WO2024046455A1 - Methods for preparing antibody-drug conjugates - Google Patents

Methods for preparing antibody-drug conjugates Download PDF

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Publication number
WO2024046455A1
WO2024046455A1 PCT/CN2023/116426 CN2023116426W WO2024046455A1 WO 2024046455 A1 WO2024046455 A1 WO 2024046455A1 CN 2023116426 W CN2023116426 W CN 2023116426W WO 2024046455 A1 WO2024046455 A1 WO 2024046455A1
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heavy chain
antibody
light chain
chain polypeptide
amino acid
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PCT/CN2023/116426
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French (fr)
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Yanfei HAN
Chengzhang SHANG
Baihong Liu
Yi Yang
Yuelei SHEN
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Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
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Publication of WO2024046455A1 publication Critical patent/WO2024046455A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This disclosure relates to antibody-drug conjugates (ADCs) and methods of preparing the ADCs.
  • ADCs antibody-drug conjugates
  • Antibody-drug conjugates are typically formed by conjugating one or more antibody cysteine thiol groups to one or more linker moieties bound to a drug, thereby forming an antibody-linker-drug complex.
  • the number of drugs coupling to a single antibody molecule is an important factor for the efficacy and safety of the resultant ADC.
  • one therapeutic antibody molecule belonging to IgG1 or IgG4 subclass has four interchain disulfide bonds, and the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8.
  • heterogeneous mixture of ADC molecules generated by conventional conjugation processes is a mixture of D0, D2, D4, D6 and D8 (referring to ADCs in which 0, 2, 4, 6, or 8 drug molecules are coupled to one single antibody molecule, respectively) . It is well known in the art that heterogeneous ADC products are generally instable and are not suitable for drug development.
  • ADC product such as WUXI BIOLOGICS’s novel antibody-drug conjugates (WO2021013068A1; Polypeptide complex for conjugation and use thereof) , in which the antibody portion comprises, from N-terminus to C-terminus, a Fab domain operably linked to a hinge region, and wherein the Fab domain and the hinge region are derived from different IgG isotypes.
  • This disclosure relates to antibody-drug conjugates (ADCs) with improved homogeneity and methods of preparing the ADCs.
  • this disclosure relates to antibody-drug coupling methods, which include mutating one or more (e.g., 2, 4, or 6) interchain cysteine residues to non-cysteine residues (e.g., glycines) in the antibody hinge region and/or between the heavy and light chains.
  • a uniform DAR value of 4 can be achieved when coupling small molecule drugs at selected cysteine thiol groups by reducing the interchain disulfide bond in the antibody.
  • the resulting ADCs exhibited unexpectedly beneficial properties, e.g., improved homogeneity.
  • the ADCs prepared using the methods described herein have a high homogeneity with conjugates having 4 drugs per antibody accounting for at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%of all conjugates.
  • the ADCs prepared using the methods described herein have an average drug-antibody ratio (DAR) of about 4, e.g., 3.8-4.2.
  • the disclosure is related to an antibody-drug conjugate comprising an antibody comprising one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and one or more cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214, in some embodiments, a therapeutic agent is linked to the antibody through the one or more cysteine residues.
  • the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide.
  • the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229.
  • the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229.
  • the antibody-drug conjugate described herein further comprises a first light chain polypeptide and a second light chain polypeptide, in some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide.
  • the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 220, and the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214.
  • the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 220
  • the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214
  • amino acid residues at heavy chain position 220 and light chain position 214 can be the same or different.
  • the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 220
  • the first and second light chain polypeptides each comprises a glycine at light chain position 214.
  • the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229
  • the first light chain polypeptide comprises a cysteine at light chain position 214
  • the second light chain polypeptide comprises a cysteine at light chain position 214
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226;
  • the antibody comprises heavy and light chain constant region sequences derived from human IgG1. In some embodiments, the antibody is a bispecific antibody or multi-specific antibody. In some embodiments, the antibody comprises knobs-into-holes (KIH) mutations.
  • the disclosure is related to an antibody-drug conjugate comprising an antibody
  • the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide
  • the first heavy chain polypeptide can interact with the first light chain polypeptide
  • the second heavy chain polypeptide can interact with the second light chain polypeptide
  • a therapeutic agent that is covalently linked to the antibody
  • the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 23
  • the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 24
  • the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22
  • the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22.
  • the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 25
  • the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 26
  • the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22
  • the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22.
  • the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 27
  • the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 28
  • the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22
  • the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22.
  • the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 29
  • the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 30
  • the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22
  • the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22.
  • the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 32
  • the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 33
  • the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31
  • the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31.
  • the first heavy chain polypeptide comprises a first heavy chain variable region (VH1)
  • the first light chain polypeptide comprises a first light chain variable region (VL1)
  • the second heavy chain polypeptide comprises a second heavy chain variable region (VH2)
  • the second light chain polypeptide comprises a second light chain variable region (VL2)
  • the VH1 and VL1 can interact with each other, forming a first antigen-binding site
  • the VH2 and VL2 can interact with each other, forming a second antigen-binding site.
  • the first antigen-binding site and the second antigen-binding site target different antigens (e.g., two different tumor-associated antigens) .
  • the first antigen-binding site targets HER2, and the second antigen-binding site targets TROP2.
  • the VH1 comprising 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 VL1 comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the V
  • the VH2 comprising 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 VL2 comprising 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
  • the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 19
  • the VH2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 20
  • the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21
  • the VL2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21.
  • the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 220 and 229 of the first heavy chain polypeptide, heavy chain positions 220 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide
  • the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 220 and 226 of the first heavy chain polypeptide, heavy chain positions 220 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain poly
  • the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain position 220 of the first heavy chain polypeptide, heavy chain position 220 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and/or
  • the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a glycine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a glycine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and/or heavy chain positions 226 and 229 of the second heavy chain polypeptide.
  • the therapeutic agent is covalently linked to the antibody, e.g., via thiolation with the one or more cysteine residues.
  • the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) .
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the therapeutic agent is MMAE or MMAF.
  • the disclosure is related to a method of conjugating a therapeutic agent to an antibody, the method comprising: (a) reducing an antibody with a reducing agent, in some embodiments, the antibody comprises one or more non-cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and one or more cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214, in some embodiments, the one or more cysteine residues form one or more thiol groups in the reduced antibody; (b) conjugating the therapeutic agent to the one or more thiol groups.
  • the reducing agent is tris (2-carboxyethyl) phosphine (TCEP) .
  • TCEP (2-carboxyethyl) phosphine
  • the reducing agent is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or more than the molar amount of the antibody.
  • the conjugation products with a drug-antibody-ratio (DAR) of 4 accounts for at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) .
  • DAR drug-antibody-ratio
  • the conjugation products with a DAR of 0, 2, 6, and/or 8 accounts for less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) .
  • the average DAR of the conjugation products is about 3.5 to about 4.5, about 3.6 to about 4.4, about 3.7 to about 4.3, about 3.8 to about 4.2, or about 3.9 to about 4.1 (e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5) .
  • the disclosure is related to an antibody-drug conjugate comprising an antibody comprising one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214; and one or more cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214, in some embodiments, a therapeutic agent is linked to the antibody through the one or more cysteine residues.
  • the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide.
  • the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226.
  • the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229.
  • the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229. In some embodiments, the antibody-drug conjugate described herein further comprises a first light chain polypeptide and a second light chain polypeptide, in some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide.
  • the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 131
  • the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214.
  • the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 131
  • the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214.
  • the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 131
  • the first and second light chain polypeptides each comprises a glycine at light chain position 214.
  • the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide.
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229;
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229;
  • the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214.
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226;
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226;
  • the first light chain polypeptide comprises a cysteine at light chain position 214;
  • the second light chain polypeptide comprises a cysteine at light chain position 214.
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131;
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131;
  • the first light chain polypeptide comprises a cysteine at light chain position 214; and
  • the second light chain polypeptide comprises a cysteine at light chain position 214.
  • the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain position 226 and 229;
  • the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain positions 226 and 229;
  • the first light chain polypeptide comprises a non-cysteine residue at light chain position 214;
  • the second light chain polypeptide comprises a non-cysteine residue at light chain position 214.
  • the antibody comprises heavy and light chain constant region sequences derived from human IgG4. In some embodiments, the antibody is a bispecific antibody or multi-specific antibody. In some embodiments, the antibody comprises knobs-into-holes (KIH) mutations. In some embodiments, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) .
  • the disclosure is related to a method of treating a condition or disorder in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody-drug conjugate or the conjugation products as described herein, to the subject.
  • the subject has a cancer, tumor, autoimmune disease, or infectious disease.
  • the subject has a solid tumor, e.g., a thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, glioma, cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, testis cancer, multiple myeloma or renal carcinoma.
  • the subject is a human.
  • the subject is a non-human animal.
  • 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 antibody-drug conjugate or the conjugation products 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 antibody-drug conjugate or the conjugation products as described herein.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the antibody-drug conjugate or the conjugation products as described herein.
  • the disclosure is related to use of the antibody-drug conjugate or the conjugation products as described herein in the manufacture of a pharmaceutical composition or a kit for treating a condition or disorder in a subject.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
  • antibody-drug conjugate refers to a conjugate formed by covalently coupling a drug to an antibody directly or indirectly via one or more suitable linkers.
  • ADC is generally in a format of antibody-linker-drug conjugate.
  • the Antibody-drug conjugates combine properties of both antibodies and cytotoxic drugs by delivering potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity.
  • the term “drug” refers to any molecule which has a desirable therapeutic property. In some embodiments, it has at least one substituted group or a partial structure allowing connection to a linker structure.
  • the drug may kill cancer cells and/or inhibit growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.
  • linker refers to a reactive molecule which contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.
  • 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.
  • antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • 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.
  • a protein is referred to as “fully-loaded” when all points of conjugation of a particular type and/or of similar reactivity are conjugated to drugs, resulting in a homogeneous population of protein-drug conjugate.
  • a protein is referred to as “partially-loaded” when only some of the possible points of conjugation of a particular type and/or of a similar reactivity are conjugated to drugs, resulting in formation of a certain isomer or isomers of the protein-drug conjugate.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • the constant regions of both the light chain and heavy chain are replaced with TCR constant regions.
  • Fab refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab’) 2 refers to a dimer of Fab’.
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.
  • “Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.
  • an “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • immunoglobulin antibodies can be assigned to different "classes” .
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Ig forms include hinge-modifications or hingeless forms (Roux et al. (1998) J. Immunol. 161 : 4083-4090; Lund et al. (2000) Eur. J. Biochem. 267: 7246-7256; US 2005/0048572; US 2004/0229310) .
  • 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
  • chimeric antibody refers to an antibody that contains a sequence present in at least two different species (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) .
  • a non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
  • humanized antibody refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity.
  • the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues.
  • humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.
  • the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin.
  • CDRs hypervariable loops
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
  • cyste engineered antibody or “cysteine engineered antibody variant” is an antibody in which one or more residues of an antibody are substituted with cysteine residues.
  • the thiol group (s) of the cysteine engineered antibodies can be conjugated to various drugs.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to the drug moiety to create an immunoconjugate, as described further herein.
  • an antibody can have a single cysteine mutation in either the heavy or light chain such that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two engineered cysteine residues.
  • Cysteine engineered antibodies and preparatory methods are disclosed by US 2012/0121615 A1, which is incorporated by reference herein in its entirety.
  • a “disulfide bond” refers to a covalent bond with the structure R-S-S-R’.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond with a second thiol group, for example from another cysteine residue.
  • the disulfide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.
  • the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the polypeptide complex complex provided herein specifically bind an antigen with a binding affinity (K D ) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 2x10 -9 M, ⁇ 10 -9 M, or ⁇ 10 -10 M) .
  • K D refers to the ratio of the dissociation rate to the association rate (koff/kon) , may be determined using surface plasmon resonance methods for example using instrument such as Biacore.
  • one antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-S bonds, each of which is formed with two -SH groups.
  • the antibody molecule can be subjected to partial or complete reduction of one or more interchain S-S bonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8.
  • D2 refers to the ADC in which two drug molecules are coupled to one single antibody molecule, where two drug molecules may be coupled to -SH groups generated by reduction of S-S bonds between heavy and light chains via linkers, or may be coupled to -SH groups generated by reduction of S-S bonds between heavy and heavy chains via linkers.
  • D4 refers to the ADC in which four drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers, or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers, or two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and light chains via linkers and the other two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and heavy chains vis linkers.
  • D6 refers to the ADC in which six drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbonds between heavy and heavy chains via linkers, or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bonds between heavy and light chains via linkers.
  • D8 refers to the ADC in which eight drug molecules are coupled to one single antibody molecule, i.e., all the four S-S bonds in one antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule.
  • the heterogeneous mixture of ADC molecules generated by conventional conjugation processes or the bio-conjugation process of the present disclosure is a mixture of D0, D2, D4, D6 and D8.
  • the “homogeneity” of antibody-drug conjugates is used to describe the property of dominance of one specific type of antibody-drug conjugate (i.e., one type selected from D0, D2, D4, D6 and D8 conjugates) in one given mixture of antibody-drug conjugates.
  • “improved homogeneity” of ADCs refers to a higher level of a specific type of ADC (e.g., D4) in the mixture of antibody-drug conjugates generated by the process of the present disclosure as compared with the mixture of ADCs generated by conventional conjugation processes.
  • D4 a specific type of ADC
  • the content of D4 is generally more than 65 wt%, for example, more than 70 wt%, while the content of D4 is normally less than 40%in the ADCs prepared by conventional conjugation processes.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • FIGS. 1A-1E are schematic diagrams showing the structure of exemplary anti-HER2/TROP2 bispecific antibodies.
  • FIG. 2 shows the binding curves of anti-HER2/TROP2 bispecific antibodies to SKOV3 cells.
  • Human IgG1, kappa isotype control (ISO) was used as a negative control.
  • FIG. 3 shows the HIC-HPLC results of H-2B2-T-6F7-229G coupled with MMAE at different antibody: TCEP ratios.
  • FIG. 4 shows the HIC-HPLC results of H-2B2-T-6F7-226G coupled with MMAE at different antibody: TCEP ratios.
  • FIG. 5 shows the HIC-HPLC results of H-2B2-T-6F7-226G-229G coupled with MMAE at different antibody: TCEP ratios.
  • FIG. 6 shows the HIC-HPLC results of H-2B2-T-6F7-214G-220G coupled with MMAE at different antibody: TCEP ratios.
  • FIG. 7 shows the HIC-HPLC results of H-2B2-T-6F7 coupled with MMAE at different antibody: TCEP ratios.
  • FIG. 8 shows the serum concentration of ADCs after administration of to C57BL/6 mice, as determined using Anti-MMAE mIgG.
  • FIG. 9 shows the serum concentration of total antibody after administration to C57BL/6 mice, as determined using G-H-IgG.
  • FIG. 10A-10B shows CDR sequences corresponding to mAbs H-2B2 and T-6F7 as defined by Kabat numbering scheme and Chothia numbering scheme.
  • FIG. 11 shows some of the relevant amino acid sequences discussed in the present disclosure.
  • This disclosure relates to ADCs with improved homogeneity, methods of making the ADCs, and methods of use thereof.
  • an ADC including an antibody and a therapeutic agent that is linked to the antibody through one or more cysteine residues.
  • the therapeutic agent cannot be linked to the mutated residues.
  • the antibody described herein includes a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide.
  • the first heavy chain polypeptide and the first light chain polypeptide can interact with each other, and the second heavy chain polypeptide and the second light chain polypeptide can interact with each other.
  • disulfide bonds in IgG antibodies are used as connection sites for the therapeutic agent.
  • the two heavy chains are connected in the hinge region by a variable number of disulfide bonds: 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3.
  • the light chain of the IgG1 is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain.
  • the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain.
  • the level of solvent exposure is different between intra-chain and inter-chain disulfide bonds. Cysteine residues that form inter-chain disulfide bonds are located in the hinge region with the exception of the third cysteine residue of the heavy chain in IgG2, IgG3 and IgG4, which is located between the interface of VH and CH1 domains. Therefore, inter-chain disulfide bonds are highly solvent exposed. On the other hand, intra-chain disulfide bonds are buried between the two layers of anti-parallel ⁇ -sheet structures within each domain and are not solvent exposed. The solvent exposure difference has important implications because exposed cysteine residues are considered more reactive than non-exposed cysteine residues.
  • C220 in the heavy chain (EU numbering) and C214 in the light chain (EU numbering) can form an inter-chain disulfide bond.
  • these amino acid residues can form two inter-chain disulfide bonds.
  • C226 in both heavy chains (EU numbering) can form one inter-chain disulfide bond
  • C229 in both heavy chains (EU numbering) can form one inter-chain disulfide bond.
  • C131 in the heavy chain (EU numbering) and C214 in the light chain (EU numbering) can form an inter-chain disulfide bond. As there are two heavy chains and two light chains, these amino acid residues can form two inter-chain disulfide bonds.
  • C226 in both heavy chains (EU numbering) can form one inter-chain disulfide bond
  • C229 in both heavy chains (EU numbering) can form one inter-chain disulfide bond.
  • interchain cysteine residues are more reactive, drugs are usually coupled to antibodies at these specific cysteine residues, forming ADCs.
  • methods of preparing ADCs by mutating one or more (e.g., 1, 2, or 3) pairs of interchain cysteine residues to non-cysteine residues, such that drugs can only be coupled to the remaining cysteine residues that are not mutated.
  • cysteine residues can be substituted by any other amino acid residues. These substituted amino acid residues can be the same or different.
  • two interchain cysteine residues corresponding to C226 in the hinge region of IgG1 (or IgG4) antibodies can be mutated.
  • drugs can only be coupled to the six interchain cysteine residues corresponding to C229 in the hinge region, C214 in the light chain, and C220 in the heavy chain of IgG1 antibodies.
  • drugs can only be coupled to the six interchain cysteine residues corresponding to C229 in the hinge region, C214 in the light chain, and C131 in the heavy chain of IgG4 antibodies.
  • An exemplary schematic structure of an IgG1 antibodies with C226 mutations described herein can be found in FIG. 1B.
  • the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229 (or heavy chain positions 131 and 229 for IgG4 antibodies) .
  • a non-cysteine residue e.g., a glycine, alanine, valine, leucine, isoleucine, or proline
  • the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229 (or heavy chain positions 131 and 229 for IgG4 antibodies) .
  • the first light chain polypeptide includes a cysteine at light chain position 214.
  • the second light chain polypeptide includes a cysteine at light chain position 214.
  • the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 25.
  • the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 26.
  • the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
  • the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 43.
  • the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 44.
  • the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
  • 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 220 and 229 of the first heavy chain polypeptide, heavy chain positions 220 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 131 and 229 of the first heavy chain polypeptide, heavy chain positions 131 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • two interchain cysteine residues corresponding to C229 in the hinge region of IgG1 (or IgG4) antibodies can be mutated.
  • drugs can only be coupled to the six interchain cysteine residues corresponding to C226 in the hinge region, C214 in the light chain, and C220 in the heavy chain of IgG1 antibodies.
  • drugs can only be coupled to the six interchain cysteine residues corresponding to C226 in the hinge region, C214 in the light chain, and C131 in the heavy chain of IgG4 antibodies.
  • An exemplary schematic structure of an IgG1 antibody with C229 mutations described herein can be found in FIG. 1C.
  • the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226 (or heavy chain positions 131 and 226 for IgG4 antibodies) .
  • a non-cysteine residue e.g., a glycine, alanine, valine, leucine, isoleucine, or proline
  • the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226 (or heavy chain positions 131 and 226 for IgG4 antibodies) .
  • the first light chain polypeptide includes a cysteine at light chain position 214.
  • the second light chain polypeptide includes a cysteine at light chain position 214.
  • the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 27.
  • the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 28.
  • the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
  • the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 45.
  • the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 46.
  • the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
  • 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 220 and 226 of the first heavy chain polypeptide, heavy chain positions 220 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 131 and 226 of the first heavy chain polypeptide, heavy chain positions 131 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG1 (or IgG4) antibodies are mutated.
  • drugs can only be coupled to the four interchain residues corresponding to C214 in the light chain and C220 in the heavy chain of IgG1 antibodies.
  • C214 is the last cysteine residue of the IgG1 light chain
  • C220 is the fifth cysteine residue of the IgG1 heavy chain.
  • drugs can only be coupled to the four interchain residues corresponding to C214 in the light chain and C131 in the Fab region of IgG4 antibodies.
  • C214 is the last cysteine residue of the IgG4 light chain and C131 is the third cysteine residue of the IgG4 heavy chain.
  • An exemplary schematic structure of an IgG1 antibody with C226/C229 mutations described herein can be found in FIG. 1D.
  • the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) .
  • the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) .
  • the first light chain polypeptide includes a cysteine at light chain position 214.
  • the second light chain polypeptide includes a cysteine at light chain position 214.
  • the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain positions 226 and 229
  • the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain positions 226 and 229.
  • the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 226, and a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229; and the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 226, and a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229.
  • a positively charged amino acid e.g., an arginine, histidine, or lysine
  • a non-cysteine residue e.g., a glycine, alanine, valine, leucine, isoleucine,
  • the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 229; and the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 229.
  • a non-cysteine residue e.g., a glycine, alanine, valine, leucine, isoleucine, or proline
  • a positively charged amino acid e.g., an arginine, histidine, or
  • the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 226, and a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 229; and the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 226 and a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 229.
  • the opposite charge between positively charged and negatively charged amino acids can compensate or partially compensate for the role of the disulfide bond in wild-type antibody.
  • the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 29.
  • the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 30.
  • the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
  • the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 47.
  • the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 48.
  • the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
  • 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain position 220 of the first heavy chain polypeptide, heavy chain position 220 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain position 131 of the first heavy chain polypeptide, heavy chain position 131 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
  • four interchain cysteine residues corresponding to C214 in the light chain and C220 in the heavy chain of IgG1 antibodies are mutated.
  • drugs can only be coupled to the four interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG1 antibodies.
  • An exemplary schematic structure of an IgG1 antibody with C214/C220 mutations described herein can be found in FIG. 1E.
  • four interchain cysteine residues corresponding to C214 in the light chain and C131 in the Fab region of IgG4 antibodies are mutated.
  • drugs can only be coupled to the four interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG4 antibodies.
  • the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) , and a cysteine at heavy chain positions 226 and 229.
  • the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) , and a cysteine at heavy chain positions 226 and 229.
  • the first light chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at light chain position 214.
  • the second light chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at light chain position 214.
  • the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 32.
  • the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 33.
  • the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 31.
  • the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 49.
  • the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 50.
  • the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 51.
  • 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and heavy chain positions 226 and 229 of the second heavy chain polypeptide.
  • 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and heavy chain positions 226 and 229 of the second heavy chain polypeptide.
  • the first and second heavy chain polypeptide described herein includes KIH mutations.
  • the first heavy chain polypeptide described herein can have S354C and/or T366W (knob mutations) within its heavy chain constant region
  • the second heavy chain polypeptide described herein can have Y349C, T366S, L368A and/or Y407V (hole mutations) within its heavy chain constant region.
  • the first heavy chain polypeptide described herein includes a heavy chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 19.
  • the second heavy chain polypeptide described herein includes a heavy chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 20.
  • the first and second light chain polypeptides described herein each includes a light chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 21.
  • the antibodies described herein can be conjugated to a therapeutic agent (a drug) .
  • the therapeutic agent can be covalently or non-covalently bind to the antibody (e.g., a bispecific antibody) .
  • antibody targets two different tumor-associated antigens.
  • the antibody is a 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, 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 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) .
  • 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 antibody described herein is coupled to the drug via a cleavable linker e.g. a SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
  • 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 antigen binding construct, 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 antigen binding construct, 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 antigen binding constructs 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 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 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. greater than 5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
  • the average drug loading for an 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 is about or at least 1, 2, 3, 4, 5, 6, 7, or 8.
  • the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.
  • the antibody described herein can be conjugated with a therapeutic agent, forming an antibody-drug conjugate (ADC) .
  • ADC antibody-drug conjugate
  • the DAR of the ADCs described herein is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5.
  • 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
  • a drug can be coupled to an antibody described herein at an activatable site, e.g., thiol groups of any of the interchain cysteine residues as described herein.
  • an IgG1 possesses many disulfide bonds, only four of which are interchain.
  • a reducing agent such as but not limited to dithiothreitol (DTT) , Tris (2-carboxyethyl) phosphine (TCEP) , or 2-Mercaptoethanol, breaks all four bonds and generates eight cysteines (i.e., containing the free thiol group) . Conjugation of all eight cysteines with the drug-linker generates a fully-loaded conjugate with approximately eight drugs per antibody.
  • DTT dithiothreitol
  • TCEP Tris (2-carboxyethyl) phosphine
  • 2-Mercaptoethanol breaks all four bonds and generates eight cysteines (i.e., containing the free thiol group) . Conjugation of all eight cysteines with the drug-linker generates a fully-loaded conjugate with approximately eight drugs per antibody.
  • the present disclosure provides methods to prepare ADCs with improved biological properties (e.g., homogeneity) by producing partially-loaded conjugates having an average of 4 drugs per antibody, which yields lower toxicity while maintaining the efficacy of fully-loaded conjugates.
  • Methods to produce partially-loaded ADCs include reduction of the antibody (e.g., any of the cysteine engineered antibodies described herein) with a reducing agent (e.g., DTT or TCEP) , wherein one or more cysteine residues form one or more thiol groups in the reduced antibody, followed by conjugating a drug to the one or more thiol groups.
  • a reducing agent e.g., DTT or TCEP
  • the methods described herein generally include reducing (e.g., fully reducing) an antibody with a reducing agent; and conjugating a drug reactive with free thiols to the reduced antibody.
  • the antibody is partially reduced with a limiting concentration of a reducing agent in a buffer.
  • the drug can be conjugated, for example, by cooling the antibody solution and dissolving the drug in a cold solvent and mixing with the antibody solution.
  • the antibody and drug solution are incubated for a period of time sufficient to form a partially loaded antibody-drug conjugate (s) .
  • the reaction can be quenched with a quenching the excess drug with a thiol-containing reagent.
  • the conjugate can be further purified.
  • the antibody is partially reduced for at least or about 1 hour at about 37 °C.
  • the reduced antibody can be cooled, for example, to about 0 °C.
  • the antibody and drug solution can be incubated, for example, for at least or about 30 minutes at about 0 °C.
  • the thiol-containing reagent can be, for example, cysteine or N-acetyl cysteine.
  • the reducing agent can be, for example, DTT or TCEP.
  • the buffer can be, for example, a sodium borate solution and the chelating agent is dethylenetriaminepentaacetic acid.
  • the chelating agent also can be, for example, ethylenetriaminepentaacetic acid or EDTA.
  • the solvent can be, for example, acetonitrile, alcohol or DMSO.
  • the drug can be, for example, a cytotoxic or a cytostatic agent.
  • the reduced antibody can be purified prior to conjugation, using for example, column chromatography, dialysis, or diafiltration.
  • the column used in column chromatography can be, for example, a desalting column, such as a PD-10 column.
  • the reduced antibody is not purified after reduction and prior to conjugation.
  • the full reduction of the antibody is controlled by addition of an excess amount of reducing agent (e.g., TCEP) , such that all interchain disulfide bonds are broken. Because of the mutation of selected interchain cysteines described herein, conjugation can only occur at interchain cysteines that are not mutated. Following conjugation of the drug to the antibody, the conjugated drug-antibody species can be separated.
  • the conjugated antibody species can be separated based on the characteristics of the antibody, the drug and/or the conjugate.
  • hydrophobic interaction chromatograph HIC
  • HIC hydrophobic interaction chromatograph
  • ADCs including an antibody having the C226 mutations (e.g., H-2B2-T-6F7-226G-ADC) described herein can be prepared.
  • the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 1.5, about 1: 2, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, 1: 2.9, about 1: 3.0, about 1: 3.1, about 1: 3.2 about 1: 3.3, about 1: 3.4, or about 1: 3.5.
  • the DAR4 peak of the ADCs including an antibody having the C226 mutations accounts for at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40%of all peaks, e.g., determined by HIC.
  • the average DAR for ADCs including an antibody having the C226 mutations (e.g., H-2B2-T-6F7-226G-ADC) described herein is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0.
  • ADCs including an antibody having the C229 mutations (e.g., H-2B2-T-6F7-229G-ADC) described herein can be prepared.
  • the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 1.5, about 1: 2, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, 1: 2.9, about 1: 3.0, about 1: 3.1, about 1: 3.2 about 1: 3.3, about 1: 3.4, or about 1: 3.5.
  • the DAR4 peak of the ADCs including an antibody having the C229 mutations accounts for at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40%of all peaks, e.g., determined by HIC.
  • the average DAR for ADCs including an antibody having the C229 mutations (e.g., H-2B2-T-6F7-229G-ADC) described herein is about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, or about 4.2.
  • ADCs including an antibody having the C226/C229 mutations (e.g., H-2B2-T-6F7-226G-229G-ADC) described herein can be prepared.
  • the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 2, about 1: 2.1, about 1: 2.2, about 1: 2.3, about 1: 2.4, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, about 1: 2.9, about 1: 3, about 1: 4, about 1: 4.5, about 1: 5, about 1: 5.5, about 1: 6, about 1: 6.5, about 1: 7, about 1: 7.5, about 1: 8, about 1: 8.5, about 1: 9, about 1: 9.5, about 1: 10, about 1: 11, about 1: 12, about 1: 13, about 1: 14, about 1: 15, about 1: 16, about 1: 17, about 1: 18, about 1: 19, about 1: 20, about 1: 30, about 1: 40, about
  • the molar amount of the reducing agent is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more than that of the antibody.
  • the DAR4 peak of the ADCs including an antibody having the C226/C229 mutations accounts for at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 97.5%, at least 98%, at least 98.5, at least 99%, or at least 99.5 of all peaks, e.g., determined by HIC.
  • the average DAR for ADCs including an antibody having the C226/C229 mutations (e.g., H-2B2-T-6F7-226G-229G-ADC) described herein is about 3.8, about 3.85, about 3.9, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.1, about 4.15, or about 4.2.
  • ADCs including an antibody having the C214/C220 mutations e.g., H-2B2-T-6F7-214G-220G-ADC
  • the C214/C131 mutations for IgG4 antibodies described herein can be prepared.
  • the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 2, about 1: 2.1, about 1: 2.2, about 1: 2.3, about 1: 2.4, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, about 1: 2.9, about 1: 3, about 1: 4, about 1: 4.5, about 1: 5, about 1: 5.5, about 1: 6, about 1: 6.5, about 1: 7, about 1: 7.5, about 1: 8, about 1: 8.5, about 1: 9, about 1: 9.5, about 1: 10, about 1: 11, about 1: 12, about 1: 13, about 1: 14, about 1: 15, about 1: 16, about 1: 17, about 1: 18, about 1: 19, about 1: 20, about 1: 30, about 1: 40, about 1: 50, about 1: 60, about 1: 70, about 1: 80, about 1: 90, or about 1: 100.
  • TCEP reducing agent
  • the DAR4 peak of the ADCs including an antibody having the C214G/C220G mutations (or the C214/C131 mutations for IgG4 antibodies) accounts for at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%of all peaks, e.g., determined by HIC.
  • the average DAR for ADCs including an antibody having the C214/C220 mutations (e.g., H-2B2-T-6F7-214G-220G-ADC) or the C214/C131 mutations for IgG4 antibodies described herein is about 3.8, about 3.85, about 3.9, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.1, about 4.15, or about 4.2.
  • antibodies can be made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non-limiting 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 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.
  • An antibody can comprise two identical copies of a light chain and/or two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody.
  • the light chains which each contain one variable domain (or variable region, VL) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding.
  • the variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound.
  • the variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) . These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody.
  • 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 region.
  • 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 antibodies or antigen-binding fragments thereof can bind to various antigens, e.g., tumor-associated antigens, an antigen of a cell that is responsible for producing autoimmune antibodies, a viral or a microbial antigen.
  • Tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art.
  • researchers In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type (s) of cancer cell as compared to on one or more normal non-cancerous cell (s) .
  • tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells.
  • the identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody -based therapies.
  • tumor-associated antigens include, but are not limited to, BMPR1B (bone morphogenetic protein receptor-type 1B) , E16 (LAT1, SLC7A5) , STEAP1 (six transmembrane epithelial antigen of prostate) , 0772P (CA125, MUC16) , MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin) , Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) , member 2, type II sodium-dependent phosphate transporter 3b) , Sema 5b (FLJ10372, KIAA1445, Mm.
  • BMPR1B bone morphogenetic protein receptor-type 1B
  • E16 LAT1, SLC7A5
  • STEAP1 six transmembrane epithelial antigen of prostate
  • 0772P CA125, MUC16
  • MPF MP
  • CD79b CD79B, CD79 ⁇ , IGb (immunoglobulin-associated beta) , B29) , FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a) , SPAP1B, SPAP1C) , HER2 (ErbB2) , NCA (CEACAM6) , MDP (DPEP1) , IL20R ⁇ (IL20R ⁇ , ZCYTOR7) , Brevican (BCAN, BEHAB) , EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) , ASLG659 (B7h) , PSCA (Prostate stem cell antigen precursor) , GEDA, BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3) , CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22
  • LY6K lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226) , GPR19 (G protein-coupled receptor 19; Mm. 4787) , GPR54 (KISS1 receptor) , ASPHD1 (aspartate beta-hydroxylase domain containing 1) , Tyrosinase, TMEM118 (ring finger protein, transmembrane 2) , GPR172A (G protein-coupled receptor 172A) , CD33, TROP2, and CLL-1 (CLEC12A, MICL, and DCAL2) .
  • Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95%sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references.
  • a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.
  • the antibodies or antigen-binding fragments can bind to programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD40, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .
  • PD-1 programmed cell death protein 1
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • LAG-3 Lymphocyte Activating 3
  • BTLA B And T Lymph
  • the 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.
  • the antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) .
  • Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • 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 antibody or an antigen binding fragment thereof can be, 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 multi-specific 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 has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the 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.
  • Antigen binding regions A and B
  • the antibodies can bind to two different antigens or two different epitopes.
  • the antibodies e.g., bispecific antibodies
  • the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
  • Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
  • purified antibody preparations e.g., purified IgG1 molecules
  • the multi-specific 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 antibodies (e.g., bispecific antibodies) 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) .
  • 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 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 antibodies can also have various forms. Many different formats of antigen binding constructs are known in the art, and are described e.g., in Suurs, et al. "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, " Pharmacology & therapeutics (2019) , which is incorporated herein by reference in the entirety.
  • the 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 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, IgG (H) -Fv, I
  • the antibody can be a TrioMab.
  • the two heavy chains are from different species, wherein different sequences restrict the heavy-light chain pairing.
  • the antibody has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knob-in-holes or some other heavy chain pairing technique.
  • CrossMAb technique can be used produce bispecific 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.
  • the CH1 in the heavy chain and the CL domain in the light chain are swapped.
  • the 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 antibody can be have a IgG-IgG format.
  • Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.
  • the 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.
  • 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 sides 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.
  • Bispecific T cell Engager ( “BiTE” ) consists of two scFvs, VLA VHA and VHB VLB on one peptide chain. It has only binding domains, no Fc region.
  • an Fc region is fused to the BiTE construct.
  • the addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.
  • 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.
  • one scFv directed against the TAA is tagged with a short recognizable peptide is assembled to a bsAb consisting of two scFvs, one directed against CD3 and one against the recognizable peptide.
  • ImmTAC In ImmTAC, a stabilized and soluble T cell receptor is fused to a scFv recognizing CD3. By using a TCR, the ImmTAC is suitable to target processed, e.g. intracellular, proteins.
  • Tri-specific nanobody has two single variable domains (nanobodies) with an additional module for half-life extension. The extra module is added to enhance half-life.
  • Trispecific Killer Engager In Trispecific Killer Engager (TriKE) , two scFvs are connected via polypeptide linkers incorporating human IL-15. The linker to IL-15 is added to increase survival and proliferation of NKs.
  • TriKE Trispecific Killer Engager
  • the 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 HER2 and TROP2, and increase the avidity to cells that co-express HER2 and TROP2, to achieve enhanced targeting function.
  • Mutations S239D and/or I332E (SI mutations) can also be introduced in antibody heavy chains to enhance the antibody affinity to Fc ⁇ RIIIA.
  • the bispecific antibody or antigen-binding fragment thereof described herein has a common light chain.
  • the ADC described herein includes an antibody, e.g., a bispecific antibody.
  • the antibody can target to 1, 2, 3, 4, 5, or 6 antigens.
  • the antigens are the same.
  • the antigens are different.
  • the antibody can target to two different antigens, e.g., two different tumor-associated antigens (TAAs) .
  • TAAs tumor-associated antigens
  • the two TAAs are HER2 and TROP2.
  • the antibody described herein is an antigen-binding fragment thereof, or a multi-specific antibody (e.g., a bispecific antibodies) .
  • the antibody can be an anti-HER2 antibody or antigen-binding fragment thereof, or an anti-TROP2 antibody or antigen-binding fragment thereof.
  • the antibody (e.g., an anti-HER2/TROP2 bispecific antibody) described herein can have various forms.
  • the antibody described herein includes one or more cysteine mutations at heavy chain positions 220, 226, and/or 229 according to EU numbering, and/or one or more cysteine mutations at light chain position 214 according to EU numbering.
  • the antibody includes a first heavy chain polypeptide and a second heavy chain polypeptide.
  • the cysteine at heavy chain position 226 in each of the two heavy chain polypeptides is mutated (e.g., to glycine) .
  • the cysteine at heavy chain position 226 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline.
  • the cysteine at heavy chain position 229 in each of the two heavy chain polypeptides is mutated (e.g., to glycine) . In some embodiments, the cysteine at heavy chain position 229 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline.
  • the antibody described herein further includes a first light chain polypeptide and a second light chain polypeptide. In some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide.
  • the cysteine at heavy chain position 220 in each of the heavy chain polypeptide is mutated (e.g., to glycine)
  • the cysteine at light chain position 214 in each of the two light chain polypeptide is mutated (e.g., to glycine)
  • the cysteine at heavy chain position 220 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline.
  • the cysteine at heavy chain position 214 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline.
  • the antibodies e.g., the anti-TROP2 antibody, the anti-HER2 antibody, or the bispecific antibody
  • ADC antibody drug conjugates
  • the antibodies 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: 22 or 31, 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 SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 38, or 39.
  • the antibodies e.g., the anti-TROP2 antibody, the anti-HER2 antibody, or the bispecific antibody
  • ADC antibody drug conjugates
  • the antibodies have a first 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 SEQ ID NO: 38, and a second 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 SEQ ID NO: 39.
  • the first and second heavy chain constant regions include KIH mutations. In some embodiments, the first and second heavy chain constant regions do not include KIH mutations.
  • Human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases.
  • HER2 is overexpressed in various cancer types such as breast cancer and gastric cancer and has been reported to be a negative prognostic factor in breast cancer.
  • anti-HER2 drugs effective for HER2-overexpressing cancers trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, and the like are known.
  • the disclosure provides several antibodies and antigen-binding fragments thereof that specifically bind to HER2.
  • the anti-HER2/TROP2 bispecific antibodies can include an antigen binding region that is derived from these antibodies.
  • the antibodies and antigen-binding fragments described herein are capable of binding to HER2.
  • the disclosure provides e.g., anti-HER2 antibody H-2B2 ( “2B2” ) , and the antibodies derived therefrom.
  • the CDR sequences for 2B2, and 2B2 derived antibodies 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 numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 10-12, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 4-6.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 7-9 and SEQ ID NOs: 10-12; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6.
  • the antibody or an 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 antibody or an 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 antibody or an 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 antibody or an antigen-binding fragment described herein can contain a light 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 insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to HER2.
  • the antibodies or antigen-binding fragments thereof 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 SEQ ID NO: 19, 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 SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen binding fragment thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the antibody or antigen binding fragment thereof 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 a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain.
  • a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain.
  • the paired polypeptides bind to HER2 (e.g., human HER2) .
  • the anti-HER2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bispecific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (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 antibodies or antigen-binding fragments thereof 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 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
  • Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody.
  • a fragment of an antibody that binds to HER2 will retain an ability to bind to HER2.
  • An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
  • Trophoblast cell-surface antigen 2 also known as Tumor-associated calcium signal transducer 2 (TACSTD2)
  • TACSTD2 Tumor-associated calcium signal transducer 2
  • TROP2 is a protein closely related to tumors. It mainly promotes tumor cell growth, proliferation and metastasis by regulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma and ovarian cancer. The protein can promote tumor cell proliferation, invasion, metastasis, spread and other processes. In addition, in breast cancer and other cancers, the high expression of TROP2 has also been found to be closely related to more aggressive diseases and poor clinical prognosis of tumors.
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to TROP2.
  • the anti-HER2/TROP2 bispecific antibodies can include an antigen binding region that is derived from these antibodies.
  • the antibodies and antigen-binding fragments described herein are capable of binding to TROP2.
  • the disclosure provides anti-TROP2 antibody T-6F7 ( “6F7” ) , and the antibodies derived therefrom.
  • the CDR sequences for 6F7, and 6F7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. Under Chothia numbering, 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: 4-6.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 13-15 and SEQ ID NOs: 16-18; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6.
  • the antibody or an 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 antibody or an 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 antibody or an 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 antibody or an antigen-binding fragment described herein can contain a light 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 insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that binds to TROP2.
  • the antibodies or antigen-binding fragments thereof 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 SEQ ID NO: 20, 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 SEQ ID NO: 21.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen binding fragments thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the antibody or antigen binding fragments thereof 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 a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain.
  • 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 TROP2.
  • the anti-TROP2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bispecific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (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 antibodies or antigen-binding fragments thereof 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 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
  • Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody.
  • a fragment of an antibody that binds to TROP2 will retain an ability to bind to TROP2.
  • An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
  • the antibody or ADC derived therefrom can include an antigen-binding region that is derived from any antibody or any antigen-binding fragment thereof as described herein.
  • the antibodies e.g., bispecific antibody
  • the ADC derived therefrom can bind to an antigen 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 0.0001 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 0.0001 s -1 , or greater than 0.00001 s -1 . In some embodiments, the dissociation rate (koff) is less than 1 x 10 -2 s -1 .
  • kinetic association rates (kon) is greater than 1 x 10 2 /Ms, greater than 1 x 10 3 /Ms, greater than 1 x 10 4 /Ms, greater than 1 x 10 5 /Ms, or greater than 1 x 10 6 /Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 5 /Ms, less than 1 x 10 6 /Ms, or less than 1 x 10 7 /Ms. In some embodiments, kinetic association rates (kon) is greater than 1 x 10 5 /Ms.
  • the antibodies e.g., bispecific antibody
  • the ADC derived therefrom can bind to an antigen with a KD of less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M, or less than 1 x 10 -10 M.
  • the KD is less than 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
  • KD is greater than 1 x 10 -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M, or greater than 1 x 10 -10 M.
  • the antibodies 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.
  • the melting curve sometimes shows two transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2.
  • Tm D1 first denaturation temperature
  • Tm D2 second denaturation temperature
  • the antibodies or ADCs 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 antibodies or ADCs 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 antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the ADCs derived therefrom have an endocytosis rate in cells (e.g., in NCI-N87 cells or NCI-H292 cells) that is at least 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%.
  • the endocytosis rate that is less than 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%.
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the binding is measured by the percentage of positive cells as determined by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the percentage of positive cells is less than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the antibodies, (e.g., bispecific antibody) , or the ADCs derived therefrom, cannot bind to dog HER2, monkey HER2, or mouse HER2.
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the binding is measured by the percentage of positive cells as determined by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the percentage of positive cells is less than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the antibodies e.g., bispecific antibody
  • the ADC derived therefrom cannot bind to dog TROP2, monkey TROP2, or mouse TROP2.
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the antibodies has a purity that is greater than 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by SEC-HPLC.
  • the antibodies the purity is less than 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, , e.g., as measured by SEC-HPLC.
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the ADCs derived therefrom has a yield that is greater than 20, 30, 40, 50, 60, 70, 80, 90, or 100 (mg/L) . In some embodiments, the yield is less than 20, 30, 40, 50, 60, 70, 80, 90, or 100 (mg/L) .
  • the stability of the antibodies is measured by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) .
  • cIEF Capillary Isoelectric Focusing
  • the percentage of the main component is greater than 10%, 20%, 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%, after being subject to various conditions e.g., as measured by cIEF.
  • the condition is storing at 40°C for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • the condition is freeze-thaw for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 50 times.
  • the condition is storing the composition at pH 3.5 for about or at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
  • the percentage of the acidic component is greater than 10%, 20%, 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 cIEF.
  • the percentage of the alkaline component is greater than 10%, 20%, 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 cIEF.
  • the percentage of the main component is less than 10%, 20%, 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 cIEF.
  • the percentage of the acidic component is less than 10%, 20%, 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 cIEF.
  • the percentage of the alkaline component is less than 10%, 20%, 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 cIEF.
  • the cell killing ability of the antibodies is measured by IC50 (ng/ml) (e.g., in NCI-N87 or NCI-H292 cells) .
  • IC50 ng/ml
  • the IC50 is greater than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40 or 50 ⁇ g/ml.
  • the IC50 is less than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40 or 50 ⁇ g/ml.
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the ADCs derived therefrom can bind to one or more Fc receptor selected from the group consisting of Fc ⁇ RI, FcRn, Fc ⁇ RIIA-R167, Fc ⁇ RIIA-H167, Fc ⁇ RIIB, Fc ⁇ RIIIA-V176, Fc ⁇ RIIIA-F176, and Fc ⁇ RIIIB-NA1, with an affinity (indicated by KD) of less than less than 1 x 10 -5 M, less than less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M, or less than 1 x 10 -10 M.
  • Fc receptor selected from the group consisting of Fc ⁇ RI, FcRn, Fc ⁇ RIIA-R167, Fc ⁇ RIIA-H167
  • the antibodies e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • TGI% tumor growth inhibition percentage
  • the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • 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, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts.
  • 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 antibody e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the antibody 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.
  • one or both mutations S239D and/or I332E are introduced in antibody Fc region to enhance the antibody affinity to Fc ⁇ RIIIA, thereby increasing ADCC effects.
  • SI mutations S239D and/or I332E
  • the antibody described herein does not have a functional Fc region.
  • the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2 , and Fv fragments.
  • the antibody described herein e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • an antibody drug conjugate e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein
  • the antibodies (a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) or the ADCs derived therefrom has a half-life after administration of more than 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days, 200 days, 210 days, 220 days, 230 days, 240 days, 250 days, 260 days, 270 days, 280 days, 290 days, 300 days, 310 days, 320 days, 330 days 340 days, 350 days, 360 days, 370 days, 380 days, 390 days, 400 days, 410 days, 420 days, 430 days, 440 days, or 450 days, as determined using any of the methods described herein.
  • 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 recombinant 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 polynucleotide
  • 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-A tail, 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-A tail
  • 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 an antibody-encoding or 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.
  • 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., 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 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 a first antigen.
  • 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 a first antigen.
  • 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 a second antigen.
  • 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 a second antigen.
  • the VL regions are identical.
  • provided herein is a vector including a nucleic acid encoding the antibody described herein.
  • the cell is a CHO cell.
  • provided herein is a cell including a nucleic acid encoding the antibody described herein.
  • 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 antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
  • the Fc region of the antibodies 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 here are designed to make 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.
  • knob-into-hole (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.
  • 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.
  • an anion-exchange chromatography can be used to purify bispecific antibodies.
  • Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE) . In solution, the resin is coated with positively charged counter-ions (cations) . Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion.
  • Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pI) . The isoelectric point is defined as the pH at which a protein has no net charge.
  • a protein When the pH > pI, a protein has a net negative charge and when the pH ⁇ pI, a protein has a net positive charge.
  • different amino acid substitution can be introduced into two heavy chains, so that the pI for the homodimer comprising two Arm A and the pI for the homodimer comprising two Arm B is different.
  • the pI for the bispecific antibody having Arm A and Arm B will be somewhere between the two pIs of the homodimers.
  • the two homodimers and the bispecific antibody can be released at different pH conditions.
  • the present disclosure shows that a few amino acid residue substitutions can be introduced to the heavy chains to adjust pI.
  • Bispecific antibodies can also include e.g., cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin.
  • Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • provided herein are methods of producing the antibody described herein, the method including (a) culturing a cell including a vector or a nucleic acid encoding the antibody described herein under conditions sufficient for the cell to produce the antibody; and (b) collecting the antibody produced by the cell.
  • the methods described herein include methods for the treatment of disorders associated with cancer.
  • the methods include administering a therapeutically effective amount of engineered antibodies (e.g., bispecific antibodies) , or the 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.
  • engineered antibodies e.g., bispecific antibodies
  • antibody drug conjugates as described herein
  • to “treat” means to ameliorate at least one symptom of the disorder associated with cancer.
  • 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.
  • 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 antibodies (e.g., bispecific antibodies) , or an antibody drug conjugate described herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, or urothelial cancer.
  • a cancer e.g., breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, or
  • 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.
  • the subject is a human.
  • the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.
  • the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, or testis cancer.
  • the cancer is pancreas cancer, lung cancer, stomach cancer, prostate cancer, breast cancer, ovary cancer, colon cancer, skin cancer, or brain cancer.
  • 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 antibody, antigen binding fragment, antibody-drug conjugates, 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 antibody, an antigen binding fragment, or an 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 antibody, antigen binding fragment, or 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 composition used.
  • Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, 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 antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, antibody-drug conjugates, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of an antibody (e.g., a bispecific antibody) or the antibody drug conjugate is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 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 antibody e.g., a bispecific antibody
  • antibody-drug conjugates, or pharmaceutical composition e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein
  • at least one additional therapeutic agent can be administered to the subject at least once a week (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) .
  • at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) .
  • At least one antibody e.g., a bispecific antibody
  • at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) .
  • the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) .
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) .
  • the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition 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 antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) or antibody-drug conjugate in the subject.
  • the subject can be administered the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) 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) .
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments, 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 antibody or antigen-binding antibody fragment (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 EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-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-1) (IDO1)
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, 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, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-B7-H3 antibody, an anti-CLDN18 antibody, an anti-SIGLEC15 antibody, an anti-41BB antibody, an anti-CD40 antibody or an anti-GITR antibody.
  • compositions that contain at least one (e.g., one, two, three, or four) of the antibodies (e.g., bispecific antibodies) , or antibody-drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antibodies, or antibody- drug conjugates described herein can be present in a pharmaceutical composition in any combination.
  • 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 antibody or antigen-binding fragment thereof 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 antibodies, or 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.
  • Exemplary doses include milligram or microgram amounts of any of the antibodies or ADCs 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
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof, or antibody-drug conjugates for various uses as described herein.
  • H-2B2-T-6F7 is a bispecific antibody including an anti-HER2 arm (H-2B2) and an anti-TROP2 arm (T-6F7) , which has a human IgG1 subtype structure. Details of this bispecific antibody can be found in PCT Application No. PCT/CN2022/074078, which is incorporated herein by reference in its entirety.
  • H-2B2-T-6F7-226G includes the mutation of a pair of cysteines to glycines at position 226 (according to EU numbering) of the hinge region of the H-2B2-T-6F7 IgG1 subtype.
  • H-2B2-T-6F7-229G includes the mutation of a pair of cysteines to glycines at position 229 (according to EU numbering) in the hinge region of the H-2B2-T-6F7 IgG1 subtype.
  • H-2B2-T-6F7-226G-229G includes the mutation of two pairs of cysteines to glycines at positions 226 and 229 (according to EU numbering) in the hinge region of the H-2B2-T-6F7 IgG1 subtype.
  • H-2B2-T-6F7-214G-220G includes the mutation of two pairs of cysteines to glycines at position 214 of the kappa light chain and position 220 of the heavy chain (according to EU numbering) .
  • H-2B2-T-6F7 is the parent bispecific antibody (H-2B2-T-6F7 IgG1 subtype) without any mutations of the interchain cysteine residues.
  • the anti-HER2 antibody H-2B2 and anti-TROP2 antibody T-6F7 can be paired to form the bispecific antibody H-2B2-T-6F7.
  • Vectors for the light chain and heavy chain, either with or without mutated interchain cysteine residues, of the antibodies were constructed.
  • CHO-S cells were co-transfected with three vectors, including a first vector encoding the H-2B2 arm heavy chain, a second vector encoding the T-6F7 arm heavy chain, and a third vector encoding the kappa light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography. Various methods can be used to reduce the chance of wrong pairing between the two heavy chains.
  • knobs-into-holes mutations were introduced to the anti-HER2 arm heavy chain and the anti-TROP2 arm heavy chain.
  • the heavy chain constant region of the H-2B2 arm has knob mutations, and the heavy chain constant region of the T-6F7 arm has hole mutations; in T-6F7-H-2B2, the heavy chain constant regions of the T-6F7 arm has knob mutations, and the heavy chain constant regions of the H-2B2 arm has hole mutations.
  • target 1 and target 2 can be HER2 and TROP2 respectively, or TROP2 and HER2 respectively.
  • the antibodies include the same light chain, and their heavy chain and light chain sequences are shown as follows:
  • Purified anti-HER2/TROP2 bispecific antibodies were analyzed by a non-reducing SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) and SEC-HPLC (size exclusion chromatography-high performance liquid chromatography) .
  • Non-reducing SDS-PAGE was performed using a 4-12%acrylamide gel.
  • the protein samples were prepared as follows. First, 2.4 ⁇ l of the protein sample was mixed with 6 ⁇ l Tris-Glycine SDS Sample Buffer (2 ⁇ ) (Invitrogen; Cat#: LC2676) and 3.6 ⁇ l distilled water. The mixture was then boiled for 2 minutes and instantly centrifuged before loading. 10 ⁇ g of each sample was loaded to the gel.
  • the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatography system (connected with XBridge TM Protein BEH SEC column ( Waters Corporation) ) was used.
  • the following parameters were used: mobile phase: 25 mmol/L phosphate buffer (PB) + 300 mmol/L NaCl, pH 6.8; flow rate: 1.8 ml/min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 ⁇ L; sample tray temperature: about 4°C; and running time: 7 minutes. Results are summarized in the table below.
  • the non-reducing SDS-PAGE results showed that the band representing H-2B2-T-6F7-214G-220G migrated faster and was slightly smaller than the parent antibody band, with both the light chain band (about 25 KD) and heavy chain band (about 50 KD) observed.
  • the SEC-HPLC result showed that the main peak percentage of H-2B2-T-6F7-214G-220G was lower than that of other tested antibodies, indicating an unstable interaction between the heavy and light chains.
  • the expression level of H-2B2-T-6F7-214G-220G was also lower than that of other tested antibodies.
  • the tested antibodies with cysteine mutations in the hinge region e.g., H-2B2-T-6F7-229G, H-2B2-T-6F7-226G and H-2B2-T-6F7-226G-229G, showed satisfactory expression results.
  • the results also indicate that introducing cysteine mutations in the hinge region did not affect purity of the antibodies.
  • CHO-S-hTROP2 (CHO-S cells expressing human TROP2; SEQ ID NO: 35) , CHO-S-fasTROP2 (CHO-S cells expressing Macaca fascicularis TROP2 (fasTROP2; SEQ ID NO: 37) , CHO-S-hHER2 (CHO-S cells expressing human HER2; amino acids 23-652 of SEQ ID NO: 34) , and CHO-S-fasHER2 (CHO-S cells expressing Macaca fascicularis (crab-eating macaque) HER2 (fasHER2, amino acids 174-804 of SEQ ID NO: 36) ) were transferred to a 96-well plate at a density of 5 ⁇ 10 4 cells/well.
  • Purified anti-HER2/TROP2 bispecific antibodies were diluted to 0.5 ⁇ g/ml and then injected into the Biacore TM 8K biosensor at 10 ⁇ L/min for about 50 seconds to achieve a desired protein density (e.g., about 120 response units (RU) ) .
  • His-tagged TROP2 protein or His-tagged HER2 protein at a concentration of 200 nM was then injected at 30 ⁇ L/min for 180 seconds. Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0) , 30 ⁇ L/min for 30 seconds) .
  • Fc ⁇ RI His-tagged Fc ⁇ RI /CD64 Protein of human, ACROBiosystems Inc., Cat#: FCA-H52H1
  • FcRn His-tagged or Tag&Strep II-tagged FCGRT&B2M Heterodimer protein, ACROBiosystems Inc., Cat#: FCM-H5286
  • Fc ⁇ RIIA-R167 His-tagged Fc gamma RIIA/CD32a (R167) protein of human, ACROBiosystems Inc., Cat#: CDA-H5221
  • Fc ⁇ RIIA-H167 His-tagged Fc gamma RIIA CD32a (H167) protein of human, ACROBiosystems Inc., Cat#: CD1-H5223
  • Fc ⁇ RIIB His-tagged mouse Fc gamma RIIB protein, ACROBiosystems Inc., Cat#: CD
  • Fc receptor proteins were diluted to 1 ⁇ g/ml and then injected into the Biacore TM 8K biosensor at 10 ⁇ L/min for about 50-100 seconds to achieve a desired protein density (e.g., about 50-200 response units (RU) ) .
  • Purified anti-HER2/TROP2 bispecific antibodies were serially diluted and then injected at 10 ⁇ g/mL for 50-100 seconds. Dissociation was monitored for 200 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 1.7, 30 ⁇ L/min for 30 seconds) .
  • H-2B2-T-6F7-226G-229G completely lost its binding ability for Fc ⁇ RIIB and Fc ⁇ RIIIB-NA1 receptors.
  • H-2B2-T-6F7-226G-229G and H-2B2-T-6F7-229G completely lost its binding ability
  • H-2B2-T-6F7-226G partially lost its binding ability, indicating that Cys229 plays an important role in maintaining the binding of Fc ⁇ RIIIA.
  • H-2B2-T-6F7-226G-229G had a reduced affinity (by about 10 folds) as compared to the parent antibody.
  • Anti-HER2/TROP2 antibodies H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were diluted to 5 mg/ml using a buffer (3 mg/ml histidine, 80 mg/ml sucrose, and 0.2 mg/ml Tween TM 80) at pH 6.0.
  • the diluted antibodies were kept in sealed Eppendorf tubes at 40 ⁇ 3 °C (hereinafter referred to as 40 °C) for 7 days, and their thermal stability was evaluated.
  • the five anti-HER2/TROP2 antibodies were diluted to 5 mg/ml using the buffer at pH 3.5.
  • the diluted antibodies were kept in sealed Eppendorf tubes at pH 3.5 for 6 hours to determine its stability in acidic conditions.
  • the five anti-HER2/TROP2 antibodies were repeatedly freeze-thawed 10 times.
  • the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatography system (connected with XBridge TM Protein BEH SEC column ( Waters Corporation) ) was used.
  • the following parameters were used: mobile phase: 25 mmol/L phosphate buffer (PB) + 300 mmol/L NaCl, pH 6.8; flow rate: 1.8 ml/min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 ⁇ L; sample tray temperature: about 4°C; and running time: 7 minutes.
  • mobile phase A 1.0 M ammonium sulfate, 20 mM sodium acetate, 10%acetonitrile pH 6.5
  • mobile phase B 20 mM sodium acetate, 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 10 °C
  • running time 30 minutes.
  • a Maurice cIEF Method Development Kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 8 ⁇ L protein sample was mixed with the following reagents in the kit: 1 ⁇ L Maurice cIEF pI Marker-4.05, 1 ⁇ L Maurice cIEF pI Marker-9.99, 35 ⁇ L 1%Methyl Cellulose Solution, 2 ⁇ L Maurice cIEF 500 mM Arginine, 4 ⁇ L Ampholytes (Pharmalyte pH ranges 3-10) , 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
  • 54 ⁇ L Sample Buffer 6 ⁇ L antibody sample, 2.4 ⁇ 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.
  • CE-SDS 54 ⁇ L Sample Buffer, 6 ⁇ L antibody sample, 2.4 ⁇ L 25 ⁇ internal standard, 3 ⁇ L 2-Mercaptoethanol (SIGMA, Cat#: M6250) 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. 50 ⁇ L supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice. The injection voltage was 4.6 kV, the injection time was 20 seconds, the separation voltage was 5.75 kV, and the separation time was 30 minutes. Detailed results are shown in the table below.
  • H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were dialyzed and concentrated in a PBS buffer by ultrafiltration. The concentration was determined by UV absorption. These antibodies were used for the subsequent antibody drug coupling reactions.
  • the purified anti-HER2/TROP2 antibodies H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were coupled with MMAE (monomethyl auristatin E) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker.
  • MMAE monomethyl auristatin E
  • VC maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl
  • a reducing agent was used to reduce the interchain disulfide bond of the bispecific antibody to couple the small molecule drug.
  • the reducing agent used was tris (2-carboxyethyl) phosphine (TCEP) .
  • ADC is added directly after the antibody name.
  • H-2B2-T-6F7 IgG1 when H-2B2-T-6F7 IgG1 is coupled to MMAE, it is named as H-2B2-T-6F7-ADC.
  • a human IgG1 isotype control was coupled to MMAE to form an isotype-control-ADC.
  • the number of conjugated drugs per antibody can be controlled by adjusting the ratio of the bispecific antibody to TCEP. As shown in FIGS. 3-7, as the amount of TCEP increased, the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-229G (FIG. 3) and H-2B2-T-6F7-226G (FIG. 4) varied from 0-6; the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-214G-220G (FIG. 6) varied from 0-4; and the number of conjugated drugs per antibody in ADCs obtained from the parent antibody H-2B2-T-6F7 (FIG. 7) varied from 0-8, each with a high level of heterogeneity.
  • the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-226G-229G was uniformly around 4.
  • the average DAR value of the ADCs obtained by coupling the antibody to the small molecule can be about 4 (see Table 9 for the specific ratios as detected by HIC-HPLC) .
  • the SEC chromatography detection results indicating the antibody purity before and after conjugation are shown in Table 10. The results showed that the DAR value of the obtained H-2B2-T-6F7-226G-229G-ADC was about 4 for when the antibody: TCEP ratio was 1: 8.
  • the average DAR value was calculated by multiplying PA% (PA%is the percentage of the 280 nm peak area to the sum of all peak areas, as measured by HIC-HPLC) with the corresponding drug load of 0, 2, 4, 6, or 8, and then divided by total PA%.
  • the anti-HER2/TROP2 bispecific antibodies or corresponding ADCs, and goat anti-human IgG secondary antibodies labeled with pH-sensitive markers were added to NCI-H292 cells (with high expression levels of human HER2 and TROP2, respectively) , and incubated for 1.5 hours. The cells were centrifuged and washed with FACS buffer. Mean fluorescent intensity (MFI) was measured by flow cytometry. Endocytosis rates of antibodies to NCI-H292 cells were calculated by determining the percentage of positively labeled cells. The results are shown in the following table. An antibody targeting an irrelevant target protein was used as an isotype control.
  • the goat anti-human IgG secondary antibodies labeled with pH-sensitive markers were used to indirectly detect endocytosis rates of the anti-HER2/TROP2 bispecific antibodies and their corresponding ADCs.
  • cysteine mutations can affect endocytosis as compared to the parent antibody H-2B2-T-6F7.
  • Each pair of cysteine mutations can reduce the endocytosis rate accumulatively.
  • H-2B2-T-6F7-226G-229G showed a further reduction of the endocytosis rate when both pairs of cysteines in the hinge region were mutated.
  • cysteine mutations between the light and heavy chains e.g., H-2B2-T-6F7-214G-220G significantly reduced endocytosis.
  • H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, and H-2B2-T-6F7-226G-229G maintained a high in vitro killing activity.
  • the ADCs (average DAR value of about 4) prepared from each mutant antibody and the parent antibody, together with the parent antibody (unconjugated) were prepared as an administration solution at 200 ⁇ g/mL in PBS and administered intravenously to 4.5-week to 8-week-old male C57BL/6 mice at a dose level of 3 mg/kg. Approximately 0.05 mL of blood was collected from the vein at 15 minutes, 6 hours, 24 hours, 96 hours, 168 hours, 240 hours, 336 hours, and 504 hours after administration. Serum was obtained by transferring blood to 1.5 mL volume polypropylene tubes, followed by centrifugation at 4 °C.
  • the serum concentration of each antibody and ADC was measured by sandwich ELISA.
  • the serum obtained in the experiment was diluted 10 times with blank serum and then 20 times with 1%BSA to prepare measurement samples.
  • the plate was washed 4 times with a PBS-T buffer, and the antibody-unbound areas were blocked with 2%BSA (bovine serum albumin, SIGMA, Cat#: A1933) for 2 hours at 37 °C. After washing the plate 4 times with the PBS-T buffer, 100 ⁇ L of blocking buffer (1%BSA) was added to each well. The wells were sealed and incubated at 37 °Cfor 1 hour. Afterwards, the plate was washed by the PBS-T buffer 4 times.
  • BSA bovine serum albumin
  • Tetramethylbenzidine (TMB) chromogenic solution (Beyotime, Cat#: P0209) was used for color development for 5-10 minutes at room temperature, and then a stop solution (Beyotime, Cat#: P0215) was added. Luminescent signals of the plate was measured at 450 nm and 630 nm.
  • the absorbance value and corresponding concentration of the calibration sample prepared by each test product was used to create a standard curve with four parameters (i.e., T 1/2 , C max , AUC 0-504h , and CL) .
  • the standard curve was used to calculate the antibody or ADC concentration of each serum sample.
  • a drug concentration-time curve was created using the calculated sample concentration at each time point.
  • Phoenix TM WinNolin 8.3 was used to calculate the pharmacokinetic parameters.
  • H-2B2-T-6F7-229G-ADC represents the concentration of H-2B2-T-6F7-229G-ADC (either conjugated or cleaved) in blood measured after administration of H-2B2-T-6F7-229G-ADC
  • H-2B2-T-6F7-229G-Tab represents the total antibody concentration of H-2B2-T-6F7-229G in blood measured after administration of H-2B2-T-6F7-229G-ADC
  • H-2B2-T-6F7 represents the concentration of total H-2B2-T-6F7 antibody in blood measured after administration of the unconjugated parent antibody H-2B2-T-6F7.

Abstract

Provided relates to antibody-drug conjugates (ADCs) having a uniform drug-to-antibody ratio of about 4, and methods of making the ADCs thereof. In some embodiments, the ADCs include an antibody (e.g., human IgG1), whose heavy chains (e.g., Fab or Fc regions), light chains, and/or hinge region include one or more cysteine mutations.

Description

METHODS FOR PREPARING ANTIBODY-DRUG CONJUGATES
CLAIM OF PRIORITY
This application claims priority to PCT/CN2022/116543, filed on September 1, 2022. The entire contents of the foregoing application are incorporated herein by reference.
TECHNICAL FIELD
This disclosure relates to antibody-drug conjugates (ADCs) and methods of preparing the ADCs.
BACKGROUND
Antibody-drug conjugates are typically formed by conjugating one or more antibody cysteine thiol groups to one or more linker moieties bound to a drug, thereby forming an antibody-linker-drug complex. The number of drugs coupling to a single antibody molecule is an important factor for the efficacy and safety of the resultant ADC. In general, one therapeutic antibody molecule belonging to IgG1 or IgG4 subclass has four interchain disulfide bonds, and the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8. Thus, the heterogeneous mixture of ADC molecules generated by conventional conjugation processes is a mixture of D0, D2, D4, D6 and D8 (referring to ADCs in which 0, 2, 4, 6, or 8 drug molecules are coupled to one single antibody molecule, respectively) . It is well known in the art that heterogeneous ADC products are generally instable and are not suitable for drug development.
In recent years, extensive efforts have been made to improve the homogeneity of ADC product. One approach involves using site-specific conjugation through specific amino acids to allow the control of the drug-to-antibody ratio. For instance, unnatural amino acids comprising keto group or azido moiety are induced into an antibody as a conjugation site resulted in highly homogeneous product due to specific reaction (Jun Y. Axup, et al., Synthesis of site-specific antibody drug conjugates using unnatural amino acids, PNAS, 2012, 109 (40) : 16101-16106; Michael P. VanBrunt, et al., Genetically Encoded Azide Containing Amino Acid in Mam malian Cells Enables Site-Specific Antibody Drug Conjugates Using Click Cycloaddition Chemistry, Bioconjugate Chem., 2015, 26 (11) : 2249-2260) . Glycoengineering technology has also been used for homogeneous conjugate production. For example, IgG  glycoengineering using endo-β-N-acetylglucosaminidase (ENGase) mutants affords antibody preparations with homogeneous glycan structures (S. Manabe, Cancer Drug Delivery Systems Based on the Tumor Microenvironment, Springer Japan, 2019, pp. 93–123; S.J. Walsh, et al., Chem. Soc. Rev. 2021, 50, 1305) . Other methods can also led to homogeneity of ADC product such as WUXI BIOLOGICS’s novel antibody-drug conjugates (WO2021013068A1; Polypeptide complex for conjugation and use thereof) , in which the antibody portion comprises, from N-terminus to C-terminus, a Fab domain operably linked to a hinge region, and wherein the Fab domain and the hinge region are derived from different IgG isotypes.
However, there remains a need for developing more methods of preparing ADCs with improved homogeneity.
SUMMARY
This disclosure relates to antibody-drug conjugates (ADCs) with improved homogeneity and methods of preparing the ADCs. In one aspect, this disclosure relates to antibody-drug coupling methods, which include mutating one or more (e.g., 2, 4, or 6) interchain cysteine residues to non-cysteine residues (e.g., glycines) in the antibody hinge region and/or between the heavy and light chains. As a result, a uniform DAR value of 4 can be achieved when coupling small molecule drugs at selected cysteine thiol groups by reducing the interchain disulfide bond in the antibody. When small molecule drugs were conjugated to specific interchain cysteine residues, the resulting ADCs exhibited unexpectedly beneficial properties, e.g., improved homogeneity.
In some embodiments, the ADCs prepared using the methods described herein have a high homogeneity with conjugates having 4 drugs per antibody accounting for at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%of all conjugates. In some embodiments, the ADCs prepared using the methods described herein have an average drug-antibody ratio (DAR) of about 4, e.g., 3.8-4.2.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising an antibody comprising one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and one or more cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214, in some embodiments, a therapeutic agent is linked to the  antibody through the one or more cysteine residues. In some embodiments, the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide.
In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229.
In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229.
In some embodiments, the antibody-drug conjugate described herein further comprises a first light chain polypeptide and a second light chain polypeptide, in some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide. In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 220, and the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 220, and the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214, in some embodiments, amino acid residues at heavy chain position 220 and light chain position 214 can be the same or different. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 220, and the first and second light chain polypeptides each comprises a glycine at light chain position 214.
In some embodiments, the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide, in some embodiments, (a) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; the second heavy  chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214; (b) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214; (c) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214; or (d) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 220, and a cysteine at heavy chain position 226 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; the first light chain polypeptide comprises a non-cysteine residue at light chain position 214; and the second light chain polypeptide comprises a non-cysteine residue at light chain position 214. In some embodiments, the antibody comprises heavy and light chain constant region sequences derived from human IgG1. In some embodiments, the antibody is a bispecific antibody or multi-specific antibody. In some embodiments, the antibody comprises knobs-into-holes (KIH) mutations.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising an antibody, in some embodiments, the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide, in some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide; and a therapeutic agent that is covalently linked to the antibody. In some embodiments, the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 23, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID  NO: 24, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 25, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 26, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 27, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 28, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 29, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 30, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22. In some embodiments, the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 32, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 33, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31.
In some embodiments, the first heavy chain polypeptide comprises a first heavy chain variable region (VH1) , the first light chain polypeptide comprises a first light chain variable region (VL1) , the second heavy chain polypeptide comprises a second heavy chain variable  region (VH2) , and the second light chain polypeptide comprises a second light chain variable region (VL2) , in some embodiments, the VH1 and VL1 can interact with each other, forming a first antigen-binding site, and the VH2 and VL2 can interact with each other, forming a second antigen-binding site. In some embodiments, the first antigen-binding site and the second antigen-binding site target different antigens (e.g., two different tumor-associated antigens) .
In some embodiments, the first antigen-binding site targets HER2, and the second antigen-binding site targets TROP2. In some embodiments, the VH1 comprising 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 VL1 comprising CDRs 1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, in some embodiments, the selected VH1 CDRs 1, 2, and 3 amino acid sequences, 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: 7-9, 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: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.
In some embodiments, the VH2 comprising 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 VL2 comprising 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: 13-15, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and (2) 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: 4-6, respectively. In some embodiments, the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 19, the VH2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 20, the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21, and the VL2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 220 and 229 of the first heavy chain polypeptide, heavy chain positions 220 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and/or light chain position 214 of the second light chain polypeptide.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy  chain positions 220 and 226; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 220 and 226 of the first heavy chain polypeptide, heavy chain positions 220 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and/or light chain position 214 of the second light chain polypeptide.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a cysteine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a cysteine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain position 220 of the first heavy chain polypeptide, heavy chain position 220 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and/or light chain position 214 of the second light chain polypeptide.
In one aspect, the disclosure is related to an antibody-drug conjugate comprising (1) an antibody comprising a first heavy chain polypeptide, in some embodiments, the first heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; a second heavy chain polypeptide, in some embodiments, the second heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; a first light chain polypeptide, in some embodiments, the first light chain polypeptide comprises a glycine at light chain position 214; and a second light chain polypeptide, in some embodiments, the second light chain polypeptide comprises a glycine at light chain position 214; and (2) a therapeutic agent that is linked to the cysteines at: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and/or heavy chain positions 226 and 229 of the second heavy chain polypeptide.
In some embodiments, the therapeutic agent is covalently linked to the antibody, e.g., via thiolation with the one or more cysteine residues. In some embodiments, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) . 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 conjugating a therapeutic agent to an antibody, the method comprising: (a) reducing an antibody with a reducing agent, in some embodiments, the antibody comprises one or more non-cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and one or more cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214, in some embodiments, the one or more cysteine residues form one or more thiol groups in the reduced antibody; (b) conjugating the therapeutic agent to the one or more thiol groups. In some embodiments, the reducing agent is tris (2-carboxyethyl) phosphine (TCEP) . In some embodiments, the reducing agent is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or more than the molar amount of the antibody. In some embodiments, the conjugation products with a drug-antibody-ratio (DAR) of 4 accounts for at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) . In some embodiments, the conjugation products with a DAR of 0, 2, 6, and/or 8 accounts for less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) . In some embodiments, the average DAR of the conjugation products is about 3.5 to about 4.5, about 3.6 to about 4.4, about 3.7 to about 4.3, about 3.8 to about 4.2, or about 3.9 to about 4.1 (e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5) .
In one aspect, the disclosure is related to an antibody-drug conjugate comprising an antibody comprising one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214; and one or more  cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214, in some embodiments, a therapeutic agent is linked to the antibody through the one or more cysteine residues. In some embodiments, the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide. In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226. In some embodiments, the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229. In some embodiments, the antibody-drug conjugate described herein further comprises a first light chain polypeptide and a second light chain polypeptide, in some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide. In some embodiments, the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 131, and the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 131, and the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214. In some embodiments, the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 131, and the first and second light chain polypeptides each comprises a glycine at light chain position 214.
In some embodiments, the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide. In some embodiments, the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229; the second heavy chain  polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214. In some embodiments, the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214. In some embodiments, the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214. In some embodiments, the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain position 226 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain positions 226 and 229; the first light chain polypeptide comprises a non-cysteine residue at light chain position 214; and the second light chain polypeptide comprises a non-cysteine residue at light chain position 214.
In some embodiments, the antibody comprises heavy and light chain constant region sequences derived from human IgG4. In some embodiments, the antibody is a bispecific antibody or multi-specific antibody. In some embodiments, the antibody comprises knobs-into-holes (KIH) mutations. In some embodiments, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) .
In one aspect, the disclosure is related to a method of treating a condition or disorder in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody-drug conjugate or the conjugation products as described herein, to the subject. In some embodiments, the subject has a cancer, tumor, autoimmune disease, or infectious disease. In some embodiments, the subject has a solid tumor, e.g., a thyroid  cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, glioma, cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, testis cancer, multiple myeloma or renal carcinoma. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.
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 antibody-drug conjugate or the conjugation products 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 antibody-drug conjugate or the conjugation products as described herein.
In one aspect, the disclosure is related to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the antibody-drug conjugate or the conjugation products as described herein.
In one aspect, the disclosure is related to use of the antibody-drug conjugate or the conjugation products as described herein in the manufacture of a pharmaceutical composition or a kit for treating a condition or disorder in a subject.
As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
As used herein, the term “antibody-drug conjugate” or ADC refers to a conjugate formed by covalently coupling a drug to an antibody directly or indirectly via one or more suitable linkers. ADC is generally in a format of antibody-linker-drug conjugate. The Antibody-drug conjugates combine properties of both antibodies and cytotoxic drugs by delivering potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity.
As used herein, the term “drug” refers to any molecule which has a desirable therapeutic property. In some embodiments, it has at least one substituted group or a partial structure allowing connection to a linker structure. The drug may kill cancer cells and/or inhibit growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.
As used herein, the term “linker” refers to a reactive molecule which contains at least two reactive groups, one of which can covalently bond a drug molecule and the other of which can covalently couple to an antibody.
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. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .
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. A protein is referred to as “fully-loaded” when all points of conjugation of a particular type and/or of similar reactivity are conjugated to drugs, resulting in a homogeneous population of protein-drug conjugate. A protein is referred to as “partially-loaded” when only some of the possible points of conjugation of a  particular type and/or of a similar reactivity are conjugated to drugs, resulting in formation of a certain isomer or isomers of the protein-drug conjugate.
“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) associating to the variable region and first constant region of a single heavy chain by a disulfide bond. In certain embodiments, the constant regions of both the light chain and heavy chain are replaced with TCR constant regions.
“Fab’” refers to a Fab fragment that includes a portion of the hinge region.
“F (ab’) 2” refers to a dimer of Fab’.
“Fc” with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.
“Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.
An “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR. Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes" . There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ,  and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al. (1998) J. Immunol. 161 : 4083-4090; Lund et al. (2000) Eur. J. Biochem. 267: 7246-7256; US 2005/0048572; US 2004/0229310) .
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 “chimeric antibody” refers to an antibody that contains a sequence present in at least two different species (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) . A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the  humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
A “cysteine engineered antibody” or “cysteine engineered antibody variant” is an antibody in which one or more residues of an antibody are substituted with cysteine residues. In accordance with the present disclosure, the thiol group (s) of the cysteine engineered antibodies can be conjugated to various drugs. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to the drug moiety to create an immunoconjugate, as described further herein. In some examples, an antibody can have a single cysteine mutation in either the heavy or light chain such that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two engineered cysteine residues. Cysteine engineered antibodies and preparatory methods are disclosed by US 2012/0121615 A1, which is incorporated by reference herein in its entirety.
A “disulfide bond” refers to a covalent bond with the structure R-S-S-R’. The amino acid cysteine comprises a thiol group that can form a disulfide bond with a second thiol group, for example from another cysteine residue. The disulfide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the polypeptide complex complex provided herein specifically bind an antigen with a binding affinity (KD) of ≤ 10-6 M (e.g., ≤ 5x10-7 M, ≤ 2x10-7 M, ≤ 10-7 M, ≤ 5x10-8 M, ≤ 2x10-8 M, ≤ 10-8 M, ≤ 5x10-9 M, ≤ 2x10-9 M, ≤ 10-9 M, or ≤ 10-10 M) . KD as used herein refers to the ratio of the dissociation rate to the association rate (koff/kon) , may be  determined using surface plasmon resonance methods for example using instrument such as Biacore.
As discussed above, a mixture of antibody-drug conjugates will be generated by the conventional conjugation processes or the bio-conjugation process of the present disclosure. In general, one antibody molecule belonging to IgG1 or IgG4 subclass has 4 inter-chain S-S bonds, each of which is formed with two -SH groups. The antibody molecule can be subjected to partial or complete reduction of one or more interchain S-S bonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and thus, the number of drugs coupling to a single antibody molecule is 2, 4, 6 or 8. In accordance with the number of drugs coupling to a single antibody molecule, the different conjugates containing different number of drug molecules are denominated as D0, D2, D4, D6 and D8. If the number of drugs coupling to a single antibody molecule is 0, the product is referred to as D0. Accordingly, D2 refers to the ADC in which two drug molecules are coupled to one single antibody molecule, where two drug molecules may be coupled to -SH groups generated by reduction of S-S bonds between heavy and light chains via linkers, or may be coupled to -SH groups generated by reduction of S-S bonds between heavy and heavy chains via linkers. D4 refers to the ADC in which four drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers, or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers, or two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and light chains via linkers and the other two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bond between heavy and heavy chains vis linkers. D6 refers to the ADC in which six drug molecules are coupled to one single antibody molecule, where four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and light chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-Sbonds between heavy and heavy chains via linkers, or four drug molecules may be coupled to four -SH groups generated by reduction of two S-S bonds between heavy and heavy chains via linkers and two drug molecules may be coupled to two -SH groups generated by reduction of one S-S bonds between heavy and light chains via linkers. And D8 refers to the ADC in which eight drug molecules are coupled to one single antibody molecule, i.e., all the four S-S bonds in one  antibody molecule are reduced to eight -SH groups and each -SH group attaches one drug molecule. In general, the heterogeneous mixture of ADC molecules generated by conventional conjugation processes or the bio-conjugation process of the present disclosure is a mixture of D0, D2, D4, D6 and D8. And thus, the “homogeneity” of antibody-drug conjugates is used to describe the property of dominance of one specific type of antibody-drug conjugate (i.e., one type selected from D0, D2, D4, D6 and D8 conjugates) in one given mixture of antibody-drug conjugates. Although antibody-drug conjugate potency in vitro has been shown to be directly dependent on drug loading (Hamblett KJ, et al., Clin Cancer Res. 2004 Oct 15; 10 (20) : 7063-70) , in-vivo therapeutical activity (e.g., antitumour) of antibody-drug conjugates with four drugs per molecule (D4) is comparable with conjugates with eight drugs per molecule (D8) at equal mAb doses, even though the conjugates contains half the amount of drug per mAb. Drug-loading also affects plasma clearance, with the D8 conjugate being cleared 3-fold faster than the D4 conjugate and 5-fold faster than a D2 conjugate.
Accordingly, “improved homogeneity” of ADCs, as used herein, refers to a higher level of a specific type of ADC (e.g., D4) in the mixture of antibody-drug conjugates generated by the process of the present disclosure as compared with the mixture of ADCs generated by conventional conjugation processes. In the ADCs prepared by the process of the present disclosure, the content of D4 is generally more than 65 wt%, for example, more than 70 wt%, while the content of D4 is normally less than 40%in the ADCs prepared by conventional conjugation processes.
The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents,  diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
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
FIGS. 1A-1E are schematic diagrams showing the structure of exemplary anti-HER2/TROP2 bispecific antibodies.
FIG. 2 shows the binding curves of anti-HER2/TROP2 bispecific antibodies to SKOV3 cells. Human IgG1, kappa isotype control (ISO) was used as a negative control.
FIG. 3 shows the HIC-HPLC results of H-2B2-T-6F7-229G coupled with MMAE at different antibody: TCEP ratios.
FIG. 4 shows the HIC-HPLC results of H-2B2-T-6F7-226G coupled with MMAE at different antibody: TCEP ratios.
FIG. 5 shows the HIC-HPLC results of H-2B2-T-6F7-226G-229G coupled with MMAE at different antibody: TCEP ratios.
FIG. 6 shows the HIC-HPLC results of H-2B2-T-6F7-214G-220G coupled with MMAE at different antibody: TCEP ratios.
FIG. 7 shows the HIC-HPLC results of H-2B2-T-6F7 coupled with MMAE at different antibody: TCEP ratios.
FIG. 8 shows the serum concentration of ADCs after administration of to C57BL/6 mice, as determined using Anti-MMAE mIgG.
FIG. 9 shows the serum concentration of total antibody after administration to C57BL/6 mice, as determined using G-H-IgG.
FIG. 10A-10B shows CDR sequences corresponding to mAbs H-2B2 and T-6F7 as defined by Kabat numbering scheme and Chothia numbering scheme.
FIG. 11 shows some of the relevant amino acid sequences discussed in the present disclosure.
DETAILED DESCRIPTION
This disclosure relates to ADCs with improved homogeneity, methods of making the ADCs, and methods of use thereof.
In one aspect, provided herein is an ADC including an antibody and a therapeutic agent that is linked to the antibody through one or more cysteine residues. In some embodiments, because of the cysteine mutations as described herein, the therapeutic agent cannot be linked to the mutated residues. In some embodiments, the antibody described herein includes a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide. The first heavy chain polypeptide and the first light chain polypeptide can interact with each other, and the second heavy chain polypeptide and the second light chain polypeptide can interact with each other. Generally, disulfide bonds in IgG antibodies are used as connection sites for the therapeutic agent. There are many similarities with regard to the disulfide bond structures in the four subclasses of IgG antibodies, IgG1, IgG2, IgG3 and IgG4. The two heavy chains are connected in the hinge region by a variable number of disulfide bonds: 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. The light chain of the IgG1 is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain. However, for IgG2, IgG3 and IgG4, the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain.
The level of solvent exposure is different between intra-chain and inter-chain disulfide bonds. Cysteine residues that form inter-chain disulfide bonds are located in the hinge region with the exception of the third cysteine residue of the heavy chain in IgG2, IgG3 and IgG4, which is located between the interface of VH and CH1 domains. Therefore, inter-chain disulfide bonds are highly solvent exposed. On the other hand, intra-chain disulfide bonds are buried  between the two layers of anti-parallel β-sheet structures within each domain and are not solvent exposed. The solvent exposure difference has important implications because exposed cysteine residues are considered more reactive than non-exposed cysteine residues. Details of the positions of the intra-chain and inter-chain disulfide bonds can be found, e.g., in Liu, H. et al. "Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. " MAbs. Vol. 4. No. 1. Taylor & Francis, 2012, which is incorporated herein by reference in its entirety.
Particularly, in IgG1, C220 in the heavy chain (EU numbering) and C214 in the light chain (EU numbering) can form an inter-chain disulfide bond. As there are two heavy chains and two light chains, these amino acid residues can form two inter-chain disulfide bonds. In addition, C226 in both heavy chains (EU numbering) can form one inter-chain disulfide bond, and C229 in both heavy chains (EU numbering) can form one inter-chain disulfide bond.
In IgG4, C131 in the heavy chain (EU numbering) and C214 in the light chain (EU numbering) can form an inter-chain disulfide bond. As there are two heavy chains and two light chains, these amino acid residues can form two inter-chain disulfide bonds. In addition, C226 in both heavy chains (EU numbering) can form one inter-chain disulfide bond, and C229 in both heavy chains (EU numbering) can form one inter-chain disulfide bond.
Because the interchain cysteine residues are more reactive, drugs are usually coupled to antibodies at these specific cysteine residues, forming ADCs. In one aspect, provided herein are methods of preparing ADCs by mutating one or more (e.g., 1, 2, or 3) pairs of interchain cysteine residues to non-cysteine residues, such that drugs can only be coupled to the remaining cysteine residues that are not mutated. These cysteine residues can be substituted by any other amino acid residues. These substituted amino acid residues can be the same or different.
C226
In some embodiments, two interchain cysteine residues corresponding to C226 in the hinge region of IgG1 (or IgG4) antibodies can be mutated. As a result, drugs can only be coupled to the six interchain cysteine residues corresponding to C229 in the hinge region, C214 in the light chain, and C220 in the heavy chain of IgG1 antibodies. Alternatively, drugs can only be coupled to the six interchain cysteine residues corresponding to C229 in the hinge region, C214  in the light chain, and C131 in the heavy chain of IgG4 antibodies. An exemplary schematic structure of an IgG1 antibodies with C226 mutations described herein can be found in FIG. 1B.
In some embodiments, the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229 (or heavy chain positions 131 and 229 for IgG4 antibodies) . In some embodiments, the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229 (or heavy chain positions 131 and 229 for IgG4 antibodies) . In some embodiments, the first light chain polypeptide includes a cysteine at light chain position 214. In some embodiments, the second light chain polypeptide includes a cysteine at light chain position 214.
In some embodiments, the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 25. In some embodiments, the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 26. In some embodiments, the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
In some embodiments, the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 43. In some embodiments, the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 44. In some embodiments, the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
For each IgG1 antibody, 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 220 and 229 of the first heavy chain polypeptide, heavy chain positions 220 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide. For each IgG4 antibody, 0, 1, 2, 3, 4, 5, or 6 therapeutic agents  can be linked to any one or combinations of the follow cysteines: heavy chain positions 131 and 229 of the first heavy chain polypeptide, heavy chain positions 131 and 229 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
C229
In some embodiments, two interchain cysteine residues corresponding to C229 in the hinge region of IgG1 (or IgG4) antibodies can be mutated. As a result, drugs can only be coupled to the six interchain cysteine residues corresponding to C226 in the hinge region, C214 in the light chain, and C220 in the heavy chain of IgG1 antibodies. Alternatively, drugs can only be coupled to the six interchain cysteine residues corresponding to C226 in the hinge region, C214 in the light chain, and C131 in the heavy chain of IgG4 antibodies. An exemplary schematic structure of an IgG1 antibody with C229 mutations described herein can be found in FIG. 1C.
In some embodiments, the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226 (or heavy chain positions 131 and 226 for IgG4 antibodies) . In some embodiments, the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226 (or heavy chain positions 131 and 226 for IgG4 antibodies) . In some embodiments, the first light chain polypeptide includes a cysteine at light chain position 214. In some embodiments, the second light chain polypeptide includes a cysteine at light chain position 214.
In some embodiments, the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 27. In some embodiments, the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 28. In some embodiments, the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
In some embodiments, the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 45. In some embodiments, the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 46. In some embodiments, the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
For each IgG1 antibody, 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 220 and 226 of the first heavy chain polypeptide, heavy chain positions 220 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide. For each IgG4 antibody, 0, 1, 2, 3, 4, 5, or 6 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 131 and 226 of the first heavy chain polypeptide, heavy chain positions 131 and 226 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
C226/C229
In some embodiments, four interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG1 (or IgG4) antibodies are mutated. As a result, drugs can only be coupled to the four interchain residues corresponding to C214 in the light chain and C220 in the heavy chain of IgG1 antibodies. Specifically, C214 is the last cysteine residue of the IgG1 light chain and C220 is the fifth cysteine residue of the IgG1 heavy chain. Alternatively, drugs can only be coupled to the four interchain residues corresponding to C214 in the light chain and C131 in the Fab region of IgG4 antibodies. Specifically, C214 is the last cysteine residue of the IgG4 light chain and C131 is the third cysteine residue of the IgG4 heavy chain. An exemplary schematic structure of an IgG1 antibody with C226/C229 mutations described herein can be found in FIG. 1D.
In some embodiments, the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) . In some embodiments, the second heavy chain polypeptide includes a non-cysteine  residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) . In some embodiments, the first light chain polypeptide includes a cysteine at light chain position 214. In some embodiments, the second light chain polypeptide includes a cysteine at light chain position 214.
In some embodiments, the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain positions 226 and 229, and the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain positions 226 and 229. In some embodiments, the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 226, and a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229; and the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 226, and a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 229. In some embodiments, the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 229; and the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 226, and a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 229. In some embodiments, the first heavy chain polypeptide includes a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 226, and a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 229; and the second heavy chain polypeptide includes a negatively charged amino acid (e.g., an aspartic acid or glutamic acid) at heavy chain position 226 and a positively charged amino acid (e.g., an arginine, histidine, or lysine) at heavy chain position 229. Without wishing to be bound by theory, it is contemplated that the opposite charge between positively charged and negatively charged amino acids can compensate or partially compensate for the role of the disulfide bond in wild-type antibody.
In some embodiments, the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less  than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 29. In some embodiments, the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 30. In some embodiments, the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 22.
In some embodiments, the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 47. In some embodiments, the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 48. In some embodiments, the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 42.
For each IgG1 antibody, 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain position 220 of the first heavy chain polypeptide, heavy chain position 220 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide. For each IgG4 antibody, 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain position 131 of the first heavy chain polypeptide, heavy chain position 131 of the second heavy chain polypeptide, light chain position 214 of the first light chain polypeptide, and light chain position 214 of the second light chain polypeptide.
C214/C220 (IgG1) or C214/C131 (IgG4)
In some embodiments, four interchain cysteine residues corresponding to C214 in the light chain and C220 in the heavy chain of IgG1 antibodies are mutated. As a result, drugs can only be coupled to the four interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG1 antibodies. An exemplary schematic structure of an IgG1 antibody with C214/C220 mutations described herein can be found in FIG. 1E. In some embodiments, four interchain cysteine residues corresponding to C214 in the light chain and C131 in the Fab region  of IgG4 antibodies are mutated. As a result, drugs can only be coupled to the four interchain cysteine residues corresponding to C226 and C229 in the hinge region of IgG4 antibodies.
In some embodiments, the first heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) , and a cysteine at heavy chain positions 226 and 229. In some embodiments, the second heavy chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at heavy chain position 220 (or heavy chain position 131 for IgG4 antibodies) , and a cysteine at heavy chain positions 226 and 229. In some embodiments, the first light chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at light chain position 214. In some embodiments, the second light chain polypeptide includes a non-cysteine residue (e.g., a glycine, alanine, valine, leucine, isoleucine, or proline) at light chain position 214.
In some embodiments, the first heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 32. In some embodiments, the second heavy chain polypeptide includes a heavy chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 33. In some embodiments, the first and second light chain polypeptides each includes a light chain constant region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, deletions, or substitutions of SEQ ID NO: 31.
In some embodiments, the first heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 49. In some embodiments, the second heavy chain polypeptide includes an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 50. In some embodiments, the first and second light chain polypeptides include an amino acid that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 51.
For each IgG1 antibody, 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations of the follow cysteines: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and heavy chain positions 226 and 229 of the second heavy chain polypeptide. For each IgG4 antibody, 0, 1, 2, 3, or 4 therapeutic agents can be linked to any one or combinations  of the follow cysteines: heavy chain positions 226 and 229 of the first heavy chain polypeptide, and heavy chain positions 226 and 229 of the second heavy chain polypeptide.
In some embodiments, the first and second heavy chain polypeptide described herein includes KIH mutations. For example, the first heavy chain polypeptide described herein can have S354C and/or T366W (knob mutations) within its heavy chain constant region, and the second heavy chain polypeptide described herein can have Y349C, T366S, L368A and/or Y407V (hole mutations) within its heavy chain constant region.
In some embodiments, the first heavy chain polypeptide described herein includes a heavy chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 19. In some embodiments, the second heavy chain polypeptide described herein includes a heavy chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 20. In some embodiments, the first and second light chain polypeptides described herein each includes a light chain variable region with an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 21.
Therapeutic Agent
The antibodies described herein can be conjugated to a therapeutic agent (a drug) . The therapeutic agent can be covalently or non-covalently bind to the antibody (e.g., a bispecific antibody) . In some embodiments, antibody targets two different tumor-associated antigens. In some embodiments, the antibody is a 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, 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) . 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) .
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.
Linker
In some embodiments, the antibody described herein 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 antibody described herein 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 antigen binding construct, 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 antigen binding constructs 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 antibodies 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.
Methods of Making ADCs
The 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 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 antigen binding construct 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 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. greater than 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 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 is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.
In some embodiments, the antibody described herein can be conjugated with a therapeutic agent, forming an antibody-drug conjugate (ADC) . In some embodiments, the DAR of the ADCs described herein is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5. 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, a drug can be coupled to an antibody described herein at an activatable site, e.g., thiol groups of any of the interchain cysteine residues as described herein. In an exemplary embodiment, an IgG1 possesses many disulfide bonds, only four of which are interchain. Because the four interchain disulfide bonds are clustered in the highly-flexible hinge region and much more solvent-accessible than other (intra-chain) disulfide bonds, reduction with an excess of, for example, a reducing agent, such as but not limited to dithiothreitol (DTT) , Tris (2-carboxyethyl) phosphine (TCEP) , or 2-Mercaptoethanol, breaks all four bonds and  generates eight cysteines (i.e., containing the free thiol group) . Conjugation of all eight cysteines with the drug-linker generates a fully-loaded conjugate with approximately eight drugs per antibody.
The present disclosure provides methods to prepare ADCs with improved biological properties (e.g., homogeneity) by producing partially-loaded conjugates having an average of 4 drugs per antibody, which yields lower toxicity while maintaining the efficacy of fully-loaded conjugates. Methods to produce partially-loaded ADCs (with 4 rather than 8 drugs per antibody) include reduction of the antibody (e.g., any of the cysteine engineered antibodies described herein) with a reducing agent (e.g., DTT or TCEP) , wherein one or more cysteine residues form one or more thiol groups in the reduced antibody, followed by conjugating a drug to the one or more thiol groups. These cysteines were analyzed by the methods described herein.
In some embodiments, the methods described herein generally include reducing (e.g., fully reducing) an antibody with a reducing agent; and conjugating a drug reactive with free thiols to the reduced antibody.
In some embodiments, the antibody is partially reduced with a limiting concentration of a reducing agent in a buffer. The drug can be conjugated, for example, by cooling the antibody solution and dissolving the drug in a cold solvent and mixing with the antibody solution. The antibody and drug solution are incubated for a period of time sufficient to form a partially loaded antibody-drug conjugate (s) . The reaction can be quenched with a quenching the excess drug with a thiol-containing reagent. The conjugate can be further purified. In a specific example, the antibody is partially reduced for at least or about 1 hour at about 37 ℃. The reduced antibody can be cooled, for example, to about 0 ℃. The antibody and drug solution can be incubated, for example, for at least or about 30 minutes at about 0 ℃.
In some embodiments, the thiol-containing reagent can be, for example, cysteine or N-acetyl cysteine. The reducing agent can be, for example, DTT or TCEP. The buffer can be, for example, a sodium borate solution and the chelating agent is dethylenetriaminepentaacetic acid. The chelating agent also can be, for example, ethylenetriaminepentaacetic acid or EDTA. The solvent can be, for example, acetonitrile, alcohol or DMSO. The drug can be, for example, a cytotoxic or a cytostatic agent.
In some embodiments, the reduced antibody can be purified prior to conjugation, using for example, column chromatography, dialysis, or diafiltration. The column used in column  chromatography can be, for example, a desalting column, such as a PD-10 column. Alternatively, the reduced antibody is not purified after reduction and prior to conjugation. In some embodiments, the full reduction of the antibody is controlled by addition of an excess amount of reducing agent (e.g., TCEP) , such that all interchain disulfide bonds are broken. Because of the mutation of selected interchain cysteines described herein, conjugation can only occur at interchain cysteines that are not mutated. Following conjugation of the drug to the antibody, the conjugated drug-antibody species can be separated. For example, in some embodiments, the conjugated antibody species can be separated based on the characteristics of the antibody, the drug and/or the conjugate. For example, hydrophobic interaction chromatograph (HIC) has been successful in isolating and separating species corresponding to 0, 2, 4, 6, and 8 drugs per antibody. Detailed methods of drug conjugation can be found, e.g., in US Patent No. US7837980 B2, which is incorporated herein by reference in its entirety.
In some embodiments, ADCs including an antibody having the C226 mutations (e.g., H-2B2-T-6F7-226G-ADC) described herein can be prepared. In some embodiments, the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 1.5, about 1: 2, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, 1: 2.9, about 1: 3.0, about 1: 3.1, about 1: 3.2 about 1: 3.3, about 1: 3.4, or about 1: 3.5. In some embodiments, the DAR4 peak of the ADCs including an antibody having the C226 mutations accounts for at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40%of all peaks, e.g., determined by HIC. In some embodiments, the average DAR for ADCs including an antibody having the C226 mutations (e.g., H-2B2-T-6F7-226G-ADC) described herein is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0.
In some embodiments, ADCs including an antibody having the C229 mutations (e.g., H-2B2-T-6F7-229G-ADC) described herein can be prepared. In some embodiments, the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 1.5, about 1: 2, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, 1: 2.9, about 1: 3.0, about 1: 3.1, about 1: 3.2 about 1: 3.3, about 1: 3.4, or about 1: 3.5. In some embodiments, the DAR4 peak of the ADCs including an antibody having the C229 mutations accounts for at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40%of all peaks, e.g., determined by HIC. In some embodiments, the  average DAR for ADCs including an antibody having the C229 mutations (e.g., H-2B2-T-6F7-229G-ADC) described herein is about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, or about 4.2.
In some embodiments, ADCs including an antibody having the C226/C229 mutations (e.g., H-2B2-T-6F7-226G-229G-ADC) described herein can be prepared. In some embodiments, the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 2, about 1: 2.1, about 1: 2.2, about 1: 2.3, about 1: 2.4, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, about 1: 2.9, about 1: 3, about 1: 4, about 1: 4.5, about 1: 5, about 1: 5.5, about 1: 6, about 1: 6.5, about 1: 7, about 1: 7.5, about 1: 8, about 1: 8.5, about 1: 9, about 1: 9.5, about 1: 10, about 1: 11, about 1: 12, about 1: 13, about 1: 14, about 1: 15, about 1: 16, about 1: 17, about 1: 18, about 1: 19, about 1: 20, about 1: 30, about 1: 40, about 1: 50, about 1: 60, about 1: 70, about 1: 80, about 1: 90, or about 1: 100. In some embodiments, the molar amount of the reducing agent (e.g., TCEP) is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more than that of the antibody. In some embodiments, the DAR4 peak of the ADCs including an antibody having the C226/C229 mutations accounts for at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 97.5%, at least 98%, at least 98.5, at least 99%, or at least 99.5 of all peaks, e.g., determined by HIC. In some embodiments, the average DAR for ADCs including an antibody having the C226/C229 mutations (e.g., H-2B2-T-6F7-226G-229G-ADC) described herein is about 3.8, about 3.85, about 3.9, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.1, about 4.15, or about 4.2.
In some embodiments, ADCs including an antibody having the C214/C220 mutations (e.g., H-2B2-T-6F7-214G-220G-ADC) or the C214/C131 mutations for IgG4 antibodies described herein can be prepared. In some embodiments, the antibody to reducing agent (e.g., TCEP) ratio is about 1: 1, about 1: 2, about 1: 2.1, about 1: 2.2, about 1: 2.3, about 1: 2.4, about 1: 2.5, about 1: 2.6, about 1: 2.7, about 1: 2.8, about 1: 2.9, about 1: 3, about 1: 4, about 1: 4.5, about 1: 5, about 1: 5.5, about 1: 6, about 1: 6.5, about 1: 7, about 1: 7.5, about 1: 8, about 1: 8.5, about 1: 9, about 1: 9.5, about 1: 10, about 1: 11, about 1: 12, about 1: 13, about 1: 14, about 1: 15, about 1: 16, about 1: 17, about 1: 18, about 1: 19, about 1: 20, about 1: 30, about 1: 40, about  1: 50, about 1: 60, about 1: 70, about 1: 80, about 1: 90, or about 1: 100. In some embodiments, the DAR4 peak of the ADCs including an antibody having the C214G/C220G mutations (or the C214/C131 mutations for IgG4 antibodies) accounts for at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%of all peaks, e.g., determined by HIC. In some embodiments, the average DAR for ADCs including an antibody having the C214/C220 mutations (e.g., H-2B2-T-6F7-214G-220G-ADC) or the C214/C131 mutations for IgG4 antibodies described herein is about 3.8, about 3.85, about 3.9, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.1, about 4.15, or about 4.2.
Antibodies and Antigen-Binding Fragments
In general, antibodies (also called immunoglobulins) can be made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting 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 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. An antibody can comprise two identical copies of a light chain and/or two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) . These 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 region.
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.
The antibodies or antigen-binding fragments thereof can bind to various antigens, e.g., tumor-associated antigens, an antigen of a cell that is responsible for producing autoimmune antibodies, a viral or a microbial antigen. Tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type (s) of cancer cell as compared to on one or more normal non-cancerous cell (s) . Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody -based therapies.
Examples of tumor-associated antigens (TAA) include, but are not limited to, BMPR1B (bone morphogenetic protein receptor-type 1B) , E16 (LAT1, SLC7A5) , STEAP1 (six transmembrane epithelial antigen of prostate) , 0772P (CA125, MUC16) , MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin) , Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) , member 2, type II sodium-dependent phosphate transporter 3b) , Sema 5b (FLJ10372, KIAA1445, Mm. 42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like) , transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B) , PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene) , ETBR (Endothelin type B receptor) , MSG783 (RNF124, hypothetical protein FLJ20315) , STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein) , TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4) , CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) , CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs. 73792) , CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta) , B29) , FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a) , SPAP1B, SPAP1C) , HER2 (ErbB2) , NCA (CEACAM6) , MDP (DPEP1) , IL20Rα (IL20Rα, ZCYTOR7) , Brevican (BCAN, BEHAB) , EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) , ASLG659 (B7h) , PSCA (Prostate stem cell antigen precursor) , GEDA, BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3) , CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) , CD79a (CD79A, CD79α, immunoglobulin-associated alpha) , CXCR5 (Burkitt’s lymphoma receptor 1) , HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) ) , P2X5 (Purinergic receptor P2X ligand-gated ion channel 5) , CD72 (B-cell differentiation antigen CD72, Lyb-2) , LY64 (Lymphocyte antigen 64 (RP105) ) , FcRH1 (Fc receptor-like protein 1) , IRTA2 (Immunoglobulin superfamily receptor translocation associated 2) , TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan) , PMEL17, TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1) , GDNF-Ra1 (GDNF family receptor alpha 1) , Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1) , TMEM46 (shisa homolog 2 (Xenopus laevis) ; SHISA2) , Ly6G6D (lymphocyte antigen 6  complex, locus G6D; Ly6-D, MEGT1) , LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67) , RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs. 168114; RET51; RET-ELE1) , LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226) , GPR19 (G protein-coupled receptor 19; Mm. 4787) , GPR54 (KISS1 receptor) , ASPHD1 (aspartate beta-hydroxylase domain containing 1) , Tyrosinase, TMEM118 (ring finger protein, transmembrane 2) , GPR172A (G protein-coupled receptor 172A) , CD33, TROP2, and CLL-1 (CLEC12A, MICL, and DCAL2) . For convenience, information relating to these antigens, all of which are known in the art, is listed herein and includes names, alternative names, Genbank accession numbers and primary reference (s) , following nucleic acid and protein sequence identification conventions of the National Center for Biotechnology Information (NCBI) (see WO2017068511A1, which is entirely incorporated herein by reference) . Nucleic acid and protein sequences are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95%sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references. For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed. The sequences and disclosure in the reference specifically recited herein are expressly incorporated by reference.
In some embodiments, the antibodies or antigen-binding fragments can bind to programmed cell death protein 1 (PD-1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD40, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .
In some embodiments, the 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 antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) . Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. 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 antibody or an antigen binding fragment thereof can be, 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 multi-specific 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 has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the 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 antibodies (e.g., bispecific antibodies) can bind to two different antigens or two different epitopes.
In some embodiments, the antibodies (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
In some embodiments, the multi-specific 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 antibodies (e.g., bispecific antibodies) 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 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 antibodies (e.g., bispecific antibodies) can also have various forms. Many different formats of antigen binding constructs are known in the art, and are described e.g., in Suurs, et al. "A review 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 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 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 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 antibody has two different heavy chains and one common light chain. Heterodimerization of heavy chains can be based on the knob-in-holes or some other heavy chain pairing technique.
In some embodiments, CrossMAb technique can be used produce bispecific 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 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 antibody can be have a IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminals of the heavy chains.
The 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, 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 sides 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.
Bispecific T cell Engager ( “BiTE” ) consists of two scFvs, VLA VHA and VHB VLB on one peptide chain. It has only binding domains, no Fc region.
In BiTE-Fc, an Fc region is fused to the BiTE construct. The addition of Fc region enhances half-life leading to longer effective concentrations, avoiding continuous IV.
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 modular scFv-scFv-scFv, one scFv directed against the TAA is tagged with a short recognizable peptide is assembled to a bsAb consisting of two scFvs, one directed against CD3 and one against the recognizable peptide.
In ImmTAC, a stabilized and soluble T cell receptor is fused to a scFv recognizing CD3. By using a TCR, the ImmTAC is suitable to target processed, e.g. intracellular, proteins.
Tri-specific nanobody has two single variable domains (nanobodies) with an additional module for half-life extension. The extra module is added to enhance half-life.
In Trispecific Killer Engager (TriKE) , two scFvs are connected via polypeptide linkers incorporating human IL-15. The linker to IL-15 is added to increase survival and proliferation of NKs.
In some embodiments, the 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 HER2 and TROP2, and increase the avidity to cells that co-express HER2 and TROP2, to achieve enhanced targeting function. Mutations S239D and/or I332E (SI mutations) can also be introduced in antibody heavy chains to enhance the antibody affinity to FcγRIIIA.
In some embodiments, the bispecific antibody or antigen-binding fragment thereof described herein has a common light chain.
In some embodiments, the ADC described herein includes an antibody, e.g., a bispecific antibody. In some embodiments, the antibody can target to 1, 2, 3, 4, 5, or 6 antigens. In some embodiments, the antigens are the same. In some embodiments, the antigens are different. In some embodiments, the antibody can target to two different antigens, e.g., two different tumor-associated antigens (TAAs) . In some embodiments, the two TAAs are HER2 and TROP2.
In some embodiments, the antibody described herein is an antigen-binding fragment thereof, or a multi-specific antibody (e.g., a bispecific antibodies) . In some embodiments, the  antibody can be an anti-HER2 antibody or antigen-binding fragment thereof, or an anti-TROP2 antibody or antigen-binding fragment thereof. The antibody (e.g., an anti-HER2/TROP2 bispecific antibody) described herein can have various forms.
In some embodiments, the antibody described herein includes one or more cysteine mutations at heavy chain positions 220, 226, and/or 229 according to EU numbering, and/or one or more cysteine mutations at light chain position 214 according to EU numbering. In some embodiments, the antibody includes a first heavy chain polypeptide and a second heavy chain polypeptide. In some embodiments, the cysteine at heavy chain position 226 in each of the two heavy chain polypeptides is mutated (e.g., to glycine) . In some embodiments, the cysteine at heavy chain position 226 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline. In some embodiments, the cysteine at heavy chain position 229 in each of the two heavy chain polypeptides is mutated (e.g., to glycine) . In some embodiments, the cysteine at heavy chain position 229 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline. In some embodiments, the antibody described herein further includes a first light chain polypeptide and a second light chain polypeptide. In some embodiments, the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide. In some embodiments, the cysteine at heavy chain position 220 in each of the heavy chain polypeptide is mutated (e.g., to glycine) , and the cysteine at light chain position 214 in each of the two light chain polypeptide is mutated (e.g., to glycine) . In some embodiments, the cysteine at heavy chain position 220 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline. In some embodiments, the cysteine at heavy chain position 214 is mutated to glycine, alanine, valine, leucine, isoleucine, or proline.
In some embodiments, the antibodies (e.g., the anti-TROP2 antibody, the anti-HER2 antibody, or the bispecific antibody) , or the related antibody drug conjugates (ADC) , 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: 22 or 31, 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 SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 38, or 39.
In some embodiments, the antibodies (e.g., the anti-TROP2 antibody, the anti-HER2 antibody, or the bispecific antibody) , or the related antibody drug conjugates (ADC) , have a first  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 SEQ ID NO: 38, and a second 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 SEQ ID NO: 39. In some embodiments, the first and second heavy chain constant regions include KIH mutations. In some embodiments, the first and second heavy chain constant regions do not include KIH mutations.
HER2
Human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases.
HER2 is overexpressed in various cancer types such as breast cancer and gastric cancer and has been reported to be a negative prognostic factor in breast cancer. As anti-HER2 drugs effective for HER2-overexpressing cancers, trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, and the like are known.
The disclosure provides several antibodies and antigen-binding fragments thereof that specifically bind to HER2. In some embodiments, the anti-HER2/TROP2 bispecific antibodies can include an antigen binding region that is derived from these antibodies.
The antibodies and antigen-binding fragments described herein are capable of binding to HER2. The disclosure provides e.g., anti-HER2 antibody H-2B2 ( “2B2” ) , and the antibodies derived therefrom.
The CDR sequences for 2B2, and 2B2 derived antibodies 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 numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 10-12, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 4-6.
Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 7-9 and SEQ ID NOs: 10-12; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6.
In some embodiments, the antibody or an 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 antibody or an 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 antibody or an 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.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light 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 insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
The disclosure also provides antibodies or antigen-binding fragments thereof that bind to HER2. The antibodies or antigen-binding fragments thereof 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 SEQ ID NO: 19, 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 SEQ ID NO: 21.
In some embodiments, the antibody or antigen binding fragment thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some  embodiments, the antibody or antigen binding fragment thereof 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 a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. 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 HER2 (e.g., human HER2) .
The anti-HER2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (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 antibodies or antigen-binding fragments thereof 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 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to HER2 will retain an ability to bind to HER2. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
TROP2
Trophoblast cell-surface antigen 2 (TROP2) , also known as Tumor-associated calcium signal transducer 2 (TACSTD2) , is a cell surface glycoprotein encoded and expressed by the  TACSTD2 gene. It has high structural sequence similarity with epithelial adhesion molecule Epcam. TROP2 is a protein closely related to tumors. It mainly promotes tumor cell growth, proliferation and metastasis by regulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma and ovarian cancer. The protein can promote tumor cell proliferation, invasion, metastasis, spread and other processes. In addition, in breast cancer and other cancers, the high expression of TROP2 has also been found to be closely related to more aggressive diseases and poor clinical prognosis of tumors.
The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to TROP2. The anti-HER2/TROP2 bispecific antibodies can include an antigen binding region that is derived from these antibodies.
The antibodies and antigen-binding fragments described herein are capable of binding to TROP2. The disclosure provides anti-TROP2 antibody T-6F7 ( “6F7” ) , and the antibodies derived therefrom.
The CDR sequences for 6F7, and 6F7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat numbering. Under Chothia numbering, 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: 4-6.
Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 13-15 and SEQ ID NOs: 16-18; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6.
In some embodiments, the antibody or an 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 antibody or an 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 antibody or an 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.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light 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 insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
The disclosure also provides antibodies or antigen-binding fragments thereof that binds to TROP2. The antibodies or antigen-binding fragments thereof 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 SEQ ID NO: 20, 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 SEQ ID NO: 21.
In some embodiments, the antibody or antigen binding fragments thereof can have 3 VH CDRs that are identical to the CDRs of any VH sequences as described herein. In some embodiments, the antibody or antigen binding fragments thereof 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 a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. 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 TROP2.
The anti-TROP2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (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 antibodies or antigen-binding fragments thereof 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 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to TROP2 will retain an ability to bind to TROP2. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
Antibody and ADC Characteristics
The antibody or ADC derived therefrom can include an antigen-binding region that is derived from any antibody or any antigen-binding fragment thereof as described herein.
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 antibodies (e.g., bispecific antibody) or the ADC derived therefrom can bind to an antigen 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 0.0001 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 0.0001 s-1, or greater than 0.00001 s-1. In some embodiments, the dissociation rate (koff) is less than 1 x 10-2 s-1.
In some embodiments, kinetic association rates (kon) is greater than 1 x 102/Ms, greater than 1 x 103/Ms, greater than 1 x 104/Ms, greater than 1 x 105/Ms, or greater than 1 x 106/Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 105/Ms, less than 1 x 106/Ms, or less than 1 x 107/Ms. In some embodiments, kinetic association rates (kon) is greater than 1 x 105/Ms.
In some embodiments, the antibodies (e.g., bispecific antibody) or the ADC derived therefrom can bind to an antigen with a KD of less than 1 x 10-6 M, less than 1 x 10-7 M, less than 1 x 10-8 M, less than 1 x 10-9 M, or less than 1 x 10-10 M. In some embodiments, the KD is less than 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10-7 M, greater than 1 x 10-8 M, greater than 1 x 10-9 M, or greater than 1 x 10-10 M.
Thermal stabilities can also be determined. The antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom 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 antibodies or ADCs 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 antibodies or ADCs 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 antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, have an endocytosis rate in cells (e.g., in NCI-N87 cells or NCI-H292 cells) that is at least 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%. In some embodiments, the endocytosis rate that is less than 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%.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, can bind to dog HER2, monkey HER2, or mouse HER2. In some embodiments, the binding is measured by the percentage of positive cells as determined by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage of positive cells is less than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the antibodies, (e.g., bispecific antibody) , or the ADCs derived therefrom, cannot bind to dog HER2, monkey HER2, or mouse HER2.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, can bind to dog TROP2, monkey TROP2, or mouse TROP2. In some embodiments, the binding is measured by the percentage of positive cells as determined by FACS. In some embodiments, the percentage of positive cells is greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage of positive cells is less than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the antibodies (e.g., bispecific antibody) , or the ADC derived therefrom, cannot bind to dog TROP2, monkey TROP2, or mouse TROP2.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, has a purity that is greater than 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, e.g., as measured by SEC-HPLC. In some embodiments, the antibodies, the purity is less than 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, , e.g., as measured by SEC-HPLC.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom,  has a yield that is greater than 20, 30, 40, 50, 60, 70, 80, 90, or 100 (mg/L) . In some embodiments, the yield is less than 20, 30, 40, 50, 60, 70, 80, 90, or 100 (mg/L) .
In some embodiments, the stability of the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, is measured by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) . In some embodiments, the percentage of the main component is greater than 10%, 20%, 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%, after being subject to various conditions e.g., as measured by cIEF. In some embodiments, the condition is storing at 40℃ for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the condition is freeze-thaw for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 50 times. In some embodiments, the condition is storing the composition at pH 3.5 for about or at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, after the treatment, the percentage of the acidic component is greater than 10%, 20%, 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 cIEF. In some embodiments, after the treatment, the percentage of the alkaline component is greater than 10%, 20%, 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 cIEF. In some embodiments, after the treatment, the percentage of the main component is less than 10%, 20%, 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 cIEF. In some embodiments, the percentage of the acidic component is less than 10%, 20%, 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 cIEF. In some embodiments, the percentage of the alkaline component is less than 10%, 20%, 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 cIEF.
In some embodiments, the cell killing ability of the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) , or the ADCs derived therefrom, is measured by IC50 (ng/ml) (e.g., in NCI-N87 or NCI-H292 cells) . In some embodiments, the IC50 is greater than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30,  40 or 50 μg/ml. In some embodiments, the IC50 is less than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40 or 50 μg/ml.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) or the ADCs derived therefrom, can bind to one or more Fc receptor selected from the group consisting of FcγRI, FcRn, FcγRIIA-R167, FcγRIIA-H167, FcγRIIB, FcγRIIIA-V176, FcγRIIIA-F176, and FcγRIIIB-NA1, with an affinity (indicated by KD) of less than less than 1 x 10-5 M, less than less than 1 x 10-6 M, less than 1 x 10-7 M, less than 1 x 10-8 M, less than 1 x 10-9 M, or less than 1 x 10-10 M.
In some embodiments, the antibodies (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) or the ADCs derived therefrom, has a tumor growth inhibition 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 antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. 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, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition 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 antibody (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) 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, one or both mutations S239D and/or I332E (SI mutations) are introduced in antibody Fc region to enhance the antibody affinity to FcγRIIIA, thereby  increasing ADCC effects. A detailed description of SI mutations can be found in US7662925, which is incorporated by reference in their entirety.
In some embodiments, the antibody described herein (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) does not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fv fragments.
In some embodiments, the antibody described herein (e.g., a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) is incorporated in an antibody drug conjugate.
In some embodiments, the antibodies (a bispecific antibody including the C226, C229, C226/C229, or C214/C220 mutations described herein) or the ADCs derived therefrom has a half-life after administration of more than 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days, 200 days, 210 days, 220 days, 230 days, 240 days, 250 days, 260 days, 270 days, 280 days, 290 days, 300 days, 310 days, 320 days, 330 days 340 days, 350 days, 360 days, 370 days, 380 days, 390 days, 400 days, 410 days, 420 days, 430 days, 440 days, or 450 days, as determined using any of the methods described herein.
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 recombinant 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-A tail, 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 an antibody-encoding or 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 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., 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 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 a first antigen. 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 a first antigen. 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 a second antigen. 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 a second antigen. In some embodiments, the VL regions are identical.
In some embodiments, provided herein is a vector including a nucleic acid encoding the antibody described herein. In some embodiments, provided herein is a cell including the vector described herein. In some embodiments, the cell is a CHO cell. In some embodiments, provided herein is a cell including a nucleic acid encoding the antibody described herein.
In some embodiments, 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 antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies 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 here are designed to make 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.
In some embodiments, knob-into-hole (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.
Furthermore, an anion-exchange chromatography can be used to purify bispecific antibodies. Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE) . In solution, the resin is coated with positively charged counter-ions (cations) . Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pI) . The isoelectric point is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. Thus, in some embodiments, different amino acid substitution can be introduced into two heavy chains, so that the pI for the homodimer comprising two Arm A and the pI for the homodimer comprising two Arm B is different. The pI for the bispecific antibody having Arm A and Arm B will be somewhere between the two pIs of the homodimers. Thus, the two homodimers and the bispecific antibody can be released at different pH conditions. The present disclosure shows that a few amino acid residue substitutions can be introduced to the heavy chains to adjust pI.
Bispecific antibodies can also include e.g., cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
In some embodiments, provided herein are methods of producing the antibody described herein, the method including (a) culturing a cell including a vector or a nucleic acid encoding the antibody described herein under conditions sufficient for the cell to produce the antibody; and (b) collecting the antibody produced by the cell.
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 engineered antibodies (e.g., bispecific antibodies) , or the 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 antibodies (e.g., bispecific antibodies) , or an antibody drug conjugate described herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testis cancer, thyroid cancer, or urothelial 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 subject is a human.
In some embodiments, the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.
In some embodiments, the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, or testis cancer.
In some embodiments, the cancer is pancreas cancer, lung cancer, stomach cancer, prostate cancer, breast cancer, ovary cancer, colon cancer, skin cancer, or brain cancer.
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 antibody, antigen binding fragment, antibody-drug conjugates, 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 antibody, an antigen binding fragment, or an 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 antibody, antigen binding fragment, or 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 composition used.
Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, 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 antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments,  antibody-drug conjugates, and/or compositions disclosed herein used and other drugs being administered to the mammal.
A typical daily dosage of an effective amount of an antibody (e.g., a bispecific antibody) or the antibody drug conjugate is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100 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 antibody (e.g., a bispecific antibody) , antibody-drug conjugates, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (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, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) . In some embodiments, at least one antibody (e.g., a bispecific antibody) , or antibody-drug conjugate, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) . In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
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 antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) . In some  embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, antibody-drug conjugate, or pharmaceutical compositions described herein) 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 antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) or antibody-drug conjugate in the subject.
In some embodiments, the subject can be administered the at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) 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 antibodies or antigen-binding antibody fragments, 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 antibody or antigen-binding antibody fragment (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 EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-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 HER3, 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, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-B7-H3 antibody, an anti-CLDN18 antibody, an anti-SIGLEC15 antibody, an anti-41BB antibody, an anti-CD40 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 antibodies (e.g., bispecific antibodies) , or antibody-drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antibodies, or antibody- drug conjugates described herein can be present in a pharmaceutical composition in any combination. 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 antibody or antigen-binding fragment thereof 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 antibodies, or 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.
Exemplary doses include milligram or microgram amounts of any of the antibodies or ADCs 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) .
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 antibodies or antigen binding fragments thereof, or antibody-drug conjugates 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. Generation and testing of anti-HER2/TROP2 bispecific antibodies
Substitution of interchain cysteine residues and preparation
H-2B2-T-6F7 is a bispecific antibody including an anti-HER2 arm (H-2B2) and an anti-TROP2 arm (T-6F7) , which has a human IgG1 subtype structure. Details of this bispecific antibody can be found in PCT Application No. PCT/CN2022/074078, which is incorporated herein by reference in its entirety.
Mutations were introduced to interchain cysteine residues of H-2B2-T-6F7. H-2B2-T-6F7-226G includes the mutation of a pair of cysteines to glycines at position 226 (according to EU numbering) of the hinge region of the H-2B2-T-6F7 IgG1 subtype. H-2B2-T-6F7-229G includes the mutation of a pair of cysteines to glycines at position 229 (according to EU numbering) in the hinge region of the H-2B2-T-6F7 IgG1 subtype. H-2B2-T-6F7-226G-229G includes the mutation of two pairs of cysteines to glycines at positions 226 and 229 (according to EU numbering) in the hinge region of the H-2B2-T-6F7 IgG1 subtype. H-2B2-T-6F7-214G-220G includes the mutation of two pairs of cysteines to glycines at position 214 of the kappa  light chain and position 220 of the heavy chain (according to EU numbering) . H-2B2-T-6F7 is the parent bispecific antibody (H-2B2-T-6F7 IgG1 subtype) without any mutations of the interchain cysteine residues.
Specifically, the anti-HER2 antibody H-2B2 and anti-TROP2 antibody T-6F7 can be paired to form the bispecific antibody H-2B2-T-6F7. Vectors for the light chain and heavy chain, either with or without mutated interchain cysteine residues, of the antibodies were constructed. CHO-S cells were co-transfected with three vectors, including a first vector encoding the H-2B2 arm heavy chain, a second vector encoding the T-6F7 arm heavy chain, and a third vector encoding the kappa light chain. After 14 days of culture, the cell supernatant was collected and purified by Protein A affinity chromatography. Various methods can be used to reduce the chance of wrong pairing between the two heavy chains. In the Fc region, knobs-into-holes mutations were introduced to the anti-HER2 arm heavy chain and the anti-TROP2 arm heavy chain. For example, in H-2B2-T-6F7, the heavy chain constant region of the H-2B2 arm has knob mutations, and the heavy chain constant region of the T-6F7 arm has hole mutations; in T-6F7-H-2B2, the heavy chain constant regions of the T-6F7 arm has knob mutations, and the heavy chain constant regions of the H-2B2 arm has hole mutations.
An exemplary antibody structure is shown in FIG. 1, where target 1 and target 2 can be HER2 and TROP2 respectively, or TROP2 and HER2 respectively.
The antibodies include the same light chain, and their heavy chain and light chain sequences are shown as follows:




Protein expression and purity detection
Purified anti-HER2/TROP2 bispecific antibodies were analyzed by a non-reducing SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) and SEC-HPLC (size exclusion chromatography-high performance liquid chromatography) .
Non-reducing SDS-PAGE was performed using a 4-12%acrylamide gel. The protein samples were prepared as follows. First, 2.4 μl of the protein sample was mixed with 6 μl Tris-Glycine SDS Sample Buffer (2 ×) (Invitrogen; Cat#: LC2676) and 3.6 μl distilled water. The mixture was then boiled for 2 minutes and instantly centrifuged before loading. 10 μg of each sample was loaded to the gel.
In the SEC-HPLC method, the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatography system (connected with XBridgeTM Protein BEH SEC column (Waters Corporation) ) was used. The following parameters were used: mobile phase: 25 mmol/L phosphate buffer (PB) + 300 mmol/L NaCl, pH 6.8; flow rate: 1.8 ml/min; column temperature: 25 ℃; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 4℃; and running time: 7 minutes. Results are summarized in the table below.
Table 1. Non-reducing SDS-PAGE and SEC-HPLC analysis results
The non-reducing SDS-PAGE results showed that the band representing H-2B2-T-6F7-214G-220G migrated faster and was slightly smaller than the parent antibody band, with both the light chain band (about 25 KD) and heavy chain band (about 50 KD) observed. In addition, the SEC-HPLC result showed that the main peak percentage of H-2B2-T-6F7-214G-220G was  lower than that of other tested antibodies, indicating an unstable interaction between the heavy and light chains. The expression level of H-2B2-T-6F7-214G-220G was also lower than that of other tested antibodies. Further, the tested antibodies with cysteine mutations in the hinge region, e.g., H-2B2-T-6F7-229G, H-2B2-T-6F7-226G and H-2B2-T-6F7-226G-229G, showed satisfactory expression results. The results also indicate that introducing cysteine mutations in the hinge region did not affect purity of the antibodies.
Cross-species binding analyses by flow cytometry
CHO-S-hTROP2 (CHO-S cells expressing human TROP2; SEQ ID NO: 35) , CHO-S-fasTROP2 (CHO-S cells expressing Macaca fascicularis TROP2 (fasTROP2; SEQ ID NO: 37) , CHO-S-hHER2 (CHO-S cells expressing human HER2; amino acids 23-652 of SEQ ID NO: 34) , and CHO-S-fasHER2 (CHO-S cells expressing Macaca fascicularis (crab-eating macaque) HER2 (fasHER2, amino acids 174-804 of SEQ ID NO: 36) ) were transferred to a 96-well plate at a density of 5 × 104 cells/well. Gradient dilutions of the sample antibodies were added into the 96-well plate, and incubated at 4℃ for 30 minutes. After that, the cells were incubated with Goat Anti-hIgG-Fc-Alex Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4℃ in the dark for 15 minutes before flow cytometry analysis. The results are shown in the following table.
Table 2. Cross-species binding results

The results showed that the cysteine mutations did not affect the binding activity of antibodies H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, and H-2B2-T-6F7-226G-229G.
In another similar experiment, binding analysis of each mutant antibody to hTROP2 and fasHER2 proteins was performed, and the results are shown in the table below.
Table 3. Cross-species binding results
The results showed that the cysteine mutations did not affect the antigen-binding activity of tested antibodies. All tested antibodies showed binding to human TROP2 and monkey HER2.
Verification of binding activity to SKOV3 cells
The binding activity of anti-HER2/TROP2 bispecific antibodies to SKOV3 cells expressing hHER2 and hTROP2 was verified by flow cytometry. The secondary antibody used in the experiment was AF647-conjugated Goat Anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., Cat #: 109-606-170) . Experiments were carried out with serially diluted antibodies to determine the EC50 value of antibody binding to cells. The results are shown in FIG. 2 and the table below.
Table 4. Combined flow cytometry test results
The results showed that each mutant antibody and the parent antibody had roughly the same trend of cell binding with concentration. The table above shows that the mutant antibodies H-2B2-T-6F7-226G and H-2B2-T-6F7-214G-220G had lower EC50 values than the parent antibody H-2B2-T-6F7, indicating their enhanced cell-binding ability. However, H-2B2-T-6F7-226G-229G, with cysteine mutations only in the hinge region, showed comparable cell-binding activity relative to the parent antibody.
Determination of antibody binding affinity to HER2 and TROP2
The affinity of the anti-HER2/TROP2 bispecific antibodies to His-tagged human TROP2 protein (hTROP2, ACROBiosystems Inc., Cat#: TR2-H5223) , His-tagged monkey TROP2 protein (fasTROP2, ACROBiosystems Inc., Cat#: R52H3) , His-tagged human HER2 protein (hHER2, ACROBiosystems Inc., Cat#: HE2-H5225; amino acids 23-652 of SEQ ID NO: 34) and His-tagged monkey HER2 protein (fasHER2, ACROBiosystems Inc., Cat#: HE2-C52Hb; SEQ ID NO: 36) were measured by surface plasmon resonance (SPR) using BiacoreTM (Biacore, INC, Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.
Purified anti-HER2/TROP2 bispecific antibodies were diluted to 0.5 μg/ml and then injected into the BiacoreTM 8K biosensor at 10 μL/min for about 50 seconds to achieve a desired protein density (e.g., about 120 response units (RU) ) . His-tagged TROP2 protein or His-tagged  HER2 protein at a concentration of 200 nM was then injected at 30 μL/min for 180 seconds. Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0) , 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.
Table 5. Affinity test results (HER2)
Table 6. Affinity test results (TROP2)
The results showed that all tested antibodies had good binding affinity to human HER2 and human TROP2. All tested antibodies also showed cross-species binding affinity to monkey HER2 and monkey TROP2. All mutant antibodies exhibited comparable affinities for HER2 and TROP2 relative to the parent antibody.
Determination of antibody binding affinity to Fc receptors
The affinity of anti-HER2/TROP2 bispecific antibodies to FcγRI (His-tagged FcγRI /CD64 Protein of human, ACROBiosystems Inc., Cat#: FCA-H52H1) , FcRn (His-tagged or Tag&Strep II-tagged FCGRT&B2M Heterodimer protein, ACROBiosystems Inc., Cat#: FCM-H5286) , FcγRIIA-R167 (His-tagged Fc gamma RIIA/CD32a (R167) protein of human, ACROBiosystems Inc., Cat#: CDA-H5221) , FcγRIIA-H167 (His-tagged Fc gamma RIIA CD32a (H167) protein of human, ACROBiosystems Inc., Cat#: CD1-H5223) , FcγRIIB (His-tagged mouse Fc gamma RIIB protein, ACROBiosystems Inc., Cat#: CDB-H5228) , FcγRIIIA-V176 (His-tagged human Fc gamma RIIIA (V176) protein, ACROBiosystems Inc., Cat#: CD8-H52H4) , FcγRIIIA-F176 (His-tagged human Fc gamma RIIIA/CD16a (F176) protein, ACROBiosystems Inc., Cat#: CDA-H5220) , FcγRIIIB-NA1 (His-tagged human Fc gamma RIIIB CD16b (NA1) , ACROBiosystems Inc., Cat#: CDB-H5227) were measured by surface plasmon resonance (SPR) using BiacoreTM (Biacore, INC, Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips.
Fc receptor proteins were diluted to 1 μg/ml and then injected into the BiacoreTM 8K biosensor at 10 μL/min for about 50-100 seconds to achieve a desired protein density (e.g., about 50-200 response units (RU) ) . Purified anti-HER2/TROP2 bispecific antibodies were serially diluted and then injected at 10 μg/mL for 50-100 seconds. Dissociation was monitored for 200 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 1.7, 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.
Table 7. Affinity test result (Fc receptors)

Note: “-” means no binding.
Compared with the parent antibody H-2B2-T-6F7, the binding of the mutated antibodies to FcγRI and FcRn receptors was almost unchanged. H-2B2-T-6F7-226G-229G completely lost its binding ability for FcγRIIB and FcγRIIIB-NA1 receptors. For FcγRIIIA-V176 and FcγRIIIA-F176 receptors, H-2B2-T-6F7-226G-229G and H-2B2-T-6F7-229G completely lost its binding ability, whereas H-2B2-T-6F7-226G partially lost its binding ability, indicating that Cys229 plays an important role in maintaining the binding of FcγRIIIA. For FcγRIIA-R167 and FcγRIIA-H167 receptors, H-2B2-T-6F7-226G-229G had a reduced affinity (by about 10 folds) as compared to the parent antibody.
Stability of anti-HER2/TROP2 bispecific antibodies
Anti-HER2/TROP2 antibodies H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were diluted to 5 mg/ml using a buffer (3 mg/ml histidine, 80 mg/ml sucrose, and 0.2 mg/ml TweenTM 80) at pH 6.0. The diluted antibodies were kept in sealed Eppendorf tubes at 40 ± 3 ℃ (hereinafter referred to as 40 ℃) for 7 days, and their thermal stability was evaluated. Alternatively, the five anti-HER2/TROP2 antibodies were diluted to 5 mg/ml using the buffer at pH 3.5. The diluted antibodies were kept in sealed Eppendorf tubes at pH 3.5 for 6 hours to determine its stability in acidic conditions. Alternatively, the five anti-HER2/TROP2 antibodies were repeatedly freeze-thawed 10 times.
After the above treatments, the following tests were performed: (1) observing the solution appearance and presence of visible non-soluble objects; (2) detecting the purity changes of antibodies 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, %)) ; (3) detecting changes in the apparent hydrophobicity of the antibodies using the Hydrophobic Interaction Chromatography-High Performance Liquid Chromatography (HIC-HPLC) method (indicated as the retention time of the main peak (HIC, min) ) ; (4) detecting the purity changes of antibodies by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under reducing (CE-SDS (R) ) and non-reducing (CE-SDS (NR) ) conditions (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) ) ; (5) detecting charge variants in the antibodies by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) .
In the SEC-HPLC experiments, the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatography system (connected with XBridgeTM Protein BEH SEC column (Waters Corporation) ) was used. The following parameters were used: mobile phase: 25 mmol/L phosphate buffer (PB) + 300 mmol/L NaCl, pH 6.8; flow rate: 1.8 ml/min; column temperature: 25 ℃; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 4℃; and running time: 7 minutes.
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: 1.0 M ammonium sulfate, 20 mM sodium acetate, 10%acetonitrile pH 6.5; mobile phase B: 20 mM sodium acetate, 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 10 ℃; and running time: 30 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 protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI Marker-4.05, 1 μL Maurice cIEF pI Marker-9.99, 35 μL 1%Methyl Cellulose Solution, 2 μL Maurice cIEF 500 mM Arginine, 4 μL Ampholytes (Pharmalyte pH ranges 3-10) , 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 experiments, Maurice (Protein simple, MauriceTM) and Maurice CE-SDS Size Application Kit (Protein simple, Cat#: PS-MAK02-S) were used.
In CE-SDS (NR) , 54 μL Sample Buffer, 6 μL antibody sample, 2.4 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.
In CE-SDS (R) , 54 μL Sample Buffer, 6 μL antibody sample, 2.4 μL 25× internal standard, 3 μL 2-Mercaptoethanol (SIGMA, Cat#: M6250) 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. 50 μL supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice. The injection voltage was 4.6 kV, the injection time was 20 seconds, the separation voltage was 5.75 kV, and the separation time was 30 minutes. Detailed results are shown in the table below.
Table 8

The results showed that all mutant antibodies and the parent antibody exhibited a good stability under various physical and chemical conditions. The cysteine mutations did not substantially affect the stability or accelerated stability of the anti-HER2/TROP2 bispecific antibodies, e.g., in acidic conditions.
Example 2. Generation and testing of antibody drug conjugates (ADCs)
After Protein A purification, H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were dialyzed and concentrated in a PBS buffer by ultrafiltration. The concentration was determined by UV absorption. These antibodies were used for the subsequent antibody drug coupling reactions.
Coupling of anti-HER2/TROP2 bispecific antibodies with drug molecules
The purified anti-HER2/TROP2 antibodies H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, H-2B2-T-6F7-226G-229G, H-2B2-T-6F7-214G-220G and H-2B2-T-6F7 were coupled with MMAE (monomethyl auristatin E) via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. A reducing agent was used to reduce the interchain disulfide bond of the bispecific antibody to couple the small molecule drug. The reducing agent used was tris (2-carboxyethyl) phosphine (TCEP) .
With respect to the names of antibody-drug conjugates, “ADC” is added directly after the antibody name. For example, when H-2B2-T-6F7 IgG1 is coupled to MMAE, it is named as H-2B2-T-6F7-ADC. A human IgG1 isotype control was coupled to MMAE to form an isotype-control-ADC.
SEC-HPLC and HIC-HPLC were used to detect the coupling of antibodies with drug molecules. The test results are shown in the tables below.
The number of conjugated drugs per antibody can be controlled by adjusting the ratio of the bispecific antibody to TCEP. As shown in FIGS. 3-7, as the amount of TCEP increased, the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-229G (FIG. 3) and H-2B2-T-6F7-226G (FIG. 4) varied from 0-6; the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-214G-220G (FIG. 6) varied from 0-4; and the number of conjugated drugs per antibody in ADCs obtained from the parent antibody H-2B2-T-6F7 (FIG. 7) varied from 0-8, each with a high level of heterogeneity. However, with the increase of TCEP and when TCEP reached a certain ratio (e.g., Antibody: TCEP = 1: 4) or in excess, the number of conjugated drugs per antibody in ADCs obtained from H-2B2-T-6F7-226G-229G (FIG. 5) was uniformly around 4. Specifically, the elution time for peaks representing DAR=0, 2, 4, 6, and 8 were about 4.39 minutes, about 5.33 minutes, about 6.49 minutes, about 7.49 minutes, and about 8.20 minutes, respectively.
According to the above experiments, it was confirmed that by adjusting the amount (or ratio) of TCEP, the average DAR value of the ADCs obtained by coupling the antibody to the small molecule can be about 4 (see Table 9 for the specific ratios as detected by HIC-HPLC) . The SEC chromatography detection results indicating the antibody purity before and after conjugation are shown in Table 10. The results showed that the DAR value of the obtained H-2B2-T-6F7-226G-229G-ADC was about 4 for when the antibody: TCEP ratio was 1: 8. The average DAR value was calculated by multiplying PA% (PA%is the percentage of the 280 nm peak area to the sum of all peak areas, as measured by HIC-HPLC) with the corresponding drug load of 0, 2, 4, 6, or 8, and then divided by total PA%.
Table 9. HIC-HPLC detection results
Table 10. SEC-HPLC detection results

The results showed that the DAR4 peak of H-2B2-T-6F7-226G-229G-ADC accounted for 98.03%of all peaks when the Antibody: TCEP was 1: 8, and the DAR value was uniformly about 4.
Endocytosis test of bispecific antibodies and ADCs
The anti-HER2/TROP2 bispecific antibodies or corresponding ADCs, and goat anti-human IgG secondary antibodies labeled with pH-sensitive markers were added to NCI-H292 cells (with high expression levels of human HER2 and TROP2, respectively) , and incubated for 1.5 hours. The cells were centrifuged and washed with FACS buffer. Mean fluorescent intensity (MFI) was measured by flow cytometry. Endocytosis rates of antibodies to NCI-H292 cells were calculated by determining the percentage of positively labeled cells. The results are shown in the following table. An antibody targeting an irrelevant target protein was used as an isotype control.
The goat anti-human IgG secondary antibodies labeled with pH-sensitive markers were used to indirectly detect endocytosis rates of the anti-HER2/TROP2 bispecific antibodies and their corresponding ADCs.
Table 11. Antibody endocytosis rates
The results showed that for unconjugated bispecific antibodies, the cysteine mutations can affect endocytosis as compared to the parent antibody H-2B2-T-6F7. Each pair of cysteine mutations can reduce the endocytosis rate accumulatively. As a result, H-2B2-T-6F7-226G-229G showed a further reduction of the endocytosis rate when both pairs of cysteines in the hinge  region were mutated. In particular, cysteine mutations between the light and heavy chains (e.g., H-2B2-T-6F7-214G-220G) significantly reduced endocytosis.
By comparing the endocytosis rates of antibodies before and after conjugation, the results showed that conjugation of mutant antibodies had little effect on endocytosis, whereas conjugation of the parent antibody (H-2B2-T-6F7) showed a relatively large change on endocytosis.
In vitro killing activity
Different concentrations of ADCs (average DAR value of about 4) were used to treat human lung cancer cell line NCI-H292 cultured in a cell culture plate, and the killing activity was detected after 72 hours of incubation within theS3 live-cell analysis system (Sartorius AG) . The results are shown in the table below.
Table 12. In vitro killing results
The results showed that H-2B2-T-6F7-226G, H-2B2-T-6F7-229G, and H-2B2-T-6F7-226G-229G maintained a high in vitro killing activity.
Pharmacokinetic (PK) test results
The ADCs (average DAR value of about 4) prepared from each mutant antibody and the parent antibody, together with the parent antibody (unconjugated) were prepared as an administration solution at 200 μg/mL in PBS and administered intravenously to 4.5-week to 8-week-old male C57BL/6 mice at a dose level of 3 mg/kg. Approximately 0.05 mL of blood was collected from the vein at 15 minutes, 6 hours, 24 hours, 96 hours, 168 hours, 240 hours, 336 hours, and 504 hours after administration. Serum was obtained by transferring blood to 1.5 mL volume polypropylene tubes, followed by centrifugation at 4 ℃.
The serum concentration of each antibody and ADC was measured by sandwich ELISA. The serum obtained in the experiment was diluted 10 times with blank serum and then 20 times with 1%BSA to prepare measurement samples.
AffiniPure Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch Inc. Cat#: 109-005-088) and Anti-MMAE mIgG (ACRO Biosystems Inc., Cat#: MME-M5252) were used to determine the serum concentration of total antibody and the serum concentration of ADCs, respectively. Specifically, 2000 ng/mL G-H-IgG or Anti-MMAE mIgG (100 μl) was added to a 96-well plate (Nunc MaxisorpTM 96-well plate, Nunc, Cat#: 468667) . The plate was incubated at 2-8℃ overnight. After the incubation, the plate was washed 4 times with a PBS-T buffer, and the antibody-unbound areas were blocked with 2%BSA (bovine serum albumin, SIGMA, Cat#: A1933) for 2 hours at 37 ℃. After washing the plate 4 times with the PBS-T buffer, 100 μL of blocking buffer (1%BSA) was added to each well. The wells were sealed and incubated at 37 ℃for 1 hour. Afterwards, the plate was washed by the PBS-T buffer 4 times. 100 μl Peroxidase AffiniPure F (ab') 2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., Cat#: 109-036-098) prepared in 1%PBS was added for determining the serum concentration of total antibody. Alternatively, G-h-IgG κ L-HRP (abcam, Cat#: ab202549) was added for determining the serum concentration of ADCs. The plate was incubated at 37 ℃for 1 hour, and then washed with the PBS-T buffer 4 times. Tetramethylbenzidine (TMB) chromogenic solution (Beyotime, Cat#: P0209) was used for color development for 5-10 minutes at room temperature, and then a stop solution (Beyotime, Cat#: P0215) was added. Luminescent signals of the plate was measured at 450 nm and 630 nm.
The absorbance value and corresponding concentration of the calibration sample prepared by each test product was used to create a standard curve with four parameters (i.e., T1/2, Cmax, AUC0-504h, and CL) . The standard curve was used to calculate the antibody or ADC concentration of each serum sample. A drug concentration-time curve was created using the calculated sample concentration at each time point. PhoenixTM WinNolin 8.3 was used to calculate the pharmacokinetic parameters.
The results are shown in FIG. 8, FIG. 9, and the table below. When the antigen G-H-IgG was used to measure the corresponding total antibody concentration in the blood, “Tab” was added to the name of corresponding groups. When the antigen Anti-MMAE mIgG was used to measure the ADC concentration in the blood, “ADC” was added to the name of corresponding  groups. For example, H-2B2-T-6F7-229G-ADC represents the concentration of H-2B2-T-6F7-229G-ADC (either conjugated or cleaved) in blood measured after administration of H-2B2-T-6F7-229G-ADC, while H-2B2-T-6F7-229G-Tab represents the total antibody concentration of H-2B2-T-6F7-229G in blood measured after administration of H-2B2-T-6F7-229G-ADC. H-2B2-T-6F7 represents the concentration of total H-2B2-T-6F7 antibody in blood measured after administration of the unconjugated parent antibody H-2B2-T-6F7.
Table 13 Pharmacokinetic parameter results
The results showed that at the dose level of 3 mg/kg, the total antibody half-life of the tested ADCs in each administration group was about 2-3 times longer than the ADC half-life.. The changes of the serum concentration of each mutant antibody ADCs were not significantly different from that of the parent antibody ADC.
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 (65)

  1. An antibody-drug conjugate comprising
    an antibody comprising
    one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and
    one or more cysteine residues at positions selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214,
    wherein a therapeutic agent is linked to the antibody through the one or more cysteine residues.
  2. The antibody-drug conjugate of claim 1, wherein the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide.
  3. The antibody-drug conjugate of claim 2, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226.
  4. The antibody-drug conjugate of claim 2 or 3, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226.
  5. The antibody-drug conjugate of any one of claims 2-4, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226.
  6. The antibody-drug conjugate of any one of claims 2-5, wherein the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229.
  7. The antibody-drug conjugate of any one of claims 2-5, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229.
  8. The antibody-drug conjugate of any one of claim 2-5 and 7, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229.
  9. The antibody-drug conjugate of any one of claims 2-5, 7, and 8, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229.
  10. The antibody-drug conjugate of any one of claims 2-9, further comprising a first light chain polypeptide and a second light chain polypeptide, wherein the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide.
  11. The antibody-drug conjugate of claim 10, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 220, and the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214.
  12. The antibody-drug conjugate of claim 10 or 11, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 220, and the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214, wherein amino acid residues at heavy chain position 220 and light chain position 214 can be the same or different.
  13. The antibody-drug conjugate of any one of claims 10-12, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 220, and the first and second light chain polypeptides each comprises a glycine at light chain position 214.
  14. The antibody-drug conjugate of claim 1, wherein the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide, wherein
    (a) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214;
    (b) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214;
    (c) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214; or
    (d) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 220, and a cysteine at heavy chain position 226 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229; the first light chain polypeptide comprises a non-cysteine residue at light chain position 214; and the second light chain polypeptide comprises a non-cysteine residue at light chain position 214.
  15. The antibody-drug conjugate of any one of claims 1-14, wherein the antibody comprises heavy and light chain constant region sequences derived from human IgG1.
  16. The antibody-drug conjugate of any one of claims 1-15, wherein the antibody is a bispecific antibody or multi-specific antibody.
  17. The antibody-drug conjugate of any one of claims 1-16, wherein the antibody comprises knobs-into-holes (KIH) mutations.
  18. A antibody-drug conjugate comprising
    an antibody, wherein the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide, wherein the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide; and
    a therapeutic agent that is covalently linked to the antibody; wherein:
    1) the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 23, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 24, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22;
    2) the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 25, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 26, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22;
    3) the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 27, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 28, the first light chain polypeptide comprises an amino acid sequence that is at least  80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22;
    4) the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 29, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 30, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22; or
    5) the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 32, the second heavy chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 33, the first light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31, and the second light chain polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 31.
  19. The antibody-drug conjugate of claim 18, wherein the first heavy chain polypeptide comprises a first heavy chain variable region (VH1) , the first light chain polypeptide comprises a first light chain variable region (VL1) , the second heavy chain polypeptide comprises a second heavy chain variable region (VH2) , and the second light chain polypeptide comprises a second light chain variable region (VL2) , wherein the VH1 and VL1 can interact with each other, forming a first antigen-binding site, and the VH2 and VL2 can interact with each other, forming a second antigen-binding site.
  20. The antibody-drug conjugate of claim 19, wherein the first antigen-binding site and the second antigen-binding site target different antigens (e.g., two different tumor-associated antigens) .
  21. The antibody-drug conjugate of claim 19 or 20, wherein the first antigen-binding site targets HER2, and the second antigen-binding site targets TROP2.
  22. The antibody-drug conjugate of claim 21, wherein
    the VH1 comprising 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 VL1 comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,
    wherein the selected VH1 CDRs 1, 2, and 3 amino acid sequences, 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: 7-9, 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: 10-12, respectively, and the selected VL1 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively.
  23. The antibody-drug conjugate of claim 22, wherein
    the VH2 comprising 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 VL2 comprising 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: 13-15, respectively, and the selected VL2 CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively; and
    (2) 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: 4-6, respectively.
  24. The antibody-drug conjugate of any one of claims 21-23, wherein the VH1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 19, the VH2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 20, the VL1 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21, and the VL2 comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 21.
  25. An antibody-drug conjugate comprising
    (1) an antibody comprising
    a first heavy chain polypeptide, wherein the first heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229;
    a second heavy chain polypeptide, wherein the second heavy chain polypeptide comprises a glycine at heavy chain position 226, and a cysteine at heavy chain positions 220 and 229;
    a first light chain polypeptide, wherein the first light chain polypeptide comprises a cysteine at light chain position 214; and
    a second light chain polypeptide, wherein the second light chain polypeptide comprises a cysteine at light chain position 214; and
    (2) a therapeutic agent that is linked to the cysteines at:
    heavy chain positions 220 and 229 of the first heavy chain polypeptide,
    heavy chain positions 220 and 229 of the second heavy chain polypeptide,
    light chain position 214 of the first light chain polypeptide, and/or
    light chain position 214 of the second light chain polypeptide.
  26. An antibody-drug conjugate comprising
    (1) an antibody comprising
    a first heavy chain polypeptide, wherein the first heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226;
    a second heavy chain polypeptide, wherein the second heavy chain polypeptide comprises a glycine at heavy chain position 229, and a cysteine at heavy chain positions 220 and 226;
    a first light chain polypeptide, wherein the first light chain polypeptide comprises a cysteine at light chain position 214; and
    a second light chain polypeptide, wherein the second light chain polypeptide comprises a cysteine at light chain position 214; and
    (2) a therapeutic agent that is linked to the cysteines at:
    heavy chain positions 220 and 226 of the first heavy chain polypeptide,
    heavy chain positions 220 and 226 of the second heavy chain polypeptide,
    light chain position 214 of the first light chain polypeptide, and/or
    light chain position 214 of the second light chain polypeptide.
  27. An antibody-drug conjugate comprising
    (1) an antibody comprising
    a first heavy chain polypeptide, wherein the first heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220;
    a second heavy chain polypeptide, wherein the second heavy chain polypeptide comprises a glycine at heavy chain positions 226 and 229, and a cysteine at heavy chain position 220;
    a first light chain polypeptide, wherein the first light chain polypeptide comprises a cysteine at light chain position 214; and
    a second light chain polypeptide, wherein the second light chain polypeptide comprises a cysteine at light chain position 214; and
    (2) a therapeutic agent that is linked to the cysteines at:
    heavy chain position 220 of the first heavy chain polypeptide,
    heavy chain position 220 of the second heavy chain polypeptide,
    light chain position 214 of the first light chain polypeptide, and/or
    light chain position 214 of the second light chain polypeptide.
  28. An antibody-drug conjugate comprising
    (1) an antibody comprising
    a first heavy chain polypeptide, wherein the first heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229;
    a second heavy chain polypeptide, wherein the second heavy chain polypeptide comprises a glycine at heavy chain position 220, and a cysteine at heavy chain positions 226 and 229;
    a first light chain polypeptide, wherein the first light chain polypeptide comprises a glycine at light chain position 214; and
    a second light chain polypeptide, wherein the second light chain polypeptide comprises a glycine at light chain position 214; and
    (2) a therapeutic agent that is linked to the cysteines at:
    heavy chain positions 226 and 229 of the first heavy chain polypeptide, and/or
    heavy chain positions 226 and 229 of the second heavy chain polypeptide.
  29. The antibody-drug conjugate of any one of claims 1-28, wherein the therapeutic agent is covalently linked to the antibody, e.g., via thiolation with the one or more cysteine residues.
  30. The antibody-drug conjugate of any one of claims 1-29, wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) .
  31. The antibody-drug conjugate of any one of claims 1-30, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
  32. The antibody-drug conjugate of any one of claims 1-31, wherein the therapeutic agent is MMAE or MMAF.
  33. A method of conjugating a therapeutic agent to an antibody, the method comprising:
    (a) reducing an antibody with a reducing agent, wherein the antibody comprises
    one or more non-cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214; and
    one or more cysteine residues selected from the group consisting of heavy chain positions 220, 226, 229 and light chain position 214,
    wherein the one or more cysteine residues form one or more thiol groups in the reduced antibody;
    (b) conjugating the therapeutic agent to the one or more thiol groups.
  34. The method of claim 33, wherein the reducing agent is tris (2-carboxyethyl) phosphine (TCEP) .
  35. The method of claim 33 or 34, wherein the reducing agent is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or more than the molar amount of the antibody.
  36. The method of any one of claims 33-35, wherein the conjugation products with a drug-antibody-ratio (DAR) of 4 accounts for at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,  at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) .
  37. The method of any one of claims 33-35, wherein the conjugation products with a DAR of 0, 2, 6, and/or 8 accounts for less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%by weight of all conjugation products (e.g., conjugation products with a DAR of 0, 2, 4, 6, and 8) .
  38. The method of any one of claims 33-37, wherein the average DAR of the conjugation products is about 3.5 to about 4.5, about 3.6 to about 4.4, about 3.7 to about 4.3, about 3.8 to about 4.2, or about 3.9 to about 4.1 (e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5) .
  39. An antibody-drug conjugate comprising
    an antibody comprising
    one or more non-cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214; and
    one or more cysteine residues at positions selected from the group consisting of heavy chain positions 131, 226, 229 and light chain position 214,
    wherein a therapeutic agent is linked to the antibody through the one or more cysteine residues.
  40. The antibody-drug conjugate of claim 39, wherein the antibody comprises a first heavy chain polypeptide and a second heavy chain polypeptide.
  41. The antibody-drug conjugate of claim 40, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 226.
  42. The antibody-drug conjugate of claim 40 or 41, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 226.
  43. The antibody-drug conjugate of any one of claims 40-42, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 226.
  44. The antibody-drug conjugate of any one of claims 40-43, wherein the first and second heavy chain polypeptides each comprises a cysteine at heavy chain position 229.
  45. The antibody-drug conjugate of any one of claims 40-43, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 229.
  46. The antibody-drug conjugate of any one of claim 40-43 and 45, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 229.
  47. The antibody-drug conjugate of any one of claims 40-43, 45, and 46, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 229.
  48. The antibody-drug conjugate of any one of claims 40-47, further comprising a first light chain polypeptide and a second light chain polypeptide, wherein the first heavy chain polypeptide can interact with the first light chain polypeptide, and the second heavy chain polypeptide can interact with the second light chain polypeptide.
  49. The antibody-drug conjugate of claim 48, wherein the first and second heavy chain polypeptides each comprises a non-cysteine residue at heavy chain position 131, and the first and second light chain polypeptides each comprises a non-cysteine residue at light chain position 214.
  50. The antibody-drug conjugate of claim 48 or 49, wherein the first and second heavy chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at heavy chain position 131, and the first and second light chain polypeptides each comprises a glycine, alanine, valine, leucine, isoleucine, or proline at light chain position 214.
  51. The antibody-drug conjugate of any one of claims 48-50, wherein the first and second heavy chain polypeptides each comprises a glycine at heavy chain position 131, and the first and second light chain polypeptides each comprises a glycine at light chain position 214.
  52. The antibody-drug conjugate of claim 39, wherein the antibody comprises a first heavy chain polypeptide, a second heavy chain polypeptide, a first light chain polypeptide, and a second light chain polypeptide, wherein
    (a) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 226, and a cysteine at heavy chain positions 131 and 229; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214;
    (b) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 229, and a cysteine at heavy chain positions 131 and 226; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214;
    (c) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain positions 226 and 229, and a cysteine at heavy chain position 131; the first light chain polypeptide comprises a cysteine at light chain position 214; and the second light chain polypeptide comprises a cysteine at light chain position 214; or
    (d) the first heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain position 226 and 229; the second heavy chain polypeptide comprises a non-cysteine residue at heavy chain position 131, and a cysteine at heavy chain positions 226 and 229; the first light chain polypeptide comprises a non-cysteine residue at light chain position 214; and the second light chain polypeptide comprises a non-cysteine residue at light chain position 214.
  53. The antibody-drug conjugate of any one of claims 39-52, wherein the antibody comprises heavy and light chain constant region sequences derived from human IgG4.
  54. The antibody-drug conjugate of any one of claims 39-53, wherein the antibody is a bispecific antibody or multi-specific antibody.
  55. The antibody-drug conjugate of any one of claims 39-54, wherein the antibody comprises knobs-into-holes (KIH) mutations.
  56. The antibody-drug conjugate of any one of claims 39-55, wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 3.8 to about 4.2 (e.g., about 3.8, about 3.9, about 4, about 4.1, or about 4.2) .
  57. A method of treating a condition or disorder in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody-drug conjugate of any one of claims 1-32 and 39-56, or the conjugation products of any one of claims 33-38, to the subject.
  58. The method of claim 57, wherein the subject has a cancer, tumor, autoimmune disease, or infectious disease.
  59. The method of claim 57, wherein the subject has a solid tumor, e.g., a thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, pancreatic cancer,  prostate cancer, liver cancer, lymphoma, glioma, cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid, skin cancer, liver cancer, testis cancer, multiple myeloma or renal carcinoma.
  60. The method of any one of claims 57-59, wherein the subject is a human.
  61. The method of any one of claims 57-59, wherein the subject is a non-human animal.
  62. 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 antibody-drug conjugate of any one of claims 1-32 and 39-56, or the conjugation products of any one of claims 33-38.
  63. A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody-drug conjugate of any one of claims 1-32 and 39-56, or the conjugation products of any one of claims 33-38.
  64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the antibody-drug conjugate of any one of claims 1-32 and 39-56, or the conjugation products of any one of claims 33-38.
  65. Use of the antibody-drug conjugate of an one of claims 1-32 and 39-56 or the conjugation products of any one of claims 33-38 in the manufacture of a pharmaceutical composition or a kit for treating a condition or disorder in a subject.
PCT/CN2023/116426 2022-09-01 2023-09-01 Methods for preparing antibody-drug conjugates WO2024046455A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015031698A1 (en) * 2013-08-28 2015-03-05 Stem Centrx, Inc. Site-specific antibody conjugation methods and compositions
WO2016064749A2 (en) * 2014-10-20 2016-04-28 Igenica Biotherapeutics, Inc. Novel antibody-drug conjugates and related compounds, compositions, and methods of use
WO2016166301A1 (en) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Site-specific antibody-drug conjugates
WO2022159984A1 (en) * 2021-01-22 2022-07-28 Bionecure Therapeutics, Inc. Anti-her-2/trop-2 constructs and uses thereof
WO2023143343A1 (en) * 2022-01-26 2023-08-03 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Anti-her2/trop2 antibodies and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015031698A1 (en) * 2013-08-28 2015-03-05 Stem Centrx, Inc. Site-specific antibody conjugation methods and compositions
WO2016064749A2 (en) * 2014-10-20 2016-04-28 Igenica Biotherapeutics, Inc. Novel antibody-drug conjugates and related compounds, compositions, and methods of use
WO2016166301A1 (en) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Site-specific antibody-drug conjugates
WO2022159984A1 (en) * 2021-01-22 2022-07-28 Bionecure Therapeutics, Inc. Anti-her-2/trop-2 constructs and uses thereof
WO2023143343A1 (en) * 2022-01-26 2023-08-03 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Anti-her2/trop2 antibodies and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHENGZHANG SHANG; LIU YANG; JIYONG ZHANG; YANFEI HAN; ZHUOLIN LI; ZHENYAN HAN; JUN LI; YING MENG; GAO AN; HAO YANG; WENQIAN AN; LE: "Abstract 4256: YH012, a novelbispecific anti-HER2 and TROP2antibody-drug conjugate, exhibitspotent antitumor efficacy", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 82, no. 12, Suppl., 15 July 2022 (2022-07-15), US, pages 4256, XP009552928, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2022-4256 *

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