WO2021257525A1 - Conditionally active anti-nectin-4 antibodies - Google Patents

Conditionally active anti-nectin-4 antibodies Download PDF

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Publication number
WO2021257525A1
WO2021257525A1 PCT/US2021/037364 US2021037364W WO2021257525A1 WO 2021257525 A1 WO2021257525 A1 WO 2021257525A1 US 2021037364 W US2021037364 W US 2021037364W WO 2021257525 A1 WO2021257525 A1 WO 2021257525A1
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Prior art keywords
seq
antibody
nos
nectin
sequence
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PCT/US2021/037364
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English (en)
French (fr)
Inventor
Jay M. Short
Gerhard Frey
Hwai Wen Chang
Jing Wang
Chao XING
Haizhen LIU
Ana Paula CUGNETTI
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Bioatla Inc
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Bioatla Inc
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Priority to IL298903A priority Critical patent/IL298903A/en
Priority to CN202180043514.3A priority patent/CN115768798A/zh
Priority to EP21826366.3A priority patent/EP4168453A4/en
Priority to JP2022577477A priority patent/JP2023531185A/ja
Priority to US18/002,064 priority patent/US20230235054A1/en
Priority to AU2021293183A priority patent/AU2021293183A1/en
Priority to MX2022016192A priority patent/MX2022016192A/es
Priority to KR1020237002055A priority patent/KR20230038711A/ko
Priority to CA3182395A priority patent/CA3182395A1/en
Publication of WO2021257525A1 publication Critical patent/WO2021257525A1/en
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
    • C07K16/2809—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 against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K47/6801—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
    • 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
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • 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
    • C07K16/2803—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This disclosure relates to anti-Nectin-4 antibodies, anti-Nectin-4 antibody fragments, anti-Nectin-4 multi-specific antibodies, immunoconjugates of such antibodies and antibody fragments and uses of these antibodies, antibody fragments, multi-specific antibodies, and immunoconjugates in pharmaceutical compositions as well as diagnostic and therapeutic methods.
  • Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which comprises four members. Nectins are cell adhesion molecules that play a key role in various biological processes such as polarity, proliferation, differentiation and migration for epithelial, endothelial, immune and neuronal cells, during development and adult life. Nectins are involved in several pathological processes in humans. Nectins are the main receptors for polio, herpes simplex and measles viruses. Mutations in the genes encoding Nectin- 1 (PVRL1) or Nectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated with other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues its expression is more restricted than that of other members of the family.
  • Nectin-4 is a tumor-associated antigen in 30%, 49%, and 86% of breast, ovarian and lung carcinomas, respectively. Nectin-4 is frequently associated with aggressive tumors. In breast tumors, Nectin-4 is expressed mainly in triple-negative carcinomas. In the serum of patients with these cancers, the detection of soluble forms of Nectin-4 is associated with a poor prognosis. Levels of serum Nectin-4 increase during metastatic progression and decrease after treatment. These results suggest that Nectin-4 could be a reliable target for the treatment of cancer.
  • Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and is currently in clinical investigation for the treatment of patients suffering from solid tumors.
  • ADC antibody-drug conjugate
  • the present invention aims at providing anti-Nectin-4 antibodies or antibody fragments with reduced or minimal side effects suitable for therapeutic and diagnostic use, especially for diagnosis and treatment of cancers.
  • Some of these anti-Nectin-4 antibodies or antibody fragments may have a higher binding or binding affinity to Nectin-4 in a tumor microenvironment in comparison with the binding or binding affinity to Nectin-4 present in a non-tumor microenvironment.
  • These anti-Nectin-4 antibodies or antibody fragments typically have at least comparable efficacy to known anti-Nectin-4 antibodies.
  • the present anti-Nectin-4 antibodies or antibody fragments may exhibit reduced side effects in comparison with monoclonal anti-Nectin-4 antibodies known in the art for having a relatively low binding affinity to Nectin-4 in normal tissues such as a non-tumor microenvironment.
  • These advantages may provide a more selective targeting of the Nectin-4 expressed in a tumor and may permit use of higher dosages of these anti-Nectin-4 antibodies or antibody fragments as a result of the selectivity of the antibodies for Nectin-4 present in a tumor microenvironment, whereby more effective therapeutic treatments can be realized without a corresponding increase in undesirable side effects.
  • the present invention provides an isolated polypeptide that specifically binds to Nectin-4.
  • the polypeptide comprises a heavy chain variable region including three complementarity determining regions (CDRs) having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • CDRs complementarity determining regions
  • the present invention provides isolated polypeptides comprising a heavy chain variable region and a light chain variable region that specifically bind to Nectin- 4, or especially human Nectin-4 protein, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 ASQGISGWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX
  • the H1 sequence may be selected from GFTFSSYNMN (SEQ ID NO: 7), and GFTFSSYNDN (SEQ ID NO: 8).
  • the H3 sequence may be selected from AYYYGMDV (SEQ ID NO: 9), AYYYGDDV (SEQ ID NO: 10), and AYYYGMDK (SEQ ID NO: 11).
  • the L1 sequence may be selected from RASQGISGWLA (SEQ ID NO: 12), RASQGISGWEA (SEQ ID NO: 13), and HASQGISGWLA (SEQ ID NO: 14).
  • the L3 sequence may be selected from QQANSFPPT (SEQ ID NO: 15), QQANSEPPT (SEQ ID NO: 16), and QQANSFPDT (SEQ ID NO: 17).
  • the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NOS: 18-30.
  • the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NOS: 31-43.
  • isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NOS: 19 and 32, SEQ ID NOS: 20 and 33, SEQ ID NOS: 21 and 34, SEQ ID NOS: 22 and 35, SEQ ID NOS: 23 and 36, SEQ ID NOS: 24 and 37, SEQ ID NOS: 25 and 38, SEQ ID NOS: 26 and 39, SEQ ID NOS: 27 and 40, SEQ ID NOS: 28 and 41 and SEQ ID NOS: 29 and 42.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: SEQ ID NOS: 19 and 32, SEQ ID NOS: 20 and 33, SEQ ID NOS: 21 and 34, SEQ ID NOS: 22 and 35, SEQ ID NOS: 23 and 36, SEQ ID NOS: 24 and 37, SEQ ID NOS: 25 and 38, SEQ ID NOS: 26 and 39, SEQ ID NOS: 27 and 40, SEQ ID NOS: 28 and 41 and SEQ ID NOS: 29 and 42, respectively; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptide may be an antibody or antibody fragment that specifically binds to Nectin-4, or especially human Nectin-4 protein.
  • the isolated polypeptide is multi-specific and specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3 and the isolated polypeptide comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 AS QGIS GWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANS X 6 P X 7 T (SEQ ID NO: 6), wherein X 4 is R
  • the L6 sequence is selected from any one of SEQ ID NOs: 50-53, and the L7 sequence is selected from SEQ ID NOs: 54 and 55.
  • [0018] comprises a heavy chain variable region that includes three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, the L2 sequence is SEQ ID NO: 5, the L3 sequence is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 wherein: the L4 sequence is GFTFNTY AMN (SEQ ID NO: 44), the L5 sequence is RIRS KYNNY ATYY AD S VKD (SEQ ID NO: 45
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • RS STGAVTTSNYDN (SEQ ID NO: 55)
  • the L8 sequence is GTNKRAP (SEQ ID NO: 48)
  • the L9 sequence is ALWYSNLWV (SEQ ID NO: 49).
  • the isolated polypeptide with nine CDRs of paragraphs [0017]-[0019] comprises a heavy chain variable region that includes three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is SEQ ID NO: 7, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is selected from SEQ ID NO: 12 and SEQ ID NO: 13, the L2 sequence is SEQ ID NO: 5, the L3 sequence is SEQ ID NO: 15, and six anti-CD3 complementarity determining regions
  • L4 sequence is GFTFNTY AMN (SEQ ID NO: 44)
  • the L5 sequence is RIRS KYNNYATYYADS VKD (SEQ ID NO: 45)
  • the L6 sequence is selected from HGNFGNSYVSWFAY (SEQ ID NO: 50)
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NOS: 18, 25, 27, and 29.
  • the isolated polypeptide may comprise a light chain variable region having a sequence selected from SEQ ID NOS: 56-60, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • the isolated polypeptide may comprise a heavy chain variable region having a sequence selected from SEQ ID NOs: 18, 25, 27, and 29, and a light chain variable region having a sequence selected from SEQ ID NOs: 56-60, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • the isolated polypeptide may comprise a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NO: 25 and SEQ ID NO: 57, SEQ ID NO: 27 and SEQ ID NO: 58, SEQ ID NO: 29 and SEQ ID NO: 59, and SEQ ID NO: 29 and SEQ ID NO: 60.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a combination of amino acid sequences selected from one of SEQ ID NOS: 18, 25, 27, and 29 in combination with one of SEQ ID NOs: 56-60, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination; and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: SEQ ID NOS: 25 and 57, SEQ ID NOS: 27 and 58, SEQ ID NOS: 29 and 59, SEQ ID NOS: 29 and 60; and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptide may be a multispecific antibody or antibody fragment that specifically binds to Nectin-4, especially human Nectin-4 protein.
  • the isolated polypeptide may be a bispecific antibody or antibody fragment that specifically binds to Nectin-4 and CD3, especially human Nectin-4 protein and CD3.
  • the isolated polypeptide or antibody or antibody fragment may have a higher binding affinity to Nectin-4 protein, especially human Nectin-4 protein, at a value of a condition in a tumor microenvironment in comparison with a different value of the same condition that occurs in a non-tumor microenvironment.
  • the condition is pH.
  • the isolated polypeptide or antibody or antibody fragment may have at least 70% of the antigen binding activity at pH 6.0 as compared to the same antigen binding activity of the parent polypeptide, antibody or antibody fragment at pH 6.0, and the polypeptide or antibody or antibody fragment may have less than 50%, or less than 40%, or less than 30%, or less than 20% or less than 10% of the antigen binding activity at pH 7.4 as compared to the same antigen binding activity of the parent polypeptide or the isolated polypeptide or antibody or antibody fragment at pH 7.4.
  • the antigen binding activity may be binding to Nectin-4 protein.
  • the isolated polypeptide, antibody or antibody fragment may have a higher binding affinity to Nectin-4 protein, especially human Nectin-4 protein, at a pH in a tumor microenvironment in comparison with a pH that occurs in a non-tumor microenvironment.
  • the pH in the tumor microenvironment may range of from 5.0 to 6.8 and the pH in the non-tumor microenvironment may range from 7.0 to 7.6.
  • the antigen binding activity of the isolated polypeptide or antibody or antibody fragment may be measured by an ELISA assay.
  • the present invention provides an immunoconjugate that includes any of the antibodies or antibody fragments of the invention described above.
  • the antibody or antibody fragment may be conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent and a cytotoxic agent.
  • the present invention provides a pharmaceutical composition that includes any of the isolated polypeptides, the antibodies or antibody fragments, or the immunoconjugates of the invention described above, together with a pharmaceutically acceptable carrier.
  • a single dose of the pharmaceutical composition of may include an amount of the isolated polypeptide, the antibody, the antibody fragment, or the immunoconjugate of about 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg,
  • a single dose of the pharmaceutical composition of may include an amount of the isolated polypeptide, the antibody or antibody fragment, or the immunoconjugate in a range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.
  • Each of the foregoing pharmaceutical compositions may further include an immune checkpoint inhibitor molecule.
  • the immune checkpoint inhibitor molecule may be an antibody or antibody fragment against an immune checkpoint.
  • the immune checkpoint may be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, CTLA4, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS or the immune checkpoint may be CTLA4, PD-1 or PD-L1.
  • Each of the foregoing pharmaceutical compositions may further include an antibody or antibody fragment against an antigen selected from PD1, PD-L1, CTLA4, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4 and a WNT protein.
  • the WNT protein may be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 and WNT16.
  • the present invention provides a kit for diagnosis or treatment including any of the isolated polypeptides, the antibodies or antibody fragments, the immunoconjugates, or the pharmaceutical compositions of the present invention described above.
  • FIG. 2 shows the binding activities of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to human Nectin-4 at pH 7.4 as measured by ELISA.
  • FIG. 3 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to human Nectin-4 at pH 6.0 as measured by ELISA.
  • Fig. 4 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to human Nectin-4 at pH 7.4 as measured by ELISA.
  • FIG. 6 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to cyno Nectin-4 at pH 7.4 as measured by ELISA.
  • FIG. 7 shows the binding activity of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to human Nectin-4 under pH range titration as measured by ELISA.
  • Fig 8 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to human Nectin-4 under pH range titration as measured by ELISA.
  • FIG. 9 shows the binding activity of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express human Nectin-4 at pH 6.0 as measured by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • FIG. 10 shows the binding activity exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express human Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 11 shows the binding activity of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express cyno Nectin-4 at pH 6.0 as measured by FACS.
  • FIG. 12 shows the binding activity of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC HEK293 cells that express cyno Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 13 shows the binding activity of exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to T47D cells that express human Nectin-4 at pH 6.0 as measured by FACS.
  • FIG. 14 shows the binding activity exemplary conditionally active anti-Nectin-4 CAB ADCs of the present invention and the WT ADC to T47D cells that express human Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 15 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express human Nectin-4 at pH 6.0 as measured by FACS.
  • FIG. 16 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express human Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 17 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express cyno Nectin-4 at pH 6.0 as measured by FACS.
  • FIG. 18 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to HEK293 cells that express cyno Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 19 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to T47D cells that express human Nectin-4 at pH 6.0 as measured by FACS.
  • FIG. 20 shows the binding activity of further exemplary conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC to T47D cells that express human Nectin-4 at pH 7.4 as measured by FACS.
  • FIG. 21 shows the cell killing activity of conditionally active anti -Nectin-4 antibody BAP143-00-01 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 22 shows the cell killing activity of conditionally active anti-Nectin-4 antibody BAP143-00-02 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 23 shows the cell killing activity of conditionally active anti-Nectin-4 antibody BAP143-00-03 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 24 shows the cell killing activity of conditionally active anti-Nectin-4 antibody BAP143-00-04 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 25 shows the cell killing activity of conditionally active anti-Nectin-4 antibody BAP143-00-05 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 26 shows the cell killing activity of conditionally active anti-Nectin-4 antibody BAP143-00-06 conditionally active anti-Nectin-4 CAB ADC and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0 and pH 7.4.
  • FIG. 27 shows the cell killing activity of representative conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 6.0.
  • FIG. 28 shows the cell killing activity of representative conditionally active anti- Nectin-4 CAB ADCs of the present invention and the WT ADC against HEK293 cells expressing human Nectin-4 at pH 7.4.
  • FIG. 29 shows the effect on tumor volumes of XXT47D xenograft mice of treatment with representative CAB ADCs of the present invention and the WT ADC of the present invention.
  • FIG. 30 shows protein sequences of the heavy and light chain variable regions of representative conditionally active anti-Nectin-4 antibodies of the present invention and the heavy and light chain variable regions of the benchmark wild type antibody.
  • FIG. 31 shows Nectin-4 in tumor tissues and downstream pathways. See Sethy C. et al., J. Cancer Res. Clin. Oncol., 146(1): 245-259 (2020).
  • FIGS. 32A-32C show the higher binding activity of CAB Nectin-4 x CAB CD3 affinity in a tumor microenvironment pH in comparison to physiological pH as measured by ELISA (FIG. 32A), the differential binding affinity of CAB Nectin-4 x CAB CD3 and WT Nectin-4 x WT CD3 in pH range 6.0-7.4 (FIG. 32B), and the in vivo efficacy of CAB Nectin4 x CAB CD3 in comparison to Isotype x WT CD3 and WT Nectin-6 x WT CD3 (FIG. 32C).
  • FIGS. 32A-32C show the higher binding activity of CAB Nectin-4 x CAB CD3 affinity in a tumor microenvironment pH in comparison to physiological pH as measured by ELISA (FIG. 32A), the differential binding affinity of CAB Nectin-4 x CAB CD3 and WT Nectin-4 x WT CD3 in pH range 6.0-7.4
  • 33A-33B show protein sequences of the heavy and light chain variable regions of representative conditionally active Nectin-4 x CD3 bispecific antibodies of the present invention and the heavy and light chain variable regions of the wild type antibody.
  • the heavy and light chains of the antibodies are: BA-150-19-01-01-BF1-VH (SEQ ID NO: 18), BA- 150-30-33-16-BF11-VH (SEQ ID NO: 25), BA-150-30-33-16-BF19-VH (SEQ ID NO: 27), BA- 150-30-03-12-BF 11 -VH (SEQ ID NO: 29) and BA-150-30-03-12-BF19-VH (SEQ ID NO: 29).
  • BA- 150-19-01-01 -BF 1 -LC (SEQ ID NO: 56), BA-150-30-33-16-BF11-LC (SEQ ID NO: 57), BA-150-30-33-16-BF19-LC (SEQ ID NO: 58), BA-150-30-03-12-BF11-LC (SEQ ID NO: 59), and BA-150-30-03-12-BF19-LC (SEQ ID NO: 60).
  • the term "about” as used herein refers to the normal variation in that measured quantity that would be expected by a skilled person making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated, “about” refers to a variation of +/- 10% of the value provided.
  • affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • affinity matured antibody refers to an antibody with one or more alterations in one or more heavy chain or light chain variable regions, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • amino acid refers to any organic compound that contains an amino group (— NH2) and a carboxyl group (— COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds.
  • the "twenty naturally encoded polypeptide-forming alpha-amino acids” are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or
  • antibody refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen.
  • antibody fragments which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., Harlow and Lane, supra), and are described further, as follows.
  • Antibodies can be used to isolate preparative quantities of the antigen by immunoaffmity chromatography.
  • neoplasia a disease that causes neoplasia.
  • autoimmune disease a disease that causes neoplasia
  • AIDS a malignant neoplasia
  • cardiovascular disease a malignant neoplasia
  • Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.
  • An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a (Fab')2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.
  • An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • anti-Nectin-4 antibody refers to an antibody that is capable of binding Nectin-4 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Nectin-4.
  • the extent of binding of an anti-Nectin-4 antibody to an unrelated, non-Nectin-4 protein is less than about 10% of the binding of an antibody to Nectin-4 as measured, e.g. by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an antibody that binds to Nectin-4 has a dissociation constant nM (e.g.
  • an anti-Nectin-4 antibody binds to an epitope of Nectin-4 that is conserved among Nectin-4 from different species, for example, the extracellular domain of Nectin-4.
  • Nectin-4 has its general meaning in the art and includes human Nectin-4, in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of human Nectin-4.
  • the amino acid sequence for native Nectin-4 includes the NCBI Reference Sequence: NP_112178.2.
  • binding refers to interaction of the variable region or an Fv of an antibody with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody variable region or Fv recognizes and binds to a specific protein structure rather than to proteins generally.
  • the term “specifically binding” or “binding specifically” means that an antibody variable region or Fv binds to or associates with more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen than with other proteins.
  • an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens.
  • an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity than it does to related proteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans).
  • polyreactive natural antibodies i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans.
  • selective binding does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term “selective binding”.
  • binding of an antibody variable region or Fv binds to an antigen
  • an antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, such as 50nM or less, for example 20nM or less, such as, 15nM or less, or 10 nM or less ,or 5nM or less, 2 nM or less, or 1 nM or less.
  • KD equilibrium constant
  • cancer and “cancerous” as used herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
  • cell proliferative disorder and “proliferative disorder” as used herein refer to disorders that are associated with some degree of abnormal cell proliferation.
  • the cell proliferative disorder is cancer.
  • chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue)
  • razoxane rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such
  • celecoxib or etoricoxib proteosome inhibitor
  • proteosome inhibitor e.g. PS341
  • bortezomib VELCADE®
  • CCI-779 tipifamib (R11577); orafenib, ABT510
  • Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®)
  • pixantrone EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine
  • Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics,” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti -estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitor
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • conditionally active antibody refers to an anti-Nectin-4 antibody which is more active under a condition in the tumor microenvironment compared to under a condition in the non-tumor microenvironment.
  • the conditions in the tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, hypoxia, lower concentration of glucose, and slightly higher temperature in comparison with non- tumor microenvironment.
  • a conditionally active antibody is virtually inactive at normal body temperature but is active at a higher temperature in a tumor microenvironment.
  • the conditionally active antibody is less active in normal oxygenated blood, but more active under a less oxygenated environment exists in tumor.
  • conditionally active antibody is less active in normal physiological pH 7.0-7.6, but more active under an acidic pH 5.0-6.8, or 6.0-6.8 that exists in a tumor microenvironment.
  • condition in the tumor microenvironment know to a person skilled in the field may also be used as the condition in the present invention under which the anti-Nectin-4 antibodies to have different binding affinity to Nectin-4.
  • cytostatic agent refers to a compound or composition which arrests growth of a cell either in vitro or in vivo.
  • a cytostatic agent may be one which significantly reduces the percentage of cells in S phase.
  • Further examples of cytostatic agents include agents that block cell cycle progression by inducing G0/G1 arrest or M-phase arrest.
  • the humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0/G1 arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Certain agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5 -fluoro uracil, and ara-C.
  • Taxanes are anticancer drugs both derived from the yew tree.
  • Docetaxel (TAXOTERE®, Rhone- Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial
  • 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 ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • detectably label refers to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, is indicative of the presence of an antigen in a sample.
  • useful detectable labels include, but are not limited to the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scatter, phosphorescence, or luminescence properties; molecules or ions detectable by their radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties.
  • the term "diagnostics” as used herein refers to determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e. g., identification of pre- metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy), and therametrics (e. g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).
  • the diagnostic method of this invention is particularly useful in detecting early stage cancers.
  • diagnostic agent refers to a molecule which can be directly or indirectly detected and is used for diagnostic purposes.
  • the diagnostic agent may be administered to a subject or a sample.
  • the diagnostic agent can be provided per se or may be conjugated to a vehicle such as a conditionally active antibody.
  • effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • an agent as used herein refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Fc region is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
  • the term “framework” or “FR” as used herein refers to variable domain residues other than complementarity determining regions (CDRs or H1 -3 in the heavy chain and L1 -3 in the light chain) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in V H (or V L ): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • full length antibody refers to an antibody which comprises an antigen-binding variable region (V H or V L ) as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different “classes”.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • function-conservative variants refers a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • a “function- conservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • human antibody as used herein is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • immunoconjugate is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • the term “individual” or “subject” as used herein refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
  • the term “inhibiting cell growth or proliferation” as used herein means decreasing a cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.
  • isolated antibody as used herein is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase High Performance Liquid Chromatography (HPLC)).
  • electrophoretic e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase High Performance Liquid Chromatography (HPLC)
  • isolated nucleic acid encoding an anti-Nectin-4 antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • metalastasis refers to all Nectin-4-involving processes that support cancer cells to disperse from a primary tumor, penetrate into lymphatic and/or blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasis) in normal tissues elsewhere in the body.
  • tumor cells refers to cellular events of tumor cells such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underlie metastasis and are stimulated or mediated by Nectin-4.
  • microenvironment means any portion or region of a tissue, organ or body that has constant or temporal, physical or chemical differences from other regions of the tissue, organ or regions of the body.
  • tumor microenvironment refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself.
  • the tumor and the tumor microenvironment are closely related and interact constantly. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads.
  • the tumor microenvironment has a low pH in the range of 5.0 to 6.8, or in the range of 5.8 to 6.8, or in the range of 6.2-6.8.
  • a normal physiological pH is in the range of 7.0-7.6.
  • the tumor microenvironment is also known to have lower concentration of glucose and other nutrients, but higher concentration of lactic acid, in comparison with blood plasma.
  • the tumor microenvironment can have a temperature that is 0.3 to 1 °C higher than the normal physiological temperature.
  • the tumor microenvironment has been discussed in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol. 16, pp.430-450, 2002, hereby incorporated by reference herein its entirety.
  • the term “non-tumor microenvironment” refers to a microenvironment at a site other than a tumor.
  • 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 and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • 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 a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel.
  • the naked antibody may be present in a pharmaceutical formulation.
  • package insert as used herein is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • percent (%) amino acid sequence identity with respect to a reference polypeptide sequence as used herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • ALIGN-2 sequence comparison computer program
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • recombinant antibody refers to an antibody (e.g. a chimeric, humanized, or human antibody or antigen-binding fragment thereof) that is expressed by a recombinant host cell comprising nucleic acid encoding the antibody.
  • host cells for producing recombinant antibodies include: (1) mammalian cells, for example, Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example plants belonging to the genus Nicotiana (e.g.
  • Nicotiana tabacum (4) yeast cells, for example, those belonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus (e.g. Aspergillus niger ); (5) bacterial cells, for example Escherichia, coli cells or Bacillus subtilis cells, etc.
  • the term “single chain Fv” (“scFv”) as used herein is a covalently linked V H ::V L heterodimer which is usually expressed from a gene fusion including V H and V L encoding genes linked by a peptide-encoding linker.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
  • terapéuticaally effective amount of the antibody of the invention is meant a sufficient amount of the antibody to treat said cancer, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • treatment refers to clinical intervention in an ahempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • variable region or “variable domain” as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (V H and V L , respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • antibodies that bind a particular antigen may be isolated using aV H or V L domain from an antibody that binds the antigen to screen a library of complementary V L or V H domains, respectively. See, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • each amount/value or range of amounts/values for each component, compound, substituent, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount/value or range of amounts/values disclosed for any other component(s), compounds(s), substituent(s), or parameter(s) disclosed herein and that any combination of amounts/values or ranges of amounts/values for two or more component(s), compounds(s), substituent(s), or parameters disclosed herein are thus also disclosed in combination with each other for the purposes of this description.
  • each lower limit of each range disclosed herein is to be interpreted as disclosed in combination with each upper limit of each range disclosed herein for the same component, compounds, substituent, or parameter.
  • a disclosure of two ranges is to be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range.
  • a disclosure of three ranges is to be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc.
  • the present invention provides an isolated polypeptide comprising a heavy chain variable region that specifically binds to Nectin-4, or especially human Nectin-4 protein.
  • the heavy chain variable region includes three complementarity determining regions (CDRs) having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • CDRs complementarity determining regions
  • the H1 sequence may be selected from GFTFSSYNMN (SEQ ID NO: 7), and GFTFSSYNDN (SEQ ID NO: 8).
  • the H3 sequence may be selected from AYYYGMDV (SEQ ID NO: 9), AYYYGDDV (SEQ ID NO: 10), and AYYYGMDK (SEQ ID NO: 11).
  • the present invention provides an isolated polypeptide comprising a light chain variable region that specifically binds to human Nectin-4.
  • the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 ASQGISGW X 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein X4 is R or H; X 5 is L or E; X 6 is F or E; and X 7 is P or D, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the L1 sequence may be selected from RASQGISGWLA (SEQ ID NO: 12), RASQGISGWEA (SEQ ID NO: 13), and HASQGISGWLA (SEQ ID NO: 14).
  • the L3 sequence may be selected from QQANSFPPT (SEQ ID NO: 15), QQANSEPPT (SEQ ID NO: 16), and QQANSFPDT (SEQ ID NO: 17).
  • the present invention provides isolated polypeptides that specifically bind to Nectin-4, or especially human Nectin-4 protein comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 ASQGISGWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX
  • the isolated polypeptide may include a heavy chain variable region having a sequence selected from SEQ ID NOS: 18-30, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the isolated polypeptide may include a light chain variable region having a sequence selected from SEQ ID NOS: 31-43, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NOS: 19 and 32, SEQ ID NOS: 20 and 33, SEQ ID NOS: 21 and 34, SEQ ID NOS: 22 and 35, SEQ ID NOS: 23 and 36, SEQ ID NOS: 24 and 37, SEQ ID NOS: 25 and 38, SEQ ID NOS: 26 and 39, SEQ ID NOS: 27 and 40, SEQ ID NOS: 28 and 41, and SEQ ID NOS: 29 and 42.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a combination of amino acid sequences selected from one of SEQ ID NOS: 18-30 in combination with one of SEQ ID NOS: 31-43; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptides of the present invention comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: : SEQ ID NOS: 19 and 32, SEQ ID NOS: 20 and 33, SEQ ID NOS: 21 and 34, SEQ ID NOS: 22 and 35, SEQ ID NOS: 23 and 36, SEQ ID NOS: 24 and 37, SEQ ID NOS: 25 and 38, SEQ ID NOS: 26 and 39, SEQ ID NOS: 27 and 40, SEQ ID NOS: 28 and 41, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination and SEQ ID NOS: 29 and 42, respectively; and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptide of the present invention specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 AS QGIS GWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein X 4 is R or H; X 5 is L
  • the L6 sequence is selected from any one of SEQ ID NOs: 50-53, and the L7 sequence is selected from SEQ ID NOs: 54 and 55.
  • the isolated polypeptide specifically binds to Nectin-4 and CD3 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is selected from SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, the L2 sequence is SEQ ID NO: 5, the L3 sequence is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 wherein: the L4 sequence is G
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • RS STGAVTTSNYDN (SEQ ID NO: 55)
  • the L8 sequence is GTNKRAP (SEQ ID NO: 48)
  • the L9 sequence is ALWYSNLWV (SEQ ID NO: 49).
  • the isolated polypeptide specifically binds to Nectin-4 and
  • CD3 and comprises a heavy chain variable region and a light chain variable region that, wherein the heavy chain variable region includes three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7, the H2 sequence is selected from SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and the light chain variable region includes three complementarity determining regions L1, L2, and L3, wherein the L1 sequence is selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, the L2 sequence is SEQ ID NO: 5, the L3 sequence is SEQ ID NO: 15, and six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 wherein: the L4 sequence is GFTFNTY AMN (SEQ ID NO: 44), the L5 sequence is RIRS KYNNY ATYY AD S VKD (SEQ ID NO: 45), the L6 sequence is selected from HGNFG
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • RS STGAVTTSNYDN (SEQ ID NO: 55)
  • the L8 sequence is GTNKRAP (SEQ ID NO: 48)
  • the L9 sequence is ALWYSNLWV (SEQ ID NO: 49).
  • H2, H3, L1, L2 and L3 sequences may further include any one of the combinations of L4,
  • the isolated polypeptide with nine CDRs comprises a heavy chain variable region having a sequence selected from SEQ ID NOS: 18, 25, 27, and 29.
  • the isolated polypeptide with nine CDRs comprises a light chain variable region having a sequence selected from SEQ ID NOS: 56-60.
  • the isolated polypeptide with nine CDRs comprises a heavy chain variable sequence of any one of SEQ ID NOs: 18, 25, 27, and 29, and a light chain variable sequence of any one of SEQ ID NOs: 56-60.
  • isolated polypeptides with nine CDRs of the present invention comprise a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NO: 25 and SEQ ID NO: 57, SEQ ID NO: 27 and SEQ ID NO: 58, SEQ ID NO: 29 and SEQ ID NO: 59, and SEQ ID NO: 29 and SEQ ID NO: 60.
  • the isolated polypeptides with nine CDRs of the present invention comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a combination of amino acid sequences selected from one of SEQ ID NOS: 18, 25, 27, and 29 in combination with one of SEQ ID NOs: 56-60; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination; and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptides with nine CDRs comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: SEQ ID NOS: 25 and 57, SEQ ID NOS: 27 and 58, SEQ ID NOS: 29 and 59, SEQ ID NOS: 29 and 60; and said isolated polypeptides specifically bind to human Nectin-4 protein.
  • the isolated polypeptide of the present invention which specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3, may also comprise the sequences described hereinabove for specific binding to Nectin-4, and a single-chain fragment variable (scFv) of any known CD3 antibody.
  • the isolated polypeptide binds CD3 independent of the conditionally active Nectin- 4 binding.
  • an isolated polypeptide of the present invention which specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3, comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 AS QGIS GW X 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein X 4 is R or H; X 5 is L or E;
  • the heavy chain variable regions and the light chain variable regions of the present invention were each obtained from a parent antibody using a method disclosed in U.S. Patent Nos. 8,709,755 and 8,859,467. This method of generating the heavy chain variable regions and the light chain variable regions, as well as the method of generating antibodies and antibody fragments disclosed in U.S. Patent Nos. 8,709,755 and 8,859,467, are hereby incorporated by reference herein.
  • the isolated polypeptide may be an antibody or antibody fragment.
  • Antibodies and antibody fragments including these heavy chain variable regions and light chain variable regions can specifically bind to Nectin-4, or especially human Nectin-4.
  • Antibodies or antibody fragments comprising a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have higher binding to Nectin-4 at a pH in the tumor microenvironment (e.g. pH 5.0-6.8, preferably, pH 6.0-6.8) than at a pH in a non-tumor microenvironment (e.g. pH 7.0-7.6).
  • the anti-Nectin- 4 antibodies or antibody fragments have a higher binding to Nectin-4 in a tumor microenvironment in comparison with their binding to Nectin-4 in a typical normal tissue microenvironment.
  • binding is measured by affinity.
  • conditionally active isolated polypeptide, antibody or antibody fragment may be less active or virtually inactive at a normal physiological condition (such as a non-tumor microenvironment) and more active at an aberrant condition (such as a tumor microenvironment), in comparison to the activity at the normal physiological condition of the parent or wild-type polypeptide, antibody or antibody fragment from which it is derived.
  • a normal physiological condition such as a non-tumor microenvironment
  • an aberrant condition such as a tumor microenvironment
  • the isolated polypeptides, anti-Nectin-4 antibodies or anti-Nectin-4 antibody fragments of the present invention may have a lower binding to Nectin-4 at a normal physiological condition (such as a non-tumor microenvironment) in comparison to the parent or wild-type polypeptide, antibody or antibody fragment from which it is derived.
  • conditionally active isolated polypeptide, anti-Nectin-4 antibody or anti-Nectin- 4 antibody fragment may be less active or virtually inactive at a pH of 7.0-7.6 in comparison to the parent or wild-type polypeptide, antibody or antibody fragment, but is active at a lower pH of 5.0-6.8 in comparison to the parent or wild-type polypeptide, antibody or antibody fragment.
  • conditionally active isolated polypeptide, antibody or antibody fragment is reversibly or irreversibly inactivated at the normal physiological condition (such as a non-tumor microenvironment) in comparison to the parent or wild-type polypeptide, antibody or antibody fragment.
  • Anti-Nectin-4 antibodies or antibody fragments of the present invention are thus expected to exhibit reduced side-effects, relative to non-conditionally active anti-Nectin-4 antibodies, due to their reduced binding to Nectin-4 in the normal tissue microenvironment.
  • Anti-Nectin-4 antibodies or antibody fragments of the present invention are also expected to have a comparable efficacy to monoclonal anti-Nectin-4 antibodies known in the art. This combination of features permits use of a higher dosage of these anti- Nectin-4 antibodies or antibody fragments due to the reduced side effects, which may provide a more effective therapy option.
  • the present invention provides an antibody or antibody fragment that specifically binds to Nectin-4, or especially human Nectin-4 protein, comprising a heavy chain variable region that includes three complementarity determining regions (CDRs) having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • CDRs complementarity determining regions
  • the H1 sequence may be selected from GFTFSSYNMN (SEQ ID NO: 7), and GFTFSSYNDN (SEQ ID NO: 8).
  • the H3 sequence may be selected from AYYYGMDV (SEQ ID NO: 9), AYYYGDDV (SEQ ID NO: 10), and AYYYGMDK (SEQ ID NO: 11).
  • the present invention provides an antibody or antibody fragment comprising a light chain variable region that specifically binds to human Nectin-4.
  • the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X4ASQGISGWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein X 4 is R or H; X 5 is L or E; X 6 is F or E; and X 7 is P or D, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the L1 sequence may be selected from RASQGISGWLA (SEQ ID NO: 12), RASQGISGWEA (SEQ ID NO: 13), and HASQGISGWLA (SEQ ID NO: 14).
  • the L3 sequence may be selected from QQANSFPPT (SEQ ID NO: 15), QQANSEPPT (SEQ ID NO: 16), and QQANSFPDT (SEQ ID NO: 17).
  • the present invention provides an antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is GFTFSSYNX 1 N (SEQ ID NO: 1); the H2 sequence is YISSSSSTIYYADSVKG (SEQ ID NO: 2); and the H3 sequence is AYYYGX 2 DX 3 (SEQ ID NO: 3); wherein X 1 is M or D; X 2 is M or D; X 3 is V or K; and the light chain variable region includes three complementarity determining regions, having sequences L1, L2, and L3, wherein: the L1 sequence is X4ASQGISGWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein: the H1 sequence is GFTFSSY
  • the heavy chain variable region may have a sequence selected from SEQ ID NOS: 18-30, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the light chain variable region may have a sequence selected from SEQ ID NOS: 31- 43, with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination.
  • the anti-Nectin-4 antibodies and antibody fragments of the present invention include the combinations of H1, H2, H3, L1, L2, and L3 CDRs or the combinations of heavy variable chain regions (selected from SEQ ID NOS: 18-30) and light chain variable regions (selected from SEQ ID NOS: 31-43) set forth above for the isolated polypeptides.
  • Preferred Nectin-4 antibodies and antibody fragments of the present invention are those that include the preferred combinations of these heavy and light chain variable regions set forth above for the isolated polypeptides.
  • preferred, antibody or antibody fragments of the present invention comprise a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NOS: 32 and 19, SEQ ID NOS: 33 and 20, SEQ ID NOS: 34 and 21, SEQ ID NOS: 35 and 22, SEQ ID NOS: 36 and 23, SEQ ID NOS: 37 and 24, SEQ ID NOS: 38 and 25, SEQ ID NOS: 39 and 26, SEQ ID NOS: 40 and 27, SEQ ID NOS: 4 land 28 and SEQ ID NOS: 42 and 29.
  • the antibody or antibody fragments of the present invention can comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a combination of amino acid sequences selected from one of SEQ ID NOS: 18-30 in combination with one of SEQ ID NOS: 31-43; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination; and said antibody or antibody fragments specifically bind to human Nectin-4 protein.
  • the antibody or antibody fragments of the present invention can comprise a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: : SEQ ID NOS: 32 and 19, SEQ ID NOS: 33 and 20, SEQ ID NOS: 34 and 21, SEQ ID NOS: 35 and 22, SEQ ID NOS: 36 and 23, SEQ ID NOS: 37 and 24, SEQ ID NOS: 38 and 25, SEQ ID NOS: 39 and 26, SEQ ID NOS: 40 and 27, SEQ ID NOS: 41and 28 and SEQ ID NOS: 42 and 29, respectively; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 31 in combination and said antibody or antibody fragments specifically bind to human Nectin-4 protein.
  • an antibody or antibody fragment of the present invention is multi- specific specifically bind to Nectin-4, or especially human Nectin-4 protein and CD3 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity determining regions having sequences H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the light chain variable region includes three complementarity determining regions having sequences L1, L2, and L3, wherein: the L1 sequence is X 4 AS QGIS GWX 5 A (SEQ ID NO: 4); the L2 sequence is AASTLQS (SEQ ID NO: 5); and the L3 sequence is QQANSX 6 PX 7 T (SEQ ID NO: 6), wherein X 4 is R or H; X 5 is L or
  • the L6 sequence is any one of SEQ ID NOs: 50-53, and the L7 sequence is selected from SEQ ID NO: 54 and 55.
  • the bi-specific anti-Nectin-4 x CD3 antibodies and antibody fragments of the present invention include the combinations of H1, H2, H3, L1, L2, L3, L4, L5, L6, L7, L8 and L9 CDRs or the combinations of a heavy variable region (selected from SEQ ID NOS: 18, 25, 27, and 29) and a light chain variable region (selected from SEQ ID NOS: 56-60) set forth above for the isolated polypeptides with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • Preferred anti- Nectin-4 antibodies and antibody fragments of the present invention are those that include the preferred combinations of these heavy and light chain variable regions set forth above for the isolated polypeptides.
  • a multi-specific antibody or antibody fragment specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3 and comprises a heavy chain variable region and a light chain variable region that wherein the heavy chain variable region includes three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7 and SEQ ID NO: 8, the H2 sequence is selected from SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and the light chain variable region includes three complementarity determining regions L1, L2, and L3, wherein: the L1 sequence is selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, the L2 sequence is SEQ ID NO: 5, the L3 sequence is selected from SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8,
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • RS STGAVTTSNYDN (SEQ ID NO: 55)
  • the L8 sequence is GTNKRAP (SEQ ID NO: 48)
  • the L9 sequence is ALWYSNLWV (SEQ ID NO: 49), with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • a multi-specific antibody or antibody fragment of the present invention specifically binds to Nectin-4, or especially human Nectin-4 protein and to CD3 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region including three complementarity determining regions, H1, H2, and H3, wherein: the H1 sequence is selected from SEQ ID NO: 7, the H2 sequence is selected from SEQ ID NO: 2, and the H3 sequence is selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and the light chain variable region includes three complementarity determining regions L1, L2, and L3, wherein: the L1 sequence is selected from SEQ ID NO: 12 and SEQ ID NO: 13, the L2 sequence is SEQ ID NO: 5, the L3 sequence is SEQ ID NO: 15, and six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9 wherein: the L4 sequence is GFTFNTY AMN (SEQ ID NO: 44),
  • HSNFGNSKV SWF AY (SEQ ID NO: 51), HGNFPNSKVSWFQY (SEQ ID NO: 52), and HSNFGNSKV SWF AY (SEQ ID NO: 53), the L7 sequence is selected from RSSTGAVTTSNYAN (SEQ ID NO: 54) and
  • RS STGAVTTSNYDN (SEQ ID NO: 55)
  • the L8 sequence is GTNKRAP (SEQ ID NO: 48)
  • the L9 sequence is ALWYSNLWV (SEQ ID NO: 49), with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • a multi-specific antibody or antibody fragment of the present invention may comprise a heavy chain variable region having a sequence selected from SEQ ID NOS: 18, 25, 27, and 29.
  • a multi-specific antibody or antibody fragment of the present invention may comprise a light chain variable region having a sequence selected from SEQ ID NOS: 56-60.
  • a multi-specific antibody or antibody fragment of the present invention comprises a heavy chain variable region having a sequence of any one of SEQ ID NOs: 18, 25, 27, and 29, and a light chain variable region having a sequence of any one of SEQ ID NOs: 56-60 with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination.
  • a multi-specific antibody or antibody fragment of the present invention comprises a heavy chain variable region and a light chain variable region having any one pair of sequences selected from: SEQ ID NO: 25 and SEQ ID NO: 57, SEQ ID NO: 27 and SEQ ID NO: 58, SEQ ID NO: 29 and SEQ ID NO: 59, and SEQ ID NO: 29 and SEQ ID NO: 60.
  • a multispecific antibody or antibody fragment of the present invention comprises a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a combination of amino acid sequences selected from one of SEQ ID NOS: 18, 25, 27, and 29 in combination with one of SEQ ID NOs: 56-60; with the proviso that the heavy and light chain variable regions cannot be SEQ ID NOS: 18 and 56 combination; and said antibody or antibody fragment specifically binds to human Nectin-4 protein.
  • a multispecific antibody or antibody fragment of the present invention comprises a heavy chain variable region and a light chain variable region each region independently having at least 80%, 85%, 90%, 95%, 98% or 99% identity to a pair of amino acid sequences selected from: SEQ ID NOS: 25 and 57, SEQ ID NOS: 27 and 58,
  • the amino acid sequence of the heavy and light chain variable regions outside of the complementarity determining regions may be mutated in accordance with the principles of substitution, insertion and deletion, as discussed in this application to provide these variants.
  • the constant regions may be modified to provide these variants.
  • both the amino acid sequence of the heavy and light chain variable regions outside of the complementarity determining regions and the constant regions may be modified to provide these variants. [0190] In deriving these variants, one is guided by the process as described herein.
  • the variants of the heavy chain and light chain variable regions may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the heavy and light chain variable regions, or by peptide synthesis.
  • Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the heavy and light chain variable regions. Any combination of deletion, insertion, and substitution can be made to arrive at the antibodies or antibody fragments of the present invention, provided that they possess the desired characteristics, e.g., antigen-binding to human Nectin-4 and/or conditional activity.
  • antibody or antibody fragment variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the CDRs and framework regions (FRs).
  • Conservative substitutions are shown in Table 1 under the heading of “conservative substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody or antibody fragment of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more complementarity determining region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display- based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and/or SDRs (a-CDRs), with the resulting variant V H or V L being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology, vol.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody or antibody fragment to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of CDR “hotspots” or SDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or poly alanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody or antibody fragment and antigen.
  • Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only CDRs in V H and V L of an antibody derived from a non- human animal in FRs of the V H and V L of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that several amino acid residues of the V H and V L of the non-human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigen binding activity.
  • substitution of these amino acid residues with different amino acid residues derived from FRs of the V H and V L of the human antibody would reduce of the binding activity.
  • attempts have to be made to identify, among amino acid sequences of the FR of the V H and V L of human antibodies, an amino acid residue which is directly associated with binding to the antibody, or which interacts with an amino acid residue of CDR, or which maintains the three-dimensional structure of the antibody and which is directly associated with binding to the antigen.
  • the reduced antigen binding activity could be increased by replacing the identified amino acids with amino acid residues of the original antibody derived from a non- human animal.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • a further object of the present invention also encompasses function-conservative variants of the antibodies of the present invention.
  • Two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 80%, preferably greater than 85%, preferably greater than 90% of the amino acids are identical, or greater than about 90%, preferably greater than 95%, are similar (functionally identical) over the whole length of the shorter sequence.
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA, etc.
  • amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define the protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, while nevertheless obtaining a protein with like properties. It is thus contemplated that various changes may be made in the sequences of the antibodies or antibody fragments of the invention, or corresponding DNA sequences which encode said antibodies or antibody fragments, without appreciable loss of their biological activity.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the anti-Nectin-4 antibodies or antibody fragments provided herein are altered to increase or decrease the extent to which the antibodies or antibody fragments are glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997.
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all gly costructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose- deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al.
  • Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986; US Pat Appl No US 2003/0157108 A; and WO 2004/056312 Al, especially at Example 11), and knockout cell lines, such as alpha-1, 6- fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al., Biotechnol. Bioeng., vol.
  • Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
  • one or more amino acid modifications may be introduced into the Fc region of the anti-Nectin-4 antibodies or antibody fragments provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • FcyRII and FcyRIII FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991.
  • Non- limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see also, e.g. Hellstrom et al. Proc. Nat'lAcad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'lAcad. Sci. USA, vol. 82, pp.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'lAcad. Sci. USA, vol. 95, pp. 652-656, 1998.
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp.163-171, 1996; Cragg, M. S. et al., Blood, vol. 101, pp. 1045-1052, 2003; and Cragg, M.
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol., vol. 18, pp. 1759-1769, 2006).
  • the variants of the antibodies or antibody fragments with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.
  • CDC Complement Dependent Cytotoxicity
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934.
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include/e those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
  • the anti-Nectin-4 antibodies or antibody fragments provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody or antibody fragment include but are not limited to water soluble polymers.
  • Non- limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight and may be branched or unbranched.
  • the number of polymers attached to the antibody or antibody fragment may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody or antibody fragment to be improved, whether the derivative will be used in a therapy under defined conditions, etc.
  • conjugates of the antibodies or antibody fragments and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody -nonproteinaceous moiety are killed.
  • the anti-Nectin-4 antibodies or antibody fragments of the invention, or their variants, have a higher binding affinity to Nectin-4 under a condition in a tumor microenvironment than under a condition in a non-tumor microenvironment.
  • the condition in tumor microenvironment and the condition in a non-tumor microenvironment are both pH.
  • the anti-Nectin-4 antibodies or antibody fragments of the invention thus can selectively bind to Nectin-4 at a pH about 5.0-6.8 but will have a lower binding affinity to Nectin-4 at a pH about 7.0-7.6 encountered in a normal, non-tumor microenvironment.
  • the exemplary anti-Nectin-4 antibodies or antibody fragments of the present invention have higher binding affinity to Nectin-4 at pH 6.0 that at pH 7.4.
  • the anti-Nectin-4 antibodies or antibody fragments of the present invention have a dissociation constant (Kd) with Nectin-4 under a condition in tumor microenvironment of about nM (e.g. 10 -8 M or less, or from 10 -8 M to 10 -13 M, or from 10 -9 M to 10 -13 M).
  • Kd dissociation constant
  • the ratio of the binding activity of the antibody or antibody fragment with Nectin-4 at the condition in tumor microenvironment to the binding activity at the same condition in non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8:1, at least about 9:1, at least about 10: 1, at least about 20: 1, at least about 30: 1, at least about 50:1, at least about 70:1, or at least about 100:1.
  • Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen using the following assay.
  • Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293: 865-881 (1999)).
  • MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ L/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in phosphate buffered saline (PBS) for two to five hours at room temperature (approximately 23 °C.).
  • a capturing anti-Fab antibody Cappel Labs
  • PBS phosphate buffered saline
  • a non-adsorbent plate (Nunc #269620) 100 pM or 26 pM [ 125 I] -antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)).
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour).
  • Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C.
  • CM5 chips immobilized antigen CM5 chips at about 10 response units (RU).
  • carboxymethylated dextran biosensor chips CM5, BIACORE, Inc.
  • EDC N- ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ L/ml CO.2 mM) before injection at a flow rate of 5 m ⁇ /minute to achieve approximately 10 response units (RU) of coupled protein.
  • 1 M ethanolamine is injected to block unreacted groups.
  • the anti-Nectin-4 antibodies of the invention may be a chimeric, humanized or human antibody.
  • an anti-Nectin-4 antibody fragment is employed, e.g., a Fv, Fab, Fab', Fab'-SH, scFv, a diabody, a triabody, a tetrabody or an F(ab')2 fragment and multi-specific antibodies formed from antibody fragments.
  • the antibody is a full length antibody, e.g., an intact IgG antibody or other antibody class or isotype as defined herein. For a review of certain antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003.
  • the diabodies of the invention may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA , vol. 90, pp. 6444-6448, 1993 for examples of diabodies. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med. , vol. 9, pp. 129-134, 2003.
  • the invention comprises single-domain antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • recombinant host cells e.g. E. coli or phage
  • the anti-Nectin-4 antibodies of the invention may be chimeric antibodies.
  • Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984).
  • the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • the chimeric antibody is a “class switched” antibody in which the class or subclass of the antibody has been changed relative to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • the chimeric antibody of the invention is a humanized antibody.
  • anon-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody may optionally also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non- human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non- human antibody e.g., the antibody from which the CDR residues are derived
  • Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and are further described, e.g., in Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'lAcad. Sci. USA, vol. 86, pp.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p.
  • the disclosure provides multi-specific anti-Nectin-4 antibodies, e.g. bispecific antibodies.
  • Multi-specific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for Nectin-4 and the other is for another antigen.
  • bispecific conditionally active antibodies may bind to two different epitopes of Nectin-4. The multi-specific antibody binds to at least Nectin-4 and another antigen with a greater activity, affinity and/or avidity at a first physiological condition than at a second physiological condition. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Nectin-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • the first physiological condition is an aberrant condition and the second physiological condition is a normal physiological condition.
  • the aberrant condition may be a condition in a tumor microenvironment.
  • the multi-specific antibody of the present invention may be referred to as a conditionally active multi-specific antibody.
  • the conditionally active multispecific antibody is virtually inactive in binding to one or both of its target antigens or epitopes at a normal physiological condition but is active at an aberrant condition, optionally having a level of activity that is higher than the activity of the conditionally active multi-specific antibody at a normal physiological condition or the activity at a normal physiological condition of the parent antibody from which it is derived.
  • the conditionally active multispecific antibody is less active or virtually inactive at a pH of 7.0-7.6, but is active at a lower pH of 5.0-6.8.
  • the conditionally active multispecific antibody is reversibly or irreversibly inactivated at the normal physiological condition.
  • the conditionally active multi-specific antibody may be more active in the lower pH environments found in the tumor microenvironment.
  • the conditionally active multi-specific antibody may be used as a drug, therapeutic agent or diagnostic agent.
  • the conditionally active multi-specific antibody or antibody fragment is less active or virtually inactive at a normal physiological condition (such as a non-tumor microenvironment) but is active at an aberrant condition (such as a tumor microenvironment), in comparison to the activity at the normal physiological condition of the parent or wild-type antibody or antibody fragment from which it is derived.
  • a normal physiological condition such as a non-tumor microenvironment
  • an aberrant condition such as a tumor microenvironment
  • the anti-Nectin-4 multi-specific antibodies or antibody fragments of the present invention may have a lower binding to Nectin-4 in normal physiological condition (such as a non-tumor microenvironment) in comparison to the parent or wild-type antibody or antibody fragment from which it is derived.
  • conditionally active multi-specific antibody or antibody fragment is less active or virtually inactive at a pH of 7.0-7.6 in comparison to the parent or wild-type antibody or antibody fragment, but is active at a lower pH of 5.0-6.8 in comparison to the parent or wild-type antibody or antibody fragment.
  • conditionally active multi-specific antibody or antibody fragment is reversibly or irreversibly inactivated at the normal physiological condition (such as a non-tumor microenvironment) in comparison to the parent or wild-type antibody or antibody fragment.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature , vol. 305, pp. 537-540, 1983), WO 93/08829, and Traunecker et al., EMBO J. vol. 10, pp. 3655-3659, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547- 1553, 1992); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.
  • Engineered antibodies with three or more functional antigen binding sites are also included herein (see, e.g. US 2006/0025576A1).
  • anti-Nectin-4 antibodies or antibody fragments of the invention may be produced using recombinant methods and compositions, which are described in detail in US 2016/0017040.
  • the invention also provides immunoconjugates comprising an isolated polypeptide or an anti-Nectin-4 antibody or antibody fragment as described herein, conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes.
  • an immunoconjugate comprises an antibody or antibody fragment as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, al pha-sarci n.ri leuriles fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP II, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria to
  • an immunoconjugate comprises an antibody or antibody fragment as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a radioactive atom to form a radioconjugate.
  • radioactive isotopes are available for the production of radioconjugates. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese and iron.
  • NMR nuclear magnetic resonance
  • the immunoconjugate comprises a radioactive agent, which may be selected from an alpha emitter, a beta emitter and a gamma emitter.
  • alpha emitters are 211 At, 210 Bi, 212 Bi, 211 Bi, 223 Ra, 224 Ra, 225 Ac and 22 7 Th.
  • beta- emitters are 67 Cu. 90 Y, 131 I, 153 Sm, 166, Ho and 186 Re.
  • gamma emitters are 60 Co, 137 Ce, 55 F e 54 Mg, 203 Kg, and 133 Ba .
  • Conjugates of an antibody/antibody fragment and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), 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 fluor
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, vol. 238, pp. 1098-, 1987.
  • Carbon-14-labeled 1-isothiocyanatobenzyl- 3-methyldi ethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
  • an acid- labile linker peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide- containing linker (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Pat. No. 5,208,020) may be used.
  • the immunuoconjugates herein expressly contemplate, but are not limited to conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo- EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S. A).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC,
  • An exemplary embodiment of an ADC includes an antibody or antibody fragment (Ab) which targets a tumor cell, a drug moiety (D), and a linker moiety (L) that attaches Ab to D.
  • the antibody is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and/or cysteine.
  • An exemplary ADC has Formula I as Ab-(L-D) p , where p is 1 to about 20.
  • the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues.
  • free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein.
  • Exemplary ADC’s of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012).
  • one or more free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to a drug.
  • an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.
  • Linkers are used to conjugate a moiety to the antibody to form an immunoconjugate such as an ADC. Suitable linkers are described in WO 2017/180842.
  • Drug moieties that may be conjugated to the antibodies are described in WO 2017/180842.
  • Drug moieties also include compounds with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease).
  • an immunoconjugate may comprise a highly radioactive atom.
  • a variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • an immunoconjugate when used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc 99 or 1 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • zirconium-89 zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • Zirconium- 89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
  • radio- or other labels may be incorporated in the immunoconjugate in known ways.
  • a peptide may be biosynthesized or chemically synthesized using suitable amino acid precursors comprising, for example, one or more fluorine-19 atoms in place of one or more hydrogens.
  • labels such as Tc 99 , 1 123 , Re 186 , Re 188 and In 111 can be attached via a cysteine residue in the antibody.
  • yttrium-90 can be attached via a lysine residue of the antibody.
  • the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes certain other methods.
  • an immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme.
  • a prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active drug, such as an anti-cancer drug.
  • ADEPT antibody-dependent enzyme-mediated prodrug therapy
  • Enzymes that may be conjugated to an antibody include, but are not limited to, alkaline phosphatases, which are useful for converting phosphate-containing prodrugs into free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase, which is useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysis, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, which are useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as b-galactosidase and neuraminidase, which are useful for converting glycosylated
  • Drug loading in the conjugates is represented by p, the average number of drug moieties per antibody. Drug loading may range from 1 to 20 drug moieties per antibody.
  • the conjugates of the present invention may have a range of drug moieties, from 1 to 20.
  • the average number of drug moieties per antibody use in the preparation of the conjugates from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • the drug loading may be limited by the number of attachment sites on the antibody.
  • the attachment is a cysteine thiol
  • 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 average drug loading for an ADC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5 (U.S. Pat. No. 7,498,298).
  • an antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below.
  • 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/antibody ratio) of an ADC may be controlled in different ways, and for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
  • any of the isolated polypeptides or anti-Nectin-4 antibodies or antibody fragments as provided herein may be used for detecting the presence of Nectin-4 in a biological sample, either quantitatively or qualitatively.
  • a biological sample comprises a cell or tissue, such as breast, pancreas, esophagus, lung and/or brain cells or tissue.
  • a further aspect of the invention relates to an isolated polypeptide or an anti-Nectin-4 antibody or antibody fragment as described herein of the invention for diagnosing and/or monitoring a cancer or another disease in which Nectin-4 expression levels are increased or decreased from a normal physiological level at least one location in the body.
  • isolated polypeptides or antibodies or antibody fragments of the invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other label known in the art as above described.
  • a detectable molecule or substance such as a fluorescent molecule, a radioactive molecule or any other label known in the art as above described.
  • an antibody or antibody fragment of the invention may be labelled with a radioactive molecule.
  • suitable radioactive molecules include but are not limited to radioactive atoms used for scintigraphic studies such as 123 I, 124 I, in In, 186 Re, and 188 Re.
  • Antibodies or antibody fragments of the invention may also be labelled with a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the distribution of the radiolabeled antibody within the patient is detected.
  • Any suitable known method can be used. Some non-limiting examples include, computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence and sonography.
  • Isolated polypeptides or antibodies or antibody fragments of the invention as described herein may be useful for diagnosing and staging of cancer and diseases associated with Nectin-4 overexpression.
  • Cancers associated with Nectin-4 overexpression may include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, sarcomas, hematological cancers (leukemias), astrocytomas, and various types of head and neck cancer or other Nectin-4 expressing or overexpressing hyperproliferative diseases.
  • Isolated polypeptides or antibodies or antibody fragments of the invention as described herein may be useful for diagnosing diseases other than cancers for which Nectin-4 expression is increased or decreased. Both the (soluble or cellular Nectin-4 forms can be used for such diagnoses.
  • diagnostic methods involve use of a biological sample obtained from the patient.
  • the biological sample encompasses a variety of sample types obtained from a subject that can be used in a diagnostic or monitoring assay.
  • Biological samples include but are not limited to blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or a tissue culture or cells derived therefrom, and the progeny thereof.
  • biological samples include cells obtained from a tissue sample collected from an individual suspected of having a cancer associated with Nectin-4 overexpression, and in preferred embodiments from glioma, gastric, lung, pancreatic, breast, prostate, renal, hepatic and endometrial.
  • Biological samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • the invention is a method of diagnosing a cancer associated with Nectin-4 overexpression in a subject by detecting Nectin-4 on cells from the subject using the antibody of the invention.
  • said method may include steps of:
  • the method according to the invention may be repeated at different times, in order to determine if antibody binding to the samples increases or decreases, wherefrom it can be determined if the cancer has progressed, regressed or stabilized.
  • the invention is a method of diagnosing a disease associated with the expression or overexpression of Nectin-4.
  • diseases may include cancers, human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases
  • an anti-Nectin-4 antibody or antibody fragment for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of Nectin-4 in a biological sample is provided.
  • a method of quantifying the amount of Nectin-4 in a biological sample is provided.
  • the method comprises contacting the biological sample with an anti-Nectin-4 antibody or antibody fragment as described herein under conditions permissive for binding of the anti-Nectin-4antibody or antibody fragment to Nectin-4 and detecting whether a complex is formed between the anti-Nectin-4 antibody or antibody fragment and Nectin-4.
  • an anti- Nectin-4 antibody or antibody fragment is used to select subjects eligible for therapy.
  • the therapy will include administration of an anti-Nectin-4 antibody or antibody fragment to the subject.
  • labeled anti-Nectin-4 antibodies or antibody fragments include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 1, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones
  • horseradish peroxidase HRP
  • alkaline phosphatase b-galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • the isolated polypeptides or anti-Nectin-4 antibodies or antibody fragments as described herein have cell killing activity. This cell killing activity extends to multiple different types of cell lines. Further, these isolated polypeptides or antibodies or antibody fragments of the present invention, once conjugated to a cytotoxic agent, can reduce tumor size and may exhibit reduced toxicity. Thus, the isolated polypeptides, anti- Nectin-4 antibodies, fragments or immunoconjugates thereof may be useful for treating proliferative diseases associated with Nectin-4 expression. The isolated polypeptides, antibodies, fragments or immunoconjugates may be used alone or in combination with any suitable agent or other conventional treatments.
  • the isolated polypeptides or anti-Nectin-4 antibodies or antibody fragments may be used to treat diseases associated with Nectin-4 expression, overexpression or activation.
  • cancers or tissue that can be treated other than the requirement for Nectin-4 expression.
  • examples include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, sarcomas, hematological cancers (leukemias), astrocytomas, and various types of head and neck cancer. More preferable cancers are glioma, gastric, lung, pancreatic, breast, prostate, renal, hepatic and endometrial cancer.
  • the isolated polypeptides or anti-Nectin-4 antibodies or antibody fragments as described herein are potential activators of the innate immune response and thus may be used in the treatment of human immune disorders, such as sepsis.
  • an anti-Nectin-4 antibody or antibody fragment of the invention may also be used as adjuvants for immunization such as for vaccines and as anti-infection agents against, for example, bacteria, viruses and parasites.
  • the isolated polypeptides or anti-Nectin-4 antibody or antibody fragment may be used to protect against, prevent or treat thrombotic diseases such as venous and arterial thrombosis and atherothrombosis.
  • anti-Nectin-4 antibody or antibody fragment may also be used to protect against, prevent or treat cardiovascular diseases as well as to prevent or inhibit the entry of viruses such as Lassa and Ebola viruses and to treat viral infections.
  • the isolated polypeptides, anti- Nectin-4 antibody, antibody fragment or anti-Nectin-4 antibody or antibody fragment immunoconjugate may be delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.
  • an effective amount of the antibody, antibody fragment or immunoconjugate is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • an aspect of the invention relates to a method for treating a disease associated with the expression of Nectin-4 comprising administering to a subject in need thereof with a therapeutically effective amount of an antibody, antibody fragment or immunoconjugate of the invention.
  • the anti-Nectin-4 antibody, antibody fragment or immunoconjugate may be formulated as a pharmaceutical composition.
  • the pharmaceutical composition including an isolated polypeptide, anti-Nectin-4 antibody, antibody fragment or immunoconjugate of the present invention can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution or composition containing a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a material that can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable pharmaceutically acceptable carriers are well-known to those in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)) Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • compositions The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. These considerations can be taken into account by a skilled person to formulate suitable pharmaceutical compositions.
  • the pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition of, for example, sterilized water or physiological saline, permit the constitution of injectable solutions.
  • Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall.
  • excipients may include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothio
  • Non-ionic surfactants or detergents may be employed to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
  • Non-ionic surfactants may be present in a concentration range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
  • Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride [0284]
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • an effective amount of the antibody or antibody fragment may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in a water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the anti-Nectin-4 antibody or antibody fragment can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with one or more of the other ingredients enumerated above, as may be required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • DMSO dimethyl sulfoxide
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the antibodies or antibody fragments may be formulated within a therapeutic mixture to deliver about 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligram, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose. Multiple doses can also be administered at selected time intervals.
  • liposomes and/or nanoparticles are contemplated for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and/or nanoparticles are known to those of skill in the art.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way.
  • ultrafme particles sized around 0.1 pm
  • Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • liposomes depend on pH, ionic strength and the presence of divalent cations
  • Pharmaceutical formulations containing an anti-Nectin-4 antibody or antibody fragment as described herein are prepared by mixing such antibody or antibody fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other may be combined into a single formulation.
  • an EGFR antagonist such as erlotinib
  • an anti- angiogenic agent such as a VEGF antagonist which may be an anti-VEGF antibody
  • a chemotherapeutic agent such as a taxoid or a platinum agent
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the anti-Nectin-4 antibody, antibody fragment or immunoconjugate of the present invention is combined in a formulation with another antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4 and a WNT protein including WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16.
  • the combination may be in the form of two separate molecules: the anti-Nectin-4 antibody, antibody fragment or immunoconjugate of the present invention, and another antibody or antibody fragment.
  • the combination may also be the form of a single molecule with binding affinity to both Nectin-4 and the other antigen, thus forming a multispecific (e.g. bispecific) antibody.
  • Active ingredients may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization.
  • hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions may be employed.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions may be employed.
  • Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • any of the isolated polypeptides, anti-Nectin-4 antibodies or antibody fragments or immunoconjugates provided herein may be used in therapeutic methods as described below.
  • an anti-Nectin-4 antibody or antibody fragment for use as a medicament is provided.
  • an anti-Nectin-4 antibody or antibody fragment for use in treating cancer e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, cancer of pancreas, brain, kidney, ovary, stomach, leukemia, uterine endometrium, colon, prostate, thyroid, liver, osteosarcoma, and/or melanoma
  • cancer e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, cancer of pancreas, brain, kidney, ovary, stomach, leukemia, uterine endometrium, colon, prostate, thyroid, liver, osteosarcoma, and/or melanoma
  • an anti-Nectin-4 antibody or antibody fragment for use in a method of treatment is provided.
  • the invention provides an anti-Nectin-4 antibody or antibody fragment for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-Nectin-4 antibody or antibody fragment.
  • the invention provides an anti-Nectin-4 antibody or antibody fragment for use in a method of treating an individual having an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) or diabetes, comprising administering to the individual an effective amount of the anti- Nectin-4 antibody or antibody fragment.
  • an immune disorder e.g., an autoimmune disorder
  • a cardiovascular disorder e.g., atherosclerosis, hypertension, thrombosis
  • an infectious disease e.g., Ebola virus, Marburg virus
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • the invention provides an anti-Nectin-4 antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function.
  • the invention provides for the use of an anti-Nectin-4 antibody or antibody fragment in the manufacture or preparation of a medicament.
  • the medicament is for treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, cancer of the pancreas, brain, kidney, ovary, stomach, leukemia, uterine endometrium, colon, prostate, thyroid, liver, osteosarcoma, and/or melanoma).
  • the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament.
  • the medicament is for use in a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) or diabetes, comprising administering to the individual an effective amount of the anti- Nectin-4 antibody or antibody fragment.
  • an immune disorder e.g., an autoimmune disorder
  • a cardiovascular disorder e.g., atherosclerosis, hypertension, thrombosis
  • an infectious disease e.g., Ebola virus, Marburg virus
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • the medicament is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function.
  • angiogenesis inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function.
  • the medicament is for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function in an individual comprising administering to the individual an amount effective of the medicament to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine section (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibit tumor stromal function.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides a method for treating a cancer.
  • the method comprises administering to an individual having such cancer an effective amount of an anti-Nectin-4 antibody or antibody fragment.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides a method for treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) or diabetes.
  • an immune disorder e.g., an autoimmune disorder
  • a cardiovascular disorder e.g., atherosclerosis, hypertension, thrombosis
  • an infectious disease e.g., Ebola virus, Marburg virus
  • the invention provides a method for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function in an individual.
  • angiogenesis inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibiting tumor stromal function in an individual.
  • the method comprises administering to the individual an effective amount of an anti-Nectin-4 antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine section (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumoral vasculature or tumor-associated vasculature), and/or inhibit tumor stromal function.
  • an “individual” is a human.
  • the invention provides pharmaceutical formulations comprising any of the anti-Nectin-4 antibodies or antibody fragments provided herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical formulation comprises any of the anti-Nectin-4antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprises any of the anti-Nectin-4antibodies or antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.
  • the antibodies or antibody fragments of the invention can be used alone, as immunoconjugates or in combination with other agents in a therapy.
  • an antibody of the invention may be co-administered with at least one additional therapeutic agent.
  • an additional therapeutic agent is an anti-angiogenic agent.
  • an additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, for example bevacizumab).
  • an additional therapeutic agent is an EGFR antagonist (in some embodiment, erlotinib).
  • an additional therapeutic agent is a chemotherapeutic agent and/or a cytostatic agent.
  • an additional therapeutic agent is a taxoid (e.g., paclitaxel) and/or a platinum agent (e.g., carboplatinum).
  • the additional therapeutic agent is an agent that enhances the patient’s immunity or immune system.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody or antibody fragment can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • Antibodies or antibody fragments can also be used in combination with radiation therapy.
  • the anti-Nectin-4 antibodies or antibody fragments may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody or antibody fragment need not be but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody or antibody fragment, and the discretion of the attending physician.
  • the antibody or antibody fragment is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g of antibody or antibody fragment/kg body weight of the patient to 40 mg of antibody or antibody fragment/kg body weight of the patient can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg of antibody or antibody fragment/kg body weight of the patient to 100 mg of antibody or antibody fragment/kg bodyweight of the patient or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g.
  • the patient receives from about two to about twenty, or e.g. about six doses of the antibody or antibody fragment).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • Specific dosages of the anti-Nectin-4 antibody or antibody fragment of the present invention that may be administered for the prevention or treatment of a disease in a subject may be about 0.3, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, 9.0, 9.6 or 10.2 mg of antibody or antibody fragment/kg bodyweight of the patient.
  • the dosage may be in a range of 0.3-2.4, 2.4-4.2, 4.2-6.0, 6.0-7.8, 7.8-10.2, 10.2-12, 12-14, 14-16, 16-18 or 18-20 mg of antibody or antibody fragment/kg bodyweight of the patient.
  • the dosage of the antibody or antibody fragment will remain the same if administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment or as an immunoconjugate. Further, a polypeptide having anti-Nectin-4 activity will be administered in the same amounts as the antibody or antibody fragment.
  • a single dose of pharmaceutical formulation of the present invention may contain an amount of the anti-Nectin-4 antibody or antibody fragment of the present invention of from about 45 pg of antibody or antibody fragment from about 13,600 mg, or from about 45 pg of antibody or antibody fragment from about 5440 mg.
  • a single dose of pharmaceutical formulation of the present invention may contain an amount of the anti- Nectin-4 antibody or antibody fragment of the present invention of from to 135 mg to 1,387 mg, or an amount such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235,
  • the amount of the anti-Nectin-4 antibody or antibody fragment of the present invention in a single dose of the pharmaceutical formulation is in the range of 135-235, 235-335, 335-435, 435-535, 535-635, 635-735, 735-835, 835-935, 935- 1035, 1035-1135, 1135-1235, 1235-1387 mg.
  • the amount of the antibody or antibody fragment in the single dose of the pharmaceutical formulation will remain the same if administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or as an immunoconjugate, or in combination with another antibody or antibody fragment against another antigen as disclosed herein.
  • a polypeptide having anti-Nectin-4 activity will be included in the single dose of the pharmaceutical formulation in the same amounts as the antibody or antibody fragment.
  • the anti-Nectin-4 antibody or antibody fragment may be conjugated to an immune checkpoint inhibitor molecule or may form part of a bispecific antibody with an immune checkpoint inhibitor.
  • the combination can be the anti-Nectin-4 antibody or antibody fragment disclosed in this application and the immune checkpoint inhibitor molecule administered as separate molecules or as a bispecific antibody.
  • a bispecific antibody has a binding activity to Nectin-4 and a second binding activity to the immune checkpoint.
  • the immune checkpoint may be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences , vol. 20, 158, 2019). Additional immune checkpoints include B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS (Manni et al., Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment, Bbacan, https://doi.Org/10.1016/j.bbcan.2018.12.002, 2018).
  • the immune checkpoint is preferably CTLA4, PD-1 or PD-L1.
  • Enhancing the host's immune function to combat tumors is the subject of increasing interest.
  • Conventional methods include (i) APC enhancement, such as (a) injection into the tumor of DNA encoding foreign MHC alloantigens, or (b) transfecting biopsied tumor cells with genes that increase the probability of immune antigen recognition (e.g., immune stimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2) of the tumor, (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T-cells.
  • APC enhancement such as (a) injection into the tumor of DNA encoding foreign MHC alloantigens, or (b) transfecting biopsied tumor cells with genes that increase the probability of immune antigen recognition (e.g., immune stimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2) of the tumor
  • immune antigen recognition e.g., immune stimulatory cytokines, GM-CSF, co
  • Adoptive cellular immunotherapy includes isolating tumor-infiltrating host T-lymphocytes, expanding the population in vitro, such as through stimulation by IL-2 or tumor or both. Additionally, isolated T-cells that are dysfunctional may be also be activated by in vitro application of the anti-PD-Ll antibodies of the invention. T-cells that are so-activated may then be readministered to the host. One or more of these methods may be used in combination with administration of the antibody, antibody fragment or immunoconjugate of the present invention.
  • Radiotherapy e.g., radiotherapy, X-ray therapy, irradiation
  • ionizing radiation to kill cancer cells and shrink tumors.
  • Radiation therapy can be administered either externally via external beam radiotherapy (EBRT) or internally via brachy therapy;
  • EBRT external beam radiotherapy
  • chemotherapy or the application of cytotoxic drug which generally affect rapidly dividing cells;
  • targeted therapies or agents which specifically affect the deregulated proteins of cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy);
  • immunotherapy or enhancement of the host's immune response (e.g., vaccine);
  • hormonal therapy or blockade of hormone (e.g., when tumor is hormone sensitive),
  • angiogenesis inhibitor or blockade of blood vessel formation and growth
  • palliative care or treatment directed to improving the quality of care to reduce pain, nausea
  • any of the previously described conventional treatments for the treatment of cancer immunity may be conducted, prior, subsequent or simultaneous with the administration of the anti-Nectin-4 antibodies or antibody fragments.
  • the anti-Nectin-4 antibodies or antibody fragments may be administered prior, subsequent or simultaneous with conventional cancer treatments, such as the administration of tumor- binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and/or the administration of chemotherapeutic agents.
  • an article of manufacture containing an isolated polypeptide, an anti- Nectin-4 antibody or antibody fragment, or an immunoconjugate as described herein and other materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided.
  • the article of manufacture comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody or antibody fragment of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody or antibody fragment; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as phosphat
  • any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-Nectin-4 antibody or antibody fragment.
  • kits comprising at least one antibody or antibody fragment of the invention.
  • Kits containing polypeptide, antibodies or antibody fragments, or antibody drug conjugate of the invention find use in detecting Nectin-4 expression (increase or decrease), or in therapeutic or diagnostic assays.
  • Kits of the invention can contain an antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads).
  • Kits can be provided which contain antibodies for detection and quantification of Nectin-4 in vitro, e.g. in an ELISA or a Western blot.
  • Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.
  • kits further contain instructions on the use thereof.
  • the instructions comprise instructions required by the U.S. Food and Drug Administration for in vitro diagnostic kits.
  • the kits further comprise instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of Nectin-4 in said sample.
  • the kits comprise one or more antibodies or antibody fragments.
  • the kits further comprise one or more enzymes, enzyme inhibitors or enzyme activators.
  • the kits further comprise one or more chromatographic compounds.
  • the kits further comprise one or more compounds used to prepare the sample for spectroscopic assay.
  • the kits further comprise comparative reference material to interpret the presence or absence of Nectin-4 according to intensity, color spectrum, or other physical attributes of an indicator.
  • Example 1 Binding activity of the anti-Nectin-4 CAB ADCs and the WT ADC to human Nectin-4
  • Example 2 Binding activity of anti-Nectin-4 CAB ADCs and the WT ADC to human Nectin-4
  • Example 4 Binding activity of anti-Nectin-4 CAB ADCs and the WT ADC to human Nectin-4
  • Example 5 Binding activity of anti-Nectin-4 CAB ADCs and the WT ADC to human Nectin-4
  • Example 7 Binding activity of anti-Nectin-4 CAB ADCs and the WT ADC measured by FACS
  • Binding activities of anti-Nectin-4 CAB ADCs and the WT ADC to HEK293 cells expressing cyno Nectin-4 cells were measured by FACS at pH 6.0 and pH 7.4.
  • the conditionally active antibodies consistently showed higher binding activities to the cyno Nectin-4 cells at pH 6.0 than at pH 7.4.
  • the EC50 values for binding to the cyno Nectin-4 cells expressing cyno Nectin-4 by the anti-Nectin-4 CAB ADCs and the WT ADC are summarized in Table 7.
  • Binding activities of anti-Nectin-4 CAB ADCs and the WT ADC to T47D cells that express human Nectin-4 were measured by FACS at pH 6.0 and pH 7.4.
  • the conditionally active antibodies consistently showed higher binding activities to the T47D cells at pH 6.0 than at pH 7.4. See Figures 13 and 14.
  • the EC50 values for binding to the T47D cells by the anti-Nectin-4 CAB ADCs and the WT ADC are summarized in Table 8.
  • Binding activities of further exemplary anti-Nectin-4 CAB ADCs and the WT ADC to HEK293 cells expressing cyno Nectin-4 were measured by FACS at pH 6.0 and pH 7.4.
  • the conditionally active antibodies consistently showed higher binding activities to the cyno Nectin-4 cells at pH 6.0 than at pH 7.4. See Figures 17 and 18.
  • the EC50 values for binding to the cyno Nectin-4 cells expressing cyno Nectin-4 by the anti-Nectin-4 CAB ADCs and the WT ADC are summarized in Table 10. Table 10
  • Example 11 Binding activity of anti-Nectin-4 CAB ADCs and the WT ADC measured by FACS
  • Binding activities of further exemplary anti-Nectin-4 CAB ADCs and the WT ADC to T47D cells that express human Nectin-4 were measured by FACS at pH 6.0 and pH 7.4.
  • the conditionally active anti-bodies consistently showed higher binding activities to the T47D cells at pH 6.0 than at pH 7.4. See Figures 19 and 20.
  • the EC50 values for binding to the T47D cells by the anti-Nectin-4 CAB ADCs and the WT ADC are summarized in Table 11.
  • Example 12 In vitro cell killing of HEK293 cells expressing human Nectin-4 [0344] In vitro cell killing of HEK293 cells expressing human Nectin-4 was similarly analyzed using HEK293 cells expressing human Nectin-4 at pH values of 6.0 and 7.4. The in vitro killing of the HEK293 cells by the anti-Nectin-4 CAB ADCs and the WT ADC is shown in Figures 21- 26. The EC50 values for the cell killing of HEK293 cells by the anti- Nectin-4 CAB ADCs and the WT ADC are shown in Table 12.
  • Example 13 In vitro cell killing of HEK293 cells expressing human Nectin-4 [0345] In vitro cell killing of HEK293 cells expressing human Nectin-4 was similarly analyzed using HEK293 cells expressing human Nectin-4 at pH values of 6.0 and 7.4. The in vitro killing of the HEK293 cells by further exemplary anti-Nectin-4 CAB ADCs and the WT ADC is shown in Figures 27- 28.
  • Example 14 In vivo test of the efficacy of representative anti-Nectin-4 CAB ADCs and the WT ADC in the subcutaneous T47D CDX model 1. Study Objective and Regulatory Compliance
  • the objective of this project is to evaluate the in vivo anti-tumor efficacy of representative anti- Nectin-4 CAB ADCs and the WT ADC in the treatment of the subcutaneous T47D breast cancer CDX model in B ALB/c nude mice.
  • LP1 represents a proprietary linker payload.
  • N number of animals per group.
  • Dose volume dosing volume was adjusted to 10 ⁇ L/g body weight.
  • B12 is isotype control
  • mice Number of animals: 63 mice plus spare
  • mice were kept in individual ventilation cages at constant temperature and humidity with 4 or 3 animals in each cage. • Temperature: 20-26 °C.
  • Cages Made of polycarbonate. The size is 300 mm x 200 mm x 180 mm. The bedding material is com cob, which was changed twice per week.
  • Cage identification The identification labels for each cage contained the following information: number of animals, sex, strain, received date, treatment, study number, group number and the starting date of the treatment.
  • Animal identification Animals were marked by ear tags.
  • T47D cells which were generated from T47D tumor cells (ATCC, Manassas, VA, cat # ATCC® HTB-133TM) by in vivo passaging twice will be used for this project.
  • T47D cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 0.2 Units/ml bovine insulin, 10% heat inactivated fetal bovine serum, 100 U/ml penicillin and 100 ⁇ L/ml streptomycin at 37 °C with 5% CO2 in air.
  • the tumor cells were routinely subcultured twice per week by trypsin-EDTA treatment.
  • the cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • mice were inoculated with 0.18 mg 17- ⁇ -estradiol pellet 3 days before subcutaneously cell inoculation on the right flank with xxT47D tumor cells (10 x 10 6 with Matrigel) in 0.2 ml of PBS for tumor development. Treatments were started on day 6 after tumor inoculation when the average tumor size reached approximately 152 mm 3 . Animals were assigned into groups according to their tumor volume using an Excel-based program for stratification and randomization. Each group consisted of 7 tumor-bearing mice. The testing articles were administrated according to the experimental design shown in Table 1-1. Testing Article Preparation
  • test article formulation was prepared before each dosing.
  • the T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day.
  • RTV relative tumor volume
  • ⁇ 50 ⁇ L serum was collected from 3 mice per group at 24 hours and 96 hours (right before the 2nd dose) post the first dose, respectively.
  • the mean tumor volume of different groups is shown in Table 3-1.
  • Gl Vehicle, G2: B12-LP1 (3 mg/kg), G3: BAP-143-00-00-LP1 (3 mg/kg), G4: BAP- 143-00-01-LPl (3 mg/kg), G5: BAP-143-00-02-LP1 (3 mg/kg), G6: BAP-143-00-03- LP1 (3 mg/kg), G7: BAP-143-00-04-LP1 (3 mg/kg), G8: BAP-143-00-05-LP1 (3 mg/kg), G9: BAP-143-00-06-LP1 (3 mg/kg).
  • Tumor growth curve is shown in Figure 29. Data presented are mean ⁇ SEM.
  • EXAMPLE 9 Binding Activity of Conditionally Active Anti-Nectin4 Antibodies Measured by SPR Analysis
  • Binding kinetics of anti-Nectin4 antibodies were measured by surface plasmon resonance using a SPR2/4 instrument (Sierra Sensors, Hamburg, Germany) and flat amine sensor chips.
  • the SPR sensor contained four flow cells (FC1-FC4), each of which was addressed individually or in groups.
  • huNectin4-His was immobilized in FC2
  • cynoNectin4-mFc in FC4 No protein was immobilized in FC1 and FC3 which were used as control surfaces for FC2 and FC4, respectively.
  • CynoNectin4 was immobilized using the same conditions as described for huNectin4-His, except that the cynoNectin4 was diluted into 10 mM NaAc buffer having pH 5.0.
  • the control surface was activated and deactivated using the same conditions, but without injecting protein.
  • PBST buffer PBS pH 7.4 with 0.05% TWEEN20TM
  • the running solution was switched to PBST with 30 mM sodium bicarbonate with the pH adjusted as indicated in the figures before the analyte injections.
  • the instrument was equilibrated with the running solution for one hour before the first analyte injection.
  • FACS fluorescence-activated cell sorting
  • HEK293-huNectin4 were maintained in stable cell line culture medium (MEM + 10% Fetal Bovine Serum (FBS) + lmg/mL G418). The cells were routinely sub-cultured twice per week. The cells were harvested during exponential growth phase and counted for plating.
  • MEM + 10% Fetal Bovine Serum (FBS) + lmg/mL G418) The cells were routinely sub-cultured twice per week. The cells were harvested during exponential growth phase and counted for plating.
  • Nectin-4 is a predictive marker for cancer diagnosis and can be a target for development of targeted therapeutics. It may play a mechanistic role in cancer metastasis and angiogenesis of serval types of primary tumors. Nectin-4 is a target for adenocarcinomas in general. Nectin-4 expression has a significant correlation with tumor grade and stages associated to tumor progression (see Fig. 31).
  • Bispecific antibodies were generated which have little to no binding to CD3 and the target antigen in healthy tissue (normal alkaline microenvironment). However, in acid conditions that mirror the tumor microenvironment (high glycolysis) the binding of the antibodies to their target molecule was strong. These molecules demonstrated binding to both recombinant TAA/CD3 and TAA/CD3 expressing cells under acidic conditions that were present in the tumor microenvironment, but not in normal tissues.
  • a dual-CAB (CAB TAA x CAB CD3) bispecific antibody was developed targeting the well-established tumor associated antigen Nectin-4. These bispecific antibodies were active against target positive human tumor xenografts. Importantly, complete tumor regression was observed upon treatment with these CAB bispecific antibodies. Reversible CAB bispecifics yielded a superior therapeutic index relative to other formats, including prodrugs.
  • EXAMPLE 16 Conditionally Active Bispecific Antibodies Targeting Nectin-4 (CAB Nectin-4 x CAB CD3)
  • CAB Nectin-4 x CAB CD3 bispecific antibody showed high affinity to recombinant human Nectin-4 ECD and CD3 epsilon/delta heterodimer protein like wild type (WT) Nectin- 4 x WT CD3 in a tumor microenvironment pH but showed lower affinity in the physiological pH (Fig. 32A). pH affinity ELISA applied human CD3 as capture antigen, human Nectin-4- mFc as detection followed by anti-mouse IgG HRP conjugated antibody. CAB Nectin-4 x CAB CD3 showed higher affinity in tumor microenvironment pH, but lower binding under the physiological pH.
  • CAB Nectin-4 x CAB CD3 pH profile showed that the affinity to human CD3 and human B7H3 were higher in an acidic tumor microenvironment pH of 6.0-6.5 and lower in a physiological pH (7.4) (Fig. 32B).
  • CAB Nectin-4 x CAB CD3 demonstrated a differential binding with human CD3 as capture antigen, human Nectin-4-mFc as detection following with anti-mouse IgGHRP conjugated antibody within the pH range 6.0-7.4.
  • the affinity binding of WT Nectin-4 x WT CD3 remained at a similar level.
  • Bispecific antibodies that bind to Nectin-4 and CD3 were constructed including the heavy and light chains as shown in the Table below.
  • the heavy and light chains of the antibodies are: BA-150-19-01-01-BF1-VH (SEQ ID NO: 18), BA-150-30-33-16-BF11-VH (SEQ ID NO: 25), BA-150-30-33-16-BF19-VH (SEQ ID NO: 27), BA-150-30-03-12-BF11-VH (SEQ ID NO: 29) and BA-150-30-03-12-BF19-VH (SEQ ID NO: 29).
  • BA-150-19-01-01-BF1-LC (SEQ ID NO: 56), BA-150-30-33-16-BF11- LC (SEQ ID NO: 57), BA-150-30-33-16-BF19-LC (SEQ ID NO: 58), BA-150-30-03-12- BF11-LC (SEQ ID NO: 59), and BA-150-30-03-12-BF19-LC (SEQ ID NO: 60).
  • CAB B7H3 x CAB CD3 and CAB Nectin-4 x CAB CD3 bispecific antibodies have increased binding under tumor conditions compared to normal conditions.
  • the pH profile ELISA confirmed the differential affinity within the pH ranges from 6.0 to 7.4.
  • CAB B7H3 x CAB CD3 and CAB Nectin-4 x CAB CD3 bispecific antibodies have similar efficacy in cancer cell line derived MiXeno models in vivo compared to the non-CAB benchmark antibodies.
  • the present invention transformed bispecific solid tumor therapies through the widening of the therapeutic index.
  • CAB Nectin-4 x CAB CD3 (BA-150-30-03-12-BF19)
  • Test articles were first diluted to 3000 ng/mL in pH 6.0 or pH 7.4 ELISA incubation buffer. Then 3000 ng/mL of test articles were 3-fold serially diluted in pH 6.0 or pH 7.4 ELISA incubation buffer.
  • CAB Nectin-4 x CAB CD3 (BA-150-30-03-12-BF19)
  • Test articles were diluted to 100 ng/mL in various pH ELISA incubation buffer range from pH 6.0 to pH 7.4.
  • CAB Nectin-4 x CAB CD3 (BA-150-30-03-12-BF19)
  • mice were inoculated subcutaneously in the right front flank region with NCI-H358 tumor cells (2 x 10 6 ), and next day followed with i.v. human PBMC (10 x 10 6 ) inoculation.
  • Mice were randomly allocated to the different study groups (8 mice/group) when the mean tumor size reaches 120mm 3 .
  • Test articles are dosed at 2.5mg/kg BIW X 4 weeks, and tumor size as well as body weight are monitored twice per week.

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EP4363453A4 (en) * 2021-06-29 2025-06-11 Elpis Biopharmaceuticals ANTI-NECTIN4 ANTIBODIES AND MULTISPECIFIC PROTEIN COMPLEXES
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WO2024012541A1 (zh) * 2022-07-14 2024-01-18 百奥泰生物制药股份有限公司 抗Nectin-4抗体药物偶联物及应用
EP4438624A1 (en) 2023-03-27 2024-10-02 LAVA Therapeutics N.V. Antibodies that bind nectin-4 and gamma-delta t cell receptors
WO2024200573A1 (en) 2023-03-27 2024-10-03 LAVA Therapeutics N.V. Nectin-4 binding agents and methods of use
US20250242065A1 (en) * 2023-09-29 2025-07-31 Aktis Oncology, Inc. Miniproteins, conjugates & uses thereof
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