Novel anti-cxcr4 antibody and its use for the detection and diagnosis of cancer

Abstract

The present invention provides a novel, isolated anti-CXCR4 antibody for use in the diagnosis of cancer. In particular, the antibody of the invention recognizes monomeric and homodimeric CXCR4, but not heterodimeric CXCR4

1 . An antibody, or an antigen-binding fragment or derivative thereof, comprising i) a heavy chain comprising the following three CDRs, respectively CDR-H1 having the sequence SEQ ID No. 1, CDR-H2 having the sequence SEQ ID No. 2 and CDR-H3 having the sequence SEQ ID No. 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 having the sequence SEQ ID No. 4, CDR-L2 having the sequence SEQ ID No. 5 and CDR-L3 having the sequence SEQ ID No. 6. 
     
     
         2 . The antibody of  claim 1 , or an antigen-binding fragment or derivative thereof, wherein the said antibody is selected among:
 a. an antibody with a heavy chain comprising the following three CDRs, respectively CDR-H1 having the sequence SEQ ID No. 1, CDR-H2 having the sequence SEQ ID No. 2 and CDR-H3 having the sequence SEQ ID No. 3; and a light-chain variable domain comprising the sequence SEQ ID No. 8;   b. an antibody with a heavy chain variable domain comprising the sequence SEQ ID No. 7; and a light chain comprising the following three CDRs, respectively CDR-L1 having the sequence SEQ ID No. 4, CDR-L2 having the sequence SEQ ID No. 5 and CDR-L3 having the sequence SEQ ID No. 6; or   c. an antibody with a heavy chain variable domain comprising the sequence SEQ ID No. 7; and a light-chain variable domain comprising the sequence SEQ ID No. 8.   
     
     
         3 . The antibody according to one of  claims 1  or  2 , or an antigen-binding fragment or derivative thereof wherein the said antibody 427aB1 comprises:
 a) a heavy chain, said heavy chain comprising:
 the following three CDRs, respectively CDR-H1 having the sequence SEQ ID No. 1, CDR-H2 having the sequence SEQ ID No. 2 and CDR-H3 having the sequence SEQ ID No. 3; and a light-chain variable domain comprising the sequence SEQ ID No. 8, and 
 a heavy chain variable domain, said heavy chain variable domain having the sequence SEQ ID No. 7; and 
 
 b) a light chain, said light chain comprising:
 the following three CDRs, respectively CDR-L1 having the sequence SEQ ID No. 4, CDR-L2 having the sequence SEQ ID No. 5 and CDR-L3 having the sequence SEQ ID No. 6; and 
 a light-chain variable domain, said light-chain variable domain having the sequence SEQ ID No. 8. 
 
 
     
     
         4 . The antibody according to one of  claims 1  to  3 , or an antigen-binding fragment or derivative thereof, wherein the said antibody is capable of binding to CXCR4 as monomer and/or homodimer. 
     
     
         5 . The antibody according to one of  claims 1  to  4 , or an antigen-binding fragment or derivative thereof, for use in diagnosing in vitro or ex vivo an oncogenic disorder associated with expression of CXCR4 or determining in vitro or ex vivo the prognosis for developing an oncogenic disorder associated with expression of CXCR4. 
     
     
         6 . The antibody according to  claim 5 , or an antigen-binding fragment or derivative thereof, characterized in that said oncogenic disorder consists of an oncogenic disorder associated with expression of CXCR4 as monomer and/or homodimer. 
     
     
         7 . The antibody according to one of  claims 1  to  6 , or an antigen-binding fragment or derivative thereof, characterized in that the said antibody is a murine antibody. 
     
     
         8 . The antibody according to one of  claims 1  to  7 , or an antigen-binding fragment or derivative thereof, characterized in that the said antibody does not block the binding of the antibody 515H7 to CXCR4. 
     
     
         9 . The antibody according to one of  claims 1  to  8 , or an antigen-binding fragment or derivative thereof, characterized in that the said antibody does not have any in vivo anti-tumoral activity. 
     
     
         10 . A murine hybridoma capable of secreting an antibody, or an antigen-binding fragment or derivative thereof, according to one of the  claims 1  to  9 . 
     
     
         11 . The marine hybridoma according to  claim 10 , said murine hybridoma being deposited at the CNCM, Institut Pasteur, Paris, France on Jun. 25, 2008 under the number 1-4018. 
     
     
         12 . An isolated nucleic acid, characterized in that it is chosen from the following nucleic acids:
 a) a nucleic acid, DNA or RNA, coding for an antibody or for a derived compound or functional fragment thereof, according to one of the  claims 1  to  8 ;   b) a nucleic acid comprising a DNA sequence comprising a sequence selected from the group consisting of the sequences SEQ ID No. 9 to 14, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences SEQ ID No. 9 to 14;   c) a nucleic acid comprising a DNA sequence comprising the sequences SEQ ID No. 15 or 16, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences SEQ ID No. 15 or 16;   d) the RNA translated from the nucleic acids as defined in a), b) or c);   e) the complementary nucleic acids of the nucleic acids as defined in a), b) and c); and   f) a nucleic acid of at least 18 nucleotides capable of hybridizing under conditions of high stringency with the sequences SEQ ID No. 15 or 16 or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequences SEQ ID 15 or 16, or a complementary sequence thereof.   
     
     
         13 . A vector comprising a nucleic acid as claimed in  claim 12 . 
     
     
         14 . A host cell transformed by or comprising a vector as claimed in  claim 13 . 
     
     
         15 . A method of producing an antibody, or an antigen-binding fragment or derivative thereof, characterized in that the method comprises the steps of a) culturing a host cell as claimed in  claim 14  in a culture medium and under appropriate culture conditions; and b) recovering said antibody, or antigen-binding fragment or derivative thereof, from the culture medium or said cultured cell. 
     
     
         16 . A method for detecting in vitro or ex vivo the presence of a tumor expressing monomeric/homodimeric CXCR4, wherein said process comprises the steps of;
 (a) contacting a biological sample from the said subject with an antibody, or an antigen-binding fragment or derivative thereof, according to anyone of  claims 1  to  9  or obtained by the method of  claim 15  or produced by hybridoma according to  claim 10  or  11 ; and   (b) detecting the binding of the said antibody, or antigen-binding fragment or derivative thereof, with the biological sample.   
     
     
         17 . A method for determining in vitro or ex vivo the percentage of cells expressing CXCR4 as monomer and/or homodimer in a tumor from a subject, said method comprising the steps of:
 (a) contacting a sample from the subject with an antibody, or an antigen-binding fragment or derivative thereof, according to anyone of  claims 1  to 9 or obtained by the method of  claim 15  or produced by hybridoma according to  claim 10  or  11 ; and   (b) quantifying the percentage of cells expressing CXCR4 as monomer and/or homodimer in the sample.   
     
     
         18 . A method for determining in vitro or ex vivo the expression level of monomeric/homodimeric CXCR4 as monomer and/or homodimer in a tumor from a subject, said method comprising the steps of:
 (a) contacting a biological sample from the subject with an antibody or a antigen-binding fragment or derivative thereof, according to anyone of  claims 1  to  9  or obtained by the method of  claim 15  or produced by hybridoma according to  claim 10  or  11 ; and   (b) quantifying the level of binding of the said antibody, or antigen-binding fragment or derivative thereof, to monomeric/homodimeric CXCR4 in the biological sample.   
     
     
         19 . The method of  claim 18 , wherein the level of binding of the said antibody, or antigen binding fragment or derivative thereof, to monomeric/homodimeric CXCR4 is measured by immunohistochemistry (IHC) or FACS, preferably by IHC. 
     
     
         20 . A method for determining in vitro or ex vivo the scoring of a tumor from a subject, said method comprising the steps of:
 (a) contacting a biological sample from the subject with an antibody, or an antigen-binding fragment or derivative thereof, according to anyone of  claims 1  to  9  or obtained by the method of  claim 15  or produced by hybridoma according to  claim 10  or  11 ;   (b) quantifying the level of binding of the said antibody, or antigen-binding fragment or derivative thereof, to monomeric/homodimeric CXCR4 as monomer and/or homodimer in the said biological sample; and   (c) scoring the tumor by comparing the quantified level of binding of the said antibody, or antigen-binding fragment or derivative thereof, from the subject to an appropriate scale.   
     
     
         21 . The method of  claim 20 , wherein the said appropriate scale is based on two parameters which are the intensity of the staining and the percentage of positive cells. 
     
     
         22 . The method of anyone of  claim 20  or  21 , wherein the said appropriate scale is a scale of 0 to 8 wherein “no reactivity” is scored 0, and a strong reactivity in a proportion of “67-100% proportion reactive” is scored 8. 
     
     
         23 . A method for determining in vitro or ex vivo the status of a tumor from a subject, said method comprising the steps of:
 (a) scoring a tumor from a subject according to anyone of  claims 20 ,  21 ; or  22 , and   (b) determining that the status of the tumor is [monomeric/homodimeric CXCR4(+)] with a score of 3 to 8; or   (c) determining that the status of the tumor is [monomeric/homodimeric CXCR4(−)] with a score of 0 to 2.   
     
     
         24 . The method of anyone of  claims 20  or  21 , wherein the said appropriate scale is a scale of 0 to 3 +  wherein no membranous reactivity of tumor cells is scored 0, and strong complete reactivity in more than 10% of tumor cells is scored 3 + . 
     
     
         25 . A method for determining in vitro or ex vivo the status of a tumor from a subject, said method comprising the steps of:
 (a) scoring a tumor from a subject according to one of  claims 20 ,  21  or  24 ; and   (b) determining that the status of the tumor is [monomeric/homodimeric CXCR4(+)] with a score of 2 +  or 3 + ; or   (c) determining that the status of the tumor is [monomeric/homodimeric CXCR4(−)] with a score of 0 or 1.   
     
     
         26 . A method for determining whether an oncogenic disorder is susceptible to treatment with a CXCR4 antagonist, said method comprising the steps of:
 (a) determining in vitro or ex vivo the status of a tumor of a subject according to  claim 23  or  25 , and   (b) determining that, if the status is [monomeric/homodimeric CXCR4(+)], the oncogenic disorder is susceptible to treatment with a CXCR4 antagonist.   
     
     
         27 . A method for determining in vitro or ex vivo the efficacy of a therapeutic regime designed to alleviate an oncogenic disorder associated with monomeric/homodimeric CXCR4 in a subject suffering from said disorder, said method comprising the steps of:
 (a) determining a first expression level of monomeric/homodimeric CXCR4 according to  claim 18  or  19  in a biological sample extracted from the said subject at a first time point;   (b) determining a second expression level of monomeric/homodimeric CXCR4 according to  claim 18  or  19  in a biological sample extracted from the said subject at a second, later time point;   (c) determining the ratio between the level obtained in (a) to the level obtained in (b); and   (d) determining that the efficacy of said therapeutic regime is high when the ratio of step (c) is greater than 1; or   (e) determining that the efficacy of said therapeutic regime is low when the ratio of step (c) is inferior or equal to 1.   
     
     
         28 . The method of  claim 27 , wherein the therapeutic regime designed to alleviate an oncogenic disorder associated with monomeric/homodimeric CXCR4 in a subject suffering from said disorder includes the administration of a CXCR4 inhibitor to the said subject. 
     
     
         29 . An in vitro or ex vivo method for selecting a cancer patient predicted to benefit or not from the administration of a therapeutic amount of a CXCR4 inhibitor, said method comprising the steps of:
 (a) determining the expression level of monomeric/homodimeric CXCR4 in the said patient according to the process of  claim 18  or  19 ;   (b) determining a reference expression level of monomeric/homodimeric CXCR4 from a healthy individual according to the process of  claim 18  or  19 ; and   (c) determining the ratio between the level obtained in (a) to the level obtained in (b); and   (d) selecting the patient as being predicted to benefit from the administration of a therapeutic amount of a CXCR4 inhibitor, if the ratio of step (c) is greater than 1; or   (e) selecting the patient as being not predicted to benefit from a administration of a therapeutic amount of a CXCR4 inhibitor, if the ratio of step (c) is inferior or equal to 1.   
     
     
         30 . The process according to  claim 28  or  29 , wherein the said CXCR4 inhibitor is the monoclonal antibody 515H7. 
     
     
         31 . A kit comprising at least an antibody, or an antigen-binding fragment or derivative thereof, according to anyone of  claims 1  to  9  or obtained by the method of  claim 15  or produced by hybridoma according to  claim 10  or  11 . 
     
     
         32 . The kit of  claim 31 , characterized in that the said antibody, or antigen-binding fragment or derivative thereof, is labeled. 
     
     
         33 . The kit of anyone of  31  or  32 , further comprising a reagent for detecting the extent of binding between the said antibody, or antigen-binding fragment or derivative thereof, and monomeric/homodimeric CXCR4. 
     
     
         34 . The kit of anyone of  claims 31  to  33 , further comprising a reagent for quantifying the level of binding between the said antibody, or antigen-binding fragment or derivative thereof, and monomeric/homodimeric CXCR4. 
     
     
         35 . The kit of anyone of  claims 31  to  34 , further comprising positive and negative control samples for the scoring of monomeric/homodimeric CXCR4 expression level. 
     
     
         36 . The kit of  claim 35 , further comprising a polyclonal antibody recognizing specifically murine antibodies, said polyclonal antibody being preferably labeled.