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

Claims

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.sup.+ wherein no membranous reactivity of tumor cells is scored 0, and strong complete reactivity in more than 10% of tumor cells is scored 3.sup.+.

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.sup.+ or 3.sup.+; 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.

Description

[0268] Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures whose legends are represented below.

[0269] FIG. 1 shows that 427aB1 Mab recognizes both CXCR4 monomers and homodimers on cell lysates.

[0270] FIGS. 2A and 2B show that 427aB1 Mab immunoprecipitates both CXCR4 monomers and homodimers.

[0271] FIGS. 3A, 3B, 3C, 3D and 3E show that 427aB1 Mab recognizes CXCR4 at the cell membrane by FACS analysis.

[0272] FIGS. 4A and 4B illustrate that 427aB1 Mab binds to CXCR4 at cell membrane even in the presence of the anti-CXCR4 515H7 therapeutic Mab by FACS analysis.

[0273] FIG. 5 shows that 427aB1 Mab does not modulate CXCR4/CXCR2 hetero-dimers conformation

[0274] FIG. 6 shows that 427aB1 Mab has no effect on MDA-MB-231 xenograft tumor growth model in Nod/Scid mice.

[0275] FIGS. 7A and 7B show FIG. 7A) IHC staining using m427aB1 and FIG. 7B) IHC staining using mIgG1 on RAMOS.

[0276] FIGS. 8A and 8B shows FIG. 8A) IHC staining using 427aB1 and FIG. 8B) IHC staining using mIgG1 on KARPAS299 xenograft tumors.

EXAMPLE 1

Anti-CXCR4 427aB1 Monoclonal Antibody (Mab) Generation (F50067-006(5C) 427aB1 cl1B, CNCM Number I-4018)

[0277] To generate monoclonal antibodies to CXCR4, Balb/c mice were immunized with recombinant NIH3T3-CXCR4 cells and/or peptides corresponding to CXCR4 extracellular N-term and loops. The mice 6-16 weeks of age upon the first immunization, were immunized once with the antigen in complete Freund's adjuvant subcutaneously (s.c.) followed by 2 to 6 immunizations with antigen in incomplete Freund's adjuvant s.c. The immune response was monitored by retroorbital bleeds. The serum was screened by ELISA (as described bellow) and mice with the higher titers of anti-CXCR4 antibodies were used for fusions. Mice were boost intravenously with antigen two days before sacrifice and removal of the spleen.

[0278] ELISA

[0279] To select the mice producing anti-CXCR4 antibodies, sera from immunized mice was tested by ELISA. Briefly, microtiter plates were coated with purified [1-41] N-terminal peptide conjugated to BSA at 5 μg equivalent peptide/mL, 100 μL/well incubated at 4° C. overnight, then blocked with 250μL/well of 0.5% gelatine in PBS. Dilutions of plasma from CXCR4-immunized mice were added to each well and incubated 2 hours at 37° C. The plates were washed with PBS and then incubated with a goat anti-mouse IgG antibody conjugated to HRP (Jackson Laboratories) for 1 hour at 37° C. After washing, plates were developed with TMB substrate, the reaction was stopped 5 min later by addition of 100 μL/well 1M H.sub.2SO.sub.4. Mice that developed the highest titers of anti-CXCR4 antibodies were used for antibody generation.

[0280] Generation of Hybridomas Producing Mobs to CXCR4

[0281] The mouse splenocytes, isolated from a Balb/c mice that developed the highest titers of anti-CXCR4 antibodies were fused with PEG to a mouse myeloma cell line Sp2/O. Cells were plated at approximately 1×10.sup.5/well in microtiter plates followed by two weeks incubation in selective medium containing ultra culture medium+2 mM L-glutamine+1 mM sodium pyruvate+1× HAT. Wells were then screened by ELISA for anti-CXCR4 monoclonal IgG antibodies. The antibody secreting hybridomas were then subcloned at least twice by limiting dilution, cultured in vitro to generate antibody for further analysis.

EXAMPLE 2

427aB1 Mab Recognizes Both CXCR4 Monomers and Homodimers on Cell Lysates

[0282] NIH3T3-hCXCR4 transfected cells, MDA-MB-231 (breast) and U937 (AML) cancer cells were washed twice in PBS. Then 100.10.sup.6 cells/ml were submitted to lysis using the following buffer: 20 mM TrisHCl pH8.5, 100 mM (NH.sub.4).sub.2SO.sub.4, 10% glycerol, 1% CHAPSO and 1% protease inhibitors cocktail for 30 min at 4° C. The cell lysate was collected by centrifugation at 10 000 g at +4° C. for 20 min and analyzed by western blot using 427aB1 Mab as primary antibody. FIG. 1 shows that Mab 427aB1 recognizes both CXCR4 monomers and homodimers in NIH3T3-CXCR4. Cancer cell lines MDA-MB-231 and U937 seem to express CXCR4 mainly as homodimers.

EXAMPLE 3

427aB1 Mab Immunoprecipitates Both CXCR4 Monomers and Homodimers

[0283] NIH3T3-CXCR4 cell pellets were washed with 20 mM TrisHCl, pH 8.5 containing 100 mM (NH4).sub.2SO.sub.4 and then suspended in lysis buffer (20 mM TrisHCl, pH 8.5 containing 100 mM (NH4).sub.2SO.sub.4, 10% glycerol, 1% CHAPSO and 10 μL/mL protease inhibitor cocktail). Cells were disrupted with Potter Elvehjem homogenizer. The solubilized membranes were collected by centrifugation at 105000 g at +4° C. for 1 h, then incubated overnight at +4° C. with 427aB1 Mab-coupled Sepharose 4B beads and mixture was poured into a glass column and washed with lysis buffer. The proteins captured by 427aB1 Mab were eluted and analyzed by western blot using 427aB1 Mab as primary antibody. Interesting fractions were pooled, concentrated and used for both WB analysis and preparative SDS-PAGE resolution (4-12% Bis-Tris gel). After silver staining, the bands of interest were excised from the gel and submitted to in-gel digestion using an automated protein digestion system, MassPREP station (Waters, Milford, Mass., USA). The gel spots were washed twice with 50 μL of 25 mM NH.sub.4HCO.sub.3 (Sigma, Steinheim, Germany) and 50 μL of acetonitrile (Carlo Erba Reactifs-SDS, Val de Reuil, France). The cysteine residues were reduced at 60° C. for 1 hour by 50 μL of 10 mM DTT prepared in 25 mM NH.sub.4HCO.sub.3 and alkylated at room temperature for 20 minutes by 50 μL of 55 mM iodoacetamide (Sigma) prepared in 25 mM NH.sub.4HCO.sub.3. After dehydration of the gel spots with acetonitrile, the proteins were digested overnight in gel by adding 10 μL of 12.5 ng/μl modified porcine trypsin (Promega, Madison, Wis., USA) in 25 mM NH.sub.4HCO.sub.3 at room temperature. The generated peptides were extracted with 35 μL of 60% acetonitrile containing 5% formic acid (Riedel-de Haën, Seelze, Denmark) followed by removing acetonitrile excess and were subjected to nano-LC-MS/MS. Mass data collected during nanoLC-MS/MS analysis were processed and converted into *.mgf files to be submitted to the MASCOT™ search engine. Searches were performed with a tolerance on measurements of 0.25 Da in MS and MS/MS modes.

[0284] FIG. 2A shows western blot analysis of eluted concentrated fractions after immunoprecipitation using 427aB1 Mab-coupled Sepharose beads. Three bands at 37-43, 75 and 150 kDa apparent molecular weights were recognized by 427aB1 Mab.

[0285] Eluted concentrated fraction after immunoprecipitation using 427aB1 Mab-coupled Sepharose beads was also resolved by SDS-PAGE and visualized by silver staining. The bands at 37-43, 75 and 150 KDa were excised from gel (FIG. 2B), digested with trypsin and analyzed by LC-MS/MS as described above. The collected peak lists were submitted to Mascot for peptide sequence database search. CXCR4 was identified in all bands:

[0286] Six CXCR4 peptides were identified in the 37-43-kDa band (band number 1) via the MASCOT™ search engine: 31-38 peptide EENANFNK, contained in N-terminal; 135-146 peptide YLAIVHATNSQR and 135-148 peptide YLAIVHATNSQRPR, and 184-188 peptide YICDR, contained in extra-cellular loop 2; 272-282 peptide QGCEFENTVHK, contained in extra-cellular loop 3 and 311-322 peptide TSAQHALTSVSR contained in C-terminal.

[0287] The 75-kDa band (band number 2) contained five CXCR4 peptides: 31-38 peptide EENANFNK, contained in N-terminal CXCR4; 135-146 peptide YLAIVHATNSQR, contained in intra-cellular loop 2; 135-148 peptide YLAIVHATNSQRPR, contained in intra-cellular loop 2; 272-282 peptide QGCEFENTVHK, contained in extra-cellular loop 3 and 311-322 peptide TSAQHALTSVSR, contained in C-terminal. Said peptides were identified via the MASCOT™ search engine

[0288] In the 150-kDa band (band number 3), two CXCR4 peptides were identified via the MASCOT™ search engine. 31-38 peptide EENANFNK, contained in N-terminal and 311-322 peptide TSAQHALTSVSR, contained in C-terminal.

[0289] The results obtained in this study clearly show that 427aB1 Mab immunoprecipitates CXCR4. In addition, 427aB1 Mab recognizes CXCR4 both as a monomer and a homodimer.

EXAMPLE 4

427aB1 Mab Recognizes CXCR4 Localized at the Cell Membrane by FACS Analysis

[0290] In this experiment, specific binding to human CXCR4 of 427aB1 Mab was assessed by FACS analysis.

[0291] NIH3T3, NIH3T3-hCXCR4 transfected cells, MDA-MB-231, Hela, HT-29 and U937 cancer cell lines were incubated with 427aB1 monoclonal antibody (0-10 μg/mL). The cells were then washed with 1% BSA/PBS/0.01% NaN3. Next, Alexa-labeled secondary antibodies were added to the cells and were allowed to incubate at 4° C. for 20 min. The cells were then washed again twice. After the second wash, FACS analysis was performed.

[0292] Results of these binding studies are provided in FIG. 3A through 3E. They show that 427aB1 binds to human CXCR4-NIH3T3 transfected cell line (FIG. 3A), but not to the parent NIH3T3 cells (not shown). This Mab was also capable to recognizing human cancer cell lines, for examples HT-29 colon cancer cells (FIG. 3B), MDA-MB-231 breast cancer cells (FIG. 3C), U937 promyelocytic cancer cells (FIG. 3D) and Hela cervix cancer cells (FIG. 3E), suggesting that these cell lines naturally express CXCR4 monomers and/or homodimers.

EXAMPLE 5

427aB1 Mab Binds to CXCR4 at Cell Membrane Even in the Presence of the Anti-CXCR4 515H7 Therapeutic Mab by FACS Analysis

[0293] In this experiment, competition of binding to human CXCR4 of anti-CXCR4 Mabs 427aB1 and 515H7 was examined by FACS analysis.

[0294] NIH3T3-hCXCR4 transfected cells, were first incubated with biotinylated 515H7 Mab (5 μg/ml) [which recognized NIH3T3-CXCR4 cells (FIG. 4A)], and then either 427aB1 Mab or 515H7 Mab (0-1 mg/mL) for 1 hour at 4° C. The cells were then washed with 1% BSA/PBS/0.01% NaN3. Next, labeled-streptavidin was added to the cells and was allowed to incubate at 4° C. for 20 min, before another couple of washes. After the second wash, FACS analysis was performed. Results of these binding studies are provided in FIG. 4B and showed that anti-CXCR4 Mab 427aB1 binds to human CXCR4-NIH3T3 transfected cells even in the presence of 515H7 Mab. In contrast, the presence of biotinylated 515H7 Mab inhibited the binding of non-labeled 515H7 Mab to CXCR4, as expected.

EXAMPLE 6

427aB1 Mab Does Not Modulate CXCR4/CXCR2 Heterodimers Conformation by BRET Analysis

[0295] This functional assay allows the evaluation of the conformational changes induced upon SDF-1 and/or 427aB1 Mab binding to CXCR4 receptor at the level of CXCR2/CXCR4 heterodimer.

[0296] Expression vectors for each of the investigated interaction partners were constructed as fusion proteins with the corresponding dye (Renato reniformis luciferase, Rluc and Yellow fluorescent protein, YFP) by applying conventional molecular biology techniques.

[0297] Two days prior performing BRET experiments, HEK293 cells were transiently transfected with expression vectors coding for the corresponding BRET partners: [CXCR4-Rluc+CXCR2-YFP]. The next day, the cells were distributed in poly-lysine pre-coated white 96 MW plates in complete culture medium [DMEM supplemented with 10% FBS]. Cells were first cultivated at 37° C. with CO.sub.2 5% in order to allow cell attachment to the plate. Cells were then starved with 200 μl DMEM/well overnight. Immediately prior to the BRET experiment, DMEM was removed and cells were quickly washed with PBS. Cells were then incubated in PBS in the presence or absence of antibody, 15 min at 37° C. prior to the addition of coelenterazine H 5 μM with or without SDF-1 in a final volume of 50 μl. After incubation for 5 minutes at 37° C. and further incubation for 20 min at room temperature, light-emission acquisition at 485 nm and 530 nm was initiated using the Mithras LB940 multilabel reader (Berthold) (1 s/wavelength/well repeated 15 times at room temperature).

[0298] Calculation of BRET ratio was performed as previously described (Angers et al., 2000): [(emission.sub.530 nm)−(emission.sub.485 nm)×Cf]/(emission.sub.485 nm), where Cf=(emission.sub.530 nm)/(emission.sub.485 nm) for cells expressing the Rluc fusion protein alone under the same experimental conditions. Simplifying this equation shows that BRET ratio corresponds to the ratio 530/485 nm obtained when the two BRET partners are present, corrected by the ratio 530/485 nm obtained under the same experimental conditions, when only the partner fused to Rluc is present in the assay. For sake of readability, results are expressed as percentage of the basal signal.

[0299] SDF1 (300 nM) decreased by about 20% the BRET signal resulting from the spatial proximity of CXCR4 and CXCR2 receptors. It is likely to indicate CXCR4/CXCR2 heterodimers formation or conformational changes of pre-existing dimers (FIG. 5). 427aB1 Mab did not modulate SDF-1-induced conformational changes for CXCR2/CXCR4 heterodimer and did not modulate by itself CXCR4/CXCR2 spatial proximity. This indicates that 427aB1 Mab has no influence on CXCR4/CXCR2 heterodimers conformation (FIG. 5).

EXAMPLE 7

427aB1 Mab Activity Evaluation in MDA-MB-231 Xenograft Tumor Growth Model in Nod/Scid Mice

[0300] The goal of this experiment was to assess the inhibitory activity of anti-CXCR4 Mab 427aB1 against an MDB-MB-231 xenograft in Nod/Scid mice.

[0301] MDA-MB-231 cells from ECACC were routinely cultured in DMEM medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Sigma, St Louis Md., USA). Cells were split 48 hours before engraftment so that they were in exponential phase of growth. Ten million MDA-MB-231 cells were engrafted in PBS to 7 weeks old Nod/Scid mice (Charles River, France). Five days after implantation, tumors were measurable (34 mm.sup.3<V.sup.3<40 mm.sup.3) and animals were divided into groups of 6 mice with comparable tumor size. Mice were treated i.p. with a 2 mg/mouse loading dose of Mab 427aB1. Then, mice were injected twice a week at 1 mg/dose/mouse of Mab 427aB1. A PBS group was introduced as a control group in this experiment. Tumor volume was measured twice a week and calculated by the formula: π/6×length×width×height. Statistical analyses were performed at each measure using a Mann-Whitney test.

[0302] No mortality was observed during treatment. Compared to the PBS control group, no significant inhibition of tumor growth at D40 (p=0.485) for 427aB1 Mab 1 mg/dose was observed. In addition, the average tumor volume after 5 weeks of treatment was not reduced by Mab 427aB1 versus PBS (FIG. 6).

EXAMPLE 8

427aB1 Mab Recognizes CXCR4 Present at the Cell Membrane (Paraffin Embedded Tumors IHC Staining)

[0303] Sections were deparaffinized, rehydrated, and placed at 98° C. for 5 minutes in pre-warm at 98° C. EDTA pH8 for heat-induced epitope retrieval and for 5 additional minutes at room temperature in the warm EDTA buffer. Slides were then rinsed in tap water for 5 minutes. After 3 washes in Tris Buffer Saline-0.05% Tween 20 (TBS-T) (Dako S3006), the endogenous peroxidase activity was blocked using Peroxidase Blocking Reagent (Dako K4007) for five minutes. Sections were washed with TBS-T and incubated in blocking reagent (UltraV block-TA-125UB-LabVision) for 5 minutes before incubation with the anti-CXCR-4 mouse monoclonal antibody (5 μg/ml, clone 427aB1, Pierre Fabre) or mouse IgG1/kappa (5 μg/ml, X0931, Dako) as an isotype control overnight at 4° C. Sections were washed with TBS-T and incubated with SignalStain Boost IHC detection Reagent (HRP, M) for 30 minutes at room temperature. Diaminobenzidine was used for development of a brown reaction product (Dako K3468). The slides were immersed in hematoxylin for 4 minutes to counterstain (Dako 53309) and washed in PBS before being mounted in Faramount mounting medium plus coverslipe. In this immunohistochemistry procedure, the brown reaction product correlates to positive staining of the cell membrane and lack of brown reaction product correlates to negative staining and no visualization of the cell membrane.

[0304] The 427aB1 Mab differentially stains the cell membrane of various tumor types. FIGS. 7 and 8 illustrated staining performed in 2 xenograft models in which an anti-tumoral activity with the therapeutic anti-CXCR-4 hz515H7 antibody has been described: RAMOS and KARPAS299.

[0305] As shown in FIGS. 7A and 7B and FIGS. 8A and 8B, the expression detected is lower in KARPAS299 (FIGS. 8A and 8B) than in RAMOS (FIGS. 7A and 7B). This data correlates nicely with the study of the CXCR-4 expression by flow cytometry. Indeed, RAMOS cells express about 5 levels more of CXCR-4 than KARPAS299 one (Antibody Binding Capacity: 200 000 for RAMOS and 40 000 for KARPAS299). Membranous staining is weaker in KARPAS299 (FIGS. 8A and 8B), whereas, membranous staining is significantly higher in RAMOS (FIGS. 7A and 7B).