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WO2024193682A1 - Anti-tpbg/met antibodies and uses thereof - Google Patents

Anti-tpbg/met antibodies and uses thereof Download PDF

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Publication number
WO2024193682A1
WO2024193682A1 PCT/CN2024/083199 CN2024083199W WO2024193682A1 WO 2024193682 A1 WO2024193682 A1 WO 2024193682A1 CN 2024083199 W CN2024083199 W CN 2024083199W WO 2024193682 A1 WO2024193682 A1 WO 2024193682A1
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WIPO (PCT)
Prior art keywords
seq
tpbg
amino acid
antigen
cdrs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/083199
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French (fr)
Inventor
Zhenyan HAN
Wenjuan DAI
Chengzhang SHANG
Yuelei SHEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xadcera Biopharmaceutical Suzhou Co Ltd
Original Assignee
Xadcera Biopharmaceutical Suzhou Co Ltd
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Publication of WO2024193682A1 publication Critical patent/WO2024193682A1/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • A61P35/00—Antineoplastic agents
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • A61K47/6835—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
    • A61K47/6859—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 the tumour determinant being from liver or pancreas cancer cell
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    • 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
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/526—CH3 domain
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This disclosure relates to multispecific anti-TPBG (trophoblast glycoprotein) /MET (tyrosine-protein kinase Met) antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) , and antibody drug conjugates derived therefrom.
  • TPBG trophoblast glycoprotein
  • MET tyrosine-protein kinase Met antibodies
  • a bispecific antibody is an artificial protein that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to targeted sites.
  • catumaxomab anti-EpCAM and anti-CD3
  • blinatumomab anti-CD19 and anti-CD3
  • bispecific antibodies have various applications, there is a need to continue to develop various therapeutics based on bispecific antibodies.
  • This disclosure relates to anti-TPBG/MET antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to TPBG and MET.
  • the antibodies or antigen-binding fragments thereof have identical light chain variable regions.
  • the antibodies or antigen-binding fragments thereof have a common light chain.
  • the disclosure also relates to antibody drug conjugates derived from these anti-TPBG/MET antibodies.
  • the disclosure is related to an anti-TPBG/MET antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET.
  • the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1)
  • the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) .
  • the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, in some embodiments, the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to
  • the second heavy chain variable region comprises CDRs 1, 2, and 3, in some embodiments, the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, in some embodiments, the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence
  • the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
  • the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
  • the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23
  • the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22
  • the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 24
  • the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  • the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23
  • the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22
  • the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 25
  • the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  • the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23
  • the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22
  • the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 43
  • the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  • the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 23
  • the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 22.
  • the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 23; and the VL1 comprising VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 22.
  • the VH2 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence
  • the VL2 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence
  • the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22; (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
  • the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22; (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
  • the VH1 comprises the sequence of SEQ ID NO: 23 and the VL1 comprises the sequence of SEQ ID NO: 22.
  • the VH2 comprises the sequence of SEQ ID NO: 24 and the VL2 comprises the sequence of SEQ ID NO: 22.
  • the VH2 comprises the sequence of SEQ ID NO: 25 and the VL2 comprises the sequence of SEQ ID NO: 22.
  • the VH2 comprises the sequence of SEQ ID NO: 43 and the VL2 comprises the sequence of SEQ ID NO: 22.
  • the first antigen-binding domain specifically binds to human or monkey TPBG; and/or the second antigen-binding domain specifically binds to human or monkey MET.
  • the first antigen-binding domain is human or humanized; and/or the second antigen-binding domain is human or humanized.
  • the antibody is a multispecific antibody (e.g., a bispecific antibody) .
  • the first antigen-binding domain is a single-chain variable fragment (scFv) ; and/or the second antigen-binding domain is a scFv.
  • the first light chain variable region and the second light chain variable region are identical.
  • the disclosure is related to an anti-TPBG/MET antibody or antigen-binding fragment thereof that cross-competes with the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein.
  • the disclosure is related to a nucleic acid comprising a polynucleotide encoding the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein.
  • the disclosure is related to a vector comprising the nucleic acid as described herein.
  • the disclosure is related to a cell comprising the vector as described herein.
  • the cell is a CHO cell.
  • the disclosure is related to a cell comprising the nucleic acid as described herein.
  • the disclosure is related to a method of producing an anti-TPBG/MET antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell as described herein under conditions sufficient for the cell to produce the anti-TPBG/MET antibody or the antigen-binding fragment thereof; and (b) collecting the anti-TPBG/MET antibody or the antigen-binding fragment thereof produced by the cell.
  • the disclosure is related to an anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein.
  • ADC anti-TPBG/MET antibody-drug conjugate
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the therapeutic agent is MMAE or MMAF.
  • the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein, to the subject.
  • the subject has a cancer expressing TPBG and/or MET (e.g., both TPBG and MET) .
  • the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, solid tumor, renal cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.
  • the subject is a human.
  • the method further comprises administering an anti-PD1 antibody to the subject.
  • the method further comprises administering a chemotherapy to the subject.
  • the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein.
  • the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein, and/or (b) the anti-TPBG/MET antibody-drug conjugate as described herein.
  • the disclosure is related to an anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET.
  • ADC anti-TPBG/MET antibody-drug conjugate
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the therapeutic agent is MMAE or MMAF.
  • the therapeutic agent is selected from:
  • the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker.
  • the linker has a structure of:
  • the antibody-drug conjugate has a structure of:
  • n 1-8; in some embodiments, “Ab” represents the antibody or antigen-binding fragment thereof.
  • the drug-to-antibody ratio (DAR) is about 4 or 8.
  • an antigen-binding domain refers to one or more protein domain (s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen (s) (e.g., an effector antigen or control antigen) .
  • an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies.
  • the antigen-binding domain can be an antibody or a fragment thereof.
  • an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer.
  • an antigen-binding domain can include an alternative scaffold.
  • the antigen-binding domain is a VHH.
  • Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.
  • an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen) .
  • an antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen) .
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) .
  • variable domains include, e.g., Fab, Fab’ , F (ab’ ) 2, and Fv fragments.
  • human antibody refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) derived from a human.
  • a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) .
  • a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) .
  • a human antibody is produced in a bacterial or yeast cell.
  • a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .
  • a transgenic non-human animal e.g., a bovine
  • human immunoglobulin locus e.g., heavy or light chain human immunoglobulin locus
  • multispecific antibody is an antibody that includes two or more different antigen-binding domains that collectively specifically bind two or more different epitopes.
  • the two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins present on the surface of the same cell or present on the surface of different cells) .
  • a multispecific antibody binds two different epitopes (i.e., a “bispecific antibody” ) .
  • a multispecific antibody binds three different epitopes (i.e., a “trispecific antibody” ) .
  • a multispecific antibody binds four different epitopes (i.e., a “quadspecific antibody” ) . In some aspects, a multispecific antibody binds five different epitopes (i.e., a “quintspecific antibody” ) . Each binding specificity may be present in any suitable valency. Non-limiting examples of multispecific antibodies are described herein.
  • bispecific antibody refers to an antibody that binds to two different epitopes.
  • the epitopes can be on the same antigen or on different antigens.
  • the term “common light chain” refers to a light chain that can interact with two or more different heavy chains, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes.
  • the term “common light chain variable region” refers to a light chain variable region that can interact with two or more different heavy chain variable regions, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes.
  • the antibody or antigen-binding fragment thereof can have a common light chain.
  • the anti-TPBG/MET antibody or antigen-binding fragment thereof can have a common light chain variable region.
  • anti-TPBG/MET antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment that binds to both MET and TPBG.
  • FIG. 1A lists heavy chain variable region and light chain variable region CDR sequences of anti-TPBG antigen binding domain (32G1) in anti-TPBG/MET antibodies as defined by Kabat definition.
  • FIG. 1B lists heavy chain variable region and light chain variable region CDR sequences of anti-MET antigen binding domain (8H10, 8D9, and 2F11) in anti-TPBG/MET antibodies as defined by Kabat definition.
  • FIG. 2A lists heavy chain variable region and light chain variable region CDR sequences of anti-TPBG antigen binding domain (32G1) in anti-TPBG/MET antibodies as defined by Chothia definition.
  • FIG. 2B lists heavy chain variable region and light chain variable region CDR sequences of anti-MET antigen binding domain (8H10, 8D9, and 2F11) in anti-TPBG/MET antibodies as defined by Chothia definition.
  • FIG. 3 lists amino acid sequences of heavy chain variable regions and light chain variable regions of anti-TPBG/MET antibodies.
  • FIG. 4 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) , and were treated with phosphate buffer saline (PBS) or ADCs.
  • PBS phosphate buffer saline
  • FIGS. 5A-5B show the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) , and were treated with PBS or ADCs.
  • FIG. 5B shows partially enlarged results of FIG. 5A.
  • FIG. 6 shows the average tumor volumes in different groups of B-NDG mice that were injected with NCI-H1975 cells, and were treated with PBS or ADCs.
  • FIG. 7 shows the average tumor volumes in different groups of B-NDG mice that were injected with NUGC-4 cells, and were treated with PBS or ADCs.
  • FIG. 8 lists certain amino acid sequences discussed in the disclosure.
  • FIG. 9 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived pancreatic tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) , and were treated with PBS or ADCs.
  • FIGs. 10A-10B show the endocytosis rates of anti-TPBG antibody, anti-MET antibodies or anti-TPBG/MET bispecific antibodies in NCI-H226 cells (FIG. 10A) or NCI-H2030 cells (FIG. 10B) .
  • FIGs. 11A-11C show the binding activity of anti-TPBG antibody, anti-MET antibodies or anti-TPBG/MET bispecific antibodies to HCC827 cells (FIG. 11A) , NCI-H226 cells (FIG. 11B) , or NCI-H2030 cell (FIG. 11C) .
  • FIG. 12 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived pancreatic tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) , and were treated with PBS or ADCs.
  • FIG. 13 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) , and were treated with PBS or ADCs.
  • a bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens) .
  • a bispecific antibody or antigen-binding fragment thereof can have two arms. Each arm can have one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or an antigen-binding region) .
  • the bispecific antibody has a common light chain.
  • anti-TPBG/MET antibodies e.g., bispecific antibodies or antigen-binding fragments thereof
  • TPBG and MET antibodies e.g., bispecific antibodies or antigen-binding fragments thereof
  • antibody drug conjugates derived from these anti-TPBG/MET antibodies e.g., bispecific antibodies or antigen-binding fragments thereof
  • Trophoblast glycoprotein also known as 5T4, Wnt-Activated Inhibitory Factor 1, or WAIF1
  • TPBG expression is associated with the directional movement of cells through epithelial mesenchymal transition, potentiation of CXCL12/CXCR4 chemotaxis and inhibition of canonical Wnt/beta-catenin while favoring non-canonical pathway signaling; all processes which help drive the spread of cancer cells.
  • TPBG tumor expression The selective pattern of TPBG tumor expression, association with a tumor-initiating phenotype plus a mechanistic involvement with cancer spread have underwritten the clinical development of different immunotherapeutic strategies including a vaccine, a tumor-targeted superantigen and an antibody drug conjugate.
  • TPBG TPBG was discovered in the context of trying to identify shared cell surface molecules that may function to allow survival of the fetus as a semi-allograft in the mother, or a tumor in its host.
  • the rationale was that such shared expression would reflect common functions relevant to growth, invasion or altered immune surveillance in the host.
  • Murine monoclonal antibodies were raised against purified glycoproteins from trophoblast membrane preparations from term human placenta and initially screened against different cancer cell lines and human peripheral blood mononuclear cells. Further screening using the TPBG monoclonal antibody (mAb) by immunohistochemistry indicated the antigen was expressed by many different cancers but with a restricted normal tissue distribution.
  • TPBG molecules as N-glycosylated proteins with an apparent molecular size of 72 kD and are encoded on chromosome 6q14-15.
  • the human gene encodes a 42 kD transmembrane protein core which contains several LRRs that are associated with protein–protein interactions of a functionally diverse set of molecules.
  • the extracellular part of the molecule has several LRRs in two domains separated by a short hydrophilic sequence; there is a transmembrane domain and a short cytoplasmic sequence.
  • the TPBG-specific mAb recognizes a conformationally dependent epitope that relies on the integrity of the intramolecular S–Sbonds and the indirect presence of the complex N-linked glycosylation.
  • human TPBG was overexpressed in murine fibroblasts, the cells became more spindle shaped and had reduced adherence while in normal epithelial cells there was E-Cadherin down-regulation, increased motility and cytoskeletal disruption.
  • TPBG-specific monoclonal antibody detected antigen expression by many different types of carcinoma but demonstrated only low-level expression in some normal adult tissue epithelia.
  • TPBG was expressed in many different primary and metastatic cancers, frequently at high levels; in some cases, there was an additional stromal expression. Linking a role for TPBG in tumor development and spread, it has been shown that TPBG expression in colorectal, gastric and ovarian cancers correlates with poorer clinical outcome.
  • TPBG E-cadherin down-regulation which is a key component of EMT; this occurs during embryonic development and is important for the metastatic spread of epithelial tumors.
  • TPBG was shown to be a marker of the early differentiation of mouse and human embryonic stem (ES) cell, and this process involves an E-to N-cadherin switch, upregulation of E-cadherin repressor molecules (Snail and Slug proteins) , increased matrix metalloproteinase (MMP-2 and MMP-9) activity and motility, all classic EMT features.
  • E-Cadherin ES cells constitutively express cell surface TPBG molecules while antibody induced down-regulation of E-Cadherin in ES cells induced TPBG membrane expression, increased motility, altered actin cytoskeleton arrangement and a mesenchymal phenotype. These observations are consistent with E-cadherin somehow preventing TPBG cell surface expression; the possible mechanism being through stabilization of cortical actin cytoskeletal organization. Co-expression of TPBG and factors involved in the epithelial-to-mesenchymal transition was also observed in undifferentiated but not in differentiated lung tumor cells.
  • TPBG molecules have been shown to be involved in the functional expression of CXCR4 at the cell surface in some embryonic and tumor cells. Both CXCL12 and CXCR4 expression have been associated with tumorigenesis in many cancers, and it is believed that CXCR4 expression facilitates the spread to tissues that highly express CXCL12 including lung, liver, lymph nodes and bone marrow.
  • TPBG is expressed by putative leukemia initiating cells in BCP-ALL, and these cells show the associated property of CXCL12/CXCR4 chemotaxis.
  • TPBG-positive leukemia-initiating cells are likely attracted by CXCL12 produced by extramedullary sites where there is decreased therapeutic bioavailability leading to disease relapse following treatment.
  • Wnt protein intracellular signaling is a central component of many aspects of cellular regulation critical to normal development, homoeostasis and regeneration, while misregulation can lead to disease, including cancer.
  • PCP planar cell polarity
  • TPBG has been shown to interfere with Wnt/ ⁇ -catenin signaling and concomitantly activate non-canonical Wnt pathways.
  • TPBG binds to the Wnt co-receptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation thereby modulating Wnt/ ⁇ -catenin signaling by regulating LRP6 subcellular localization.
  • TPBG enhances ⁇ -catenin-independent Wnt signaling in promoting a non-canonical function of Dickkopf1.
  • MET also called c-Met, tyrosine-protein kinase Met, or hepatocyte growth factor receptor (HGFR)
  • HGFR hepatocyte growth factor receptor
  • HGF methyl mesenchymal growth factor
  • MET binding is unclear, but it is generally believed that two HGF molecules bind to two MET molecules leading to receptor dimerization and autophosphorylation at tyrosines 1230, 1234, and 1235.
  • Ligand-independent MET autophospliorylation can also occur due to gene amplification, mutation or receptor over-expression.
  • MET is frequently amplified, mutated or over-expressed in many types of cancer including gastric, lung, colon, breast, bladder, head and neck, ovarian, prostate, thyroid, pancreatic, and CNS cancers. Missense mutations typically localized to the kinase domain are commonly found in hereditary papillary renal cell carcinomas (PRCC) and in 13%of sporadic PRCCs, MET mutations localized to the semaphorin or juxtamembrane domains of MET are frequently found in gastric, head and neck, liver, ovarian, NSCLC and thyroid cancers. MET amplification has been detected in brain, colorectal, gastric, and lung cancers, often correlating with disease progression.
  • PRCC hereditary papillary renal cell carcinomas
  • MET mutations localized to the semaphorin or juxtamembrane domains of MET are frequently found in gastric, head and neck, liver, ovarian, NSCLC and thyroid cancers.
  • NSCLC non-small cell lung cancer
  • gastric cancers gastric cancers
  • MET overexpression is also frequently observed in lung cancer.
  • nearly half of lung adenocarcinomas exhibited high levels of MET and HGF, both of which correlated with enhanced tumor growth rate, metastasis and poor prognosis.
  • MET amplification was first identified in cultured cells that became resistant to gefitinib, an EGFR kinase inhibitor, and exhibited enhanced survival through the HER3 pathway. This was further validated in clinical samples where nine of 43 patients with acquired resistance to either erlotinib or gefitinib exhibited MET amplification.
  • MET signaling Aberrant MET signaling has been implicated in the development/progression of many human cancers. This results from the overexpression of MET, activating mutations in MET, transactivation, autocrine or paracrine signaling, or MET gene amplification. Binding of HGF to MET stimulates receptor dimerization, autophosphorylation, activation of the receptor's internal, cytoplasmic tyrosine kinase domain, and initiation of multiple signal transduction and transactivation pathways involved in regulation of DNA synthesis (gene activation) and cell cycle progression or division, inhibition of MET signaling may result in inhibition of one or more MET downstream signaling pathways and therefore neutralizing MET may have various effects, including inhibition of cell proliferation and differentiation, angiogenesis, cell motility and metastasis.
  • the bispecific anti-TPBG/MET antibody described herein can be designed to have an IgG1 subtype structure with knobs-into-holes (KIH) mutations, which can promote heterodimerization and avoid mispairing between the two heavy chains.
  • the bispecific anti-TPBG/MET antibody has a higher endocytosis rate than the corresponding monoclonal antibodies or the control bispecific antibodies.
  • the bispecific anti-TPBG/MET antibody described herein can be conjugated with a therapeutic agent, forming an antibody drug conjugate (ADC) .
  • ADC antibody drug conjugate
  • the drug-to-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7.
  • the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.4, about 3.7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.3, about 3.8
  • the DAR of the ADCs described herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5, about 7.5 to about 8.4, about 7.6 to about 8.4, about 7.7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to about 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.5,
  • the anti-TPBG/MET ADC described herein can effectively inhibit in vitro cancer cell growth at a concentration of less than 10 ⁇ g/mL, less than 3.33 ⁇ g/mL, less than 1.11 ⁇ g/mL, less than 0.37 ⁇ g/mL, less than 0.12 ⁇ g/mL, less than 0.04 ⁇ g/mL, or less than 0.01 ⁇ g/mL.
  • the anti-TPBG/MET ADC described herein can inhibit in vivo cancer cell growth (e.g., lung cancer, gastric cancer, or skin cancer) in a xenograft mouse model at a dose level of less than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1.5 mg/kg, or 1 mg/kg.
  • in vivo cancer cell growth e.g., lung cancer, gastric cancer, or skin cancer
  • a xenograft mouse model at a dose level of less than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1.5 mg/kg, or 1 mg/kg.
  • the anti-TPBG/MET antibody described herein has a common light chain.
  • the anti-TPBG/MET antibody includes an anti-TPBG antigen-binding domain (e.g., 32G1) or an anti-MET antigen-binding domain (e.g., 8H10, 8D9, or 2F11) .
  • the anti-TPBG/MET antibodies have a heavy chain variable region targeting TPBG (e.g., any one of the VH targeting TPBG described herein) , a heavy chain variable region targeting MET (e.g., any one of the VH targeting MET described herein) , and two identical common light chain variable regions.
  • the CDR sequences for 32G1 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 4-6, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3 as defined by Kabat definition.
  • the CDRs can also be defined by Chothia definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 13-15, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 1-3.
  • the human light chain variable region and human heavy chain variable region for 32G1 are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively.
  • the CDR sequences for 8H10 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 16-18, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3.
  • the human light chain variable region and human heavy chain variable region for 8H10 are shown in SEQ ID NO: 22 and SEQ ID NO: 24, respectively.
  • the CDR sequences for 8D9 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 10-12, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 19-21, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3.
  • the human light chain variable region and human heavy chain variable region for 8D9 are shown in SEQ ID NO: 22 and SEQ ID NO: 25, respectively.
  • the CDR sequences for 2F11 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 37-39, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 40-42, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3.
  • the human light chain variable region and human heavy chain variable region for 2F11 are shown in SEQ ID NO: 22 and SEQ ID NO: 43, respectively.
  • the anti-TPBG/MET antibodies described herein can contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 37-39, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, and SEQ ID NOs: 40-42; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region
  • VH
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the anti-TPBG/MET antibodies contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence is SEQ ID NO: 23, 24, 25, or 43
  • the selected VL sequence is SEQ ID NO: 22.
  • the anti-TPBG/MET antibody or antigen-binding fragment can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment can have 3 VL CDRs that are identical to the CDRs of any VL sequences as described herein.
  • the disclosure also provides nucleic acid comprising a polynucleotide encoding an anti-TPBG/MET antibody.
  • the immunoglobulin heavy chain or immunoglobulin light chain in the anti-TPBG/MET antibody comprises CDRs as shown in FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B.
  • the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region)
  • the paired polypeptides bind to MET and/or TPBG.
  • the anti-TPBG/MET antibodies can also be anti-TPBG/MET antibody variants (including derivatives and conjugates) of anti-TPBG/MET antibodies or antibody fragments.
  • Additional anti-TPBG/MET antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof.
  • the anti-TPBG/MET antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass.
  • the anti-TPBG/MET antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or antigen-binding fragment thereof.
  • Fragments of anti-TPBG/MET antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity to both MET and TPBG. Thus, a fragment of an anti-TPBG/MET antibody will retain an ability to bind to MET and TPBG.
  • the multispecific anti-TPBG/MET antibody (e.g., bispecific antibody) includes an antigen-binding domain that is derived from an anti-TPBG antibody, and an antigen-binding domain that is derived from an anti-MET antibody.
  • anti-TPBG/MET antibodies and antigen-binding fragments thereof can have various forms.
  • antibodies can be made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non-limiting anti-TPBG/MET antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the anti-TPBG/MET antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • the hypervariable regions known as the complementary determining regions (CDRs)
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the anti-TPBG/MET antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) .
  • the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions.
  • the anti-TPBG/MET antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, or camelid) .
  • the antigen-binding domain or antigen binding fragment is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab’, F (ab’) 2 , and variants of these fragments.
  • an anti-TPBG/MET antibody or antigen binding fragment thereof can comprise e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain.
  • Non-limiting examples of antigen-binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • the scFv in an anti-TPBG/MET antibody has two heavy chain variable domains, and two light chain variable domains.
  • the anti-TPBG/MET scFv has two antigen binding regions (Antigen binding regions: A and B) , and the two antigen binding regions can bind to the respective target antigens with different affinities.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can comprises one, two, or three heavy chain variable region CDRs selected from FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B.
  • the anti-TPBG/MET antibodies described herein can be conjugated to a therapeutic agent.
  • the anti-TPBG/MET antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .
  • cytotoxic or cytostatic agent e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalas
  • the therapeutic agent is MMAE or MMAF.
  • the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.
  • the anti-TPBG/MET antibody is a bispecific antibody.
  • Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • any of the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) .
  • a stabilizing molecule e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution
  • stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) .
  • the conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an anti-TPBG/MET antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., in Suurs, et al. "Areview of bispecific antibodies and antibody constructs in oncology and clinical challenges, " Pharmacology &therapeutics (2019) , which is incorporated herein by reference in the entirety.
  • the anti-TPBG/MET antibody is a BiTe, a (scFv) 2 , a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv.
  • the anti-TPBG/MET antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F (ab’) 2 , a diabody, a crossMab, a DAF (two-in-one) , a DAF (four-in-one) , a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a ⁇ -body, an orthogonal Fab, a DVD-IgG, a IgG (H) -scFv, a scFv- (H) IgG, IgG (L) -scFv, scFv-(L)IgG, IgG (L, H) -Fv, Ig
  • the anti-TPBG/MET antibody can be a TrioMab.
  • the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.
  • the anti-TPBG/MET antibody has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.
  • CrossMAb technique can be used produce bispecific anti-TPBG/MET antibodies.
  • CrossMAb technique can be used enforce correct light chain association in bispecific heterodimeric IgG antibodies, this technique allows the generation of various bispecific antibody formats, including bi- (1+1) , tri- (2+1) and tetra- (2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) -based antibodies.
  • These formats can be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association.
  • the anti-TPBG/MET antibody can be a Duobody.
  • the Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy-light chains.
  • Dual-variable-domain antibody (DVD-Ig) , additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting.
  • VH and VL variable light chain domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.
  • scFv-IgG In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3) .
  • the scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.
  • the anti-TPBG/MET antibody can have a IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.
  • the anti-TPBG/MET antibody can also have a Fab-scFv-Fc format.
  • Fab-scFv-Fc format a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.
  • the anti-TPBG/MET antibody can be a TF.
  • Three Fab fragments are linked by disulfide bridges. Two fragments target the tumor associated antigen (TAA) and one fragment targets a hapten.
  • TAA tumor associated antigen
  • the TF format does not have an Fc region.
  • ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.
  • Dual affinity retargeting has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together.
  • the sulfur bond can improve stability over BiTEs.
  • an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.
  • tetravalent DART In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetravalent molecule.
  • Tandem diabody comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.
  • the ScFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.
  • the anti-TPBG/MET antibody is a bispecific antibody.
  • the bispecific antibody in present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the avidity to cells with low expression levels of TPBG and MET, and increase the avidity to cells that co-express TPBG and MET, to achieve enhanced targeting function.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof have a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 26, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 27 and 28.
  • the anti-TPBG/MET antibodies include KIH mutations.
  • the anti-TPBG/MET antibody includes a first antigen-binding domain that specifically binds to TPBG, and a second antigen-binding domain that specifically binds to MET.
  • the first antigen-binding domain includes a heavy chain that including one or more knob mutations (aknob heavy chain)
  • the second antigen-binding domain includes a heavy chain including one or more hole mutations (ahole heavy chain) .
  • the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (ahole heavy chain)
  • the second antigen-binding domain that includes a heavy chain including one or more knob mutations (aknob heavy chain)
  • the anti-TPBG/MET antibody includes a knob heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 27.
  • the anti-TPBG/MET antibody includes a hole heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 28.
  • the anti-TPBG/MET antibodies can include an anti-TPBG antigen-binding domain and any anti-MET antigen-binding domain as described herein.
  • the anti-TPBG/MET antibodies, or antigen-binding fragments thereof can initiate CDC or ADCC.
  • the disclosure provides anti-TPBG/MET antibodies and antigen-binding fragments thereof that can specifically bind to TPBG.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can block the binding between TPBG and its ligands (e.g., GIPC PDZ domain containing family, member 1 (GIPC1) ) .
  • the anti-TPBG/MET antibody can inhibit TPBG-associated signaling pathways, thus treating cancer.
  • the anti-TPBG/MET antibody described herein is a TPBG agonist.
  • the anti-TPBG/MET antibody described herein is a TPBG antagonist.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to TPBG (e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s - 1 .
  • TPBG e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG
  • Koff dissociation rate
  • the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s - 1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • kinetic association rates (kon) is greater than 1 ⁇ 10 2 /Ms, greater than 1 ⁇ 10 3 /Ms, greater than 1 ⁇ 10 4 /Ms, greater than 1 ⁇ 10 5 /Ms, or greater than 1 ⁇ 10 6 /Ms. In some embodiments, kinetic association rates (kon) is less than 1 ⁇ 10 5 /Ms, less than 1 ⁇ 10 6 /Ms, or less than 1 ⁇ 10 7 /Ms.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to TPBG (e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG) with a KD of less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M.
  • TPBG e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG
  • the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, or greater than 1 ⁇ 10 -10 M.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can also include an antigen-binding domain that can specifically bind to MET.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can block the binding between MET and its ligands (e.g., Hepatocyte growth factor (HGF) ) .
  • HGF Hepatocyte growth factor
  • the anti-TPBG/MET antibody can inhibit MET-associated signaling pathways, thus treating cancer.
  • the anti-TPBG/MET antibody described herein is a MET agonist.
  • the anti-TPBG/MET antibody described herein is a MET antagonist.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to MET (e.g., human MET, monkey MET, mouse MET, and/or chimeric MET) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • kinetic association rates (kon) is greater than 1 ⁇ 10 2 /Ms, greater than 1 ⁇ 10 3 /Ms, greater than 1 ⁇ 10 4 /Ms, greater than 1 ⁇ 10 5 /Ms, or greater than 1 ⁇ 10 6 /Ms. In some embodiments, kinetic association rates (kon) is less than 1 ⁇ 10 5 /Ms, less than 1 ⁇ 10 6 /Ms, or less than 1 ⁇ 10 7 /Ms.
  • KD kinetic rate constants
  • KD is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M.
  • the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, or greater than 1 ⁇ 10 -10 M.
  • the anti-TPBG/MET antibody e.g., bispecific antibody
  • binds to both MET and TPBG for cells that express both MET and TPBG
  • the antibody has a higher binding affinity to these cells.
  • Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the accumulated strength of multiple affinities of individual non-covalent binding interactions.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to human TPBG or monkey TPBG. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof cannot bind to human TPBG or monkey TPBG. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to human MET or monkey MET. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof cannot bind to human MET or monkey MET.
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity that is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.
  • the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity of above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96%, above 97%, or above 98%, as determined by size exclusion chromatography (SEC) .
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a hydrophobic interaction chromatography (HIC) retention time that is longer than 2 minutes, longer than 3 minutes, longer than 4 minutes, longer than 5 minutes, longer than 6 minutes, longer than 7 minutes, or longer than 8 minutes. In some embodiments, the HIC retention time is less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, less than 6 minutes, less than 7 minutes, or less than 8 minutes.
  • HIC hydrophobic interaction chromatography
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity of above 85%, above 86%, above 87%, above 88%, above 89%, above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96%, above 97%, above 98%, or above 99%, as determined by capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) .
  • CE-SDS capillary electrophoresis-sodium dodecyl sulphate
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a main peak that constitutes more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%of the total sample, as determined by capillary isoelectric focusing (cIEF) .
  • cIEF capillary isoelectric focusing
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has an acidic peak or alkaline peak that constitutes less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, or less than 75%of the total sample, as determined by capillary isoelectric focusing (cIEF) .
  • cIEF capillary isoelectric focusing
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • TGI% tumor growth inhibition rate or percentage
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%.
  • the TGI (%) can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the treatment starts.
  • TGI% [1- (Ti-T0) / (Vi-V0) ] ⁇ 100%
  • Ti is the average tumor volume in the treatment group on day i.
  • T0 is the average tumor volume in the treatment group on day zero.
  • Vi is the average tumor volume in the control group on day i.
  • V0 is the average tumor volume in the control group on day zero.
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a functional Fc region.
  • effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • effector function of a functional Fc region is phagocytosis.
  • effector function of a functional Fc region is ADCC and phagocytosis.
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • the anti-TPBG/MET antibody, antigen-binding fragment, or ADC does not have a functional Fc region.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof are Fab, Fab’, F (ab’) 2 , and Fv fragments.
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein have an Fc region without effector function.
  • the Fc is a human IgG4 Fc.
  • the Fc does not have a functional Fc region.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • Fc region a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric fusion protein thus generated may have any increased half-life in vitro and/or in vivo.
  • the IgG4 has S228P mutation (EU numbering) .
  • the S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.
  • anti-TPBG/MET 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 glycostructures 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; or position 314 in Kabat numbering) ; 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.
  • the Fc region of the anti-TPBG/MET antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
  • the main peak of HPLC-SEC accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100%of the protein complex described herein after purification by protein A-based affinity chromatography and/or size-exclusive chromatography.
  • the bispecific anti-TPBG/MET antibody described herein has a higher endocytosis rate than the corresponding monoclonal antibodies and/or control bispecific antibodies described herein. In some embodiments, the anti-TPBG/MET antibody described herein has a higher endocytosis rate than Telisotuzumab analog and/or PF06263507 analog.
  • ADC Antibody Drug Conjugates
  • the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (adrug) .
  • the therapeutic agent can be covalently or non-covalently bind to the anti-TPBG/MET antibody.
  • the anti-TPBG/MET antibody is an anti-TPBG/MET bispecific antibody.
  • the bispecific antibody has a common light chain.
  • the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .
  • cytotoxic or cytostatic agent e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalas
  • the therapeutic agent can include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents.
  • the therapeutic agent to be conjugated can be selected from, but not limited to, MMAE (monomethyl auristatin E) , MMAD (monomethyl auristatin D) , or MMAF (monomethyl auristatin F) .
  • C 1-6 is intended to encompass, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 .
  • the compounds or any formula depicting and describing the compounds of the present disclosure may have one or more chiral (asymmetric) centers.
  • the present invention encompasses all stereoisomeric forms of the compounds or any formula depicting and describing the compounds of the present invention. Centers of asymmetry that are present in the compounds or any formula depicting and describing the compounds of the present invention can all independently of one another have (R) or (S) configuration.
  • bonds to a chiral carbon are depicted as straight lines in the structural formulas, or when a compound name is recited without an (R) or (S) chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of each such chiral carbon, and hence each enantiomer or diastereomer and mixtures thereof, are embraced within the formula or by the name.
  • the disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios.
  • enantiomers are a subject of the disclosure in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios.
  • the disclosure includes both the cis form and the trans form as well as mixtures of these forms in all ratios.
  • the preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis.
  • a derivatization can be carried out before a separation of stereoisomers.
  • the separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound or it can be done on a final racemic product.
  • Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration.
  • absolute stereochemistry may be determined by Vibrational Circular Dichroism (VCD) spectroscopy analysis.
  • VCD Vibrational Circular Dichroism
  • the structures depicted herein are also meant to include the compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, the compounds wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Such compounds are referred to as a “isotopic variant” .
  • the present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds or any formula depicting and describing the compounds of the present invention.
  • isotopes suitable for inclusion in the compounds of the present invention include, but not limited to, isotopes of hydrogen, such as 2 H (i.e., D) and 3 H; carbon, such as 11 C, 13 C, and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; iodine, such as 123 I and 125 I; nitrogen, such as 13 N and 15 N; oxygen, such as 15 O, 17 O, and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S.
  • isotopic variants of the compounds or any formula depicting and describing the compounds of the present disclosure, for example those incorporating a radioactive isotope may be useful in drug and/or substrate tissue distribution studies.
  • compounds having the depicted structures that differ only in the replacement with heavier isotopes can afford certain therapeutic advantages, for example, resulting from greater metabolic stability, increased in vivo half-life, or reduced dosage requirements and, hence, may be utilized in some particular circumstances.
  • Isotopic variants of compounds or any formula depicting and describing the compounds of the present disclosure can generally be prepared by techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and synthesis using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
  • the compounds as provided herein are described with reference to both generic formulas and specific compounds.
  • the compounds of the present disclosure may exist in a number of different forms or derivatives, all within the scope of the disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystal forms or polymorphs, and active metabolites, etc.
  • the term “pharmaceutically acceptable salt” includes salts that retain the biological effectiveness of the free acid/base form of the specified compound and that are not biologically or otherwise undesirable.
  • Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids.
  • the disclosure also comprises their corresponding pharmaceutically acceptable salts.
  • the compounds of the present invention which contain acidic groups, such as carboxyl groups, can be present in salt form, and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts or as ammonium salts.
  • salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids.
  • a suitable base e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide.
  • base salts of compounds of the present disclosure include but are not limited to copper (I) , copper (II) , iron (II) , iron (III) , manganese (II) , and zinc salts.
  • Compounds of the present disclosure which contain one or more basic groups, e.g., groups which can be protonated, can be present in salt form, and can be used according to the disclosure in the form of their addition salts with inorganic or organic acids.
  • acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, cit
  • the salts which are formed are, inter alia, hydrochlorides, chlorides, hydrobromides, bromides, iodides, sulfates, phosphates, methanesulfonates (mesylates) , tosylates, carbonates, bicarbonates, formates, acetates, sulfoacetates, triflates, oxalates, malonates, maleates, succinates, tartrates, malates, embonates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates, and glutamates.
  • the stoichiometry of the salts formed from the compounds of the disclosure may moreover be an integral or non-integral multiple of one.
  • Compounds of the present disclosure which contain basic nitrogen-containing groups can be quaternized using agents such as C 1-4 alkyl halides, for example, methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; diC 1-4 alkyl sulfates, for example, dimethyl, diethyl, and diamyl sulfate; C 10-18 alkyl halides, for example, decyl, dodecyl, lauryl, myristyl, and stearyl chloride, bromide, and iodide; and arylC 1-4 alkyl halides, for example, benzyl chloride and phenethyl bromide.
  • agents such as C 1-4 alkyl halides, for example, methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; diC
  • the disclosure also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions) .
  • the respective salts can be obtained by customary methods which are known to those skilled in the art, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.
  • the present disclosure also includes all salts of the compounds of the present disclosure which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
  • Stahl and Wermuth Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) .
  • solvate refers to a molecular complex comprising the compound of Formula (I) , or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules.
  • hydrate is employed when the solvent is water.
  • compositions in accordance with the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, e.g., D 2 O, d 6 -acetone, d 6 -DMSO.
  • the therapeutic agent is conjugated via a linker (or a linking agent compound) .
  • linker or “linking agent compound” refers to a compound that can connect a ligand (e.g., the antibodies or the antigen-binding fragments thereof described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) together to form a ligand-drug conjugate by reacting with a group of the ligand compound and the therapeutic agent compound respectively by, for example, a coupling reaction.
  • the linker described herein is a compound having the following formula: Q-L Formula (I) ,
  • Q denotes to a junction moiety capable of being coupled to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide and hydrazone bond
  • L denotes to a linker moiety capable of connecting Q to a therapeutic agent.
  • the junction moiety (Q in Formula (I) ) has the following structure:
  • the linker moiety (L in Formula (I) ) has the following formula:
  • L 1 is a polypeptide residue consisting of three to eight amino acid residues which comprises at least one amino acid residue with a side chain carboxyl group, for example, glutamic acid residue or aspartic acid residue, where “-COOH” denotes carboxyl group of an amino acid residue at C-terminal of the polypeptide residue;
  • L 2 is absent or a monodentate, bidentate or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of the polypeptide residue L 1 , and L 2 has a structure of -NHC (R L2a ) (R L2b ) (R L2c ) , where R L2a , R L2b , and R L2c are each independently selected from the group consisting of H, - (CH 2 O) (CH 2 CH 2 O) m (CH 2 ) p C (O) OH, and - (CH 2 O) (CH 2 CH 2 O) m (CH 2 ) p C (O) NHR L2d , R L2d is H or C 1-6 alkyl optionally substituted with 1 to 6 hydroxy groups, each m is independently an integer from 0 to 10, preferably 0 to 4, for example 0, 1, 2, 3, or 4, especially preferably m is 0, and each p is independent an integer from 1 to 4, for example, 1, 2, 3, or 4
  • the polypeptide residue L 1 is NH -Glu-Val-Ala- COOH .
  • the hydrophilic group L 2 has the following structure:
  • the linker described herein is a compound having the following structure:
  • the linker is a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.
  • the therapeutic agent that is conjugated to the antibodies or the antigen-binding fragments thereof described herein is discussed as follows.
  • the therapeutic agent described herein is a cytotoxic agent.
  • the cytotoxic agent is a camptothecin compound, an analogue or a derivative thereof.
  • the camptothecin compound is a compound having the following structure:
  • X is selected from the group consisting of -CH2-, O and S; Y is selected from the group consisting of H, D, and F.
  • the therapeutic agent is CPT-1.
  • the structure of CPT-1 is shown below:
  • the therapeutic agent is CPT-2.
  • the structure of CPT-2 is shown below:
  • the therapeutic agent is CPT3.
  • the structure of CPT-3 is shown below:
  • the therapeutic agent is CPT-4.
  • the structure of CPT-4 is shown below:
  • the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof.
  • the auristatin can be, for example, an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatins include AFP, MMAF, and MMAE.
  • Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity. Auristatins bind tubulin and can exert a cytotoxic or cytostatic effect on cancer cell. There are a number of different assays, known in the art, which can be used for determining whether an auristatin or resultant antibody-drug conjugate exerts a cytostatic or cytotoxic effect on a desired cell.
  • the therapeutic agent is a chemotherapeutic agent.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN TM ) ; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide
  • paclitaxel Bristol-Myers Squibb Oncology, Princeton, N.J.
  • doxetaxel Rhone-Poulenc Rorer, Antony, France
  • chlorambucil gemcitabine
  • 6-thioguanine platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • etoposide VP-16
  • ifosfamide mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston)
  • anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin
  • chemotherapeutic agents can be found in, e.g., US20180193477A1, which is incorporated by reference in its entirety.
  • the anti-TPBG/MET antibody is coupled to the drug via a cleavable linker, e.g. a SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
  • a cleavable linker e.g. a SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
  • the anti-TPBG/MET antibody is coupled to the drug via a non-cleavable linker e.g. a MCC linker formed using SMCC or sulfo-SMCC.
  • an appropriate linker for a given ADC can be readily made by the skilled person having knowledge of the art and taking into account relevant factors, such as the site of attachment to the anti-TPBG/MET antibody, any structural constraints of the drug and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer) .
  • relevant factors such as the site of attachment to the anti-TPBG/MET antibody, any structural constraints of the drug and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer) .
  • a number of specific linker-toxin combinations have been described and may be used with the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein to prepare ADCs in certain embodiments.
  • Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicins and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4, and bis-maleimido-trioxyethylene glycol (BMPEO) -based linkers with maytansinoid DM1.
  • auristatins such as MMAE and MMAF
  • camptothecins such as SN-38, duocarmycins and PBD dimers
  • non-cleavable MC-based linkers with auristatins MMAF and MMAE acid-labile hydrazone-based linkers
  • a drug-maleimide complex i.e., maleimide linking drug
  • maleimide Most common reactive group capable of bonding to thiol group in ADC preparation is maleimide.
  • organic bromides, iodides also are frequently used.
  • the anti-TPBG/MET ADC can be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press) .
  • conjugation can be achieved by (1) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; or (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody.
  • Conjugation methods (1) and (2) can be employed with a variety of antibodies, drug moieties, and linkers to prepare the anti-TPBG/MET ADCs described here.
  • linkers, linker components and toxins are commercially available or may be prepared using standard synthetic organic chemistry techniques. These methods are described e.g., in March’s Advanced Organic Chemistry (Smith &March, 2006, Sixth Ed., Wiley) ; Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press) ; US20210379193A1, and US20180193477A1, which are incorporated herein by reference in the entirety.
  • linker-toxins comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, N.Y. ) .
  • Drug loading is represented by the number of drug moieties per antibody in a molecule of ADC.
  • the drug loading may be limited by the number of attachment sites on the antibody.
  • the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, the drug loading may range from 0 to 8 drug moieties per antibody.
  • higher drug loading e.g. p ⁇ 5
  • the average drug loading for an anti-TPBG/MET antibody-drug conjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5.
  • the optimal ratio of drug moieties per antibody can be around 4.
  • the DAR for an anti-TPBG/MET ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8.
  • the average DAR in the anti-TPBG/MET ADC composition is about 1 ⁇ about 2, about 2 ⁇ about 3, about 3 ⁇ about 4, about 3 ⁇ about 5, about 4 ⁇ about 5, about 5 ⁇ about 6, about 6 ⁇ about 7, or about 7 ⁇ about 8.
  • the Fc region of the anti-TPBG/MET antibodies or antigen-binding fragments thereof was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) .
  • S228P serine at position 228
  • a detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
  • the methods described herein are designed to make a bispecific anti-TPBG/MET antibody.
  • Bispecific anti-TPBG/MET antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • knobs-into-holes technology can be used, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization.
  • the KIH technique is described e.g., in Xu, Yiren, et al. "Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system. " MAbs. Vol. 7. No.1. Taylor &Francis, 2015, which is incorporated by reference in its entirety.
  • one heavy chain has a T366W, and/or S354C (knob) substitution (EU numbering)
  • the other heavy chain has an Y349C, T366S, L368A, and/or Y407V (hole) substitution (EU numbering)
  • one heavy chain has one or more of the following substitutions Y349C and T366W (EU numbering)
  • the other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V (EU numbering) .
  • a substitution (-ppcpScp-->-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.
  • the present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of anti-TPBG/MET antibody polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleot
  • a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-Atail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus.
  • viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y.
  • the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter e.g., a heterologous promoter
  • a heterologous promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters are known to the skilled artisan.
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., an anti-TPBG/MET antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
  • the disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any amino acid sequence as described herein.
  • the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.
  • the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
  • the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology” ) .
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine percentage of sequence homology is known in the art.
  • amino acid residues conserved with similar physicochemical properties e.g. leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein.
  • the nucleic acid e.g., cDNA
  • the nucleic acid includes a polynucleotide encoding a polypeptide of a heavy chain as described herein.
  • the nucleic acid includes a polynucleotide encoding a polypeptide of a light chain as described herein.
  • the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.
  • the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to TPBG.
  • a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to TPBG.
  • the vector includes two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to MET.
  • a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to MET.
  • the methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of anti-TPBG/MET antibodies or anti-TPBG/MET antibody-drug conjugates as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • tumor refers to cancerous cells, e.g., a mass of cancerous cells.
  • Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the agents described herein are designed for treating or diagnosing a carcinoma in a subject.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the cancer is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • the cancer is a chemotherapy resistant cancer.
  • the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of anti-TPBG/MET antibodies or an anti-TPBG/MET antibody drug conjugates disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC) , mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or renal cancer.
  • a cancer e.g., bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC) , mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or renal cancer.
  • the cancer is a solid tumor, lung cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated by the present invention.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the anti-TPBG/MET antibody, anti-TPBG/MET antigen binding fragment, anti-TPBG/MET antibody-drug conjugates, anti-TPBG/MET antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of an anti-TPBG/MET antibody, an anti-TPBG/MET antigen binding fragment, or an anti-TPBG/MET antibody-drug conjugate is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective amount of an anti-TPBG/MET antibody, anti-TPBG/MET antigen binding fragment, or anti-TPBG/MET antibody-drug conjugate may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the agent used.
  • Effective amounts and schedules for administering the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments thereof, anti-TPBG/MET antibody-encoding polynucleotides, anti-TPBG/MET antibody-drug conjugates, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage that must be administered will vary depending on, for example, the mammal that will receive the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments thereof, anti-TPBG/MET antibody-encoding polynucleotides, anti-TPBG/MET antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of the agent or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of an anti-TPBG/MET antibody or anti-TPBG/MET ADC is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 30 mg/kg, 20 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg.
  • the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg.
  • the dosage is about or at least 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
  • the at least one anti-TPBG/MET antibody, the anti-TPBG/MET antigen-binding fragment thereof, anti-TPBG/MET antibody-drug conjugates, or pharmaceutical composition e.g., comprising any of the anti-TPBG/MET antibodies, anti- TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC
  • at least one additional therapeutic agent can be administered to the subject (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, anti-TPBG/MET antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC) .
  • the one or more additional therapeutic agents and the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, or anti-TPBG/MET antibody-drug conjugate are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding fragment, or anti-TPBG/MET ADC in the subject.
  • the subject can be administered the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, anti-TPBG/MET antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • pharmaceutical composition e.g., comprising any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC
  • an extended period of time e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of anti-TPBG/MET antibodies or anti-TPBG/MET antigen-binding antibody fragments, anti-TPBG/MET antibody-drug conjugates (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, or anti-TPBG/MET ADC (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .
  • one or more additional therapeutic agents can be administered to the subject.
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an TPBG inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of MET, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) .
  • the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of TPBG, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, prala
  • therapeutic agents
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.
  • compositions that contain at least one (e.g., one, two, three, or four) of the anti-TPBG/MET antibodies (e.g., bispecific antibodies) , anti-TPBG/MET antigen-binding fragments, or anti-TPBG/MET antibody-drug conjugates described herein.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) .
  • the compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) .
  • Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant.
  • Absorption of the anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) .
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.
  • compositions containing one or more of any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments, anti-TPBG/MET antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) .
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) .
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • a therapeutically effective amount of the anti-TPBG/MET antibodies, an anti-TPBG/MET antigen-binding fragment thereof, or an anti-TPBG/MET ADC will be an amount that treats the disease (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) .
  • any of the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .
  • Exemplary doses include milligram or microgram amounts of any of the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC described herein per kilogram of the subject’s weight (e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; or about 0.1 mg/kg to about 0.5 mg/kg) .
  • weight e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; or about 0.1 mg/kg to about 0.5 mg/kg
  • therapeutic agents vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the therapeutic agent in vivo.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the disclosure also provides methods of manufacturing the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC for various uses as described herein.
  • bispecific antigen-binding molecules targeting TPBG and MET are referred to as anti-TPBG/MET bispecific antibody below.
  • Anti-TPBG antibody (32G1, VH SEQ ID NO: 23; VL SEQ ID NO: 22) and anti-MET antibodies (8H10, VH SEQ ID NO: 24; VL SEQ ID NO: 22; 8D9, VH SEQ ID NO: 25; VL SEQ ID NO: 22 and 2F11, VH SEQ ID NO: 43; VL SEQ ID NO: 22) can be paired to form bispecific antibodies.
  • Vectors encoding the light chain and heavy chain of the antibodies were constructed.
  • CHO-Scells were co-transfected with three vectors, including a first vector encoding the heavy chain of an anti-TPBG antibody, a second vector encoding the heavy chain of an anti-MET antibody, and a third vector encoding the common light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography.
  • Exemplary bispecific antibodies obtained include 32G1-8H10, 32G1-2F11, 8D9-32G1 and 32G1-8
  • knobs-into-holes mutations were introduced in the Fc regions.
  • the heavy chain constant region of 32G1 includes knob mutations
  • the heavy chain constant region of 8H10 includes hole mutations.
  • the heavy chain constant region of 32G1 includes knob mutations
  • the heavy chain constant region of 2F11 includes hole mutations.
  • 8D9-32G1 the heavy chain constant region of 8D9 includes knob mutations
  • the heavy chain constant region of 32G1 includes hole mutations.
  • the heavy chain constant region of 32G1 includes knob mutations
  • the heavy chain constant region of 8D9 includes hole mutations.
  • the sequences of the light chain constant region, the heavy chain constant region with knob mutations, and the heavy chain constant region with hole mutations are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively.
  • CHO-hTPBG cells, CHO-fasTPBG cells, NIH3T3-hMET cells or NIH3T3-fasMET cells were transferred to a 96-well plate at a density of 5 ⁇ 10 4 cells/well, respectively.
  • the anti-TPBG/MET bispecific antibody at a concentration of 5 ⁇ g/mL was added to the 96-well plate, and incubated at 4°C for 30 minutes. Then, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4°C in the dark for 15 minutes before flow cytometry analysis.
  • CHO-hTPBG cells and CHO-fasTPBG cells were obtained by transfecting CHO-Scells with vectors expressing human TPBG (SEQ ID NO: 29) and monkey (Macaca fascicularis) TPBG (SEQ ID NO: 30) , respectively.
  • NIH3T3-hMET cells and NIH3T3-fasMET cells were obtained by transfecting NIH3T3 cells with vectors expressing human MET (SEQ ID NO: 31) and monkey (Macaca fascicularis) MET (SEQ ID NO: 32) , respectively.
  • Telisotuzumab (ABT-700) is a humanized IgG1 monoclonal antibody targeting MET, which is in early clinical development at AbbVie for the treatment of advanced solid tumors with c-MET overexpression, and its heavy chain variable region and light chain variable region sequences are shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.
  • PF06263507 is a humanized IgG1 monoclonal antibody targeting TPBG, and the VH and VL sequences are shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively.
  • 32G1-8H10, 32G1-2F11 and 8D9-32G1 can bind to human TPBG, monkey TPBG, human MET and monkey MET.
  • Anti-TPBG antibody, anti-MET antibody, or anti-TPBG/MET bispecific antibodies together with the pHAb-AffiniPure Fab Goat Anti-Human IgG secondary antibody were added to NCI-H226 cells (ATCC, Cat#: CRL-5826) or SNU-5 cells (ATCC, Cat#: CRL-5973) , respectively, and incubated for 3.5-6.5 hours. The cells were centrifuged and washed with FACS buffer. MFI (mean fluorescence intensity) was measured using a flow cytometer. Endocytosis rates of antibodies were calculated. Human IgG1 was used as an isotype control (ISO) . The expression levels of TPBG and MET in NCI-H226 cells were 46.11 and 131.33 by RNA-Sequence, respectively. The expression levels of TPBG and MET in SNU-5 cells were 8.79 and 871.42, respectively.
  • ISO isotype control
  • NCI-H226 cells or NCI-H2030 ATCC, Cat#: CRL-5914
  • the expression levels of TPBG and MET in NCI-H2030 cells were 21.38 and 70.31 by RNA-Sequence, respectively.
  • anti-TPBG antibody, anti-MET antibodies, or anti-TPBG/MET bispecific antibodies with the concentration of 2.5 ⁇ g/mL together with the pHAb-AffiniPure Fab Goat Anti-Human IgG secondary antibody were added to NCI-H226 cells or NCI-H2030, respectively, and incubated for 6 hours.
  • MFI mean fluorescence intensity
  • binding activity of anti-TPBG/MET bispecific antibodies to human TPBG, human MET, monkey TPBG and monkey MET were verified by surface plasmon resonance (SPR) using Biacore TM (Biacore, Inc., Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.
  • hTPBG-His ACROBiosystems Inc., Cat#: TPG-H52E5
  • fasTPBG-His ACROBiosystems Inc., Cat#: TPG-C52H3
  • hMET-His Sino Biological, Inc., Cat#: 10692-H08H
  • fasMET-His Sino Biological, Inc., Cat#: 90304-C08H
  • hTPBG-His, fasTPBG-His, hMET-His or fasMET-His was injected into the Biacore TM 8K biosensor at 10 ⁇ L/min for about 50 seconds to achieve a desired protein density (e.g., about 100 -1000 response units (RU) ) .
  • Purified antibodies at concentrations of 2 ⁇ g/mL and 10 ⁇ g/mL in 1 ⁇ HBS-EP+ buffer (pH 7.4) were then injected at 10 ⁇ L/min for 50 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with a glycine solution (pH 2.0) at 30 ⁇ L/min for 30 seconds.
  • Anti-TPBG/MET bispecific antibodies 32G1-8H10, 32G1-2F11 and 8D9-32G1 were buffer exchanged into pH 6.0 (3 mg/mL histidine, 80 mg/mL sucrose, and 0.2 mg/mL Tween 80) .
  • the antibodies were kept in sealed Eppendorf tubes at 40 ⁇ 2 °C for 7 days, and the thermal stability were evaluated.
  • the bispecific antibodies were loaded into a protein A column and eluted with a buffer (0.1 mol/L HAc) at pH 3.5.
  • Half of the antibodies were added with a 2 M Tris buffer to make the pH into 7.5 immediately. The remaining half were kept at pH 3.5 for 6 hours and then the pH was adjusted to 7.5.
  • the diluted antibodies were kept in sealed Eppendorf tubes at pH 3.5 ⁇ 0.1, 25 ⁇ 2°C (hereinafter referred to as pH 3.5) for 6 hours to test stability at low pH.
  • the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatograph system (connected with XBridge TM Protein BEH SEC column ( Waters Corporation) ) was used.
  • the following parameters were used: mobile phase: 0.1 M phosphate buffer (PB) + 10%acetonitrile (CAN) , pH 7.4; flow rate: 1.8 mL/min; column temperature: 25°C; detection wavelength: 280 nm, 220 nm; injection volume: 10 ⁇ L; sample tray temperature: about 8°C; and running time: 7 minutes.
  • mobile phase A 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5
  • mobile phase B 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5
  • flow rate 0.8 mL/min
  • gradient 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and 45 min 100%A
  • column temperature 30°C
  • detection wavelength 280 nm, 220 nm
  • injection amount 10 ⁇ g
  • sample tray temperature about 10°C
  • running time 50 minutes.
  • a Maurice cIEF Method Development Kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 8 ⁇ L of 30 ⁇ g protein sample was mixed with the following reagents in the kit: 1 ⁇ L Maurice cIEF pI Marker-7.05, 1 ⁇ L Maurice cIEF pI Marker-10.10, 35 ⁇ L 1%Methyl Cellulose Solution, 2 ⁇ L Maurice cIEF 500 mM Arginine, 1.33 ⁇ L Ampholytes (Pharmalyte pH ranges 3-10) , 6.66 ⁇ L Ampholytes (Pharmalyte pH ranges 8-10.5) and water (added to make a final volume of 100 ⁇ L) .
  • Maurice cIEF Cartridges PS-MC02-C were used to generate imaging capillary isoelectric focusing spectra. The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to integrate the absorbance of the 280 nm-focused protein.
  • CE-SDS (NR) experiments Maurice (Protein simple, Maurice TM ) and Maurice CE-SDS Size Application Kit (Protein simple, Cat#: PS-MAK02-S) were used.
  • CE-SDS (NR) 30 ⁇ L Sample Buffer, 30 ⁇ L 30ug antibody sample, 1.5 ⁇ L 25 ⁇ internal standard, 3 ⁇ L 250 nM Iodoacetamide (SIGMA, Cat#: 16125) were add to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The samples were then cooled to room temperature followed by centrifugation at 10000 rpm for 3 minutes. Supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.
  • Each purified antibody (32G1, 8H10, 8D9, 2F11, 32G1-8H10, 32G1-2F11 or 8D9-32G1) was coupled with MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) through a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • ADC is added directly after the antibody name.
  • 32G1-8H10 with IgG1 constant region is coupled to MMAE, it is named as 32G1-8H10-ADC.
  • Antibody-drug conjugates produced by similar methods also included Telisotuzumab-ADC and PF06263507-ADC.
  • isotype control human IgG1 was coupled to MMAE to form ISO-ADC.
  • HIC-HPLC were used to detect the coupling of antibodies with drug molecules.
  • an Agilent 1260 chromatography system (connected with ProPac TM HIC-10 column (4.6 ⁇ 250 mm, Thermo Scientific) ) was used, and samples were diluted using mobile phase A to 0.5 mg/mL.
  • mobile phase A 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5
  • mobile phase B 0.1 M PB, 10%acetonitrile pH 6.5
  • flow rate 0.8 mL/min
  • gradient 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and 45 min 100%A
  • column temperature 30°C
  • detection wavelength 280 nm
  • injection volume 10 ⁇ L
  • sample tray temperature about 6°C
  • running time 45 minutes.
  • the HIC-HPLC detection results show that the drug-to-antibody ratio (DAR) of each ADC is about 4.
  • the ADCs were tested for their effects on tumor growth in vivo in a model of lung cancer patient-derived xenograft model. Immunofluorescence staining of patient-derived tumor fragments was performed and the images were analyzed via a image analysis platform (3.2 version) . The results showed that TPBG positive cells constituted 58.86%of total cells and MET positive cells constituted 24.08%of total cells in the human lung tumor tissues.
  • the patient-derived tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) were engrafted in the right flank of B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: B-CM-002) .
  • B-NDG mice Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: B-CM-002
  • the tumors in the mice reached a volume of about 200-300 mm 3
  • the mice were randomly placed into different groups based on tumor volumes.
  • the mice were then injected with PBS, ISO-ADC, 32G1-2F11-ADC, 32G1-8H10-ADC, 8D9-32G1-ADC or Telisotuzumab-ADC. Details are shown in the table below.
  • the body weights were measured twice a week. During the experiment, there was no significant difference in body weights among groups, indicating that the tested ADCs were well tolerated and were not obviously toxic to the mice.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 28 days after grouping (Day 28) ; TGI (%) ; and the statistical differences (P value) of tumor volumes between the treatment and control groups.
  • the tumor volumes of mice in different groups are shown in FIG. 4.
  • the treatment groups (G2-G6) showed different tumor inhibition effects compared with PBS group (G1) .
  • 32G1-2F11-ADC (G3) , 32G1-8H10-ADC (G4) and 8D9-32G1-ADC (G5) showed a higher TGI%on Day 28 (45.9%, 96.4%and 64.5%) than that of the positive control Telisotuzumab-ADC (G6, 11.3%) and ISO-ADC (G2, -0.7%) .
  • the patient-derived lung tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) were engrafted in the right flank of B-NDG mice.
  • the mice were randomly placed into different groups based on tumor volumes.
  • the mice were then injected with PBS (G1) , 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) , 8D9-32G1-ADC (G4) or PF06263507-ADC (G5) at 3 mg/kg.
  • Immunofluorescence staining of patient-derived tumor fragments showed that TPBG positive cells constituted 31.01%of total cells and MET positive cells constituted 76.96%of total cells in the human lung tumor tissues.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 11 days after grouping (Day 11) , 21 days after grouping (Day 21) , 35 days after grouping (Day 35) and 49 days after grouping (Day 49) ; TGI (%) ; and the statistical differences (P value) of body weight and tumor volumes between the treatment and control groups.
  • mice in different groups are shown in FIG. 5A and FIG. 5B.
  • the tumor growth in the treatment groups (G2-G5) was significantly inhibited as compared to that of the control group (G1) .
  • 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) and 8D9-32G1-ADC (G4) all inhibited tumor growth with a higher TGI% (e.g., on Day 35) than that of the positive control PF06263507-ADC (G5) .
  • the ADCs were tested for their effects on tumor growth in vivo in a lung cancer model of NCI-H1975 cell xenograft. Specifically, about 1 ⁇ 10 6 NCI-H1975 cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of about 200 mm 3 , the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS, ISO-ADC, 32G1-8H10-ADC, 32G1-2F11-ADC, 8D9-32G1-ADC or PF06263507-ADC by intravenous (i. v. ) administration. The frequency of administration was once a week (2 administrations in total) . Details are shown in the table below.
  • the tumor volumes were measured twice a week and body weights of the mice were recorded as well. Euthanasia was performed when tumor volume of a mouse reached 3000 mm 3 .
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) , 28 days after grouping (Day 28) and 39 days after grouping (Day 39) ; the survival rate of the mice; TGI (%) ; and the statistical differences (P value) of body weights and tumor volumes between the treatment and control groups. As all mice died in G1 group and G2 group on Day 39. Thus, the tumor volume, TGI (%) and P value for Day 39 was not available.
  • FIG. 6.32G1-2F11-ADC, 32G1-8H10-ADC and 8D9-32G1-ADC at a dosage of 3 mg/kg (G3-G5) showed better anti-tumor activities comparing to the positive control PF06263507-ADC (G8) and ISO-ADC (G2) in the lung cancer model.
  • 32G1-2F11-ADC and 32G1-8H10-ADC exhibited a dose-dependent antitumor activity (G3, G6 and G4, G7) .
  • the ADCs were tested for their effects on tumor growth in vivo in a gastric cancer model of NUGC-4 cell xenograft. Specifically, about 5 ⁇ 10 6 NUGC-4 cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of about 200 mm 3 , the mice were randomly placed into different groups based on tumor volumes (5 mice per group) . The mice were then injected with PBS (G1) , 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) , 8D9-32G1-ADC (G4) or PF06263507-ADC (G5) at 3 mg/kg by intravenous (i. v. ) administration. The frequency of administration was once a week (2 administrations in total) .
  • the tumor volumes of mice in different groups are shown in FIG. 7.
  • the treatment groups (G2-G5) showed different tumor inhibition effects compared with PBS groups (G1) .
  • 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) and 8D9-32G1-ADC (G4) showed a higher TGI%on Day 35 (97.7%, 87.6%and 94.5%respectively) comparing to the positive control PF06263507-ADC (G5, 20.2%) .
  • the anti-TPBG/MET bispecific ADCs showed robust and sustained anti-tumor effects in the gastric cancer model.
  • B-NDG mice were engrafted in the right flank with patient-derived pancreatic tumor fragments (2 mm ⁇ 2 mm ⁇ 2 mm) .
  • the tumors in the mice reached a volume of about 200-300 mm 3 .
  • the mice were randomly placed into different groups based on the volume of the tumor.
  • the mice were then injected with PBS or ADCs by intravenosus (i. v. ) administration. Details of the administration scheme are shown in the table below.
  • 32G1-8H10-ADC and 32G1-8D9-ADC had better in vitro killing activity than their parental monoclonal antibody ADCs (32G1-ADC, 8H10-ADC or 8D9-ADC) and the positive controls PF06263507-ADC and Telisotuzumab-ADC.
  • NCI-H226 cells, NCI-H2030 cells or HCC827 cells were incubated with serial diluted purified anti-TPBG/MET antibodies (maximum concentration: 5 ⁇ g/mL, 2-fold dilutions, 10 gradients) for 30 min at 4°C. Then, after one wash with PBS, the cells were incubated with the secondary antibody Alexa 647 anti human IgG Fc ⁇ (Jackson ImmunoResearch Laboratories, Inc., Cat #: 109-606-170) at 4°C for 15 minutes before flow cytometry analysis. The cells were collected, and the mean fluorescence intensity (MFI) was determined.
  • MFI mean fluorescence intensity
  • FIGs. 11A-11C A fitting curve was obtained using antibody concentration ( ⁇ g/mL) as the X-axis and MFI as the Y-axis. The results are shown in FIGs. 11A-11C, which showed that 32G1-8H10 and 32G1-8D9 showed better binding activity to NCI-H226 cells, NCI-H2030 cells and HCC827 cells than the monoclonal antibodies (32G1, 8H10 or 8D9) and the positive controls (PF06263507 analog or Telisotuzumab analog) .
  • the purified antibodies were coupled with CPT-1, CPT-2, CPT-3, or CPT-4 through a CPT-L linker.
  • 32G1-8D9 is coupled to CPT-1
  • 32G1-8D9-CPT1 is named as 32G1-8D9-CPT2.
  • 32G1-8D9-CPT2 is named as 32G1-8D9-CPT2.
  • Exemplary ADCs obtained by this method included: 32G1-8D9-CPT1, 32G1-8D9-CPT2, 32G1-8H10-CPT1, 32G1-8H10-CPT2, 32G1-2F11-CPT1, 32G1-2F11-CPT2, 32G1-CPT2, 2F11-CPT2.
  • MS Mass Spectrometry
  • a human IgG1 molecule was coupled to CPT-2 to form isotype-CPT2 (ISO-CPT2) , as an isotype control.
  • ISO-CPT2 isotype-CPT2
  • the MS detection results showed that the drug-to-antibody ratio (DAR) of the ADCs was about 4.
  • SYD-1875 is an antibody-drug conjugate comprising a humanized IgG1 monoclonal antibody directed against the 5T4 oncofetal antigen, covalently conjugated to a duocarmycin-based linker-drug.
  • the VH and VL sequences of the antibody in SYD-1875 are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
  • the VH and VL were linked to the IgG1 constant region, resulting in a monoclonal antibody named as SYD-1875 analog.
  • Telisotuzumab analog, PF06263507 analog and SYD-1875 analog were also coupled with CPT-2 through the CPT-L linker to form Telisotuzumab-CPT2, PF06263507 -CPT2 and SYD-1875-CPT2, respectively; these serve as the reference ADCs in the following experiments.
  • mice were engrafted in the right flank with gastric cancer patient-derived tumor tissue fragments (2 mm ⁇ 2 mm ⁇ 2 mm) .
  • the immunofluorescence staining results showed that TPBG-positive cells and MET-positive cells in the gastric tumor fragments were 10.11%and 1.19%, respectively.
  • the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i.v. administration. Details are shown in the table below.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 17 days after grouping (Day 17) , and 35 days after grouping (Day 35) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
  • mice were engrafted in the right flank with pancreatic cancer patient-derived tumor tissue fragments (2 mm ⁇ 2 mm ⁇ 2 mm) .
  • pancreatic cancer patient-derived tumor tissue fragments (2 mm ⁇ 2 mm ⁇ 2 mm) .
  • the immunofluorescence staining results showed that TPGB-positive cells and MET-positive cells in the pancreatic tumor fragments were 19.54%and 8.31%, respectively.
  • the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i. v. administration. Details are shown in the table below.
  • mice The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 18 days after grouping (Day 18) and 32 days after grouping (Day 32) ; survial of the mice; TGI (%) and the statistical differences (P value) of mice body weight and tumor volume between the treatment and control groups.
  • mice in different groups are shown in FIG. 12.
  • the results showed that 32G1-8D9-CPT2, 32G1-8H10-CPT2 and 32G1-2F11-CPT2 exhibited better tumor inhibitory effects than that of the positive control PF06263507-CPT2.
  • the ADCs were tested for their effect in two human lung PDX (PDX001 and PDX002) models.
  • the immunofluorescence staining results in PDX001 showed that TPGB-positive cells and MET-positive cells in the pancreatic tumor fragments were 58.86%and 24.08%, respectively.
  • PDX002 TPBG positive cells and MET positive cells were 31.01%and 76.96%, respectively.
  • B-NDG mice were engrafted in the right flank with pancreatic cancer patient-derived tumor tissue fragments (2 mm ⁇ 2 mm ⁇ 2 mm) .
  • mice In PDX001 model, when the tumors in the mice reached a volume of about 150-200 mm 3 , the mice were randomly placed into different groups based on the tumor volume. One day after grouping, the mice were injected with PBS or ADCs by i. v. administration one day after grouping. Details of PDX001 model are shown in the table below.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 25 days after grouping (Day 25) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
  • PDX002 model when the tumors in the mice reached a volume of about 200-300 mm 3 , the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i. v. administration one day after grouping. Details of PDX002 model are shown in the table below.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 25 days after grouping (Day 25) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
  • the tumor size in groups treated with the ADCs are shown in FIG. 13.
  • the results showed that 32G1-8D9-CPT2, 32G1-8H10-CPT2 and 32G1-2F11-CPT2 all exhibited good tumor inhibitory effects with higher TGI%than the corresponding monoclonal ADC 32G1-CPT2.
  • 32G1-2F11-CPT2 exhibited good tumor inhibitory effects with higher TGI%than its parental antibody ADCs 32G1-CPT2 and 2F11-CPT2.

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Abstract

Provided are anti-TPBG/MET antibodies, and antibody drug conjugates derived therefrom, and the use thereof.

Description

ANTI-TPBG/MET ANTIBODIES AND USES THEREOF
CLAIM OF PRIORITY
This application claims priority to PCT/CN2023/083313, filed on March 23, 2023, and PCT/CN2024/078153, filed on February 22, 2024. The entire contents of the foregoing application are incorporated herein by reference.
TECHNICAL FIELD
This disclosure relates to multispecific anti-TPBG (trophoblast glycoprotein) /MET (tyrosine-protein kinase Met) antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) , and antibody drug conjugates derived therefrom.
BACKGROUND
A bispecific antibody is an artificial protein that can simultaneously bind to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual targeting of different disease mediators, and delivering payloads to targeted sites. The approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) has become a major milestone in the development of bispecific antibodies.
As bispecific antibodies have various applications, there is a need to continue to develop various therapeutics based on bispecific antibodies.
SUMMARY
This disclosure relates to anti-TPBG/MET antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof specifically bind to TPBG and MET. In some embodiments, the antibodies or antigen-binding fragments thereof have identical light chain variable regions. In some embodiments, the antibodies or antigen-binding fragments thereof have a common light chain. The disclosure also relates to antibody drug conjugates derived from these anti-TPBG/MET antibodies.
In one aspect, the disclosure is related to an anti-TPBG/MET antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET. In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1) ; and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) .
In some embodiments, the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1  amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, in some embodiments, the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence, in some embodiments, the selected VH1 CDRs 1, 2, and 3 amino acid sequences, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
In some embodiments, the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, in some embodiments, the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, in some embodiments, the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence, in some embodiments, the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37-39, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (5) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and (6) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40- 42, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
In some embodiments, (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37-39, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; (5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; or (6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40-42, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 24, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region  comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 25, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 43, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22. In some embodiments, the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 23, and the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 22.
In some embodiments, the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 23; and the VL1 comprising VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 22.
In some embodiments, the VH2 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22; (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
In some embodiments, the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22; (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
In some embodiments, the VH1 comprises the sequence of SEQ ID NO: 23 and the VL1 comprises the sequence of SEQ ID NO: 22. In some embodiments, the VH2 comprises the sequence of SEQ ID NO: 24 and the VL2 comprises the sequence of SEQ ID NO: 22. In some embodiments, the VH2 comprises the sequence of SEQ ID NO: 25 and the VL2 comprises the sequence of SEQ ID NO: 22. In some embodiments, the VH2 comprises the sequence of SEQ ID NO: 43 and the VL2 comprises the sequence of SEQ ID NO: 22.
In some embodiments, the first antigen-binding domain specifically binds to human or monkey TPBG; and/or the second antigen-binding domain specifically binds to human or monkey MET. In some embodiments, the first antigen-binding domain is human or humanized;  and/or the second antigen-binding domain is human or humanized. In some embodiments, the antibody is a multispecific antibody (e.g., a bispecific antibody) . In some embodiments, the first antigen-binding domain is a single-chain variable fragment (scFv) ; and/or the second antigen-binding domain is a scFv. In some embodiments, the first light chain variable region and the second light chain variable region are identical.
In one aspect, the disclosure is related to an anti-TPBG/MET antibody or antigen-binding fragment thereof that cross-competes with the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein.
In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein.
In one aspect, the disclosure is related to a vector comprising the nucleic acid as described herein.
In one aspect, the disclosure is related to a cell comprising the vector as described herein. In some embodiments, the cell is a CHO cell.
In one aspect, the disclosure is related to a cell comprising the nucleic acid as described herein.
In one aspect, the disclosure is related to a method of producing an anti-TPBG/MET antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell as described herein under conditions sufficient for the cell to produce the anti-TPBG/MET antibody or the antigen-binding fragment thereof; and (b) collecting the anti-TPBG/MET antibody or the antigen-binding fragment thereof produced by the cell.
In one aspect, the disclosure is related to an anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.
In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein, to the subject. In some embodiments, the subject has a cancer expressing TPBG and/or MET (e.g., both TPBG and MET) . In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, solid tumor, renal cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering an anti-PD1 antibody to the subject. In some embodiments, the method further comprises administering a chemotherapy to the subject.
In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a  composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein.
In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof or the anti-TPBG/MET antibody-drug conjugate as described herein.
In one aspect, the disclosure is related to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) the anti-TPBG/MET antibody or antigen-binding fragment thereof as described herein, and/or (b) the anti-TPBG/MET antibody-drug conjugate as described herein.
In one aspect, the disclosure is related to an anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic agent is MMAE or MMAF.
In some embodiments, the therapeutic agent is selected from
In some embodiments, the therapeutic agent is linked to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker has a structure of:
In some embodiments, the antibody-drug conjugate has a structure of:
in some embodiments, n =1-8; in some embodiments, “Ab” represents the antibody or antigen-binding fragment thereof.
In some embodiments, the drug-to-antibody ratio (DAR) is about 4 or 8.
As used herein, the term “antigen-binding domain” refers to one or more protein domain (s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids  from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen (s) (e.g., an effector antigen or control antigen) . In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. In some embodiments, an antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is a VHH. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art. In some examples, an antigen-binding domain can bind to a single antigen (e.g., one of an effector antigen and a control antigen) . In other examples, an antigen-binding domain can bind to two different antigens (e.g., an effector antigen and a control antigen) .
As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’ , F (ab’ ) 2, and Fv fragments.
As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) derived from a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) . In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) . In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .
As used herein, the term “multispecific antibody” is an antibody that includes two or more different antigen-binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins  present on the surface of the same cell or present on the surface of different cells) . In some aspects, a multispecific antibody binds two different epitopes (i.e., a “bispecific antibody” ) . In some aspects, a multispecific antibody binds three different epitopes (i.e., a “trispecific antibody” ) . In some aspects, a multispecific antibody binds four different epitopes (i.e., a “quadspecific antibody” ) . In some aspects, a multispecific antibody binds five different epitopes (i.e., a “quintspecific antibody” ) . Each binding specificity may be present in any suitable valency. Non-limiting examples of multispecific antibodies are described herein.
As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.
As used herein, the term “common light chain” refers to a light chain that can interact with two or more different heavy chains, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. Similarly, the term “common light chain variable region” refers to a light chain variable region that can interact with two or more different heavy chain variable regions, forming different antigen-binding sites, wherein these different antigen-binding sites can specifically bind to different antigens or epitopes. In some embodiments, the antibody or antigen-binding fragment thereof can have a common light chain. In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment thereof can have a common light chain variable region.
As used herein, the term “anti-TPBG/MET antibody or antigen-binding fragment thereof” refers to an antibody or antigen-binding fragment that binds to both MET and TPBG.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1A lists heavy chain variable region and light chain variable region CDR sequences of anti-TPBG antigen binding domain (32G1) in anti-TPBG/MET antibodies as defined by Kabat definition.
FIG. 1B lists heavy chain variable region and light chain variable region CDR sequences of anti-MET antigen binding domain (8H10, 8D9, and 2F11) in anti-TPBG/MET antibodies as defined by Kabat definition.
FIG. 2A lists heavy chain variable region and light chain variable region CDR sequences of anti-TPBG antigen binding domain (32G1) in anti-TPBG/MET antibodies as defined by Chothia definition.
FIG. 2B lists heavy chain variable region and light chain variable region CDR sequences of anti-MET antigen binding domain (8H10, 8D9, and 2F11) in anti-TPBG/MET antibodies as defined by Chothia definition.
FIG. 3 lists amino acid sequences of heavy chain variable regions and light chain variable regions of anti-TPBG/MET antibodies.
FIG. 4 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) , and were treated with phosphate buffer saline (PBS) or ADCs.
FIGS. 5A-5B show the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) , and were treated with PBS or ADCs. FIG. 5B shows partially enlarged results of FIG. 5A.
FIG. 6 shows the average tumor volumes in different groups of B-NDG mice that were injected with NCI-H1975 cells, and were treated with PBS or ADCs.
FIG. 7 shows the average tumor volumes in different groups of B-NDG mice that were injected with NUGC-4 cells, and were treated with PBS or ADCs.
FIG. 8 lists certain amino acid sequences discussed in the disclosure.
FIG. 9 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) , and were treated with PBS or ADCs.
FIGs. 10A-10B show the endocytosis rates of anti-TPBG antibody, anti-MET antibodies or anti-TPBG/MET bispecific antibodies in NCI-H226 cells (FIG. 10A) or NCI-H2030 cells (FIG. 10B) .
FIGs. 11A-11C show the binding activity of anti-TPBG antibody, anti-MET antibodies or anti-TPBG/MET bispecific antibodies to HCC827 cells (FIG. 11A) , NCI-H226 cells (FIG. 11B) , or NCI-H2030 cell (FIG. 11C) .
FIG. 12 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived pancreatic tumor fragments (2 mm × 2 mm × 2 mm) , and were treated with PBS or ADCs.
FIG. 13 shows the average tumor volumes in different groups of B-NDG mice that were engrafted with patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) , and were treated with PBS or ADCs.
DETAILED DESCRIPTION
A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens) . In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms.  Each arm can have one heavy chain variable region and one light chain variable region, forming an antigen-binding domain (or an antigen-binding region) . In some embodiments, the bispecific antibody has a common light chain.
The present disclosure relates to anti-TPBG/MET antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to TPBG and MET, and antibody drug conjugates derived from these anti-TPBG/MET antibodies.
Anti-TPBG/MET Antibody
Trophoblast glycoprotein (TPBG) , also known as 5T4, Wnt-Activated Inhibitory Factor 1, or WAIF1, is expressed by many different cancers but rarely in normal adult tissues. TPBG expression is associated with the directional movement of cells through epithelial mesenchymal transition, potentiation of CXCL12/CXCR4 chemotaxis and inhibition of canonical Wnt/beta-catenin while favoring non-canonical pathway signaling; all processes which help drive the spread of cancer cells. The selective pattern of TPBG tumor expression, association with a tumor-initiating phenotype plus a mechanistic involvement with cancer spread have underwritten the clinical development of different immunotherapeutic strategies including a vaccine, a tumor-targeted superantigen and an antibody drug conjugate.
TPBG was discovered in the context of trying to identify shared cell surface molecules that may function to allow survival of the fetus as a semi-allograft in the mother, or a tumor in its host. The rationale was that such shared expression would reflect common functions relevant to growth, invasion or altered immune surveillance in the host. Murine monoclonal antibodies were raised against purified glycoproteins from trophoblast membrane preparations from term human placenta and initially screened against different cancer cell lines and human peripheral blood mononuclear cells. Further screening using the TPBG monoclonal antibody (mAb) by immunohistochemistry indicated the antigen was expressed by many different cancers but with a restricted normal tissue distribution.
A series of biochemical and genomic studies identified TPBG molecules as N-glycosylated proteins with an apparent molecular size of 72 kD and are encoded on chromosome 6q14-15. The human gene encodes a 42 kD transmembrane protein core which contains several LRRs that are associated with protein–protein interactions of a functionally diverse set of molecules. The extracellular part of the molecule has several LRRs in two domains separated by a short hydrophilic sequence; there is a transmembrane domain and a short cytoplasmic sequence. Importantly, the TPBG-specific mAb recognizes a conformationally dependent epitope that relies on the integrity of the intramolecular S–Sbonds and the indirect presence of the complex N-linked glycosylation. When human TPBG was overexpressed in murine fibroblasts, the cells became more spindle shaped and had reduced adherence while in normal epithelial cells there was E-Cadherin down-regulation, increased motility and cytoskeletal disruption.
Immunohistochemistry (IHC) of frozen sections established that the TPBG-specific monoclonal antibody detected antigen expression by many different types of carcinoma but  demonstrated only low-level expression in some normal adult tissue epithelia. Importantly, TPBG was expressed in many different primary and metastatic cancers, frequently at high levels; in some cases, there was an additional stromal expression. Linking a role for TPBG in tumor development and spread, it has been shown that TPBG expression in colorectal, gastric and ovarian cancers correlates with poorer clinical outcome.
Overexpression of TPBG in normal murine epithelial cells is associated with E-cadherin down-regulation which is a key component of EMT; this occurs during embryonic development and is important for the metastatic spread of epithelial tumors. TPBG was shown to be a marker of the early differentiation of mouse and human embryonic stem (ES) cell, and this process involves an E-to N-cadherin switch, upregulation of E-cadherin repressor molecules (Snail and Slug proteins) , increased matrix metalloproteinase (MMP-2 and MMP-9) activity and motility, all classic EMT features. Undifferentiated knock-out (KO) E-Cadherin ES cells constitutively express cell surface TPBG molecules while antibody induced down-regulation of E-Cadherin in ES cells induced TPBG membrane expression, increased motility, altered actin cytoskeleton arrangement and a mesenchymal phenotype. These observations are consistent with E-cadherin somehow preventing TPBG cell surface expression; the possible mechanism being through stabilization of cortical actin cytoskeletal organization. Co-expression of TPBG and factors involved in the epithelial-to-mesenchymal transition was also observed in undifferentiated but not in differentiated lung tumor cells.
TPBG molecules have been shown to be involved in the functional expression of CXCR4 at the cell surface in some embryonic and tumor cells. Both CXCL12 and CXCR4 expression have been associated with tumorigenesis in many cancers, and it is believed that CXCR4 expression facilitates the spread to tissues that highly express CXCL12 including lung, liver, lymph nodes and bone marrow. TPBG is expressed by putative leukemia initiating cells in BCP-ALL, and these cells show the associated property of CXCL12/CXCR4 chemotaxis. TPBG-positive leukemia-initiating cells are likely attracted by CXCL12 produced by extramedullary sites where there is decreased therapeutic bioavailability leading to disease relapse following treatment.
Wnt protein intracellular signaling is a central component of many aspects of cellular regulation critical to normal development, homoeostasis and regeneration, while misregulation can lead to disease, including cancer. There are two pathways, the most characterized being the canonical Wnt/β-catenin pathway while non-canonical Wnt signaling through a cell autonomous planar cell polarity (PCP) type pathway can drive the modulation of actin and microtubular skeletons facilitating cell movement in development of cancer. TPBG has been shown to interfere with Wnt/β-catenin signaling and concomitantly activate non-canonical Wnt pathways. TPBG binds to the Wnt co-receptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation thereby modulating Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, TPBG enhances β-catenin-independent Wnt signaling in promoting a non-canonical function of Dickkopf1. These results suggest that TPBG facilitates pathway selection in Wnt-receiving cells, inhibiting Wnt/β- catenin canonical while concomitantly activating the non-canonical Wnt signaling pathway associated with increased motility. resolution crystal of the extracellular domain of TPBG and associated cell biological studies have provided a structural basis for inhibition of Wnt/β-catenin signaling.
A detailed review of TPBG and its functions can be found in Stern, P. L., et al. "5T4 oncofetal antigen: an attractive target for immune intervention in cancer. " Cancer Immunology, Immunotherapy 66.4 (2017) : 415-426; and Harrop, R., et al. "Cancer stem cell mobilization and therapeutic targeting of the 5T4 oncofetal antigen. " Therapeutic Advances in Vaccines and immunotherapy 7 (2019) : 2515135518821623; each of which is incorporated by reference in its entirety.
MET, also called c-Met, tyrosine-protein kinase Met, or hepatocyte growth factor receptor (HGFR) , is a protein that in humans is encoded by the MET gene. The protein possesses tyrosine kinase activity. The primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor. Activation of MET by its ligand hepatocyte growth factor (HGF) stimulates a plethora of cell processes including growth, motility, invasion, metastasis, epithelial-mesenchymal transition, angiogenesis/wound healing, and tissue regeneration. The exact stoichiometry of HGF: MET binding is unclear, but it is generally believed that two HGF molecules bind to two MET molecules leading to receptor dimerization and autophosphorylation at tyrosines 1230, 1234, and 1235. Ligand-independent MET autophospliorylation can also occur due to gene amplification, mutation or receptor over-expression.
MET is frequently amplified, mutated or over-expressed in many types of cancer including gastric, lung, colon, breast, bladder, head and neck, ovarian, prostate, thyroid, pancreatic, and CNS cancers. Missense mutations typically localized to the kinase domain are commonly found in hereditary papillary renal cell carcinomas (PRCC) and in 13%of sporadic PRCCs, MET mutations localized to the semaphorin or juxtamembrane domains of MET are frequently found in gastric, head and neck, liver, ovarian, NSCLC and thyroid cancers. MET amplification has been detected in brain, colorectal, gastric, and lung cancers, often correlating with disease progression. Up to 4%and 20%of non-small cell lung cancer (NSCLC) and gastric cancers, respectively, exhibit MET amplification. MET overexpression is also frequently observed in lung cancer. Moreover, in clinical samples, nearly half of lung adenocarcinomas exhibited high levels of MET and HGF, both of which correlated with enhanced tumor growth rate, metastasis and poor prognosis.
Nearly 60%of all tumors that become resistant to EGFR tyrosine kinase inhibitors increase MET expression, amplify MET, or increase MET only known ligand, HGF, suggesting the existence of a compensatory pathway for EGFR through MET. MET amplification was first identified in cultured cells that became resistant to gefitinib, an EGFR kinase inhibitor, and exhibited enhanced survival through the HER3 pathway. This was further validated in clinical samples where nine of 43 patients with acquired resistance to either erlotinib or gefitinib exhibited MET amplification.
Aberrant MET signaling has been implicated in the development/progression of many human cancers. This results from the overexpression of MET, activating mutations in MET, transactivation, autocrine or paracrine signaling, or MET gene amplification. Binding of HGF to MET stimulates receptor dimerization, autophosphorylation, activation of the receptor's internal, cytoplasmic tyrosine kinase domain, and initiation of multiple signal transduction and transactivation pathways involved in regulation of DNA synthesis (gene activation) and cell cycle progression or division, inhibition of MET signaling may result in inhibition of one or more MET downstream signaling pathways and therefore neutralizing MET may have various effects, including inhibition of cell proliferation and differentiation, angiogenesis, cell motility and metastasis.
A detailed review of MET and its functions can be found in Huang, X., et al. "Targeting the HGF/MET axis in cancer therapy: challenges in resistance and opportunities for improvement. " Frontiers in Cell and Developmental Biology 8 (2020) : 152; and Santarpia, M., et al. "A narrative review of MET inhibitors in non-small cell lung cancer with MET exon 14 skipping mutations. " Translational Lung Cancer Research 10.3 (2021) : 1536; each of which is incorporated by reference in its entirety.
In some embodiments, the bispecific anti-TPBG/MET antibody described herein can be designed to have an IgG1 subtype structure with knobs-into-holes (KIH) mutations, which can promote heterodimerization and avoid mispairing between the two heavy chains. In some embodiments, the bispecific anti-TPBG/MET antibody has a higher endocytosis rate than the corresponding monoclonal antibodies or the control bispecific antibodies.
In some embodiments, the bispecific anti-TPBG/MET antibody described herein can be conjugated with a therapeutic agent, forming an antibody drug conjugate (ADC) . In some embodiments, the drug-to-antibody ratio (DAR) of the ADCs described herein is about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7. In some embodiments, the DAR of the ADCs described herein is about 3.5 to about 4.5, about 3.6 about 4.5, about 3.7 to about 4.5, about 3.8 to about 4.5, about 3.9 to about 4.5, about 4.0 to about 4.5, about 4.1 to about 4.5, about 4.2 to about 4.5, about 4.3 to about 4.5, about 4.4 to about 4.5, about 3.5 to about 4.4, about 3.6 to about 4.4, about 3.7 to about 4.4, about 3.8 to about 4.4, about 3.9 to about 4.4, about 4.0 to about 4.4, about 4.1 to about 4.4, about 4.2 to about 4.4, about 4.3 to about 4.4, about 3.5 to about 4.3, about 3.6 to about 4.3, about 3.7 to about 4.3, about 3.8 to about 4.3, about 3.9 to about 4.3, about 4.0 to about 4.3, about 4.1 to about 4.3, about 4.2 to about 4.3, about 3.5 to about 4.2, about 3.6 to about 4.2, about 3.7 to about 4.2, about 3.8 to about 4.2, about 3.9 to about 4.2, about 4.0 to about 4.2, about 4.1 to about 4.2, about 3.5 to about 4.1, about 3.6 to about 4.1, about 3.7 to about 4.1, about 3.8 to about 4.1, about 3.9 to about 4.1, about 4.0 to about 4.1, about 3.5 to about 4.0, about 3.6 to about 4.0, about 3.7 to about 4.0, about 3.8 to about 4.0, about 3.9 to about 4.0, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6. In some embodiments, the DAR of the ADCs described  herein is about 7.5 to about 8.5, about 7.6 to about 8.5, about 7.7 to about 8.5, about 7.8 to about 8.5, about 7.9 to about 8.5, about 8.0 to about 8.5, about 8.1 to about 8.5, about 8.2 to about 8.5, about 8.3 to about 8.5, about 8.4 to about 8.5, about 7.5 to about 8.4, about 7.6 to about 8.4, about 7.7 to about 8.4, about 7.8 to about 8.4, about 7.9 to about 8.4, about 8.0 to about 8.4, about 8.1 to about 8.4, about 8.2 to about 8.4, about 8.3 to about 8.4, about 7.5 to about 8.3, about 7.6 to about 8.3, about 7.7 to about 8.3, about 7.8 to about 8.3, about 7.9 to about 8.3, about 8.0 to about 8.3, about 8.1 to about 8.3, about 8.2 to about 8.3, about 7.5 to about 8.2, about 7.6 to about 8.2, about 7.7 to about 8.2, about 7.8 to about 8.2, about 7.9 to about 8.2, about 8.0 to about 8.2, about 8.1 to about 8.2, about 7.5 to about 8.1, about 7.6 to about 8.1, about 7.7 to about 8.1, about 7.8 to about 8.1, about 7.9 to about 8.1, about 8.0 to about 8.1, about 7.5 to about 8.0, about 7.6 to about 8.0, about 7.7 to about 8.0, about 7.8 to about 8.0, about 7.9 to about 8.0, about 7.5 to about 7.9, about 7.6 to about 7.9, about 7.7 to about 7.9, about 7.8 to about 7.9, about 7.5 to about 7.8, about 7.6 to about 7.8, about 7.7 to about 7.8, about 7.5 to about 7.7, about 7.6 to about 7.7, or about 7.5 to about 7.6.
In some embodiments, the anti-TPBG/MET ADC described herein can effectively inhibit in vitro cancer cell growth at a concentration of less than 10 μg/mL, less than 3.33 μg/mL, less than 1.11 μg/mL, less than 0.37 μg/mL, less than 0.12 μg/mL, less than 0.04 μg/mL, or less than 0.01 μg/mL. In some embodiments, the anti-TPBG/MET ADC described herein can inhibit in vivo cancer cell growth (e.g., lung cancer, gastric cancer, or skin cancer) in a xenograft mouse model at a dose level of less than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1.5 mg/kg, or 1 mg/kg.
In some embodiments, the anti-TPBG/MET antibody described herein has a common light chain. In some embodiments, the anti-TPBG/MET antibody includes an anti-TPBG antigen-binding domain (e.g., 32G1) or an anti-MET antigen-binding domain (e.g., 8H10, 8D9, or 2F11) . In some embodiments, the anti-TPBG/MET antibodies have a heavy chain variable region targeting TPBG (e.g., any one of the VH targeting TPBG described herein) , a heavy chain variable region targeting MET (e.g., any one of the VH targeting MET described herein) , and two identical common light chain variable regions.
The CDR sequences for 32G1 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 4-6, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3 as defined by Kabat definition. The CDRs can also be defined by Chothia definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 13-15, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 32G1 are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively.
The CDR sequences for 8H10 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 16-18, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human light chain variable region and human  heavy chain variable region for 8H10 are shown in SEQ ID NO: 22 and SEQ ID NO: 24, respectively.
The CDR sequences for 8D9 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 10-12, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 19-21, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 8D9 are shown in SEQ ID NO: 22 and SEQ ID NO: 25, respectively.
The CDR sequences for 2F11 antigen-binding domain include CDRs of the heavy chain variable domain, SEQ ID NOs: 37-39, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 40-42, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 1-3. The human light chain variable region and human heavy chain variable region for 2F11 are shown in SEQ ID NO: 22 and SEQ ID NO: 43, respectively.
In some embodiments, the anti-TPBG/MET antibodies described herein can contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 37-39, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, and SEQ ID NOs: 40-42; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3.
In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions;  SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions;  SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
In some embodiments, the anti-TPBG/MET antibodies contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 23, 24, 25, or 43, and the selected VL sequence is SEQ ID NO: 22.
In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment can have 3 VL CDRs that are identical to the CDRs of any VL sequences as described herein.
The disclosure also provides nucleic acid comprising a polynucleotide encoding an anti-TPBG/MET antibody. The immunoglobulin heavy chain or immunoglobulin light chain in the anti-TPBG/MET antibody comprises CDRs as shown in FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to MET and/or TPBG.
The anti-TPBG/MET antibodies can also be anti-TPBG/MET antibody variants (including derivatives and conjugates) of anti-TPBG/MET antibodies or antibody fragments. Additional anti-TPBG/MET antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The anti-TPBG/MET antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the anti-TPBG/MET antibody or antigen-binding fragment is an IgG (e.g., IgG1) antibody or antigen-binding fragment thereof.
Fragments of anti-TPBG/MET antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity to both MET and TPBG. Thus, a fragment of an anti-TPBG/MET antibody will retain an ability to bind to MET and TPBG.
Antibodies and Antigen Binding Fragments thereof
In some embodiments, the multispecific anti-TPBG/MET antibody (e.g., bispecific antibody) includes an antigen-binding domain that is derived from an anti-TPBG antibody, and an antigen-binding domain that is derived from an anti-MET antibody. These anti-TPBG/MET antibodies and antigen-binding fragments thereof can have various forms.
In general, antibodies (also called immunoglobulins) can be made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting anti-TPBG/MET antibody of  the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the anti-TPBG/MET antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.
The hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.
Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991) ; Morea et al., Biophys Chem. 68 (1-3) : 9-16 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan . 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety.
The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
In some embodiments, the anti-TPBG/MET antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.
The anti-TPBG/MET antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, or camelid) . The antigen-binding domain or antigen binding fragment is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab’, F (ab’) 2, and variants of these fragments. Thus, in some embodiments, an anti-TPBG/MET antibody or antigen binding fragment thereof can comprise e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen-binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
In some embodiments, the scFv in an anti-TPBG/MET antibody has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the anti-TPBG/MET scFv has two antigen binding regions (Antigen binding regions: A and B) , and the two antigen binding regions can bind to the respective target antigens with different affinities.
In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can comprises one, two, or three heavy chain variable region CDRs selected from FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B.
In some embodiments, the anti-TPBG/MET antibodies described herein can be conjugated to a therapeutic agent. The anti-TPBG/MET antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) . In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, e.g., a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.
In some embodiments, the anti-TPBG/MET antibody is a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first  antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
Any of the anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an anti-TPBG/MET antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
The anti-TPBG/MET antibodies or antigen-binding fragments thereof can also have various forms. Many different formats of bispecific antibodies or antigen-binding fragments thereof are known in the art, and are described e.g., in Suurs, et al. "Areview of bispecific antibodies and antibody constructs in oncology and clinical challenges, " Pharmacology &therapeutics (2019) , which is incorporated herein by reference in the entirety.
In some embodiments, the anti-TPBG/MET antibody is a BiTe, a (scFv) 2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. In some embodiments, the anti-TPBG/MET antibody is a VHH-scAb, a VHH-Fab, a Dual scFab, a F (ab’) 2, a diabody, a crossMab, a DAF (two-in-one) , a DAF (four-in-one) , a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, a DVD-IgG, a IgG (H) -scFv, a scFv- (H) IgG, IgG (L) -scFv, scFv-(L)IgG, IgG (L, H) -Fv, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F (ab’) 2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a lmmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFv1-PEG-scFv2.
In some embodiments, the anti-TPBG/MET antibody can be a TrioMab. In a TrioMab, the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.
In some embodiments, the anti-TPBG/MET antibody has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knobs-into-holes or some other heavy chain pairing technique.
In some embodiments, CrossMAb technique can be used produce bispecific anti-TPBG/MET antibodies. CrossMAb technique can be used enforce correct light chain association in bispecific heterodimeric IgG antibodies, this technique allows the generation of various  bispecific antibody formats, including bi- (1+1) , tri- (2+1) and tetra- (2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) -based antibodies. These formats can be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association. The method is described in Klein et al., "The use of CrossMAb technology for the generation of bi-and multispecific antibodies. " MAbs. Vol. 8. No. 6. Taylor &Francis, 2016, which is incorporated by reference in its entirety. In some embodiments, the CH1 in the heavy chain and the CL domain in the light chain are swapped.
The anti-TPBG/MET antibody can be a Duobody. The Fab-exchange mechanism naturally occurring in IgG4 antibodies is mimicked in a controlled matter in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy-light chains.
In Dual-variable-domain antibody (DVD-Ig) , additional VH and variable light chain (VL) domain are added to each N-terminus for bispecific targeting. This format resembles the IgG-scFv, but the added binding domains are bound individually to their respective N-termini instead of a scFv to each heavy chain N-terminus.
In scFv-IgG, the two scFv are connected to the C-terminus of the heavy chain (CH3) . The scFv-IgG format has two different bivalent binding sites and is consequently also called tetravalent. There are no heavy-chain and light-chain pairing problem in the scFv-IgG.
In some embodiments, the anti-TPBG/MET antibody can have a IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.
The anti-TPBG/MET antibody can also have a Fab-scFv-Fc format. In Fab-scFv-Fc format, a light chain, heavy chain and a third chain containing the Fc region and the scFv are assembled. It can ensure efficient manufacturing and purification.
In some embodiments, the anti-TPBG/MET antibody can be a TF. Three Fab fragments are linked by disulfide bridges. Two fragments target the tumor associated antigen (TAA) and one fragment targets a hapten. The TF format does not have an Fc region.
ADAPTIR has two scFvs bound to each side of an Fc region. It abandons the intact IgG as a basis for its construct, but conserves the Fc region to extend the half-life and facilitate purification.
Dual affinity retargeting (DART) has two peptide chains connecting the opposite fragments, thus VLA with VHB and VLB with VHA, and a sulfur bond at their C-termini fusing them together. In DART, the sulfur bond can improve stability over BiTEs.
In DART-Fc, an Fc region is attached to the DART structure. It can be generated by assembling three chains, two via a disulfide bond, as with the DART. One chain contains half of the Fc region which will dimerize with the third chain, only expressing the Fc region. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.
In tetravalent DART, four peptide chains are assembled. Basically, two DART molecules are created with half an Fc region and will dimerize. This format has bivalent binding to both targets, thus it is a tetravalent molecule.
Tandem diabody (TandAb) comprises two diabodies. Each diabody consists of an VHA and VLB fragment and a VHA and VLB fragment that are covalently associated. The two diabodies are linked with a peptide chain. It can improve stability over the diabody consisting of two scFvs. It has two bivalent binding sites.
The ScFv-scFv-toxin includes toxin and two scFv with a stabilizing linker. It can be used for specific delivery of payload.
In some embodiments, the anti-TPBG/MET antibody is a bispecific antibody. In some embodiments, the bispecific antibody in present disclosure is designed to be 1+1 (monovalent for each target) and has an IgG1 subtype structure. This can reduce the avidity to cells with low expression levels of TPBG and MET, and increase the avidity to cells that co-express TPBG and MET, to achieve enhanced targeting function.
In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof have a light chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 26, and a heavy chain constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 27 and 28.
In some embodiments, the anti-TPBG/MET antibodies include KIH mutations. In some embodiments, the anti-TPBG/MET antibody includes a first antigen-binding domain that specifically binds to TPBG, and a second antigen-binding domain that specifically binds to MET. In some embodiments, the first antigen-binding domain includes a heavy chain that including one or more knob mutations (aknob heavy chain) , and the second antigen-binding domain includes a heavy chain including one or more hole mutations (ahole heavy chain) . In some embodiments, the first antigen-binding domain includes a heavy chain that includes one or more hole mutations (ahole heavy chain) , and the second antigen-binding domain that includes a heavy chain including one or more knob mutations (aknob heavy chain) . In some embodiments, the anti-TPBG/MET antibody includes a knob heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 27. In some embodiments, the anti-TPBG/MET antibody includes a hole heavy chain comprising a constant region that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 28.
Antibody Characteristics
The anti-TPBG/MET antibodies can include an anti-TPBG antigen-binding domain and any anti-MET antigen-binding domain as described herein. In some embodiments, the anti-TPBG/MET antibodies, or antigen-binding fragments thereof can initiate CDC or ADCC.
The disclosure provides anti-TPBG/MET antibodies and antigen-binding fragments thereof that can specifically bind to TPBG. The anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can block the binding between TPBG and its ligands (e.g., GIPC PDZ domain containing family, member 1 (GIPC1) ) . In some embodiments, by binding to TPBG or blocking the TPBG interaction with its ligands, the anti-TPBG/MET antibody can inhibit TPBG-associated signaling pathways, thus treating cancer. Thus, in some embodiments, the anti-TPBG/MET antibody described herein is a TPBG agonist. In some embodiments, the anti-TPBG/MET antibody described herein is a TPBG antagonist.
General techniques can be used to measure the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some implementations, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to TPBG (e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG) with a dissociation rate (koff) of less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, or less than 0.00001 s- 1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s- 1, greater than 0.0001 s-1, greater than 0.00001 s-1, or greater than 0.000001 s-1.
In some embodiments, kinetic association rates (kon) is greater than 1 × 102/Ms, greater than 1 × 103/Ms, greater than 1 × 104/Ms, greater than 1 × 105/Ms, or greater than 1 × 106/Ms. In some embodiments, kinetic association rates (kon) is less than 1 × 105/Ms, less than 1 × 106/Ms, or less than 1 × 107/Ms.
In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to TPBG (e.g., human TPBG, monkey TPBG, mouse TPBG, and/or chimeric TPBG) with a KD of less than 1 × 10-6 M, less than 1 × 10-7 M, less than 1 × 10-8 M, less than 1 ×10-9 M, or less than 1 × 10-10 M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 × 10-7 M, greater than 1 × 10-8 M, greater than 1 × 10-9 M, or greater than 1 × 10-10 M.
The anti-TPBG/MET antibodies or antigen-binding fragments thereof can also include an antigen-binding domain that can specifically bind to MET. The anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can block the binding between MET and its ligands (e.g., Hepatocyte growth factor (HGF) ) . In some embodiments, by binding to MET or blocking the MET/HGF interaction, the anti-TPBG/MET antibody can inhibit MET-associated signaling pathways, thus treating cancer. Thus, in some embodiments, the anti-TPBG/MET antibody described herein is a MET agonist. In some embodiments, the anti-TPBG/MET antibody described herein is a MET antagonist.
In some implementations, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to MET (e.g., human MET, monkey MET, mouse MET, and/or chimeric MET) with a dissociation rate (koff) of less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, or less than 0.00001 s-1. In some embodiments, the dissociation rate (koff) is greater  than 0.01 s-1, greater than 0.001 s-1, greater than 0.0001 s-1, greater than 0.00001 s-1, or greater than 0.000001 s-1.
In some embodiments, kinetic association rates (kon) is greater than 1 × 102/Ms, greater than 1 × 103/Ms, greater than 1 × 104/Ms, greater than 1 × 105/Ms, or greater than 1 × 106/Ms. In some embodiments, kinetic association rates (kon) is less than 1 × 105/Ms, less than 1 × 106/Ms, or less than 1 × 107/Ms.
Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 × 10-6 M, less than 1 × 10-7 M, less than 1 × 10-8 M, less than 1 × 10-9 M, or less than 1 × 10-10 M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.In some embodiments, KD is greater than 1 × 10-7 M, greater than 1 × 10-8 M, greater than 1 × 10-9 M, or greater than 1 × 10-10 M.
Because the anti-TPBG/MET antibody (e.g., bispecific antibody) binds to both MET and TPBG, for cells that express both MET and TPBG, the antibody has a higher binding affinity to these cells. Avidity can be used to measure the binding affinity of an antibody to these cells. Avidity is the accumulated strength of multiple affinities of individual non-covalent binding interactions.
Thermal stabilities can also be determined. The anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2. The presence of these two peaks often indicate the denaturation of the Fc domains (Tm D1) and Fab domains (Tm D2) , respectively. When there are two peaks, Tm usually refers to Tm D2. Thus, in some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.
In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to human TPBG or monkey TPBG. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof cannot bind to human TPBG or monkey TPBG. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof can bind to human MET or monkey MET. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof cannot bind to human MET or monkey MET.
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity that is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by HPLC.
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity of above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96%, above 97%, or above 98%, as determined by size exclusion chromatography (SEC) . In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a hydrophobic interaction chromatography (HIC) retention time that is longer than 2 minutes, longer than 3 minutes, longer than 4 minutes, longer than 5 minutes, longer than 6 minutes, longer than 7 minutes, or longer than 8 minutes. In some embodiments, the HIC retention time is less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, less than 6 minutes, less than 7 minutes, or less than 8 minutes.
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a purity of above 85%, above 86%, above 87%, above 88%, above 89%, above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96%, above 97%, above 98%, or above 99%, as determined by capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) .
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a main peak that constitutes more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%of the total sample, as determined by capillary isoelectric focusing (cIEF) . In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has an acidic peak or alkaline peak that constitutes less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, or less than 75%of the total sample, as determined by capillary isoelectric focusing (cIEF) .
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. The TGI (%) can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the treatment starts. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula:
TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100%
Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC has a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
In some embodiments, the anti-TPBG/MET antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the anti-TPBG/MET antibodies or antigen-binding fragments thereof are Fab, Fab’, F (ab’) 2, and Fv fragments. In some embodiments, the anti-TPBG/MET antibodies or antigen-binding fragments thereof as described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
Some other modifications to the Fc region can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and/or in vivo.
In some embodiments, the IgG4 has S228P mutation (EU numbering) . The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.
In some embodiments, anti-TPBG/MET antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, 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 glycostructures 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; or position 314 in Kabat numbering) ; 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. In some embodiments, to reduce glycan heterogeneity, the Fc region of the anti-TPBG/MET antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
In some embodiments, the main peak of HPLC-SEC accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100%of the protein complex described  herein after purification by protein A-based affinity chromatography and/or size-exclusive chromatography.
In some embodiments, the bispecific anti-TPBG/MET antibody described herein has a higher endocytosis rate than the corresponding monoclonal antibodies and/or control bispecific antibodies described herein. In some embodiments, the anti-TPBG/MET antibody described herein has a higher endocytosis rate than Telisotuzumab analog and/or PF06263507 analog.
Antibody Drug Conjugates (ADC)
The anti-TPBG/MET antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent (adrug) . The therapeutic agent can be covalently or non-covalently bind to the anti-TPBG/MET antibody. In some embodiments, the anti-TPBG/MET antibody is an anti-TPBG/MET bispecific antibody. In some embodiments, the bispecific antibody has a common light chain.
In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) . Useful classes of cytotoxic, cytostatic, or immunomodulatory agents include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.
In some embodiments, the therapeutic agent can include, but not limited to, cytotoxic reagents, such as chemo-therapeutic agents, immunotherapeutic agents and the like, antiviral agents or antimicrobial agents. In some embodiments, the therapeutic agent to be conjugated can be selected from, but not limited to, MMAE (monomethyl auristatin E) , MMAD (monomethyl auristatin D) , or MMAF (monomethyl auristatin F) .
Definitions of specific functional groups and chemical terms are described in more detail below. For purpose of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition, inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5th Edition, John Wiley &Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
All ranges cited herein are inclusive, unless expressly stated to the contrary. When a range of values is listed, it is intended to encompass each value and sub-range within the range.  For example, “C1-6” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6.
The compounds or any formula depicting and describing the compounds of the present disclosure may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds or any formula depicting and describing the compounds of the present invention. Centers of asymmetry that are present in the compounds or any formula depicting and describing the compounds of the present invention can all independently of one another have (R) or (S) configuration. When bonds to a chiral carbon are depicted as straight lines in the structural formulas, or when a compound name is recited without an (R) or (S) chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of each such chiral carbon, and hence each enantiomer or diastereomer and mixtures thereof, are embraced within the formula or by the name.
The disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios. Thus, enantiomers are a subject of the disclosure in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism the disclosure includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Alternatively, absolute stereochemistry may be determined by Vibrational Circular Dichroism (VCD) spectroscopy analysis.
Unless otherwise stated, the structures depicted herein are also meant to include the compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, the compounds wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Such compounds are referred to as a “isotopic variant” . The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds or any formula depicting and describing the compounds of the present invention. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but not limited to, isotopes of hydrogen, such as 2H (i.e., D) and 3H; carbon, such as 11C, 13C, and 14C; chlorine, such as 36Cl; fluorine, such as 18F; iodine, such as 123I and 125I; nitrogen, such as 13N and 15N; oxygen, such as 15O, 17O, and 18O; phosphorus, such as 32P; and sulfur, such as 35S. Certain isotopic variants of the compounds or any formula depicting and describing the  compounds of the present disclosure, for example those incorporating a radioactive isotope, may be useful in drug and/or substrate tissue distribution studies. Particularly, compounds having the depicted structures that differ only in the replacement with heavier isotopes, such as the replacement of hydrogen by deuterium (2H, or D) , can afford certain therapeutic advantages, for example, resulting from greater metabolic stability, increased in vivo half-life, or reduced dosage requirements and, hence, may be utilized in some particular circumstances. Isotopic variants of compounds or any formula depicting and describing the compounds of the present disclosure can generally be prepared by techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and synthesis using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
The compounds as provided herein are described with reference to both generic formulas and specific compounds. In addition, the compounds of the present disclosure may exist in a number of different forms or derivatives, all within the scope of the disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, solvated forms, different crystal forms or polymorphs, and active metabolites, etc.
As used herein, the term “pharmaceutically acceptable salt” , unless otherwise stated, includes salts that retain the biological effectiveness of the free acid/base form of the specified compound and that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. In cases where the compounds of the present disclosure contain one or more acidic or basic groups, the disclosure also comprises their corresponding pharmaceutically acceptable salts. Thus, the compounds of the present invention which contain acidic groups, such as carboxyl groups, can be present in salt form, and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts or as ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts are readily available, for instance, by reacting the compound having an acidic group with a suitable base, e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of compounds of the present disclosure include but are not limited to copper (I) , copper (II) , iron (II) , iron (III) , manganese (II) , and zinc salts. Compounds of the present disclosure which contain one or more basic groups, e.g., groups which can be protonated, can be present in salt form, and can be used according to the disclosure in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic  acid, fumaric acid, malonic acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. The salts which are formed are, inter alia, hydrochlorides, chlorides, hydrobromides, bromides, iodides, sulfates, phosphates, methanesulfonates (mesylates) , tosylates, carbonates, bicarbonates, formates, acetates, sulfoacetates, triflates, oxalates, malonates, maleates, succinates, tartrates, malates, embonates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates, and glutamates. The stoichiometry of the salts formed from the compounds of the disclosure may moreover be an integral or non-integral multiple of one.
Compounds of the present disclosure which contain basic nitrogen-containing groups can be quaternized using agents such as C1-4alkyl halides, for example, methyl, ethyl, isopropyl, and tert-butyl chloride, bromide, and iodide; diC1-4alkyl sulfates, for example, dimethyl, diethyl, and diamyl sulfate; C10-18alkyl halides, for example, decyl, dodecyl, lauryl, myristyl, and stearyl chloride, bromide, and iodide; and arylC1-4alkyl halides, for example, benzyl chloride and phenethyl bromide.
If the compounds of the present disclosure simultaneously contain acidic and basic groups in the molecule, the disclosure also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions) . The respective salts can be obtained by customary methods which are known to those skilled in the art, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present disclosure also includes all salts of the compounds of the present disclosure which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review on more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002) .
The compound or any formula depicting and describing the compounds of the present disclosure and pharmaceutically acceptable salts thereof may exist in unsolvated and solvated forms. As used herein, the term “solvate” refers to a molecular complex comprising the compound of Formula (I) , or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. For example, the term “hydrate” is employed when the solvent is water.
Pharmaceutically acceptable solvates in accordance with the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, e.g., D2O, d6-acetone, d6-DMSO.
Linker (linking agent compound)
In some embodiments, the therapeutic agent is conjugated via a linker (or a linking agent compound) . As used herein, the term “linker” or “linking agent compound” refers to a compound  that can connect a ligand (e.g., the antibodies or the antigen-binding fragments thereof described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) together to form a ligand-drug conjugate by reacting with a group of the ligand compound and the therapeutic agent compound respectively by, for example, a coupling reaction.
In some embodiments, the linker described herein is a compound having the following formula:
Q-L
Formula (I) ,
or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, wherein Q denotes to a junction moiety capable of being coupled to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide and hydrazone bond; L denotes to a linker moiety capable of connecting Q to a therapeutic agent.
In some embodiments, the junction moiety (Q in Formula (I) ) has the following structure:
In some embodiments, the linker moiety (L in Formula (I) ) has the following formula:
where L1 is a polypeptide residue consisting of three to eight amino acid residues which comprises at least one amino acid residue with a side chain carboxyl group, for example, glutamic acid residue or aspartic acid residue, where “-COOH” denotes carboxyl group of an amino acid residue at C-terminal of the polypeptide residue;
L2 is absent or a monodentate, bidentate or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of the polypeptide residue L1, and L2 has a structure of -NHC (RL2a) (RL2b) (RL2c) , where RL2a, RL2b, and RL2c are each independently selected from the group consisting of H, - (CH2O) (CH2CH2O) m (CH2) pC (O) OH, and - (CH2O) (CH2CH2O) m (CH2) pC (O) NHRL2d, RL2d is H or C1-6 alkyl optionally substituted with 1 to 6 hydroxy groups, each m is independently an integer from 0 to 10, preferably 0 to 4, for example 0, 1, 2, 3, or 4, especially preferably m is 0, and each p is independent an integer from 1 to 4, for example, 1, 2, 3, or 4; and
denotes to the N-terminal side of the polypeptide residue covalently attached to the junction moiety Q.
In some embodiments, the polypeptide residue L1 is NH-Glu-Val-Ala-COOH. In some embodiments, the hydrophilic group L2 has the following structure:
wherein “*” denotes the site covalently attached to polypeptide residue L1, e.g., side chain of the Glu residue in NH-Glu-Val-Ala-COOH.
In some embodiments, the linker described herein is a compound having the following structure:
In some embodiments, the linker is a VC linker. Details of the linkers used for ADCs can be found, e.g., in Su, Z. et al. "Antibody–drug conjugates: Recent advances in linker chemistry. " Acta Pharmaceutica Sinica B (2021) , which is incorporated herein by reference in its entirety.
Therapeutic agent
In some embodiments, the therapeutic agent that is conjugated to the antibodies or the antigen-binding fragments thereof described herein is discussed as follows.
In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, an analogue or a derivative thereof. In some preferred embodiments, the camptothecin compound is a compound having the following structure:
wherein X is selected from the group consisting of -CH2-, O and S; Y is selected from the group consisting of H, D, and F.
In some embodiments, the therapeutic agent is CPT-1. The structure of CPT-1 is shown below:
In some embodiments, the therapeutic agent is CPT-2. The structure of CPT-2 is shown below:
In some embodiments, the therapeutic agent is CPT3. The structure of CPT-3 is shown below:
In some embodiments, the therapeutic agent is CPT-4. The structure of CPT-4 is shown below:
In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04/010957, International Patent Publication No. WO 02/088172, and U.S. Pat. Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein in its entirety and for all purposes.
Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity. Auristatins bind tubulin and can exert a cytotoxic or cytostatic effect on cancer cell. There are a number of different assays, known in the art, which can be used for determining whether an auristatin or resultant antibody-drug conjugate exerts a cytostatic or cytotoxic effect on a desired cell.
In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM) ; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2’ , 2’ , 2’ -trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; taxanes, e.g. paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J. ) and doxetaxel (Rhone-Poulenc Rorer, Antony, France) ; chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston) ; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. A detailed description of the chemotherapeutic agents can be found in, e.g., US20180193477A1, which is incorporated by reference in its entirety.
In some embodiments, the anti-TPBG/MET antibody is coupled to the drug via a cleavable linker, e.g. a SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the anti-TPBG/MET antibody is coupled to the drug via a non-cleavable linker e.g. a MCC linker formed using SMCC or sulfo-SMCC. Selection of an appropriate linker for a given ADC can be readily made by the skilled person having knowledge of the art and taking into account relevant factors, such as the site of attachment to the anti-TPBG/MET antibody, any structural constraints of the drug and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer) . A number of specific linker-toxin combinations have been described and may be used with the anti-TPBG/MET  antibodies or antigen-binding fragments thereof described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicins and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4, and bis-maleimido-trioxyethylene glycol (BMPEO) -based linkers with maytansinoid DM1. Some these therapeutic agents and linkers are described, e.g., in Peters &Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9: 35-65; US Patent Publication No. US 2015/0374847, and US20180193477A1; which are incorporated herein by reference in the entirety.
Depending on the desired drug and selected linker, those skilled in the art can select suitable method for coupling them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker complex which still contains reactive groups for conjugating to the anti-TPBG/MET antibodies or antigen-binding fragments thereof through covalent linkage. In some embodiments, a drug-maleimide complex (i.e., maleimide linking drug) can be used for the payload bearing reactive group in the present disclosure. Most common reactive group capable of bonding to thiol group in ADC preparation is maleimide. Additionally, organic bromides, iodides also are frequently used.
The anti-TPBG/MET ADC can be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press) . For example, conjugation can be achieved by (1) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; or (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody. Conjugation methods (1) and (2) can be employed with a variety of antibodies, drug moieties, and linkers to prepare the anti-TPBG/MET ADCs described here. Various prepared linkers, linker components and toxins are commercially available or may be prepared using standard synthetic organic chemistry techniques. These methods are described e.g., in March’s Advanced Organic Chemistry (Smith &March, 2006, Sixth Ed., Wiley) ; Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press) ; US20210379193A1, and US20180193477A1, which are incorporated herein by reference in the entirety. In addition, a number of pre-formed drug-linkers suitable for reaction with a selected anti-TPBG/MET antibody or antigen-binding fragment are also available commercially, for example, linker-toxins comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, N.Y. ) .
Several specific examples of methods of preparing anti-TPBG/MET ADCs are known in the art and are described in U.S. Pat. No. 8,624,003 (pot method) , U.S. Pat. No. 8,163,888 (one- step) , and U.S. Pat. No. 5,208,020 (two-step method) , and US20180193477A1, which are incorporated herein by reference in the entirety. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed. ) , Springer.
Drug loading is represented by the number of drug moieties per antibody in a molecule of ADC. For some antibody-drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, the drug loading may range from 0 to 8 drug moieties per antibody. In certain embodiments, higher drug loading, e.g. p≥5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an anti-TPBG/MET antibody-drug conjugate 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 antibody-drug conjugates, the optimal ratio of drug moieties per antibody can be around 4. In some embodiments, the DAR for an anti-TPBG/MET ADC composition is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the anti-TPBG/MET ADC composition is about 1～ about 2, about 2～ about 3, about 3～about 4, about 3～ about 5, about 4～ about 5, about 5～ about 6, about 6～ about 7, or about 7～about 8.
In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the anti-TPBG/MET antibodies or antigen-binding fragments thereof was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
In some embodiments, the methods described herein are designed to make a bispecific anti-TPBG/MET antibody. Bispecific anti-TPBG/MET antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
In some embodiments, knobs-into-holes (KIH) technology can be used, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote  heterodimerization. The KIH technique is described e.g., in Xu, Yiren, et al. "Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system. " MAbs. Vol. 7. No.1. Taylor &Francis, 2015, which is incorporated by reference in its entirety. In some embodiments, one heavy chain has a T366W, and/or S354C (knob) substitution (EU numbering) , and the other heavy chain has an Y349C, T366S, L368A, and/or Y407V (hole) substitution (EU numbering) . In some embodiments, one heavy chain has one or more of the following substitutions Y349C and T366W (EU numbering) . The other heavy chain can have one or more the following substitutions E356C, T366S, L368A, and Y407V (EU numbering) . Furthermore, a substitution (-ppcpScp-->-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.
Recombinant Vectors
The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of anti-TPBG/MET antibody polypeptides or fragments thereof by recombinant techniques.
As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al.,  1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6: 616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
For expression, the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley &Sons, New York, N. Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997) .
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
Transcription of DNA encoding an anti-TPBG/MET antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide (e.g., an anti-TPBG/MET antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
The disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to  any nucleotide sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any amino acid sequence as described herein.
In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.
In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology” ) . The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine percentage of sequence homology is known in the art. In some embodiments, amino acid residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,  methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.
The disclosure provides one or more nucleic acid encoding any of the polypeptides as described herein. In some embodiments, the nucleic acid (e.g., cDNA) includes a polynucleotide encoding a polypeptide of a heavy chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide of a light chain as described herein. In some embodiments, the nucleic acid includes a polynucleotide encoding a scFv polypeptide as described herein.
In some embodiments, the vector can have two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to TPBG. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to TPBG.
In some embodiments, the vector includes two of the nucleic acids as described herein, wherein the vector encodes the VL region and the VH region that together bind to MET. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids as described herein, wherein together the pair of vectors encodes the VL region and the VH region that together bind to MET.
Methods of Treatment
The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of anti-TPBG/MET antibodies or anti-TPBG/MET antibody-drug conjugates as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years) . Administration of a therapeutically effective amount of an agent described herein for the treatment of a condition associated with cancer will result in decreased number of cancer cells and/or alleviated symptoms.
As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular  tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
In some embodiments, the cancer is a chemotherapy resistant cancer.
In one aspect, the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
In one aspect, the disclosure features methods that include administering a therapeutically effective amount of anti-TPBG/MET antibodies or an anti-TPBG/MET antibody drug conjugates disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC) , mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or renal cancer. In some embodiments, the cancer is a solid tumor, lung cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.
As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the anti-TPBG/MET antibody, anti-TPBG/MET antigen binding fragment, anti-TPBG/MET antibody-drug conjugates, anti-TPBG/MET antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
An effective amount can be administered in one or more administrations. By way of example, an effective amount of an anti-TPBG/MET antibody, an anti-TPBG/MET antigen binding fragment, or an anti-TPBG/MET antibody-drug conjugate is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective amount of an anti-TPBG/MET antibody, anti-TPBG/MET antigen binding fragment, or anti-TPBG/MET antibody-drug conjugate may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of the agent used.
Effective amounts and schedules for administering the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments thereof, anti-TPBG/MET antibody-encoding polynucleotides, anti-TPBG/MET antibody-drug conjugates, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments thereof, anti-TPBG/MET antibody-encoding polynucleotides, anti-TPBG/MET antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of the agent or compositions disclosed herein used and other drugs being administered to the mammal.
A typical daily dosage of an effective amount of an anti-TPBG/MET antibody or anti-TPBG/MET ADC is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 mg/kg, 30 mg/kg, 20 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about or at least 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
In any of the methods described herein, the at least one anti-TPBG/MET antibody, the anti-TPBG/MET antigen-binding fragment thereof, anti-TPBG/MET antibody-drug conjugates, or pharmaceutical composition (e.g., comprising any of the anti-TPBG/MET antibodies, anti- TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC) and, optionally, at least one additional therapeutic agent can be administered to the subject (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, anti-TPBG/MET antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC) . In some embodiments, the one or more additional therapeutic agents and the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, or anti-TPBG/MET antibody-drug conjugate are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding fragment, or anti-TPBG/MET ADC in the subject.
In some embodiments, the subject can be administered the at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, anti-TPBG/MET antibody-drug conjugate, or pharmaceutical composition (e.g., comprising any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding antibody fragments, or anti-TPBG/MET ADC) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of anti-TPBG/MET antibodies or anti-TPBG/MET antigen-binding antibody fragments, anti-TPBG/MET antibody-drug conjugates (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding antibody fragment, or anti-TPBG/MET ADC (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .
In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an TPBG inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of MET, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) . In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .
In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of TPBG, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-TIM3 antibody, or an anti-GITR antibody.
Pharmaceutical Compositions and Routes of Administration
Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the anti-TPBG/MET antibodies (e.g., bispecific antibodies) , anti-TPBG/MET antigen-binding fragments, or anti-TPBG/MET antibody-drug conjugates described herein. The pharmaceutical compositions may be formulated in any manner known in the art.
Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No.  4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the anti-TPBG/MET antibody, anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .
Compositions containing one or more of any of the anti-TPBG/MET antibodies, anti-TPBG/MET antigen-binding fragments, anti-TPBG/MET antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the anti-TPBG/MET antibodies, an anti-TPBG/MET antigen-binding fragment thereof, or an anti-TPBG/MET ADC will be an amount that treats the disease (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .
Exemplary doses include milligram or microgram amounts of any of the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC described herein per kilogram of the subject’s weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; or about 0.1 mg/kg to about 0.5 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the therapeutic agent in vivo.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the anti-TPBG/MET antibodies, the anti-TPBG/MET antigen-binding fragment thereof, or the anti-TPBG/MET ADC for various uses as described herein.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1. Preparation of anti-TPBG/MET bispecific antibodies
Provided herein are bispecific antigen-binding molecules targeting TPBG and MET. These antigen-binding molecules are referred to as anti-TPBG/MET bispecific antibody below.
Anti-TPBG antibody (32G1, VH SEQ ID NO: 23; VL SEQ ID NO: 22) and anti-MET antibodies (8H10, VH SEQ ID NO: 24; VL SEQ ID NO: 22; 8D9, VH SEQ ID NO: 25; VL SEQ ID NO: 22 and 2F11, VH SEQ ID NO: 43; VL SEQ ID NO: 22) can be paired to form bispecific antibodies. Vectors encoding the light chain and heavy chain of the antibodies were constructed. CHO-Scells were co-transfected with three vectors, including a first vector encoding the heavy chain of an anti-TPBG antibody, a second vector encoding the heavy chain of an anti-MET antibody, and a third vector encoding the common light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography. Exemplary bispecific antibodies obtained include 32G1-8H10, 32G1-2F11, 8D9-32G1 and 32G1-8D9.
Various methods can be used to reduce the chance of wrong pairing between the two heavy chains. For example, knobs-into-holes mutations were introduced in the Fc regions. For example, in 32G1-8H10, the heavy chain constant region of 32G1 includes knob mutations, and  the heavy chain constant region of 8H10 includes hole mutations. In 32G1-2F11, the heavy chain constant region of 32G1 includes knob mutations, and the heavy chain constant region of 2F11 includes hole mutations. In 8D9-32G1, the heavy chain constant region of 8D9 includes knob mutations, and the heavy chain constant region of 32G1 includes hole mutations. In 32G1-8D9, the heavy chain constant region of 32G1 includes knob mutations, and the heavy chain constant region of 8D9 includes hole mutations. The sequences of the light chain constant region, the heavy chain constant region with knob mutations, and the heavy chain constant region with hole mutations are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively.
Example 2. Cross-species binding activity of anti-TPBG/MET bispecific antibodies
CHO-hTPBG cells, CHO-fasTPBG cells, NIH3T3-hMET cells or NIH3T3-fasMET cells were transferred to a 96-well plate at a density of 5 × 104 cells/well, respectively. The anti-TPBG/MET bispecific antibody at a concentration of 5 μg/mL was added to the 96-well plate, and incubated at 4℃ for 30 minutes. Then, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4℃ in the dark for 15 minutes before flow cytometry analysis.
CHO-hTPBG cells and CHO-fasTPBG cells were obtained by transfecting CHO-Scells with vectors expressing human TPBG (SEQ ID NO: 29) and monkey (Macaca fascicularis) TPBG (SEQ ID NO: 30) , respectively. NIH3T3-hMET cells and NIH3T3-fasMET cells were obtained by transfecting NIH3T3 cells with vectors expressing human MET (SEQ ID NO: 31) and monkey (Macaca fascicularis) MET (SEQ ID NO: 32) , respectively.
Telisotuzumab (ABT-700) is a humanized IgG1 monoclonal antibody targeting MET, which is in early clinical development at AbbVie for the treatment of advanced solid tumors with c-MET overexpression, and its heavy chain variable region and light chain variable region sequences are shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.
PF06263507 is a humanized IgG1 monoclonal antibody targeting TPBG, and the VH and VL sequences are shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively.
The test results are shown in the table below. 32G1-8H10, 32G1-2F11 and 8D9-32G1 can bind to human TPBG, monkey TPBG, human MET and monkey MET.
Table 1

Example 3. Internalization of anti-TPBG/MET bispecific antibodies
Anti-TPBG antibody, anti-MET antibody, or anti-TPBG/MET bispecific antibodies together with the pHAb-AffiniPure Fab Goat Anti-Human IgG secondary antibody were added to NCI-H226 cells (ATCC, Cat#: CRL-5826) or SNU-5 cells (ATCC, Cat#: CRL-5973) , respectively, and incubated for 3.5-6.5 hours. The cells were centrifuged and washed with FACS buffer. MFI (mean fluorescence intensity) was measured using a flow cytometer. Endocytosis rates of antibodies were calculated. Human IgG1 was used as an isotype control (ISO) . The expression levels of TPBG and MET in NCI-H226 cells were 46.11 and 131.33 by RNA-Sequence, respectively. The expression levels of TPBG and MET in SNU-5 cells were 8.79 and 871.42, respectively.
Table 2
The results are shown in Table 2, which indicate that 32G1-8H10, 32G1-2F11 and 8D9-32G1 exhibited a good endocytosis efficiency both in NCI-H226 cells and SNU-5 cells.
In another experiment, the internalization of the antibodies in NCI-H226 cells or NCI-H2030 (ATCC, Cat#: CRL-5914) was determined. The expression levels of TPBG and MET in NCI-H2030 cells were 21.38 and 70.31 by RNA-Sequence, respectively. Specifically, anti-TPBG antibody, anti-MET antibodies, or anti-TPBG/MET bispecific antibodies with the concentration of 2.5μg/mL together with the pHAb-AffiniPure Fab Goat Anti-Human IgG secondary antibody were added to NCI-H226 cells or NCI-H2030, respectively, and incubated for 6 hours. MFI (mean fluorescence intensity) was measured using a flow cytometer and the results are shown in FIGs. 10A-10B, which showed that 32G1-8H10 and 32G1-8D9 exhibited a good endocytosis efficiency both in NCI-H226 cells and NCI-H2030 cells, much better than the positive control PF06263507 analog. In addition, 32G1-8H10 exhibited a better endocytosis efficiency than its parental monoclonal antibodies 32G1 and 8H10.
Example 4. Binding activity of anti-TPBG/MET bispecific antibodies
The binding activity of anti-TPBG/MET bispecific antibodies to human TPBG, human MET, monkey TPBG and monkey MET were verified by surface plasmon resonance (SPR) using BiacoreTM (Biacore, Inc., Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.
Specifically, hTPBG-His (ACROBiosystems Inc., Cat#: TPG-H52E5) and fasTPBG-His (ACROBiosystems Inc., Cat#: TPG-C52H3) were diluted to 1000 nM with 1× HBS-EP+ buffer (pH 7.4) . hMET-His (Sino Biological, Inc., Cat#: 10692-H08H) and fasMET-His (Sino Biological, Inc., Cat#: 90304-C08H) were diluted to 400 nM with 1× HBS-EP+ buffer (pH 7.4) . Then hTPBG-His, fasTPBG-His, hMET-His or fasMET-His was injected into the BiacoreTM 8K biosensor at 10 μL/min for about 50 seconds to achieve a desired protein density (e.g., about 100 -1000 response units (RU) ) . Purified antibodies at concentrations of 2 μg/mL and 10 μg/mL in 1× HBS-EP+ buffer (pH 7.4) were then injected at 10 μL/min for 50 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with a glycine solution (pH 2.0) at 30 μL/min for 30 seconds.
Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6. 99-110) using BiacoreTM 8K Evaluation Software 3.0. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon) .
As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody.
The results for the tested antibodies are summarized in the table below, which showed that the anti-TPBG/MET bispecific antibodies 32G1-8H10, 32G1-2F11 and 8D9-32G1 had a good binding affinity to human TPBG, monkey TPBG, human MET and monkey MET.
Table 3
Example 5. Stability of anti-TPBG/MET bispecific antibodies
Anti-TPBG/MET bispecific antibodies 32G1-8H10, 32G1-2F11 and 8D9-32G1 were buffer exchanged into pH 6.0 (3 mg/mL histidine, 80 mg/mL sucrose, and 0.2 mg/mL Tween 80) . The antibodies were kept in sealed Eppendorf tubes at 40 ± 2 ℃ for 7 days, and the thermal stability were evaluated. Alternatively, the bispecific antibodies were loaded into a protein A column and eluted with a buffer (0.1 mol/L HAc) at pH 3.5. Half of the antibodies were added with a 2 M Tris buffer to make the pH into 7.5 immediately. The remaining half were kept at pH 3.5 for 6 hours and then the pH was adjusted to 7.5. The diluted antibodies were kept in sealed Eppendorf tubes at pH 3.5 ± 0.1, 25 ± 2℃ (hereinafter referred to as pH 3.5) for 6 hours to test stability at low pH.
After the above treatments, the following tests were performed: (1) the purity of antibodies were measured by Size-Exclusion High Performance Liquid Chromatography (SEC-HPLC) (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) ) ; (2) the hydrophobicity of the antibodies measured by Hydrophobic Interaction Chromatography-High Performance Liquid Chromatography (HIC-HPLC) method (indicated as the retention time of the main peak (HIC, min) ) ; (3) the pI (isoelectric point) and charge variants of the antibodies were measured by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) ; (4) the purity changes of antibodies by capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) under non-reducing (CE-SDS (NR) ) conditions (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) ) ; and (5) the appearance and presence of visible non-soluble particles.
In the SEC-HPLC experiments, the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatograph system (connected with XBridgeTM Protein BEH SEC column (Waters Corporation) ) was used. The following parameters were used: mobile phase: 0.1 M phosphate buffer (PB) + 10%acetonitrile (CAN) , pH 7.4; flow rate: 1.8  mL/min; column temperature: 25℃; detection wavelength: 280 nm, 220 nm; injection volume: 10 μL; sample tray temperature: about 8℃; and running time: 7 minutes.
In the HIC-HPLC experiments, an Agilent 1260 chromatograph system (connected with ProPacTM HIC-10 column (4.6 × 250 mm, Thermo Scientific) ) was used, and samples were 10 times diluted by using mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5; mobile phase B: 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5; flow rate: 0.8 mL/min; gradient: 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and 45 min 100%A; column temperature: 30℃; detection wavelength: 280 nm, 220 nm; injection amount: 10 μg; sample tray temperature: about 10℃; and running time: 50 minutes.
In the cIEF experiments, a Maurice cIEF Method Development Kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 8 μL of 30 μg protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI Marker-7.05, 1 μL Maurice cIEF pI Marker-10.10, 35 μL 1%Methyl Cellulose Solution, 2 μL Maurice cIEF 500 mM Arginine, 1.33 μL Ampholytes (Pharmalyte pH ranges 3-10) , 6.66 μL Ampholytes (Pharmalyte pH ranges 8-10.5) and water (added to make a final volume of 100 μL) . On the Maurice analyzer (Protein Simple, Santa Clara, CA) , Maurice cIEF Cartridges (PS-MC02-C) were used to generate imaging capillary isoelectric focusing spectra. The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to integrate the absorbance of the 280 nm-focused protein.
In the CE-SDS (NR) experiments, Maurice (Protein simple, MauriceTM) and Maurice CE-SDS Size Application Kit (Protein simple, Cat#: PS-MAK02-S) were used. In CE-SDS (NR) , 30 μL Sample Buffer, 30 μL 30ug antibody sample, 1.5 μL 25× internal standard, 3 μL 250 nM Iodoacetamide (SIGMA, Cat#: 16125) were add to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70℃ water bath for 10 minutes. The samples were then cooled to room temperature followed by centrifugation at 10000 rpm for 3 minutes. Supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.
Detailed results of anti-TPBG/MET bispecific antibodies are shown in the table below. The results showed that 32G1-8H10, 32G1-2F11 and 8D9-32G1 had good stability as well as physical and chemical properties.
Table 4

Example 6. Preparation of anti-TPBG/MET Antibody Drug Conjugates (ADC)
Each purified antibody (32G1, 8H10, 8D9, 2F11, 32G1-8H10, 32G1-2F11 or 8D9-32G1) was coupled with MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F) through a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
For the names of antibody-drug conjugates, “ADC” is added directly after the antibody name. For example, if 32G1-8H10 with IgG1 constant region is coupled to MMAE, it is named as 32G1-8H10-ADC. Antibody-drug conjugates produced by similar methods also included Telisotuzumab-ADC and PF06263507-ADC. For isotype control, human IgG1 was coupled to MMAE to form ISO-ADC.
HIC-HPLC were used to detect the coupling of antibodies with drug molecules. In the HIC-HPLC experiments, an Agilent 1260 chromatography system (connected with ProPacTM HIC-10 column (4.6 × 250 mm, Thermo Scientific) ) was used, and samples were diluted using mobile phase A to 0.5 mg/mL. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB) , 10%acetonitrile pH 6.5; mobile phase B: 0.1 M PB, 10%acetonitrile pH 6.5; flow rate: 0.8 mL/min; gradient: 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and 45 min 100%A; column temperature: 30℃; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 6℃; and running time: 45 minutes.
The HIC-HPLC detection results show that the drug-to-antibody ratio (DAR) of each ADC is about 4.
Example 7. Anti-Tumor Activity in lung cancer patient-derived xenograft model
The ADCs were tested for their effects on tumor growth in vivo in a model of lung cancer patient-derived xenograft model. Immunofluorescence staining of patient-derived tumor fragments was performed and the images were analyzed via aimage analysis platform (3.2 version) . The results showed that TPBG positive cells constituted 58.86%of total cells and MET positive cells constituted 24.08%of total cells in the human lung tumor tissues.
Specifically, the patient-derived tumor fragments (2 mm × 2 mm × 2 mm) were engrafted in the right flank of B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: B-CM-002) . When the tumors in the mice reached a volume of about 200-300 mm3, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected  with PBS, ISO-ADC, 32G1-2F11-ADC, 32G1-8H10-ADC, 8D9-32G1-ADC or Telisotuzumab-ADC. Details are shown in the table below.
Table 5
The body weights were measured twice a week. During the experiment, there was no significant difference in body weights among groups, indicating that the tested ADCs were well tolerated and were not obviously toxic to the mice.
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 28 days after grouping (Day 28) ; TGI (%) ; and the statistical differences (P value) of tumor volumes between the treatment and control groups.
Table 6
The tumor volumes of mice in different groups are shown in FIG. 4. The treatment groups (G2-G6) showed different tumor inhibition effects compared with PBS group (G1) . In addition, 32G1-2F11-ADC (G3) , 32G1-8H10-ADC (G4) and 8D9-32G1-ADC (G5) showed a higher TGI%on Day 28 (45.9%, 96.4%and 64.5%) than that of the positive control Telisotuzumab-ADC (G6, 11.3%) and ISO-ADC (G2, -0.7%) .
In a similar experiment, the patient-derived lung tumor fragments (2 mm × 2 mm × 2 mm) were engrafted in the right flank of B-NDG mice. When the tumors in the mice reached a volume of about 200-250 mm3, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS (G1) , 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) , 8D9-32G1-ADC (G4) or PF06263507-ADC (G5) at 3 mg/kg. Immunofluorescence staining of patient-derived tumor fragments showed that TPBG positive  cells constituted 31.01%of total cells and MET positive cells constituted 76.96%of total cells in the human lung tumor tissues.
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 11 days after grouping (Day 11) , 21 days after grouping (Day 21) , 35 days after grouping (Day 35) and 49 days after grouping (Day 49) ; TGI (%) ; and the statistical differences (P value) of body weight and tumor volumes between the treatment and control groups.
Table 7
The tumor volumes of mice in different groups are shown in FIG. 5A and FIG. 5B. The tumor growth in the treatment groups (G2-G5) was significantly inhibited as compared to that of the control group (G1) . In particular, 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) and 8D9-32G1-ADC (G4) all inhibited tumor growth with a higher TGI% (e.g., on Day 35) than that of the positive control PF06263507-ADC (G5) . In addition, the experiment was then continued until 63 days after grouping (Day 63) and 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) and 8D9-32G1-ADC (G4) still showed better tumor inhibitory effect than PF06263507-ADC (G5) .
Example 8. Anti-Tumor Activity in NCI-H1975 xenograft model
The ADCs were tested for their effects on tumor growth in vivo in a lung cancer model of NCI-H1975 cell xenograft. Specifically, about 1 × 106 NCI-H1975 cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of about 200 mm3, the mice were randomly placed into different groups based on tumor volumes. The mice were then injected with PBS, ISO-ADC, 32G1-8H10-ADC, 32G1-2F11-ADC, 8D9-32G1-ADC or PF06263507-ADC by intravenous (i. v. ) administration. The frequency of administration was once a week (2 administrations in total) . Details are shown in the table below.
Table 8

The tumor volumes were measured twice a week and body weights of the mice were recorded as well. Euthanasia was performed when tumor volume of a mouse reached 3000 mm3.
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) , 28 days after grouping (Day 28) and 39 days after grouping (Day 39) ; the survival rate of the mice; TGI (%) ; and the statistical differences (P value) of body weights and tumor volumes between the treatment and control groups. As all mice died in G1 group and G2 group on Day 39. Thus, the tumor volume, TGI (%) and P value for Day 39 was not available.
Table 9
The tumor volumes in groups treated with the ADCs are shown in FIG. 6.32G1-2F11-ADC, 32G1-8H10-ADC and 8D9-32G1-ADC at a dosage of 3 mg/kg (G3-G5) showed better anti-tumor activities comparing to the positive control PF06263507-ADC (G8) and ISO-ADC (G2) in the lung cancer model. In addition, 32G1-2F11-ADC and 32G1-8H10-ADC exhibited a dose-dependent antitumor activity (G3, G6 and G4, G7) .
Example 9. Anti-Tumor Activity in NUGC-4 xenograft model
The ADCs were tested for their effects on tumor growth in vivo in a gastric cancer model of NUGC-4 cell xenograft. Specifically, about 5 × 106 NUGC-4 cells were injected subcutaneously in B-NDG mice. When the tumors in the mice reached a volume of about 200 mm3, the mice were randomly placed into different groups based on tumor volumes (5 mice per group) . The mice were then injected with PBS (G1) , 32G1-2F11-ADC (G2) , 32G1-8H10-ADC  (G3) , 8D9-32G1-ADC (G4) or PF06263507-ADC (G5) at 3 mg/kg by intravenous (i. v. ) administration. The frequency of administration was once a week (2 administrations in total) .
The tumor volumes of mice in different groups are shown in FIG. 7. The treatment groups (G2-G5) showed different tumor inhibition effects compared with PBS groups (G1) . In addition, 32G1-2F11-ADC (G2) , 32G1-8H10-ADC (G3) and 8D9-32G1-ADC (G4) showed a higher TGI%on Day 35 (97.7%, 87.6%and 94.5%respectively) comparing to the positive control PF06263507-ADC (G5, 20.2%) . The anti-TPBG/MET bispecific ADCs showed robust and sustained anti-tumor effects in the gastric cancer model.
Example 10. Anti-Tumor Activity in human pancreatic PDX model
The ADCs were tested for their effects on tumor growth in vivo in human pancreatic PDX model. Immunofluorescence staining of patient-derived pancreatic tumor fragments was performed and the images were analyzed via HALO 3.2 version. The results showed that TPBG positive cells constituted 19.5%of total cells and MET positive cells constituted 8.3%of total cells in the human pancreatic tumor tissues.
Specifically, B-NDG mice were engrafted in the right flank with patient-derived pancreatic tumor fragments (2 mm×2 mm×2 mm) . When the tumors in the mice reached a volume of about 200-300 mm3, the mice were randomly placed into different groups based on the volume of the tumor. The mice were then injected with PBS or ADCs by intravenosus (i. v. ) administration. Details of the administration scheme are shown in the table below.
Table 10
During the experiment, there was no significant difference in body weights among groups. The tumor volume was measured twice a week and the results are shown in Table 11 and FIG. 9, which show that 32G1-8H10-ADC and 32G1-8D9-ADC showed better anti-tumor activity compared with the control group PF06263507-ADC at a dose of 3 mg/kg. In addition, 32G1-8H10-ADC and 32G1-8D9-ADC showed dose-dependent antitumor activity.
Table 11

Example 11. In vitro killing activity
Series diluted ADCs (maximum concentration: 20 μg/mL, 3-fold dilutions, 10 gradients) were used to treat human lung squamous carcinoma NCI-H226 cells (5 × 103) cultured in a cell culture plate, and the killing activity was detected after 72 hours of incubation in IncuCyte (Sartorius AG, S3) . The results are shown in the table below.
Table 12

( “NA” means no in vitro killing activity)
The above results showed that 32G1-8H10-ADC and 32G1-8D9-ADC had better in vitro killing activity than their parental monoclonal antibody ADCs (32G1-ADC, 8H10-ADC or 8D9-ADC) and the positive controls PF06263507-ADC and Telisotuzumab-ADC.
Example 12. Binding activity verification of anti-TPBG/MET antibodies to NCI-H226 cells, NCI-H2030 cells and HCC827 cells
The binding activities of anti-TPBG/MET antibodies to NCI-H226 cells, NCI-H2030 cells or HCC827 cells (ATCC, Cat#: CRL-2868) were verified by flow cytometry.
NCI-H226 cells, NCI-H2030 cells or HCC827 cells were incubated with serial diluted purified anti-TPBG/MET antibodies (maximum concentration: 5 μg/mL, 2-fold dilutions, 10 gradients) for 30 min at 4℃. Then, after one wash with PBS, the cells were incubated with the secondary antibody Alexa647 anti human IgG Fcγ (Jackson ImmunoResearch Laboratories, Inc., Cat #: 109-606-170) at 4℃ for 15 minutes before flow cytometry analysis. The cells were collected, and the mean fluorescence intensity (MFI) was determined. A fitting curve was obtained using antibody concentration (μg/mL) as the X-axis and MFI as the Y-axis. The results are shown in FIGs. 11A-11C, which showed that 32G1-8H10 and 32G1-8D9 showed better binding activity to NCI-H226 cells, NCI-H2030 cells and HCC827 cells than the monoclonal antibodies (32G1, 8H10 or 8D9) and the positive controls (PF06263507 analog or Telisotuzumab analog) .
Example 13. Antibody Drug Conjugates
The purified antibodies were coupled with CPT-1, CPT-2, CPT-3, or CPT-4 through a CPT-L linker. For the names of antibody-drug conjugates, CPTx (x = 1, 2, 3, or 4) is added directly after the antibody name. For example, when 32G1-8D9 is coupled to CPT-1, it is named as 32G1-8D9-CPT1. As another example, when 32G1-8D9 is coupled to CPT-2, it is named as 32G1-8D9-CPT2. Exemplary ADCs obtained by this method included: 32G1-8D9-CPT1, 32G1-8D9-CPT2, 32G1-8H10-CPT1, 32G1-8H10-CPT2, 32G1-2F11-CPT1, 32G1-2F11-CPT2, 32G1-CPT2, 2F11-CPT2.
MS (Mass Spectrometry) was used to detect the coupling of antibodies with drug molecules. A human IgG1 molecule was coupled to CPT-2 to form isotype-CPT2 (ISO-CPT2) , as an isotype control. The MS detection results showed that the drug-to-antibody ratio (DAR) of the ADCs was about 4.
SYD-1875 is an antibody-drug conjugate comprising a humanized IgG1 monoclonal antibody directed against the 5T4 oncofetal antigen, covalently conjugated to a duocarmycin-based linker-drug. The VH and VL sequences of the antibody in SYD-1875 are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively. The VH and VL were linked to the IgG1 constant region, resulting in a monoclonal antibody named as SYD-1875 analog.
Telisotuzumab analog, PF06263507 analog and SYD-1875 analog were also coupled with CPT-2 through the CPT-L linker to form Telisotuzumab-CPT2, PF06263507 -CPT2 and SYD-1875-CPT2, respectively; these serve as the reference ADCs in the following experiments.
Example 14. Anti-Tumor Activity in Patient-Derived Gastric Cancer Xenograft Model
B-NDG mice were engrafted in the right flank with gastric cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) . The immunofluorescence staining results showed that TPBG-positive cells and MET-positive cells in the gastric tumor fragments were 10.11%and 1.19%, respectively. When the tumors in the mice reached a volume of about 200-300 mm3, the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i.v. administration. Details are shown in the table below.
Table 13
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 17 days after grouping (Day 17) , and 35 days after grouping  (Day 35) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
Table 14
The results showed that 32G1-8D9-CPT2, 32G1-8H10-CPT2 and 32G1-2F11-CPT2 all exhibited good tumor inhibitory effects.
Example 15. Anti-Tumor Activity in Patient-Derived Pancreatic Cancer Xenograft Model
B-NDG mice were engrafted in the right flank with pancreatic cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) . The immunofluorescence staining results showed that TPGB-positive cells and MET-positive cells in the pancreatic tumor fragments were 19.54%and 8.31%, respectively. When the tumors in the mice reached a volume of about 200-300 mm3, the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i. v. administration. Details are shown in the table below.
Table 15
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 18 days after grouping (Day 18) and 32 days after grouping (Day 32) ; survial of the mice; TGI (%) and the statistical differences (P value) of mice body weight and tumor volume between the treatment and control groups.
Table 16

The tumor volumes of mice in different groups are shown in FIG. 12. The results showed that 32G1-8D9-CPT2, 32G1-8H10-CPT2 and 32G1-2F11-CPT2 exhibited better tumor inhibitory effects than that of the positive control PF06263507-CPT2.
Example 16. Anti-Tumor Activity in Patient-Derived Lung Cancer Xenograft Model
The ADCs were tested for their effect in two human lung PDX (PDX001 and PDX002) models. The immunofluorescence staining results in PDX001 showed that TPGB-positive cells and MET-positive cells in the pancreatic tumor fragments were 58.86%and 24.08%, respectively. In PDX002, TPBG positive cells and MET positive cells were 31.01%and 76.96%, respectively. B-NDG mice were engrafted in the right flank with pancreatic cancer patient-derived tumor tissue fragments (2 mm × 2 mm × 2 mm) .
In PDX001 model, when the tumors in the mice reached a volume of about 150-200 mm3, the mice were randomly placed into different groups based on the tumor volume. One day after grouping, the mice were injected with PBS or ADCs by i. v. administration one day after grouping. Details of PDX001 model are shown in the table below.
Table 17
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 25 days after grouping (Day 25) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
Table 18
The results showed that both 32G1-8H10-CPT2 and 32G1-2F11-CPT2 exhibited good tumor inhibitory effects in lung tumors, better than the positive controls SYD-1875-CPT2 and Telisotuzumab-CPT2.
In PDX002 model, when the tumors in the mice reached a volume of about 200-300 mm3, the mice were randomly placed into different groups based on the tumor volume. The mice were then injected with PBS or ADCs by i. v. administration one day after grouping. Details of PDX002 model are shown in the table below.
Table 19
The table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after grouping (Day 14) and 25 days after grouping (Day 25) ; survial of the mice; TGI (%) and the statistical differences (P value) of tumor volume between the treatment and control groups.
Table 20
The tumor size in groups treated with the ADCs are shown in FIG. 13. The results showed that 32G1-8D9-CPT2, 32G1-8H10-CPT2 and 32G1-2F11-CPT2 all exhibited good tumor inhibitory effects with higher TGI%than the corresponding monoclonal ADC 32G1-CPT2. Specifically, 32G1-2F11-CPT2 exhibited good tumor inhibitory effects with higher TGI%than its parental antibody ADCs 32G1-CPT2 and 2F11-CPT2.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (46)

  1. An anti-TPBG/MET antibody or antigen-binding fragment thereof, comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET.
  2. The anti-TPBG/MET antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1) ; and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) .
  3. The anti-TPBG/MET antibody or antigen-binding fragment thereof of claim 2, wherein
    the first heavy chain variable region (VH1) comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH1 CDR3 amino acid sequence; and
    the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL1 CDR3 amino acid sequence,
    wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, and the selected VL1 CDRs 1, 2, and 3 amino acid sequences are one of the following:
    (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and
    (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
  4. The anti-TPBG/MET antibody or antigen-binding fragment thereof of claim 2 or 3, wherein
    the second heavy chain variable region (VH2) comprises CDRs 1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH2 CDR3 amino acid sequence; and
    the second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein the VL2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL2 CDR3 amino acid sequence,
    wherein the selected VH2 CDRs 1, 2, and 3 amino acid sequences, and the selected VL2 CDRs 1, 2, and 3 amino acid sequences are one of the following:
    (1) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (2) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (3) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37-39, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (4) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (5) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and
    (6) the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40-42, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
  5. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein
    (1) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7-9, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (2) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10-12, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (3) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37-39, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (4) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16-18, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively;
    (5) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19-21, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; or
    (6) the selected VH1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13-15, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and the selected VH2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40-42, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively.
  6. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 24, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  7. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 25, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  8. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-5, wherein the first heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 23, the first light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22, the second heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 43, and the second light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 22.
  9. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-8, wherein the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 23, and the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 22.
  10. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-9, wherein the VH1 comprises VH1 CDR1, VH1 CDR2, and VH1 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 23; and the VL1 comprising VL1 CDR1, VL1 CDR2, and VL1 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 22.
  11. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-10, wherein the VH2 comprises an amino acid sequence that is at least 90%identical to a selected VH sequence, and the VL2 comprises an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:
    (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22;
    (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and
    (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
  12. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-11, wherein the VH2 comprises VH2 CDR1, VH2 CDR2, and VH2 CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and the VL2 comprising VL2 CDR1, VL2 CDR2, and VL2 CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:
    (1) the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22;
    (2) the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22; and
    (3) the selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 22.
  13. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-12, wherein the VH1 comprises the sequence of SEQ ID NO: 23 and the VL1 comprises the sequence of SEQ ID NO: 22.
  14. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-13, wherein the VH2 comprises the sequence of SEQ ID NO: 24 and the VL2 comprises the sequence of SEQ ID NO: 22.
  15. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-13, wherein the VH2 comprises the sequence of SEQ ID NO: 25 and the VL2 comprises the sequence of SEQ ID NO: 22.
  16. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-13, wherein the VH2 comprises the sequence of SEQ ID NO: 43 and the VL2 comprises the sequence of SEQ ID NO: 22.
  17. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the first antigen-binding domain specifically binds to human or monkey TPBG; and/or the second antigen-binding domain specifically binds to human or monkey MET.
  18. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the first antigen-binding domain is human or humanized; and/or the second antigen-binding domain is human or humanized.
  19. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein the antibody is a multispecific antibody (e.g., a bispecific antibody) .
  20. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-19, wherein the first antigen-binding domain is a single-chain variable fragment (scFv) ; and/or the second antigen-binding domain is a scFv.
  21. The anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 2-20, wherein the first light chain variable region and the second light chain variable region are identical.
  22. An anti-TPBG/MET antibody or antigen-binding fragment thereof that cross-competes with the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-21.
  23. A nucleic acid comprising a polynucleotide encoding the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22.
  24. A vector comprising the nucleic acid of claim 23.
  25. A cell comprising the vector of claim 24.
  26. The cell of claim 25, wherein the cell is a CHO cell.
  27. A cell comprising the nucleic acid of claim 23.
  28. A method of producing an anti-TPBG/MET antibody or an antigen-binding fragment thereof, the method comprising:
    (a) culturing the cell of any one of claims 25-27 under conditions sufficient for the cell to produce the anti-TPBG/MET antibody or the antigen-binding fragment thereof; and
    (b) collecting the anti-TPBG/MET antibody or the antigen-binding fragment thereof produced by the cell.
  29. An anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22.
  30. The anti-TPBG/MET antibody drug conjugate of claim 29, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
  31. The anti-TPBG/MET antibody drug conjugate of claim 29 or 30, wherein the therapeutic agent is MMAE or MMAF.
  32. The antibody-drug conjugate of claim 29, wherein the therapeutic agent is selected from
  33. The antibody-drug conjugate of claim 29 or 32, wherein the therapeutic agent is linked to the antibody or antigen-binding fragment thereof, or the antigen-binding protein construct via a linker.
  34. The antibody-drug conjugate of claim 33, wherein the linker has a structure of:
  35. The antibody-drug conjugate of any one of claims 27 and 32-34, wherein the antibody-drug conjugate has a structure of:
    wherein n = 1-8; wherein “Ab” represents the antibody or antigen-binding fragment thereof, or the antigen-binding protein construct.
  36. A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22, or the anti-TPBG/MET antibody-drug conjugate of any one of claims 29-35, to the subject.
  37. The method of claim 36, wherein the subject has a cancer expressing TPBG and/or MET (e.g., both TPBG and MET) .
  38. The method of claim 36 or claim 37, wherein the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, solid tumor, renal cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.
  39. The method of any one of claims 36-38, wherein the subject is a human.
  40. The method of any one of claims 36-39, wherein the method further comprises administering an anti-PD1 antibody to the subject.
  41. The method of any one of claims 36-40, wherein the method further comprises administering a chemotherapy to the subject.
  42. A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22, or the anti-TPBG/MET antibody-drug conjugate of any one of claims 29-35.
  43. A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22, or the anti-TPBG/MET antibody-drug conjugate of any one of claims 29-35.
  44. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and
    (a) the anti-TPBG/MET antibody or antigen-binding fragment thereof of any one of claims 1-22, and/or
    (b) the anti-TPBG/MET antibody-drug conjugate of any one of claims 29-35.
  45. An anti-TPBG/MET antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to a bispecific antibody or antigen-binding fragment thereof comprising: a first antigen-binding domain that specifically binds to TPBG; and a second antigen-binding domain that specifically binds to MET.
  46. The anti-TPBG/MET ADC of any one of claims 29-35 and 45, wherein the drug-to-antibody ratio (DAR) is about 4 or 8.
PCT/CN2024/083199 2023-03-23 2024-03-22 Anti-tpbg/met antibodies and uses thereof Pending WO2024193682A1 (en)

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