Antibodies

Abstract

The present invention relates to antibodies binding to B7H4, including bispecific antibodies binding to B7H4 and CD3. The invention further provides pharmaceutical compositions comprising the antibodies and use of the antibodies for therapeutic and diagnostic procedures, in particular in cancer therapy.

1 - 78 . (canceled) 
     
     
         79 . An antibody comprising a first binding arm which comprises a first antigen-binding region which binds to human CD3, and a second binding arm which comprises a second antigen-binding region which binds to human B7H4, wherein the first and second antigen-binding regions comprise a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH and VL regions are humanized and/or human. 
     
     
         80 . The antibody of  claim 79 , wherein the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18, 19, and 20, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, the sequence GTN, and SEQ ID NO: 24, respectively, and   (b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18, 19, and 21, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, the sequence GTN, and SEQ ID NO: 24, respectively.   
     
     
         81 . The antibody of  claim 79 , wherein the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (a) a VH region comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22; and   (b) a VH region comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22.   
     
     
         82 . The antibody of  claim 79 , wherein the first antigen-binding region comprises:
 a VH region comprising the amino acid sequence of SEQ ID NO: 16, with the exception of an amino acid substitution at a position selected from the group consisting of T31, N57, H101, G105, S110, and Y114, such as T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, and Y114V, wherein the numbering of amino acid positions is according to the sequence of SEQ ID NO: 16, and   a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, the sequence GTN, and SEQ ID NO: 24, respectively.   
     
     
         83 . The antibody of  claim 79 , wherein the second antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 27, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively;   (b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 30, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively;   (c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 37, 38, and 39, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 41, the sequence DTS, and SEQ ID NO: 42, respectively;   (d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 44, 45, and 46, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 48, the sequence YTS, and SEQ ID NO: 49, respectively;   (e) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 51, 52, and 53, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 55, the sequence GAS, and SEQ ID NO: 56, respectively;   (f) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 32, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively; and   (g) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 66, 67, and 68, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70, the sequence GAS, and SEQ ID NO: 71, respectively.   
     
     
         84 . The antibody of  claim 79 , wherein the second antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (a) a VH region comprising the amino acid sequence of SEQ ID NO: 25, and a VL region comprising the amino acid sequence of SEQ ID NO: 33;   (b) a VH region comprising the amino acid sequence of SEQ ID NO: 29, and a VL region comprising the amino acid sequence of SEQ ID NO: 33;   (c) a VH region comprising the amino acid sequence of SEQ ID NO: 36, and a VL region comprising the amino acid sequence of SEQ ID NO: 40;   (d) a VH region comprising the amino acid sequence of SEQ ID NO: 43, and a VL region comprising the amino acid sequence of SEQ ID NO: 47;   (e) a VH region comprising the amino acid sequence of SEQ ID NO: 50, and a VL region comprising the amino acid sequence of SEQ ID NO: 54;   (f) a VH region comprising the amino acid sequence of SEQ ID NO: 31, and a VL region comprising the amino acid sequence of SEQ ID NO: 33; and   (g) a VH region comprising the amino acid sequence of SEQ ID NO: 65, and a VL region comprising the amino acid sequence of SEQ ID NO: 69.   
     
     
         85 . The antibody of  claim 79 , wherein:
 (a) the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (i) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18, 19, and 20, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, the sequence GTN, and SEQ ID NO: 24, respectively, and 
 (ii) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18, 19, and 21, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, the sequence GTN, and SEQ ID NO: 24, respectively, and 
   (b) the second antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (i) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 27, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively; 
 (ii) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 30, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively; 
 (iii) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 37, 38, and 39, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 41, the sequence DTS, and SEQ ID NO: 42, respectively; 
 (iv) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 44, 45, and 46, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 48, the sequence YTS, and SEQ ID NO: 49, respectively; 
 (v) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 51, 52, and 53, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 55, the sequence GAS, and SEQ ID NO: 56, respectively; 
 (vi) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 26, 32, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively; and 
 (vii) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 66, 67, and 68, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70, the sequence GAS, and SEQ ID NO: 71, respectively. 
   
     
     
         86 . The antibody of  claim 79 , wherein:
 (a) the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (i) a VH region comprising the amino acid sequence of SEQ ID NO: 16, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and 
 (ii) a VH region comprising the amino acid sequence of SEQ ID NO: 17, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and 
   (b) the second antigen-binding region comprises a VH region and a VL region selected from the group consisting of:
 (i) a VH region comprising the amino acid sequence of SEQ ID NO: 25, and a VL region comprising the amino acid sequence of SEQ ID NO: 33; 
 (ii) a VH region comprising the amino acid sequence of SEQ ID NO: 29, and a VL region comprising the amino acid sequence of SEQ ID NO: 33; 
 (iii) a VH region comprising the amino acid sequence of SEQ ID NO: 36, and a VL region comprising the amino acid sequence of SEQ ID NO: 40; 
 (iv) a VH region comprising the amino acid sequence of SEQ ID NO: 43, and a VL region comprising the amino acid sequence of SEQ ID NO: 47; 
 (v) a VH region comprising the amino acid sequence of SEQ ID NO: 50, and a VL region comprising the amino acid sequence of SEQ ID NO: 54; 
 (vi) a VH region comprising the amino acid sequence of SEQ ID NO: 31, and a VL region comprising the amino acid sequence of SEQ ID NO: 33; and 
 (vii) a VH region comprising the amino acid sequence of SEQ ID NO: 65, and a VL region comprising the amino acid sequence of SEQ ID NO: 69. 
   
     
     
         87 . The antibody of  claim 85 , wherein the first binding arm comprises a first heavy chain and the second binding arm comprises a second heavy chain, each of the first and second heavy chains comprising at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain, at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in the second heavy chain, at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, wherein the substitutions in the first and second heavy chains are not in the same positions, and wherein the amino acid positions are numbered according to EU numbering. 
     
     
         88 . The antibody of  claim 87 , wherein (a) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain, or (b) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in the first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in the second heavy chain. 
     
     
         89 . The antibody of  claim 85 , wherein the first binding arm comprises a first heavy chain and the second binding arm comprises a second heavy chain, wherein in both first and second heavy chains,
 (a) the amino acid residues at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively,   (b) the amino acid residue at the position corresponding to position D265 in a human IgG1 heavy chain is A, or   (c) the amino acid residues at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively,   wherein the amino acid positions are numbered according to EU numbering.   
     
     
         90 . The antibody of  claim 85 , wherein (a) the first binding arm comprises a lambda light chain constant region, such as a lambda light chain constant region comprising the amino acid sequence of SEQ ID NO: 64, and the second binding arm comprises a kappa light chain constant region, such as a kappa light chain constant region comprising the amino acid sequence of SEQ ID NO: 63, or (b) the first binding arm comprises a kappa light chain constant region, such as a kappa light chain constant region comprising the amino acid sequence of SEQ ID NO: 63, and the second binding arm comprises a lambda light chain constant region, such as a lambda light chain constant region comprising the amino acid sequence of SEQ ID NO: 64. 
     
     
         91 . The antibody of  claim 90 , wherein (a) the first binding arm comprises a heavy chain comprising an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 60, and the second binding arm comprises a heavy chain comprising an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 61, or (b) the first binding arm comprises a heavy chain comprising an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 61 and the second binding arm comprises a heavy chain comprising an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 60, optionally wherein the IgG1 heavy chain constant regions do not comprise a terminal lysine. 
     
     
         92 . A pharmaceutical composition comprising the antibody of  claim 85 , and a pharmaceutically acceptable carrier. 
     
     
         93 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of  claim 85 . 
     
     
         94 . The method of  claim 93 , wherein the cancer is selected from the group consisting of: lung cancer (such as lung squamous cell carcinoma), NSCLC (ADC or SQCC), stomach cancer, pancreas cancer (such as pancreatic adenocarcinoma), cholangiocarcinoma, bladder cancer (such as bladder urothelial carcinoma), cervical cancer, head and neck cancer, breast cancer (such as breast invasive carcinoma), ovarian cancer (such as ovarian serous cystadenocarcinoma), and uterine cancer (such as uterine carcinosarcoma or uterine corpus endometrial carcinoma). 
     
     
         95 . A nucleic acid, or set of nucleic acids, encoding the antibody of  claim 85 . 
     
     
         96 . An expression vector comprising the nucleic acid, or set of nucleic acids, of  claim 95 . 
     
     
         97 . A host cell comprising (a) the nucleic acid, or set of nucleic acids, of  claim 95 , or (b) an expression vector comprising the nucleic acid, or set of nucleic acids. 
     
     
         98 . A method of producing an antibody which binds to human CD3 and human B7H4, the method comprising:
 (a) providing an antibody which binds to human CD3,   (b) providing an antibody which binds to human B7H4,   (c) incubating the antibody which binds to human CD3 together with the antibody which binds to human B7H4 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide-bond isomerization, and   (d) obtaining an antibody which binds to human CD3 and human B7H4,   wherein the antibody which binds to human CD3 and the antibody which binds to human B7H4 comprise the CDR sequences of the first and second antigen-binding regions, respectively, of the antibody of  claim 85 .   
     
     
         99 . A kit-of-parts, such as a kit for use as a companion diagnostic or for identifying within a population of patients those patients which have a propensity to respond to treatment, comprising the antibody of  claim 85 , and instructions for use. 
     
     
         100 . A bispecific antibody comprising a first binding arm comprising a first heavy chain and a first light chain and a second binding arm comprising a second heavy chain and a second light chain, wherein the first binding arm binds to human CD3 and the second binding arm binds to human B7H4, and wherein:
 (a) the first heavy chain comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 17, and the first light chain comprises a variable light chain (VL) region comprising the amino acid sequence of SEQ ID NO: 22, and   (b) the second heavy chain comprises a VH region comprising the amino acid sequence of SEQ ID NO: 29, and the second light chain comprises a VL region comprising the amino acid sequence of SEQ ID NO: 33,   wherein the first heavy chain and second heavy chain comprise heavy chain constant regions comprising the amino acid sequences set forth in SEQ ID NOs: 60 and 61, respectively, and   wherein the first light chain and second light chain comprise light chain constant regions comprising the amino acid sequences set forth in SEQ ID NOs: 64 and 63, respectively, and   wherein optionally the heavy chain constant regions do not comprise a terminal lysine.   
     
     
         101 . An antibody which binds to human B7H4, wherein the antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region selected from the group consisting of:
 (a) a VH region comprising the CDR1, CDR2, and CDR3 sequences within SEQ ID NO: 25, and a VL region comprising the CDR1, CDR2, and CDR3 sequences within SEQ ID NO: 33;   (b) a VH region comprising the CDR1, CDR2, and CDR3 sequences within SEQ ID NO: 29, and a VL region comprising the CDR1, CDR2, and CDR3 sequences within SEQ ID NO: 33;   (c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26, 27, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively;   (d) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 26, 30, and 28, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, the sequence GAS, and SEQ ID NO: 35, respectively;   (e) a VH region comprising the amino acid sequence of SEQ ID NO: 25, and a VL region comprising the amino acid sequence of SEQ ID NO: 33; and   (f) a VH region comprising the amino acid sequence of SEQ ID NO: 29, and a VL region comprising the amino acid sequence of SEQ ID NO: 33,   wherein optionally the antibody comprises an Fc region in which the amino acid residues at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively, wherein the amino acid positions are numbered according to EU numbering, and   wherein optionally the Fc region further comprises a K409R or F405L mutation.   
     
     
         102 . The antibody of  claim 101 , wherein the antibody comprises:
 (a) a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 60 or 61, and/or   (b) a light chain constant region comprising the amino acid sequence of SEQ ID NO: 63 or 64, and   optionally wherein the heavy chain constant regions do not comprise a terminal lysine.   
     
     
         103 . A pharmaceutical composition comprising the antibody of  claim 101  and a pharmaceutically acceptable carrier. 
     
     
         104 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of  claim 101 . 
     
     
         105 . The method of  claim 104 , wherein the cancer is selected from the group consisting of: lung cancer (such as lung squamous cell carcinoma), NSCLC (ADC or SQCC), stomach cancer, pancreas cancer (such as pancreatic adenocarcinoma), cholangiocarcinoma, bladder cancer (such as bladder urothelial carcinoma), cervical cancer, head and neck cancer, breast cancer (such as breast invasive carcinoma), ovarian cancer (such as ovarian serous cystadenocarcinoma), and uterine cancer (such as uterine carcinosarcoma or uterine corpus endometrial carcinoma). 
     
     
         106 . A nucleic acid, or set of nucleic acids, encoding the VH region and/or VL region of the antibody of  claim 101 . 
     
     
         107 . An expression vector comprising the nucleic acid, or set of nucleic acids, of  claim 106 . 
     
     
         108 . A host cell comprising (a) the nucleic acid, or set of nucleic acids, of  claim 107 , or (b) an expression vector comprising the nucleic acid, or set of nucleic acids.