure, or fragments thereof, include cloning vectors and expression
vectors. While the
cloning vector selected may vary according to the host cell intended to be
used, useful cloning
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vectors generally have the ability to self-replicate, may possess a single
target for a particular
restriction endonuclease, and/or may carry genes for a marker that can be used
in selecting
clones containing the vector. Examples include plasmids and bacterial viruses,
e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322,
pMB9, ColE1,
pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. Cloning
vectors are
available from commercial vendors such as BioRad, Stratagene, and Invitrogen.
[00373] Expression vectors generally are replicable polynucleotide constructs
that contain a
nucleic acid of the present disclosure. The expression vector may replicate in
the host cells either
as episomes 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 vector.
Expression of Recombinant Antibodies
[00374] Antibodies are typically produced by recombinant expression.
Recombinant
polynucleotide constructs typically include an expression control sequence
operably linked to
the coding sequences of antibody chains, including naturally-associated or
heterologous
promoter regions. Preferably, the expression control sequences are eukaryotic
promoter systems
in vectors capable of transforming or transfecting eukaryotic host cells. Once
the vector has
been incorporated into the appropriate host, the host is maintained under
conditions suitable for
high level expression of the nucleotide sequences, and the collection and
purification of the
cross-reacting antibodies.
[00375] Mammalian cells are a preferred host for expressing nucleotide
segments encoding
immunoglobulins or fragments thereof See Winnacker, From Genes to Clones, (VCH
Publishers, NY, 1987). A number of suitable host cell lines capable of
secreting intact
heterologous proteins have been developed in the art, and include CHO cell
lines (e.g., DG44),
various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-
producing
myelomas including 5p2/0 and NSO. Preferably, the cells are nonhuman.
Expression vectors
for these cells can include expression control sequences, such as an origin of
replication, a
promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and
necessary processing
information sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites, and
transcriptional terminator sequences. Preferred expression control sequences
are promoters
derived from endogenous genes, cytomegalovirus, 5V40, adenovirus, bovine
papillomavirus,
and the like. See Co etal.,i Immunol. 148:1149 (1992).
[00376] Once expressed, antibodies can be purified according to standard
procedures of the
art, including HPLC purification, column chromatography, gel electrophoresis
and the like (see
generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
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Antibody Characterization
[00377] Methods for analyzing binding affinity, binding kinetics, and cross-
reactivity are
known in the art. See, e.g., Ernst et at., 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 assay),
immunoprecipitation, surface
plasmon resonance (SPR, e.g., BiacoreTM (GE Healthcare, Piscataway, NJ)),
kinetic exclusion
assays (e.g. KinExAg), flow cytometry, fluorescence-activated cell sorting
(FACS), BioLayer
interferometry (e.g., OctetTM (ForteBio, Inc., Menlo Park, CA)), and Western
blot analysis. SPR
techniques are reviewed, e.g., in Hahnfeld et at. Determination of Kinetic
Data Using SPR
Biosensors, Molecular Diagnosis of Infectious Diseases (2004). In a typical
SPR experiment,
one interactant (target or targeting agent) is immobilized on an SPR-active,
gold-coated glass
slide in a flow cell, and a sample containing the other interactant is
introduced to flow across the
surface. When light of a given wavelength is shined on the surface, the
changes to the optical
reflectivity of the gold indicate binding, and the kinetics of binding. In
some embodiments,
kinetic exclusion assays are used to determine affinity. This technique is
described, e.g., in
Darling et at., Assay and Drug Development Technologies Vol. 2, number 6 647-
657 (2004). In
some embodiments, BioLayer interferometry assays are used to determine
affinity. This
technique is described, e.g., in Wilson et al., Biochemistry and Molecular
Biology Education,
38:400-407 (2010); Dysinger et al., I Immunol. Methods, 379:30-41 (2012).
IV. Therapeutic Methods
[00378] In some embodiments, methods for treating cancer in a subject are
provided. In some
embodiments, the method comprises administering to the subject (1) an antibody-
drug conjugate
(ADC) that comprises a first antibody that binds a tumor-associated antigen
and a cytotoxic
agent, wherein the cytotoxic agent is a tubulin disrupter; and (2) a second
antibody that binds to
an immune cell engager, wherein the second antibody comprises an Fc with
enhanced binding to
one or more activating FcyRs. In some embodiments, the Fc of the second
antibody has
enhanced binding to one or more of FcyRIIIa, FcyRIIa, and/or FcyRI. In some
embodiments,
the Fc of the second antibody has reduced binding to one or more inhibitory
FcyRs. In some
embodiments, the Fc of the second antibody has reduced binding to FcyRIIb.
[00379] In some embodiments, a method of treating cancer comprises
administering to a
subject with 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 tubulin disrupter, and (2) a second antibody that binds an immune
cell engager,
wherein the second antibody comprises an Fc with enhanced ADCC activity
relative to a
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corresponding wild-type Fc of the same isotype. In some embodiments, the
second antibody
comprises an Fc with 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 has
enhanced
binding to one or more of FcyRIIIa, FcyRIIa, and/or FcyRI. In some
embodiments, the Fc of the
second antibody has reduced binding to one or more inhibitory FcyRs. In some
embodiments,
the Fc of the second antibody has reduced binding to FcyRIIb.
[00380] In various embodiments, the second antibody is a nonfucosylated
antibody. In
various such embodiments, the second antibody is comprised in a composition of
antibodies,
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
nonfucosylated.
[00381] In some embodiments, the second antibody binds TIGIT. In some
embodiments, the
second antibody binds CD40. In some embodiments, the second antibody binds an
immune cell
engager provided herein.
[00382] In various embodiments, the tubulin disrupter conjugated to the first
antibody in the
ADC is an auristatins, a tubulysin, a colchicine, a vinca alkaloid, a taxane,
a cryptophycin, a
maytansinoid, or a hemiasterlin. In some embodiments, the ADC comprises MMAE
or MMAF.
In various embodiments, the first antibody binds a tumor-associated antigen,
such as a tumor-
associated antigen provided herein.
[00383] Any of the ADCs described herein may be combined with any of the
antibodies that
bind 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
lifastuzumab vedotin, 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-
LIVIA, 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
brentuximab vedotin (SGN-35), and the second antibody is SEA-BCMA. In some
embodiments, the ADC is enfortumab vedotin, and the second antibody is SEA-
BCMA. In
some embodiments, the ADC is disitamab vedotin, and the second antibody is SEA-
BCMA. In
some embodiments, the ADC is tisotumab vedotin, and the second antibody is SEA-
BCMA.
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[00384] 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 lifastuzumab vedotin, 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 SEA-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 brentuximab vedotin (SGN-35), and the second antibody
is SEA-
CD40. In some embodiments, the ADC is enfortumab vedotin, and the second
antibody is SEA-
CD40. In some embodiments, the ADC is disitamab vedotin, and the second
antibody is SEA-
CD40. In some embodiments, the ADC is tisotumab vedotin, and the second
antibody is SEA-
CD40.
[00385] 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 lifastuzumab vedotin, 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 brentuximab vedotin (SGN-35), and the second antibody
is SEA-
CD70. In some embodiments, the ADC is enfortumab vedotin, and the second
antibody is SEA-
CD70. In some embodiments, the ADC is disitamab vedotin, and the second
antibody is SEA-
CD70. In some embodiments, the ADC is tisotumab vedotin, and the second
antibody is SEA-
CD70.
[00386] 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 lifastuzumab vedotin, and the second antibody is
SEA-TGT. In
some embodiments, the ADC is SEA-CD40, and the second antibody is SEA-TGT. In
some
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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 brentuximab vedotin (SGN-35), and the second antibody
is SEA-
TGT. In some embodiments, the ADC is enfortumab vedotin, and the second
antibody is SEA-
TGT. In some embodiments, the ADC is disitamab vedotin, and the second
antibody is SEA-
TGT. In some embodiments, the ADC is tisotumab vedotin, and the second
antibody is SEA-
TGT.
[00387] In some embodiments, the subject is a human.
[00388] 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, clear cell renal carcinoma, head and neck cancer, lung cancer, lung
adenocarcinoma,
stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,
skin cancer,
melanoma, neoplasm of the central nervous system, 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 clear cell renal
carcinoma. In some
embodiments, the cancer is a lymphoma or a 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 lymphoma. In some embodiments, the cancer is a metastatic cancer.
[00389] In some embodiments, the cancer is one with high tumor mutation burden
as such
cancers have more antigen to drive T cell responses. Thus, in some
embodiments, the cancer is
a high mutational burden cancer such as lung, melanoma, bladder, or gastric
cancer. In some
embodiments, the cancer has microsatellite instability.
[00390] In various embodiments, the second antibody depletes T regulatory
(Treg) cells,
activates antigen presenting cells (APCs), enhances CD8 T cell responses,
upregulates co-
stimulatory receptors, and/or promotes release of immune activating cytokines
(such as
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CXCL10 and/or IFNy). In some embodiments, the second antibody promotes release
of immune
activating cytokines to a greater extent than immune suppressive cytokines
(such as IL10 and/or
MDC).
[00391] The ADC and second antibody may be administered concurrently or
sequentially.
For sequential administration, a first dose of the ADC may be administered
before the first dose
of the second antibody, or a first dose of the second antibody may be
administered before the
ADC. For concurrent administration, in some embodiments, the ADC and second
antibody may
be administered as separate pharmaceutical composition or in the same
pharmaceutical
composition.
[00392] In some embodiments, a therapeutic agent is administered at a
therapeutically
effective amount or dose. A daily dose range of about 0.01 mg/kg to about 500
mg/kg, or about
0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10
mg/kg to
about 50 mg/kg, can be used. The dosages, however, may be varied according to
several factors,
including the chosen route of administration, the formulation of the
composition, patient
response, the severity of the condition, the subject's weight, and the
judgment of the prescribing
physician. The dosage can be increased or decreased over time, as required by
an individual
patient. In certain instances, a patient initially is given a low dose, which
is then increased to an
efficacious dosage tolerable to the patient. Determination of an effective
amount is well within
the capability of those skilled in the art.
[00393] In some embodiments, the enhanced activity observed with the
particular
combination therapies described herein have certain benefits as compared to
corresponding
monotherapy treatment. For example, in some embodiments, administration of the
ADC and the
second antibody in combination has a toxicity profile comparable to that of
the ADC or the
second antibody when either is administered as monotherapy. In some
embodiments, the
effective dose of the ADC and/or the second antibody when dosed in combination
is less than
when administered as monotherapy. In some embodiments, administration of the
ADC and the
second antibody in combination provide a longer duration of response as
compared to
corresponding monotherapy treatment. In some embodiments, administration of
the ADC and
the second antibody in combination results in longer progression-free survival
as compared to
corresponding monotherapy. In some embodiments, the administration of the ADC
and the
second antibody can be used to treat recurrent cancer that recurs following
monotherapy
treatment with either agent individually.
[00394] The route of administration of a pharmaceutical composition can be
oral,
intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular,
inhalational, topical,
intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular
or otic delivery, or any
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other methods known in the art. In some embodiments, one or more therapeutic
agents are
administered orally, intravenously, or intraperitoneally.
[00395] Co-administered therapeutic agents can be administered together or
separately,
simultaneously or at different times. When administered, the therapeutic
agents independently
can be administered once, twice, three, four times daily or more or less
often, as needed. In
some embodiments, the administered therapeutic agents are administered once
daily. In some
embodiments, the administered therapeutic agents are administered at the same
time or times,
for instance as an admixture. In some embodiments, one or more of the
therapeutic agents is
administered in a sustained-release formulation.
[00396] In some embodiments, therapeutic agents are administered concurrently.
In some
embodiments, the therapeutic agents are administered sequentially. For
example, in some
embodiments, a first therapeutic agent is administered, for example for 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 prior to
administering a second
therapeutic agent.
[00397] In some embodiments, the treatment provided herein is administered to
the subject
over an extended period of time, e.g., for at least 30, 40, 50, 60, 70, 80,
90, 100, 150, 200, 250,
300, 350 days or longer.
V. Compositions and Kits
[00398] In another aspect, compositions and kits for use in treating or
preventing a cancer in a
subject are provided.
Pharmaceutical Compositions
[00399] In some embodiments, pharmaceutical compositions for use in the
present methods
are provided. In some embodiments, the ADC is administered in a first
pharmaceutical
composition and the antibody that binds an immune cell engager is administered
in a second
pharmaceutical composition. In some embodiments, the ADC and the antibody that
binds an
immune cell engager are administered in a single pharmaceutical composition.
[00400] Guidance for preparing formulations for use in the present invention
is found in, for
example, 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
herein by reference. The pharmaceutical compositions described herein can be
manufactured in
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a manner that is known to those of skill in the art, i.e., by means of
conventional mixing,
dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping
or lyophilizing
processes. The following methods and excipients are merely exemplary and are
in no way
limiting.
[00401] In some embodiments, one or more therapeutic agents are prepared for
delivery in a
sustained-release, controlled release, extended-release, timed-release or
delayed-release
formulation, for example, in semi-permeable matrices of solid hydrophobic
polymers containing
the therapeutic agent. Various types of sustained-release materials have been
established and are
well known by those skilled in the art. Current extended-release formulations
include film-
coated tablets, multiparticulate or pellet systems, matrix technologies using
hydrophilic or
lipophilic materials and wax-based tablets with pore-forming excipients (see,
for example,
Huang, et at. Drug Dev. Ind. Pharm. 29:79 (2003); Pearnchob, et at. Drug Dev.
Ind. Pharm.
29:925 (2003); Maggi, et al. Eur. I Pharm. Biopharm. 55:99 (2003); Khanvilkar,
et al., Drug
Dev. Ind. Pharm. 228:601 (2002); and Schmidt, et at., Int. I Pharm. 216:9
(2001)). Sustained-
release delivery systems can, depending on their design, release the compounds
over the course
of hours or days, for instance, over 4, 6, 8, 10, 12, 16, 20, 24 hours or
more. Usually, sustained
release formulations can be prepared using naturally-occurring or synthetic
polymers, for
instance, polymeric vinyl pyrrolidones, such as polyvinyl pyrrolidone (PVP);
carboxyvinyl
hydrophilic polymers; hydrophobic and/or hydrophilic hydrocolloids, such as
methylcellulose,
ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose; and
carboxypolymethylene.
[00402] For oral administration, a therapeutic agent can be formulated readily
by combining
with pharmaceutically acceptable carriers that are well known in the art. Such
carriers enable
the compounds to be formulated as tablets, pills, dragees, capsules,
emulsions, lipophilic and
hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the
like, for oral
ingestion by a patient to be treated. Pharmaceutical preparations for oral use
can be obtained by
mixing the compounds with a solid excipient, optionally grinding a resulting
mixture, and
processing the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets
or dragee cores. Suitable excipients include, for example, fillers such as
sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone
(PVP). If desired, disintegrating agents can be added, such as a cross-linked
polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
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[00403] A therapeutic agent can be formulated for parenteral administration by
injection, e.g.,
by bolus injection or continuous infusion. For injection, the compound or
compounds can be
formulated into preparations by dissolving, suspending or emulsifying them in
an aqueous or
nonaqueous solvent, such as vegetable or other similar oils, synthetic
aliphatic acid glycerides,
esters of higher aliphatic acids or propylene glycol; and if desired, with
conventional additives
such as solubilizers, isotonic agents, suspending agents, emulsifying agents,
stabilizers and
preservatives. In some embodiments, compounds can be formulated in aqueous
solutions,
preferably in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or
physiological saline buffer. Formulations for injection can be presented in
unit dosage form,
e.g., in ampules or in multi-dose containers, with an added preservative. The
compositions can
take such forms as suspensions, solutions or emulsions in oily or aqueous
vehicles, and can
contain formulatory agents such as suspending, stabilizing and/or dispersing
agents.
[00404] A therapeutic agent can be administered systemically by transmucosal
or transdermal
means. For transmucosal or transdermal administration, penetrants appropriate
to the barrier to
be permeated are used in the formulation. For topical administration, the
agents are formulated
into ointments, creams, salves, powders and gels. In one embodiment, the
transdermal delivery
agent can be DMSO. Transdermal delivery systems can include, e.g., patches.
For
transmucosal administration, penetrants appropriate to the barrier to be
permeated are used in the
formulation. Such penetrants 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 are
hereby
incorporated herein by reference.
[00405] In some embodiments, a pharmaceutical composition comprises an
acceptable carrier
and/or excipients. A pharmaceutically acceptable carrier includes any
solvents, dispersion
media, or coatings that are physiologically compatible and that 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 can contain
one or more
physiologically acceptable compound(s) that act, for example, to stabilize the
composition or to
increase or decrease the absorption of the active agent(s). Physiologically
acceptable compounds
can include, for example, carbohydrates, such as glucose, sucrose, or
dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low molecular weight
proteins,
compositions that reduce the clearance or hydrolysis of the active agents, or
excipients or other
stabilizers and/or buffers. Other pharmaceutically acceptable carriers and
their formulations are
well-known and generally described in, for example, Remington: The Science and
Practice of
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Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005.
Various
pharmaceutically acceptable excipients are well-known in the art and can be
found in, for
example, Handbook of Pharmaceutical Excipients (5th ed., Ed. Rowe et at.,
Pharmaceutical
Press, Washington, D.C.).
[00406] Dosages and desired drug concentration of pharmaceutical compositions
of the
disclosure may vary depending on the particular use envisioned. The
determination of the
appropriate dosage or route of administration is well within the skill of one
in the art. Suitable
dosages are also described herein.
Kits
[00407] In some embodiments, kits for use in treating a subject having a
cancer are provided.
In some embodiments, the kit comprises:
an antibody-drug conjugate comprising a first antibody conjugated to a tubulin
disrupter, as provided herein; and
a second antibody that binds an immune cell engager, as provided herein.
[00408] In some embodiments, the kits can further comprise instructional
materials
containing directions (i.e., protocols) for the practice of the methods of
this invention (e.g.,
instructions for using the kit for treating a cancer). While the instructional
materials typically
comprise written or printed materials, they are not limited to such. Any
medium capable of
storing such instructions and communicating them to an end user is
contemplated by this
invention. Such media include, but are not limited to electronic storage media
(e.g., magnetic
discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
Such media may
include addresses to internet sites that provide such instructional materials.
VI. Examples
[00409] The examples discussed below are intended to be purely
exemplary of the
invention and should not be considered to limit the invention in any way. The
examples are not
intended to represent that the experiments below are all or the only
experiments performed.
Efforts have been made to ensure accuracy with respect to numbers used (for
example, amounts,
temperature, etc.) but some experimental errors and deviations should be
accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is average
molecular weight,
temperature is in degrees Centigrade, and pressure is at or near atmospheric.
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Example 1: Non-directed Chemotherapeutic Agents Impair T Cell Responses
1.1 Materials and Methods
[00410] Human primary T cells were induced to undergo proliferation using
CD3/CD28
coated beads. 20,000 carboxyfluorescein diacetate succinimidyl ester (CSFE)
labeled, enriched
CD3+ T cells were incubated with anti-CD3 CD28 beads (1 bead per 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
[00411] As shown in FIG. 1, proliferation of primary human T cells was
significantly reduced
by all the single free agent chemotherapeutics tested. These data suggest that
systemic exposure
to chemotherapeutic agents may limit T cell mediated activity in patients
including responses
from immune-oncology agents (e.g., antibodies that bind immune cell engagers).
Example 2: Vedotin ADCs Do Not Inhibit T Cell Proliferation, Despite Directed
Delivery
to T Cells (BV (SGN-35) Treatment of CD30+ CD8 T Cells)
2.1 Materials and Methods
[00412] Human primary CD8 T cells were labeled with CSFE and induced to
undergo
proliferation with anti-CD3-CD28 beads (1 bead per 4 T cells) + 10 ng/mL IL-2
for 4 days.
During activation, CD30 was upregulated on the surface of T cells. CD30+ CD8 T
cells were
treated with either CD30 directed vc-MMAE (brentuximab vedotin; BV; SGN-35) or
an isotype
control. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain
(ThermoFisher) and live
cells were counted via flow cytometry.
2.2 Results
[00413] As shown in FIG. 2, cell proliferation of primary human CD30+ CD8 T
cells was not
significantly altered by treatment with By. These data suggest that systemic
exposure of a
vedotin ADC, even if targeted directly to CD8 T cells, does not impact CD8
mediated anti-
tumor responses.
Example 3: Endoplasmic Reticulum Stress Induction is Superior for Vedotin ADCs
3.1 Materials and Methods
[00414] Induction of endoplasmic reticulum (ER) stress is one of the first and
required steps
for the initiation of immunogenic cell responses (FIG. 3B). MIA-PaCa-2
pancreatic cancer cell
lines were treated with ADCs conjugated to distinct payloads, including
vedotin (MMAE),
emtansine (DM1), the Exatecan DS-8201 (Ex), as well as the free microtubule
stabilizing agent
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 (FIG. 3B) was assessed by Western blot.
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3.1.1 Western blot
[00415] Treated cells were centrifuged at 14,000-16,000 rpm for 10 mins and
stored at -20 C.
Cell pellets were resuspended in 4X BOLTTm LDS sample buffer (Thermo Fisher
Catalog #
B0007) and lysed by heating at 95 C for 5-10 minutes to produce cell lysates.
Sample lysates
were run on a Bis-Tris 4-12% gradient gel at 140V for 1 hr 40 mins in MOPS
buffer. The Bis-
Tris gel was then transferred onto a nitrocellulose membrane using an iBlot2.
Membranes were
washed once in 1X TBS and incubated overnight at 4 C in Licor Blocking buffer.
Membranes
were washed four times in lx TB S-T for 5-10 mins each. Images were developed
on the Licor
Odyssey System using the 84 p.m resolution and auto intensities.
3.1.2 CHOP luciferase induction assay
[00416] Assessment of downstream pathways to ER stress induction was performed
using
MIA-PaCa-2 cells transduced with the CHOP driven luciferase reporter cell
line. CHOP is the
last step in the ER stress response cascade and its expression levels are
increased by ER stress.
Several ADC payloads in clinical development (FIG. 3A) were used in the
assessment.
Induction of CHOP was measured using a reporter system for CHOP activity
according to the
manufacturer's instructions (Bright-GbTM Luciferase Assay System, Promega). In
brief,
100,000 cells/well were plated in a 96-well, flat-bottom clear plate (aliquot
150 tL per well).
200 tL of media were aliquoted to outer wells of the plate to provide a media
"blanket" around
the wells of cells. At 24, 48, and 72 hours, plates were removed from the
incubator and allowed
to come to room temperature. 100 tL of media was removed from the wells. 100
tL of
BrightGlo Reagent was added to each well. Plate was shaken for at least two
minutes before
reading. The Envision CTG 96-well Standard Protocol was used to read the
plate.
3.2 Results
[00417] As shown in FIG. 3C-E, auristatin-based ADCs (IV:MAE-ADC or MMAF-ADC)
treatment was the only condition found to induce the early ER stress response
pJNK signal of
the different ADC payloads tested. ER stress induction is tied to microtubule
disruptions as the
ER requires intact microtubules to expand and contract to accommodate the
protein translational
needs of the cell. The ability of MIVIAE as a microtubule disrupting agent to
induce this ER
stress is exemplified in the data shown.
[00418] FIG. 3D-E demonstrate that CHOP, a downstream signal in the ER stress
pathway, is
significantly induced by IVINIAE ADCs and that downstream pathways to ER
stress are also
driven differently by MMAE ADCs as compared to other payloads.
Example 4: ICD potential of Different Clinical ADC Payloads
4.1 Materials and Methods
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[00419] Classical markers of ICD include surface exposure of calreticulin and
release of ATP
and HMGB1 which occur concomitantly with the induction of ER stress response.
These
molecules are considered danger signals and activate innate immune cells and
increase tumor
antigen specific T cell responses. MIA-PaCa-2 cancer cells were treated with
IC50
concentrations of ADC-bearing payloads that are currently at the clinical
stage, i.e., MMAE,
DM1, and Exatecan (Ex). Treated cells were then analyzed for ICD marker
induction. Cisplatin
was used as a negative control because it is able to drive cell death but is
not known to induce
ICD.
[00420] 100,000-150,000 cells were plated per well in a 96-well dish. Cells
were allowed to
reach 50-60% confluence. The media was removed and fresh culture media was
added per well
of cells. 1 g/mL of 1 M of drug was added to each well of cells. After 24
hours, 250 tL (for the
ATP release assay) or 200
(for the HMGB1 assay) of media was collected and transferred
into a labeled 1.5 mL Eppendorf tube. Each tube of sample was centrifuged at
10,000 rpm for 1
min. 50 of media was transferred to a well in a 96-well clear bottom plate.
50 of CTG
was added to each well. The plate was shaken for 1-2 mins. The Envision Plate
Reader was used
to read the plate.
[00421] HMGB1 release levels were monitored by luminescent intensity per well
using the
Envision Plate Reader. HMGB1 and ATP release levels were reported as the fold
change over
the background values for untreated samples. Acquired values were converted to
text file and
exported and analyzed using Excel and GraphPad Prism.
4.2 Results
[00422] As shown in FIG. 4A, vc-MMAE was potent in driving ATP release
compared to the
other payloads tested. While HMGB1 release is associated with induction of ICD
its release is
also seen when cells begin to undergo necrosis and is not directly associated
with robust immune
cell engagement. Treatment of MIA-PaCa-2 cells with microtubilin disrupting
agents vc-MMAE
and DM1 resulted in robust HMGB1 release, which contrasts to the topoisomerase
inhibitor
Exatecan (Ex) (FIG. 4B).
Example 5: Immune Activation Assessment of ADC Payloads
5.1 Materials and Methods
5.1.1 Cells
[00423] As shown in FIG. 5A, ADCs conjugated to MMAE disrupts microtubules,
resulting
in ER stress response, leading to immunogenic cell death (ICD). The dying
cells in turn release
immune-activating 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
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immune system. This activation results in, for example, upregulation of
proteins such as CD80,
CD86, HLA-DR, and CD40, an increase of MHCII expression on monocytes, and the
release of
cytokines such as CXCL-10/IP10 and IL-12, thereby initiating antitumor T cell
responses. Such
T cell responses can be further augmented by PD-1/L1 inhibitors. Here, the
immunologic
consequence of ICD was assessed in human peripheral blood mononuclear cell
(PBMC)
cultures. Cancer cells exposed to ADCs conjugated to distinct payloads were
added to PBMCs.
[00424] L540cy cancer cells exposed to EC50 concentrations of ADC or free
drug, for 18
hours (at 37 C in 5% CO2. were washed and 250 ul of PBMCs suspended at 10x106
cell/mL
were added to the cancer cell lines killed cells for 48 hrs. Tissue culture
media was taken and
cytokines measured by Luminex assessment.
[00425] Treatment was performed in triplicate for 2 independent PBMC donors.
5.1.2 Co-stimulatory molecule surface expression
[00426] After treatment, cell pellets were resuspended in 50 mL of BD FACs
buffer and
transferred to 96 well round-bottom microtiter plates. Fc receptors were
blocked with human
100 pg/mL Fc-fragments for 30 minutes on ice. A master mix composed of PE-HLA-
DR
(MHCII) and APC-CD14 diluted at 1:100 was prepared in BD FACs buffer
containing 100
mg/mL human purified Fc-fragments. 1011.1 of the master mix was added to each
well containing
90 11.1 of re-suspended cells and samples were incubated for 1 hour on ice.
Cells were then
centrifuged at 400 xg in a pre-cooled Eppendorf 5810R centrifuge for 5
minutes. The
supernatant was removed and cells were washed with 200 mL of BD FACs buffer.
The wash
was performed twice and cells were resuspended in 200 mL of FACs buffer and
samples were
analyzed on an Attune flow cytometer. HLA-DR mean fluorescence was determined
using
FlowJo analysis software.
5.1.3 Cytokine production
[00427] After treatment, PBMCs/cancer cell co-cultures were spun with a plate
adapter in an
Eppendorf 5810R at 800 rpm for 5 minutes. Serum or tissue culture supernatant
were removed
and transferred to a 96-strip tube rack and samples were frozen at ¨80 C until
processing.
Frozen tissue culture supernatants and serum were thawed overnight at 4 C and
processed for
cytokine production using a Luminex Multiplex Kit from Millipore
[00428] Tissue culture supernatant and serum samples were processed as per the
manufacturer's instructions. Briefly, assay plates were washed with 200 tL of
wash buffer per
well, followed by addition of 25 tL standard or buffer, 25 tL matrix or
sample, and 25 tL of
multiplexed analyte beads to each well. Samples were incubated overnight with
vigorous
shaking at 4 C. Plates are washed the assay plates twice with wash buffer.
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[00429] Detection antibodies (25 ilL) were added to each well and incubated at
room
temperature for 1 hour. 25 tL of streptavidin-phycoerythrin (SA-PE) was added
and samples
incubated at room temperature for 30 minutes. The plate was washed twice with
wash buffer,
and beads were resuspended with 150 tL of sheath fluid. The samples were
analyzed using
Luminex MagPix systems in combination with the Xponent software system.
Cytokine levels
were calculated from the standard curve.
5.2 Results
[00430] Innate cell activation was observed, as evidenced by increased surface
activation
markers (MHCII) and the release of inflammatory cytokines (CXCL-10/IP10) (FIG.
5B-C).
Innate immune cells become activated when exposed to vc-MMAE treated tumor
cells. Immune
cell activation by vc-MMAE was more robust than activation by other ADC
payloads (FIG. 5B-
C).
[00431] Vc-MMAE-mediated ICD is regulated cell death that activates adaptive
immune
responses against antigens from dead and dying tumor cells and allows for the
generation of
robust innate immune cell activation and subsequent cytotoxic T-cell responses
targeted towards
specific tumor cell antigens. Here, it was demonstrated that vc-MMAE killed
cancer cells
elicited an increase of surface MHCII and release of the innate cytokine
CXCL10 a strong
chemotactic and inflammatory mediator, from monocyte/macrophages after uptake
of dead cells.
Example 6: Payload Evaluation on Trastuzumab Backbone
6.1 Materials and Methods
[00432] The ability of trastuzumab ADC conjugates bearing various clinical
stage payloads to
induce ER stress and downstream ICD markers ATP and HMGB1 was assessed. The
payloads
used were DM1, MMAE, and Exatecan (Ex).
6.2 Results
[00433] Two observations were made: (1) trastuzumab conjugated to vcMNIAE
drove the
most robust ER stress response which was associated with induction of ATP and
HMGB1; and
(2) the late cell death marker HMGB1 seemed elevated for the other payload
classes indicating
that secondary necrosis maybe associated with these payload classes rather
than frank ICD (FIG.
6C-E). The findings here are similar to findings described above in Example 4,
which used Mia-
PaCa-2 cells (FIG. 4A-B).
Example 7: Induction of Early Stage ER Stress Markers (JNK Signaling
Activation) is
Generally Superior for MMAE ADCs
7.1 Materials and Methods
[00434] As described in Example 5 and shown in FIG. 5A, the ICD pathway
involves various
aspects. This pathway is illustrated further in FIG. 7A. As shown in FIG. 7A
and noted above,
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a tubulin disrupter such MMAE in an initial stage disrupts microtubules,
thereby causing ER
stress and ICD. ICD in turn causes release of immune-activating molecules such
as DAMPs,
ATP, HMGB1, and CRT. These molecules can subsequently activate innate cells
that are
capable of initiating antitumor T cell responses and can induce T cell memory.
Such T cell
responses can be further augmented by combination with other immune
modulators, such as the
immune cell engagers described herein. Several of the following examples
report the results of
studies showing the effectiveness of MMAE compared to other ADC payloads to
induce the
foregoing different aspects of the ICD pathway.
[00435] Induction of endoplasmic reticulum (ER) stress is one of the first
steps for the
initiation of immunogenic cell responses, and JNK signaling activation is an
indicator of ER
stress (see FIG. 3B). To assess this indicator, MIA-PaCa-2 pancreatic cancer
cells were treated
with 1 g/mL ADCs conjugated to distinct payloads, as shown in FIG. 7B. After
24 or 48 hours
of treatment, cells were harvested for western blot analysis, and the upstream
ER stress marker
pJNK was assessed by Simple Western immunoassay (WesTM, Protein Simple).
[00436]
Treated cells were dissociated from culture plates using cell scrapers.
Suspended
cells were centrifuged for 10 minutes at 1000 rpm, 4 C. Supernatant was
removed and cell
pellets were resuspended in lysis buffer (containing protease and phosphatase
inhibitors). After a
minimum of 10 minutes on ice, samples were centrifuged at 13,500 g for 10 min
to pellet out
cellular debris. Lysis solution was relocated to separate tubes and stored at -
80 C. Lysate
protein amount was quantified using Bio-Rad DC Protein Assay Kit (Cat. #
5000112), to allow
for equal lane loading. Sample lysates and reagents were loaded into an assay
plate and placed in
WesTM. Phospho-JNK was identified using a primary antibody (Cell Signaling
Technologies
Cat. # 9251S) and immunoprobed using an HRP-conjugated secondary antibody
(Protein Simple
Cat. # 042-206) and chemiluminescent substrate. The resulting chemiluminescent
signal was
detected, quantitated, and displayed by the integrated Compass software.
7.2 Results
[00437] As shown in FIG. 7C-F, of the different ADC payloads tested, MMAE-ADCs
(SGD-
1006) treatment was one of the strongest inducers of JNK phosphorylation, an
early ER stress
response. In general, MMAE-ADC treatment generated stronger pJNK signals
compared to
treatment with maytansine-ADCs (FIG. 7C), camptothecin-ADCs (FIG. 7D),
anthracycline-
ADCs (FIG. 7E), and calicheamicin-ADCs (FIG. 7F). (hIgGs in FIG. 7C-F are non-
targeted
conjugates with the same payload as the corresponding ADC.) The sole exception
was
treatment with an ADC containing the anthracycline mp-EDA-PNU (SGD-8335),
which
generated a pJNK signal comparable to that from treatment with MMAE-ADCs (FIG.
7E). ER
stress induction is tied to microtubule disruptions as the ER requires intact
microtubules to
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expand and contract to accommodate the protein translational needs of the
cell. The ability of
MMAE as a microtubule disrupting agent to induce this ER stress is exemplified
in the data
shown.
Example 8: Induction of Late Stage ER Stress Markers (CHOP Induction) is
Generally
Superior for MMAE ADCs
8.1 Materials and Methods
[00438] CHOP is the last step in the ER stress response cascade and its
expression levels are
increased by ER stress (see FIG. 3B). Assessment of this downstream pathway to
ER stress was
performed using MIA-PaCa-2 cells transduced with a CHOP-driven luciferase
reporter
(Signosis, Inc.). Several ADCs comprising distinct payloads were used in the
assessment. See
FIG. 7A.
[00439] Induction of CHOP in the MIA-PaCa-2 cells was measured by detection of
luciferase
signal (Bright-GbTM Luciferase Assay System, Promega). In brief, 10,000
cells/well were plated
in 96-well, black-walled, flat-bottom clear plates in 75 per
well. ADCs were dosed in 25
per well to achieve a final ICso concentration. At 36, 48, and 72 hours,
plates were removed
from the incubator and allowed to come to room temperature. 100 tL of Bright-
Glo Reagent
was added to each well. Plate was shaken for at least five minutes before
reading. The Envision
CTG 96-well Standard Protocol was used to read the plate.
8.2 Results
[00440] As shown in FIG. 8A-D, treatment with ADCs containing vc-MMAE (SGD-
1006)
resulted in CHOP induction that was comparable to CHOP induction from
treatment with ADCs
containing mertansine (SPP-5351) or ravtansine (SPDB-5352) (FIG. 8A) and from
treatment
with ADCs containing mp-EDA-PNU (SGD-8335) or mp-Gluc-DXZ (SGD-8248) (FIG.
8C).
Also, treatment with ADCs containing MMAE (SGD-1006) resulted in CHOP
induction that
was stronger than CHOP induction from treatment with ADCs containing
camptothecins (FIG.
8B), AT (SGD-4830) (FIG. 8D), or teserine (SGD-7455) (FIG. 8D). In contrast,
treatment with
ADCs containing ozogamycin (SGD-8677) resulted in CHOP induction that was
slightly
stronger than that from treatment with ADCs containing vc-MMAE (SGD-1006) FIG.
8D).
Example 9: Induction of Immunostimulatory DAMPs is Generally Superior for MMAE
ADCs
9.1 Materials and Methods
[00441] ICD causes release of immune-activating molecules¨damage-associated
molecular
patterns (DAMPs)¨such as ATP, HMGB1, and CRT. To measure ICD, ATP and HMGB1
release was assessed as follows. MIA-PaCa-2 cancer cells were treated with
ICso concentrations
of ADCs with various payloads to assess in vitro ICD marker induction. See
FIG. 7A.
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[00442] 200,000 cells were plated per well in 6-well TC plates and allowed to
attach to plate
ON. Cells reached 50-60% confluence. ICso concentrations of ADCs were added to
each
treatment well. After 72 hours, 500 (for the ATP
release assay) or 750 (for the HMGB1
assay) of culture supernatant was collected and transferred into a labeled 1.5
mL Eppendorf
tube. Each tube of sample was centrifuged at 13,000 rpm for 1 minute. 50 tL of
media was
transferred to triplicate wells in a 96-well clear bottom plate. 50 of
CellTiter-Glog
(Promega) was added to each well. The plate was shaken for 1-2 minutes. The
CTG 96-well
Standard Protocol on the Envision Plate Reader was used to read the plate.
Each tube sample
supernatant was then used to measure HMGB1 release levels, which were
quantified by ELISA
(IBL). HMGB1 and ATP release levels were reported as the fold change over the
background
values for untreated samples. Acquired values were converted to text file and
exported and
analyzed using Excel and GraphPad Prism.
9.2 Results
[00443] As shown in FIG. 9A-D, treatment with ADCs containing vc-MMAE (SGD-
1006)
resulted in ATP release and HMGB1 release that was stronger than that from
treatment with
ADCs containing maytansines (FIG. 9A) and from treatment with ADCs containing
camptothecins (FIG. 9B). Also, treatment with ADCs containing vc-MMAE (SGD-
1006)
resulted in ATP release and HMGB1 release that is stronger than that from
treatment with ADCs
containing teserine (SGD-7455) or the auristatin AT (SGD-4830) (FIG. 9D).
[00444] Treatment with ADCs containing vc-MMAE (SGD-1006) resulted in ATP
release
and HMGB1 release that is comparable to that from treatment with ADCs
containing
anthracyclines (compared to HMGB1, ATP release is less robust) (FIG. 9C) and
from treatment
with ADCs containing ozogamycin (SGD-8677) (FIG. 9D).
Example 10: Activation of Innate Cells (Cytokine Release) is Generally
Superior for
MMAE ADCs
10.1 Materials and Methods
[00445] DAMPs activate innate cells that can initiate antitumor 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 was assessed
as
follows.
[00446] L540cy cancer cells exposed to ICso concentrations of ADCs or
paclitaxel for 24
hours (at 37 C in 5% CO2) were washed, and 0.2 x 106 cells/well PBMCs were
added to the
killed cancer cells for a 1:10 L540cy:PBMC ratio. The payloads of the ADCs
used in this
experiment are described FIG. 7A. Co-cultures were incubated for 48 hours.
Cell culture
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supernatants were collected at 24 hours, and cytokines were measured by
Luminex assessment,
including the innate cytokine CXCL-10/IP10.
[00447] Following the 48-hour co-culture incubation, cell pellets were
resuspended in 50 [IL
of BD FACs buffer and transferred to 96-well round-bottom microtiter plates.
Fc receptors were
blocked with human Fc-fragments at 100m/mL for 30 minutes on ice. A master mix
including
PE-Cy7 anti-HLA-DR (MHCII), PE anti-CD14, PE-Dazzle 594 anti-CD11b, BV605 anti-
CD3,
and BV421 anti-CD19, diluted at 1:100, was prepared in BD FACs buffer
containing 100m/mL
human purified Fc-fragments. 10 [EL of the master mix was added to each well
containing 90 [IL
of re-suspended cells and samples were incubated for 1 hour on ice. Cells were
then centrifuged
at 400 x g in a pre-cooled Eppendorf 5810R centrifuge for 5 minutes. The
supernatant was
removed, and the cells were washed with 200 mL of BD FACs buffer. The wash was
performed
twice, and the cells were resuspended in 200 mL of FACs buffer. Samples were
analyzed on an
Attune flow cytometer. Monocytes were defined as CD14+CD11b+CD3-CD19-. HLA-DR
mean
fluorescence was determined using FlowJo analysis software.
10.2 Results
[00448] As shown in FIG. 10A-D, treatment with ADCs containing MMAE (SGD-1006)
resulted in monocyte MHC II expression that was comparable to or higher than
that from
treatment with ADCs containing other payloads, including maytansines (FIG.
10A),
camptothecins (FIG. 10B), anthracyclines (FIG. 10C), and the calicheamicin
ozogamycin (SGD-
8677) and the PBD teserine (SGD-7455) (FIG. 10D). Treatment with ADCs
containing MMAE
(SGD-1006) also resulted in release of innate cytokine CXCL-10/IP10 that was
consistently
higher than that from treatment with ADCs containing the same payloads (FIG.
10A-D).
10.3 Summary of Superior ICD potential of WAE ADCs
[00449] As shown in these experiments, MMAE-ADCs can induce various ICD
hallmarks
and various immunogenic cell responses, including induction of early stage ER
stress (e.g., JNK
activation), induction of late ER stress (e.g., CHOP induction), induction of
immune-activating
molecules (e.g., ATP and HMGB1 release), and activation of innate immune cells
(e.g.,
macrophage activation). None of the other ADC payloads tested induced these
ICD hallmarks
consistently. FIG. 10E provides a summary of the ICD potential (as measured by
the above
hallmarks) of ADCs with different types of payloads, and illustrate the
overall superiority of a
tubulin disrupter, particularly MMAE.
Example 11: Differential FcyR Binding to FcyRIIa, FcyRIIb or FcyRIIIa based
upon Fc
backbone.
11.1 Materials and Methods
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[00450] Antibodies SEA-CD40, APX005M, ADC-1013, and Selicrelumab (FIG. 11A)
were
assessed for FcyR binding using flow cytometry to CHO cells that were
transfected with human
FcyRIIa, FcyRIIb or FcyRIIIa. CHO cells were incubated with increasing
concentrations of
antibodies and a secondary antibody used to assess binding as monitored by
flow cytometry.
[00451] For each cell line, 50 million cells were washed once in 50 mL of PBS.
Cells were
counted again and resuspended at 2.2 million cells/mL in BD stain buffer.
Cells were plated in a
96-well round bottom plate at 0.1 mL of cells per well.
[00452] Antibody solutions were diluted to make the following final
concentrations: 3
mg/mL, 1 mg/mL, 0.3 mg/mL, 0.1 mg/mL, 0.03 mg/mL, 0.01 mg/mL, 0.003 mg/mL,
0.001
mg/mL, 0.0003 mg/mL. Each antibody solution was diluted at 10x (i.e., 11 tL
each antibody
solution was added to 89 tL of media) to produce the following concentrations:
300, 100, 30,
10, 3, 1, 0.03. 0.01, 0.003, 0.001, and 0.0003 [tg/ml. Media was removed from
the cells and the
cells were washed with media. 100 tL of antibody solution was added to each
well. Antibody
solutions were added with decreasing concentrations in the vertical direction.
After a 1-hour
incubation at 4 C, the plate was centrifuged, and each well of cells was
washed twice with 200
BD stain buffer. Pelleted cells were resuspended by vortexing the plate.
[00453] Next, PE conjugated anti-human IgG Fc antibody was prepared (1/50
dilution of 1
mg/ml concentration = 33 g/mL saturating concentration) in BD stain buffer.
The solution was
incubated at 30 min in a dark fridge. After incubation, the plate was
centrifuged, the supernatant
was removed, and the cells were washed twice with 200
BD stain buffer per well. Cells were
resuspended in PBS + 1% paraformaldehyde and kept at 4 C until they were
analyzed by flow
cytometry. Samples were analyzed using the Attune flow cytometer. Data points
for the
geometric mean of fluorescence intensity (GEO mean fluorescence) were graphed
using
Graphpad Prism.
11.2 Results
[00454] APX005 5267E exhibited the highest affinity for FcyRIIa and Fcyllb
(FIG. 11B-D).
SEA-CD40 had the highest affinity for FcyRIIIa with lowest affinity for
FcyRIIb (FIG. 11B-D).
The data demonstrate the potential of different Fc backbones to impact binding
to different
FcyRs. The SEA-CD40 nonfucosylated backbone shows differential binding as
compared to the
other CD40 antibodies in development in that it bound activating but not
inhibitory FcyRs (FIG.
11B-D).
Example 12: Induced Cell Death in MIA-PaCa-2 Cells
12.1 Materials and Methods
[00455] MIA-PaCa-2 pancreatic tumor cells were induced to undergo cell death
with EC50
concentrations of the non-ICD inducing agent Abraxane (which acts similarly to
paclitaxel in
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Example 3 above), or the 2 ICD inducing agents oxaliplatin or vc-MMAE. Cell
were incubated
with each agent for 18 hrs. Tumor cells were then added to human PBMCs plus
various CD40-
directed agonists (1 [tg/m1) with differing Fc backbones (FIG. 11A) and immune
activation
assessed 48 hrs later.
12.2 Results
[00456] SEA-CD40 combined with vcMMAE ADC killed tumor cells, at least in
part, by
inducing superior release of immune activating cytokines (CXCL10 and IFNy;
FIG. 12A-B),
while other CD40 agonists with differing Fc backbones amplified the immune
dampening
cytokines (IL-10 and MDC; FIG 12C-D). This example illustrates the improved
immune
response observed with an Fc backbone that has enhanced binding to FcyRIIIa.
Example 13: Differential Immune Activation to Apoptotic Melanoma Cells as a
Function
of Fc Backbone
13.1 Materials and Methods
[00457] Two melanoma cell lines, SK-MEL 12 and SK-MEL 28 were treated with
Abraxane,
oxaloplatin, or vc-MMAE at EC50 concentrations for 18 hrs. Human PBMCs plus 1
pg/m1 of
various CD40-directed agonists with differing Fc backbones (Fig. 11A) were
added to the
treated melanoma/tumor cell. Immune activation was assessed 48 hrs later.
13.2 Results
[00458] SEA-CD40 combined with vc-MMAE ADC induced release of immune
activating
cytokines (CXCL10; FIG. 13A-B) while other CD40 agonists amplified the immune
dampening
cytokines (IL-10; FIG. 13C-D).
Example 14: Differential Immune Activation to a Variety of Apoptotic Tumor
Cell Types
as a Function of Fc Backbone
14.1 Materials and Methods
14.1.1 Cells
[00459] Cell death was induced in tumor cells from melanoma, lung, breast and
pancreas
using the ICD-inducing agent oxaliplatin or vc-MMAE at EC50 concentrations and
18 hours of
incubation. Treated tumor cells were added to human PBMCs and various CD40-
directed
agonists with differing Fc backbones (Fig. 11A). Immune activation was
assessed 48 hrs later.
PBMCs and tumor cell lines were treated as described above in Example 5.
Instead of MIA-
PaCa-2 cancer cells (which was used in Example 5), the following cell lines
were used: the
melanoma cell lines SK-MEL 12 and SK-MEL 28, the lung cancer cell line A549,
the breast
cancer cell line MDA-MB-468, and the pancreatic cancer cell line MIA-PaCa-2.
Cells were
treated in triplicate.
14.1.2 Cytokine production
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[00460] Cytokine production was assessed as described above in Example 5.
14.2 Results
[00461] SEA-CD40 combined with vc-IVIIVIAE ADC drove superior release of
immune
activating cytokines (CXCL10 and IFNy; FIG. 14A and 14C) while other CD40
agonists
amplified the immune dampening cytokines (IL-10; FIG. 14B). As with Examples
12 and 13,
this example demonstrates the improved immune response observed with an Fc
backbone that
has enhanced binding to FcyRIIIa as compared to other Fc backbones.
Example 15: Synergistic Effect with Combination of a Nonfucosylated SEA-CD40
Antibody and a Auristatin Based ADC
15.1 Materials and Methods
[00462] Human CD40 transgenic mice were implanted with A20 cells engineered to
express
the antigen Thy 1.1. Tumor cells were implanted subcutaneously in the flank on
day 0. When
mean tumor size (measured by using the formula Volume (mm3) = 0.5 * Length *
Width2, where
length is the longer dimension) of 100 mm3 was reached, mice were randomized
into treatment
groups of 5 mice per group. Animals were then treated with indicated
treatments
intraperitoneally; each treatment was given once every three days for a total
of three treatments.
Stock concentrations of antibody were diluted to the appropriate concentration
and injected into
animals in 10011.1 volumes. Final dosages were 1 mg/kg for the SEA-CD40 and 1
mg/kg for the
vc-IVIIVIAE ADC directed to Thy1.1. Tumor length, tumor width, and mouse
weight were
measured throughout the study and tumor volume was calculated using the
formula above.
Animals were followed until tumor volume was measured until they reached
¨1,000 mm3 when
animals were then euthanized.
15.2 Results
[00463] As shown in FIG. 15, treatment of the A20 tumor model with a
subtherapeutic dose
of SEA-CD40 resulted in reduced tumor growth and tumor growth delay.
Similarly, the vc-
IVIIVIAE containing antibody-drug conjugate showed mild tumor growth delay.
However, when
the two agents were administered in tandem to the animals, curative antitumor
responses were
observed. This data demonstrates the synergistic benefit in combining both the
enhanced SEA-
CD40 antibody with immunogenic cell death induction chemotherapy delivered via
an ADC,
including the possibility of achieving curative response.
Example 16: Differential Activities in Various Tumor Cell Lines Treated with
an
Auristatin Based ADC Targeted to a Tumor-Associated Antigen and TIGIT
Antibodies
with Different Fc Backbones
16.1 Materials and Methods
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[00464] Five different human cancer cell lines, SK-MEL 28 (melanoma) MDA-MB-
468
(breast), CORL23 (lung), A549 (lung), and HT-26 (colon), were used in this
example. Each cell
line was treated for 18 hrs with 11.tg/m1 of a tumor-targeting antibody-vcMMAE
ADC with a
drug-to-antibody ratio (DAR) of 4. After incubation at 37 C, the tumor cells
were washed and
human PBMCs were added with 11.tg/m1 of the anti-TIGIT antibody treatments as
indicated in
FIG. 16. Anti-TIGIT antibodies used had various levels of backbone effector
function, with the
LALA TIGIT antibody having no FcyR binding, and the SEA-TIGIT antibody having
enhanced
FcyR binding (increased binding to the activating FcyIIIaR, and decreased
binding to the
inhibitory FcyRIIbR. Table D below shows the relative activities of the
different TGT
antibodies. These cultures of immune cells, anti-TIGIT antibody treatment, and
dead/dying
tumor cells were incubated for an additional 48 hrs. The supernatant for each
condition was
harvested and evaluated using ELISA per the manufacturer's instructions for
cytokine induction.
Table D: TGT antibodies
CD155 T reg CD8 T cell APC
depletion response activation
Anti-TIGIT
LALA ++
Anti-TIGIT IgG1
++ ++ ++
SEA-TGT
++ ++++ +++ +++
16.2 Results
[00465] As shown in FIG. 16, dead/dying tumor cells with PBMC incubation that
didn't
comprise any further immunomodulating treatments ("Untreated" in FIG. 16) had
some
stimulation of the immune cells as seen by the production of the innate type I
interferon related
cytokine IP10. The level of IP10 activation was dependent on tumor cell type
as the SK-MEL
28, MDA-MB-468 and CORL23 induced much more activation of PBMCs than the A549
or
HT-26 cells. Regardless of tumor cells though, subsequent addition of the
enhanced effector
function anti-TIGIT mAb SEA-TGT to the co-cultures resulted in further
enhanced immune cell
activation across the board. For the cell lines where co-incubation with dead
cells alone wasn't
enough to drive substantial activation, the inclusion of the nonfucosylated
TIGIT mAb SEA-
TGT showed the most substantial increases demonstrating its strong activation
ability. In these
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cells the IgG1 backbone TIGIT antibody was able to drive immune cell
activation that was
muted when compared to the enhanced mAb. The LALA version of the anti-TIGIT,
which has
no FcyR engaging ability, was inactive at driving any immune activation.
Example 17: Differential Activities in Various Tumor Cell Lines Treated with a
Auristatin
Based ADC Targeted to the Tumor-Associated Antigen and TIGIT Antibodies with
Different Fc Backbones
17.1 Materials and Methods
[00466] 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 in this
example. Each cell line was treated for 18 hrs with 1 [tg/m1 of a tumor-
targeting antibody-
vcMMAE ADC with a drug-to-antibody ratio (DAR) of 4. After incubation at 37 C,
the tumor
cells were washed and human PBMCs were added with 1 [tg/m1 of the anti-TIGIT
antibody
treatments as indicated in FIG. 16. These cultures of immune cells, anti-TIGIT
antibody
treatment, and dead/dying tumor cells were incubated for an additional 48 hrs.
The supernatant
for each condition was harvested and evaluated using ELISA per the
manufacturer's instructions
for cytokine induction.
17.2 Results
[00467] As shown in FIG. 17, dead/dying tumor cells with PBMC incubation that
didn't
comprise any further immunomodulating treatments ("Untreated" in FIG. 16) had
some
stimulation of the immune cells as seen by the production of the adaptive
cytokine IFNy. The
level of IFNy activation was dependent upon tumor cell type as SK-MEL 28, MDA-
MB-468 and
CORL23 induced more activation of PBMCs than A549 or HT-26 cells. Regardless
of tumor
cells though, subsequent addition of the nonfucosylated SEA-TGT mAb which has
enhanced
effector function to the co-cultures resulted in further enhanced immune cell
activation across
the board. For cell lines where co-incubation with dead cells alone wasn't
enough to drive
substantial activation, inclusion of the nonfucosylated TIGIT mAb SEA-TGT
showed the most
substantial increases demonstrating its strong activation ability. In these
cells the IgG1 backbone
TIGIT antibody was able to drive some immune cell activation but it was
greatly muted vs. the
enhanced mAb. The LALA version of the anti-TIGIT, which has no FcyR engaging
ability, was
inactive at driving any immune activation.
Example 18: Synergistic Effect with Combination of a Nonfucosylated TIGIT
Antibody
and an Auristatin Based ADC
18.1 Materials and Methods
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18.1.1 In vitro evaluation of a TIGIT antibody and an auristatin based ADC
[00468] A549 non-small cell lung cancer carcinoma cells were induced to
undergo cell death
with an ECso concentration of the ICD-inducing agent vc-MMAE conjugated to a
tumor-cell-
targeting antibody. The cells were incubated with the agent for 18 hours and
then added to
human PBMCs in concert with various concentrations (1, 0.1, 0.01 [tg/m1) of
anti-TIGIT
antibodies with different Fc backbones, including anti-TIGIT LALA, SEA-TGT,
and antibody
3106 H4/L1, which is an IgG1 antibody (US 2018/0066055 Al). Then, immune
activation was
assessed by measuring cytokine (IP10) levels 48 hours after co-culture.
18.1.2 In vivo evaluation of a TIGIT antibody and an auristatin based ADC
[00469] Balb/c mice were implanted with the CT26 syngeneic tumor cell line
that expresses
the tumor antigen Thy1.1 subcutaneously in the flank on day 0. When mean tumor
size
(measured by using the formula Volume (mm3) = 0.5 * Length * Width2, where
length is the
longer dimension) of 100 mm3 was reached, mice were randomized into treatment
groups of 5
mice per group. Animals were then treated with indicated treatments
intraperitoneally; each
treatment was given once every three days for a total of three treatments.
Stock concentrations of
antibody were diluted to the appropriate concentration and injected into
animals in 100 .1
volumes. Final dosages were 0.1 mg/kg for the SEA-TGT mIgG2a and 5 mg/kg for
the tumor-
targeting vc-MMAE Thy1.1 ADC. Both antibodies used were on mIgG2a backbones,
and SEA-
TGT mIgG2a is nonfucosylated. Tumor length, tumor width, and mouse weight were
measured
throughout the study, and tumor volume was calculated using the formula above.
Animals were
followed until tumor volume was measured until they reached ¨ 1,000 mm3 when
animals were
then euthanized.
[00470] Balb/c mice were implanted with Renca syngeneic tumor cell line that
expresses the
tumor antigen EphA2 subcutaneously in the flank on day 0. When mean tumor size
(measured
by using the formula Volume (mm3) = 0.5 * Length * Width2, where length is the
longer
dimension) of 100 mm3 was reached, mice were randomized into treatment groups
of 5 mice per
group. Animals were then treated with indicated treatments intraperitoneally;
each treatment was
given once every three days for a total of three treatments. Stock
concentrations of antibody
were diluted to the appropriate concentration and injected into animals in 100
11.1 volumes. Final
dosages were 0.1 mg/kg for the SEA-TGT and 1 mg/kg for the tumor-targeting vc-
MMAE
EphA2 ADC. Both antibodies used were on mIgG2a backbones. Tumor length, tumor
width,
and mouse weight were measured throughout the study, and tumor volume was
calculated using
the formula above. Animals were followed until tumor volume was measured until
they reached
¨ 1,000 mm3 when animals were then euthanized.
18.2 Results
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[00471] As shown in FIG. 18A, incubation of the ADC killed tumor cells with an
immune
population of cells, resulting in some activation of the immune cells because
of the
immunogenic cell death induced via the IVIMAE as measured by induction of the
cytokine IP 1 0
(see bars marked with "0" on X axis). Further, addition of increasing
concentrations of SEA-
TGT to the co-culture resulted in substantial increases in the induction of
this cytokine. This was
not seen with either the effector-null anti-TIGIT antibody (LALA) or the
standard anti-TIGIT
IgG1 antibody (3106 H4/L1), showing that the nonfucosylated backbone of SEA-
TGT drives
synergy with immunogenic cell-death-inducing MMAE to provide superior immune
cell
activation.
[00472] As shown in FIG. 18B and FIG. 18C, treatment of the CT26 tumor model
and the
Renca tumor model, respectively, with a subtherapeutic dose of 0.1 mg/kg of
SEA-TGT resulted
in reduced tumor growth and tumor growth delay. The vc-MMAE (vedotin) ADC
showed mild
tumor growth delay on its own. When the two agents were administered in tandem
to the
animals, a substantial reduction in tumor growth was observed, as well as a
curative response in
40% of the animals. These data demonstrate the synergistic benefit in
combining the SEA-TGT
mIgG2a antibody (the SEA-TGT antibody reformatted as a nonfucosylated mouse
IgG2a that
corresponds to a nonfucosylated human IgG1 backbone), with its enhanced
effector function,
with an ADC that induces immunogenic cell death.
[00473] The fact that such observations were made in two different tumor
models with ADCs
targeting two different tumor cell antigens suggests the anti-tumor activity
of this combination
may be broadly applicable to different tumor types.
Example 19: Synergistic Effect with Combination of a Nonfucosylated TIGIT
Antibody
and Another Auristatin Based ADC
19.1 Materials and Methods
[00474] Renca cells engineered to express murine B7H4 were implanted
subcutaneously in
Balb/c mice. Tumors were allowed to grow to reach 100 mm3, at which time mice
were treated
with subtherapeutic doses of SEA-TGT and SGN-B7H4 MMAE ADC (B7H4V), or a
subtherapeutic dose of SEA-TGT and a therapeutic dose of oxaliplatin.
Compounds were given
on the same day, and mice were treated for a total of 3 doses 7 days apart.
19.2 Results
[00475] As shown in Fig. 19, SEA-TGT combinatorial activity extended to
increased anti-
tumor activity when a subtherapeutic dose of SEA-TGT was combined with a
subtherapeutic
dose of B7H4V. The combinatorial activity of SEA-TGT (subtherapeutic dose) and
B7H4V
(subtherapeutic dose) was similar to the combinatorial activity of SEA-TGT
(subtherapeutic
dose) and oxaliplatin (therapeutic dose), a known ICD inducer. Oxaliplatin,
however, is
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associated with warnings of anaphylaxis and renal toxicity, and when given at
the therapeutic
dose, is not very active on its own and is often used in combination with a
variety of other
chemotherapies.
[00476] Also as shown in Fig. 19, the curative effect of SEA-TGT was
significantly increased
when it was combined with the B7H4V. Although not intending to be bound by
theory, this
effect is thought to be due to induction of ICD and the long-lived memory T
cell response that is
induced with such a combination (see also Example 23 below).
[00477] In summary, the results of this experiment are consistent with the
other results
described herein showing that a nonfucosylated antibody directed against an
immune cell
engager (in this experiment, an nonfucosylated anti-TIGIT antibody such as SEA-
TGT)
combines well with an agent that induces immune cell death, which in this
particular example
were both an MMAE ADC (i.e., B7H4V) and oxaliplatin. The combination with an
MMAE
ADC, however, is preferred because of the toxicity associated with oxaliplatin
and because
comparable therapeutic effects were observed at a subtherapeutic dose of the
ADC as compared
to a therapeutic dose of oxaliplatin.
Example 20: Synergistic Effect with Combination of a Nonfucosylated SEA-CD70
Antibody and an Auristatin Based ADC
[00478] SEA-CD70 (SEA-h1F6) is a nonfucosylated antibody targeting the CD70
antigen.
The CD70 molecule is a member of the tumor necrosis factor (TNF) ligand
superfamily
(TNF SF) and it binds to the related receptor, CD27 (TNFRSF7). The interaction
between the
two molecules activates intracellular signals from both receptors. In normal
conditions, CD70
expression is transient and limited to activated T and B cells, mature
dendritic, and natural killer
(NK) cells. Similarly, CD27 is expressed on both naive and activated effector
T cells, as well as
NK and activated B cells. However, CD70 is also aberrantly expressed in
various hematologic
cancers, including acute myeloid leukemia (AML), myelodysplastic syndrome
(MDS), and non-
Hodgkin's lymphoma (NHL), as well as carcinomas, and plays a role in both
tumor cell survival
and/or tumor immune evasion. SEA-CD70 (which comprises VH and VL of SEQ ID
NOs: 41
and 42, respectively, and CDRs of SEQ ID NOs: 53-58), acts through blocking
CD70/CD27 axis
signaling, eliciting antibody dependent cellular phagocytosis (ADCP) and
complement
dependent cytotoxicity (CDC), and enhancing antibody dependent cellular
cytotoxicity (ADCC).
As described below, SEA-CD70 was tested in combination with brentuximab
vedotin (BV,
SGN-35, cAC10-MMAE) in a subcutaneous NHL model. Brentuximab vedotin, also
referred to
as SGN-35, is a CD30-targeting ADC containing MMAE conjugated to the
monoclonal
antibody cAC10. CD30 is expressed in Hodgkin's lymphoma as well as in a subset
of NHL
patients.
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20.1 Materials and Methods
In vivo evaluation of subcutaneous tumor growth in an NHL xenograft model
[00479] Farage cells (2.5x106 cells/animal) were resuspended in 0.1 mL of 25%
matrigel and
injected subcutaneously into SCID mice, which contain active innate immune
effector cells to
mediate ADCP and ADCC. When mean tumor size of 100 mm3 was reached (measured
by using
the formula: volume (mm3) = 0.51ength*width2, where the length is the longer
dimension),
mice were randomized into treatment groups of 6 mice per group. Treatments
were given
intraperitoneally. Stock concentrations of antibody and chemotherapy were
diluted to the
appropriate concentration and injected into animals at 10 pL/g of body weight.
Tumor length
and width and animal weight were measured at least two times weekly throughout
the study.
Nineteen days post implant, dosing was initiated with 3 mg/kg SEA-CD70 and/or
1 mg/kg SGN-
35. SEA-CD70 was dosed intraperitoneally (IP) every 4 days for 5 times and SGN-
35 was dosed
once IP on day 19. Animals were followed until tumor volume measured more than
500 mm3, at
which time the animals were euthanized. A tumor size endpoint of 500 mm3 was
chosen due to
the tendency of tumors to become ulcerated at a larger size.
20.2 Results
[00480] As shown in FIG. 20A, combining SGN-35 and SEA-CD70 delayed tumor
growth
compared to single agent SGN-35 or SEA-CD70 treatments. All untreated tumors
increased
volume more than 5 times their original size by day 28.5 (9.5 days post
treatment), while tumors
treated with single agent SEA-CD70 or single agent SGN-35 reached an average 5
fold increase
at days 34.5 and 46 respectively (15.5 and 27 days post treatment). Notably,
none of the tumors
treated with the combination of SEA-CD70 and SGN-35 reached the established
size endpoint
by the time the experiment was concluded (day 50) (Fig 20B). Compared to
single SEA-CD70
or SGN-35 treatments, no overt toxicity or additional loss of weight was
observed when
combining SEA-CD70 and SGN-35. These data indicated that the combination of an
ADC
carrying an MMAE payload (SGN-35) and a nonfucosylated antibody (SEA-CD70) is
efficacious and well-tolerated.
Example 21: Synergistic Effect with Combination of a Nonfucosylated SEA-BCMA
Antibody and a Auristatin ADC
[00481] SEA-BCMA is a nonfucosylated antibody targeting B-cell maturation
antigen
(BCMA), which is expressed on multiple myeloma (MM). SEA-BCMA (which has VH
and VL
of SEQ ID NOs: 45 and 46, respectively, and CDRs of SEQ ID NOs: 47-52), acts
through
blocking ligand mediated BCMA cell signaling, antibody dependent cellular
phagocytosis
(ADCP), and enhanced antibody dependent cellular cytotoxicity (ADCC). As
described below,
SEA-BCMA was tested in combination with SGN-CD48A in disseminated MM tumor
xenograft
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models. SGN-CD48A is a CD48-targeting ADC containing a glucuronide linked
IVIMAE. CD48
is broadly expressed in MM.
21.1 Materials and Methods
21.1.1 In vivo survival evaluation of xenograft model
[00482] MIM1S MM cells were injected IV into SCID animals, which contain
active innate
immune effector cells to mediate ADCP and ADCC. Seven days post implant,
dosing was
initiated with 0.1 mg/kg SEA-BCMA and/or 0.01 mg/kg SGN-CD48A, and animals
were
monitored for survival. SEA-BCMA was dosed weekly for 5 weeks IP and SGN-CD48A
was
dosed once IP. Animals were followed for 160 days for survival (N=8/group). By
day 51, all
untreated animals had been humanely euthanized according to IACUC protocols.
21.1.2 In vivo luciferase evaluation of xenograft model
[00483] L363 luciferase MM cells were injected IV into SCID animals, and MINI
cells were
allowed to home to the bone marrow. The luciferase signal was monitored over
time. At thirty
days post implant, dosing was initiated with 3 mg/kg SEA-BCMA and/or 0.3 mg/kg
SGN-
CD48A. SEA-BCMA was dosed weekly for 5 weeks IP and SGN-CD48A was dosed once
IP.
Animals were followed for 175 days (N=5/group). By day 58, all untreated
animals had been
humanely euthanized according to IACUC protocols.
21.2 Results
[00484] As shown in FIG. 21A-B, SGN-CD48A combined with SEA-BCMA induced
complete remissions and prolonged survival in the mouse models tested. In FIG.
21A, by day
160, five of eight animals remained alive in the group that received the
combination therapy,
compared to zero animals treated with SEA-BCMA alone and one animal treated
with SGN-
CD8A alone. In FIG. 21B, by day 37, all animals treated with the combination
therapy
displayed no detectable luciferase signal, which remained absent until the end
of study, at day
175. This striking synergy may be due to the unique combination of an ICD
inducing auristatin
ADC with the innate immune cell engaging SEA-BCMA.
Example 22: Vedotin ADC Induces Immune Cell Recruitment and Activation in vivo
22.1 Materials and Methods
[00485] Tumor xenografts were isolated from animals treated with a vc-MMAE ADC
or non-
binding vc-MMAE isotype ADC for 8 days, and subject to flow cytometry or
cytokine profiling.
CD45 positive immune cells were stained for CD11 c and activation observed by
staining for the
expression of WIC-Class II on the cell surface. Intratumoral cytokines were
measured by
Luminex.
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22.2 Results
[00486] As shown in Fig. 22, Tumor-bearing mice treated with MMAE-based ADCs
targeting a common tumor antigen (vc-MMAE ADC) resulted in the promotion of
immune cell
recruitment and activation in tumors. Dendritic cell infiltration and
dendritic cell antigen-
presenting were both significantly enhanced when treated with MMAE-based ADCs
targeting
the tumors (vcMMAE ADC) compared to the non-binding control (non-binding ADC)
(Fig.
22B). Intratumoral cytokine levels were also significantly enhanced when
treated with MMAE-
based ADCs targeting the tumors (vc-MMAE ADC) (Fig. 22C). These data suggest
that ADCs
comprising tubulin disrupter induce ER stress and tumor cell death in a manner
that results in
promotion of immune cell recruitment and activation in tumors.
[00487] These results suggest that MMAE-based ADCs as preferred partners for
immune
checkpoint blockade agents.
Example 23: Induction of T Cell Memory by Vedotin ADC
23.1 Materials and Methods
[00488] Balb/c mice were subcutaneously implanted with Renca syngeneic tumor
cells,
which express the tumor antigen Epha2, in the flank on day 0. When mean tumor
size (measured
by using the formula Volume (mm3) = 0.5 * Length * Width2, where length is the
longer
dimension) of 100 mm3 was reached, mice were randomized into treatment groups
of 5 mice per
group. Animals were then treated with indicated treatments intraperitoneally
(FIG. 21A). Each
treatment was given once. Stock concentrations of antibody were diluted to the
appropriate
concentration and injected into animals in 100 tL volumes. Final dosages were
5 mg/kg for the
tumor-targeting ADCs (ADC-vcMMAE) and the non-binding ADCs (isotype-vcMMAE,
also
known as h00-vcMMAE). Tumor length, tumor width, and mouse weight were
measured
throughout the study, and tumor volume was calculated using the formula above.
Animals were
followed until tumor volume reached ¨ 1,000 mm3, when animals were then
euthanized.
[00489] Mice who achieved a curative anti-tumor response were monitored. Then,
30 days
after achieving a cure, mice were rechallenged with Renca tumor cells (FIG.
21B), and
outgrowth and rejection of the new tumors were assessed.
23.2 Results
[00490] As shown in FIG. 23A, in the Renca syngeneic model, treatment with a
single dose
of tumor-targeting MMAE ADCs (ADC-vcMMAE) resulted in strong anti-tumor
activity and
curative responses. As shown in FIG. 23B, when mice cured with the MMAE ADC
treatment
were rechallenged with Renca tumor cells to assess the induction of immune
memory, such mice
were able to reject the subsequently implanted tumor cells. Such results
demonstrate an ability
of MMAE ADCs to elicit a specific anti-tumor T cell response.
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Example 24: Brentuximab vedotin (BV; SGN-35)-treated Cells Confer Protective
Anti-
tumor Immunity
24.1 Materials and Methods
[00491] A20 cells expressing human CD30 were treated with CD30-Auristatin ADC
(BV;
SGN-35) or MMAE for 18 hrs. Alternatively, one aliquot of cells was flash
frozen. Ficoll
centrifugation of treated samples was performed to remove live 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 intraperitoneally injected into mice. The
mice were
immunized 2 times, 7 days apart. Immunized mice rested for 7 additional days,
and then were
challenged with A20 lymphoma cells, and tumor growth or rejection was
monitored over time.
24.2 Results
[00492] As shown in FIG. 24, mice immunized with CD30-expressing A20 cells
that were
killed using BV or MMAE displayed stronger immune responses rejecting
implanted A20 cells,
compared to mice immunized with CD30-expressing A20 cells that were killed
with flash
freezing, a non-ICD method of cell death. These results indicate an induction
of a memory T cell
response. Induction of immunologic memory is considered the gold standard for
assessing the
ICD activity of a molecule.
[00493] Collectively, the results presented in the forgoing Examples support
the unique
ability of auristatin based ADCs (e.g., MIVIAE and MMAF) to induce immunogenic
cell death.
As demonstrated by the foregoing examples, the mechanism of action of
auristatins and their
ability to disrupt microtubule networks appears associated with induction of
ER stress responses
that leads to the exposure and secretion of danger signals (DAMPs). Exposure
of these DAMPs
initiate an innate immune cell response that can lead to an antigen specific T
cell response.
Induction of new antigen specific T cells that can recognize tumor antigens
can lead to curative
anti-tumor activity preclinically that is associated with long term memory T
cells responses that
provide long term immune protection.
[00494] This population of memory T cells, which are induced by MMAE ADCs, may
be
further increased and/or enhanced by nonfucosylated antibodies. After
establishing
immunological memory (e.g., through the above memory T cell responses)
resulting from
MMAE ADC induced ICD, nonfucosylated antibodies such as SEA-TIGIT can further
augment
immune responses through tumor-blocking/inhibitory mechanisms, similar to the
checkpoint
inhibitory mechanisms of PD-1/PD-Ll.
[00495] Further, 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 the
anti-tumor activity. Immune agonism can be amplified by the use of
nonfucosylated antibodies
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or antibodies that have been engineered to have enhanced binding to activating
FcyRs and/or
decreased binding to inhibitory FcyRs (e.g., as shown in the examples above
with
nonfucosylated CD40 and BCMA antibodies). Nonfucosylated antibodies can have
increased
binding to the activating FcyRIIIa receptor with and decreased or minimal
binding to the
inhibitory FcyRIIb receptor. This attribute is multimodal, depending on the
nature of the
antibody target. In the case of receptors like CD40 that are optimally active
when clustered,
nonfucosylated antibody binding to FcyRIIA+ cells increases receptor
clustering and immune
agonism and activation. See FIG. 25. As in the case of TIGIT, nonfucosylated
antibodies
increase the strength of an immune synapse between an antigen (+) T cells and
an antigen
presenting cells (FIG. 25). Engagement of the FcyRIIIa on the innate cell
increases their
activation and production of factors that can enhance an antigen specific T
cell response. Lastly,
the nonfucosylated backbone can, independently of the target antigen, bind to
innate immune
cells or other FcyRIIIa cells such as gamma delta T cells to induce an
activated state that can
help elicit a secondary antigen specific T cell response. All these mechanisms
by which the
nonfucosylated antibody work can lead to a T cell response that drives anti-
tumor activity and
long lived immune protection. The decreased or lack of binding to FcyRIIb
means that there are
no counter or inhibitory signals that reduce the immune activation driven by
the nonfucosylated
antibodies.
[00496] The mechanism of action of auristatin ADCs such as MMAE ADCs, coupled
with
the immune modulation by nonfucosylated mAbs, results in synergistic and
complementary
activity that was shown to result in enhanced immune activation and curative
anti-tumor
response as demonstrated herein.
[00497] All
publications, patents, patent applications or 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, or other document was
individually indicated
to be incorporated by reference for all purposes.
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Table of Sequences
Name SEQ Sequence
ID
NO
Anti-TIGIT 1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG
antibody Clone 13 SIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSE
VH Protein VGAILGYVWFDPWGQGTLVTVSS
Anti-TIGIT 2 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGS
antibody Clone 13A LIPYFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSE
VH VGAILGYVWFDPWGQGTLVTVSS
Anti-TIGIT 3 QVQLVQSGAEVKKPGS SVKVSCKASGGTFSAWAISWVRQAPGQGLEWMG
antibody Clone 13B SIIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPS
VH EVSGILGYVWFDPWGQGTLVTVSS
Anti-TIGIT 4 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGS
antibody Clone 13C IIPLFGKANYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCARGPSE
VH VKGILGYVWFDPWGQGTLVTVSS
Anti-TIGIT 5 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGS
antibody Clone 13D IIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSE
VH VKGILGYVWFDPWGQGTLVTVSS
Clones 13, 13A, 6 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLL
13B, 13C, and 13D IYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARRIPITFG
VL Protein GGTKVEIK
Clone 13 VH CDR1 7 GTFSSYAIS
Clones 13A, 13C, 8 GTFLSSAIS
and 13D VH CDR1
Clone 13B VH 9 GTFSAWAIS
CDR1
Clone 13 VH CDR2 10 SIIPIFGTANYAQKFQG
Clone 13A VH 11 SLIPYFGTANYAQKFQG
CDR2
Clones 13B and 13D 12 SIIPYFGKANYAQKFQG
VH CDR2
Clone 13C VH 13 SIIPLFGKANYAQKFQG
CDR2
Clones 13 and 13A 14 ARGPSEVGAILGYVWFDP
VH CDR3
Clone 13B VH 15 ARGPSEVSGILGYVWFDP
CDR3
Clones 13C and 13D 16 ARGPSEVKGILGYVWFDP
VH CDR3
Clones 13, 13A, 17 RSSQSLLHSNGYNYLD
13B, 13C, and 13D
VL CDR1
Clones 13, 13A, 18 LGSNRAS
13B, 13C, and 13D
VL CDR2
Clones 13, 13A, 19 MQARRIPIT
13B, 13C, and 13D
VL CDR3
Clone 13 heavy 20 QVQLVQSGAEVKI(PGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEW
chain hIgG1 (and MGSIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCA
hIgG1 RGPSEVGAILGYVWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
nonfucosylated) AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
amino acid SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
sequence; bold FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
indicates VH; SEA- EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
TGT PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGK
Clone 13A heavy 21 QVQLVQSGAEVKI(PGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEW
chain hIgG1 (and MGSLIPYFGTANYAQKFQGRVTITADES TSTAYMELSSLRSEDTAVYYC
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hIgG1 ARGPSEVGAILGYVWFDPWGQGTLVTVSS A S TKGP S VFPL AP S
SKSTSGG
nonfucosylated) TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
amino acid S S SLGTQTYI CNVNHKP SNTKVDKKVEPK S CDKTHT CPP CPAPELLGGP
S VF
sequence; bold LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
indicates VH REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPP SRDELTKNQVSL TCLVKGFYP SDIAVEWESNGQPENNYK
TTPPVLD SD G S FFLY SKLTVDK SRWQQ GNVF SCSVMHEALHNHYTQKSLS
LSPGK
Clone 13B heavy 22 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAWAISWVRQAPGQGLEW
chain hIgG1 (and MGSIIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
hIgG1 ARGPSEVSGILGYVWFDPWGQGTLVTVSSASTKGPSVFPLAPS SKS TS GG
nonfucosylated) TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
amino acid S S SLGTQTYI CNVNHKP SNTKVDKKVEPK S CDKTHT CPP CPAPELLGGP
S VF
sequence; bold LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
indicates VH REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPP SRDELTKNQVSL TCLVKGFYP SDIAVEWESNGQPENNYK
TTPPVLD SD G S FFLY SKLTVDK SRWQQ GNVF SCSVMHEALHNHYTQKSLS
LSPGK
Clone 13C heavy 23 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEW
chain hIgG1 (and MGSIIPLFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
hIgG1 ARGPSEVKGILGYVWFDPWGQGTLVTVS SASTKGP SVFPL AP S SKSTSGG
nonfucosylated) TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
amino acid S S SLGTQTYI CNVNHKP SNTKVDKKVEPK S CDKTHT CPP CPAPELLGGP
S VF
sequence; bold LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
indicates VH REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPP SRDELTKNQVSL TCLVKGFYP SDIAVEWESNGQPENNYK
TTPPVLD SD G S FFLY SKLTVDK SRWQQ GNVF SCSVMHEALHNHYTQKSLS
LSPGK
Clone 13D heavy 24 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEW
chain hIgG1 (and MGSIIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC
hIgG1 ARGPSEVKGILGYVWFDPWGQGTLVTVS SASTKGP SVFPL AP S SKSTSGG
nonfucosylated) TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
amino acid S S SLGTQTYI CNVNHKP SNTKVDKKVEPK S CDKTHT CPP CPAPELLGGP
S VF
sequence; bold LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
indicates VH REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPP SRDELTKNQVSL TCLVKGFYP SDIAVEWESNGQPENNYK
TTPPVLD SD G S FFLY SKLTVDK SRWQQ GNVF SCSVMHEALHNHYTQKSLS
LSPGK
Clone 13, 13A, 13B, 25 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP
13C, and 13D light QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAR
chain hkappa (and RIPITFGGGTKVEIKRTVAAP SVF1FPP SDEQLKS GTASVVCLLNNFYPREA
nonfucosylated) KVQWKVDNALQSGNSQESVTEQD SKD S TY SL S STLTL SKADYEKHKVYAC
amino acid EVTHQGL S SPVTKSFNRGEC
sequence; bold
indicates VL; SEA-
TGT
SEA-CD40 26 EVQLVESGGGLVQPGGSLRLSCAASGYS FTGYY I HWVRQAPGKGLEWVAR
(nonfucosylated VI PNAGGT SYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREG
hS2C6) heavy chain IYWWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDY FPEP
VTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVN
HKPSNT KVDKKVEPKSCDKT HTCP PCPAPELLGGPSVFL FPPKPKDTLMI
SRT PEVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKT KPRE EQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAP I E KT I S KAKGQPRE PQVYTL PP
SREEMT KNQVSLTCLVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEA-CD40 27 DIQMTQ SP SSLSASVGDRVT ITCRSSQSLVHSNGNT FLHWYQQKPGKAPK
(nonfucosylated LL IYIVSNRFSGVPSRFSGSGSGTDFILT I SSLQPEDFATYFCSQTTHVP
hS2C6) light chain WT FGQGTKVE I KRTVAAP SVFI FP PS DEQLKSGTASVVCLLNNFY
PREAK
VQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGEC
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VH of SEA-CD40 28 EVQLVESGGGLVQPGGSLRLSCAASGYS FTGYY I HWVRQAPGKGLEWVAR
VI PNAGGT SYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREG
IYWWGQGTLVTVSS
VL of SEA-CD40 29 DIQMTQ SP SSLSASVGDRVT ITCRSSQSLVHSNGNT FLHWYQQKPGKAPK
LL IYIVSNRFSGVPSRFSGSGSGTDFTLT I SSLQPEDFATY FCSQTTHVP
WT FGQGTKVE 1K
SEA-CD40 VH 30 GYY I H
CDR1
SEA-CD40 VH 31 RVIPNAGGTSYNQKFKG
CDR2
SEA-CD40 VH 32 EGIYW
CDR3
SEA-CD40 VL 33 RS SQ SLVH SNGNT FLH
CDR1
SEA-CD40 VL 34 TVSNRFS
CDR2
SEA-CD40 VL 35 SQTTHVPWT
CDR3
Alternative anti- 36 RVIPQAGGTSYNQKFKG
CD40 antibody
CDR2
SGN-B6A heavy 37 QFQLVQSGAEVKKPGASVKVSCKASGYS FT DYNVNWVRQAPGQGLEWI GV
chain variable region INPKYGTTRYNQKFKGRATLTVDKST STAYMELSSLRSEDTAVYYCTRGL
NAWDYWGQGTLVTVSS
SGN-B6A light 38 DIQMTQ SP SSLSASVGDRVT ITCGASENIYGALNWYQQKPGKAPKLL I YG
chain variable region ATNLEDGVPSRFSGSGSGRDYT FT I S SLQPEDIATYYCQNVLTT PYT
FGQ
GT KLE I K
SGN-STNV heavy 39 EVQLVQSGAEVKKPGASVKVSCKASGYT FT DHAI HWVRQAPGQGLEWMGY
chain variable region FS PGNDDI KYNE KFRGRVTMTADKS S STAYMELRSLRS DDTAVY
FCKRSL
ST PYWGQGTLVTVSS
SGN-STNV heavy 40 DIVMTQSPDSLAVSLGERAT INCKSSQSLLNRGNHKNYLTWYQQKPGQ PP
chain variable region KLL I YWASTRESGVPDRFSGSGSGTDFTLT I S
SLQAEDVAVYYCQNDYTY
PYT FGQGT KVE I K
SEA-CD70 heavy 41 QVQLVQSGAEVKKPGASVKVSCKASGYT FTNYGMNWVRQAPGQGLKWMGW
chain variable region INTYTGEPTYADAFKGRVIMIRDT SI STAYMELSRLRSDDTAVYYCARDY
GDYGMDYWGQGTTVTVSS
SEA-CD70 light 42 DIVMTQSPDSLAVSLGERAT INCRASKSVSTSGY SFMHWYQQKPGQPPKL
chain variable region L I YLASNLESGVPDRFSGSGSGTDFTLT I S
SLQAEDVAVYYCQHSREVPW
T FGQGT KVE I K
SGN-CD228A 43
heavy chain variable
QVQLQSGPGLVJ'S1fLSLTCTVSGDS17SGFWNW1RQPPGKGLEVIGVISDSGflYYNP
region SLKSRVTISRDTSKN Q YSI.KI.SSVTA A DTAV YYCARRTLA TITTUD
YWGQGTINTVSS
SGN-CD228A light 44
chain variable region
DFVMTQSPLSLPVTLGQPASISCRASOSLVH.CDGNTYLHWYQQAPGQSPRELINRVSNRF
SGVPDRFSGSGSGMFTLKIS RVEAEDVGVYYCSOSTHI'PPTFGQOTKLEI-k. (SEQ ID
SGN-BCMA heavy 45 QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYIHWVRQAPGQGL
chain variable region EWIGYINPNSGYTNYAQKFQGRATMTADKSINTAYVELSRLRSDDT
AVYFCTRYMWERVTGFFDFWGQGTMVTVSS
SGN-BCMA light 46 DIQMTQSPSSVSASVGDRVTITCLASEDISDDLAWYQQKPGKAPKV
chain variable region LVYTTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYKF
PPTFGGGTKVEIK
SGN-BCMA VH 47 DYYIH
CDR1
SGN-BCMA VH 48 YINPNSGYTNYAQKFQG
CDR2
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SGN-BCMA VH 49 YMWERVTGFFDF
CDR3
SGN-BCMA VL 50 LASEDISDDLA
CDR1
SGN-BCMA VL 51 TTSSLQS
CDR2
SGN-BCMA VL 52 QQTYKFPPT
CDR3
SEA-CD70 VH 53 NYGMN
CDR1
SEA-CD70 VH 54 WINTYTGEPTYADAFKG
CDR2
SEA-CD70 VH 55 DYGDYGMDY
CDR3
SEA-CD70 VL 56 RASKSVSTSGYSFMH
CDR1
SEA-CD70 VL 57 LASNLES
CDR2
SEA-CD70 VL 58 QHSREVPWT
CDR3
Zolbetuximab 59 QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGL
(175D10) heavy EWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSA
chain variable region VYYCTRSWRGNSFDYWGQGTTLTVSS
Zolbetuximab 60 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKP
(175D10) light chain GQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYY
variable region CQNDYSYPFTFGSGTK
Zolbetuximab 61 SYWIN
(175D10) VH CDR1
Zolbetuximab 62 NIYPSDSYTNYNQKFKD
(175D10) VH CDR2
Zolbetuximab 63 SWRGNSFDY
(175D10) VH CDR3
Zolbetuximab 64 KSSQSLLNSGNQKNYLT
(175D10) VL CDR1
Zolbetuximab 65 WASTRES
(175D10) VL CDR2
Zolbetuximab 66 QNDYSYPFT
(175D10) VL CDR3
163E12 heavy chain 67 QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGL
variable region KWMGWINTNTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDT
ATYFCARLGFGNAMDYWGQGTSVTVSS
163E12 light chain 68 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKP
variable region GQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYY
CQNDYSYPLTFGAGTKLELK
163E12 VH CDR1 69 NYGMN
163E12 VH CDR2 70 WINTNTGEPTYAEEFKG
163E12 VH CDR3 71 LGFGNAMDY
163E12 VL CDR1 72 KSSQSLLNSGNQKNYLT
163E12 VL CDR2 73 WASTRES
163E12 VL CDR3 74 QNDYSYPLT
SGN-PDL1V heavy 75 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLE
chain variable region WMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAV
YFCARKFHFVSGSPFGMDVWGQGTTVTV SS
SGN-PDL1V light 76 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI
chain variable region YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP
TFGQGTKVEIK
SGN-PDL1V VH 77 TAAIS
CDR1
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SGN-PDL1V VH 78 GIIPIFGKAHYAQKFQG
CDR2
SGN-PDL1V VH 79 KFHFVSGSPFGMDV
CDR3
SGN-PDL1V VL 80 RASQSVSSYLA
CDR1
SGN-PDL1V VL 81 DASNRAT
CDR2
SGN-PDL1V VL 82 QQRSNWPT
CDR3
SGN-ALPV heavy 83 EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGL
chain variable region EWLALIRNKATGYTTEYTASVKGRFTISRDNSKSILYLQMNSLKTED
TAVYYCARASFYYDGKVLAYWGQGTLVTVS S
SGN-ALPV light 84 DTQMTQ SP S SLSA SVGDRVTITCQASQDINKYLAWYQYKPGKAPKL
chain variable region LIHYTS SLQ SGVPSRF SGSGSGRDYTFTIS SLQPEDIATYYCLQYDNL
YTFGQGTKLEIK
SGN-ALPV VH 85 DYYMS
CDR1
SGN-ALPV VH 86 LIRNKATGYTTEYTASVKG
CDR2
SGN-ALPV VH 87 ASFYYDGKVLAY
CDR3
SGN-ALPV VL 88 QASQDINKYLA
CDR1
SGN-ALPV VL 89 YTSSLQS
CDR2
SGN-ALPV VL 90 LQYDNLYT
CDR3
SGN-B7H4V heavy 91 QLQLQESGPGLVKP SETLSLTCTVSGGSIKSGSYYWGWIRQPPGKGL
chain variable region EWIGNIYYSGSTYYNP SLRSRVTISVDTSKNQF SLKLS SVTAADTAV
YYCAREGSYPNQFDPWGQGTLVTV S S
SGN-B7H4V light 92 EIVMTQ SPATLSVSPGERATLSCRASQ SVS SNLAWYQQKPGQAPRL
chain variable region LIYGA S TRATGIPARF S GS GSGTEFTLTI S SLQ
SEDFAVYYCQQYHSF
PFTFGGGTKVEIK
SGN-B7H4V VH 93 GSIKSGSYYWG
CDR1
SGN-B7H4V VH 94 NIYYSGSTYYNPSLRS
CDR2
SGN-B7H4V VH 95 AREGSYPNQFDP
CDR3
SGN-B7H4V VL 96 RASQSVSSNLA
CDR1
SGN-B7H4V VL 97 GASTRAT
CDR2
SGN-B7H4V VL 98 QQYHSFPFT
CDR3
Disitamab vedotin 99 EV Q LV Q SGAEVKKPGATVKIS CKVSGYTFTDYYIHWVQ QAPGKGL
heavy chain EWMGRVNPDHGD SYYNQKFKDKATITADKSTDTAYMEL S SLRSED
TAVYFCARNYLFDHWGQGTLVTV S SA STKGP SVFPLAP S SKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ S SGLYSLS S
VVTVP S S SLGTQTYICNVNHKP SNTKVDKKVEPKS CDKTHTCPPCP
APELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KV SNKALPAPIEKTI SKAKGQPREP QVYTLPP S REEMTKNQV S LTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDK
SRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK
Disitamab vedotin 100 DIQMTQ SP S SV SA SVGDRVTITCKASQDVGTAVAWYQQKPGKAPK
light chain LLIYWA SIRHTGVPSRF SGSGSGTDFTLTIS SLQPEDFATYYCHQFAT
YTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPR
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EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLS SPVTKSFNRGEC
Lifastuzumab 101 EVQLVE S GGGLVQPGGSLRL S CAA SGF SF SDFAMSWVRQAPGKGLE
vedotin heavy chain WVATIGRVAFHTYYPD SMKGRFTISRDNSKNTLYLQMNSLRAEDT
AVYYCARHRGFDVGHFDFWGQGTLVTVS SA S TKGP SVFPLAP S SKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ S SGLY
SLS SVVTVPS S SLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKV SNKALPAPIEKTI S KAKGQPREP QVYTLPP S REEMTKNQV S LT
CLVKGFYP SDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTV
DKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK
Lifastuzumab 102 DIQMTQ SPS SL SA SVGDRVTITCRS SETLVHS SGNTYLEWYQQKPGK
vedotin light chain APKLLIYRV SNRFSGVPSRF SGSGSGTDFTLTIS SLQPEDFATYYCFQ
GSFNPLTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQ SGNSQESVTEQD SKD S TY SL S STLTL S KA
DYEKHKVYACEVTHQGLS SPVTKSFNRGEC
Enfortumab Vedotin 103 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGL
(EV) heavy chain EWVSYIS SSSSTIYYAD SVKGRFTISRDNAKNSLSLQMNSLRDEDTA
variable region VYYCARAYYYGMDVWGQGTTVTVS S
Enfortumab Vedotin 104 DIQMTQ SP S SV SA SVGDRVTITCRA S QGISGWLAWYQQKPGKAPKF
(EV) light chain LIYAASTLQ SGVPSRF SGSGSGTDFTLTIS SLQPEDFATYYCQQANSF
variable region PPTFGGGTKVEIK
Enfortumab Vedotin 105 SYNMN
(EV) VH CDR1
Enfortumab Vedotin 106 YISSSSSTIYYADSVKG
(EV) VH CDR2
Enfortumab Vedotin 107 AYYYGMDV
(EV) VH CDR3
Enfortumab Vedotin 108 RASQGISGWLA
(EV) VL CDR1
Enfortumab Vedotin 109 AASTLQS
(EV) VL CDR2
Enfortumab Vedotin 110 QQANSFPPT
(EV) VL CDR3
h2A2 heavy chain 111 QFQLVQ SGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQG
variable region LEWIGVINPKYGTTRYNQKFKGRATLTVDKSTSTAYMELS SLRSED
TAVYYCTRGLNAWDYWGQGTLVTVS S
h2A2 light chain 112 DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKL
variable region LIYGATNLEDGVP SRFSGSGSGRDYTFTIS SLQPEDIATYYCQNVLTT
PYTFGQGTKLEIK
h2A2 VH CDR1 113 DYNVN
h2A2 VH CDR2 114 VINPKYGTTRYNQKFKG
h2A2 VH CDR3 115 GLNAWDY
h2A2 VL CDR1 116 GA S ENIYGALN
h2A2 VL CDR2 117 GATNLED
h2A2 VL CDR3 118 QNVLTTPYT
h15H3 heavy chain 119 QVQLVQ SGAEVKKPGASVKVSCKASGYSFSGYFMNWVRQAPGQG
variable region LEWMGLINPYNGD S FYNQKFKGRVTMTRQ TS TS TVYMEL S SLRSED
TAVYYCVRGLRRDFDYWGQGTLVTVS S
hl 5H3 light chain 120 DVVMTQ S PL SLPVTLGQPA S I S CKS SQ S LLD
SDGKTYLNWLFQRPGQ
variable region SPRRLIYLVSELD SGVPDRF SGS GS GTDFTLKI S RVEAEDVGVYYCW
QGTHFPRTFGGGTKLEIK
h15H3 VH CDR1 121 GYFMN
h15H3 VH CDR2 122 LINPYNGDSFYNQKFKG
h15H3 VH CDR3 123 GLRRDFDY
h15H3 VL CDR1 124 KSSQSLLDSDGKTYLN
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h15H3 VL CDR2 125 LVSELDS
h15H3 VL CDR3 126 WQGTHFPRT
SGN-CD228A 127 QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEY
heavy chain variable IGYISDSGITYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYC
region ARRTLATYYAMDYWGQGTLVTVSS
SGN-CD228A light 128 DFVMTQSPLSLPVTLGQPASISCRASQSLVHSDGNTYLHWYQQRPG
chain variable region QSPRLLTYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
SQSTHVPPTFGQGTKLEIK
SGN-CD228A VH 129 SGYWN
CDR1
SGN-CD228A VH 130 YISDSGITYYNPSLKS
CDR2
SGN-CD228A VH 131 RTLATYYAMDY
CDR3
SGN-CD228A VL 132 RASQSLVHSDGNTYLH
CDR1
SGN-CD228A VL 133 RVSNRFS
CDR2
SGN-CD228A VL 134 SQSTHVPPT
CDR3
SGN-LIV1A 135 QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMIHWVRQAPGQG
Ladiratuzumab LEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSD
Vedotin (LV) heavy DTAVYYCAVHNAHYGTWFAYWGQGTLVTVSS
chain variable region
SGN-LIV1A 136 DVVMTQ SPLSLPVTLGQPASIS CRS S Q SLLHS SGNTYLEWYQQRPGQ
Ladiratuzumab SPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQ
Vedotin (LV) light GSHVPYTFGGGTKVEIK
chain variable region
SGN-LIV1A 137 DYYMH
Ladiratuzumab
Vedotin (LV) VH
CDR1
SGN-LIV1A 138 WIDPENGDTEYGPKFQG
Ladiratuzumab
Vedotin (LV) VH
CDR2
SGN-LIV1A 139 HNAHYGTWFAY
Ladiratuzumab
Vedotin (LV) VH
CDR3
SGN-LIV1A 140 RSSQSLLHSSGNTYLE
Ladiratuzumab
Vedotin (LV) VL
CDR1
SGN-LIV1A 141 KISTRFS
Ladiratuzumab
Vedotin (LV) VL
CDR2
SGN-LIV1A 142 FQGSHVPYT
Ladiratuzumab
Vedotin (LV) VL
CDR3
Tisotumab Vedotin 143 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGL
(TV) heavy chain EWVSSISGSGDYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDT
variable region AVYYCARSPWGYYLDSWGQGTLVTVSS
Tisotumab Vedotin 144 DIQMTQSPPSLSASAGDRVTITCRASQGISSRLAWYQQKPEKAPKSLI
(TV) light chain YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP
variable region YTFGQGTKLEIK
Tisotumab Vedotin 145 GFTFSNYA
(TV) VH CDR1
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Tisotumab Vedotin 146 ISGSGDYT
(TV) VH CDR2
Tisotumab Vedotin 147 ARSPWGYYLDS
(TV) VH CDR3
Tisotumab Vedotin 148 .. QGIS SR
(TV) VL CDR1
Tisotumab Vedotin 149 .. AAS
(TV) VL CDR2
Tisotumab Vedotin 150 QQYNSYPYT
(TV) VL CDR3
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