WO2024258743A1 - Méthodes et compositions associées à des anticorps et conjugués anticorps-médicament (adcs) qui se lient à des protéines de nectine-4 - Google Patents

Méthodes et compositions associées à des anticorps et conjugués anticorps-médicament (adcs) qui se lient à des protéines de nectine-4 Download PDF

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Publication number
WO2024258743A1
WO2024258743A1 PCT/US2024/033036 US2024033036W WO2024258743A1 WO 2024258743 A1 WO2024258743 A1 WO 2024258743A1 US 2024033036 W US2024033036 W US 2024033036W WO 2024258743 A1 WO2024258743 A1 WO 2024258743A1
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antibody
nectin
seq
binding fragment
antigen binding
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Inventor
Pia Challita-Eid
Sabine G. ROTTMANN
Dong Jun Lee
Andrew M. HAU
Maria SHAHMORADGOLI
Richard H. LI
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Adcentrx Therapeutics Inc
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Adcentrx Therapeutics Inc
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Priority to KR1020267000884A priority Critical patent/KR20260026054A/ko
Priority to AU2024304180A priority patent/AU2024304180A1/en
Priority to CN202480040082.4A priority patent/CN121620528A/zh
Publication of WO2024258743A1 publication Critical patent/WO2024258743A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61K47/6849—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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • SEQUENCE LISTING XML FILE The content(s) of the following submissions are fully incorporated by reference herein in their entirety: the content of a computer readable form (CRF) of the Sequence Listing in XML file (file name: 9300-20000.40 – SEQ LIST - XML – 07-June-2024, date recorded June 06, 2024, size: 136 KB) . STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.
  • the invention described herein relates to antibodies, antigen-binding fragments, and antibody drug conjugates (ADCs) thereof that bind NECTIN-4 proteins.
  • the invention further relates to the prognostic, prophylactic, and therapeutic methods, and compositions useful in the treatment of cancers and other immunological and neurological disorders.
  • BACKGROUND OF THE INVENTION Cancer is the second leading cause of death next to coronary disease worldwide. Although cancer therapy has improved over the past decades and survival rates have increased, the heterogeneity of cancer still demands new therapeutic strategies utilizing a plurality of treatment modalities.
  • ADCs Antibody-drug conjugates
  • Drugs and linkers have been the focus of ADC development, in addition to (monoclonal) antibody (mAb) and target selection. Recently, however, the importance of conjugate homogeneity has been explored. It has been reported that the pharmacological profile of ADCs may be improved by applying site-specific conjugation technologies that make use of surface-exposed cysteine residues engineered into antibodies that are then conjugated to a linker drug, resulting in site-specifically conjugated ADCs with defined drug-to-antibody ratios (DARs).
  • DARs drug-to-antibody ratios
  • the invention provides antibodies, antigen-binding fragments, antibody drug conjugates (ADCs), antibody immune modifying conjugates, antibody fusion proteins, and antibody fragment fusion proteins that bind to NECTIN-4 proteins and polypeptide fragments of NECTIN-4 proteins.
  • ADCs antibody drug conjugates
  • the invention comprises fully human antibodies conjugated with a therapeutic agent.
  • the entire nucleic acid sequence of Table IV is not encoded and/or the entire amino acid sequence of Table V is not prepared.
  • the entire nucleic acid sequence of Table IV is encoded and/or the entire amino acid sequence of Table V is prepared, either of which are in respective human unit dose forms.
  • the invention further provides various immunogenic or therapeutic compositions, such as antibodies, antibody drug conjugates, and strategies for treating cancers that express NECTIN-4 such as those cancers listed in Table I.
  • the present disclosure teaches an antibody composition denoted CL.Z.
  • the present disclosure teaches an antibody composition denoted CL.X.
  • the present disclosure teaches an antibody composition denoted CL.N.
  • the present disclosure teaches an antibody composition denoted CL.I. In another embodiment, the present disclosure teaches an antibody composition denoted CL.B. In another embodiment, the present disclosure teaches an antibody composition denoted CL.D. In another embodiment, the present disclosure teaches methods of synthesizing antibodies. In another embodiment, the present disclosure teaches methods of synthesizing antibodies and conjugating a drug moiety to the antibody to form an ADC. In another embodiment, the present disclosure teaches methods of treating cancer(s) in humans. In another embodiment, the present disclosure teaches methods of treating immunological or neurological disorder(s) in humans.
  • the present disclosure teaches uses of one or more compositions herein in the manufacture of a medicament for treating cancer(s), immunological and/or neurological disorder(s) in humans.
  • BRIEF DESCRIPTION OF THE FIGURES Figure 1. Antibody Binding Specificity of Nectin-4 Abs on Multiple Cell Lines.
  • Figure 2(A). Shows Ab1.
  • Figure 2(B). Shows Ab2.
  • Figure 2(C). Shows Ab3.
  • Figure 2(D). Shows Ab4.
  • Figure 2(E). Shows Ab5.
  • Figure 4 In Vitro Cytotoxicity of Nectin4 ADCs Across Multiple Cancer Cell Lines.
  • Figure 4(A). Shows NCI-H322 Lung Adenocarcinoma cell line.
  • Figure 4(B). Shows PC3-NECTIN-4 recombinant NECTIN-4 expressing PC3 prostate cancer cell line.
  • Figure 4(C). Shows NECTIN-4 negative cell line PC3.
  • Figure 5. In Vitro Cytotoxicity of NECTIN-4 ADCs Using Multiple Payloads on Sum190PT Breast Cancer Cell Line.
  • Figure 6(A). Shows NECTIN-4 negative cell line in co-culture with NECTIN-4 expressing cell line.
  • Figure 10(P). Shows the structure denoted DL-16.
  • Figure 11. In Vitro Cytotoxicity of NECTIN-4 ADCs Across Multiple Primary Cultures of Normal Human Cells.
  • Figure 11(A). Shows HCEpC cells.
  • Figure 11(B). Shows HDFa cells.
  • Figure 11(C). Shows HEKa cells.
  • Figure 12. Flow Cytometry Histograms of NECTIN-4 with Enfortumab and Ab5 Across Multiple Primary Cultures of Normal Human Cells.
  • Figure 12(A). Shows HCEpC cells.
  • Figure 12(B). Shows HDFa cells.
  • Figure 12(C). Shows HEKa cells.
  • Figure 13(A). Shows the % combined G2 and sub-G1 phases of HT-1376 cells following 72-hour treatment with Ab5-ADC2.
  • Figure 13(B). Shows Representative flow cytometry histogram of Ab5-ADC2-treated cells compared to non- treated control cells.
  • Figure 14. Induction of Immunogenic Cell Death of Free Payload Compared to MMAE in NCI- H292 Cancer cells.
  • ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
  • ADCP Antibody-Dependent Cell-Mediated Phagocytosis
  • PK Pharmacokinetic
  • Figure 19(A). Shows tumor growth kinetics.
  • Figure 20 Shows survival probabilities in Kaplan-Meier plot.
  • Figure 20 In Vivo Efficacy of NECTIN-4 ADC Ab5-ADC2 in a Mouse Clinical Trial of Six (6) Cancer Models.
  • Figure 20(A). Shows PDX10.
  • Figure 20(B). Shows PDX12.
  • Figure 20(C). Shows PDX13.
  • Figure 20(D). Shows PDX16.
  • Figure 20(E). Shows PDX34.
  • Figure 20(F) Shows PDX36.
  • Figure 21 NECTIN-4 Expression in Patient-Derived Cervical Cancer Xenograft Models via Immunohistochemistry.
  • Figure(s) 21(A), 21(C), 21(E), 21(G), 21(I), and 21(K) show expression(s) at high resolution.
  • Figure(s) 21(B), 21(D), 21(F), 21(H), 21(J), and 21(L) show expression(s) at low resolution.
  • Figure 21(A) and 21(B). Shows PDX12.
  • Figure 21(C) and 21(D). Shows PDX10.
  • Figure 21(E) and 21(F). Shows PDX16.
  • Figure 21(G) and 21(H). Shows PDX13.
  • Figure 21(I) and 21(J). Shows PDX34.
  • Figure 21(K) and 21(L). Shows PDX36.
  • FIG 22(A). Shows Free Payload Concentration in Normal Tissues.
  • Figure 22(B). Shows Payload Concentration of Ab5-ADC2 and Enfortumab Vedotin.
  • Figure 23(A). Shows stability of the conjugation and percent of initial drug-to-antibody ratio (DAR) of Ab5-ADC2 compared to Enfortumab Vedotin.
  • Figure 23(B). Shows deconvoluted MS profiles of the heavy and light chains of affinity purified Ab5-ADC2.
  • advanced cancer “locally advanced cancer”, “advanced disease” and “locally advanced disease” mean cancers that have extended through the relevant tissue capsule and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1- C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system.
  • AUA American Urological Association
  • stage C1- C2 disease under the Whitmore-Jewett system
  • T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system.
  • surgery is not recommended for patients with locally advanced disease and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) cancer.
  • substituted means that the specified group or moiety bears one or more substituents.
  • unsubstituted means that the specified group bears no substituents.
  • optionally substituted means that the specified group is unsubstituted or substituted by one or more substituents. Where the term “substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system.
  • analog refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g., a NECTIN-4 related protein). For example, an analog of a NECTIN-4 protein can be specifically bound by an antibody or T cell that specifically binds to NECTIN-4.
  • antibody is used in the broadest sense unless clearly indicated otherwise.
  • an “antibody” can be naturally occurring or synthetic such as monoclonal antibodies produced by conventional hybridoma or transgenic mice technology.
  • NECTIN-4 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
  • the term “antibody” refers to any form of antibody or fragment thereof that specifically binds NECTIN-4 and/or exhibits the desired biological activity and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and antibody fragments so long as they specifically bind NECTIN-4 and/or exhibit the desired biological activity.
  • the term “antibody” encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen.
  • the antibody is an IgG antibody.
  • the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.
  • an antibody of the present invention is a mammalian antibody.
  • Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art.
  • the antibody is produced by recombinant means known in the art.
  • a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody.
  • One or more vectors can be used to transfect the DNA sequence expressing at least one VL and at least one VH region in the host cell.
  • Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); SHEPARD, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); GODING, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition).
  • An antibody of the present invention can be modified by recombinant means to increase efficacy of the antibody in mediating the desired function.
  • antibodies can be modified by substitutions using recombinant means.
  • the substitutions will be conservative substitutions.
  • at least one amino acid in the constant region of the antibody can be replaced with a different residue.
  • the modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity.
  • the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both scientific and patent literature.
  • These antibodies can be screened for binding to normal or defective NECTIN-4. See e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996).
  • Suitable antibodies with the desired biologic activities can be identified using the following in vitro assays including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and the following in vivo assays such as the inhibition of tumor growth.
  • the antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. They can also be used to quantify the NECTIN-4 and/or its receptor.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (WARD et al., (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarily determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and C H1 domains
  • F(ab′) 2 fragment a bivalent fragment comprising two
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., BIRD et. al. (1988) Science 242:423-426; and HUSTON et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody.
  • the term “Fc,” as used herein, refers to a region comprising a hinge region, CH 2 and/or CH 3 domains.
  • any form of the “antigen” can be used to generate an antibody that is specific for NECTIN-4 of the invention.
  • the eliciting antigen may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art.
  • the eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein).
  • the antigen may be produced in a genetically modified cell.
  • the DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain.
  • portion in the context of an antigen, refers to the minimal number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest.
  • any genetic vectors suitable for transformation of the cells of interest may be employed, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids.
  • the antibody of the methods and compositions herein specifically bind at least a portion of the extracellular domain of the target of interest.
  • the antibodies or antigen binding fragments thereof provided herein may constitute or be part of a “bioactive agent.”
  • bioactive agent refers to any synthetic or naturally occurring compound that binds the antigen and/or enhances or mediates a desired biological effect to enhance cell-killing toxins.
  • the binding fragments useful in the present invention are biologically active fragments.
  • biologically active refers to an antibody or antibody fragment that is capable of binding the desired antigenic epitope and directly or indirectly exerting a biologic effect.
  • Direct effects include, but are not limited to the modulation, stimulation, and/or inhibition of a growth signal, the modulation, stimulation, and/or inhibition of an anti-apoptotic signal, the modulation, stimulation, and/or inhibition of an apoptotic or necrotic signal, modulation, stimulation, and/or inhibition the ADCC cascade, and modulation, stimulation, and/or inhibition the CDC cascade and/or Fc silencing.
  • proteins specifically binds to the target as well as a discrete domain, or discrete amino acid sequence, within the target with no or insignificant binding to other (for example, unrelated) proteins. This term, however, does not exclude the fact that the antibodies or binding fragments thereof may also be cross-reactive with closely related molecules.
  • the antibodies and fragments thereof as well as antibody drug conjugates comprising these described herein may specifically bind to NECTIN-4 disclosed herein, with at least 2, 5, 10, 50, 100, or 1000-fold greater affinity than they bind to closely related molecules. “Bispecific” antibodies are also useful in the present methods and compositions.
  • bispecific antibody refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes.
  • the epitopes are from the same antigen.
  • the epitopes are from two different antigens.
  • Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., MILSTEIN et. al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., BRENNAN, et.
  • Bispecific antibodies include bispecific antibody fragments. See, e.g., HOLLINGER, et. al., Proc. Natl. Acad. Sci. U.S.A.90:6444-48 (1993), GRUBER, et. al., J. Immunol.152:5368 (1994).
  • the monoclonal antibodies described herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity (U.S. Pat. No.4,816,567; and MORRISON et. al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
  • cancer As used herein, the terms “cancer,” “neoplasm,” and “tumor,” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a “clinically detectable” tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • Tumors may be hematopoietic tumor, for example, tumors of blood cells or the like, meaning liquid tumors.
  • therapeutic agent refers to all agents that provide a therapeutic benefit and/or are therapeutically effective as defined herein.
  • a therapeutic agent may, for example, reverse, ameliorate, alleviate, inhibit, or limit the progress of, or lessen the severity of, a disease, disorder, or condition, affect, improve, or ameliorate one or more symptoms of disease, such as cancer.
  • Such an agent may be cytotoxic or cytostatic.
  • chemotherapeutic agents include, but is not limited to, chemotherapeutic agents, anti- neoplastic agents and “Drug Unit” agents as defined herein.
  • anti-neoplastic agent refers to all agents that provide a therapeutic benefit and/or are therapeutically effective, as defined herein, in the treatment of a neoplasm or cancer.
  • Cyhemotherapeutic Agent refers to all chemical compounds that are effective in inhibiting tumor growth.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents; for example, nitrogen mustards, ethyleneimine compounds and alkyl sulphonates; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; mitotic inhibitors, for example, anti-tubulin agents such as vinca alkaloids, auristatins and derivatives of podophyllotoxin; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, for example, DNA minor groove binders; and growth factor receptor antagonists.
  • chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.
  • CDR complementarity determining region
  • CDR-H1, CDR-H2, CDR-H3 three (3) CDRs in each heavy chain variable region
  • CDR-L1, CDR-L2, CDR-L3 three (3) CDRs in each light chain variable region
  • the precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • PLICKTHUN A “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun.8; 309(3):657-70, (AHo numbering scheme).
  • the boundaries of a given CDR may vary depending on the scheme used for identification.
  • the Kabat scheme is based structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies.
  • the two schemes place certain insertions and deletions (“indels”) at various positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., “CDR- H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above.
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method.
  • conservative substitution refers to substitutions of amino acids and/or amino acid sequences that are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., WATSON, et. al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co., p.224 (4th Ed.1987)).
  • Such exemplary substitutions are preferably made in accordance with those set forth in Table II and Table(s) III.
  • such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
  • substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V).
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g., Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); HENIKOFF et. al., PNAS 1992 Vol 8910915-10919; LEI et.
  • cytotoxic agent refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • cytotoxic agents include, but are not limited to auristatins, auristatin derivatives, auromycins, camptothecins (Topoisomerase 1 inhibitors), maytansinoids, ricin, ricin A-chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,
  • Antibodies may also be conjugated to any of the aforementioned cytotoxic agents and also to an anti-cancer prodrug activating enzyme capable of converting the prodrug to its active form.
  • the term “diabodies” refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H —V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and HOLLINGER et. al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).
  • the term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
  • identity identity indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWS gap dna CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of Meyers, et al., Comput. Appi. Biosci., 4:11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percentage identity between two amino acid sequences can be determined using the NEEDLEMAN, et. al., J. Mol.
  • a polynucleotide sequence may be identical to a reference polynucleotide sequence that is 100% identical to the reference sequence, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, such as at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical.
  • Such alterations are selected from at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleotide alterations is determined by multiplying the total number of nucleotides in the reference polynucleotide sequence as described herein by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in the reference polynucleotide sequence, or: n n ⁇ x n -(x ny ), wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in the reference polynucleotide sequence as described herein (see the nucleic acid sequences in the “Sequence Listing” for exemplary reference polynucleotides sequences), and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99% or 1.00 for 100%, is the symbol for the multiplication operator
  • a polypeptide sequence may be identical to a polypeptide reference sequence as described herein, that is 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%, such as at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by the polypeptide reference sequence by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the polypeptide reference sequence as described herein, or: n a ⁇ x a -(x ay ), wherein n a is the number of amino acid alterations, x a is the total number of amino acids in the reference polypeptide sequence, and y is, 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • the percent identity may be determined across the length of the sequence.
  • the term “over 75% identical” includes over 75%, 80%, 85%, 95% and 99% identity as well as all discrete values, and discrete subranges, within this range.
  • the antibody provided herein is a “human antibody.”
  • the term “human antibody” refers to an antibody in which essentially the entire sequences of the light chain and heavy chain sequences, including the complementary determining regions (CDRs), are from human genes.
  • human monoclonal antibodies are prepared by the trioma technique, the human B-cell technique (see, e.g., KOZBOR, et. al., Immunol.
  • the human antibody is generated in a transgenic mouse.
  • Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used.
  • fully human antibody sequences are made in a transgenic mouse engineered to express human heavy and light chain antibody genes.
  • WO 02/43478 and U.S. Pat. No.6,657,103 (Abgenix) and its progeny.
  • B cells from transgenic mice that produce the desired antibody can then be fused to make hybridoma cell lines for continuous production of the antibody.
  • humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See e.g., CABILLY, U.S. Pat.
  • the terms “inhibit” or “inhibition of” as used herein means to reduce by a measurable amount, or to prevent entirely.
  • the term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
  • metal cancer and “metastatic disease” mean cancers that have spread to regional lymph nodes or to distant sites and are meant to include stage D disease under the AUA system and stage T ⁇ N ⁇ M+ under the TNM system.
  • modified refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide.
  • Such changes, or modifications may be obtained by post synthesis modifications of natural amino acids, non-natural amino acids, natural amino acid polypeptide or a non-natural amino acid polypeptide, or by co-translation, or by post-translational modifications of a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide.
  • “Molecular recognition” means a chemical event in which a host molecule is able to form a complex with a second molecule (i.e., the guest). This process occurs through non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, ionic interaction.
  • the term “monoclonal antibody,” as used herein, 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. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a plurality of monoclonal antibodies with different epitope specificities, affinities, or avidities within a single antigen that contains multiple antigenic epitopes.
  • 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., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in CLACKSON et. al., Nature 352: 624-628 (1991) and MARKS et.
  • nnAA non-natural amino acid
  • nnAA non-natural encoded amino acid
  • unnatural amino acid amino acid
  • non-naturally occurring amino acid amino acid
  • the term nnAA includes, but is not limited to, amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of non-natural amino acids.
  • a “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
  • polypeptide means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter (See, Table II) or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein.”
  • single-chain Fv or “scFv” or “single chain” antibody refers to antibody fragments comprising the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • the terms “specific,” “specifically binds” and “binds specifically” refer to the selective binding of the antibody to the target antigen epitope.
  • Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific.
  • a specific antibody is one that only binds the NECTIN-4 antigen but does not bind to any other irrelevant antigen.
  • a specific antibody is one that binds human NECTIN-4 antigen but does not bind a non-human NECTIN-4 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology with the NECTIN-4 antigen.
  • a specific antibody is one that binds human NECTIN-4 antigen but does not bind a non-human NECTIN-4 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater percent identity with the amino acid sequence of the NECTIN-4 antigen.
  • a specific antibody is one that binds human NECTIN-4 antigen and binds murine NECTIN-4 antigen, but with a higher degree of binding the human antigen.
  • a specific antibody is one that binds human NECTIN-4 antigen and binds primate NECTIN-4 antigen, but with a higher degree of binding the human antigen.
  • the specific antibody binds to human NECTIN-4 antigen and any non-human NECTIN-4 antigen, but with a higher degree of binding the human antigen or any combination thereof.
  • to treat or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; as is readily appreciated in the art, full eradication of disease is a preferred but albeit not a requirement for a treatment act.
  • variant refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g., NECTIN-4 protein as shown in Table IV).
  • An analog is an example of a variant protein.
  • Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
  • isolated or biologicalcally pure refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in-situ environment.
  • a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the NECTIN-4 genes or that encode polypeptides other than NECTIN-4 gene product or fragments thereof.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated NECTIN-4 polynucleotide.
  • a protein is said to be “isolated,” for example, when physical, mechanical, or chemical methods are employed to remove the NECTIN-4 proteins from cellular constituents that are normally associated with the protein.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated NECTIN-4 protein.
  • an isolated protein can be prepared by chemical means.
  • Suitable “labels” include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • the antibodies provided herein can be useful as the antigen-binding component of fluorobodies. (See, e.g., Zeytun et al., Nat. Biotechnol.21:1473-79 (2003).
  • NECTIN-4 proteins and/or “NECTIN-4 related proteins” of the invention include those specifically identified herein (see, Table IV), as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different NECTIN-4 proteins or fragments thereof, as well as fusion proteins of a NECTIN-4 protein and a heterologous polypeptide are also included. Such NECTIN-4 proteins are collectively referred to as the NECTIN-4-related proteins, the proteins of the invention, or NECTIN-4.
  • NECTIN-4-related protein refers to a polypeptide fragment or a NECTIN-4 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 515, 516, 517, 518, 519 or more amino acids.
  • NECTIN-4 antibodies Another aspect of the invention provides antibodies that bind to NECTIN-4 as disclosed herein.
  • NECTIN-4 antibodies of the invention are particularly useful in cancer (see, e.g., Table I), for prognostic assays, imaging, diagnostic, and therapeutic methodologies.
  • a NECTIN-4 binding assay is disclosed herein for use in detection of cancer, for example, in an immunoassay.
  • NECTIN-4 antibodies are useful (e.g., when combined with a therapeutic agent, such as in an ADC, in the treatment, and/or prognosis of cancer (for example, the cancers set forth in Table I) to the extent NECTIN-4 is also expressed or overexpressed in these other cancers.
  • intracellularly expressed antibodies e.g., single chain antibodies
  • Various methods for the preparation of antibodies, specifically monoclonal antibodies, are well known in the art.
  • antibodies can be prepared by immunizing a suitable mammalian host using a NECTIN-4-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
  • fusion proteins of NECTIN-4 can also be used, such as a NECTIN-4 GST-fusion protein.
  • a GST fusion protein comprising all or most of the amino acid sequence of NECTIN-4 is produced, and then used as an immunogen to generate appropriate antibodies.
  • a NECTIN-4-related protein is synthesized and used as an immunogen.
  • naked DNA immunization techniques known in the art are used (with or without purified NECTIN-4-related protein or NECTIN-4 expressing cells) to generate an immune response to the encoded immunogen (for review, see DONNELLY et. al., 1997, Ann. Rev. Immunol.15: 617-648).
  • Preferred methods for the generation of NECTIN-4 antibodies are further illustrated by way of the examples provided herein.
  • Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art.
  • methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or another carrier protein are well known in the art.
  • NECTIN-4 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
  • NECTIN-4 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody- producing B cells, as is generally known.
  • Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a NECTIN-4-related protein.
  • the cells When the appropriate immortalized cell culture is identified, the cells can be expanded, and antibodies produced either from in vitro cultures or from ascites fluid.
  • the antibodies or fragments of the invention can also be produced by recombinant means. Regions that bind specifically to the desired regions of a NECTIN-4 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human NECTIN-4 antibodies can also be produced and are preferred for use in therapeutic contexts.
  • CDR complementarity-determining region
  • human monoclonal antibodies of the invention can be prepared using VelocImmune mice into which genomic sequences bearing endogenous mouse variable segments at the immunoglobulin heavy chain (VH, DH, and JH segments) and/or kappa light chain (VK and JK) loci have been replaced, in whole or in part, with human genomic sequences bearing unrearranged germline variable segments of the human immunoglobulin heavy chain (VH, DH, and JH) and/or kappa light chain (VK and JK) loci (Regeneron, Tarrytown, N.Y.). See, for example, U.S. Pat.
  • human antibodies of the invention can be generated using the HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin gene miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see e.g., LONBERG, et. al. (1994) Nature 368(6474): 856-859).
  • HuMAb mouse Medarex, Inc.
  • Fully human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
  • KM mice such mice are described in TOMIZUKA et. al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02/43478 to TOMIZUKA, et. al.
  • Human monoclonal antibodies of the invention can also be prepared using phage or yeast display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art.
  • Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Pat. Nos.5,476,996 and 5,698,767 to WILSON, et. al. Additionally, human antibodies of the present invention can be made with techniques using transgenic mice, inactivated for antibody production, engineered with human heavy and light chains loci referred to as Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of preparing transgenic mice that produce human antibodies can be found in U.S. Pat. No.6,657,103. See, also, U.S. Pat.
  • the binding affinity (K D ) of the antibodies, binding fragments thereof, and antibody drug conjugates comprising the same for NECTIN-4 may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively, the K D may be between 5 and 10 nM; or between 1 and 2 nM. The K D may be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.
  • the binding affinity may be measured by BIACORE for example, by capture of the test antibody onto a protein-A coated sensor surface and flowing NECTIN-4 over this surface.
  • the binding affinity can be measured by FORTEBIO for example, with the test antibody receptor captured onto a protein-A coated needle and flowing NECTIN-4 over this surface.
  • Engineered antibodies of the invention include those in which modifications have been made to framework residues within V H and/or V L (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence.
  • an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis (e.g., “backmutated” from leucine to methionine). Such “backmutated” antibodies are also intended to be encompassed by the invention. Engineering of the VH and/or VL can also be made to modify the binding affinity to the antigen.
  • frameworks and/or CDR regions changing residues within the frameworks and/or CDR regions to increase affinity, or reduce affinity to Nectin-4 are also intended to be encompassed by the invention.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No.2003/0153043 by CARR, et. al.
  • antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • a NECTIN-4 antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • the number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the NECTIN-4 antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the NECTIN-4 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat.
  • the NECTIN-4 antibody is modified to increase its biological half-life.
  • the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022 by PRESTA et. al.
  • the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(A).
  • the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(B). In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(C). In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(D). In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(E). In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence set forth in Table VI(F). In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(G). In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(H). In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(I).
  • the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(J). In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(K). In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence set forth in Table VI(L). In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain variable region sequence(s) set forth in Table VIII. In another embodiment, the NECTIN-4 antibody comprises the antibody light chain variable region sequence(s) set forth in Table IX. In another embodiment, the NECTIN-4 antibody comprises the antibody CDR sequence(s) set forth in Table X. In another embodiment, the NECTIN-4 antibody is conjugated to a therapeutic agent.
  • NECTIN-4 antibodies can be established by a number of well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, NECTIN-4-related proteins, NECTIN-4 expressing cells or extracts thereof.
  • a NECTIN-4 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator, or an enzyme.
  • the invention provides antibody-drug conjugates (ADCs), comprising an antibody (preferably a NECTIN-4 antibody disclosed herein) conjugated to a therapeutic agent.
  • the therapeutic agent maybe a cytotoxic agent, a cytostatic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radio-conjugate).
  • the invention further provides methods of using the ADCs.
  • an ADC comprises any of the above NECTIN-4 antibodies covalently attached or attached via oxime bond to a cytotoxic agent or a detectable agent.
  • an ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using self-hydrolyzing maleimides for cysteine modification (See, WO 2013/173337).
  • an ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using a cysteine modification and further comprises reducing the cysteine residue to form a sulfhydryl moiety.
  • an ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent using a polypeptide moiety and a self-immolative moiety.
  • an ADC comprises a NECTIN-4 antibody conjugated to a therapeutic agent wherein the ADC has a high drug antibody ratio (DAR).
  • DAR drug antibody ratio
  • Toxins used in antibody- toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (MANDLER et. al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; MANDLER et. al.
  • toxins may affect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
  • cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • the FDA has approved twelve (12) ADCs, including gemtuzumab ozogamicin (MYLOTARG, Wyeth Pharmaceuticals), which was the first ADC approved by the FDA in 2000.
  • MYLOTARG gemtuzumab ozogamicin
  • ADCETRIS (brentuximab vedotin, Seattle Genetics,), ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec), KADCYLA® (ado- trastuzumab emtansine, Genentech), BESPONSA® (inotuzumab ozogamicin, Pfizer/Wyeth), POLIVY (polatuzumab vedotin, Genentech/Roche), Cantuzumab mertansine (Immunogen, Inc.), MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), and PADCEV (enfortumab vedotin-ejfv, Seattle Genetics / Astellas (Agensys, Inc.,
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radio-conjugated antibodies. Examples include 177 Lu, 89 Zr, 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis- active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3
  • Carbon-14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO94/11026).
  • Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.5,877,296).
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha-s
  • a ricin immunotoxin can be prepared as described in Vitetta et al (1987) Science, 238:1098. See, for example, WO 93/21232 (published Oct.28, 1993).
  • the present invention further contemplates an ADC formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
  • the antibody may comprise a highly radioactive atom.
  • a variety of radioactive isotopes are available for the production of radio-conjugated antibodies.
  • Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 88 , Sm 53 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the conjugate may comprise a radioactive atom for scintigraphic studies, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the radio- or other labels may be incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as tc 99m or I 123 , Re 186 , Re 188 and In 111 can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (FRAKER et. al. (1978) Biochem. Biophys. Res. Commun.80: 49-57 can be used to incorporate iodine-123.
  • the present invention provides, inter alia, antibody-drug conjugate compounds for targeted delivery of therapeutic agents.
  • the inventors have made the discovery that the antibody-drug conjugate compounds have potent cytotoxic and/or cytostatic activity against cells expressing NECTIN-4 and variants thereof.
  • the antibody-drug conjugate compounds comprise an Antibody unit covalently linked to at least one Drug unit.
  • the Drug units can be covalently linked directly to the Antibody unit or via a Linker unit (- LU-).
  • the antibody drug conjugate compound has the following formula: Ab-(LU-D) p ADC Schema (I) or a pharmaceutically acceptable salt or solvate thereof; wherein: • Ab is the Antibody unit, e.g., a NECTIN-4 antibody of the present invention; • (LU-D) is a Linker Unit-Drug unit moiety, wherein: • LU- is a Linker unit, and • -D is a drug unit having cytostatic or cytotoxic activity against a target cell; and • p ranges from 1 to 20 or alternatively 1-50.
  • the antibody drug conjugate compound has the following formula: Ab-(A a -W w —Y y -D) p ADC Schema (II) or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is the Antibody unit, e.g., a NECTIN-4 antibody of the present invention; and • -A a -W w —Y y — is a Linker unit (LU), wherein: • -A- is a Stretcher unit, • a is 0 or 1 or 2 or 3, • each —W— is independently an Amino Acid unit, • w is an integer ranging from 0 to 12, • —Y— is a self-immolative spacer unit, • y is 0, 1 or 2; • -D is a drug unit having cytostatic or cytotoxic activity against the target cell; and • p is an integer from 1 to 20 or alternatively 1-50.
  • Ab is the Antibody unit, e.g., a NECTIN-4
  • the antibody drug conjugate compound has the following formula: ADC Schema (III) or ADC Schema (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is the Antibody unit, e.g., a NECTIN-4 antibody of the present invention; • each R is independently selected from N, CH, or C; • R’ is C or CH • W is selected from: In some embodiments, the antibody drug conjugate compound has the following formula: • Ab is the antibody unit, e.g., anti-NECTIN-4 antibody of the present invention. • Each R is independently selected from N, CH, or C.
  • • W is selected from: • Xb is a spacer moiety selected from the group consisting of an alkyl, a heteroalkyl, polyethylene glycol (PEG), and a peptide. • b is 0, 1 or 2. • Y b is a polypeptide moiety that comprises about 1 to about 6 amino acids that are natural and/or unnatural amino acids. • Z b is a self-immolative moiety including but not limited to: • D is a drug unit having cytostatic or cytotoxic activity against the target cell. • p is an integer from 1 to 20 or alternatively 1-50.
  • the antibody drug conjugate compound has the following formula: ADC Schema (VII) or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is the unit, e.g., a NECTIN-4 antibody of the present invention; 1 • R is , wherein R 2 is a unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; • each of R x and R y is independently selected from R and L-R z , provided that when one of R x and R y is NR z , the other is R; • R 5 is H or CR’ 3 , wherein each R’ is independently H or F; • R 6 is H or CH 2 CN; • LU is a linker unit; and • R is H or a C 1 -C 3 alkyl; and • i is an integer in the range of 1 to about 20.
  • the antibody drug conjugate compound has the following formula: ADC Schema (VIII) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is the Antibody unit, e.g., a NECTIN-4 antibody of the present invention; 1 R is , wherein R 2 is a unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; each of R x and R y is independently selected from R and L-R z , provided that when one of R x and R y is NR z , the other is R; R 5 is H or CR’ 3 , wherein each R’ is independently H or F; R 6 is H or CH 2 CN; LU is a linker unit; and R is H or a C 1 -C 3 alkyl; and j is an integer in the range of 1 to about 20.
  • Ab is the Antibody unit, e.g., a NECTIN-4
  • the antibody drug conjugate compound has the following formula: ADC Schema (IX) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is the Antibody unit, e.g., a NECTIN-4 antibody of the present invention; 1 R is , wherein R 2 is a unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; each of R x and R y is independently selected from R and L-R z , provided that when one of R x and R y is NR z , the other is R; R 5 is H or CR’ 3 , wherein each R’ is independently H or F; R 6 is H or CH 2 CN; LU is a linker unit; and R is H or a C 1 -C 3 alkyl; and k is an integer in the range of 1 to about 20.
  • Ab is the Antibody unit, e.g., a NECTIN-4
  • the antibody drug conjugate compound has the following formula: • a or or cycloheteroalkyl; • R 3 is H or C 1 -C 3 alkyl; • R 5 is H or CR’ 3 wherein each R’ is independently H or F; • R 6 is H or CH 2 CN; • Ab is the antibody unit, e.g., anti-NECTIN-4 antibody of the present invention. • Each R is independently selected from N, CH, or C. • J is a conjugation moiety.
  • X b is a spacer moiety selected from the group consisting of an alkyl, a heteroalkyl, polyethylene glycol (PEG), and a peptide. • b is 0, 1 or 2.
  • Y b is a polypeptide moiety that comprises about 1 to about 6 amino acids that are natural and/or unnatural amino acids.
  • Z b is a self-immolative moiety including but not limited to:
  • the drug loading is represented by p, the average number of drug molecules per Antibody. Drug loading may range from 1 to 24 drugs (D) per Antibody. The average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Antibody-Drug-Conjugates in terms of p may also be determined.
  • the Antibody-drug conjugate compounds comprise NECTIN-4 antibodies.
  • the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(A).
  • the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(B).
  • the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(C). In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(D). In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(E). In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence set forth in Table VI(F). In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(G). In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(H). In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(I).
  • the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(J). In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(K). In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence set forth in Table VI(L). In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain variable region sequence(s) set forth in Table VIII. In another embodiment, the NECTIN-4 ADC comprises the antibody light chain variable region sequence(s) set forth in Table IX. In another embodiment, the NECTIN-4 ADC comprises the antibody CDR sequence(s) set forth in Table X. In another embodiment, the aforementioned NECTIN-4 antibodies are conjugated to a therapeutic agent.
  • the therapeutic agent is a Drug-Linker (DL) payload set forth in Figure 10.
  • the DL payload is set forth in Figure 10(A) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(B) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(C) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(D) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(E) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(F) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(G) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(H) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(I) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof. In one embodiment, the DL payload is set forth in Figure 10(J) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof. In one embodiment, the DL payload is set forth in Figure 10(K) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof. In one embodiment, the DL payload is set forth in Figure 10(L) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(M) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(N) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(O) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • the DL payload is set forth in Figure 10(P) and has the following chemical structure: or a pharmaceutically acceptable salt or solvate form thereof.
  • a number of different reactions are available for covalent attachment of drugs and/or linkers to binding agents.
  • the amino acid residues of the binding agent e.g., antibody molecule
  • the amino acid residues of the binding agent including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids.
  • One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody.
  • bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody molecule.
  • the Schiff base reaction also available for attachment of drugs to binding agents.
  • This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent.
  • Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present invention.
  • an intermediate which is the precursor of the linker, is reacted with the drug under appropriate conditions.
  • reactive groups are used on the drug and/or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the NECTIN-4 antibodies under appropriate conditions.
  • the antibody-drug conjugate compounds comprise a Linker unit between the drug unit and the antibody unit.
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment.
  • the linker unit is not cleavable, and the drug is released, for example, by antibody degradation.
  • the linker is conjugated to a NECTIN-4 antibody described herein.
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea).
  • the linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the linker can also be cleaved by a cleaving agent that is present in the extracellular environment (e.g., in the vicinity to the cellular membrane or tissue space).
  • the linker can be, e.g., a peptidyl linker that is cleaved by an extracellular peptidase or protease enzyme, including, but not limited to, a cathepsin family enzyme or matrix metalloproteinases).
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker hydrolyzable under acidic conditions.
  • an acid- labile linker that is hydrolyzable in the lysosome e.g., an oxime, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • an acid- labile linker that is hydrolyzable in the lysosome (e.g., an oxime, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.
  • U.S. Pat. Nos.5,122,368; 5,824,805; 5,622,929; DUBOWCHIK AND WALKER 1999, Pharm.
  • the linker is cleavable under reducing conditions known in the art.
  • the linker can also be cleaved under reducing conditions found intra-cellularly (or extra-cellularly).
  • the specific linker N—O bond may be formally reduced and broken to result in a cleavage of the linker.
  • the linker is a malonate linker (Johnson et al., 1995, Anticancer Res.15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med- Chem.3(10):1299-1304), or a 3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem.3(10):1305-12).
  • the linker unit is not cleavable, and the drug is released by antibody degradation. (See PCT Publication No.
  • the linker is not substantially sensitive to the extracellular environment.
  • “not substantially sensitive to the extracellular environment,” in the context of a linker means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody- drug conjugate compound presents in an extracellular environment (e.g., in plasma).
  • Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.
  • the linker promotes cellular internalization as known in the art.
  • a variety of exemplary linkers that can be used with the present compositions and methods are described in WO 2004/010957, U.S. Publication No.2006/0074008, U.S. Publication No.20050238649, and U.S. Publication No.2006/0024317 (each of which is incorporated by reference herein in its entirety and for all purposes).
  • a “Linker unit” is a bifunctional compound that can be used to link a Drug unit and an Antibody unit to form an antibody-drug conjugate compound.
  • the Linker unit has the formula: -A a -W w —Y y — o wherein: -A- is a Stretcher unit, o a is 0 or 1, o each —W— is independently an Amino Acid unit, o w is an integer ranging from 0 to 12, o —Y— is a self-immolative Spacer unit, and o y is 0, 1 or 2.
  • a is 0 or 1
  • w is 0 or 1
  • y is 0, 1 or 2.
  • a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0. V.)
  • the Stretcher Unit (A) when present, is capable of linking an Antibody unit to an Amino Acid unit (—W—), if present, to a Spacer unit (—Y—), if present; or to a Drug unit (-D).
  • Useful functional groups that can be present on a NECTIN-4 antibody, either naturally or via chemical manipulation include, but are not limited to, keto, aldehyde, sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl, and amino.
  • the keto group is on a non-natural amino acid (nnAA) incorporated into the antibody of the invention.
  • the aldehyde group is on a nnAA incorporated into the antibody of the invention.
  • sulfhydryl groups can be generated by reduction of the intramolecular disulfide bonds of a NECTIN-4 antibody.
  • sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a NECTIN-4 antibody with 2- iminothiolane (Traut's reagent) or other sulfhydryl generating reagents.
  • the NECTIN-4 antibody is a recombinant antibody and is engineered to carry one or more lysines.
  • the recombinant NECTIN-4antibody is engineered to carry additional sulfhydryl groups, e.g., additional cysteines.
  • the Stretcher unit forms a bond with a sulfur atom of the Antibody unit.
  • the sulfur atom can be derived from a sulfhydryl group of an antibody.
  • the Stretcher unit is linked to the Antibody unit via a disulfide bond between a sulfur atom of the Antibody unit and a sulfur atom of the Stretcher unit.
  • the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of an antibody.
  • the Stretcher contains a reactive site that is reactive to a modified carbohydrate's (—CHO) group that can be present on an antibody.
  • a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (—CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko et al., 1991, Bioconjugate Chem.2:133-41.
  • a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko et al., 1991, Bioconjugate Chem.2:133-41.
  • the Amino Acid Unit (—W—), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug moiety if the Spacer unit is absent, and links the Antibody unit to the Drug unit if the Stretcher unit and Spacer unit are absent.
  • the Amino Acid unit can comprise natural amino acids. In other embodiments, the Amino Acid unit can comprise non-natural amino acids.
  • the Amino Acid unit can be enzymatically cleaved by one or more enzymes, including a cancer or tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
  • the Amino Acid unit is valine-citrulline (vc or Val-Cit).
  • the Amino Acid unit is phenylalanine-lysine.
  • the Amino Acid unit is N-methylvaline-citrulline.
  • the Amino Acid unit is 5- aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.
  • the Spacer Unit (—Y—), when present, links an Amino Acid unit to the Drug unit when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug unit when the Amino Acid unit is absent.
  • Spacer unit also links the Drug unit to the Antibody unit when both the Amino Acid unit and Stretcher unit are absent.
  • Spacer units are of two general types: non self- immolative or self-immolative. Examples of possible spacers of the invention are known in the art. See, TOKI et. al., 2002, J. Org. Chem.67:1866-1872 and Nature Biotechnology 21(7):778-784). Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (HAY et. al., 1999, Bioorg. Med. Chem. Lett.9:2237) and ortho or para-aminobenzylacetals.
  • Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (RODRIGUES et. al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (STORM et. al., 1972, J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (AMSBERRY et. al., 1990, J. Org. Chem.55:5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (KINGSBURY et. al., 1984, J.
  • the Drug unit (D) can be any cytotoxic, cytostatic, or immunomodulatory (e.g., immunosuppressive) drug.
  • D is a Drug unit (moiety) having an atom that can form a bond with the Spacer unit, with the Amino Acid unit, with the Stretcher unit or with the Antibody unit.
  • the Drug unit D has a nitrogen atom that can form a bond with the Spacer unit.
  • drug unit and “Drug moiety” are synonymous and used interchangeably.
  • cytotoxic, cytostatic, or immunomodulatory agents include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.
  • the Drug is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) 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.
  • the Drug Unit is a calicheamicin, camptothecin, a maytansinoid, or an anthracycline.
  • the drug is a taxane, a topoisomerase inhibitor, a vinca alkaloid.
  • suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBI compound; see also U.S. Pat. No.6,130,237), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, and vinca alkaloids.
  • cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.
  • the Drug is an anti-tubulin agent.
  • anti-tubulin agents examples include, auristatins, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).
  • Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.
  • the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents.
  • the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.52:127-131).
  • the cytotoxic or cytostatic agent is a dolastatin.
  • the cytotoxic or cytostatic agent is of the auristatin class, for example, Dolastatin-10, Auristatin e, or Auristatin PHE, etc.
  • Drug Loading Drug loading is represented by p and is the average number of Drug moieties per antibody in a molecule. Drug loading may range from 1 to 24 drug moieties (D) per antibody.
  • ADCs of the invention include collections of antibodies conjugated with a range of drug moieties, from 1 to 24. The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy and, ELISA assay. The quantitative distribution of ADC in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as electrophoresis.
  • p may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • higher drug loading e.g., p>5
  • the drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.
  • the optimal ratio of drug moieties per antibody may be less than 8 and may be about 2 to about 5. See U.S. Pat. No.7,498,298 (herein incorporated by reference in its entirety). In certain embodiments, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction.
  • an antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug to antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachments (such as thioMab or thioFab prepared as disclosed herein and in WO2006/034488 (herein incorporated by reference in its entirety)).
  • linker-drug attachments such as thioMab or thioFab prepared as disclosed herein and in WO2006/034488 (herein incorporated by reference in its entirety)
  • the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug.
  • Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., Hamblett, K. J., et al.
  • a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the Antibody Drug conjugate in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability.
  • Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the Antibody Drug conjugate.
  • a thymidine incorporation assay may be used.
  • cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 ⁇ Ci of 3 H-thymidine during the final 8 hours of the 72-hour period.
  • the incorporation of 3 H-thymidine into cells of the culture is measured in the presence and absence of the ADC.
  • necrosis, or apoptosis programmeed cell death
  • Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane.
  • Apoptosis is typically characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.
  • Cell viability can be measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMARTM blue (see, e.g., PAGE et. al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.
  • a dye such as neutral red, trypan blue, or ALAMARTM blue
  • the protein-binding dye sulforhodamine B can also be used to measure cytotoxicity (SKEHAN et. al., 1990, J. Natl. Cancer Inst.82:1107-12).
  • a tetrazolium salt such as MTT, or CellTiter-Glo ⁇ , is used in a quantitative assay for mammalian cell survival and proliferation by detecting living, but not dead, cells (see, e.g., MOSMANN, 1983, J. Immunol. Methods 65:55-63).
  • Apoptosis can be quantitated by measuring, for example, DNA fragmentation. Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available.
  • Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide).
  • a fluorescent dye such as, for example, acridine orange or ethidium bromide.
  • Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane.
  • a DNA dye e.g., acridine orange, ethidium bromide, or propidium iodide
  • Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage. The presence of apoptotic cells can be measured in both the attached and “floating” compartments of the cultures.
  • both compartments can be collected by removing the supernatant, trypsinizing the attached cells, combining the preparations following a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation).
  • a centrifugation wash step e.g. 10 minutes at 2000 rpm
  • detecting apoptosis e.g., by measuring DNA fragmentation.
  • PCT Patent Application WO98/16628 and U.S. Pat. No.6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micro-metastasis, and the formation of osteoblastic metastases characteristic of late-stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like. In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions.
  • xenografts from tumor bearing mice treated with therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice.
  • the extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
  • the therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system.
  • Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site.
  • Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intra-organ, orthotopic, and the like.
  • a preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP.
  • Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection. Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
  • the pharmaceutical composition of the present invention may comprise more than one species of ADC of the invention due to modification of a NECTIN-4 antibody.
  • the present invention includes a pharmaceutical composition comprising the ADC of the invention, wherein the NECTIN-4 antibody is an antibody with a C-terminal lysine partially removed or completely removed an antibody having N-terminal post-translational modification, an antibody lacking heavy chain C-terminal lysine and having N-terminal post-translational modification, and/or an antibody having heavy chain C-terminal lysine and not having N-terminal post-translational modification.
  • NECTIN-4 antibody is set forth in Table(s) VI and Table VII.
  • NECTIN-4 Treatment of Cancer(s) Expressing NECTIN-4
  • targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues or cells, even vital normal organ tissues.
  • a vital organ is one that is necessary to sustain life, such as the heart or colon.
  • a non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
  • Immunoprivileged organs are organs that are protected from blood by a blood-organ barrier and thus are not accessible to immunotherapy. Examples of immunoprivileged organs are the brain and testis.
  • therapeutic approaches that inhibit the activity of a NECTIN-4 protein are useful for patients suffering from cancer that expresses NECTIN-4 (such as, for example, those cancers set forth in Table I).
  • These therapeutic approaches generally fall into three classes.
  • the first class modulates NECTIN-4 function as it relates to tumor cell growth leading to inhibition or retardation of tumor cell growth or inducing its killing.
  • the second class comprises various methods for inhibiting the binding or association of a NECTIN-4 protein with its binding partner or with other proteins.
  • the third class comprises a variety of methods for inhibiting the transcription of a NECTIN-4 gene or translation of NECTIN-4 mRNA.
  • cancer patients can be evaluated for the presence and level of NECTIN-4 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative NECTIN-4 imaging, or other techniques that reliably indicate the presence and degree of NECTIN-4 expression.
  • Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose, if applicable. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
  • NECTIN-4 ADC Cocktails Therapeutic methods of the invention contemplate the administration of single NECTIN-4 ADCs as well as combinations, or cocktails, of different antibodies (i.e., NECTIN-4 antibodies or antibodies that bind another protein).
  • NECTIN-4 antibodies can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic and biologic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF, PD1, PD-L1), surgery or radiation.
  • the NECTIN-4 antibodies are administered in conjugated form.
  • the NECTIN-4 antibodies are set forth in Table VI and Table VII.
  • NECTIN-4 ADC formulations are administered via any route capable of delivering the antibodies to a tumor cell.
  • Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like.
  • Treatment generally involves repeated administration of the NECTIN-4 ADC preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range, including but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight.
  • IV intravenous injection
  • doses in the range of 10-1000 mg MAb per week are effective and well tolerated.
  • an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the MAb preparation represents an acceptable dosing regimen.
  • the initial loading dose is administered as a 90-minute or longer infusion.
  • the periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated.
  • numerous factors can influence the ideal dose regimen in a particular case.
  • Such factors include, for example, the binding affinity and half-life of the antibodies used, the degree of NECTIN-4 expression in the patient, the extent of circulating shed NECTIN-4 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
  • patients should be evaluated for the levels of NECTIN-4 in a given sample (e.g., the levels of circulating NECTIN-4 antigen and/or NECTIN-4 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc.
  • An object of the present invention is to provide NECTIN-4 ADCs, which inhibit or retard the growth of tumor cells expressing NECTIN-4.
  • a further object of this invention is to provide methods to inhibit angiogenesis and other biological functions and thereby reduce tumor growth in mammals, preferably humans, using such NECTIN-4 ADCs, and in particular using such NECTIN-4 ADCs combined with other drugs or immunologically active treatments.
  • NECTIN-4 ADCs in conjunction with chemotherapeutic agents or radiation or combinations thereof.
  • the inhibition of tumor growth by a NECTIN-4 ADC is enhanced more than expected when combined with chemotherapeutic agents or radiation or combinations thereof.
  • Synergy may be shown, for example, by greater inhibition of tumor growth with combined treatment than would be expected from a treatment of only NECTIN-4 ADC or the additive effect of treatment with a NECTIN-4 ADC and a chemotherapeutic agent or radiation.
  • synergy is demonstrated by remission of the cancer where remission is not expected from treatment either from a NECTIN-4 ADC or with treatment using an additive combination of a NECTIN-4 ADC and a chemotherapeutic agent or radiation or an immunotherapy such as CAR-T or NK cell therapy.
  • the method for inhibiting growth of tumor cells using a NECTIN-4 ADC and a combination of chemotherapy or radiation or both comprises administering the NECTIN-4 ADC before, during, or after commencing chemotherapy or radiation therapy, as well as any combination thereof (i.e. before and during, before and after, during and after, or before, during, and after commencing the chemotherapy and/or radiation therapy).
  • the NECTIN-4 ADC is typically administered between 1 and 60 days, preferably between 3 and 40 days, more preferably between 5 and 12 days before commencing radiation therapy and/or chemotherapy.
  • the method is performed in a manner that will provide the most efficacious treatment and ultimately prolong the life of the patient.
  • the administration of chemotherapeutic agents can be accomplished in a variety of ways including systemically by the parenteral and enteral routes.
  • the NECTIN-4 ADCs and the chemotherapeutic agent are administered as separate molecules.
  • chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin,
  • the source of radiation used in combination with a NECTIN-4 ADC, can be either external or internal to the patient being treated.
  • the therapy is known as external beam radiation therapy (EBRT).
  • EBRT external beam radiation therapy
  • the treatment is called brachytherapy (BT).
  • BT brachytherapy
  • the radiation therapy is boron neutron capture therapy.
  • the radiation is Proton Boron Fusion Therapy.
  • additional cancer treating agents and/or regimes for example additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies (e.g.
  • Anti- CTLA-4 antibodies as described in WO/2005/092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.
  • chemotherapeutic agents described above may be used.
  • growth factor inhibitors, biological response modifiers, anti- hormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti- androgens may be used.
  • anti-hormones for example anti-estrogens such as Nolvadex (tamoxifen) or, anti-androgens such as Casodex (4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2- methyl-3-′-(trifluoromethyl)propionanilide) may be used.
  • anti-estrogens such as Nolvadex (tamoxifen) or, anti-androgens such as Casodex (4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2- methyl-3-′-(trifluoromethyl)propionanilide
  • the above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens.
  • the therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic
  • kits are within the scope of the invention.
  • kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein.
  • the container(s) can comprise a NECTIN-4 antibody or several NECTIN-4 antibodies (See, Tables VI and VII) of the disclosure.
  • Kits can comprise a container comprising a drug unit.
  • the kit can include all or part of the NECTIN-4 ADCs and/or diagnostic assays for detecting cancer and/or other immunological disorders.
  • the kit of the invention will typically comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
  • a label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit.
  • the label can be on or associated with the container.
  • a label can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • the label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a cancer or other immunological disorder.
  • kit and “article of manufacture” can be used as synonyms.
  • the article of manufacture typically comprises at least one container and at least one label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers can be formed from a variety of materials such as glass, metal, or plastic.
  • the container can hold one or several NECTIN-4 ADCs and/or one or more therapeutics doses of NECTIN-4 ADCs.
  • the container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agents in the composition can be a NECTIN-4 antibody or ADC of the present disclosure.
  • the article of manufacture can further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution.
  • EXEMPLARY EMBODIMENTS 1) An antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 1 further comprising a light chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 1 wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 28.
  • the antibody or antigen binding fragment thereof of claim 2 wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 34.
  • the antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 5.
  • the antibody or antigen binding fragment thereof of claim 2 wherein the light chain comprises the sequence set forth in SEQ ID NO: 17.
  • the antibody or antigen binding fragment thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 34.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 1-15 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 1-16.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 16.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 17.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 1-15.
  • the method of any of claim 18-20 wherein the subject is a human subject.
  • the method of claim 21 wherein the cancer is set forth in Table I.
  • An antibody or antigen binding fragment thereof wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 24, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 35.
  • the antibody or antigen binding fragment thereof of claim 24, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 6.
  • the antibody or antigen binding fragment thereof of claim 24, wherein the light chain comprises the sequence set forth in SEQ ID NO: 18.
  • the antibody or antigen binding fragment thereof of claim 24, comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 29 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 35.
  • the antibody or antigen binding fragment thereof of claim 24 wherein the antigen binding fragment thereof is a Fab, F(ab′)2, Fv or scFv.
  • the antibody or antigen binding fragment thereof of claim 24, wherein the antibody is a fully human antibody.
  • the antibody or antigen binding fragment thereof of claim 24, wherein the antibody or antigen binding fragment thereof is conjugated to a drug via a linker.
  • the antibody or antigen binding fragment thereof of claim 34 wherein the drug is an auristatin analog.
  • the antibody or antigen binding fragment thereof of claim 34 further comprising a spacer unit.
  • the antibody or antigen binding fragment thereof of claim 34 further comprising an amino acid unit.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 24-38 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 24-39.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 39.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 40.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 24-39.
  • the method of any of claim 41-43 wherein the subject is a human subject.
  • the method of claim 43 wherein the cancer is set forth in Table I.
  • the method of claim 43 wherein the method further comprises administering radiation or a chemotherapeutic agent.
  • An antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 47 further comprising a light chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 47, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 30.
  • the antibody or antigen binding fragment thereof of claim 47, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 36.
  • the antibody or antigen binding fragment thereof of claim 47, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 8.
  • the antibody or antigen binding fragment thereof of claim 47 wherein the light chain comprises the sequence set forth in SEQ ID NO: 20.
  • the antibody or antigen binding fragment thereof of claim 47 comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 36.
  • the antibody or antigen binding fragment thereof of claim 47 wherein the antigen binding fragment thereof is a Fab, F(ab′)2, Fv or scFv.
  • the antibody or antigen binding fragment thereof of claim 47 wherein the antibody is a fully human antibody.
  • the antibody or antigen binding fragment thereof of claim 47 wherein the antibody or antigen binding fragment thereof is recombinantly produced.
  • the antibody or antigen binding fragment thereof of claim 47 wherein the antibody or antigen binding fragment thereof is conjugated to a drug via a linker.
  • the antibody or antigen binding fragment thereof of claim 47, wherein the drug is an auristatin analog.
  • the antibody or antigen binding fragment thereof of claim 47 further comprising a stretcher unit.
  • the antibody or antigen binding fragment thereof of claim 47 further comprising a spacer unit.
  • the antibody or antigen binding fragment thereof of claim 47 further comprising an amino acid unit.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 47-61 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 47-62.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 62.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 63.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 47-65.
  • the method of any of claim 64-66 wherein the subject is a human subject.
  • the method of claim 66 wherein the cancer is set forth in Table I.
  • the method of claim 66, wherein the method further comprises administering radiation or a chemotherapeutic agent.
  • An antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 70 further comprising a light chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 70, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31.
  • the antibody or antigen binding fragment thereof of claim 70, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 37.
  • the antibody or antigen binding fragment thereof of claim 70, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 10.
  • the antibody or antigen binding fragment thereof of claim 70 wherein the light chain comprises the sequence set forth in SEQ ID NO: 22.
  • the antibody or antigen binding fragment thereof of claim 70 comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 37.
  • the antibody or antigen binding fragment thereof of claim 70 wherein the antigen binding fragment thereof is a Fab, F(ab′)2, Fv or scFv.
  • the antibody or antigen binding fragment thereof of claim 70 wherein the antibody is a fully human antibody.
  • the antibody or antigen binding fragment thereof of claim 70 wherein the antibody or antigen binding fragment thereof is recombinantly produced.
  • the antibody or antigen binding fragment thereof of claim 70 wherein the antibody or antigen binding fragment thereof is conjugated to a drug via a linker.
  • the antibody or antigen binding fragment thereof of claim 70, wherein the drug is an auristatin analog.
  • the antibody or antigen binding fragment thereof of claim 70 further comprising a stretcher unit.
  • the antibody or antigen binding fragment thereof of claim 70 further comprising a spacer unit.
  • the antibody or antigen binding fragment thereof of claim 70 further comprising an amino acid unit.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 70-84 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 70-84.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 85.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 86.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 70-88.
  • the method of any of claim 87-89, wherein the subject is a human subject.
  • the method of claim 89, wherein the cancer is set forth in Table I.
  • the method of claim 89, wherein the method further comprises administering radiation or a chemotherapeutic agent.
  • An antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 93 further comprising a light chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84.
  • CDRs complementarity determining regions
  • the antibody or antigen binding fragment thereof of claim 93, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 32.
  • the antibody or antigen binding fragment thereof of claim 93, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 38.
  • the antibody or antigen binding fragment thereof of claim 93, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 12.
  • the antibody or antigen binding fragment thereof of claim 93 wherein the light chain comprises the sequence set forth in SEQ ID NO: 24.
  • the antibody or antigen binding fragment thereof of claim 93 comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 38.
  • the antibody or antigen binding fragment thereof of claim 93 wherein the antigen binding fragment thereof is a Fab, F(ab′)2, Fv or scFv.
  • the antibody or antigen binding fragment thereof of claim 93 wherein the antibody is a fully human antibody.
  • the antibody or antigen binding fragment thereof of claim 93 wherein the antibody or antigen binding fragment thereof is recombinantly produced.
  • the antibody or antigen binding fragment thereof of claim 93, wherein the antibody or antigen binding fragment thereof is conjugated to a drug via a linker.
  • the antibody or antigen binding fragment thereof of claim 93, wherein the drug is an auristatin analog.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 93-107 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 93- 108. 0) A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 108.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 109.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 93-107.
  • the method of claim 112, wherein the cancer is set forth in Table I.
  • An antibody or antigen binding fragment thereof wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.
  • the antibody or antigen binding fragment thereof of claim 116 further comprising a light chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87.
  • the antibody or antigen binding fragment thereof of claim 116, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33.
  • the antibody or antigen binding fragment thereof of claim 116 wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 39.
  • the antibody or antigen binding fragment thereof of claim 116, wherein the light chain comprises the sequence set forth in SEQ ID NO: 26.
  • the antibody or antigen binding fragment thereof of claim 116 comprising a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO: 39.
  • the antibody or antigen binding fragment thereof of claim 116 wherein the antigen binding fragment thereof is a Fab, F(ab′)2, Fv or scFv.
  • the antibody or antigen binding fragment thereof of claim 116 wherein the antibody is a fully human antibody.
  • the antibody or antigen binding fragment thereof of claim 116 wherein the antibody or antigen binding fragment thereof is recombinantly produced.
  • the antibody or antigen binding fragment thereof of claim 116, wherein the antibody or antigen binding fragment thereof is conjugated to a drug via a linker.
  • the antibody or antigen binding fragment thereof of claim 116, wherein the drug is an auristatin analog.
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any of claims 116-130 and a pharmaceutically acceptable excipient.
  • a kit comprising the antibody or antigen binding fragment thereof of any of claims 116- 131.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 131.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the kit of claim 132.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any of claims 116-130.
  • An antibody drug conjugate comprising a NECTIN-4 antibody or antigen binding fragment thereof conjugated to a Drug-Linker (DL) payload, wherein the antibody or antigen binding fragment thereof comprises a heavy chain CDR region comprising an amino acid sequence set forth in any of SEQ ID NO: 52 through SEQ IS NO: 69.
  • the ADC of claim 139 further comprising a NECTIN-4 antibody or antigen binding fragment thereof wherein the antibody or antigen binding fragment thereof comprises a light chain CDR region comprising an amino acid sequence set forth in any of SEQ ID NO: 70 through SEQ ID NO: 87.
  • the ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: .
  • the ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 143) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 144) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 145) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 146) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: 147) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: N . 8) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: .
  • the ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 0) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: N . 1) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 2) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: N . 3) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: N . 4) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: . 5) The ADC of claim 139 or 140, wherein the DL payload comprises the following chemical structure: N .
  • ADC of claim 139 or 140 wherein the DL payload comprises the following chemical structure: N .
  • a pharmaceutical composition comprising the ADCs of any of claims 139 through 156 and a pharmaceutically acceptable excipient.
  • a kit comprising the ADC of any of claims 139-156.
  • a kit comprising the pharmaceutical composition of claim 157. 0)
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an ADC of any of claims 139-156. 161)
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 157. 162)
  • the method of claim 160 wherein the subject is a human.
  • the NECTIN-4 antibodies were generated by de novo discovery campaign using yeast display human Fab antibody libraries. After several rounds of enrichment followed by clone screening, clones that specifically recognize human Nectin-4 expressed on cancer cells were identified. Subsequently, the variable heavy and light chains of the antibody were sequenced from DNA isolated from the yeast clones. To express the nectin-4 antibodies recombinantly, the antibody variable heavy and light chain sequences were cloned upstream of the human heavy chain IgG1 and human light chain Ig ⁇ constant regions, respectively. Signal peptides were inserted upstream of the heavy and light chains to allow secretion of the antibodies.
  • the complete anti-nectin-4 antibody human heavy chain and light chain cassettes were cloned downstream of the CMV promoter/enhancer in a cloning vector. A polyadenylation site was included downstream of the MAb coding sequence.
  • the recombinant anti- nectin-4 antibody heavy and light chain expressing constructs were transfected into CHO cells. Stably transfected Chinese Hamster Ovary (CHO) cells underwent selection and recovery process for the generation of stable pools expressing recombinant antibodies and Fc variants. For the antibody generation, fed-batch production process with typical culture duration of 8 to 15 days was used for stably transfected pools prior to harvest of the culture media.
  • transiently transfected cells were cultured for a typical duration of 3-15 days prior to harvest of the culture media.
  • Protein-A affinity purification was performed for harvested cell culture fluid and purified materials were buffer exchanged into phosphate-buffered saline (PBS) or other preferred antibody formulation buffer.
  • PBS phosphate-buffered saline
  • the quality of recombinant antibodies was assessed by size-exclusion chromatography, SDS-PAGE and other methods known in the art.
  • the resulting NECTIN-4 antibodies are set forth in Table VI, Table VII and comprise (SEQ ID NO: 4) through (SEQ ID NO: 27).
  • Example 2 Binding Assays of NECTIN-4 Antibodies. The binding affinity of NECTIN-4 antibodies of the invention were assessed using the following protocols.
  • Tumor cell lines were harvested, and cells re-suspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were plated into 96-well round bottom plates and incubated with antibody (10 ⁇ g/mL) for one (1) hour on ice. For cell binding specificity determinations, antibodies were added at 10 ⁇ g/mL. After incubation, cells were pelleted by centrifugation at 200 ⁇ g for 5 minutes, washed twice with FACS buffer, and counterstained with R-PE labeled goat anti-human Fc gamma fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA) for an additional one (1) hour on ice, protected from light.
  • FACS buffer 2% FBS + 5 mM EDTA in PBS.
  • NECTIN-4 antibodies bind specifically to NECTIN-4 on multiple cancer cell lines (T-47D, RT4, NCI-H1781, NCI-H322, PC-3, L-540, SU-DHL-1, and K562). (See, Figure(s) 1).
  • Example 3 Binding Assays of NECTIN-4 Antibodies. In another example, the binding affinity of NECTIN-4 antibodies of the invention were assessed using the following protocols.
  • Tumor cell lines were harvested, and cells re-suspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were plated into 96-well round bottom plates and incubated with antibody (10 ⁇ g/mL) for one (1) hour on ice. For cell binding specificity determinations, antibodies were added at 10 ⁇ g/mL with 3-fold dilutions. After incubation, cells were pelleted by centrifugation at 200 ⁇ g for 5 minutes, washed twice with FACS buffer, and counterstained with R-PE labeled goat anti-human Fc gamma fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA) for an additional one (1) hour on ice, protected from light.
  • FACS buffer 2% FBS + 5 mM EDTA in PBS.
  • NECTIN-4 antibodies bind specifically to NECTIN-4 on T-47D breast cancer cell line. (See, Figure(s) 2 and Table XII).
  • Example 4 Binding Assays of NECTIN-4 Antibodies.
  • the binding affinity of NECTIN-4 antibodies of the invention were assessed using the following protocols. Briefly, Tumor cell lines were harvested, and cells re-suspended in FACS buffer (2% FBS + 5 mM EDTA in PBS).
  • NECTIN-4 ADCs were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA). The results show that the NECTIN-4 antibodies bind specifically to NECTIN-4 on NCI-H292 lung cancer cell line and also bind comparatively to NCI-H292 in their respective ADC format. (See, Figure(s) 3 and Table XIII).
  • Example 5 Cytotoxicity of NECTIN-4 ADCs In Vitro. The in vitro cytotoxicity of the NECTIN-4 ADCs was determined using the following protocols.
  • tumor cell lines were harvested, plated into 384-well white flat-bottom plates, and allowed to re- attach for 2-4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test articles over a dose-titration (500 nM max with 5-fold dilutions). After 5-day treatment, the remaining cell viability was determined by CellTiter Glo assay based on the manufacturer’s instructions (Promega; Madison, WI). Data were normalized to non-treated control cells and dose-response curves were fitted using a 4-parameter logistic equation using GraphPad Prism software (version 9; La Jolla, CA).
  • NECTIN-4 ADCs utilizing multiple linker / payloads have cytotoxic effects in vitro on the NECTIN-4 positive cell line PC3- NECTIN-4.
  • Example 6 Cytotoxicity of NECTIN-4 ADC Payloads In Vitro. The in vitro cytotoxicity of the NECTIN-4 ADC payloads was determined using the following protocols. Briefly, tumor cell lines were harvested, plated into 384-well white flat-bottom plates, and allowed to re-attach for 2-4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test articles over a dose-titration (500 nM max with 5-fold dilutions).
  • Example 7 Bystander Activity of NECTIN-4 ADCs Compared to enfortumab vedotin.
  • Bystander activity of the NECTIN-4 ADCs compared to commercial enfortumab vedotin (PADCEV) was determined using the following protocols. Briefly, using a co-culture model of Nectin-4 expressing PC3-Nectin4 recombinant cells and Nectin-4 non-expressing SU-DHL-1 cells labeled with Cell Trace Violet (Invitrogen; Waltham, MA) was conducted in 24-well plates. The ratio of target- positive to target-negative cells was 1:2.
  • SU-DHL-1 cells were harvested and collected into 96-well round bottom plates and sequentially stained with Fixable Viability Dye (FVD) eFluor780 (Invitrogen) and APC-Annexin V (BioLegend; San Diego, CA) before analysis by flow cytometry.
  • FVD Fixable Viability Dye
  • APC-Annexin V BioLegend; San Diego, CA
  • Monocultures of SU-DHL-1 cells treated with ADC served as controls to demonstrate non-target specific killing, or absence thereof.
  • Early apoptotic (Annexin V single positive), late apoptotic (FVD eFluor780 single positive), and necrotic (Annexin V and FVD eFluor780 double positive) cells were considered non-viable.
  • NECTIN-4 ADCs In vivo efficacy of the NECTIN-4 ADCs was performed using the following protocols. Briefly, HT-1376 cell suspension was mixed 1:1 with Matrigel.5,000,000 viable cells were subcutaneously injected into the rear flank of female BALB/c nude mice. When mean tumor size reached approximately 100 mm 3 , mice were randomized into five (5) groups. Test articles were dosed once at 5mg/kg. The study was terminated on day 28. The results show that all NECTIN-4 ADCs inhibited tumor growth when compared to the control groups. (See, Figure(s) 7).
  • Example 9 Efficacy of NECTIN-4 ADC in a Sum190PT Breast Cancer Xenograft Model In Vivo.
  • NECTIN-4 ADC (Ab5-ADC2) was performed using the following protocols. Briefly, Sum190PT cell suspension was mixed 1:1 with Cultrex ECM. 3,000,000 viable cells were subcutaneously injected into the rear flank of female NSG mice. When mean tumor size reached approximately 130 mm 3 , mice were randomized into groups, five (5) mice per group. Test articles were dosed at 10mg/kg twice one week apart. The study was terminated on day 28. The results show in vivo efficacy of a Nectin-4 ADC (filled circle) compared to the respective isotype control ADC (open square) and PBS group (open circle) in a Nectin4-positive Sum190PT breast cancer xenograft model.
  • Example 10 Efficacy of NECTIN-4 ADC Compared to PADCEV in a Patient Derived Head and Neck Cancer Model In Vivo. Further In vivo efficacy of a NECTIN-4 ADC (Ab5-ADC2) compared to commercial enfortumab vedotin (PADCEV) was performed using the following protocols. Briefly, tumors derived from a head and neck squamous cell carcinoma patient were propagated in vivo in immunocompromised mice as a patient-derived xenograft (PDX). Tumor fragments (2-3mm in diameter) from stock mice were harvested and used for subcutaneous inoculation into female NOD/SCID mice.
  • PDX patient-derived xenograft
  • mice When mean tumor size reached approximately 150 mm 3 , mice were randomized into groups, five (5) mice per group. Test articles were dosed at 5 mg/kg or 10 mg/kg as single dose or twice, two (2) weeks apart. The results show Nectin-4 ADC (circles) displays better efficacy and complete tumor eradication compared to enfortumab vedotin (squares) in a Nectin4-positive patient-derived head and neck cancer model. (See, Figure(s) 9).
  • Example 11 Use of Chimeric Antigen Receptor (CAR) T Cell Therapy in Cancers Expressing NECTIN-4. Generally speaking, T cells help find and fight off infections and diseases, such as cancer in the body.
  • CAR Chimeric Antigen Receptor
  • CAR-T therapy a chimeric antigen receptor (CAR) is designed to recognize specific markers that are expressed in cancer (e.g., NECTIN-4). Studies have shown that when a CAR is attached to a specific antigen an immune response is induced and the T cells recognize cancer and can inhibit cancer growth.
  • CAR-T therapy a chimeric antigen receptor (CAR) is designed to recognize specific markers that are expressed in cancer (e.g., NECTIN-4).
  • NECTIN-4 a chimeric antigen receptor
  • Studies have shown that when a CAR is attached to a specific antigen an immune response is induced and the T cells recognize cancer and can inhibit cancer growth.
  • blood is collected from a patient having cancer that expresses NECTIN-4.
  • the T cells from the patient’s blood are isolated and genetically engineered to generate CAR-T cells.
  • the CAR-T cells are cultivated and expanded using techniques known in the art.
  • NK cells are not tailored to specific antigens. However, while NK cells can recognize and attack cancer cells, the NK cells do not live long enough or multiple quickly enough to fight cancer cells entirely. However, studies have shown that NK cells can be enhanced by treating them with immune system proteins called cytokines. Studies have shown that enhancing NK cells with cytokines allows for a more robust immune response.
  • One advantage of NK cell therapy is the lack of side effects versus CAR-T therapy. In some instances, NK cells are also enhanced with CARs to make them more attuned to fighting cancer. See, LU, et. al., Frontiers in Oncology, vol.11, Art.720501 (Aug.2021).
  • NK cells are generated from peripheral blood (PB), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), and NK92 cell lines. After isolation from the aforementioned sources, NK cells are stimulated by cytokines such as IL-2, IL-15, and/or IL-18. Moreover, NK cells can be modified ex vivo to express CARs, allowing NK cells to recognize specific tumor associated antigens such as NECTIN-4. Finally, the NK cells are infused into the patients’ bloodstream. See, MEHTA, et. al., Int. J.
  • NECTIN-4 antibodies and NECTIN-4 ADCs Characterization Assays Nectin-4 antibodies and NECTIN-4 ADC compositions of the invention were further characterized using assays known in the art. The results shown in Figure 11 and Figure 12 confirm better safety profile and stronger efficacy, leading to an improved therapeutic window relative to other NECTIN-4 antibodies and NECTIN-4 ADCs known in the art.
  • Example 14 Safety assessment of NECTIN-4 ADCs Across Multiple Primary Cultures of Human Normal Cells In Vitro.
  • the in vitro cytotoxicity of the NECTIN-4 ADCs was determined using the following protocols. Briefly, normal human-derived primary cells including corneal epithelial cells, adult dermal fibroblasts and adult epidermal keratinocytes were harvested, plated into 384-well white flat-bottom plates, and allowed to re-attach for 2-4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test articles over a dose-titration (1000 nM max with 5-fold dilutions).
  • NECTIN-4 ADC Ab5-ADC2 has significantly weaker cytotoxic effects in vitro than enfortumab vedotin on multiple normal primary cells, such as human corneal epithelial cells (See, Figure(s) 11(A)), adult dermal fibroblasts (See, Figure(s) 11(B)), and adult epidermal keratinocytes (See, Figure(s) 11(C)).
  • human corneal epithelial cells See, Figure(s) 11(A)
  • adult dermal fibroblasts See, Figure(s) 11(B)
  • adult epidermal keratinocytes See, Figure(s) 11(C)
  • Table XVI which shows the toxicokinetic parameters of ADC and total IgG at a repeat dose of up to 18 mg/kg and thus provides evidence of an increased HNSTD in NHP as an outcome of the described improved in vitro safety profile.
  • HT-1376 tumor cells were harvested and plated into 6-well plates and incubated overnight at 37°C. The next day, cells were treated with Ab5-ADC2 at 5 nM for 72 hours and then processed for propidium iodide staining with FxCycle PI/RNase Staining Solution (Invitrogen, Waltham, MA, USA). DNA content was measured by flow cytometry and cell cycle data modeling were analyzed using the Watson Pragmatic algorithm with FlowJo software (version 10; BD Biosciences, Franklin Lakes, NJ, USA). The results show the cell cycle analysis of the percent (%) combined G2 and sub-G1 phases of HT-1376 cells following 72-hour treatment with Ab5-ADC2.
  • FIG. 13(A) shows representative flow cytometry histogram of Ab5-ADC2-treated cells that show increased cell populations in both sub-G1 and G2 phases of the cell cycle compared to non-treated control cells as expected for this payload class.
  • Example 16 Immunogenic Cell Death (ICD) Analysis of Free Payload Compared to MMAE.
  • ICD Immunogenic Cell Death
  • ICD immunogenic cell death
  • Example 17 Complement Dependent Cytotoxicity (CDC) Analysis of Ab5-ADC2 and Ab5.
  • CDC Complement Dependent Cytotoxicity
  • HT-1376 tumor cells were harvested and plated into 96-well white flat-bottom plates and incubated with indicated serially diluted test articles for one (1) hour on ice to allow binding of antibody or ADC to cells. Cells were then opsonized with baby rabbit complement for 1 hour at 37°C, and remaining cell viability following cell lysis due to CDC activity was determined by CellTiter-Glo 2.0 Assay (Promega). The results show lack of any significant CDC activity for Ab5-ADC2 as well as enfortumab. (See, Figure(s) 15)).
  • Example 18 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Analysis of Ab5-ADC2 and Ab5.
  • ADCC Antibody-Dependent Cell-Mediated Cytotoxicity
  • Example 19 Antibody-Dependent Cell-Mediated Phagocytosis (ADCP) Analysis of Ab5-ADC2 and Ab5.
  • ADCP Antibody-Dependent Cell-Mediated Phagocytosis
  • tumor cells were co-incubated with engineered effector cells (6:1 to 7.5:1 ratio of effector to target cells) in the presence of indicated antibody or ADC test articles for 6 hours, and ADCP activity was measured using commercial Fc ⁇ RI ADCP bioassay kit (Promega).
  • Assay control reagent isoCTRL Ab in RAMOS cells expressing the target of the isoCTRL Ab
  • weak ADCP activity was observed for Ab5-ADC2 in the NECTIN-4 expressing SUM190PT and HT-1376 cancer cell lines. (See, Figure(s) 17)).
  • Example 20 Pharmacokinetics Profile of ADC and Free Payload of Ab5-ADC2 in Cynomolgus Monkeys.
  • the pharmacokinetics profile of Ab5-ADC2 and its corresponding free payload was determined using the following protocols. Briefly, Cynomolgus monkeys were dosed with ADC test article Ab5-ADC2 at different doses levels for 2 doses. Blood was collected at different timepoints and processed into serum for total antibody and ADC quantitation, and plasma for free payload analysis.
  • Enzyme-linked immunosorbent assay was used to quantitate the total antibody and ADC concentrations by capturing with soluble recombinant NECTIN-4 and detecting with either biotin-labelled anti-human IgG, or biotin-labelled anti-payload, respectively, followed by binding of “Streptavidin-HRP”. TMB was used as the colorimetric substrate, and the color reaction was stopped by H2SO4 solution. The absorbance was measured at 450 nm-630 nm on a plate reader. The calibration curve was generated by plotting the response (the mean absorbance of each calibration curve sample minus the mean absorbance value of blank) of serially diluted test article against the calibration sample concentrations and fitting by a four-parameter logistic model.
  • Example 21 Efficacy of NECTIN-4 ADC Compared to Enfortumab Vedotin in a Patient Derived Cervical Cancer Model PDX36 In Vivo. Further In vivo efficacy of a NECTIN-4 ADC (Ab5-ADC2) compared to commercial Enfortumab Vedotin was performed using the following protocols.
  • PDX in this instance PDX36
  • Tumor fragments (2-3mm in diameter) from stock mice were harvested and used for subcutaneous inoculation into female NOD/SCID mice.
  • mean tumor size reached approximately 150 mm 3
  • mice were randomized into groups, 5 mice per group.
  • Test articles were dosed at 2.5, 5 or 10mg/kg.
  • Ab5-ADC2 dark circles
  • Enfortumab Vedotin dark triangles
  • Example 22 Efficacy of NECTIN-4 ADC in Multiple Patient Derived Cervical Cancer Models (PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) In Vivo. Further In vivo efficacy of NECTIN-4 ADC (Ab5-ADC2) was performed in multiple cervical cancer model(s) using the following protocols. Briefly, PDX (in this instance PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) derived from patients with cervical cancer were propagated in vivo in immunocompromised mice.
  • tissue samples were fixed in 10% buffered neutral formalin, processed, and embedded into paraffin wax, then prepared as 4 ⁇ m tissue sections. After deparaffinization and re-hydration, sections were treated for antigen retrieval. The tissue sections after antigen retrieval were then incubated with mouse anti-nectin-4 primary antibody or IgG (antibody control). The tissue sections bound with primary antibodies were washed and detected with an enzyme-labeled, secondary antibody. The bound secondary antibody was color-developed, using the Leica Bond Refine Polymer Detection system. The stained tissue sections were then scanned, imaged, and evaluated with light microscopy, using an Aperio ScanScope CS (Aperio; Vista, CA).
  • Aperio ScanScope CS Aperio; Vista, CA
  • the PDX12 (21(A) and 21(B)), PDX10 (21(C) and 21(D)), PDX16 (21(E) and 21(F)), PDX13 (21(G) and 21(H)), PDX34 (21(I) and 21(J)), and PDX36 (21(K) and 21(L)) are shown in the respective figures with images shown at high resolution (21(A), (C), (E), (G), (I) and (K)), as well as at low resolution (21(B), (D), (F), (H), (J) and (L)).
  • Example 24 Free Payload Concentration Analysis of Ab5-ADC2 or Enfortumab Vedotin via LC- MS/MS. Free payload concentration of Ab5-ADC2 or Enfortumab Vedotin in multiple tissues of NECTIN- 4 positive breast cancer tumor bearing mice was assessed using the following protocols. Briefly, a Sum190PT inflammatory breast cancer xenograft mouse model was dosed with either Ab5-ADC2 or Enfortumab Vedotin.
  • Example 25 Stability and DAR Retention Analysis Ab5-ADC2. Stability assessment and DAR retention analysis were performed using the following protocols.
  • Sprague Dawley rats 10-12 weeks old upon arrival, were acclimated for one week and then given a single intravenous dose of Ab5-ADC2 or Enfortumab Vedotin.
  • Blood samples were collected post-dose at various timepoints: 1 hour, 4 hours, day 3, 7, 14 and 21 days.
  • Blood collected in EDTA coated tubes was processed to plasma by centrifuging at 10,000g for 10 minutes at 4°C. Plasma was aliquoted and stored frozen at -80°C until analysis. Plasma samples were diluted in TBS then combined with biotin-labelled capture anti-human antibody coated beads, followed by mixing with streptavidin coated DynaBeads (Invitrogen) and incubated for about 2 hours.
  • NECTIN-4 antibodies and NECTIN-4 ADCs are synthesized in accordance with the present invention which specifically accumulate in a tumor cell and are used in the treatment of certain tumors and other immunological disorders and/or other diseases (See, Table I). In connection with each of these indications, two clinical approaches are successfully pursued. I.) Adjunctive therapy: In adjunctive therapy, patients are treated with NECTIN-4 antibodies and NECTIN-4 ADCs in combination with a chemotherapeutic or pharmaceutical or biopharmaceutical agent or a combination thereof. Primary cancer targets are treated under standard protocols by the addition of NECTIN-4 antibodies and NECTIN-4 ADCs.
  • Protocol designs address effectiveness as assessed by the following examples, including but not limited to, reduction in tumor mass of primary or metastatic lesions, increased progression free survival, overall survival, improvement of patient’s health, disease stabilization, as well as the ability to reduce usual doses of standard chemotherapy and other biologic agents. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic or biologic agent.
  • Monotherapy In connection with the use of the NECTIN-4 antibodies and NECTIN-4 ADCs in monotherapy of tumors, the NECTIN-4 antibodies and NECTIN-4 ADCs are administered to patients without a chemotherapeutic or pharmaceutical or biological agent. In one embodiment, monotherapy is conducted clinically in end-stage cancer patients with extensive metastatic disease.
  • Dosage Unit Form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the NECTIN-4 antibodie(s) and NECTIN-4 ADC, the individual mechanics of the irradiation mechanism (reactor) and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an compound for the treatment of sensitivity in individuals.
  • CDP Clinical Development Plan
  • the CDP follows and develops treatments of cancer(s) and/or immunological disorders (See, Table I) using NECTIN-4 antibodies and NECTIN-4 ADCs of the disclosure.
  • Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses.
  • Trials are open label comparing standard chemotherapy with standard therapy plus NECTIN-4 antibodie(s) and NECTIN-4 ADCs.
  • concentration of NECTIN-4 antibodie(s) and NECTIN-4 ADCs in a tumor as determined by standard detection methods known in the art.
  • the nucleotide sequence encoding the variable region is underlined. 1 – CAGGTCCAGTTAGTTCAATCAGGTGCCGAGGTCAAAAAGCCAGGTTCTTCCGTCAAAGTG – 60 1 - Q V Q L V Q S G A E V K K P G S S V K V - 20 61 – TCATGCAAGGCTAGCGGTGGCACCTTTTCTTCTTACGCTTTTTCTTGGGTGAGACAGGCA – 120 21 - S C K A S G G T F S S W V R Q A - 40 121 – – 180 41 - P G Q G L E W M G R F G T T N Y - 60 181 – – 240 61 - A Q K F Q G R V T I S T S T A Y - 80 241 – ATGGAATTAAGTTCTCTGAGATCCGAGGACACC
  • the nucleotide sequence encoding the variable region is underlined. 1 - CAGGTTCAGTTAGTCGAGTCTGGTGGCGGTGTCGTCCAGCCTGGTAGATCCTTAAGGCTG - 60 1 - Q V Q L V E S G G G V V Q P G R S L R L - 20 61 - TCATGTGCCGCTAGCGGATTTACCTTTGGTGGTTACGGTATGCATTGGGTGAGACAAGCC - 120 21 - S C A A S G F T F G G G Y G M H W V R Q A - 40 121 - CCCGGTAAAGGCCTGGAATGGGTCGCTGTGATAAGTTACTCTGGTTCTAACAAATACTAC - 180 661 - GAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCTGCCCTGCCCCTGAACTGCTG - 720 221 -
  • the nucleotide sequence encoding the variable region is underlined.
  • the nucleotide sequence encoding the variable region is underlined. 1 - GAGGTTCAGTTAGTCGAGAGCGGGGGGGGTTTAGTTAAGCCAGGTGGCAGTTTAAGACTG - 60 1 - E V Q L V E S G G G L V K P G G S L R L - 20 61 - TCTTGTGCCGCTTCCGGTTTTACCTTTTCTTCTGCTTGGATGTCTTGGGTGAGACAGGCA - 120 21 - S C A A S G F T F S S A W M S W V R Q A - 40 121 - CCTGGTAAAGGCCTGGAATGGGTTGGAAGGATAAAATCTGAAACCTACGGAGGGACTACA - 180 S E T Y G G T T - 60 - 240 S R D D S K N T - 80 241 - TTGTACTTACAGATGAACT
  • the nucleotide sequence encoding the variable region is underlined. 1 - GAGGTTCAGTTGTTAGAGAGCGGGGGGGGTCTGGTTCAGCCTGGTGGCAGCTTAAGACTG - 60 1 - E V Q L L E S G G G L V Q P G G S L R L - 20 61 - AGTTGTGCCGCTTCTGGTTTTACTTTCTCTAATTATGCAATGTCCTGGGTGAGGCAAGCC - 120 241 - TTACAGATGAACTCTTTGAGAGCTGAGGACACCGCAGTGTATTACTGTGCTAGAGGTTTT - 300 81 - L Q M N S L R A E D T A V Y Y C A R G F - 100 301 - TCTAGATTTGATCATTGGGGACAAGGAACTTTGGTCACAGTGTCATCTGCCAGCACTAAA - 360 101 - S
  • the nucleotide sequence encoding the variable region is underlined. 1 - GAAATCGTCTTGACCCAGTCCCCAGGTACTCTGTCTTTAAGTCCCGGGGAAAGAGCTACT - 60 1 - E I V L T Q S P G T L S L S P G E R A T - 20 61 - CTGTCCTGTAGGGCAAGTCAGTCTGTTTCTTCTAATTATCTGGCCTGGTATCAGCAAAAA - 120 21 - L S C R A S Q S V S S N Y L A W Y Q Q K - 40 121 - CCCGGTCAAGCTCCAAGACTGTTGATCTATGGTGCAAGCAGTAGAGCCACCGGAATCCCT - 180 41 - P G Q A P R L L I Y G A S S R A T G I P - 60 181 - GATAGGTTTTCCGGTTC
  • the nucleotide sequence encoding the variable region is underlined. 1 - CAGTCCGTGTTGACCCAGCCTCCATCAGTCTCAGGAGCCCCAGGCCAGAGAGTGACCATT - 60 1 - Q S V L T Q P P S V S G A P G Q R V T I - 20 61 - TCTTGTACTGGATCTTCCTCAAATATCGGGGCCGGTTACGATGTTCATTGGTATCAGCAA - 120 21 - S C T G S S S N I G A G Y D V H W Y Q Q - 40 121 - CTGCCCGGTACAGCTCCAAAACTGTTGATCTATGATAATAGAAACAGACCTAGCGGTGTGTG - 180 41 - L P G T A P K L L I Y D N R N R P S G V - 60 181 - CCCGACAGGTTCTCCG
  • the nucleotide sequence encoding the variable region is underlined. 1 - GACATACAAATGACCCAGTCTCCTTCTTCTCTGAGCGCATCTGTCGGCGATAGAGTGACT - 60 S L S A S V G D R V T - 20 - 120 S R Y L N W Y Q Q K P - 40 121 - GGCAAGGCTCCAAAACTGTTGATCAATGCTGCTTCAAGCCTGCAATCCGGAGTTCCCTCA - 180 41 - G K A P K L L L I N A A S S L Q S G V P S - 60 181 - AGATTTTCTGGTTCCGGCTCAGGAACTGATTTCACCCTGACTATAAGTTCTTTGCAGCCT - 240 61 - R F S G S G T D F T L T I S S L Q P - 80 241 - GAAGACTTTGC
  • the nucleotide sequence encoding the variable region is underlined. 1 - GACATACAAATGACCCAGTCTCCTTCTTCTCTGAGCGCATCTGTCGGCGATAGAGTGACT - 60 S L S A S V G D R V T - 20 - 120 Y T Y L N W Y Q Q K P - 40 121 - GGCAAGGCTCCAAAACTGTTGATCTATGCTGCTTCAAGCCTGCAATCCGGAGTTCCCTCA - 180 41 - G K A P K L L L I Y A A S S L Q S G V P S - 60 181 - AGATTTTCTGGTTCCGGCTCAGGAACTGATTTCACCCTGACTATAAGTTCTTTGCAGCCT - 240 61 - R F S G S G T D F T L T I S S L Q P - 80 241 - GAAGACT
  • the nucleotide sequence encoding the variable region is underlined. 1 - GAAATCGTCTTGACCCAGTCCCCAGGTACTCTGTCTTTAAGTCCCGGGGAAAGAGCTACT - 60 1 - E I V L T Q S P G T L S L S P G E R A T - 20 61 - CTGTCCTGTAGGGCAACTCAGTCTGTTTCTGGTTCTTATCTGGCCTGGTATCAGCAAAAA - 120 21 - L S C R A T Q S V S G S Y L A W Y Q Q K - 40 121 - CCCGGTCAAGCTCCAAGACTGTTGATCTATGGTGCAAGCAGTAGAGCCACCGGAATCCCT - 180 41 - P G Q A P R L L I Y G A S S R A T G I P - 60 181 - GATAGGTTTTCCGGTTC
  • Amino Acid Sequences of Antibody Heavy Chains Table VII(A). Amino acid sequence (SEQ ID NO: 28) of CL.Z heavy chain. The variable region is underlined, and the Kabat CDR regions are boxed.
  • QVQLVESGGGVVQPGRSLRLSCAASGFSFSAYGMHWVRQAPGKGLEWVAVISYDGSYKYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVGAFPWVFFDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • Amino Acid Sequences of Antibody Light Chains Table VII(G). Amino acid sequence (SEQ ID NO: 34) of CL.Z light chain. The variable region is underlined, and the Kabat CDR regions are boxed. DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGYSTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Table VII(H).
  • the variable region is underlined, and the Kabat CDR regions are boxed.
  • variable region is underlined, and the Kabat CDR regions are boxed.

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Abstract

L'invention concerne des conjugués anticorps-médicament (ADC) qui se lient à la/aux protéine(s) de nectine-4 et des variants associés. La nectine-4 présente une expression spécifique au tissu dans un tissu adulte normal, et est exprimée de manière aberrante dans les cancers listés dans le tableau I. Par conséquent, les ADC de l'invention fournissent une composition thérapeutique pour le traitement du cancer.
PCT/US2024/033036 2023-06-13 2024-06-07 Méthodes et compositions associées à des anticorps et conjugués anticorps-médicament (adcs) qui se lient à des protéines de nectine-4 Pending WO2024258743A1 (fr)

Priority Applications (3)

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KR1020267000884A KR20260026054A (ko) 2023-06-13 2024-06-07 넥틴-4 단백질에 결합하는 항체 및 항체 약물 접합체 (adc)와 관련된 방법 및 조성물
AU2024304180A AU2024304180A1 (en) 2023-06-13 2024-06-07 Methods and compositions related to antibodies and antibody drug conjugates (adcs) that bind nectin-4 proteins
CN202480040082.4A CN121620528A (zh) 2023-06-13 2024-06-07 与结合至nectin-4蛋白的抗体和抗体药物缀合物(adc)相关的方法和组合物

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