Anti-S100A4 Humanized Antibodies, Uses and Methods

Abstract

Herein are provided isolated, humanized, anti-S100A4 antibody molecules and methods of producing said antibodies. Also provided are nucleic acids, vectors, isolated host cells and pharmaceutical compositions containing the antibody, and methods of treatment comprising administering said antibody.

1 . An isolated antibody comprising:
 a) a heavy chain variable (VH) region comprising:
 i. a heavy chain complementarity-determining region 1 (CDR-H1) comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; 
 ii. a heavy chain complementarity-determining region 2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and 
 iii. a heavy chain complementarity-determining region 3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6; 
   and
 b) a light chain variable (VL) region comprising:
 i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10; 
 ii. a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11; and 
 iii. a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12; 
 
 wherein the VH region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and a variant of any one of SEQ ID NO:s 13 to 17, wherein any one amino acid not part of the CDR sequences as defined by SEQ ID NO:s 1 to 6 has been altered for another amino acid, with the proviso that no more than 5 amino acids have been so altered, for example wherein 5, 4, 3, 2, or 1 amino acid has been so altered in each amino acid sequence,
 and/or wherein the VL region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and a variant of any one of SEQ ID NO:s 18 to 22, wherein any one amino acid not part of the CDR sequences as defined by SEQ ID NO:s 7 to 12 has been altered for another amino acid, with the proviso that no more than 5 amino acids have been so altered, for example wherein 5, 4, 3, 2, or 1 amino acid has been so altered in each amino acid sequence. 
 
   
     
     
         2 . The antibody according to  claim 1 , wherein the heavy chain variable (VH) region comprises:
 (a) a heavy chain complementarity-determining region 1 (CDR-H1) comprising or consisting of the amino acid sequence of SEQ ID NO: 1;   (b) a heavy chain complementarity-determining region 2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and   (c) a heavy chain complementarity-determining region 3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 3;   and wherein the light chain variable (VL) region comprising a light chain variable (VL) region comprises:   i. a light chain complementarity-determining region 1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO: 7;   ii. a light chain complementarity-determining region 2 (CDR-L2) comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and   iii. a light chain complementarity-determining region 3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ ID NO: 9.   
     
     
         3 . The antibody according to  claim 1 , wherein the heavy chain variable (VH) region comprises:
 (a) a heavy chain complementarity-determining region 1 (CDR-H1) comprising or consisting of the amino acid sequence of SEQ ID NO: 4;   (b) a heavy chain complementarity-determining region 2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and   (c) a heavy chain complementarity-determining region 3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 6;   and wherein the light chain variable (VL) region comprising a light chain variable (VL) region comprises:   i. a light chain complementarity-determining region 1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO: 10;   ii. a light chain complementarity-determining region 2 (CDR-L2) comprising or consisting of the amino acid sequence of SEQ ID NO: 11; and   iii. a light chain complementarity-determining region 3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ ID NO: 12.   
     
     
         4 . An isolated antibody, comprising:
 (a) a heavy chain variable (VH) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and a variant of any one of SEQ ID NO:s 13 to 17, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 5 amino acids have been so altered, for example wherein 5, 4, 3, 2, or 1 amino acid has been so altered in each amino acid sequence,   and   (b) a light chain variable (VL) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and a variant of any one of SEQ ID NO:s 18 to 22, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 5 amino acids have been so altered, for example wherein 5, 4, 3, 2, or 1 amino acid has been so altered in each amino acid sequence.   
     
     
         5 . The antibody according to any one of the preceding claims, wherein the antibody is a bispecific antibody. 
     
     
         6 . The antibody according to any one of the preceding claims, wherein the antibody is selected from the group consisting of a full-length antibody, a Fab fragment, a F(ab′) fragment, a F(ab′) 2  fragment, a scFv, a diabody, and a triabody. 
     
     
         7 . The antibody according to any one of the preceding claims, wherein the antibody is an immunoglobulin subclass antibody selected from the group consisting of human IgG1, human IgG2, human IgG3 and human IgG4. 
     
     
         8 . The antibody according to any one of the preceding claims, wherein the antibody is a human IgG4 immunoglobulin subclass antibody. 
     
     
         9 . The antibody according to  claim 8 , wherein the antibody comprises a human heavy chain constant (CH) region comprising or consisting of the sequence as set forth in SEQ ID NO: 56 or a variant thereof with at least 80% sequence identity thereto, and a human light chain constant (CL) region comprising or consisting of the sequence as set forth in SEQ ID NO: 57 or a variant thereof with at least 80% sequence identity thereto. 
     
     
         10 . The antibody according to any one of the preceding claims, wherein the antibody comprises an Fc domain with a mutated human IgG constant region. 
     
     
         11 . The antibody of  claim 10 , wherein the antibody comprises a mutant human IgG4 heavy chain constant region. 
     
     
         12 . The antibody of  claim 11 , wherein the mutant human IgG4 heavy chain constant region comprises an S228P substitution, numbering according to EU numbering. 
     
     
         13 . The antibody according to any one of the preceding claims, wherein the antibody comprises:
 (a) an amino acid substitution of the amino acid at position 40 of the VH region of any one of SEQ ID NO:s 13 to 17 to phenylalanine, an amino acid substitution of the amino acid at position 43 of the VH region of any one of SEQ ID NO:s 13 to 17 to serine, and/or an amino acid substitution of the amino acid at position 44 of the VH region of any one of SEQ ID NO:s 13 to 17 to lysine; and/or   (b) an amino acid substitution of the amino acid at position 42 of the VL region of any one of SEQ ID NO:s 18 to 22 to glycine, an amino acid substitution of the amino acid at position 43 of the VL region of any one of SEQ ID NO:s 18 to 22 to threonine, and/or an amino acid substitution of the amino acid at position 44 of the VL region of any one of SEQ ID NO:s 18 to 22 to leucine.   
     
     
         14 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of a VH region selected from the group consisting of SEQ ID NO:s 24 to 38. 
     
     
         15 . The antibody according to any one of the preceding claims, wherein the VL region comprises or consists of a VL region selected from the group consisting of SEQ ID NO:s 39 to 53. 
     
     
         16 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of SEQ ID NO: 24 (VH1_H40Phe) and the VL region comprises or consists of SEQ ID NO: 47 (VL3_L44Leu). 
     
     
         17 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of SEQ ID NO: 24 (VH1_H40Phe) and the VL region comprises or consists of SEQ ID NO: 20 (VL3). 
     
     
         18 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of SEQ ID NO: 26 (VH1_H44Lys) and the VL region comprises or consists of SEQ ID NO: 20 (VL3). 
     
     
         19 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of SEQ ID NO: 13 (VH1) and the VL region comprises or consists of SEQ ID NO: 47 (VL3_L44Leu). 
     
     
         20 . The antibody according to any one of the preceding claims, wherein the VH region comprises or consists of SEQ ID NO: 13 (VH1) and the VL region comprises or consists of SEQ ID NO: 20 (VL3). 
     
     
         21 . The antibody according to any one of the preceding claims, wherein the antibody is PEGylated. 
     
     
         22 . The antibody according to any one of the preceding claims, wherein the antibody is capable of binding to native conformation S100A4 protein. 
     
     
         23 . The antibody according to any one of the preceding claims, wherein the antibody is capable of binding to dimeric, oligomeric and/or multimeric forms of S100A4 protein. 
     
     
         24 . The antibody according to any one of the preceding claims, wherein the antibody is capable of binding to a polypeptide having at least 80% sequence identity to amino acids 1 to 101 as set out in SEQ ID NO: 23. 
     
     
         25 . The antibody according to any one of the preceding claims, wherein the antibody is capable of binding to human S100A4 polypeptide of SEQ ID NO: 23. 
     
     
         26 . The antibody according to any one of the preceding claims, wherein administration of the antibody to a subject results in reduced or no S100A4-dependent FcγIIaR clustering and activation. 
     
     
         27 . The antibody according to any one of the preceding claims, wherein administration of the antibody to a subject results in reduced or no increase in TNFα. 
     
     
         28 . The antibody according to any one of the preceding claims, wherein the antibody is capable of reducing S100A4-mediated fibrosis. 
     
     
         29 . The antibody according to any one of the preceding claims, wherein the antibody is capable of neutralizing a biological activity of S100A4. 
     
     
         30 . The antibody according to  claim 29 , wherein the biological activity of S100A4 is in promoting tumor progression and/or in inducing tumor metastasis. 
     
     
         31 . The antibody according to  claim 30 , wherein the antibody is capable of inhibiting the biological activity of S100A4 in promoting tumor progression and/or in inducing tumor metastasis and/or in inflammation. 
     
     
         32 . The antibody according to any one of the preceding claims, wherein the antibody is capable of inhibiting T-cell recruitment mediated by S100A4. 
     
     
         33 . The antibody according to any one of the preceding claims, wherein the antibody is capable of inhibiting macrophage recruitment and/or infiltration mediated by S100A4. 
     
     
         34 . The antibody according to any one of the preceding claims, wherein the antibody is capable of inhibiting the biological activity of S100A4 protein in stimulating cell invasion. 
     
     
         35 . The antibody according to  claim 34 , wherein the biological activity of S100A4 protein in stimulating cell invasion is determined in a 3D Matrigel matrix assay or a T cell invasion assay where S100A4 stimulates T cell infiltration into a fibroblasts monolayer or wherein the biological activity of S100A4 in inducing tumor metastasis is determined in an in vivo mouse xenograft model. 
     
     
         36 . An isolated nucleic acid molecule encoding the antibody according to any one of  claims 1  to  35 . 
     
     
         37 . The nucleic acid molecule according to  claim 36 , wherein the nucleic acid molecule is codon-optimized for the cell of expression. 
     
     
         38 . An expression vector comprising the nucleic acid molecule encoding an anti-S100A4 antibody molecule according to  claim 36  or  37 . 
     
     
         39 . The expression vector according to  claim 38 , wherein the nucleic acid molecule is operably linked to control sequences to direct its expression. 
     
     
         40 . An isolated host cell comprising the isolated nucleic acid molecule according to  claim 36 , or the expression vector according to any one of  claim 38  or  39 . 
     
     
         41 . The isolated host cell of  claim 40 , wherein the cell is a human cell. 
     
     
         42 . The isolated host cell of  claim 40 , wherein the cell is Chinese hamster ovary (CHO) cell. 
     
     
         43 . A method of producing an anti-S100A4 antibody molecule, the method comprising culturing the host cell according to any one of  claims 40  to  42  under conditions wherein the antibody is expressed. 
     
     
         44 . The method according to  claim 43 , further comprising purifying the antibody and isolating the anti-S100A4 antibody thus produced. 
     
     
         45 . A pharmaceutical composition comprising the antibody according to any one of  claims 1  to  35 , the nucleic acid molecule according to  claim 36  or  37 , the expression vector according to  claim 38  or  39 , and/or the host cell according to any one of  claims 40  to  42 , and a pharmaceutically acceptable diluent, carrier and/or excipient. 
     
     
         46 . A method of treatment of an individual with an S100A4-mediated condition comprising administering the antibody according to any one of  claims 1  to  35 , the nucleic acid or expression vector of any one of  claims 36  to  39 , or the host cell according to any one of  claims 40  to  42  to an individual in need thereof. 
     
     
         47 . The method of treatment according to  claim 46 , wherein the S100A4-mediated condition is cancer or an inflammatory condition. 
     
     
         48 . The method of treatment according to  claim 47 , wherein the cancer is metastatic cancer or the inflammatory condition is psoriasis, rheumatoid arthritis or inflammatory myopathy. 
     
     
         49 . The method of treatment according to  claim 47  or  48 , wherein the cancer is gastric cancer, pancreatic cancer, colorectal cancer, thyroid cancer, breast cancer, squamous cell carcinoma, non-small cell lung cancer, prostate cancer, lung cancer, head and neck cancer, brain cancer (including glioblastoma multiforme), renal cell carcinoma (including clear cell renal carcinoma), melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, colon cancer, esophageal cancer, ovary cancer, cervical cancer or hepatoma. 
     
     
         50 . A method for diagnosis or prognosis of an S100A4-related condition in an individual, the method comprising
 (a) contacting a biological sample from the individual with an anti-S100A4 antibody according to any one of  claims 1  to  35  which is capable of binding to S100A4 polypeptide present in the sample; and   (b) determining the presence and/or amount of the complex formed between the antibody molecule and the S100A4 polypeptide.   
     
     
         51 . The method according to  claim 50 , wherein the biological sample is blood, plasma, serum, tissue samples, interstitial tissue fluids, cerebrospinal fluid, synovia or saliva.