Novel anti cxcr4 antibodies and their use for the treatment of cancer

Abstract

The present invention relates to a novel isolated antibody, or the derived compounds or functional fragments of same, capable of binding to CXCR4 but also of inducing conformational changed of the CXCR4 homodimers and/or heterodimers. More particularly, the present invention relates to the 414H5 and 515H7 antibodies, specific to the CXCR4 protein, as well as their use for the treatment of cancer. Pharmaceutical compositions composed of such antibodies and a process for the selection of such antibodies are also covered.

1 . A method for detecting in vitro the presence of a CXCR4 expressing tumor cell in a subject, the method comprising the steps of (a) contacting a sample from the subject with an anti-CXCR4 antibody, or a derived compound, or functional fragment thereof, and (b) detecting the binding of the anti-CXCR4 antibody, derived compound, or functional fragment thereof with the sample, wherein the anti-CXCR4 antibody is selected from:
 a) an antibody that comprises i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 1, 2, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 4, 5, and 6, respectively;   b) an antibody that comprises, according to IMGT numbering, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 1, 2, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 7, 5, and 8, respectively;   c) an antibody that comprises, according to Kabat numbering, i) a light chain comprising the CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 9, 10, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 11, 12, and 6, respectively;   d) an antibody which comprises i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 40, 2, and 41, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 42, 5, and 43, respectively;   e) an antibody which comprises, according to IMGT numbering, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 40, 2, and 41, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 44, 5, and 45, respectively;   
       and
 f) an antibody which comprises, according to Kabat numbering, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 46, 47, and 41, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 48, 49, and 43, respectively. 
 
     
     
         2 . The method according to  claim 1 , wherein the antibody is a murine antibody and is selected from:
 a) an antibody that comprises i) a light chain variable domain comprising the sequence of SEQ ID No. 13 and ii) a heavy chain variable domain comprising the sequence of SEQ ID No. 14;   and   b) an antibody that comprises i) a light chain variable domain comprising the sequence of SEQ ID No. 50 and ii) a heavy chain variable domain comprising the sequence of SEQ ID No. 51.   
     
     
         3 . The method according to  claim 1 , wherein the antibody is a chimeric antibody and is selected from:
 a) an antibody that comprises i) a light chain variable domain comprising the sequence of SEQ ID No. 64 and ii) a heavy chain variable domain comprising the sequence of SEQ ID No. 65;   and   b) an antibody that comprises i) a light chain variable domain comprising the sequence of SEQ ID No. 66 and ii) a heavy chain variable domain comprising the sequence of SEQ ID No. 67.   
     
     
         4 . The method according to  claim 1 , wherein the antibody is a humanized antibody that comprises i) a light chain variable domain comprising a sequence selected from SEQ ID Nos. 76 to 82 and ii) a heavy chain variable domain comprising a sequence selected from SEQ ID Nos. 72 to 75. 
     
     
         5 . The method according to  claim 1 , wherein the antibody is a humanized antibody that comprises i) a light chain comprising a sequence selected from SEQ ID Nos. 87 to 93 and ii) a heavy chain comprising a sequence selected from SEQ ID Nos. 83 to 86. 
     
     
         6 . The method according to  claim 1 , wherein the sample is a biological fluid from the subject, a tissue biopsy from the subject, cells from the subject adapted to tissue culture, or a subcellular fraction of cells from the subject. 
     
     
         7 . A method of determining in vitro the expression level of CXCR4 in a tumor from a subject, wherein the method comprises the steps of (a′) detecting the presence of a CXCR4 expressing tumor cell according to  claim 1 , and (b′) quantifying the level of antibody binding to CXCR4 in the sample. 
     
     
         8 . The method according to  claim 7 , wherein the CXCR4 expression level is measured by a method selected from Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence, immunohistochemistry (IHC), and radio-immunoassay (RIA). 
     
     
         9 . The method according to  claim 8 , wherein the CXCR4 expression level is measured by IHC. 
     
     
         10 . A method of diagnosing in vitro a CXCR4 expressing tumor in a subject, wherein the method comprises (i) determining the expression level of CXCR4 in a tumor sample from the subject by the method of  claim 6 , (ii) comparing the CXCR4 expression level of the tumor sample of step (i) with a reference expression level of CXCR4 from normal tissue, and (iii) diagnosing the CXCR4-expressing tumor based on the difference between the CXCR4 expression level of the tumor sample and that of the normal tissue. 
     
     
         11 . A method of determining in vitro the prognosis for developing a CXCR4-expressing tumor in a subject, wherein the method comprises (i) determining the expression level of CXCR4 in a tumor sample from the subject by the method of claim  6 , (ii) comparing the CXCR4 expression level of the tumor sample of step (i) with a reference expression level of CXCR4 from normal tissue, and (iii) determining the prognosis for developing a CXCR4-expressing tumor, wherein the level of CXCR4 expression indicates the degree of susceptibility to developing a CXCR4-expressing tumor 
     
     
         12 . A method of determining in vitro the CXCR4 status of a tumor of a subject, wherein the method comprises the steps of (1) determining the expression level of CXCR4 in the tumor cells of the tumor according to  claim 6 , (2) scoring the tumor for CXCR4 expression level, and (3) comparing the scoring to that obtained from a normal control sample. 
     
     
         13 . The method according to  claim 12 , wherein the scoring comprises using a scale based on two parameters, which are the intensity of the CXCR4 staining and the percentage of CXCR4 positive cells. 
     
     
         14 . The method according to  claim 13 , wherein the scale is a scale from 0 to 3 + , wherein no membranous CXCR4 reactivity in tumor cells is scored as 0, and strong complete CXCR4 reactivity in more than 10% of tumor cells is scored as 3 + . 
     
     
         15 . The method according to  claim 13 , wherein the scale is a scale of 0 to 3 wherein no membranous CXCR4 reactivity in tumor cells is scored as 0; faint perceptible CXCR4 membranous reactivity in more than 10% of the tumor cells is scored as 1 + ; weak to moderate complete membranous CXCR4 reactivity in more than 10% of the tumor cells is scored as 2 + ; and strong complete CXCR4 reactivity in more than 10% of tumor cells is scored as 3 + . 
     
     
         16 . The method according to  claim 12 , wherein the tumor is determined to be CXCR4(+) (CXCR4 positive) if the score is 2 +  or 3 + . 
     
     
         17 . A method for determining whether an oncogenic disorder in a subject is susceptible to treatment with a anti-CXCR4 antibody, a derived compound, or functional fragment thereof, wherein the method comprises (a) determining in vitro the CXCR4 status of a tumor of the subject by the method of  claim 12 , and (b) determining that, if the status is CXCR4(+), the oncogenic disorder is susceptible to treatment with an anti-CXCR4 antibody, or a derived compound, or functional fragment thereof. 
     
     
         18 . A kit for diagnosis or prognosis of a CXCR4 expressing tumor, the kit comprising at least one anti-CXCR4 antibody, or a derived compound, or functional fragment thereof, wherein the anti-CXCR4 antibody is selected from one or more of:
 a) an antibody that comprises i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 1, 2, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 4, 5, and 6, respectively;   b) an antibody that comprises, according to IMGT numbering, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 1, 2, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 7, 5, and 8, respectively;   c) an antibody that comprises, according to Kabat numbering, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 9, 10, and 3, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 11, 12, and 6, respectively;   d) an antibody which comprises i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 40, 2, and 41, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 42, 5, and 43, respectively;   e) an antibody which comprises, according to IMGT, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 40, 2, and 41, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 44, 5, and 45, respectively; and   f) an antibody which comprises, according to Kabat, i) a light chain comprising CDR regions CDR-L1, CDR-L2, and CDR-L3 comprising sequences SEQ ID Nos. 46, 47, and 47, respectively and ii) a heavy chain comprising CDR regions CDR-H1, CDR-H2, and CDR-H3 comprising sequences SEQ ID Nos. 48, 49, and 43, respectively.   
     
     
         19 . The kit according to  claim 18 , wherein the anti-CXCR4 antibody is labeled. 
     
     
         20 . The kit according to  claim 19 , wherein the label is chosen from fluorescein isothiocyanate, chromophores, radionuclides, and enzymes. 
     
     
         21 . The kit according to  claim 19  for determining in vitro the CXCR4 status of a tumor, the kit further comprising:
 i) a reagent useful for detecting the binding between the labeled anti-CXCR4 antibody and CXCR4; and 
 ii) positive and negative control samples. 
 
     
     
         22 . The kit according to  claim 18 , the kit further comprising a labeled reagent useful for detecting the binding between the anti-CXCR4 antibody and CXCR4. 
     
     
         23 . The kit according to  claim 22 , wherein the label is chosen from fluorescein isothiocyanate, chromophores, radionuclides, and enzymes. 
     
     
         24 . The method of  claim 1 , wherein the anti-CXCR4 antibody is selected from an antibody secreted by a murine hybridoma selected from a hybridoma filed at the CNCM, Institut Pasteur, Paris, on Oct. 22, 2007, under number 1-3860 and a hybridoma filed at the CNCM, Institut Pasteur, Paris, on Jun. 25, 2008, under number I-4019. 
     
     
         25 . The method of  claim 2 , wherein the murine antibody is selected from an antibody secreted by a murine hybridoma selected from a hybridoma filed at the CNCM, Institut Pasteur, Paris, on Oct. 22, 2007, under number 1-3860 and a hybridoma filed at the CNCM, Institut Pasteur, Paris, on Jun. 25, 2008, under number I-4019.