FAPalpha -specific antibody with improved producibility

Abstract

Recombinant antibody proteins are provided that specifically bind fibroblast activation protein alpha (FAPα) and comprise framework modifications resulting in the improved producibility in host cells. The invention also relates to the use of said antibodies for diagnostic and therapeutic purposes and methods of producing said antibodies.

What is claimed is:  
     
         1 . An antibody protein having the complementary determining regions of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said antibody protein specifically binding to fibroblast activation protein, characterised in that it has framework modifications resulting in the improved producibility in host cells as compared to a chimeric antibody having the variable regions of F19 and foreign constant regions.  
     
     
         2 . An antibody protein characterized in that it has a variable light chain region and a variable heavy chain region according to  claim 1 , each joined to a human constant region.  
     
     
         3 . The antibody protein of  claim 2 , wherein said human constant region of the light chain is a human kappa constant region.  
     
     
         4 . The antibody protein of  claim 2 , wherein said human constant region of the heavy chain is a human gamma-1 constant region.  
     
     
         5 . An antibody protein according to any one of  claims 1  to  4 , characterised in that its expression level in crude media samples as determined by ELISA and/or purified antibody yield exceeds the expression levels and/or purification yield of the chimeric antibody without framework modifications by at least a factor of 10.  
     
     
         6 . An antibody protein according to any one of  claims 1  to  4 , characterised in that its expression levels in crude media samples as determined by ELISA and/or purified antibody yield exceeds the expression levels and/or purification yield of the chimeric antibody without framework modifications by at least a factor of 20.  
     
     
         7 . An antibody protein according to any one of  claims 1  to  4 , characterised in that its expression level in crude media samples as determined by ELISA and/or purified antibody yield exceeds the expression levels and/or purification yield of the chimeric antibody without framework modifications by at least a factor of 100.  
     
     
         8 . An antibody protein according to any one of  claims 1  to  7 , characterised in that it displays improved producibility in eukaryotic cells.  
     
     
         9 . The antibody protein according to  claim 8  wherein said eukaryotic cell is a Chinese hamster ovary cell (CHO cell).  
     
     
         10 . An antibody protein according to any one of  claims 1  to  9 , wherein the amino acid in Kabat position 87 of the light chain region is not asparagine.  
     
     
         11 . The antibody protein of  claim 10 , wherein the amino acid in Kabat position 87 of the light chain region is selected from aromatic or aliphatic amino acids.  
     
     
         12 . The antibody protein of  claim 11 , wherein said aromatic amino acid in Kabat position 87 of the light chain region is a tyrosine or phenylalanine.  
     
     
         13 . The antibody protein according to any one of  claims 1  to  12 , wherein the amino acid in Kabat position 36 of the light chain region is selected from aromatic amino acids.  
     
     
         14 . An antibody protein according to any one of  claims 1  to  13  that contains the variable region of the light chain as set forth in SEQ ID NO:2.  
     
     
         15 . An antibody protein of  claim 14  characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1.  
     
     
         16 . An antibody protein according to any one of  claims 1  to  13  that contains the variable region of the light chain as set forth in SEQ ID NO:6.  
     
     
         17 . An antibody protein of  claim 16  characterized in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:5.  
     
     
         18 . An antibody protein according to any one of  claims 1  to  17  containing a variable region of the heavy chain as set forth in any one of SEQ ID NOs: 8, 10, 12, 14.  
     
     
         19 . An antibody protein according to  claim 18  characterised in that the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NOs:7, 9, 11, 13.  
     
     
         20 . An antibody protein according to any one of  claims 1  to  14  containing the variable region of the light chain as set forth in SEQ ID NO:2 and the variable region of the heavy chain as set forth in SEQ ID NOs:12.  
     
     
         21 . The antibody protein of  claim 20  characterised in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1 and the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:11.  
     
     
         22 . An antibody protein according to  claim 20  or  claim 21  containing the constant region of the light chain as set forth in SEQ ID NO:20 and the constant region of the heavy chain as set forth in SEQ ID NO:22.  
     
     
         23 . An antibody protein according to any one of  claims 1  to  13 ,  18  or  19  containing the variable region of the light chain as set forth in SEQ ID NO:2 and the variable region of the heavy chain as set forth in SEQ ID NOs:8.  
     
     
         24 . The antibody protein of  claim 23  characterised in that the variable region of the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:1 and the variable region of the heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NO:7.  
     
     
         25 . A nucleotide sequence encoding an antibody protein according to any one of  claims 1  to  24 .  
     
     
         26 . A recombinant DNA vector that contains a nucleotide sequence of  claim 25 .  
     
     
         27 . The recombinant DNA vector of  claim 26 , said vector being an expression vector.  
     
     
         28 . A host cell carrying a vector according to claims  26  or  27 .  
     
     
         29 . The host cell of  claim 28 , wherein said host cell is a eukaryotic cell.  
     
     
         30 . The host cell of  claim 29 , wherein said eukaryotic host cell is a mammalian cell.  
     
     
         31 . The host cell of  claim 30 , wherein said eukaryotic host cell is a CHO or a COS cell.  
     
     
         32 . A method of producing antibody proteins according to any one of  claims 1  to  24 , said method comprising the steps of: 
 (a) cultivating a host cell according to any one of  claims 28  to  31  under conditions where said antibody protein is expressed by said host cell, and  
 (b) isolating said antibody protein.  
 
     
     
         33 . The method of  claim 32 , wherein said host cell is a mammalian cell, preferably a CHO or COS cell.  
     
     
         34 . The method of  claim 32  or  33 , wherein said host cell is cotransfected with two plasmids carrying the expression units for light and heavy chains respectively.  
     
     
         35 . An antibody protein according to any one of  claims 1  to  24 , wherein said antibody protein is conjugated to a therapeutic agent.  
     
     
         36 . The antibody protein of  claim 35 , wherein said therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, inflammatory agents and chemotherapeutic agents.  
     
     
         37 . The antibody protein of  claim 36 , wherein said radioisotope is a β-emitting radioisotope.  
     
     
         38 . The antibody protein of  claim 37 , wherein said radioisotope is selected from the group consisting of  186 Rhenium,  188 Rhenium,  131 Iodine and  90 Yttrium.  
     
     
         39 . An antibody protein according to any one of  claims 1  to  24 , characterised in that it is labelled.  
     
     
         40 . The antibody protein of  claim 39 , wherein said label is a detectable marker.  
     
     
         41 . The antibody protein of  claim 40 , wherein the detectable marker is a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.  
     
     
         42 . An antibody protein according to any one of  claims 1  to  24  conjugated to an imageable agent.  
     
     
         43 . The antibody protein of  claim 42 , wherein the imageable agent is a radioisotope.  
     
     
         44 . The antibody protein of  claim 43 , wherein said radioisotope is a γ-emitting radioisotopes.  
     
     
         45 . The antibody protein of  claim 44 , wherein said radioisotope is  125 I.  
     
     
         46 . A pharmaceutical composition containing an antibody protein according to any one of  claims 1  to  24  and a pharmaceutically acceptable carrier.  
     
     
         47 . A pharmaceutical composition containing an antibody protein according to any one of  claims 35  to  38  and a pharmaceutically acceptable carrier.  
     
     
         48 . A pharmaceutical composition containing an antibody protein according to any one of  claims 42  to  45  and a pharmaceutically acceptable carrier.  
     
     
         49 . The pharmaceutical composition of  claims 46  to  48 , for use in the treatment or imaging of tumors, wherein said tumors are associated with activated stromal fibroblasts, preferably wherein said tumors are tumors selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.  
     
     
         50 . Use of an antibody protein according to anyone of  claims 1  to  24  for the treatment of cancer.  
     
     
         51 . Use of an antibody protein according to anyone of  claims 35  to  38  for the treatment of cancer.  
     
     
         52 . Use of an antibody protein according to anyone of  claims 42  to  45  imaging activated stromal fibroblasts.  
     
     
         53 . Use of an antibody protein according to anyone of  claims 39  to  41  for detecting the presence of activated stromal fibroblasts in a sample.  
     
     
         54 . A method of treating tumors, wherein the tumor is associated with activated stromal fibroblasts capable of specifically forming a complex with antibody proteins according to any one of  claims 1  to  24  or  35  to  38 , which comprises contacting the tumor with an amount of said antibody proteins effective to treat the tumor.  
     
     
         55 . The method of  claim 54 , wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.  
     
     
         56 . The method of  claim 54 , wherein the contacting is effected in vitro.  
     
     
         57 . The method of  claim 54 , wherein the contacting is effected in vivo.  
     
     
         58 . A method of detecting the presence of activated stromal fibroblasts in wound healing, inflammation or a tumor, characterised in that 
 (a) a sample, possibly containing activated stromal fibroblasts, is contacted with an antibody protein according to any one of  claims 1  to  24  or  39  to  41  under conditions suitable for the formation of a complex between said antibody and antigen,    (b) detecting the presence of said complex, thereby detecting the presence of activated stromal fibroblasts in wound healing, inflammation or a tumor.    
     
     
         59 . The method of  claim 58 , wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.  
     
     
         60 . The method of  claim 58  or  59 , wherein the antibody protein is a protein according to any one of  claims 39  to  41 .  
     
     
         61 . A method of imaging the presence of activated stromal fibroblasts in a healing wound, inflamed skin or a tumor, in a human patient, characterised in that 
 (a) an antibody protein according to any one of  claims 1  to  24  conjugated to an imageable agent is administered to a human patient under conditions suitable for the formation of an antibody-antigen complex,    (b) imaging any complex formed in this manner.    
     
     
         62 . The method of  claim 61 , wherein the tumor is a tumor having cancer cells selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.  
     
     
         63 . A method of detecting tumor-stroma, characterised in that 
 (a) a suitable sample is contacted with an antibody protein according to any one of  claims 1  to  24 , under conditions suitable for the formation of an antibody-antigen complex,    (b) detecting the presence of any complex so formed,    (c) relating the presence of said complex to the presence of tumor-stroma.    
     
     
         64 . The method of  claim 62 , wherein said antibody is labelled with a detectable marker.  
     
     
         65 . A method of imaging tumor-stroma in a human patient, which comprises 
 (a) administering to the patient an antibody protein according to any one of  claims 42  to  45 , under conditions suitable for the formation of an antibody-antigen complex,    (b) imaging any complex so formed, and thereby imaging the presence of tumor-stroma in a human patient.    
     
     
         66 . An antibody protein containing an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         67 . An antibody protein according to  claim 66  further containing an amino acid sequence as set forth in SEQ ID NO:12.  
     
     
         68 . An antibody protein according to  claim 66  or  67  further containing an amino acid sequence as set forth in SEQ ID NO:20 and an amino acid sequence as set forth in SEQ ID NO:22.  
     
     
         69 . A DNA molecule coding for an antibody protein according to any one of  claims 66  to  68 .  
     
     
         70 . A host cell carrying a DNA molecule according to  claim 69 .  
     
     
         71 . A method of producing an antibody protein of any one of  claims 66  to  68 , said method comprising the steps of 
 (a) cultivating the host cell of  claim 70  under conditions where said antibody protein is expressed by said host cell, and  
 (b) isolating said protein.  
 
     
     
         72 . An antibody protein according to any one of  claims 66  to  68  which is conjugated to a radioisotope, preferably  131 I,  125 I,  186 Re,  188 Re, or  90 Y.  
     
     
         73 . A pharmaceutical composition comprising an antibody protein according to any one of  claims 66  to  68 , or  72 , and a pharmaceutically acceptable carrier.