EP4240415A1 - Polythérapie à base d'un conjugué anticorps-médicament avec un nhibiteur de cellules immunitaires - Google Patents

Polythérapie à base d'un conjugué anticorps-médicament avec un nhibiteur de cellules immunitaires

Info

Publication number
EP4240415A1
EP4240415A1 EP21816258.4A EP21816258A EP4240415A1 EP 4240415 A1 EP4240415 A1 EP 4240415A1 EP 21816258 A EP21816258 A EP 21816258A EP 4240415 A1 EP4240415 A1 EP 4240415A1
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
amino acid
acid sequence
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21816258.4A
Other languages
German (de)
English (en)
Inventor
Shyra Gardai
Alyson SMITH
Kerry Klussman
Bernard LIU
Heather VAN EPPS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seagen Inc
Original Assignee
Seagen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seagen Inc filed Critical Seagen Inc
Publication of EP4240415A1 publication Critical patent/EP4240415A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • 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 Fc ⁇ Rs, wherein the activating Fc ⁇ Rs include one or more of Fc ⁇ RIIIa, Fc ⁇ RIIa, and/or Fc ⁇ RI.
  • ADC antibody-drug conjugate
  • Embodiment 2 The method of embodiment 1, wherein the second antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa.
  • Embodiment 3 The method of embodiment 1, wherein second antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa and Fc ⁇ RIIa.
  • Embodiment 4 The method of embodiment 1, wherein the second antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa and Fc ⁇ RI.
  • Embodiment 5 The method of embodiment 1, wherein the second antibody comprises an Fc with enhanced binding to Fc ⁇ RIIIa, Fc ⁇ RIIa, and Fc ⁇ RI. [0009] Embodiment 6.
  • Embodiment 7 The method of embodiment 6, wherein the Fc of the second antibody has reduced binding to Fc ⁇ RIIb.
  • Embodiment 8 The method of any one of embodiments 1-7, wherein the Fc of the second antibody has reduced fucose levels and/or has been engineered to comprise one or more mutations such that the Fc has enhanced binding to the one or more activating Fc ⁇ Rs.
  • Embodiment 9. The method of embodiment 8, wherein the second antibody is nonfucosylated.
  • Embodiment 10 Embodiment 10.
  • 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.
  • Embodiment 12 The method of any one of embodiments 1-11, wherein the first antibody binds a tumor-associated antigen.
  • Embodiment 13 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.
  • 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 Fc with enhanced ADCC activity relative to a corresponding wild-type Fc of the same isotype.
  • ADC antibody-drug conjugate
  • Embodiment 16 The method of any one of embodiments 13-15, wherein the second antibody comprises an Fc with enhanced binding to one or more activating Fc ⁇ Rs, wherein the activating Fc ⁇ Rs include one or more of Fc ⁇ RIIIa, Fc ⁇ RIIa, and/or Fc ⁇ RI.
  • Embodiment 17 The method of embodiment 16, wherein the second antibody comprise an Fc with enhanced binding to at least Fc ⁇ RIIIa.
  • Embodiment 18 The method of embodiment 16, wherein second antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa and Fc ⁇ RIIa.
  • Embodiment 20 The method of embodiment 16, wherein the second antibody comprises an Fc with enhanced binding to Fc ⁇ RIIIa, Fc ⁇ RIIa, and Fc ⁇ RI.
  • 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 Fc ⁇ Rs.
  • Embodiment 22 The method of embodiment 21, wherein the Fc of the second antibody has reduced binding to Fc ⁇ RIIb.
  • Embodiment 23 Embodiment 23.
  • 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
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the first antibody does not bind Nectin-4.
  • 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.
  • Embodiment 26 Embodiment 26.
  • the first antibody binds an antigen selected from CD71, Axl, AMHRII, and LGR5, Axl, CA9, CD142, CD20, CD22, CD228, CD248, CD30, CD33, CD37, CD48, CD7, CD71, CD79b, CLDN18.2, CLDN6, c-MET, EGFR, EphA2, ETBR, FCRH5, GCC, Globo H, gpNMB, HER-2, IL7R, Integrin beta-6, KAAG-1, LGR5, LIV-1, LRRC15, Ly6E, Mesothelin (MSLN), MET, MRC2, MUC16, NaPi2b, Nectin-4, OT-MUC1 (onco-tethered-MUC1), PSMA, ROR1, SLAMF7, SLC44A4, SLITRK6, STEAP-1, STn, TIM-1, TRA-1-60, and Tumor-associated antigen selected from CD71, Axl, A
  • 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.
  • 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 ⁇ V ⁇ 6), CD22, CD79b, CD30, CD19, CD70, CD228, CD47, and CD48.
  • Embodiment 29 Embodiment 29.
  • 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, SLC44A4, CD30, and sTn.
  • 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, SLC44A4, CD30, and sTn.
  • tubulin disrupter is an auristatins, a tubulysin, a colchicine, a vinca alkaloid, a taxane, a cryptophycin, a maytansinoid, or a hemiasterlin.
  • Embodiment 31 The method of embodiment 30, wherein the tubulin disrupter is an auristatin.
  • Embodiment 32 The method of embodiment 30, wherein the tubulin disrupter is an auristatin.
  • 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).
  • 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).
  • MMAE N-methylvaline-valine-dolaisoleuine-
  • 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; ⁇ MSLN-MMAE, targeting Mesothelin (MSLN) (Roche-Genentech), RC68, also known as Remegen EGFR ADC, targeting EGFR (RemeGen (Rongchang Biopharmaceutical (Yantai)
  • 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.
  • 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
  • Embodiment 34 The method of embodiment 33, wherein the MMAE is conjugated to the first antibody through a linker that comprises valine and citrulline.
  • Embodiment 35 The method of embodiment 34, wherein the linker-MMAE is vcMMAE.
  • 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.
  • Embodiment 37 Embodiment 37.
  • Embodiment 38 The method of any one of embodiments 1-32, wherein the tubulin disrupter is MMAF. [0043] Embodiment 38-1.
  • 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,
  • Embodiment 39 The method of embodiment any one of embodiments 1-30, wherein the tubulin disrupter is a tubulysin.
  • Embodiment 40 The method of embodiment 39, wherein the tubulysin is selected from tubulysin D, tubulysin M, tubuphenylalanine, and tubutyrosine.
  • Embodiment 41 Embodiment 41.
  • 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 ozo
  • 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.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • Embodiment 46 Embodiment 46.
  • Embodiment 47 The method of any one of embodiments 1-41, 33-1, and 38-1, wherein the first antibody is an anti-PD-L1 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.
  • Embodiment 48 Embodiment 48.
  • 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.
  • Embodiment 50 Embodiment 50.
  • 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.
  • Embodiment 52 Embodiment 52.
  • 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.
  • Embodiment 54 The method of embodiment 53, wherein the antibody-drug conjugate is disitamab vedotin.
  • Embodiment 55 Embodiment 55.
  • Embodiment 56 The method of embodiment 55, wherein the antibody-drug conjugate is lifastuzumab vedotin.
  • Embodiment 57 Embodiment 57.
  • 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.
  • Embodiment 59 Embodiment 59.
  • 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.
  • Embodiment 61 Embodiment 61.
  • 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.
  • Embodiment 63 Embodiment 63.
  • 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.
  • Embodiment 65 Embodiment 65.
  • 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.
  • Embodiment 67 Embodiment 67.
  • 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.
  • Embodiment 69 Embodiment 69.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 70 The method of embodiment 69, wherein the antibody-drug conjugate is tisotumab vedotin.
  • Embodiment 71 Embodiment 71.
  • Embodiment 72 The method of any one of embodiments 1-71, 33-1, and 38-1, wherein the second antibody binds TIGIT.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 77 The method of any one of embodiments 1-71, wherein the second antibody binds CD40.
  • Embodiment 78 The method of any one of embodiments 1-71, wherein the second antibody binds CD40.
  • 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.
  • 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.
  • Embodiment 80 Embodiment 80.
  • Embodiment 81 The method of any one of embodiments 1-71, 33-1, and 38-1, wherein the second antibody binds CD70.
  • 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.
  • Embodiment 83 Embodiment 83.
  • Embodiment 84 The method of any one of embodiments 1-71, 33-1, and 38-1, wherein the second antibody binds BCMA.
  • 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.
  • Embodiment 86 Embodiment 86.
  • 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.
  • Embodiment 90 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.
  • 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.
  • Embodiment 90 Embodiment 90.
  • Embodiment 95 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 Fc ⁇ Rs as compared to a corresponding wild-type Fc of the same isotype.
  • Embodiment 93 The method of embodiment 92, wherein the Fc of the second antibody has reduced binding to Fc ⁇ RIIb.
  • Embodiment 94 The method of any one of embodiments 1-93, 33-1, and 38-1, wherein the Fc of the second antibody has enhanced binding to Fc ⁇ RIIIa and reduced binding to Fc ⁇ RIIb.
  • Embodiment 95 Embodiment 95.
  • 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.
  • Embodiment 97 Embodiment 97.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 101 Embodiment 101.
  • 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.
  • 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).
  • Embodiment 104 Embodiment 104.
  • Embodiment 105 The method of embodiment 104, wherein the induction of immune memory comprises induction of memory T cells.
  • 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).
  • 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.
  • 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.
  • Embodiment 110 The method of embodiment 109, wherein the immune activating cytokine is CXCL10 or IFN ⁇ .
  • 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.
  • 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.
  • Embodiment 114 The method of any one of embodiments 1-113, 33-1, and 38-1, wherein the cancer has high tumor mutation burden.
  • Embodiment 115 Embodiment 115.
  • FIG.1 shows that non-directed chemotherapeutic agents impair T cell responses.
  • 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.
  • 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.
  • ADCs auristatin antibody-drug conjugates
  • 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.
  • 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 ⁇ g/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.
  • 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.
  • 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.
  • FIG.7A further illustrates the immunogenic cell death (ICD) pathway.
  • FIG.7B provides information regarding the payloads of certain ADCs used in FIG. 7B-E.
  • 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).
  • 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).
  • 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.
  • 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).
  • FIG.10E summarizes the results in FIG.7B-E, FIG.8A-D, FIG.9A-D, and FIG. 10A-D.
  • FIG.11A-D show antibody binding to Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIIa in Chinese hamster ovary (CHO cells).
  • FIG.11A shows antibodies assessed.
  • FIG.11B shows binding to Fc ⁇ RIIa.
  • FIG.11C shows binding to Fc ⁇ RIIIa.
  • FIG.11D shows binding to Fc ⁇ RIIb.
  • FIG.12A-D show levels of CXCL10 (FIG.12A), IFN ⁇ (FIG.12B), IL10 (FIG.
  • 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.
  • FIG.14A-C show levels of CXCL14 (FIG.14A), IL14 (FIG.14B), and IFN ⁇ (FIG. 14C) in tumor cells from melanoma, lung, breast, and pancreas.
  • 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.
  • 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.
  • FIG.17 shows IFN ⁇ 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.
  • 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 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 vc-MMAE ADC.
  • SEA-TGT enhanced (nonfucosylated) IgG1 Fc backbone
  • 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 vc-MMAE 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.
  • ADC Thy1.1 vc-MMAE ADC in FIG.18B and EphA2 vc-MMAE ADC in FIG.18C
  • mIgG2a SEA-TGT the SEA-TGT antibody reformatted as a nonf
  • 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.
  • SEA-TGT nonfucosylated TIGIT antibody
  • B7H4V SGN-B7H4 vedotin ADC
  • 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 mm 3 tumor size endpoint.
  • NHL non-Hodgkin lymphoma
  • FIG.21A-B show synergistic effects of SEA-BCMA, a nonfucosylated anti-BCMA antibody, and SGN-CD48A, an anti-CD48 ADC containing MMAE.
  • FIG.21A shows in vivo survival evaluation of a xenograft model
  • FIG.21B shows in vivo luciferase evaluation of a xenograft model.
  • 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 CD11c and activation observed by staining for the expression of MHC-Class II on the cell surface.
  • FIG.22C Intratumoral cytokines were measured by Luminex.
  • 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).
  • 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.
  • 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.
  • the SEA-CD40 antibody can bind CD-40 expressed on antigen presenting cells (APCs) with the Fc portion of the antibody binding to Fc ⁇ RIIIa expressed on natural killer (NK) cells or on monocytes, which promotes receptor clustering.
  • APCs antigen presenting cells
  • the SEA-TGT antibody in contrast, binds to TIGIT expressed on T-cells and the Fc region of the antibody binds to Fc ⁇ RIIIa expressed on APCs.
  • ICD Immunogenic Cell Death
  • 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.
  • 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.
  • tumor cells undergoing ICD in response to auristatins display a unique set of 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.
  • DAMPS danger-associated molecular patterns
  • ICD insulin receptor mediated cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic containing apoptosis.
  • auristatins such as auristatins
  • 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).
  • calreticulin 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.
  • 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.
  • 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.
  • auristatins e.g., MMAE and MMAF
  • 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 Fc ⁇ Rs, decreased binding to inhibitory Fc ⁇ Rs, enhanced ADCC activity, and/or enhanced ADCP activity. Certain such antibodies with the desired activities were nonfucosylated.
  • 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 Fc ⁇ Rs.
  • the second antibody is nonfucosylated.
  • the second antibody has enhanced ADCC and/or ADCP activity.
  • 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.
  • the “variable region” contains the antigen-binding region of the antibody and is involved in specificity and affinity of binding. See, Fundamental Immunology 7 th Edition, Paul, ed., Wolters Kluwer Health/Lippincott Williams & Wilkins (2013). Light chains are typically classified as either kappa or lambda.
  • antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J. Immunol.148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al. (1993), PNAS. USA 90:6444, Gruber et al.
  • antibody includes an antibody by itself (naked antibody) or an antibody conjugated to a cytotoxic or cytostatic drug.
  • 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.
  • 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.
  • the antibodies described herein are monoclonal antibodies.
  • Specific binding of a monoclonal antibody to its target antigen means an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 . 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.
  • 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.
  • a light chain mature variable region means a light chain variable region without the light chain signal peptide.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • 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 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]
  • 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.
  • Binding affinity is used to describe monovalent interactions (intrinsic activity).
  • 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 (KD).
  • 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).
  • SPR surface plasmon resonance
  • BiacoreTM surface plasmon resonance
  • the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd (or koff), respectively.
  • 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).
  • 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).
  • 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 KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform).
  • SPR Surface Plasmon Resonance
  • BiacoreTM system kinetic exclusion assays
  • KinExA® KinExA®
  • BioLayer interferometry e.g., using the ForteBio® Octet platform.
  • binding affinity is determined using a BioLayer interferometry assay.
  • cross-reacts refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised.
  • 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.
  • 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).
  • 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.
  • LAE refers to the tripeptide linker leucine-alanine-glutamic acid.
  • dLAE refers to the tripeptide linker D-leucine-alanine-glutamic acid, wherein the leucine in the tripeptide linker is in the D-configuration.
  • Subject “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.
  • 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.
  • Treatment and prevention 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.
  • 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.
  • a “therapeutic amount” or “therapeutically effective amount” of an agent 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.
  • an agent e.g., an antibody as described herein
  • the terms “administer,” “administered,” or “administering” refer to methods of delivering agents, compounds, or compositions to the desired site of biological action.
  • 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.
  • 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.
  • an “immune cell engager” refers to a molecule (e.g., a transmembrane protein) that is involved in modulating an immune cell response, either positively or negatively.
  • the antibody binds to a receptor on immune cells or tumors and results in direct immune cell engagement or releases a negative inhibitory signal.
  • the immune cell engager is a molecule involved in T-cell signaling.
  • the immune cell engager modulates (e.g. activates) antigen-present cells (APCs).
  • APCs antigen-present cells
  • the immune cell engager in certain embodiments is an immune checkpoint protein. Other examples of potential immune cell engagers are listed below.
  • 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.
  • the antibody that binds the target involved in immune regulation 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 Fc ⁇ Rs, 2) reduced binding to inhibitory Fc ⁇ Rs, 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.
  • APCs antigen presenting cells
  • the antibody comprises an Fc with enhanced binding to one or more activating Fc ⁇ Rs and/or reduced binding to one or more inhibitory Fc ⁇ Rs to obtain the desired enhanced Fc ⁇ R binding profile.
  • Activating Fc ⁇ Rs include one or more of Fc ⁇ RIIIa, Fc ⁇ RIIa, and/or Fc ⁇ RI.
  • Inhibitory Fc ⁇ Rs include, for example, Fc ⁇ RIIb.
  • the antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa. In other embodiments, the antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa and Fc ⁇ RIIa.
  • the antibody comprises an Fc with enhanced binding to at least Fc ⁇ RIIIa and Fc ⁇ RI. In certain embodiments, the antibody comprises an Fc with enhanced binding to Fc ⁇ RIIIa, Fc ⁇ RIIa, and Fc ⁇ RI. [00186] In some embodiments, the antibody, in addition to or separately from enhanced binding to an activating Fc ⁇ R, has reduced binding to one or more inhibitory Fc ⁇ Rs. Thus, in some embodiments, the antibody has reduced binding to Fc ⁇ RIIa and/or Fc ⁇ RIIb. [00187] In some embodiments, the antibody is nonfucosylated. In some embodiments, the antibody further has one of the Fc ⁇ R binding profiles described above.
  • the Fc of the antibody comprises amino acid changes relative to a wild-type Fc to enhance binding to an activating Fc ⁇ R, and/or reduce binding to one or more inhibitory Fc ⁇ Rs to obtain an Fc ⁇ R binding profile such as described above.
  • the Fc of the antibody comprises the substitutions S293D, A330L, and I332E in the heavy chain constant region.
  • 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), Bst1/CD157, C5 complement, CC chemokine receptor 4 (CCR4), CD123, CD137, CD19, CD20, CD25 (IL2RA), CD276, CD278, CD3, CD32, CD33, CD37, CD38, CD4 and HIV-1 gp120-binding 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 T
  • an antibody is an agonist of an immune cell engager.
  • an antibody is an agonist of an immune cell engager selected from CD80, CD86, OX40 (CD134), GITR, CD137, CD40, VTCN1, CD276, IFNAR2, IFNAR1, CSF1R, VSIR (VISTA), and HLA.
  • an antibody is an antagonist of an immune cell engager.
  • 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 IDO1.
  • the antibody that binds an immune cell engager for the methods provided herein can be an inhibitor against a checkpoint protein.
  • the antibody that binds an immune cell engager for the methods provided herein can be a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, a B7 inhibitor, a TIM3 (HAVCR2) inhibitor, an OX40 (CD134) inhibitor, a GITR agonist, a CD137 agonist, or a CD40 agonist, a VTCN1 inhibitor, an IDO1 inhibitor, a CD276 inhibitor, a PVRIG inhibitor, a TIGIT inhibitor, a CD25 (IL2RA) inhibitor, an IFNAR2 inhibitor, an IFNAR1 inhibitor, a CSF1R inhibitor, a VSIR (VISTA) inhibitor, or a therapeutic agent targeting HLA.
  • a PD-1 inhibitor a PD-L1 inhibitor
  • a PD-L2 inhibitor
  • the antibody that binds an immune cell engager may be an antibody comprising an Fc with enhanced binding to one or more activating Fc ⁇ Rs.
  • the antibody that binds an immune cell engager is a nonfucosylated antibody.
  • the antibody that binds an immune cell engager is a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is an anti-CTLA-4 antibody.
  • 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.
  • the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206) or a nonfucosylated version thereof.
  • the anti-CTLA-4 antibody is ipilimumab (also known 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.
  • the antibody that binds an immune cell engager is a PD- 1/PD-L1 inhibitor.
  • PD-l/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.
  • the antibody that binds an immune cell engager is a PD-1 inhibitor.
  • the PD-1 inhibitor is an anti-PD-1 antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-PD-1 antibody is AMP-224, a fusion protein, or a nonfucosylated version thereof.
  • 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.
  • the PD-1 antibody is cemiplimab or a nonfucosylated version thereof.
  • the PD-1 antibody is camrelizumab or a nonfucosylated version thereof.
  • the PD-1 antibody is sintilimab or a nonfucosylated version thereof.
  • the PD-1 antibody is tislelizumab or a nonfucosylated version thereof.
  • 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-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab) or a nonfucosylated version thereof.
  • the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01) or a nonfucosylated version thereof.
  • the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriq®) or a nonfucosylated version thereof.
  • the PD-L1 inhibitor is avelumab or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a PD-L2 inhibitor.
  • the PD-L2 inhibitor is an anti-PD-L2 antibody.
  • the anti-PD-L2 antibody is rHIgM12B7A or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a lymphocyte activation gene-3 (LAG-3) inhibitor.
  • the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211), or a nonfucosylated version thereof.
  • the LAG-3 inhibitor is BMS-986016 or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a B7 inhibitor.
  • the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor.
  • the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834), or a nonfucosylated version thereof.
  • 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.
  • the antibody that binds an immune cell engager is a TIM3 (T- cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).
  • the antibody that binds an immune cell engager is an OX40 (CD134) agonist antibody.
  • the anti-OX40 antibody is MEDI6469 or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a GITR agonist.
  • 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-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab or a nonfucosylated version thereof. In another embodiment, the anti-CD137 antibody is PF-05082566 or a nonfucosylated version thereof. [00205] In one embodiment, the antibody that binds an immune cell engager is a CD40 agonist.
  • the antibody that binds an immune cell engager is an anti-CD40 antibody.
  • the anti-CD40 antibody is CF-870,893 or a nonfucosylated version thereof.
  • the anti-CD40 antibody is MP0317 (Molecular Partners) or a nonfucosylated version thereof.
  • the anti-CD40 antibody is YH003 (Eucure Biopharma) or a nonfucosylated version thereof.
  • the anti-CD40 antibody is CDX-1140 (Celldex Therapeutics) or a nonfucosylated version thereof.
  • the anti-CD40 antibody is YH003 (Eucure Biopharma) or a nonfucosylated version thereof.
  • 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.
  • the anti-CD40 antibody is selicrelumab (Roche) or a nonfucosylated version thereof.
  • 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.
  • the antibody that binds an immune cell engager is an antibody that binds CD70.
  • the antibody is SEA-CD70. See, e.g., US Patent No. 8,067,546; Table of Sequences herein.
  • the antibody that binds an immune cell engager is an antibody that binds BCMA.
  • 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.
  • the antibody that binds an immune cell engager is an anti- interleukin-15 antibody.
  • the antibody that binds an immune cell engager is a VTCN inhibitor.
  • the VTCN inhibitor is FPA150 or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is an anti-IDO antagonist antibody.
  • the antibody that binds an immune cell engager is a TIGIT inhibitor.
  • the TIGIT inhibitor is an anti-TIGIT antibody.
  • the TIGIT inhibitor is MTIG7192A or a nonfucosylated version thereof.
  • the TIGIT inhibitor is BMS-986207 (BMS) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is OMP-313M32 or a nonfucosylated version thereof.
  • the TIGIT inhibitor is MK-7684.
  • the TIGIT inhibitor is AB154 or a nonfucosylated version thereof.
  • the TIGIT inhibitor is CGEN-15137 or a nonfucosylated version thereof.
  • the TIGIT inhibitor is SEA-TGT.
  • 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.
  • 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 COM902 (Compugen) or a nonfucosylated version thereof. In yet another embodiment, the TIGIT inhibitor is domvanalimab (Arcus Biosciences) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is etigilimab (Mereo BioPharma) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is GSK4428859 (GSK) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is HL186 (HanAll Biopharma) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is MIL-100 (Beijing Mabworks Biotech) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is YH-29143 (Yu Han) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is HLX53 (Shanghai Henlius Biotech) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is IBI939 (Innovent Biologics) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is JS006 (Junshi Biosciences) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is M6223 (Merck KGaA) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is MG1131 (Mogam Institute) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is ociperlimab (BeiGene) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is tiragolumab (Roche; described in US Patent No.10,047,158) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is TJT6 (I-Mab Biopharma) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is vibostolimab (MSD; described in US Patent No.10,618,958) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is YBL-012 (Y Biologics) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is IBI-939 (Innovent) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is AZD2936 (AstraZeneca) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is EOS-448 (iTeos/GSK) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is BAT6005 (Bio Thera) or a nonfucosylated version thereof.
  • the TIGIT inhibitor is AGEN1777 (BMS/Agenus) or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a VSIR inhibitor.
  • the VSIR inhibitor is an anti-VSIR antibody.
  • the VSIR inhibitor is MTIG7192A or a nonfucosylated version thereof.
  • the VSIR inhibitor is CA-170 or a nonfucosylated version thereof.
  • the VSIR inhibitor is JNJ 61610588 or a nonfucosylated version thereof.
  • the VSIR inhibitor is HMBD-002 or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a TIM3 inhibitor.
  • the TIM3 inhibitor is an anti-TIM3 antibody.
  • the TIM3 inhibitor is AJUD009 or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a CD25 (IL2RA) inhibitor.
  • the CD25 (IL2RA) inhibitor is an anti-CD25 (IL2RA) antibody.
  • the CD25 (IL2RA) inhibitor is daclizumab or a nonfucosylated version thereof.
  • the CD25 (IL2RA) inhibitor is basiliximab or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is an IFNAR1 inhibitor.
  • the IFNAR1 inhibitor is an anti-IFNAR1 antibody.
  • the IFNAR1 inhibitor is anifrolumab or a nonfucosylated version thereof.
  • the IFNAR1 inhibitor is sifalimumab or a nonfucosylated version thereof.
  • the antibody that binds an immune cell engager is a CSF1R inhibitor.
  • the CSF1R inhibitor is an anti-CSF1R antibody.
  • the CSF1R inhibitor is pexidartinib or a nonfucosylated version thereof. In 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.
  • 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.
  • 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 IFN ⁇ ).
  • the antibody that binds an immune cell engager promotes release of immune-activating cytokines (e.g., CXCL10 and IFN ⁇ ) to a greater extent than immune suppressive cytokines (such as IL10 and/or MDC).
  • antibodies that bind to human TIGIT are provided as the antibodies against the immune cell engager.
  • the anti-TIGIT antibody inhibits interaction between TIGIT and one or both of the ligands CD155 and CD112.
  • the anti-TIGIT antibody inhibits the interaction between TIGIT and CD155 in a functional bioassay, allowing CD155-CD226 signaling to occur.
  • the anti-TIGIT antibody exhibits synergy with an anti-PD-1 agent (e.g., an anti-PD-1 antibody) or an anti-PD-L1 agent (e.g., an anti-PD-L1 antibody).
  • 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.
  • 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.
  • the antibody has a binding affinity (K D ) 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 (K D ) for human TIGIT of less than 50 pM.
  • the antibody has a K D 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 5 pM to about 250 pM, or about 10 pM to about 100 pM.
  • a nonfucosylated anti-TIGIT antibody 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 .
  • the anti-TIGIT antibody binds to mouse TIGIT with a binding affinity (K D ) 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.
  • 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).
  • a cell e.g., a cell line that expresses human TIGIT, cynomolgus monkey TIGIT, or mouse TIGIT
  • a primary cell that endogenously expresses TIGIT e.g., primary T cells that endogenously express human TIGIT, cyno TIGIT, or mouse TIGIT.
  • 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).
  • TIGIT e.g., purified or recombinant human TIGIT, purified or recombinant cyno TIGIT, or purified or recombinant mouse TIGIT
  • a chimeric protein comprising TIGIT e.g., an Fc-fusion protein comprising human TIGIT, cy
  • 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. [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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 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.
  • 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: [00229]
  • 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.
  • VH heavy chain variable region
  • 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.
  • a VH sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • VL light chain variable region
  • an anti- TIGIT antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:6.
  • a VL sequence having at least 90% sequence identity to a reference sequence 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.
  • 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 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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., J. Clin. Oncol.27:4371-4377 (2009) and De Vos et al., J. Hematol. Oncol.7:1-9 (2014). 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.
  • a nonfucosylated anti-CD40 antibody is provided for use in the present methods.
  • 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.
  • S2C6 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 S2C6 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, CD40L. See, e.g., US Patent No.6,946,129.
  • S2C6 was humanized and this humanized antibody is referred to as humanized S2C6, herein, and alternatively as dacetuzumab, which is fucosylated humanized S2C6 (fhS2C6, or SGN-40). See, e.g., WO 2006/128103, which is incorporated herein by reference for any purpose.
  • SEA-CD40 is a nonfucosylated humanized S2C6 antibody.
  • Other versions of humanized S2C6 are disclosed at WO2008/091954; these can be nonfucosylated and used in the methods disclosed herein.
  • 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; 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VH heavy chain variable region
  • an anti- CD40 antibody comprises a VH comprising the amino acid sequence of NO:28.
  • a VH sequence having at least 90% sequence identity to a reference sequence 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.
  • 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 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.
  • VL light chain variable region
  • an anti-CD40 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:29.
  • a VL sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • 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
  • 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.
  • the anti-CD40 antibody comprises the heavy chain variable region and light chain variable region disclosed as SEQ ID NO:28 and 29, respectively.
  • 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]
  • a nonfucosylated anti-CD70 antibody is provided for use in the present methods as the antibody that binds an immune cell engager.
  • 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.
  • TNF tumor necrosis factor
  • TNFRSF7 CD27
  • CD70 expression is transient and limited to activated T and B cells, mature dendritic, and natural killer (NK) cells.
  • NK natural killer
  • CD27 is expressed on both na ⁇ ve and activated effector T cells, as well as NK and activated B cells.
  • CD70 is also aberrantly expressed in 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.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • NHL non-Hodgkin lymphoma
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VH heavy chain variable region
  • an anti- CD70 antibody comprises a VH comprising the amino acid sequence of NO:41.
  • a VH sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • VL light chain variable region
  • an anti-CD70 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:42.
  • a VL sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • 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
  • 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.
  • the anti-CD70 antibody comprises the heavy chain variable region and light chain variable region disclosed as SEQ ID NO:41 and 42, respectively.
  • a nonfucosylated anti-BCMA antibody is provided for use in the present methods as the antibody that binds an immune cell engager.
  • the nonfucosylated anti-BCMA antibody is SEA-BCMA, which is an antibody targeting B-cell 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VH heavy chain variable region
  • an anti- BCMA antibody comprises a VH comprising the amino acid sequence of NO:45.
  • a VH sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • VL light chain variable region
  • an anti- BCMA antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:46.
  • a VL sequence having at least 90% sequence identity to a reference sequence 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.
  • 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.
  • 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
  • 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.
  • 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 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 Fc ⁇ Rs; reduced binding to inhibitory Fc ⁇ Rs; enhanced ADCC activity; and/or enhanced ADCP activity.
  • Antibodies having Fc 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 Fc 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).
  • oligosaccharide side chains of the immunoglobulins affect the protein’s function (Boyd et al., (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.
  • CAMPATH-1H a recombinant humanized murine monoclonal IgG1 antibody which recognizes the CDw52 antigen of human lymphocytes
  • CHO Chinese Hamster Ovary
  • CHO cells with tetracycline-regulated expression of ⁇ (1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al. (1999) Mature Biotech.17:176-180).
  • Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • 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.
  • 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 O-linked glycosylation sites).
  • glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.
  • 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.
  • the glycosylation (including glycosylation pattern) of antibodies may also be altered without altering the amino acid sequence or the underlying nucleotide sequence.
  • 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 al., (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.
  • 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).
  • Endo H endoglycosidase H
  • the recombinant host cell can be genetically engineered, e.g., make defective in processing certain types of polysaccharides.
  • 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 digestion, and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge.
  • Methods for releasing oligosaccharides for analytical purposes include, without limitation, enzymatic treatment (commonly performed using peptide-N-glycosidase F/endo- ⁇ -galactosidase), elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-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.
  • a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the number of Kabat).
  • the complex N-glycoside-linked sugar chain has a biantennary composite sugar chain, mainly having the following structure: where + indicates the sugar molecule can be present or absent, and the numbers indicate the position of linkages between the sugar molecules.
  • 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 ⁇ 1,6 bond (the 6-position of GlcNAc is linked to the 1- position of fucose).
  • GlcNAc N-acetylglucosamine
  • Man refers to mannose.
  • 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.
  • only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s) of the antibodies.
  • less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibodies in a composition have core fucosylation by fucose.
  • about 2% of the antibodies in the composition have core fucosylation by fucose.
  • the antibodies of the composition 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).
  • 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.
  • 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 G0, G1, or G2 glycan structure.
  • a fucose residue on a G0, G1, or G2 glycan structure See, e.g., Raju et al., 2012, MAbs 4: 385-391, Figure 3.
  • about 2% of the antibodies in the composition have a fucose residue on a G0, G1, or G2 glycan structure.
  • the antibodies of the composition when less than 60% of the antibodies in a composition have a fucose residue on a G0, G1, 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 G0, G1, or G2 glycan structure. It should be noted that G0 glycans include G0-GN glycans.
  • G0-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.
  • G0-GN and G1- GN glycans can be fucosylated or nonfucosylated. [00279]
  • 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.
  • a fucose analogue can inhibit an enzyme(s) in the fucose salvage pathway.
  • a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of fucokinase, or GDP-fucose-pyrophosphorylase.
  • 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).
  • 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.
  • 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.
  • the fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit a fucose transporter (e.g., GDP-fucose transporter).
  • 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.
  • 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.
  • Other strategies that may be used include GlycoMAb ® (US Patent No.6,602,684) and Potelligent ® (BioWa).
  • GlycoMAb ® US Patent No.6,602,684
  • Potelligent ® BioWa
  • Any of these methods can be used to generate a cell line that would be able to produce a nonfucosylated antibody.
  • Various engineering approaches can also be utilized to obtain Fc regions with the desired Fc ⁇ R activity and effector function.
  • the Fc is engineered to have the following combination of mutations: S239D, A330L and I332E, which increases the affinity of the Fc domain for Fc ⁇ RIIIA and consequently increases ADCC.
  • substitutions that enhance affinity for Fc ⁇ RIIIa include, for example, T256A, K290A, S298A, E333A, and K334A.
  • substitutions that enhance binding to activating Fc ⁇ RIIIa and reduced binding to inhibitory Fc ⁇ RIIIb include, for example, F243L/R292P/Y300L/V305I/P396L and F243L/R292P/Y300L/L235V/P396L.
  • the substitutions are in an IgG1 Fc background.
  • Oligosaccharides covalently attached to the conserved Asn297 are involved in the ability of the Fc region of an IgG to bind Fc ⁇ R (Lund et al., 1996, J. Immunol.157:4963-69; Wright and Morrison, 1997, Trends Biotechnol.15:26-31). Engineering of this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of bisecting N- acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol.17:176-180; Davies et al., 2001, Biotech.
  • a systemic substitution of solvent-exposed amino acids of human IgG1 Fc region has generated IgG variants with altered Fc ⁇ R binding affinities (Shields et al., 2001, J. Biol. Chem. 276:6591-604).
  • IgG variants with altered Fc ⁇ R binding affinities
  • a subset of these variants involving substitutions at Thr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 to Ala demonstrate increased in both binding affinity toward Fc ⁇ R and ADCC activity (Shields et al., 2001, J. Biol. Chem.276:6591-604; Okazaki et al., 2004, J. Mol.
  • an antibody-drug conjugate 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.
  • the antibody-drug conjugate comprises an antibody conjugated to a cytotoxic agent.
  • the cytotoxic agent is a tubulin disrupter.
  • the antibody binds an antigen expressed on a tumor cell.
  • 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, 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,
  • 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, AMHRII, 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, SLC
  • TAG-72 Tumor-
  • 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.
  • the ADC binds an antigen selected from Her2, TROP2, BCMA, cMet, integrin alphVbeta6 (integrin ⁇ V ⁇ 6), CD22, CD79b, CD30, CD19, CD70, CD228, and CD47.
  • 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, SLC44A4, CD30, and sTn.
  • 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, SLC44A4, CD30, and sTn.
  • 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,
  • 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, 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).
  • an antigen selected from AMHRII, Axl, CA9,
  • 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.
  • the antibody of the ADC does not bind Nectin-4.
  • 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.
  • the methods provided herein comprise administering an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody conjugated to a tubulin disrupting agent.
  • tubulin disrupting agent 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.
  • 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.
  • the cytotoxic agent conjugated to the ADC is MMAF.
  • 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.
  • Colchicines include, but are not limited to, colchicine and CA-4.
  • Vinca alkaloids include, but are not limited to, Vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindfesine (VDS).
  • Taxanes include, but are not limited to, Taxol ® (paclitaxel) and Taxotere ® (docetaxel).
  • Cryptophycins include but are not limited to cryptophycin-1 and cryptophycin-52.
  • 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.
  • 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 H.sub.25 or P.sub.2S.sub.5); C-14-alkoxymethyl(demethoxy/CH.sub.20R) (U.S. Pat. No.4,331,598); C-14- hydroxymethyl or acyloxymethyl (CH.sub.20H or CH.sub.2OAc) (U.S. Pat. No.4,450,254) (prepared 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.
  • Hemiasterlins include but are not limited to, hemiasterlin and HTI-286.
  • 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, colcimid, estramustine, cemadotin, combretastatins, discodermolide, eleutherobin, eribulin, prolabolin, phomopsin, and laulimalide.
  • the ADC for use in the methods herein may, in some embodiments, comprise linker units.
  • the ADC may comprise a linker region between the cytotoxic agent and the antibody.
  • the linker is a protease cleavable linker, an acid-cleavable linker, a disulfide linker, or a self-stabilizing linker.
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment.
  • 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).
  • the therapeutic agent e.g., tubulin disrupter
  • 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.
  • 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.
  • the linker may comprise a cleavable unit.
  • 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).
  • 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.
  • 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.
  • 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 linker. Accordingly, the linker can be cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease.
  • 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).
  • the linker will comprise a cleavable unit (e.g., a peptidyl unit) and the cleavable unit will be directly conjugated to the therapeutic agent.
  • 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.
  • a cleavable unit e.g., amino acid
  • an independent hydrolysis reaction takes place within the target cell to cleave the spacer unit from the drug.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody will be linked to the drug via a disulfide, for example the disulfide linked maytansinoid conjugates SPDB-DM4 and SPP-DM1.
  • 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.
  • 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.
  • the interchain bonds of the antibody are converted to free thiol groups prior to reaction with the functional group of the linker.
  • a cysteine residue is an introduced into the heavy or light chain of an antibody and reacted with the linker.
  • 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.
  • 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.
  • the subscript p will range from 1 to 20 (i.e., both integer and non-integer values from 1 to 20).
  • the subscript p will be an integer from 1 to 20, and will represent the number of drug-linkers on a singular antibody.
  • 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.
  • p represents the average drug loading of the antibody-drug conjugates in the composition, and p ranges from 1 to 20.
  • p is from about 1 to about 8 drugs per antibody.
  • p is 1.
  • p is 2.
  • p is from about 2 to about 8 drugs per antibody.
  • p is from about 2 to about 6, 2 to about 5, or 2 to about 4 drugs per antibody.
  • p is about 2, about 4, about 6 or about 8 drugs per antibody.
  • 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 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.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • auristatins include MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E).
  • MMAF monomethyl auristatin F
  • MMAE monomethyl auristatin E
  • 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: 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.
  • the antibody is conjugated to the linker via a sulfur atom of a cysteine residue.
  • the cysteine residue is one that is engineered into the antibody.
  • the cysteine residue is an interchain disulfide cysteine residue.
  • the antibody-drug conjugates have the linker units disclosed in the application US20160310612A1 (PCT/US2014/060477) herein incorporated in its entirety by reference.
  • the antibody- drug conjugates have following formula (II): wherein D is a drug unit, PEG is the polyethylene glycol unit that masks the hydrophobicity of the drug-linker, L p 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 L p is bonded to L, which is the antibody.
  • D is a drug unit
  • PEG is the polyethylene glycol unit that masks the hydrophobicity of the drug-linker
  • L p 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
  • the antibody-drug conjugates have the following formula III: wherein AD is a drug attachment unit that allows for additional attachment of X-D moieties indicated by t in parallel orientation to the PEG Unit and L, L p , Z, A, X, D, m, p and s are as defined for Formula II [00332]
  • an LDC of the present invention is represented by the structure of Formula IV below: wherein AD, L, L p , PEG, Z, A, X, D, m, p, s and t are as defined for Formula III.
  • the antibody-drug conjugates have the following formula 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 1A: 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
  • 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: wherein L B ’ is capable of transformation to L B 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.
  • 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 A1.
  • Ab comprises an antigen-binding protein (e.g., any antibody as described herein)
  • mc represents a maleimidocaproyl group
  • mp refers to maleimidopropionyl:
  • val-cit (vc) represents a valine-citrulline dipeptide
  • PABC represents a p- aminobenzyloxycarbonyl group
  • dLAE represents a D-leucine-alanine-glutamic acid tripeptide: mp-dLAE-PABC-MMAE.
  • the drug loading is represented by p, the number of drug-linker molecules per antibody.
  • 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 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.
  • the antigen-binding protein e.g,. an antibody
  • 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 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.
  • 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.
  • 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
  • the ADC is SGN-PDL1V, comprising an anti-PD-L1 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the ADC is SGN-ALPV, comprising an anti-ALP antibody and MMAE, 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the ADC is SGN-B7H4V, comprising an anti-B7H4 antibody and MMAE, 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • 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.
  • 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.
  • 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.
  • the ADC is SGN-B6A, which comprises an antibody that binds to AVB6 and MMAE.
  • 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.
  • 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 comprising the amino acid sequences of SEQ ID NOs:113-118.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • 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; WO2020/163225A1.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • SGN-LIV1A comprises the anti-LIV-1 antibody conjugated to mc-v
  • the ADC is tisotumab vedotin (TV), which comprises an antibody that binds tissue factor (TF) and MMAE.
  • TV tisotumab vedotin
  • TF tissue factor
  • MMAE MMAE
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • an ADC comprises MMAE and binds a target 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, 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).
  • a target selected from AMHRII, Ax
  • 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; ⁇ MSLN-MMAE, targeting Mesothelin (MSLN) (Roche-Genentech), RC68, also known as Remegen EGFR ADC, targeting EGFR (RemeGen (Rongchang Biopharmaceutical (Yantai) Co.
  • 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.
  • 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
  • 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.
  • 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
  • an ADC is selected from the ADCs in Table A, Table B, or Table C.
  • Table A, Table B, and Table C the ADCs with sequences provided in the Table of Sequences are marked with an asterisk (*).
  • the ADC is not enfortumab vedotin.
  • the ADC is not brentuximab vedotin.
  • the ADC is not tisotumab vedotin.
  • the ADC is not ladiratuzumab vedotin.
  • the ADC is not SGN-CD228A.
  • 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.
  • 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, 1:755-768 (2006)).
  • 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 Sel, 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 (3 rd 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 al., EMBO J.10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)).
  • Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or antibodies covalently bound to immunotoxins (see, e.g., U.S. Patent No. 4,676,980, WO 91/00360; and WO 92/200373).
  • Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems.
  • the expression system is a mammalian cell, such as a hybridoma, or a CHO cell. Many such systems are widely available from commercial suppliers.
  • 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.
  • 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.
  • 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., J. Biochem. Biophys. Meth., 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries.
  • Fab’-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab’) 2 fragments (see, e.g., Carter et al., BioTechnology, 10:163-167 (1992)).
  • 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.
  • 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.
  • 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.
  • PEGylation polyethylene glycol
  • serum albumin serum albumin
  • 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 Sel, 2000, 13:361-367); “knobs-into-holes” engineering (see, e.g., Ridgway et al., Protein Eng Des Sel, 1996, 9:617-721); “diabody” technology (see, e.g., Hollinger et al., PNAS (USA), 1993, 90:6444-6448); and intramolecular trimerization (see, e.g., Alvarez-Cienfuegos et al., Scientific Reports, 2016, doi:/10.1038/srep28643); See also, Spiess et al., Molecular Immunology, 2015, 67(2), Part A:95-106.
  • Constant Region 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.
  • human isotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity
  • human isotype IgG2 weak complement-dependent cytotoxicity
  • 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.
  • 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.
  • One or several amino acids at the amino or carboxy terminus of the light and/or 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.
  • 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 S239C 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-Fc ⁇ R 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.
  • 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-Fc ⁇ R binding interaction.
  • FcRn is a receptor that is structurally similar to MHC Class I antigen that non-covalently associates with ⁇ 2-microglobulin. FcRn regulates the catabolism of IgGs and their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev.
  • IgG1 Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem.276:6591-604). IgG1 molecules 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 IgG1.
  • Other exemplary substitutions for increasing binding to FcRn include a Gln at position 250 and/or a Leu at position 428.
  • Complement fixation activity of antibodies can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575).
  • the same substitutions on a human IgG2 backbone can convert an antibody isotype that binds poorly to C1q and is severely deficient in complement activation activity to one that can both bind C1q and mediate CDC (Idusogie et al., 2001, J. Immunol.166:2571-75).
  • Several other methods have also been applied to improve complement fixation activity of antibodies.
  • 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 al., 1995, J. Immunol. 154:2226-36). Also, substituting Ser444 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 al., 1992, J. Immunol.148:2918-22).
  • a bispecific diabody construct with specificity for C1q also confers CDC activity (Kontermann et al., 1997, Nat. Biotech.15:629-31).
  • Complement activity can be reduced by mutating at least one of the amino acid residues 318, 320, and 322 of the heavy chain to a residue 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 C1q binding.
  • Ser, Thr, Cys, and Met can be used at residues 320 and 322, but not 318, to reduce or abolish C1q binding activity.
  • Replacement of the 318 (Glu) residue by a polar residue may modify but not abolish C1q binding activity.
  • Replacing residue 297 (Asn) with Ala results in removal of lytic activity but only slightly reduces (about three fold weaker) affinity for C1q. 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 C1q binding: D270A, K322A, P329A, and P311S (see WO 06/036291).
  • 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 Fc ⁇ 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.
  • 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.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell; or a human cell.
  • a polynucleotide e.g., an isolated polynucleotide
  • 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.
  • 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.
  • a host cell as described herein e.g., a host cell expressing a polynucleotide or vector as described herein
  • 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.
  • the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof include cloning vectors and expression vectors.
  • useful cloning 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, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • 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.
  • 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.
  • 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).
  • suitable host cell lines capable of secreting intact heterologous proteins include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0.
  • 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, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol.148:1149 (1992).
  • 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)).
  • Antibody Characterization [00377] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are known in the art.
  • 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 Fc ⁇ Rs.
  • ADC antibody-drug conjugate
  • the Fc of the second antibody has enhanced binding to one or more of Fc ⁇ RIIIa, Fc ⁇ RIIa, and/or Fc ⁇ RI.
  • the Fc of the second antibody has reduced binding to one or more inhibitory Fc ⁇ Rs.
  • 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 corresponding wild-type Fc of the same isotype.
  • the second antibody comprises an Fc with enhanced ADCC and ADCP activity relative to a corresponding wild-type Fc of the same isotype.
  • the Fc of the second antibody has enhanced binding to one or more of Fc ⁇ RIIIa, Fc ⁇ RIIa, and/or Fc ⁇ RI. In some embodiments, the Fc of the second antibody has reduced binding to one or more inhibitory Fc ⁇ Rs. In some embodiments, the Fc of the second antibody has reduced binding to Fc ⁇ RIIb. [00380] In various embodiments, the second antibody is a nonfucosylated antibody.
  • 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.
  • the second antibody binds TIGIT.
  • the second antibody binds CD40.
  • the second antibody binds an immune cell engager provided herein.
  • 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.
  • the ADC comprises MMAE or MMAF.
  • the first antibody binds a tumor-associated antigen, such as a tumor- associated antigen provided herein.
  • Any of the ADCs described herein may be combined with any of the antibodies that bind an immune cell engager described herein.
  • the ADC is SGN-PDL1V, and the second antibody is SEA-BCMA.
  • the ADC is SGN-ALPV, and the second antibody is SEA-BCMA.
  • the ADC is SGN- B7H4V, and the second antibody is SEA-BCMA.
  • the ADC is lifastuzumab vedotin, and the second antibody is SEA-BCMA.
  • the ADC is SEA-CD40, and the second antibody is SEA-BCMA.
  • the ADC is SEA-CD70, and the second antibody is SEA-BCMA.
  • the ADC is SGN- B6A, and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN- CD228A, and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN- LIV1A, and the second antibody is SEA-BCMA. In some embodiments, the ADC is SGN- STNV, and the second antibody is SEA-BCMA. In some embodiments, the ADC is 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.
  • 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. [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.
  • 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.
  • the ADC is brentuximab vedotin (SGN-35), and the second antibody is SEA- CD40.
  • the ADC is enfortumab vedotin, and the second antibody is SEA- CD40.
  • the ADC is disitamab vedotin, and the second antibody is SEA- CD40.
  • the ADC is tisotumab vedotin, and the second antibody is SEA- CD40.
  • the ADC is SGN-PDL1V, and the second antibody is SEA- CD70.
  • 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.
  • 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.
  • the ADC is tisotumab vedotin, and the second antibody is SEA- CD70.
  • the ADC is SGN-PDL1V, and the second antibody is SEA- TGT.
  • the ADC is SGN-ALPV, and the second antibody is SEA-TGT.
  • the ADC is SGN-B7H4V, and the second antibody is SEA-TGT.
  • the ADC is lifastuzumab vedotin, and the second antibody is SEA-TGT.
  • the ADC is SEA-CD40, and the second antibody is SEA-TGT.
  • the ADC is SEA-CD70, and the second antibody is SEA-TGT.
  • the ADC is SGN-B6A, and the second antibody is SEA-TGT.
  • the ADC is SGN-CD228A, and the second antibody is SEA-TGT.
  • the ADC is SGN-LIV1A, and the second antibody is SEA-TGT.
  • the ADC is SGN-STNV, and the second antibody is SEA-TGT.
  • the ADC is brentuximab vedotin (SGN-35), and the second antibody is SEA- TGT.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 CXCL10 and/or IFN ⁇ ).
  • the second antibody promotes release of immune activating cytokines to a greater extent than immune suppressive cytokines (such as IL10 and/or MDC).
  • 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.
  • the ADC and second antibody may be administered as separate pharmaceutical composition or in the same pharmaceutical composition.
  • a therapeutic agent is administered at a therapeutically effective amount or dose.
  • the dosages 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.
  • 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.
  • the enhanced activity observed with the particular combination therapies described herein have certain benefits as compared to corresponding monotherapy treatment.
  • 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.
  • the effective dose of the ADC and/or the second antibody when dosed in combination is less than when administered as monotherapy.
  • 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.
  • 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 other methods known in the art.
  • one or more therapeutic agents are administered orally, intravenously, or intraperitoneally.
  • 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.
  • 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.
  • 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.
  • compositions and kits for use in treating or preventing a cancer in a subject are provided.
  • Pharmaceutical Compositions [00399]
  • pharmaceutical compositions for use in the present methods are provided.
  • 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.
  • Guidance for preparing formulations for use in the present invention is found in, for example, Remington: The Science and Practice of Pharmacy, 21 st 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.
  • compositions described herein can be manufactured in 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.
  • 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.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • 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.
  • 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.
  • PVP polyvinyl pyrrolidone
  • carboxyvinyl hydrophilic polymers such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose
  • carboxypolymethylene 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).
  • disintegrating agents can be added, such as a cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • a therapeutic agent can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • 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.
  • 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.
  • 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.
  • the agents are formulated into ointments, creams, salves, powders and gels.
  • the transdermal delivery agent can be DMSO.
  • Transdermal delivery systems can include, e.g., patches.
  • 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.
  • 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.
  • 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.
  • carbohydrates such as glucose, sucrose, or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • 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 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
  • kits for use in treating a subject having a cancer comprise: 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.
  • 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).
  • 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.
  • Example 1 Non-directed Chemotherapeutic Agents Impair T Cell Responses
  • 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.
  • CSFE carboxyfluorescein diacetate succinimidyl ester
  • 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.
  • CSFE carb
  • 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 BV. 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.
  • MMAE vedotin
  • DM1 emtansine
  • Ex Exatecan DS-8201
  • FIG.3A 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-GloTM Luciferase Assay System, Promega). In brief, 100,000 cells/well were plated in a 96-well, flat-bottom clear plate (aliquot 150 ⁇ L per well). 200 ⁇ L 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 ⁇ L of media was removed from the wells.100 ⁇ L of BrightGlo Reagent was added to each well.
  • auristatin-based ADCs (MMAE-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 MMAE as a microtubule disrupting agent to induce this ER stress is exemplified in the data shown.
  • FIG.3D-E demonstrate that CHOP, a downstream signal in the ER stress pathway, is significantly induced by MMAE 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 [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.
  • 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.
  • 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 immune system.
  • DAMPs damage-associated molecular patterns
  • 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.
  • T cell responses can be further augmented by PD-1/L1 inhibitors.
  • 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% CO 2.
  • 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.10 ⁇ l of the master mix was added to each well containing 90 ⁇ l 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.
  • assay plates were washed with 200 ⁇ L of wash buffer per well, followed by addition of 25 ⁇ L standard or buffer, 25 ⁇ L matrix or sample, and 25 ⁇ L 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. [00429] Detection antibodies (25 ⁇ L) were added to each well and incubated at room temperature for 1 hour. 25 ⁇ L 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 ⁇ L of sheath fluid.
  • SA-PE streptavidin-phycoerythrin
  • 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.
  • 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 vcMMAE 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).
  • 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, 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.
  • T cell responses can be further augmented by combination with other immune modulators, such as the immune cell engagers described herein.
  • immune modulators such as the immune cell engagers described herein.
  • 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).
  • 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).
  • MMAE-ADCs SGD- 1006 treatment was one of the strongest inducers of JNK phosphorylation, an early ER stress response.
  • 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).
  • 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.
  • 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.
  • Induction of CHOP in the MIA-PaCa-2 cells was measured by detection of luciferase signal (Bright-GloTM Luciferase Assay System, Promega).
  • Bright-GloTM Luciferase Assay System Promega.
  • 10,000 cells/well were plated in 96-well, black-walled, flat-bottom clear plates in 75 ⁇ L per well.
  • ADCs were dosed in 25 ⁇ L per well to achieve a final IC 50 concentration.
  • plates were removed from the incubator and allowed to come to room temperature.100 ⁇ L 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.
  • ICD 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.
  • DAMPs damage-associated molecular patterns
  • MIA-PaCa-2 cancer cells were treated with IC 50 concentrations of ADCs with various payloads to assess in vitro ICD marker induction. See FIG.7A.
  • 200,000 cells were plated per well in 6-well TC plates and allowed to attach to plate ON. Cells reached 50-60% confluence.
  • IC 50 concentrations of ADCs were added to each treatment well. After 72 hours, 500 ⁇ L (for the ATP release assay) or 750 ⁇ L (for the HMGB1 assay) of culture supernatant was collected and transferred into a labeled 1.5 mL Eppendorf tube.
  • HMGB1 release levels 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.
  • MHCII expression and CXCL-10/IP10 was assessed as follows. [00446] L540cy cancer cells exposed to IC 50 concentrations of ADCs or paclitaxel for 24 hours (at 37 °C in 5% CO 2 ) were washed, and 0.2 x 10 6 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 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 ⁇ L of BD FACs buffer and transferred to 96-well round-bottom microtiter plates. Fc receptors were blocked with human Fc-fragments at 100 ⁇ g/mL for 30 minutes on ice.
  • 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).
  • JNK activation e.g., JNK activation
  • late ER stress e.g., CHOP induction
  • immune-activating molecules e.g., ATP and HMGB1 release
  • innate immune cells e.g., macrophage activation
  • 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 Fc ⁇ R Binding to Fc ⁇ RIIa, Fc ⁇ RIIb or Fc ⁇ RIIIa based upon Fc backbone.
  • Antibodies SEA-CD40, APX005M, ADC-1013, and Selicrelumab were assessed for Fc ⁇ R binding using flow cytometry to CHO cells that were transfected with human Fc ⁇ RIIa, Fc ⁇ RIIb or Fc ⁇ RIIIa. 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.
  • 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 ⁇ L each antibody solution was added to 89 ⁇ L of media) to produce the following concentrations: 300, 100, 30, 10, 3, 1, 0.03.0.01, 0.003, 0.001, and 0.0003 ⁇ g/ml.
  • SEA-CD40 had the highest affinity for Fc ⁇ RIIIa with lowest affinity for Fc ⁇ RIIb (FIG.11B-D).
  • the data demonstrate the potential of different Fc backbones to impact binding to different Fc ⁇ Rs.
  • 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 Fc ⁇ Rs (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 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 ⁇ g/ml) with differing Fc backbones (FIG.11A) and immune activation assessed 48 hrs later.
  • Abraxane which acts similarly to paclitaxel in Example 3 above
  • 2 ICD inducing agents oxaliplatin or vc-MMAE oxaliplatin
  • vc-MMAE 2 ICD inducing agents
  • SEA-CD40 combined with vcMMAE ADC killed tumor cells, at least in part, by inducing superior release of immune activating cytokines (CXCL10 and IFN ⁇ ; 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 Fc ⁇ RIIIa.
  • 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 ⁇ g/ml 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.
  • 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.
  • 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 [00460] Cytokine production was assessed as described above in Example 5.
  • Example 15 Synergistic Effect with Combination of a Nonfucosylated SEA-CD40 Antibody and a Auristatin Based ADC 15.1
  • 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 [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 1 ⁇ g/ml 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 ⁇ g/ml 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 Fc ⁇ R binding, and the SEA-TIGIT antibody having enhanced Fc ⁇ R binding (increased binding to the activating Fc ⁇ IIIaR, and decreased binding to the inhibitory Fc ⁇ RIIbR. Table D below shows the relative activities of the different TGT antibodies.
  • 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 ⁇ g/ml of a tumor-targeting antibody- vcMMAE ADC with a drug-to-antibody ratio (DAR) of 4.
  • DAR drug-to-antibody ratio
  • the level of IFN ⁇ 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.
  • Example 18 Synergistic Effect with Combination of a Nonfucosylated TIGIT Antibody and an Auristatin Based ADC 18.1 Materials and Methods 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 EC50 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 ⁇ g/ml) of anti-TIGIT antibodies with different Fc backbones, including anti-TIGIT LALA, SEA-TGT, and antibody 31C6 H4/L1, which is an IgG1 antibody (US 2018/0066055 A1). Then, immune activation was assessed by measuring cytokine (IP10) levels 48 hours after co-culture.
  • IP10 cytokine
  • mice were implanted with the CT26 syngeneic tumor cell line that expresses the tumor antigen Thy1.1 subcutaneously in the flank on day 0.
  • 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 mm 3 , 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.
  • 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 is 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.
  • 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 (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.
  • TNF tumor necrosis factor
  • TNFRSF7 CD27
  • CD27 is expressed on both na ⁇ ve and activated effector T cells, as well as NK and activated B cells.
  • 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.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • NHL non- Hodgkin’s lymphoma
  • 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).
  • ADCP antibody dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • 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.
  • 20.1 Materials and Methods In vivo evaluation of subcutaneous tumor growth in an NHL xenograft model [00479] Farage cells (2.5x10 6 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.
  • IP intraperitoneally
  • SEA-BCMA is a nonfucosylated antibody targeting B-cell maturation antigen (BCMA), which is expressed on multiple myeloma (MM).
  • BCMA B-cell maturation antigen
  • SEA-BCMA acts through blocking ligand mediated BCMA cell signaling, antibody dependent cellular phagocytosis (ADCP), and enhanced antibody dependent cellular cytotoxicity (ADCC).
  • SGN-CD48A is a CD48-targeting ADC containing a glucuronide linked MMAE.
  • CD48 is broadly expressed in MM. 21.1 Materials and Methods 21.1.1 In vivo survival evaluation of xenograft model [00482] MM1S 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.
  • CD45 positive immune cells were stained for CD11c and activation observed by staining for the expression of MHC-Class II on the cell surface. Intratumoral cytokines were measured by Luminex. 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).
  • MMAE-based ADCs 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.
  • ADC-vcMMAE tumor-targeting ADCs
  • isotype-vcMMAE also known as h00-vcMMAE
  • 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.
  • 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.
  • 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.
  • 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.
  • auristatin based ADCs e.g., MMAE and MMAF
  • DAMPs danger signals
  • 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.
  • This population of memory T cells which are induced by MMAE ADCs, may be further increased and/or enhanced by nonfucosylated antibodies.
  • 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-L1.
  • auristatin based ADCs e.g., MMAE and MMAF
  • 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 or antibodies that have been engineered to have enhanced binding to activating Fc ⁇ Rs and/or decreased binding to inhibitory Fc ⁇ Rs (e.g., as shown in the examples above with nonfucosylated CD40 and BCMA antibodies).
  • Nonfucosylated antibodies can have increased binding to the activating Fc ⁇ RIIIa receptor with and decreased or minimal binding to the inhibitory Fc ⁇ RIIb 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 Fc ⁇ RIIA+ cells increases receptor clustering and immune agonism and activation. See FIG.25.
  • nonfucosylated antibodies increase the strength of an immune synapse between an antigen (+) T cells and an antigen presenting cells (FIG.25). Engagement of the Fc ⁇ RIIIa on the innate cell increases their activation and production of factors that can enhance an antigen specific T cell response.
  • the nonfucosylated backbone can, independently of the target antigen, bind to innate immune cells or other Fc ⁇ RIIIa 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.

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  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne des méthodes de traitement du cancer avec un anticorps qui se lie à un dispositif d'activation de cellules immunitaires en combinaison avec un conjugué anticorps-médicament.
EP21816258.4A 2020-11-08 2021-11-05 Polythérapie à base d'un conjugué anticorps-médicament avec un nhibiteur de cellules immunitaires Pending EP4240415A1 (fr)

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US202063111045P 2020-11-08 2020-11-08
US202163172411P 2021-04-08 2021-04-08
US202163208179P 2021-06-08 2021-06-08
PCT/US2021/058208 WO2022098972A1 (fr) 2020-11-08 2021-11-05 Polythérapie à base d'un conjugué anticorps-médicament avec un nhibiteur de cellules immunitaires

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AU (1) AU2021376218A1 (fr)
CA (1) CA3200974A1 (fr)
IL (1) IL302402A (fr)
MX (1) MX2023004941A (fr)
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JP2023548538A (ja) 2023-11-17
AU2021376218A1 (en) 2023-06-15
KR20230104659A (ko) 2023-07-10
CA3200974A1 (fr) 2022-05-12
WO2022098972A1 (fr) 2022-05-12
TW202233248A (zh) 2022-09-01
IL302402A (en) 2023-06-01
MX2023004941A (es) 2023-07-12

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