Claudin-6 antibodies and drug conjugates

Abstract

The present disclosure provides antigen-binding proteins which bind to Claudin-6 (CLDN6). In various aspects, the antigen-binding proteins bind to Extracellular Loop 2 (EL2) of the extracellular domain of CLDN6. Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions comprising such entities are moreover provided. Also provided are methods of making an antigen-binding protein and methods of treating a subject having cancer.

What is claimed is: 
     
         1 . An antigen-binding protein comprising:
 a. a heavy chain CDR1 amino acid sequence of: SEQ ID NO: 504 or SEQ ID NO: 507, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   b. a heavy chain CDR2 amino acid sequence of: SEQ ID NOs: 505 or SEQ ID NO: 508, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   c. a heavy chain CDR3 amino acid sequence of: SEQ ID NO: 506 or SEQ ID NO: 509, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   d. a light chain CDR1 amino acid sequence of: SEQ ID NO: 449 or SEQ ID NO: 476, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   e. a light chain CDR2 amino acid sequence of: SEQ ID NO: 450 or SEQ ID NO: 477, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   f. a light chain CDR3 amino acid sequence of: SEQ ID NO: 451 or SEQ ID NO: 454, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; and/or   g. a combination of any two or more of (a)-(f).   
     
     
         2 . The antigen-binding protein of  claim 1 , wherein the variant sequence has at least about 80% or at least or about 85% sequence identity. 
     
     
         3 . The antigen-binding protein of  claim 2 , wherein the variant sequence has at least about 90% sequence identity or at least or about 95% sequence identity. 
     
     
         4 . The antigen-binding protein of claim of any one of  claims 1 - 3 , wherein:
 a. the antigen-binding protein binds to a human Claudin6 (CLDN6) protein (SEQ ID NO: 200);   b. the antigen-binding protein binds to Extracellular Loop 2 (EL2) of an extracellular domain (ECD) of CLDN6 and does not bind to Extracellular Loop 1 (EL1) of the ECD of CLDN6;   c. does not bind to any of Claudin3 (CLDN3), Claudin4 (CLDN4), and Claudin9 (CLDN9) and inhibits binding of a reference antibody to CLDN6 endogenously expressed by OVCA429 cells with less than about 1200 nM; or   d. a combination thereof.   
     
     
         5 . The antigen-binding protein of any one of  claims 1 - 4 , which binds to an epitope within the amino acid sequence of WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 2). 
     
     
         6 . The antigen-binding protein of  claim 5 , which binds to the amino acid sequence of TAHAIIRDFYNPL (SEQ ID NO: 3) or LVAEAQKREL (SEQ ID NO: 4) of CLDN 6. 
     
     
         7 . The antigen-binding protein of any one of  claims 1  to  6 , which does not bind to any one or more of Claudin3 (CLDN3), Claudin4 (CLDN4), and Claudin9 (CLDN9). 
     
     
         8 . The antigen-binding protein of  claim 7 , which does not bind to CLDN3. 
     
     
         9 . The antigen-binding protein of  claim 8 , which binds to CLDN6, CLDN4, and CLDN9. 
     
     
         10 . The antigen-binding protein of  claim 8 , which does not bind to CLDN9. 
     
     
         11 . The antigen-binding protein of  claim 10 , which binds to CLDN6 and CLDN4. 
     
     
         12 . The antigen-binding protein of  claim 8 , which does not bind to CLDN4. 
     
     
         13 . The antigen-binding protein of  claim 12 , which binds to CLDN6 and CLDN9. 
     
     
         14 . The antigen-binding protein of any one of the previous claims, comprising a light chain CDR1 amino acid sequence of SEQ ID NO: 449, a light chain CDR2 amino acid sequence or SEQ ID NO: 450, and a light chain CDR3 amino acid sequence or SEQ ID NO: 451 and one or two of a heavy chain CDR1 amino acid sequence of SEQ ID NO: 504, a heavy chain CDR2 amino acid sequence or SEQ ID NO: 505, and a heavy chain CDR3 amino acid sequence or SEQ ID NO: 506. 
     
     
         15 . The antigen-binding protein of claim any one of the previous claims, comprising a light chain CDR1 amino acid sequence of SEQ ID NO: 476, a light chain CDR2 amino acid sequence or SEQ ID NO: 477, and a light chain CDR3 amino acid sequence or SEQ ID NO: 454 and one or two of a heavy chain CDR1 amino acid sequence of SEQ ID NO: 507, a heavy chain CDR2 amino acid sequence or SEQ ID NO: 508, and a heavy chain CDR3 amino acid sequence or SEQ ID NO: 509. 
     
     
         16 . The antigen-binding protein of any one of  claims 11 - 15 , comprising six CDR amino acid sequences selected from the group consisting of:
 a. SEQ ID NOs: 449-451 and 504-506; and   b. SEQ ID NOs: 476, 477, 454 and 507-509.   
     
     
         17 . The antigen-binding protein of any one of  claims 11 - 16 , comprising:
 a. a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence labeled as S1-S12 in  FIG. 22 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   b. a light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence labeled as S1-S12 in  FIG. 22 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   c. both (a) and (b).   
     
     
         18 . The antigen-binding protein of  claim 17 , wherein the variant sequence has at least about 80% or at least about 85% sequence identity. 
     
     
         19 . The antigen-binding protein of  claim 18 , wherein the variant sequence has at least about 90% or at least about 95% sequence identity. 
     
     
         20 . The antigen-binding protein of any one of  claims 17 - 19 , comprising a pair of amino acid sequences:
 a. SEQ ID NOs: 389 and 490;   b. SEQ ID NOs: 389 and 491;   c. SEQ ID NOs: 389 and 492;   d. SEQ ID NOs: 389 and 493;   e. SEQ ID NOs: 389 and 494;   f. SEQ ID NOs: 389 and 495;   g. SEQ ID NOs: 383 and 496;   h. SEQ ID NOs: 383 and 497;   i. SEQ ID NOs: 383 and 498;   j. SEQ ID NOs: 383 and 499;   k. SEQ ID NOs: 383 and 500,   l. SEQ ID NOs: 383 and 501;   m. SEQ ID NOs: 383 and 503;   n. SEQ ID NOs: 389 and 502;   o. the heavy chain variable region sequence labeled as S1 in  FIG. 22  and the light chain variable region sequence labeled as S1 in  FIG. 22 ;   p. the heavy chain variable region sequence labeled as S2 in  FIG. 22  and the light chain variable region sequence labeled as S2 in  FIG. 22 ;   q. the heavy chain variable region sequence labeled as S3 in  FIG. 22  and the light chain variable region sequence labeled as S3 in  FIG. 22 ;   r. the heavy chain variable region sequence labeled as S4 in  FIG. 22  and the light chain variable region sequence labeled as S4 in  FIG. 22 ;   s. the heavy chain variable region sequence labeled as S5 in  FIG. 22  and the light chain variable region sequence labeled as S5 in  FIG. 22 ;   t. the heavy chain variable region sequence labeled as S6 in  FIG. 22  and the light chain variable region sequence labeled as S6 in  FIG. 22 ;   u. the heavy chain variable region sequence labeled as S7 in  FIG. 22  and the light chain variable region sequence labeled as S7 in  FIG. 22 ;   v. the heavy chain variable region sequence labeled as S78 in  FIG. 22  and the light chain variable region sequence labeled as S8 in  FIG. 22 ;   w. the heavy chain variable region sequence labeled as S89 in  FIG. 22  and the light chain variable region sequence labeled as S9 in  FIG. 22 ;   x. the heavy chain variable region sequence labeled as S910 in  FIG. 22  and the light chain variable region sequence labeled as S10 in  FIG. 22 ;   y. the heavy chain variable region sequence labeled as S11 in  FIG. 22  and the light chain variable region sequence labeled as S11 in  FIG. 22 ; or   z. the heavy chain variable region sequence labeled as S12 in  FIG. 22  and the light chain variable region sequence labeled as S12 in  FIG. 22 .   
     
     
         21 . The antigen-binding protein of any one of the previous claims, which is an antibody. 
     
     
         22 . The antigen-binding protein of  claim 21 , which is a monoclonal antibody. 
     
     
         23 . The antigen-binding protein of  claim 21  or  22 , which is an IgG. 
     
     
         24 . The antigen-binding protein of any one of the previous claims, which inhibits at least about 50% colony growth in a soft agar 3D proliferation assays. 
     
     
         25 . The antigen-binding protein of any one of the previous claims, which inhibits tumor growth in xenograft mice injected with human cancer cells. 
     
     
         26 . The antigen-binding protein of  claim 25 , which inhibits tumor growth of in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. 
     
     
         27 . The antigen-binding protein of  claim 26 , which inhibits at least 50% tumor growth in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells. 
     
     
         28 . An antigen-binding protein comprising
 a. a heavy chain CDR1 amino acid sequence of: SEQ ID NO: 504 or SEQ ID NO: 507, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   b. a heavy chain CDR2 amino acid sequence of: SEQ ID NOs: 505 or SEQ ID NO: 508, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   c. a heavy chain CDR3 amino acid sequence of: SEQ ID NO: 506 or SEQ ID NO: 509, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   d. a light chain CDR1 amino acid sequence of: SEQ ID NO: 449 or SEQ ID NO: 476, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   e. a light chain CDR2 amino acid sequence of: SEQ ID NO: 450 or SEQ ID NO: 477, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity;   f. a light chain CDR3 amino acid sequence of: SEQ ID NO: 451 or SEQ ID NO: 454, or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   g. a combination of any two or more of (a)-(f).   
     
     
         29 . An antigen-binding protein comprising six CDR amino acid sequences selected from the group consisting of:
 a. SEQ ID NOs: 449-451 and 504-506; and   b. SEQ ID NOs: 476, 477, 454 and 507-509.   
     
     
         30 . An antigen-binding protein comprising:
 a. a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 490-503, or a heavy chain variable region amino acid sequence labeled as S1-S12 in  FIG. 22 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   b. a light chain variable region amino acid sequence of any one of SEQ ID NOs: 380-383, 388-390, 479, and 481, or a light chain variable region amino acid sequence labeled as S1-S12 in  FIG. 22 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   c. both (a) and (b).   
     
     
         31 . The antigen-binding protein of  claim 30 , wherein the variant sequence has at least about 85% sequence identity or about 90% or about 95% sequence identity. 
     
     
         32 . An antigen-binding protein comprising a pair of amino acid sequences selected from the group consisting of:
 a. SEQ ID NOs: 389 and 490;   b. SEQ ID NOs: 389 and 491;   c. SEQ ID NOs: 389 and 492;   d. SEQ ID NOs: 389 and 493;   e. SEQ ID NOs: 389 and 494;   f. SEQ ID NOs: 389 and 495;   g. SEQ ID NOs: 383 and 496;   h. SEQ ID NOs: 383 and 497;   i. SEQ ID NOs: 383 and 498;   j. SEQ ID NOs: 383 and 499;   k. SEQ ID NOs: 383 and 500;   l. SEQ ID NOs: 383 and 501;   m SEQ ID NOs: 383 and 503;   n. SEQ ID NOs: 389 and 502;   o. the heavy chain variable region sequence labeled as S1 in  FIG. 22  and the light chain variable region sequence labeled as S1 in  FIG. 22 ;   p. the heavy chain variable region sequence labeled as S2 in  FIG. 22  and the light chain variable region sequence labeled as S2 in  FIG. 22 ;   q. the heavy chain variable region sequence labeled as S3 in  FIG. 22  and the light chain variable region sequence labeled as S3 in  FIG. 22 ;   r. the heavy chain variable region sequence labeled as S4 in  FIG. 22  and the light chain variable region sequence labeled as S4 in  FIG. 22 ;   s. the heavy chain variable region sequence labeled as S5 in  FIG. 22  and the light chain variable region sequence labeled as S5 in  FIG. 22 ;   t. the heavy chain variable region sequence labeled as S6 in  FIG. 22  and the light chain variable region sequence labeled as S6 in  FIG. 22 ;   u. the heavy chain variable region sequence labeled as S7 in  FIG. 22  and the light chain variable region sequence labeled as S7 in  FIG. 22 ;   v. the heavy chain variable region sequence labeled as S78 in  FIG. 22  and the light chain variable region sequence labeled as S8 in  FIG. 22 ;   w. the heavy chain variable region sequence labeled as S89 in  FIG. 22  and the light chain variable region sequence labeled as S9 in  FIG. 22 ;   x. the heavy chain variable region sequence labeled as S910 in  FIG. 22  and the light chain variable region sequence labeled as S10 in  FIG. 22 ;   y. the heavy chain variable region sequence labeled as S11 in  FIG. 22  and the light chain variable region sequence labeled as S11 in  FIG. 22 ; or   z. the heavy chain variable region sequence labeled as S12 in  FIG. 22  and the light chain variable region sequence labeled as S12 in  FIG. 22 .   
     
     
         33 . An antigen-binding protein comprising:
 a. a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 or 513 or in  FIG. 23  or  FIG. 25 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   b. a light chain variable region amino acid sequence set forth as SEQ ID NO: 511 or 512 or in  FIG. 24  or  FIG. 26 , or a variant sequence thereof which differs by only one or two amino acids or which has at least or about 70% sequence identity; or   c. both (a) and (b).   
     
     
         34 . The antigen-binding protein of  claim 33 , wherein the variant sequence has at least about 85% sequence identity. 
     
     
         35 . The antigen-binding protein of  claim 34 , wherein the variant sequence has at least about 90% or about 95% sequence identity. 
     
     
         36 . An antigen-binding protein comprising a pair of amino acid sequences wherein the pair comprises
 a. a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 510 and a light chain variable region amino acid sequence set forth as SEQ ID NO: 511, or a variant sequence thereof which differs by only 1-5 amino acids or which has at least or about 70% sequence identity; optionally, wherein the 1-5 amino acids which differ are as shown in  FIG. 23  for the heavy chain or  FIG. 24  for the light chain, or   b. a heavy chain variable region amino acid sequence set forth as SEQ ID NO: 513 and a light chain variable region amino acid sequence set forth as SEQ ID NO: 512, or a variant sequence thereof which differs by only 1-5 amino acids or which has at least or about 70% sequence identity; or optionally, wherein the 1-5 amino acids which differ are as shown in  FIG. 25  for the heavy chain or  FIG. 26  for the light chain.   
     
     
         37 . The antigen-binding protein of any one of the previous claims, comprising a Fc polypeptide comprising an afucosylated glycan. 
     
     
         38 . A conjugate comprising an antigen-binding protein of any one of the previous claims or described herein. 
     
     
         39 . The conjugate of  claim 38 , wherein the antigen-binding protein comprises the amino acid sequence set forth in SEQ ID NO: 387 and SEQ ID NO: 389. 
     
     
         40 . The conjugate of  claim 38 , wherein the antigen-binding protein comprises the amino acid sequence set forth in SEQ ID NO: 379 and SEQ ID NO: 383. 
     
     
         41 . The conjugate of any one of  claims 38 - 40  comprising a cytotoxic agent or a chemotherapeutic agent. 
     
     
         42 . The conjugate of  claim 41 , wherein the chemotherapeutic agent is an anti-mitotic agent which inhibits cell division by blocking tubulin polymerization. 
     
     
         43 . The conjugate of  claim 42 , wherein the anti-mitotic agent is an auristatin. 
     
     
         44 . The conjugate of  claim 43 , wherein the auristatin is MMAE. 
     
     
         45 . The conjugate of any one of  claims 38 - 44 , wherein the agent is conjugated to the antigen-binding protein via a cleavable linker. 
     
     
         46 . The conjugate of  claim 45 , wherein the cleavable linker is VC-PAB-MMAE. 
     
     
         47 . The conjugate of any one of  claims 38 - 46 , wherein the antigen-binding protein is an antibody, 
     
     
         48 . The conjugate of  claim 47 , wherein the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is an IgG antibody. 
     
     
         49 . The conjugate of  claim 47  or  48 , wherein the antibody is a human antibody, humanized antibody, or a chimeric antibody. 
     
     
         50 . The conjugate of any one of  claims 38 - 49 , wherein an average number of units of the agent conjugated per antibody is in a range of 1 to 8, preferably wherein the average number of units of the agent conjugated per antibody is in a range of 3-8. 
     
     
         51 . The conjugate of any one of  claims 38 - 50 , wherein the conjugate is a heterogeneous conjugate. 
     
     
         52 . The conjugate of any one of  claims 38 - 50 , wherein the conjugate is a homogeneous conjugate. 
     
     
         53 . The conjugate of any one of  claims 38 - 50  and  52 , wherein the agent is conjugated at a specific site of the antigen-binding protein. 
     
     
         54 . The conjugate of  claim 53 , wherein the specific site is an unpaired cysteine residue. 
     
     
         55 . The conjugate of any one of  claims 47 - 54 , wherein the conjugate comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 387 and SEQ ID NO: 389 conjugated to MC-VC-PAB-MMAE. 
     
     
         56 . The conjugate of any one of  claims 47 - 54 , wherein the conjugate comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 379 and SEQ ID NO: 383 conjugated to MC-VC-PAB-MMAE. 
     
     
         57 . A fusion protein comprising an antigen-binding protein of any one of the previous claims. 
     
     
         58 . A nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of any one of the previous claims, a conjugate of  claim 38 - 56 , or a fusion protein of  claim 57 . 
     
     
         59 . A vector comprising the nucleic acid of  claim 58 . 
     
     
         60 . A host cell comprising the nucleic acid of  claim 58  or the vector of  claim 59 . 
     
     
         61 . A method of producing an antigen-binding protein that binds to a Claudin6 (CLDN6) protein, comprising (i) culturing the host cell of  claim 60  in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of any one of the previous claims, and (ii) harvesting the antigen-binding protein from the cell culture medium. 
     
     
         62 . A method of producing a fusion protein comprising an antigen-binding protein that binds to a Claudin6 (CLDN6) protein, comprising (i) culturing the host cell of  claim 60  in a cell culture medium, wherein the host cell comprises a nucleic acid comprising a nucleotide sequence encoding a fusion protein of  claim 43 , and (ii) harvesting the fusion protein from the cell culture medium. 
     
     
         63 . A method of producing a pharmaceutical composition comprising combining an antigen-binding protein of any one of  claims 1  to  37 , a conjugate of any one of  claims 38 - 56 , a fusion protein of  claim 57 , a nucleic acid of  claim 58 , a vector of  claim 59 , a host cell of  claim 60 , or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient 
     
     
         64 . A pharmaceutical composition comprising an antigen-binding protein of any one of  claims 1  to  37 , a conjugate of any one of  claims 38 - 56 , a fusion protein of  claim 57 , a nucleic acid of  claim 58 , a vector of  claim 59 , a host cell of  claim 60 , and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         65 . A method of treating a subject with a CLDN6-expressing cancer comprising administering to the subject a pharmaceutical composition of  claim 64  in an amount effective to treat the cancer. 
     
     
         66 . A method of inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition of  claim 64  in an amount effective to inhibit tumor growth. 
     
     
         67 . A method of reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition of  claim 64  in an amount effective to reduce tumor size. 
     
     
         68 . A method of preventing the recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition of  claim 64  in an amount effective to prevent the recurrence of cancer. 
     
     
         69 . A method of treating cancer in a subject diagnosed to be a low over-expresser of CLDN6, comprising administering to the subject a pharmaceutical composition of  claim 64  in an amount effective to prevent the recurrence of cancer 
     
     
         70 . The method of any one of  claims 65 - 69  wherein the administering induces apoptosis in tumor cells. 
     
     
         71 . The method of any one of  claims 65 - 69  wherein the administering induces apoptosis in cells expressing CLDN6. 
     
     
         72 . A method of detecting Claudin6 (CLDN6) in a sample, comprising contacting the sample with an antigen-binding protein of any one of  claims 1  to  37 , a conjugate of any one of  claims 38 - 56 , or a fusion protein of  claim 57 , and assaying for an immunocomplex comprising the antigen-binding protein, conjugate or fusion protein bound to CLDN6. 
     
     
         73 . A method of diagnosing a Claudin6 (CLDN6)-positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein of any one of  claims 1  to  37 , a conjugate of any one of  claims 38 - 56 , or a fusion protein of  claim 57 , and assaying for an immunocomplex comprising the antigen-binding protein, conjugate or fusion protein bound to CLDN6.