WO2024184812A1 - Anticorps anti-cldn6 et procédés d'utilisation - Google Patents

Anticorps anti-cldn6 et procédés d'utilisation Download PDF

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WO2024184812A1
WO2024184812A1 PCT/IB2024/052127 IB2024052127W WO2024184812A1 WO 2024184812 A1 WO2024184812 A1 WO 2024184812A1 IB 2024052127 W IB2024052127 W IB 2024052127W WO 2024184812 A1 WO2024184812 A1 WO 2024184812A1
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antibody
seq
amino acid
acid sequence
antigen
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PCT/IB2024/052127
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English (en)
Inventor
Dan Li
Xiaoyan Tang
Ming Lei
Ting Shao
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Beigene Switzerland Gmbh
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Priority to US18/620,353 priority Critical patent/US20240301051A1/en
Publication of WO2024184812A1 publication Critical patent/WO2024184812A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

  • compositions comprising the antibodies or antigen-binding fragments thereof, as well as methods for the treatment of cancer.
  • CLDN claudin
  • TJ tight junctions
  • CLDNs By interacting with each other in both cis (intracellular) and trans (intercellular) interactions, CLDNs exert important roles in regulating paracellular permeability and maintaining cell polarity(Tsukita et al., Trends in Biochemical Sciences. 2019). In additional, CLDNs could server as protein scaffolds for assembling complexes at cell junctions, and transmit signals to the cell interior to modulate gene expression and cell behavior(Matter et al., Nat Rev Mol Cell Biol. 2003; Singh etal., Pflugers Arch. 2017).
  • CLDN6 was first identified and characterized in 2001(Turksen et al., Developmental Dynamics. 2001). The expression of CLDN6 is dynamically modulated by various factors and mechanisms(Du etal., Mol Med Rep. 2021). CLDN6 is one of the earliest proteins expressed in embryonic stem cells committed to the epithelial fate and a cell-surface-specific marker of human pluripotent stem cells (hPSCs)(Ben-David etal., Nat Commun. 2013). Interestingly, CLDN6 expression could be detected in fetal tissues including the stomach, pancreas, lung, and kidney, but not in the corresponding adult tissue samples(Reinhard et al., Science.
  • hPSCs human pluripotent stem cells
  • CLDN6 claudin 9
  • the present disclosure provides anti-CLDN6 antibodies and antigen-binding fragments thereof.
  • the present disclosure encompasses the following embodiments.
  • the present disclosure provides, an antibody or antigen-binding fragment thereof, comprising an antigen binding domain that specifically binds to human claudin 6 (CLDN6)
  • the antigen binding domain does not bind to other claudin (CLDN) protein family members.
  • CLDN9 human claudin 9
  • the antigen binding domain has high selectivity for human CLDN6 over human CLDN9.
  • the antigen binding domain that specifically binds to human CLDN6 comprises: (a) a heavy chain variable region that comprises: (i) a heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 2, (iii) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises :(iv) a light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 4, (v) a LCDR2 of SEQ ID NO: 5, and (vi) a LCDR3 of SEQ ID NO: 6; (b) a heavy chain variable region that comprises: (i) a HCDR1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 23, (iii) a HCDR3 of SEQ ID NO: 3; and a light chain variable region that comprises: (iv) a LCDR1 of SEQ ID NO:
  • the antigen binding domain comprises: (a) a heavy chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 8; (b) a heavy chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 12; (c) a heavy chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94,
  • the antigen binding domain comprises: (a) a heavy chain variable region has an amino acid sequence comprising SEQ ID NO:7, and a light chain variable region has an amino acid sequence comprising SEQ ID NO: 8; (b) a heavy chain variable region as an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region has an amino acid sequence comprising SEQ ID NO: 12; (c) a heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region has an amino acid sequence comprising SEQ ID NO: 42; (d) a heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region has an amino acid sequence comprising SEQ ID NO: 44; (e) a heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region has an amino acid sequence comprising SEQ ID NO: 47; or (f) a heavy chain variable region has an amino acid sequence comprising SEQ ID NO:
  • the antibody or antigen-binding fragment as disclosed herein is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.
  • the antibody is a multi-specific antibody.
  • the antibody is a bispecific antibody.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the antibody or antigen-binding fragment as disclosed herein has reduced glycosylation or no glycosylation or is hypofucosylated.
  • the antibody or antigen-binding fragment as disclosed herein comprises increased bisecting GlcNac structures.
  • the Fc domain of the antibody or antigen-binding fragment as disclosed herein is an IgGl .
  • the Fc domain is an IgGl with reduced effector function.
  • the Fc domain is an IgG4.
  • the present disclosure provides, a pharmaceutical composition comprising the antibody or antigen-binding fragment as disclosed herein
  • the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprising a histidine/histidine HC1, a trehalose dihydrate, and/or a polysorbate 20.
  • the present disclosure provides for a method of treating cancer comprising administering to a patient in need an effective amount of the antibody or antigenbinding fragment as disclosed herein.
  • the cancer is a solid cancer.
  • the cancer is selected from gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor and/or glioma.
  • the antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agent.
  • the one or more therapeutic agent is selected from paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide or 5-azacytidine.
  • the one or more therapeutic agent is a paclitaxel agent, lenalidomide or 5-azacytidine.
  • the therapeutic agent an anti-PDl or anti-PDLl antibody.
  • the anti-PDl antibody is Tislelizumab.
  • the present disclosure provides, an isolated nucleic acid that encodes the antibody or antigen-binding fragment as disclosed herein.
  • the present disclosure provides, a vector comprising the nucleic acid.
  • the present disclosure provides a host cell comprising the nucleic acid or the vector.
  • the present disclosure provides, a process for producing the antibody or antigen-binding fragment as disclosed herein comprising cultivating the host cell as disclosed herein and recovering the antibody or antigen-binding fragment from the culture.
  • the antibody or antigen-binding fragment of as disclosed herein is for use in a method for treating cancer.
  • the present disclosure provides for the use of the antibody or antigenbinding fragment as disclosed herein in the manufacture of a medicament for the treatment of cancer.
  • composition as disclosed herein is for use in a method for treating cancer.
  • An antibody or antigen-binding fragment thereof comprising an antigen binding domain that specifically binds to human CLDN6.
  • the antibody or antigen-binding fragment, wherein the antigen binding domain that specifically binds to human CLDN6 comprises:
  • a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 2, (c) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 4, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6;
  • a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 23, (c) a HCDR3 of SEQ ID NO: 3; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 4, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6;
  • a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 39, (c) a HCDR3 of SEQ ID NO: 3; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 40, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6; or
  • a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 45, (c) a HCDR3 of SEQ ID NO: 3; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 40, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6.
  • the antibody or antigen-binding fragment of the instant invention, wherein the antigen binding domain comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 46
  • a light chain variable region comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 47; or
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment of the instant invention wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids within SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 have been inserted, deleted or substituted.
  • the antibody or antigen-binding fragment of the instant invention, wherein the antigen binding domain comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment of the instant invention which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.
  • the antibody of the instant invention wherein the antibody is a multi-specific antibody.
  • the antibody of the instant invention wherein the antibody is a bispecific antibody.
  • the antibody or antigen-binding fragment of the instant invention wherein the antibody or antigen-binding fragment thereof has antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the antibody or antigen-binding fragment of the instant invention wherein the antibody or antigen-binding fragment thereof comprises increased bisecting GlcNac structures.
  • the antibody or antigen-binding fragment of the instant invention wherein the Fc domain is an IgGl .
  • the antibody or antigen-binding fragment of the instant invention, wherein the Fc domain is an IgGl with reduced effector function.
  • a pharmaceutical composition comprising The antibody or antigen-binding fragment of the instant invention, which further comprises a pharmaceutically acceptable carrier.
  • composition further comprising histidine/histidine HC1, trehalose dihydrate, and polysorbate 20.
  • a method of treating cancer comprising administering to a patient in need an effective amount The antibody or antigen-binding fragment of the instant invention.
  • the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma.
  • the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide or 5 -azacytidine.
  • the therapeutic agent is a paclitaxel agent, lenalidomide or 5- azacytidine.
  • a vector comprising the nucleic acid of the instant invention.
  • a host cell comprising the nucleic acid or the vector of the instant invention .
  • a process for producing an antibody or antigen-binding fragment thereof comprising cultivating the host cell and recovering the antibody or antigen-binding fragment from the culture.
  • the antibody or an antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45.
  • CDRs complementarity determining regions
  • the antibody or an antigen-binding fragment thereof comprises:
  • HCDRs complementarity determining regions
  • a light chain variable region comprising one or more complementarity determining regions (LCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 40.
  • LCDRs complementarity determining regions
  • the antibody or an antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs) which are HCDR1 comprising an amino acid sequence of SEQ ID NO: 1; HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 45, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and/or (b) a light chain variable region comprising three complementarity determining regions (LCDRs) which are LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 40, LCDR2 comprising an amino acid sequence of SEQ ID NO: 5,; and LCDR3 comprising an amino acid sequence of SEQ ID NO: 6.
  • HCDRs heavy chain variable region comprising three complementarity determining regions
  • the antibody or an antigen-binding fragment thereof comprises:
  • HCDRs heavy chain variable region comprising three complementarity determining regions (HCDRs) which are HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3;
  • HCDR1 comprising an amino acid sequence of SEQ ID NO: 1
  • HCDR2 comprising an amino acid sequence of SEQ ID NO: 23
  • HCDR3 comprising an amino acid sequence of SEQ ID NO: 3;
  • HCDR1 comprising an amino acid sequence of SEQ ID NO: 1
  • HCDR2 comprising an amino acid sequence of SEQ ID NO: 39
  • HCDR3 comprising an amino acid sequence of SEQ ID NO: 3;
  • HCDR1 comprising an amino acid sequence of SEQ ID NO: 15, HCDR2 comprising an amino acid sequence of SEQ ID NO: 45, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3; and/or (b) a light chain variable region comprising three complementarity determining regions (LCDRs) which are
  • LCDR1 comprising an amino acid sequence of SEQ ID NO: 4
  • LCDR2 comprising an amino acid sequence of SEQ ID NO: 5
  • LCDR3 comprising an amino acid sequence of SEQ ID NO: 6;
  • LCDR1 comprising an amino acid sequence of SEQ ID NO: 40
  • LCDR2 comprising an amino acid sequence of SEQ ID NO: 5
  • LCDR3 comprising an amino acid sequence of SEQ ID NO: 6.
  • the antibody or the antigen-binding fragment comprises: a antigen binding domain comprising: a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 2, (c) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 4, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6.
  • the antibody or the antigen-binding fragment comprises: a antigen binding domain comprising: a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 23, (c) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 4, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6.
  • the antibody or the antigen-binding fragment comprises: a antigen binding domain comprising: a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 39, (c) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 40, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6.
  • the antibody or the antigen-binding fragment comprises: a antigen binding domain comprising: a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO: 45, (c) a HCDR3 of SEQ ID NO: 3 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 40, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6.
  • the antibody of the present disclosure or an antigen-binding fragment thereof comprises: (a) a heavy chain variable region having an amino acid sequence of HCDRs or VHs listed in Table 1; and/or (b) a light chain variable region comprising an amino acid sequence of LCDRs or VLs listed in Table 1.
  • the antibody of the present disclosure or an antigen-binding fragment thereof comprises: (a) an amino acid sequence comprising one, two, or three amino acid substitutions in the amino acid sequence of HCDRs or VHs listed in Table 1; and/or (b) a light chain variable region comprising an amino acid sequence comprising one, two, three, four, or five amino acid substitutions in the amino acid of LCDRs or VLs listed in Table 1.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibody of the present disclosure is of IgGl, IgG2, IgG3, or IgG4 isotype.
  • the antibody of the present disclosure comprises Fc domain of wild-type human IgGl (also referred as human IgGlwt or huIgGl) or IgG2.
  • the antibody of the present disclosure binds to CLDN6 with a binding affinity (KD) of from 1 x 10' 6 M to 1 x 10' 10 M. In another embodiment, the antibody of the present disclosure binds to CLDN6 with a binding affinity (KD) of about 1 x 10' 6 M, about 1 x 10' 7 M, about 1 x 10' 8 M, about 1 x 10' 9 M or about 1 x 10' 10 M. [085] In another embodiment, the anti-human CLDN6 antibody of the present disclosure shows a cross-species binding activity to cynomolgus CLDN6.
  • antibodies of the present disclosure have strong Fc-mediated effector functions.
  • the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against CLDN6 expressing target cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • FIGS. 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, and 1J show the cell binding activity of chBG87P engineered variants.
  • FIG. 1 A shows cell binding activity of the 1 st round of BG87P humanization back mutation variants (BG87P-zO, BG87P-BzO, BG87P-Bzl, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7, and BG87P-Bz8) compared with anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T/human CLDN6.
  • FIG. IB shows cell binding activity of combination humanized variants (BG87P-21, BG87P-22, BG87P- 23, and BG87P-24) compared with anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T/human CLDN6.
  • FIG. 1C shows cell binding activity of combination humanized variants (BG87P-25, BG87P-26, and BG87P-27) compared with anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T/human CLDN6.
  • FIG. IE shows cell binding activity of combination humanized variants (BG87P-25, BG87P-26, and BG87P-27) compared with anti-CLDN6 chimeric BG87P (chBG87P) against the cancer cell line PA-1.
  • FIG. IE shows cell binding activity of combination humanized variants (BG87P-25, BG87P-26, and BG87P-27) compared with anti-CLDN6 chimeric BG87P (chBG87P) against the cancer cell line PA-1.
  • IF shows cell binding activity of post translational modifications (PTM) removal engineering variants (BG87P-ml, BG87P-m2, BG87P-m3, BG87P-m4, BG87P-m5, BG87P-m6, BG87P-m7, and BG87P-m8) compared to anti-CLDN6 chimeric BG87P (chBG87P) and BG87P-BzO against the HEK293T/human CLDN6.
  • PTM post translational modifications
  • FIG. 1G shows cell binding activity of BG87P solubility engineering variants (BG87P-21, BG87P-34, and BG87P-33) compared to anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T/human CLDN6.
  • FIG. 1H shows non-specific binding activity of solubility engineering variants (BG87P-21, BG87P-34, and BG87P-33) compared to anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T-human CLDN9.
  • FIG. 1G shows cell binding activity of BG87P solubility engineering variants (BG87P-21, BG87P-34, and BG87P-33) compared to anti-CLDN6 chimeric BG87P (chBG87P) against the HEK293T-human CLDN9.
  • FIG. 1 J shows crossreactivity of humanized variants (BG87P-21, BG87P-34, and BG87P-33) compared to anti-CLDN6 chimeric BG87P (chBG87P) against CH0K1 -mouse CLDN6.
  • FIG. 2 depicts the predicated a hydrophobic patch in a homology model of chimeric BG87P by Schroedinger.
  • I97-Y98-Y100-V100a of HCDR3 are predicated to form an exposed hydrophobic patch, along with Y49-W50 of HCDR2 (Y49 is the last residue of FR2 of variable region of light chain while W50 is the first residue of LCDR2).
  • FIG. 3 shows the hydrophobicity of selected humanized BG87P variants after engineering (BG87-33, BG87-34, BG87P-21) as determined by HIC-HPLC.
  • FIGS. 4A and 4B show the comparison of binding activity between chimeric and humanized sp34s in Hut78 cells.
  • FIG. 4A shows comparison of binding affinities between chimeric sp34 (ch-sp34) and humanized sp34 BG53P (BG53P) measured by melt flow index (MFI) in Hut78 cells.
  • FIG. 4B shows the comparison of binding affinities among chimeric sp34 (ch-sp34), humanized sp34 BG53P (BG53P) and BG56P.
  • FIG. 5 shows the comparison of binding activity between humanized sp34 BG56P (BG56P) and humanized sp34 scFv BG561p (BG561P).
  • FIGS. 6A, 6B, and 6C show the comparison of binding affinities among humanized sp34 scFvs in Hut78 cells.
  • FIG. 6A shows comparison of binding affinities between humanized sp34 scFv BG561p (BG561P) and humanized scFv BG562P (BG562P) in Hut78 cells.
  • FIG. 6B shows comparison of binding affinities between humanized scFv BG562P (BG562P) and humanized scFv BG563P (BG563P) in Hut78 cells.
  • FIG. 6C shows comparison of binding affinities between humanized scFv BG563P (BG563P) and humanized scFv BG564P (BG564P) in Hut78 cells.
  • FIGS. 8A and 8B show the target-binding activity of CLDN6> ⁇ CD3 BsAb BG143P.
  • FIG. 8A shows CD3 binding activity of BG143P in CD3 -expressing Jurkat cells.
  • FIG. 8B shows CLDN6 binding activity of BG143P in CLDN6-expressoing PA-1 cells.
  • FIGS. 9A, 9B, and 9C shows on-target functional activity of CLDN6*CD3 BsAb BG143P in tumor cell lines with different CLDN6 expression.
  • FIG. 9A shows redirected T cell cytotoxicity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI- 111299 cells by cell lysis assay.
  • FIG. 9A shows redirected T cell cytotoxicity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI- 111299 cells by cell lysis assay.
  • FIG. 9A shows redirected T cell
  • FIG. 9B shows IFN-y induction activity of BG143P in CLDN6- expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells.
  • FIG. 9C shows IL-2 induction activity of BG143P in CLDN6-expressing PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells.
  • FIGS. 10A, 10B, and 10C show functional specificity of CLDN6xCD3 BsAb BG143P against human CLDN6 and CLDN9.
  • FIG. 10A shows binding specificity of BG143P against human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells.
  • FIG. 10B shows killing specificity (cell lysis activity) of BG143P against human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells.
  • FIG. 10C shows cytokine (IFN- y) induction of BG143P against human CLDN6 (left graph) and CLDN9 (right graph) in NCI-H1299 cells.
  • FIGS. 11 A and 1 IB show the in vivo efficacy of CLDN6*CD3 BsAb BG143P in an OV-90 xenograft model in PBMC-humanized mice.
  • FIG. 11 A shows the tumor volume along with time. Mice were left untreated (no PBMC), treated with PBS (PBS i.p QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p), treated with 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p) or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, i.p).
  • FIG. 1 IB shows the percentage of hCD45+ cells in the peripheral blood of the mice left untreated (no PBMC), treated with PBS (PBS i.p QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p), treated with 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p) or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, i.p) at day 13, day 21, and day 27 post PBMC injection, indicating human PBMC reconstitution.
  • FIGS 12A and 12B show the in vivo efficacy of CLDN6*CD3 BsAb BG143P in aB16F10/human CLDN6 syngeneic model in hCD3EDG-transgenic mice.
  • FIG. 12A shows the tumor volume along with time. Mice were treated with PBS (PBS i.p QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p), treated with 0.03 mg/kg of BG143P (BG143P - 0.01 mg/kg, i.p) or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, i.p).
  • FIG. 12B shows the body weight of the mice treated with PBS (PBS i.p QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p), treated with 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, i.p) or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, i.p) over the course of day 11 to 27 post inoculation, as an indicator of tolerability of the mice to the antibody.
  • PBS PBS i.p QW
  • the term “or” is used to mean, and is used interchangeably with, the term “and/or” unless the context clearly dictates otherwise. As also used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
  • a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).
  • anti-cancer agent refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
  • CLDN6 refers to a member of the CLDN family.
  • CLDN6 has a molecular weight of 23 kDa.
  • CLDN6 has four transmembrane domains and a PDZ-binding region at the carboxyl end of the cytoplasm.
  • the amino acid sequence of human CLDN6 can be found at UniPort ID P56747.
  • An exemplary human CLDN6 sequence is SEQ ID NO: 87.
  • CLDN9 refers to another member of the CLDN family.
  • CLDN9 has a molecular weight of 23 kDa, and the amino acid sequence of which can be found at UniPort ID 095484.
  • An exemplary human CLDN9 sequence is SEQ ID NO: 88.
  • cluster of differentiation 3 refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3s, CD3y, CD3a, and CD30 chains.
  • the term encompasses “full-length,” unprocessed CD3 (e.g., unprocessed or unmodified CD3s or CD3y), as well as any form of CD3 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants.
  • CD3 includes, for example, human CD3s protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length, and human CD3y protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length.
  • NCBI RefSeq No. NP_000724 human CD3s protein
  • NP_000064 human CD3y protein
  • Non-limiting examples include an animal.
  • the animal is a mammal (e.g., primate, higher primate, human, rat, mouse, dog, cat, rabbit).
  • the mammal is a human.
  • the subject is a patient comprising, or at risk of having, a disorder described herein.
  • treating any disease or disorder refers to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.
  • the terms “prevent,” “preventing” or “prevention” as used herein with reference to a cancer refer to precluding or reducing the risk of developing cancer. Prevention may also refer to the prevention of recurrence or a secondary cancer once an initial cancer has been treated or cured.
  • the terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a human.
  • antibody refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen non-covalently, reversibly, and in a specific manner.
  • a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL or VK) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four framework regions (FRs) arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.
  • the antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • full-length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region.
  • the anti-CLDN6 antibodies comprise at least one antigen-binding site, at least a variable region. In some embodiments, the anti-CLDN6 antibodies comprise an antigen-binding fragment from an CLDN6 antibody described herein. In some embodiments, the anti-CLDN6 antibody is isolated or recombinant.
  • the term “monoclonal antibody” or “mAb” or “Mab” herein means a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts.
  • conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their complementarity determining regions (CDRs), which are often specific for different epitopes.
  • CDRs complementarity determining regions
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example Kohler et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993.
  • the antibodies disclosed herein can be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclass thereof such as IgGl, IgG2, IgG3, IgG4.
  • a hybridoma producing a monoclonal antibody can be cultivated in vitro or in vivo.
  • High titers of monoclonal antibodies can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired antibodies.
  • Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of the heavy chain can define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as a, 5, s, y, or p, and define the antibody's isotypes as IgA, IgD, IgE, IgG, and IgM, respectively.
  • the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
  • variable regions of each light/heavy chain (VL/VH) pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the two binding sites are, in general, the same in primary sequence.
  • variable domains of both the heavy and light chains comprise three hypervariable regions, also called “complementarity determining regions” or “CDRs,” which are located between relatively conserved framework regions (FR).
  • CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chain variable domains comprise FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4).
  • the positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, AbM and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikam et al., J. Mol.
  • ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme)). Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M. P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.
  • HCDR1 amino acid residues 26-35
  • LCDR2 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • hypervariable region means the amino acid residues of an antibody that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “CDR” (e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain).
  • CDR e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain
  • HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain.
  • an “antigen-binding fragment” means antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions.
  • antigen-binding fragments include, but not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single chain Fv (ScFv); nanobodies and multi-specific antibodies formed from antibody fragments.
  • an antibody “specifically binds” to a target protein, meaning the antibody exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity.
  • An antibody “specifically binds” or “selectively binds,” is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, or antigen binding antibody fragment, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, for example, in a biological sample, blood, serum, plasma or tissue sample.
  • the antibodies or antigen-binding fragments thereof specifically bind to a particular antigen at least two times when compared to the background level and do not specifically bind in a significant amount to other antigens present in the sample.
  • the antibody or antigen- binding fragment thereof specifically bind to a particular antigen at least ten (10) times when compared to the background level of binding and does not specifically bind in a significant amount to other antigens present in the sample.
  • Antigen-binding domain as used herein, comprise at least three CDRs and specifically bind to an epitope.
  • An “antigen-binding domain” of a multi-specific antibody e.g., a bispecific antibody
  • Multi-specific antibodies can be bispecific, trispecific, tetraspecific etc., with antigen binding domains directed to each specific epitope.
  • Multi-specific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) that comprises multiple antigen binding domains, for example, 2, 3, 4 or more antigen binding domains that specifically bind to a first epitope and 2, 3, 4 or more antigen binding domains that specifically bind a second epitope.
  • multivalent e.g., a bispecific tetravalent antibody
  • comprises multiple antigen binding domains for example, 2, 3, 4 or more antigen binding domains that specifically bind to a first epitope and 2, 3, 4 or more antigen binding domains that specifically bind a second epitope.
  • human antibody herein means an antibody that comprises human immunoglobulin protein sequences only.
  • a human antibody can contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell.
  • mouse antibody or “rat antibody” mean an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.
  • humanized or “humanized antibody” means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the prefix “hum,” “hu,” “Hu,” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies.
  • the humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions can be included to increase affinity, increase stability of the humanized antibody, remove a post-translational modification or for other reasons.
  • epitope refers to the particular site on an antigen to which an antibody binds.
  • the particular site on an antigen to which an antibody binds can be determined, for example, by crystallography. Methods such as hydroxyl radical protein footprinting and alanine scanning mutagenesis can also be used but may provide less resolution.
  • the term “monospecific antibody” refers to an antibody that specifically binds to only one antigen.
  • a monospecific antibody can bind to only one epitope of an antigen or can bind to two or more epitopes of an antigen.
  • a monospecific antibody that binds to two or more epitopes of an antigen is a monospecific polyepitopic antibody.
  • the term “multispecific antibody” or “multi-specific antibody” refers to an antibody that specifically binds two or more antigens (e.g., a bispecific antibody, a trispecific antibody, etc.).
  • Non-limiting examples of multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH/VL unit has polyepitopic specificity, antibodies having two or more VL and VH domains with each VH/VL unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, diabodies, triabodies, etc., as well as full-length antibodies and/or antibody fragments that have been linked covalently or non-covalently.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • polyepitopic antibody and “antibody having polyepitopic specificity” are used herein interchangeably to refer to an antibody that binds to two or more epitopes on the same or different antigen.
  • Fc region is used herein to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof.
  • the C-terminal lysine (residue 447 according to the Eu numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all Lys447 residues removed, antibody populations with no Lys447 residues removed, and antibody populations having a mixture of antibodies with and without the Lys447 residue.
  • a “functional Fc region” possesses an effector function of a native sequence Fc region.
  • exemplary effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays disclosed herein or otherwise known in the art.
  • a functional Fc region may possess effector function substantially similar to a wild-type IgG, reduced effector function compared to a wild-type IgG, or enhanced effector function compared to a wild-type IgG.
  • the comparison is typically to a wild- type human IgGl .
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., from about one to about ten amino acid modifications, and in some embodiments from about one to about five amino acid modifications), preferably one or more amino acid substitution(s).
  • the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, preferably at least about 90% homology therewith, or preferably at least about 95% homology therewith.
  • variant Fc regions may possess reduced or enhanced effector function, as compared to a wild-type IgG. For antibodies comprising a human Fc region, the comparison is typically to a wild-type human IgGl.
  • Fc component refers to a hinge region, a CH2 domain or a CH3 domain of an Fc region.
  • Hae region is generally defined as stretching from about residue 216 to 230 of an IgG (Eu numbering), from about residue 226 to 243 of an IgG (Kabat numbering), or from about residue 1 to 15 of an IgG (IMGT unique numbering).
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, a F(ab) c , an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a triabody, a tetrabody, a single-chain antibody, an scFv, an scFv dimer, a single domain antibody, a single-domain antibody, and a multivalent domain antibody.
  • binding fragments compete with the intact antibody from which they were derived for specific binding. Binding fragments can be produced by
  • Fab refers to that portion of an antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F(ab')2 refers to a dimer of Fab'.
  • F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Fv refers to the smallest fragment of an antibody to bear the complete antigen binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • single-chain antibody refers to an antibody consisting of a heavy chain variable region and a light chain variable region connected by a linker.
  • the linker may be a peptide.
  • the length of the linker varies depending upon the type of single-chain antibody. Covalently or non-covalently linking two or more single-chain antibodies together results in higher order forms.
  • Single-chain antibodies may include, but are not limited to, single-domain antibodies, multivalent domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra- scFvs), diabodies, and triabodies and tetrabodies.
  • scFvs single chain variant fragments
  • divalent scFvs divalent scFvs
  • tri-scFvs trivalent scFvs
  • tetravalent scFvs tetra- scFvs
  • diabodies and triabodies and tetrabodies.
  • single-chain Fv antibody and “scFv” are used herein interchangeably to refer to a single-chain antibody consisting of heavy variable region and a light chain variable region connected by a linker.
  • the linker may be a peptide.
  • the linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length.
  • the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site.
  • a linker peptide is rich in glycine, as well as serine or threonine.
  • Covalently or non- covalently linking two or more scFvs together results in higher order forms di-scFvs, tri- scFvs, tetra- scFvs, etc.
  • the antigen-binding sites of each scFv in a higher order form can target the same or different antigen or epitope.
  • single-chain Fv-Fc antibody or “scFv-Fc” refers to a full-length antibody consisting of a scFv connected to an Fc region.
  • a “diabody” is a higher order variant of a single-chain antibody consisting of two singlechain antibodies.
  • a linker is used that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with the complementary domains of another chain, thereby creating two antigen-binding sites.
  • the linker may be a peptide.
  • the antigen-binding sites can target the same or different antigens or epitopes. Triabodies (three single chain antibodies assembled to form three antigenbinding sites), tetrabodies (four single chain antibodies assembled to form four antigen-binding sites), and higher order variants can similarly be produced. See, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP404097; WO93/11161.
  • a “single-domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more VH domains are covalently joined with a peptide linker to create a multivalent domain antibody.
  • the two or more VH domains of a multivalent domain antibody can target the same or different antigens or epitopes.
  • heavy chain antibody refers to an antibody that consists of two heavy chains.
  • a heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. December 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4):277-302 (2001); WO94/04678; WO94/25591; or U.S. Pat. No. 6,005,079.
  • Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428):446-8 (1993); Nguyen V. K. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. April; 54(l):39-47 (2002); Nguyen V. K. et al. Immunology. May; 109(l):93-101 (2003)).
  • variable domain of a heavy chain antibody represents the smallest known antigen- binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun. 15 (2007)).
  • corresponding human germline sequence refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences.
  • the corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences.
  • the corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
  • the corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.
  • the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57- 86, 2000.
  • Equilibrium dissociation constant refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-l). Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies of the present disclosure generally will have an equilibrium dissociation constant of less than about 10- 7 or 10-8 M, for example, less than about 10-9 M or 10-10 M, in some aspects, less than about 10-11 M, 10-12 M or 10-13 M.
  • cancer or “tumor” herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of the present disclosure, the cancer is not limited to certain type or location.
  • conservative substitution means substitution of the original amino acid by a new amino acid that does not substantially alter the chemical, physical and/or functional properties of the antibody or fragment, e.g., its binding affinity to CLDN6. Specifically, common conservative substations of amino acids are well known in the art.
  • knob-into-hole refers to amino acids that direct the pairing of two polypeptides together either in vitro or in vivo by introducing a spatial protuberance (knob) into one polypeptide and a socket or cavity (hole) into the other polypeptide at an interface in which they interact.
  • knob-into-holes have been introduced in the Fc:Fc binding interfaces, CL:CHI interfaces or VH/VL interfaces of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al, 1997, Protein Science 6:781-788).
  • knob-into-holes insure the correct pairing of two different heavy chains together during the manufacture of multi-specific antibodies.
  • multi-specific antibodies having knob-into-hole amino acids in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains.
  • Knob-into-hole technology can also be used in the VH or VL regions to also insure correct pairing.
  • knock as used herein in the context of “knob-into-hole” technology refers to an amino acid change that introduces a protuberance (knob) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide.
  • the other polypeptide has a hole mutation.
  • hole as used herein in the context of “knob-into-hole” refers to an amino acid change that introduces a socket or cavity (hole) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide.
  • the other polypeptide has a knob mutation.
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as values for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). Lor amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), algorithm which has been incorporated into the GAP program in the GCG software package using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • compositions e.g., pharmaceutically acceptable compositions, which include anti-CLDN6 antibodies as described herein, formulated together with at least one pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g. by injection or infusion).
  • compositions disclosed herein can be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions e.g., injectable and infusion solutions
  • dispersions or suspensions e.g., dispersions or suspensions
  • liposomes e.g., liposomes, and suppositories.
  • a suitable form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusion solutions.
  • One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous infusion or injection.
  • the antibody is administered by intramuscular or subcutaneous injection.
  • the term “therapeutically effective amount” as herein used refers to the amount of an antibody that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom.
  • the “therapeutically effective amount” can vary with the antibody, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. An appropriate amount in any given instance can be apparent to those skilled in the art or can be determined by routine experiments.
  • the “therapeutically effective amount” refers to the total amount of the combination objects for the effective treatment of a disease, a disorder or a condition.
  • composition therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Powders and/or liquids can be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
  • an anti-CLDN6xCD3 multi-specific antibody is administered to the subject at the same time as, just before, or just after administration of an additional therapeutic agent.
  • an anti-CLDN6xCD3 multi-specific antibody is administered as a co-formulation with an additional therapeutic agent.
  • the present disclosure provides for antibodies, antigen-binding fragments, and anti- CLDN6 antibodies. Furthermore, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable attributes, and thus can be used for reducing the likelihood of cancer or used for treating cancer. The present disclosure further provides pharmaceutical compositions comprising the antibodies and methods of making and using such pharmaceutical compositions for the prevention and treatment of cancer and associated disorders.
  • the present disclosure provides for antibodies or antigen-binding fragments thereof that specifically bind to CLDN6.
  • Antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, the antibodies or antigen-binding fragments thereof, generated as described, below.
  • the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein said antibodies or antibody fragments (e.g., antigenbinding fragments) comprise a VH domain having an amino acid sequences listed in Table 1.
  • the present disclosure also provides antibodies or antigen-binding fragments that specifically bind CLDN6, wherein said antibodies or antigen-binding fragments comprise a HCDR having an amino acid sequence of any one of the HCDRs listed in Table 1.
  • the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein said antibodies comprise (or alternatively, consist of) one, two, three, or more HCDRs having an amino acid sequence of any of the HCDRs listed in Table 1.
  • the present disclosure provides for antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein said antibodies or antigen-binding fragments comprise a VL domain having an amino acid sequences listed in Table 1.
  • the present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein said antibodies or antigen-binding fragments comprise a LCDR having an amino acid sequence of any one of the LCDRs listed in Table 1.
  • said disclosure provides for antibodies or antigen-binding fragments that specifically bind to CLDN6, said antibodies or antigen-binding fragments comprise (or alternatively, consist of) one, two, three or more LCDRs having an amino acid sequence of any of the LCDRs listed in Table 1.
  • antibodies or antigen-binding fragments thereof of the present disclosure include amino acids that have been changed, yet have at least 60%, 70%, 80%, 90%, 95% or 99% identity in the CDR regions with the CDR regions disclosed in Table 1.
  • the amino acid sequence has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity.
  • it includes amino acid changes wherein no more than 1, 2, 3, 4 or 5 amino acids have been changed in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 1.
  • amino acids or nucleic acids encoding the amino acids have been changed; yet have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences described in Table 1.
  • the amino acid sequence has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity.
  • it includes changes in the amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been changed in the variable regions when compared with the variable regions depicted in the sequence described in Table 1, while retaining substantially the same therapeutic activity.
  • the present disclosure also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to CLDN6. Such nucleic acid sequences can be optimized for expression in mammalian cells.
  • the present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CLDN6.
  • the antibodies and antigen-binding fragments can bind to the same epitope of CLDN6.
  • the present disclosure also provides for antibodies and antigen-binding fragments thereof that bind to the same epitope as do the anti-CLDN6 antibodies described in Table 1. Additional antibodies and antigen-binding fragments thereof can therefore be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies in binding assays.
  • the ability of a test antibody to inhibit the binding of antibodies and antigen-binding fragments thereof of the present disclosure to CLDN6 demonstrates that the test antibody can compete with that antibody or antigen-binding fragments thereof for binding to CLDN6.
  • Such an antibody can, without being bound to any one theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on CLDN6 as the antibody or antigen-binding fragments thereof with which it competes.
  • the antibody that binds to the same epitope on CLDN6 as the antibodies or antigen-binding fragments thereof of the present disclosure is a human or humanized monoclonal antibody.
  • Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.
  • the anti-CLDN6 antibodies as disclosed herein can be an anti- CLDN6 multi-specific antibody.
  • An antibody molecule is a multi-specific antibody molecule, for example, it comprises a number of antigen binding domains, wherein at least one antigen binding domain sequence specifically binds CLDN6 as a first epitope and a second antigen binding domain sequence specifically binds a second epitope.
  • the multi-specific antibody comprises a third, fourth or fifth antigen binding domain.
  • the multi-specific antibody is a bispecific antibody, a tri-specific antibody, or tetra-specific antibody.
  • the multi-specific antibody comprises at least one anti-CLDN6 antigen binding domain and at least one anti-CD3 antigen binding domain.
  • the multi- specific antibody is a bispecific antibody.
  • a bispecific antibody specifically binds only two antigens.
  • the bispecific antibody comprises a first antigen binding domain which specifically binds CLDN6 and a second antigen binding domain that specifically binds another epitope.
  • the bispecific antibody that comprises antigen binding fragments, the antigen-binding fragment can be a Fab, F(ab’)2, Fv, or a single chain Fv (ScFv) or a scFv.
  • the domains and/or regions of the polypeptide chains of the bispecific tetravalent antibody can be separated by linker regions of various lengths.
  • the antigen binding domains are separated from each other, a CL, CHI, hinge, CH2, CH3, or the entire Fc region by a linker region.
  • a linker region For example, VLl-CL-(linker) VH2-CH1, VH-linker-VL.
  • Such linker region may comprise a random assortment of amino acids, or a restricted set of amino acids.
  • Such linker regions can be flexible or rigid (see US2009/0155275).
  • Multi-specific antibodies have been constructed by genetically fusing two single chain Fv (scFv) or Fab fragments with or without the use of flexible linkers (Mallender et al., J. Biol. Chem. 1994269:199-206; Macket al., Proc. Natl. Acad. Sci. USA. 1995 92:7021-5; Zapata et al., Protein Eng. 1995 8.1057-62), via a dimerization device such as leucine Zipper (Kostelny et al., J. Immunol. 1992148:1547-53; de Kruifetal J. Biol. Chem.
  • the bispecific tetravalent antibodies as disclosed herein comprise a linker region of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues between one or more of its antigen binding domains, CL domains, CHI domains, Hinge region, CH2 domains, CH3 domains, or Fc regions.
  • the amino acids glycine and serine comprise the amino acids within the linker region.
  • the linker can be GS, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA(G4S)4, SAKTTP, SAKTTPKLGG, SAKTTPKLEEGEFSEARV, ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALM
  • the multivalent antibody comprises at least one dimerization specific amino acid change.
  • the dimerization specific amino acid changes result in “knobs into holes” interactions, and increases the assembly of correct multivalent antibodies.
  • the dimerization specific amino acids can be within the CHI domain or the CL domain or combinations thereof.
  • the dimerization specific amino acids used to pair CHI domains with other CHI domains (CHI-CHI) and CL domains with other CL domains (CL-CL) and can be found at least in the disclosures of WO2014082179, W02015181805 family and
  • the dimerization specific amino acids can also be within the Fc domain and can be in combination with dimerization specific amino acids within the CHI or CL domains.
  • the disclosure provides a bispecific antibody comprising at least one dimerization specific amino acid pair.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in, e.g., U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are changed to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the publication WO 94/29351 by Bodmer et al.
  • one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced by one or more allotypic amino acid residues, for the IgGl subclass and the kappa isotype.
  • Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl , IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described in, e.g., the publication WO00/42072 by Presta.
  • the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).
  • the glycosylation of the multi-specific antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks or has reduced glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for “antigen.”
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation can increase the affinity of the antibody for antigen.
  • Such an approach is described in, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation.
  • EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation.
  • WO99/54342 by Umana et al., describes cell lines engineered to express glycoproteinmodifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase HI (GnTIH)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17: 176-180, 1999).
  • glycoproteinmodifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransferase HI (GnTIH)
  • Reduced ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with combinations of alterations that reduce FcyR binding or Clq binding activities, thereby reducing or eliminating ADCC and CDC effector functions.
  • IgG4 Fc engineered with combinations of alterations that reduce FcyR binding or Clq binding activities thereby reducing or eliminating ADCC and CDC effector functions.
  • IgG4 Fc engineered with combinations of alterations that reduce FcyR binding or Clq binding activities thereby reducing or eliminating ADCC and CDC effector functions.
  • Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production.
  • Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
  • the disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions as described herein.
  • the polynucleotide encoding the heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 56, SEQ ID NO: 60, and SEQ ID NO: 64.
  • the polynucleotide encoding the light chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 10, 57, 61 or 65.
  • the polynucleotides of the present disclosure can encode the variable region sequence of an anti-CLDN6 antibody. They can also encode both a variable region and a constant region of the antibody. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of the exemplified anti-CLDN6 antibodies.
  • expression vectors and host cells for producing the anti-CLDN6 antibodies are also provided in the present disclosure.
  • the choice of expression vector depends on the intended host cells in which the vector is to be expressed.
  • the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an anti-CLDN6 antibody chain or antigen-binding fragment.
  • an inducible promoter is employed to prevent expression of inserted sequences except under the control of inducing conditions.
  • Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter.
  • Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells.
  • other regulatory elements can also be required or desired for efficient expression of an anti-CLDN6 antibody or antigen-binding fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences.
  • the efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987).
  • the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.
  • the host cells for harboring and expressing the anti-CLDN6 antibody chains can be either prokaryotic or eukaryotic.
  • E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a betalactamase promoter system, or a promoter system from phage lambda.
  • the promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes such as yeast, can also be employed to express anti-CLDN6 antibodies.
  • Insect cells in combination with baculovirus vectors can also be used.
  • mammalian host cells are used to express and produce the anti-CLDN6 antibodies of the present disclosure.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector.
  • a hybridoma cell line expressing endogenous immunoglobulin genes
  • mammalian cell line harboring an exogenous expression vector include any normal mortal or normal or abnormal immortal animal or human cells.
  • suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B-cells and hybridomas.
  • the use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986)
  • necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasoneinducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
  • the current standard for an engineered heterodimeric antibody Fc domain is the knobs- into-holes (KiH) design, which introduced mutations at the core CH3 domain interface.
  • the resulted heterodimers have a reduced CH3 melting temperature (69°C or less).
  • the ZW heterodimeric Fc design has a thermal stability of 81 ,5°C, which is comparable to the wild-type CH3 domain.
  • the antibodies or antigen-binding fragments of the present disclosure are useful in a variety of applications including, but not limited to, methods for the detection of CLDN6.
  • the antibodies or antigen-binding fragments are useful for detecting the presence of CLDN6 in a biological sample.
  • the term “detecting” as used herein includes quantitative or qualitative detection.
  • a biological sample comprises a cell or tissue.
  • such tissues include normal and/or cancerous tissues that express CLDN6 at higher levels relative to other tissues.
  • the present disclosure provides a method of detecting the presence of CLDN6 in a biological sample.
  • the method comprises contacting the biological sample with an anti-CLDN6 antibody under conditions permissive for binding of the antibody to the antigen and detecting whether a complex is formed between the antibody and the antigen.
  • the biological sample can include, without limitation, urine, tissue, sputum or blood samples.
  • the method comprises contacting a test cell with an anti-CLDN6 antibody; determining the level of expression (either quantitatively or qualitatively) of CLDN6 expressed by the test cell by detecting binding of the anti-CLDN6 antibody to the CLDN6 polypeptide; and comparing the level of expression by the test cell with the level of CLDN6 expression in a control cell (e.g., a normal cell of the same tissue origin as the test cell or a non- CLDN6 expressing cell), wherein a higher level of CLDN6 expression in the test cell as compared to the control cell indicates the presence of a disorder associated with expression of CLDN6.
  • a control cell e.g., a normal cell of the same tissue origin as the test cell or a non- CLDN6 expressing cell
  • the antibodies or antigen-binding fragments of the present disclosure are useful in a variety of applications including, but not limited to, methods for the treatment of a CLDN6- associated disorder or disease.
  • the CLDN6-associated disorder or disease is a cancer.
  • the present disclosure provides a method of treating cancer.
  • the method comprises administering to a patient in need thereof an effective amount of an anti-CLDN6 antibody or antigen-binding fragment.
  • the cancer is a solid tumor.
  • the cancer can include, without limitation, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor, and/or glioma.
  • the antibody or antigen-binding fragment as disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Antibodies or antigen-binding fragments of the disclosure can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an antibody or antigen-binding fragment of the disclosure will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to 100 mg/kg of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • Such doses can be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody).
  • An initial higher loading dose, followed by one or more lower doses can be administered.
  • other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • anti-CLDN6 antibodies of the present disclosure can be used in combination with other therapeutic agents.
  • Other therapeutic agents that can be used with the anti-CLDN6 antibodies of the present disclosure include: but are not limited to, a chemotherapeutic agent (e.g., paclitaxel or a paclitaxel agent; (e.g.
  • Abraxane® docetaxel
  • carboplatin topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), multikinase inhibitor (e.g., MGCD265, RGB-286638), CD-20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g.,
  • Anti-CLDN6 antibodies of the present disclosure can be used in combination with other therapeutics, for example, immune checkpoint antibodies.
  • immune checkpoint antibodies can include anti-PDl antibodies.
  • Anti-PDl antibodies can include, without limitation, Tislelizumab, Pembrolizumab or Nivolumab. Tislelizumab is disclosed in US 8,735,553. Pembrolizumab (formerly MK-3475), is disclosed in US 8,354,509 and US 8,900,587 and is a humanized lgG4-K immunoglobulin which targets the PD1 receptor and inhibits binding of the PD1 receptor ligands PD-L1 and PD-L2.
  • Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is under clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC), and refractory Hodgkin's lymphoma (cHL).
  • NSCLC metastatic non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL refractory Hodgkin's lymphoma
  • Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human lgG4-K monoclonal antibody.
  • Nivolumab (clone 5C4) is disclosed in US Patent No. US 8,008,449 and WO 2006/121168.
  • Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.
  • anti-HGIT antibodies can include without limitation, anti- HGIT antibodies as disclosed in WO2019/129261.
  • anti-OX40 antibodies can include without limitation, anti- 0X40 antibodies as disclosed in WO2019/223733.
  • anti-HM3 antibodies can include without limitation, anti- HM3 antibodies as disclosed in WO2018/036561.
  • compositions and formulations comprising an anti-CLDN6 antibody or antigen-binding fragment thereof, or polynucleotides comprising sequences encoding an anti-CLDN6 antibody or antigen-binding fragment.
  • compositions comprise one or more anti-CLDN6 antibodies or antigen-binding fragments, or one or more polynucleotides comprising sequences encoding one or more anti- CLDN6 antibodies or antigen-binding fragments.
  • suitable carriers such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
  • compositions of an anti-CLDN6 antibody or antigen-binding fragment as described herein are prepared by mixing such antibody or antigen-binding fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • the formulation is composed of L-histidine/L-histidine hydrochloride monohydrate, trehalose and polysorbate 20.
  • concentration of the anti-CLDN6 antibody drug product after constitution with sterile water for injection, is an isotonic solution consisting of 10 mg/mL anti-CLDN6 antibody, 20 mM histidine/histidine HC1, 240 mM trehalose dihydrate, and 0.02% polysorbate 20, at a pH of approximately 5.5.
  • Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.
  • Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • Lymphoid organs including spleens and lymph nodes, were isolated from mice immunized as described above. Hybridomas were generated by fusions with immortalized mouse myeloma cells derived from the SP2/0 by PEG based fusion. The resulting cells were plated in 96 well cell culture plates using regular 1640 medium supplemented with HAT for selection of hybridomas.
  • Hybridomas were generated as described in Example 1. After 10-13 days of culture and growth media replacement, hybridoma culture supernatants were collected from individual wells and screened to identify wells with secreted CLDN6-specific antibodies. All supernatants were initially screened against at least two overexpression cell lines, including CHOKl/human CLDN6 and CHOKl/human CLDN9 (internally prepared). Antibody binding on overexpression cell lines were measured by FACS. Supernatants from over approximately 20000 culture wells in 4 hybridoma fusions were screened for CLDN6 antibodies.
  • hybridoma culture supernatant and a CLDN6-expressing cancer cell line such as PA-1 or CHOKl/human CLDN6 stable cell lines
  • control cells such as parental CHOK1
  • fluorescence was measured by flow cytometry.
  • Hybridomas from positive wells were transferred to 24-well plates with fresh culture media to grow for 2-3 days before screening again by flow cytometry to confirm antibody binding to the cyno CLDN6 overexpression cell line and the human CLDN6 positive cancer cell line (PA-1).
  • Antibody binding to the cyno CLDN6 overexpression cell line and the human CLDN6 positive cancer cell line (PA-1) was measured by flow cytometry.
  • hybridoma culture supernatant and CLDN-expressing cancer cell line such as PA-1 or CHOKl/human CLDN6 and CHOKl/human CLDN9 stable cell lines
  • control cells such as parental CHOK1
  • fluorescence was measured by flow cytometry.
  • Selected CLDN6 antibody-secreting hybridomas were subcloned once or twice to ensure monoclonality. Briefly, approximately 80-100 viable hybridoma cells were plated in 3 mb of semi-solid methylcellulose medium (Stem Cell Technologies) in 6 well plate. After 7-10 days, hybridoma colonies arising from single cells as visible clones were picked to 96-well plate, and were further cultured for 2-4 days in fresh medium. Culture supernatant was screened by ELISA and flow cytometry as previously described to confirm human and cyno CLDN6 binding. Stable hybridoma subclones were cultured in vitro for cell cryopreservation, antibody producing and antibody VH and VL gene cloning and sequencing.
  • the ChBG87P antibody was generated by subcloning variable region of mouse BG87P (SEQ ID NO: 7 and 8) into an in-house developed expression vectors which contains constant regions of a human wildtype IgGl and kappa chain.
  • the antibody was expressed by co- transfection of the above two constructs into HEK293T cells and purified using a protein A column (Cat: 17-5438-02, GE Life Sciences ®.).
  • the purified chimeric antibody was concentrated to 0.5-10mg/ml in PBS and stored in aliquots in -80°C- Freezer.
  • chBG87P human germline IgG genes were searched for sequences that share high degrees of homology to the protein sequences of chBG87P variable regions by sequence comparison against the human immunoglobulin gene database in IMGT.
  • Humanization was carried out by CDR-grafting following with critical back mutations incorporated.
  • the humanized antibodies were engineered as human IgGl wild type format by using an in-house developed expression vector.
  • mutations from murine variable region to human amino acid residues in framework regions were guides by 3D structures analysis and the murine framework residues with structural importance for maintaining the canonical structural of CDRs were retained in the first round of the humanization design.
  • BG87P-Bzl VH SEQ ID NO: 15 and VL: SEQ ID NO: 14
  • BG87P-Bz2 VH SEQ ID NO: 16 and VL: SEQ ID NO: 14
  • BG87P-Bz3 VH SEQ ID NO: 17 and VL: SEQ ID NO: 14
  • BG87P- Bz4 VH SEQ ID NO: 18 and VL: SEQ ID NO: 14
  • BG87P-Bz5 VH SEQ ID NO: 19 and VL: SEQ ID NO: 14
  • BG87P-Bz6 VH SEQ ID NO: 13 and VL: SEQ ID NO: 20
  • BG87P-Bz7 VH SEQ ID NO: 13 and VL: SEQ ID NO: 20
  • BG87P-Bz8 VH SEQ ID NO: 13 and VL SEQ ID NO: 22.
  • BG87P-BzO (VH: SEQ ID NO: 13 and VL: SEQ ID NO: 14) is the variant with all theoretical back mutations incorporated and the binding capacity of BG87P-BzO should be comparable to parental chBG87P. Comparison of the binding data reveals which back mutations affect binding significantly. Specifically, LCDRs of chBG87P (SEQ ID NO: 4 to 6) were grafted into the framework of human germline variable gene IGKV1-5 and 01-IGKJ4*01 with A43S, L78V and Y87F murine framework residues (resulted as SEQ ID NO: 14).
  • HCDRs of chBG87P (SEQ ID NO: 1 to 3) were grafted into the framework of human germline variable gene IGHV1- 3 and 01-JH6c with V2I, T28S, I69L, R71V and Y91F murine framework residues retained (resulted as SEQ ID NO: 13);
  • BG87P-zO VH: SEQ ID NO: 11 and VL: SEQ ID NO: 12
  • VH SEQ ID NO: SEQ ID NO: 11 and VL: SEQ ID NO: 12
  • All first round of BG87P humanized variants (BG87P-zO, BG87P-BzO, BG87P-Bzl, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7, and BG87P- Bz8) were constructed as a humanized full-length antibody using in-house developed expression vectors that contain constant regions of a human wildtype IgGl and kappa chain, respectively, with easy adapting sub-cloning sites.
  • CLDN6 over-expressing HEK293T cells and cancer cell line PA-1 which express high levels of human CLDN6 were used to evaluate the binding activity of BG87P related engineering variants. Live cells were seeded in 96-well plate, and were incubated with a series of dilutions of chBG87P and engineered variants thereof. Goat anti-human IgG was used as second antibody to detect antibody binding to the cell surface. EC50 values for dosedependent binding to CLDN6 expressing cell lines were determined by fitting the dose-response data to the four-parameter logistic model with GraphPad Prism.
  • BG87P-ml VH SEQ ID NO: 31 and VL SEQ ID NO: 8
  • BG87P-m2 VH SEQ ID NO: 32 and VL SEQ ID NO: 8
  • BG87P-m3 VH SEQ ID NO: 33 and VL SEQ ID NO: 8
  • BG87P-m4 VH SEQ ID NO: 34 and VL SEQ ID NO: 8)
  • BG87P-m5 VH SEQ ID NO: 35 and VL SEQ ID NO: 14
  • BG87P-m6 VH SEQ ID NO: 36 and VL SEQ ID NO 14
  • BG87P-m7 VH SEQ ID NO: 37 and VL SEQ ID NO: 14
  • BG87P-m8 VH SEQ ID NO: 38 and VL SEQ ID NO: 14
  • VH and VL sequences of resulted 2 nd round humanized variants BG87P-21, BG87P-22, BG87P-23, BG87P-24, BG87P-25, BG87P-26, and BG87P-27 are given in Table 1.
  • BG87P-21 was selected as the top humanized candidate for further consideration (VH and VL amino acid sequences are SEQ ID NO: 24 and 12 respectively).
  • BG87P-21 included critical back mutation site VH: T28S and PTM site VH: V65G, which revealed a comparable cell binding affinity as compared to chBG87P.
  • the Emax was reduced of 22% in HEK293T/human CLDN6 and 40% in PA-1 (Table 7 and Table 8).
  • thermostability assessment the thermal stability of BG87P related engineering variants were described by the thermal unfolding transition midpoint Tm (°C), which was measured by extrinsic fluorescence.
  • the Tm was determined using QuantStudioTM 6 Flex System from Applied Biosystems. 20 pL of 1 mg/ml sample was mixed with 20 pL of 40XSYPRO orange. The plate was scanned from 25°C to 95°C at a rate of 0.9°C/min. The Tm was assigned using the first derivative of the raw data from the QuantStudioTM 6 Flex System Analysis software. The results were summarized in Table 9, which indicates both chBG87P and humanized variant BG87P-21 showed good thermostability.
  • CIC is a technique to identify antibody candidates with poor solubility or non-specific binding propensity.
  • IgG from human serum or other ligands were chemically coupled to an NHS-activated chromatography resin. The retention times of proteins were tested on this resin using a HPLC to evaluate proteins solubility.
  • HPLC mobile phase
  • the method is used to study weak protein-protein interactions, to predict the aggregation trend, to reveal the influence of formulation ingredients on intermolecular interactions and support the formulation buffer selection.
  • Antibodies with buffer exchange samples were diluted to Img/mL and centrifuge at 14000rpm for 30min, then check Tm, Tagg and DLS. Loading samples onto the Uni. 9pL/well. Each sample set a duplicate hole. Set equipment parameters following the guide of Uncle and run the experiment. In this experiment we used B22 & Kd mode. Run information: Temperature (°C): 25. Incubation Time (sec): 120. No. of Acquisitions: 4. Acquisition Time (sec): 5. Attenuator Control: Auto. Laser. Control: Auto Run.
  • AC- SINS is the assay to obtain the self-interaction for the sample to predict the aggregation possibility. It is based on concentrating antibodies from diluted solutions around gold nanoparticles pre-coated with polyclonal capture. Interactions between immobilized antibodies lead to reduced inter-particle distances and increased plasmon wavelengths (wavelength of maximum absorbance), which can be readily measured by optical means. Dilute the antibodies with offering buffer to 0.05 mg/mL, respectively. After gold nanoparticle preparation, a 9:1 volume ratio was used to mix gold nanopartical solution with coating solution. After room temperature incubation for Ih, thilolated PEG (final concentration 0.1 uM) was used to block empty sites in AuNP.
  • the hydrophobic patch causes the HIC retention time of the chBG87P to be over 25 minutes and the humanized BG87P-21 to be 21.9 minutes, both of them are higher than the acceptable threshold which is 21.1 minutes in IgG format.
  • the root cause is the hydrophobic patches in HCDR3, in particular the I97-Y98-Y100-V100a part together with Y49-W50 (mainly W50) at the edge of FR2 (framework region 2) and LCDR2 of light chain (FIG. 2A).
  • Y49-W50 mainly W50
  • chBG87P has been engineered to a humanized antibody and we have identified BG87P-21 as a final top clone.
  • a potential developability risk of a hydrophobic patch driven from HCDR3 of chBG87P has not been solved (FIG. 2).
  • chBG87P shows promising binding activity and superior CLDN6 selectivity (FIG. 1A, FIG. IB, FIG. 1C, FIG ID, FIG. IE, FIG. IF, FIG, 1G, and FIG. 1H)
  • additional engineering of BG87P-21 has been done to remove the hydrophobic patch for optimal manufacturability and the mitigation of potential ADA risk.
  • Combination variants BG87P-31 and BG87P-32 (VH and VL amino acid sequences are SEQ ID NO: 46 and 42 respectively) were selected as top candidates with comparable binding affinity to BG87P-21 and improved HIC-retention 17.4min for BG87P-31 and 18.49min for BG87P-32, both of them are superior to the parental BG87P-21 22.3min (Table 14 and FIG 3).
  • mouse clone sp34 (Blumberg 1990 PNAS 87 (18):7220-24) was an optimal clone for developing anti-CD3 based therapeutics due to its cyno CD3 cross-reactivity.
  • human germline IgG genes were searched for sequences that share high degrees of homology to the protein sequences (SEQ ID NO: 48 - 57) of sp34 variable regions by blasting the human immunoglobulin gene database in IMGT (http://www(dot)imgt(dot)org/IMGT_vquest/share/textes/ index(dot)html) and NCBI (http://www(dot)ncbi(dot)nlm(dot)nih(dot)gov/igblast/) websites.
  • IMGT http://www(dot)imgt(dot)org/IMGT_vquest/share/textes/ index(dot)html
  • NCBI http://www(dot)ncbi(dot)nlm(dot)nih(dot)gov/igblast/
  • CDRs of sp34 VL (SEQ ID NO: 51 ⁇ 53) were grafted into the framework of human germline variable gene IGVK3-15 with several murine framework residues (QI, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, 185, and F87) retained.
  • CDRs of sp34 VH (SEQ ID NO: 48 - 50) were grafted into the framework of human germline variable gene IGVH3-7 with several murine framework residues (D73, S76, M89, V93) retained.
  • Humanized sp34 (hu-sp34) and chimeric sp34 (ch-sp34) were constructed as human full- length antibody format using in-house developed expression vectors that contain constant regions of a human IgGl and kappa chain, respectively, with easy adapting sub-cloning sites.
  • Expression and preparation of humanized sp34 and chimeric sp34 antibodies was achieved by co-transfection of the heavy chain and corresponding light chain constructs into 293 G cells (developed in house) and by purification using a protein A column. The purified antibodies were concentrated to 0.5-5 mg/mL in PBS and stored in aliquots in -80°C freezer for using in the assays below.
  • hu-sp34 variant antibodies were tested in binding assays as described previously. Comparing to hu-sp34-lA-lf, mutations of V36Y, G46L and G49Y (Kabat numbering) on VK significantly impaired binding affinities of the humanized variants and while the rest versions of hu-sp34 humanization variants had comparable binding activities to hu-sp34-l A- If. D73N in VH significantly reduced expression level (data not shown).
  • the purified scFv-Fc format antibodies were concentrated to 0.5-5 mg/mL in PBS and stored in aliquots in -80°C freezer for using in the assays below.
  • the scFv-ized BG56P (referred as BG561P, SEQ ID NO: 48 - 53 and 62 - 65) showed comparable binding affinities with that of antibody version of BG56P in SPR and FACS (Table 17 and FIG. 5).
  • BG563P humanized scFv
  • SEQ ID NO: 48, 71, 50, 51 - 53, 73, and 74 showed improved thermal and colloidal stabilities compared with BG561P while showed slightly improved binding affinity to human CD3 in FACS assay.
  • the potential PTM sites include potential deamidation site N30 (NT) in the junction region of FR1 and HCDR1 (Kabat CDR definition) and N100 (NS) in HCDR3. Each of the N was mutated to S to remove the potential deamidation sites.
  • Tm Melting temperature
  • the aggregation temperature Tagg (°C) is representative of the colloidal stability of the samples and was obtained by monitoring the onset of aggregation by SLS266 using UNCLETM (Unchained lab, Pleasanton, CA). Samples were loaded into Uni, and subjected to a temperature ramping from 15°C to 95°C. The back-reflection optics cannot detect near UV light scattering by protein aggregates, and thus only non-scattered light reaches the detector. The reduction of back reflected light is therefore a direct measure for aggregation in the sample , which suggested the Tagg of BG564P was improved compared with BG561P (Table 20).
  • Agonistic anti-CD3 antibodies have demonstrated toxicity in the clinical setting, which may indicate that systemic FcyR cross-linking is not ideal for CD3 activation.
  • the aim was to achieve potent CD3 stimulation specifically at the tumor site without systemic CD3 activation for a broad range of cancers.
  • CLDN6xCD3 BsAb BG143P we generated a CLDN6xCD3 BsAb BG143P with the following features as shown in FIG. 7.
  • This specific construct BG143P includes an IgG-fusion like multi-specific antibody format with a module ratio of 1 : 1, a well- engineered Fab fragment BG87P-34 that binds to CLDN6 and a scFv of BG564P that binds to CD3 fusion at the N terminal of CH2, and a Fc null version of huIgGl, which has no FcyR binding but retains FcRn binding. Knob into Hole (KIH ) was also introduced in Fc to increase heterodimerization.
  • the sequence information of BG143P is listed in SEQ ID NO: 79 - 84.
  • the FACS results further confirmed the binding activity of BG143P to CD3 and CLDN6.
  • the BsAb showed strong binding activities to CD3 -expressing Jurkat in a dose- responsive manner with ECso of 6.98 nM.
  • BG143P showed strong binding activities to CLDN6-expressing PA-1 in a dose-responsive manner with ECso of 81.26 nM (FIG. 8B).
  • T cell-redirected cytotoxicity of BG143P against PA-1 (cancer cell line with high CLDN6 expression), Hutu80 (cancer cell line with medium CLDN6 expression), AGS (cancer cell line with low and heterogeneous CLDN6 expression) and NCI-H1299 (cancer cell line negative for CLDN6 expression) was evaluated using human PBMCs as effector cells.
  • the supernatant was collected for cytokine detection.
  • Target cell killing was measured by Nano-Gio detection kit (Promega).
  • A represents the average luminescence signal of wells with untreated target cells only
  • B represents the average luminescence signal of wells with antibody and PBMCs
  • “C” represents the average luminescence signal of wells with target cells totally lysed with Triton- Xi 00.
  • IFN-y and IL-2 were detected in supernatants by HTRF kit (Cisbio).
  • BG143P showed potent T cell-redirected killing and cytokine release induction potency in a dose-dependent manner with pM EC50 levels.
  • reaction solution was removed, and the cells were washed twice with 200 pL/well FACS buffer. Then, APC-anti human Fey was diluted 500-fold with FACS buffer and added as secondary antibodies to the cells. The cells were incubated at 4°C for 30 mins, then washed twice as above, and suspended in 100 pL FACS buffer. The cell suspension was subjected to flow cytometry.
  • A represents the average luminescence signal of wells with untreated target cells only
  • B represents the average luminescence signal of wells with antibody and PBMCs
  • C represents the average luminescence signal of wells with target cells totally lysed with Triton-XIOO. IFN-y was detected by HTRF kit (Cisbio).
  • BG143P was an antibody with specific binding (FIG. 10A), cell killing (e.g. lysis) (FIG. 10B) and IFN-y induction activity (FIG. 10B) against human CLDN6, but not human CLDN9.
  • FIG. 11 A shows the in vivo antitumor efficacy of BG143P, which showed strong efficacy at 0.03 mg/kg and 0.1 mg/kg, with TGI% (Tumor Growth Inhibition Ratio, %) of 115.43% and 125.92%.
  • Test article or PBS were intraperitoneally injected into the mice every week.
  • the length (L) and width (W) of the tumor mass and body weight of each mouse were measured three times per week.
  • BG143P at 0.1 mg/kg presented strong efficacy, with a TGI% of 93.54%, which is shown in FIG. 12A. No obvious body weight loss observed in the study, as illustrated in FIG. 12B.
  • Claudin-6 is a single prognostic marker and functions as a tumorpromoting gene in a subgroup of intestinal type gastric cancer. Gastric Cancer, 2020. 23(3): p. 403-417.

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Abstract

La présente divulgation concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui se lient à la CLDN6 humaine, une composition pharmaceutique comprenant ledit anticorps ou des fragments de liaison à l'antigène de celui-ci, et l'utilisation de l'anticorps ou de fragments de liaison à l'antigène de celui-ci ou de la composition pour le traitement d'une maladie, telle que le cancer.
PCT/IB2024/052127 2023-03-06 2024-03-05 Anticorps anti-cldn6 et procédés d'utilisation WO2024184812A1 (fr)

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