Antibody directed against the endothelin receptor beta sub-type

Abstract

Antibodies directed against the endothelin receptor sub-type B, in particular monoclonal antibodies, a fragment or derivative thereof. The present disclosure also relates to the therapeutic, diagnostic use or as a research tool of such an antibody in the field of cancers and in particular glioblastoma.

Claims

1. An antibody directed against an endothelin receptor sub-type B comprising: a heavy chain variable region comprising a CDR1 (hereinafter designated CDR1.sub.H) the amino acid sequence of which is GYTFISYW (SEQ ID NO: 5); a CDR2 (hereinafter designated CDR2.sub.H) the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3 (hereinafter designated CDR3.sub.H) the amino acid sequence of which is AREGDYAWFAY (SEQ ID NO: 15); and a light chain variable region comprising a CDR1 (hereinafter designated CDR1.sub.L) the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2 (hereinafter designated CDR2.sub.L) the amino acid sequence of which is KVS; and a CDR3 (hereinafter designated CDR3.sub.L) the amino acid sequence of which is FQGSHVPWT (SEQ ID NO: 27); or ii) a heavy chain variable region comprising a CDR1.sub.H the amnio acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2.sub.H the amnio acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3.sub.H the amnio acid sequence of which is VREGWDAWFVY (SEQ ID NO: 17); and a light chain variable region comprising a CDR1.sub.L the amnio acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2.sub.L the amnio acid sequence of which is KVF; and a CDR3.sub.L the amnio acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); or iii) a heavy chain variable region comprising a CDR1.sub.H the amnio acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2.sub.H the amnio acid sequence of which is IDPNSGGT (SEQ ID NO: 12); and a CDR3.sub.H the amnio acid sequence of which is AREGEFAWFAY (SEQ ID NO: 19); and a light chain variable region comprising a CDR1.sub.L the amnio acid sequence of which is QNIVHSNGNTY (SEQ ID NO: 24); a CDR2.sub.L the amnio acid sequence of which is KVS; and a CDR3.sub.L the amnio acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); a fragment or derivative thereof, wherein said fragment has at least one antigen-binding site, wherein said derivative is a single chain Fv or a single domain antibody, and wherein said antibody and said fragment or derivative thereof are capable of recognising particular conformational isomers of the endothelin receptor sub-type B expressed at the surface of glioblastoma cells.

2. The antibody according to claim 1, wherein said antibody comprises a heavy chain variable region the amino acid sequence of which exhibits at least 80% identity with the following sequence: TABLE-US-00020 (SEQ ID NO: 31) QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIG RIDPDSGGTKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR EGDYAWFAYWGQGTLVPVSA. 

3. The antibody according to claim 1, wherein said antibody comprises a light chain variable region the amino acid sequence of which exhibits at least 80% identity with the following sequence: TABLE-US-00021 (SEQ ID NO: 37) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKLEIK.

4. The antibody according to claim 1, wherein said antibody is an IgG1/kappa type or IgG3/kappa type immunoglobulin.

5. The antibody according to claim 1, wherein said antibody is monoclonal.

6. The antibody according to claim 1, wherein said antibody is a monoclonal murine antibody obtained from a hybridoma chosen from a hybridoma deposited with CNCM on the 19 May 2016 under accession number CNCM I-5084, a hybridoma deposited with the CNCM on the 7 Jun. 2016 under accession number CNCM 1-5104 and a hybridoma deposited with the CNCM on the 7 Jun. 2016 under accession number CNCM I-5103.

7. The antibody according to claim 1, wherein said antibody is a chimerized antibody.

8. The antibody according to claim 1, wherein said antibody is a humanized antibody.

9. A pharmaceutical composition comprising, as an active ingredient, an antibody according to claim 1 and a pharmaceutically acceptable vehicle.

10. A compound comprising an antibody according to claim 1 conjugated with an element chosen from the group consisting of a cytotoxic group, an easily detectable group, or an effector group, wherein the cytotoxic group is selected from the group consisting of alkylating agents, methotrexate, 5-fluoro-uraci, vinblastine, gemcitabine, fludarabine, nicotinamide, doxorubicin, mitomycin, L-asparaginase, cisplatin, taxol and analogues/derivatives thereof, ricin, abrin, Pseudomonas exotoxin, TNF-alpha, interleukin 2, methotrexate-alanine, mitomycin phosphate, 5-fluorocytosine, photofrin, capecitabine, a carboxypeptidase, an aminopeptidase or endopeptidase, phosphatase, a sulphatase, an amidase, a kinase, a glycosidase, a deaminase, a reductase, an oxidase, an anti-sense oligonucleotide and an aptamer; wherein the easily detectable group is selected in the group consisting of biotin, digoxigenin, 5-bromodeoxiuridin, an alkaline phosphatase, a peroxidase, an acetylcholine esterase (AChE), a glucose amylase, a lysozyme, fluorescein and derivatives thereof, rhodamine and derivatives thereof, GFP (Green Fluorescent Protein) and derivatives thereof and umbelliferone; luminol; luciferase and luciferin; iodine-123, iodine-125, iodine-126, iodine-133, indium- Ill, indium-113m, bromine-77, gallium-67, gallium-68, ruthenium-95, ruthenium-97, technetium-99m, fluorine-19, fluorine-18, carbon-13, nitrogen-15, oxygen-17, scandium- 47, tellurium-122m, thulium-165 and yttrium-199; and wherein the effector group is selected from the group consisting of a ligand of a cancer marker, an antibody identical to or different from the antibody according to claim 1, a protein, a peptide, a DNA, an RNA, an RNAi, an aptamer, a PNA and an LNA.

11. A pharmaceutical composition comprising, as an active ingredient, a compound according to claim 10 and a pharmaceutically acceptable vehicle.

12. A process for diagnosing a glioblastoma in vitro comprising the steps of: a.sub.1′) contacting a biological sample taken from a subject with a compound according to claim 10; b.sub.1′) detecting the signal emitted by the easily detectable group and c.sub.1′) determining the presence or absence of a glioblastoma in said subject based on the signal detected in step (b.sub.1′).

13. An antibody directed against the endothelin receptor sub-type B comprising: i) a heavy chain variable region comprising a CDR1 (hereinafter designated CDR1H) the amino acid sequence of which is GYTFISYW (SEQ ID NO: 5); a CDR2 (hereinafter designated CDR2H) the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3 (hereinafter designated CDR3H) the amino acid sequence of which is AREGDYAWFAY (SEQ ID NO: 15); and a light chain variable region comprising a CDR1 (hereinafter designated CDR1L) the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2 (hereinafter designated CDR2L) the amino acid sequence of which is KVS; and a CDR3 (hereinafter designated CDR3L) the amino acid sequence of which is FQGSHVPWT (SEQ ID NO: 27); or ii) a heavy chain variable region comprising a CDR1H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2H the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3H the amino acid sequence of which is VREGWDAWFVY (SEQ ID NO: 17); and a light chain variable region comprising a CDR1L the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2L the amino acid sequence of which is KVF; and a CDR3L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); or iii) a heavy chain variable region comprising a CDR1H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2H the amino acid sequence of which is IDPNSGGT (SEQ ID NO: 12); and a CDR3H the amino acid sequence of which is AREGEFAWFAY (SEQ ID NO: 19) and a light chain variable region comprising a CDR1L the amino acid sequence of which is QNIVHSNGYTY (SEQ ID NO: 24); a CDR2L the amino acid sequence of which is KVS; and a CDR3L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); a fragment or derivative thereof, wherein said fragment has at least one antigen-binding site, and wherein said derivative is a single chain Fv or a single domain antibody.

14. A compound comprising the antibody according to claim 13, conjugated with a cytotoxic group or an effector group wherein an effector group is selected from the group consisting of a ligand of a cancer marker; an antibody identical to or different from the antibody according to claim 1; a protein; a peptide; a DNA, an RNA, an RNAi, an aptamer, a PNA and an LNA.

15. The antibody according to claim 13, wherein the amino acid sequence of the heavy chain variable region exhibits at least 80% identity with sequence: TABLE-US-00022 (SEQ ID NO: 31)  QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIG RIDPDSGGTKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR EGDYAWFAYWGQGTLVPVSA;  and/or wherein the amino acid sequence of the light chain variable region exhibits at least 80% identity with sequence: TABLE-US-00023 (SEQ ID NO: 37) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKLEIK.

16. A compound comprising the antibody according to claim 15, conjugated with a cytotoxic group or an effector group wherein an effector group is selected from the group consisting of a ligand of a cancer marker; an antibody identical to or different from the antibody according to claim 1; a protein; a peptide; a DNA, an RNA, an RNAi, an aptamer, a PNA and an LNA.

17. A method for treating a cancer associated with ETBR overexpression, which method comprises administering to a subject in need thereof a compound comprising an antibody against endothelin receptor sub-type B, or an antigen-binding fragment thereof or derivative thereof, wherein antibody against endothelin receptor sub-type B, antigen-binding fragment thereof or derivative thereof, is conjugated with a cytotoxic group, and wherein the antibody against endothelin receptor sub-type B comprises: i) a heavy chain variable region comprising a CDR1 (hereinafter designated CDR1H) the amino acid sequence of which is GYTFISYW (SEQ ID NO: 5); a CDR2 (hereinafter designated CDR2H) the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3 (hereinafter designated CDR3H) the amino acid sequence of which is AREGDYAWFAY (SEQ ID NO: 15); and a light chain variable region comprising a CDR1 (hereinafter designated CDR1L) the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2 (hereinafter designated CDR2L) the amino acid sequence of which is KVS; and a CDR3 (hereinafter designated CDR3L) the amino acid sequence of which is FQGSHVPWT (SEQ ID NO: 27); or ii) a heavy chain variable region comprising a CDR1H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2H the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3H the amino acid sequence of which is VREGWDAWFVY (SEQ ID NO: 17); and a light chain variable region comprising a CDR1L the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2L the amino acid sequence of which is KVF; and a CDR3L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); or iii) a heavy chain variable region comprising a CDR1H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2H the amino acid sequence of which is IDPNSGGT (SEQ ID NO: 12); and a CDR3H the amino acid sequence of which is AREGEFAWFAY (SEQ ID NO: 19) and a light chain variable region comprising a CDR1L the amino acid sequence of which is QNIVHSNGYTY (SEQ ID NO: 24); a CDR2L the amino acid sequence of which is KVS; and a CDR3L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29), wherein said derivative is a single chain Fv or a single domain antibody.

18. The method according to claim 17, wherein said cancer is a melanoma, glioblastoma, or bladder cancer.

19. The method according to claim 17, wherein said cancer is a melanoma.

20. The method according to claim 17, wherein said cancer is a glioblastoma.

21. The method according to claim 17, wherein said antibody against endothelin receptor sub-type B, the amino acid sequence of the heavy chain variable region exhibits at least 80% identity with sequence: QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKY NEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVPVSA (SEQ ID NO: 31); and/or the amino acid sequence of the light chain variable region exhibits at least 80% identity with sequence: TABLE-US-00024 (SEQ ID NO: 37) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKLEIK.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows binding curves of Rendomab-B49 (FIG. 1A), Rendomab-B41 (FIG. 1B) and Rendomab-B36 (FIG. 1C) on CHO cells overexpressing ETB-R with (squares) or without (dots) pre-incubation of the cells in the presence of 300 nM of endothelin 1. In both cases, an affinity in the order of one nanomole has been measured.

(2) FIG. 2 shows images obtained in confocal microscopy on neurospheres from a biopsy of a patient having a high grade glioblastoma in the presence of 1 μg/mL of labelled Rendomab-B1 (FIG. 2A) or 1 μg/mL of labelled Rendomab-B49 (FIG. 2B). Only the nuclei of DAPI labelled cells are viewed in FIG. 2A.

(3) FIG. 3 shows images obtained in confocal microscopy on tumor cells from a biopsy of a patient having a low grade glioblastoma in the presence of DAPI (FIG. 3A) or 1 μg/mL of Rendomab-B49 (FIG. 3B).

(4) FIG. 4 shows images obtained in confocal microscopy on tumor cells from a biopsy of a patient having a glioblastoma in the presence of 1 μg/mL Rendomab-B36.

(5) FIG. 5 shows the nucleic sequences deduced in amino acids from the variable domains of the light chain (VL) (FIG. 5A), in particular, VLRendoMabB49 (nucleic acid) that corresponds to SEQ ID NO: 36 and VLRendoMabB49 (amino acid) that corresponds to SEQ ID NO: 37, and the heavy chain (VH) (FIG. 5B), in particular, VHRendoMabB49 (nucleic acid) that corresponds to SEQ ID NO: 30 and VHRendoMabB49 (amino acid) that corresponds to SEQ ID NO: 31, of the IgG1/kappa murine antibody Rendomab-B49 specific to the endothelin receptor B.

(6) FIG. 6 shows the nucleic sequences deduced in amino acids from the variable domains of the light chain (VL) (FIG. 6A), in particular, VLRendoMabB41 (nucleic acid) that corresponds to SEQ ID NO: 38 and VLRendoMabB41 (amino acid) that corresponds to SEQ ID NO: 39, and the heavy chain (VH) (FIG. 6B), in particular, VHRendoMabB41 (nucleic acid) that corresponds to SEQ ID NO: 32 and VHRendoMabB41 (amino acid) that corresponds to SEQ ID NO: 33, of the IgG1/kappa murine antibody Rendomab-B41 specific to the endothelin receptor B.

(7) FIG. 7 shows the nucleic sequences deduced in amino acids of the variable domains of the light chain (VL) (FIG. 7A), in particular, VLRendoMabB36 (nucleic acid) that corresponds to SEQ ID NO: 40 and VLRendoMabB36 (amino acid) that corresponds to SEQ ID NO: 41, and the heavy chain (VH) (FIG. 7B), in particular, VHRendoMabB36 (nucleic acid) that corresponds to SEQ ID NO: 34 and VHRendoMabB36 (amino acid) that corresponds to SEQ ID NO: 35, of the IgG3/kappa murine antibody Rendomab-B36 specific to the endothelin receptor B.

(8) FIG. 8 shows the epitopic mapping. FIG. 8A presents the revealed “Pep scan” membrane. FIG. 8B presents the sequences of the peptides recognized by Rendomab-B49 that corresponds to SEQ ID NO: 42 with high intensity (C5, C6, C7, C8, C9 and C10 peptides). FIG. 8C presents the location of the epitope recognized by Rendomab-B49 in the sequence of the human endothelin receptor sub-type B.

DETAILED DISCLOSURE OF PARTICULAR EMBODIMENTS

(9) I. Materials and Methods

(10) I.1. Immunisation

(11) The so-called “gene immunisation” strategy developed in the laboratory of the inventors consists in combining DNA injections with protein boosts as an injection of cells overexpressing ETB-R (Allard et al, 2011, “Electroporation-aided DNA immunisation generates polyclonal antibodies against the native conformation of human endothelin B receptor”, DNA and Cell Biology, vol. 30, pages 727-737).

(12) Briefly, three injections, in a mouse tibial muscle, of 50 μg of plasmid DNA pcDNA3/ETB-R, were made with a periodicity of 14 days. Each DNA injection was followed by an electrostimulation according to the following characteristics: 8 pulses each of 20 ms, 80 Volts, 1 Hz. Three immunisation boosts were then made, by injection by the intra-peritoneal route of 2.10.sup.6 COS cells transiently overexpressing ETB-R.

(13) The best responder mice were sacrificed in order to conduct cell fusion of the lymphoid cells of their spleens with the murine myeloma NS-1.

(14) I.2. Screening Hybridomas

(15) The hybridomas obtained were first screened by ELISA on CHO cells stably expressing ETB-R, with as a negative control, CHO cells expressing the irrelevant receptor NK1 (CHO-WT).

(16) The hybridomas retained were then screened in flow cytometry (apparatus Guava, Millipore). Three hybridomas called “Rendomab-B49”, “Rendomab-B41” and “Rendomab-B36” were finally retained at the end of both these screens. The antibodies secreted were then produced from liquid tumors (ascites) induced in the mouse by injection by the intra-peritoneal route of the selected hybridomas.

(17) II. Biochemical Characterisation

(18) After purifying Rendomab-B49, Rendomab-B41 and Rendomab-B36, the characterisation of their biochemical properties was conducted.

(19) The isotyping of the heavy and light chains of Rendomab-B49 was made using the “Rapid ELISA Mouse mAb Isotyping” kit from Piercell. It is an immunoglobulin of isotype 1, G type, for the heavy chain and kappa for the light chain. Rendomab-B49 is thus an IgG1/kappa type immunoglobulin. Rendomab-B41 and Rendomab-B36 are, an IgG1/kappa type immunoglobulin and an IgG3/kappa type immunoglobulin, respectively.

(20) After purifying Rendomab-B49, Rendomab-B41 and Rendomab-B36, their binding specificity was established by flow cytometry (Facs Calibur, Becton Dickinson BD Bioscience), by indirect labelling using a commercial fluorescent anti-mouse secondary antibody (Life Technologie: Alexa Fluor® 488 F(ab′)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*).

(21) CHO-ETBR cells were separated into 2 groups. One of the groups was incubated beforehand in a culture medium for 2 h at 37° C. in the presence of endothelin 1 at a final concentration of 300 nM so as to internalise the ETBR receptor for the purpose of demonstrating the recognition specificity of the 3 Rendomab antibodies to ETB-R.

(22) Then, the cells were aliquoted in a 15 ml falcon tube (300 000 cells/tube) in the presence of an increasing concentration (concentration range from 0.04 nM to 800 nM two by two) of antibody Rendomab-B49. After 2 h incubation at 4° C., the cells are washed three times in a PBS buffer and then incubated for 1 h at 4° C. with the secondary antibody at a final concentration of 4 μg/mL. The cells are then washed 3 times with a PBS buffer and then analyzed with FACs Calibur after counting 30 000 cells.

(23) III. Binding Properties on Glioblastoma Cells

(24) III.1. Protocol

(25) The cells are cultured either in neurospheres (suspended culture) or in adherence on glass slides covered with poly D Lysine/Laminine. The cells are then bound by a 4% paraformaldehyde solution for 15 min at room temperature, and then washed twice with PBS (SigmaAldrich). The non-specific sites are blocked and the cells are permeabilized by a PBS solution+5% donkey serum+0.1% Triton, for 30 min.

(26) The primary antibody Rendomab-B49, diluted in the previous solution at 1 μg/mL is contacted with the cells overnight at 8° C. After 2 rinses with PBS, the cells are incubated with a commercial secondary antibody at a final concentration of 4 μg/ml either coupled with Alexa 488 (Life Technologie: Alexa Fluor® 488 F(ab′)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*) or coupled with Alexa 680 (Life Technologie Alexa Fluor® 680 F(ab′)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*) for 2 h according to the recommendations of the provider, and then rinsed twice with PBS.

(27) The cells are briefly incubated with a 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole) solution to view the nuclei, and then after washing with PBS, the coverslips are mounted between slip and slide with an ad hoc assembling medium for observation. The photographs are taken with a Zeiss microscope provided with a 400 magnification apotome module.

(28) III.2. Results

(29) The affinity close to the nanomolar range for Rendomab-B49, Rendomab-B41 and Rendomab-B36 and their exclusive specificity for the human endothelin receptor sub-type B are illustrated in FIG. 1.

(30) The binding curve observed for Rendomab-B49 (FIG. 1A), Rendomab-B41 (FIG. 1B) and Rendomab-B36 (FIG. 1C) is characteristic of the binding of an antibody to its target with a saturation plateau.

(31) In addition, the pre-incubation of CHO-ETBR cells for 2 h at 37° C. in the presence of 300 nM of endothelin 1 causes the internalisation of ETB-R, which results in a drop of more than 50% of the Rendomab-B49, Rendomab-B41 and Rendomab-B36 binding under these conditions thus demonstrating the binding specificity of these antibodies for ETB-R. The apparent dissociation constant K.sub.D of the antibodies is determined by taking the value of the concentration giving a MFI equal to 50% the value of the MFI at the plateau. This constant is close to 1 nM for the 3 antibodies.

(32) Then, the absence of binding of Rendomab-B49, Rendomab-B41 and Rendomab-B36 on CHO-WT cells non transfected by ETBR is noted showing that the binding observed on CHO-ETBR cells is not due to a membrane protein of the CHO cells.

(33) Binding experiments on tumor cells isolated from biopsies of patients with a glioblastoma.

(34) In FIG. 2, there is an absence of labelling of neurospheres isolated from a high grade glioblastoma tumor with the antibody Rendomab-B1 (FIG. 2A) whereas it is observed, on the same cells, a very strong fluorescent labelling with the antibody Rendomab-B49 (FIG. 2B).

(35) Likewise, FIG. 3 shows a strong fluorescent labelling by the antibody Rendomab-B49 (FIG. 3B) of tumor cells isolated from a low grade glioblastoma tumor, these cells being also labelled with DAPI (FIG. 3A).

(36) Comparable results are obtained with the antibodies Rendomab-B41 and Rendomab-B36. To that end, FIG. 4 shows a strong fluorescent labelling with the antibody Rendomab-B36 on tumor cells isolated from a glioblastoma tumor.

(37) IV. Molecular Cloning

(38) The cloning of nucleic precursors coding the heavy chain and the light chain of Rendomab-B1 was made using the kits: “Gene-Elute/total RNA” (Sigma) and “RACE-PCR” (Invitrogen).

(39) The nucleic sequences deduced in amino acids of the variable domains of the light chain (VL) and the heavy chain (VH) of Rendomab-B49, Rendomab-B41 and Rendomab-B36 are given in FIGS. 5 to 7 respectively.

(40) V. Epitopic Mapping

(41) V.1. Materials and Methods

(42) The mapping of the epitope recognised by Rendomab-B49, Rendomab-B41 and Rendomab-B36 at the ETB-R surface was made by a “Pep-scan” technique in collaboration with and according to the protocols developed within UMR 3145 “SysDiag” CNRS/BioRad located in Montpellier (Dr. Claude Granier). The sequence of the human endothelin receptor sub-type B exhibits the following amino acid sequence:

(43) TABLE-US-00019 (SEQ ID NO: 42) MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPT KTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETF KYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLH IVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRY RAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGS YLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM LRKKSGMQIALNDHLKORREVAKTVFCLVLVFALCWLPLHLSRILKLTLY NQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSC LCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS.

(44) For this, 144 peptides of the 12 amino acids each offset by one amino acid were used. These peptides correspond to all the ETB-R sequences displayed on the extra-cytoplasmic side of the membrane.

(45) In order to reveal the epitopic peptide(s) recognized by Rendomab-B49, the “Pep-scan” membrane was treated according to the following protocol: humidification of the membrane in an ethanol bath; 3 washes in 25 ml of TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4) for 10 min under agitation at ambient temperature; saturation of the membrane with 25 ml of the saturation buffer (TBS, 5% skimmed milk powder, 0.1% Tween 20) for 30 min under agitation at ambient temperature; incubation with 25 ml of saturation buffer containing the Rendomab-B49 antibody at a final concentration of 1 μg/ml overnight at 4° C. under agitation; 3 short washes (30 seconds) with TBS buffer then 3 washes of 10 min under agitation with 25 ml of TBST buffer (TBST=TBS+0.1% Tween 20); incubation with 25 ml of saturation buffer containing the goat anti-mouse secondary antibody diluted at 1/5,000 for 30 min at ambient temperature under agitation; 3 short washes (30 seconds) with TBS buffer then 3 washes of 10 min under agitation with 25 ml of TBST buffer; revelation of the membrane by immersing it in the revelation solution from Pierce (Pierce ECL Plus Western Blotting Ref: 32132) for 5 min and by the signal acquisition in automatic mode by the system ChemiDoc™ of BioRad).

(46) V.2. Results

(47) The results of the epitopic analysis of Rendomab-B49 are presented at FIG. 8. The peptide sequences hybridizing with high intensity are C5, C6, C7, C8, C9 and C10 peptides (FIG. 8A).

(48) Their alignment makes it possible to identify the epitope predominantly recognized by Rendomab-B49: the latter is EVPKGDR corresponding to the sequence from amino acid 70 to amino acid 76 in SEQ ID NO: 42 (FIG. 8B). As soon as the glutamic acid E70 is lacking in peptide C11 of sequence VPKGDRTAGSPP corresponding to the sequence from amino acid 71 to amino acid 82 in SEQ ID NO: 42, the antibody binding decreases significantly (FIG. 8A).

(49) The location of the epitopic peptide in the sequence of the human endothelin receptor sub-type B is presented at FIG. 8C. This sequence is at the N-terminal end of the receptor.