FR2965810A1 - ANTAGONIST ANTIBODY OF ENDOTHELIN RECEPTOR B-SUBTYPE B AND USES THEREOF - Google Patents

Abstract

The present invention relates to a particular monoclonal endothelin receptor B subtype antagonist antibody, a fragment or a derivative thereof. The present invention also relates to the therapeutic, diagnostic or research use of such an antibody.

Description

ANTAGONISTIC ANTIBODY OF SUBType B OF ENDOTHELIN RECEPTORS AND USES THEREOF

TECHNICAL FIELD The present invention belongs to the technical field of antibodies, their therapeutic and diagnostic use as well as their use as a research tool. More particularly, the present invention provides an antibody, advantageously monoclonal, specific for the native and functional conformation of endothelin receptor subtype B and in particular human endothelin receptors, said antibody being capable of blocking the pharmacological properties of such endothelin receptors. receptors. The present invention also relates to the use of this antibody for therapeutic and diagnostic purposes as well as for research purposes. STATE OF THE PRIOR ART The receptors of the various endothelins (designated ET1, ET2 and ET3 in humans) belong to the family of receptors with 7 transmembrane domains also designated GPCRs for "G protein-coupled receptors". Endothelin receptors have two main subtypes in humans: subtype A (ETA-R) and subtype B (ETB-R). 2 The endothelin axis and its receptors is involved in several pathophysiological functions and dysfunctions. As non-limiting examples, mention may be made of arterial hypertension, atherosclerosis, coronary heart disease, renal dysfunction, cerebrovascular diseases, Crohn's disease, pulmonary fibrosis, asthma, etc. ( See R. Shah's review, 2007, "Endothelins in Health and Disease," Eur.

Int. Med., Vol. 18, pp. 272-282). In addition, endothelin receptors have also been shown to be associated with the development of many cancers, promoting the proliferation, survival and dissemination of cancer cells as well as angiogenesis (Bagnato & Rosano, 2008, "The endothelin axis in cancer J. of Biochem. & Cell Biology, Vol 40, pp. 1443-1451). Concerning the endothelin receptor subtype B, the latter exhibits a modification of its level of expression, in particular in melanomas, colon cancer, Kaposi's sarcoma, glioblastomas (brain tumors), and in cases of bladder cancer. It is also established that endothelin receptor subtype B is involved in the lack of recognition of certain cancer cells, especially ovarian cancer cells by the immune system, by inducing a strong reduction in infiltration. lymphocyte (Buckanovich et al., 2008, "Endothelin B receptor mediates the endothelial barrier to the eye homing to tumors and disables immune therapy," Nature Medecine, 14, pages 28-36). Thus, blocking the physiological and / or pharmacological properties of the endothelin receptors and, more particularly, the endothelin receptor subtype B therefore appears particularly relevant in human clinical biology (Kandalaft et al., 2009, "Endothelin B receptor, a new target in cancer immune therapy ", Clin Cancer Res, 15, pages 4521-4528).

Different strategies exist to block receptors, particularly for therapeutic purposes, such as the use of peptide or non-peptide antagonists and the use of monoclonal antibodies antagonizing these receptors. However, in the passive immunotherapy arsenal of cancers using monoclonal antibodies (15 antibodies have been approved to date), none targets GPCRs. Indeed, the difficulty in obtaining monoclonal antibodies specific to GPCRs is a consequence of the problems related to obtaining these same receptors, in a native and functional form, outside of their membrane context.

The article by Kondoh et al., 1990, entitled "Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization" in BBRC, vol. 172, pp. 503-510 discloses the binding properties of 4 monoclonal antibodies (A2, G9, E7 and G10) to solubilized endothelin receptor complexes present on the surface of lung membranes of rats. The G9 and G10 antibodies are immunoglobulins of type G and isotype 2a (IgG2a), whereas antibodies A2 and E7 are immunoglobulins IgG1. If these four antibodies are specific for endothelin-solubilized receptors, the article by Kondoh et al. provides no information as to the specificity of these antibodies (ETA-R and / or ETB-R), as to the recognition of receptors of human origin, or as to any possible antagonistic property. Patent Application JP 2008280266 in the name of Seikisui Chemical Co Ltd and published November 20, 2008 relates to a monoclonal antibody called ha21. It is a mouse immunoglobulin of IgG2a / kappa isotype, which is exclusively specific for the human endothelin receptor subtype A, and which does not recognize murine or rat receptors. The analysis of the ETA-R sequence, carried out via overlapping peptides, made it possible to identify a region of the second extracytoplasmic loop of ETA-R as part of the epitope recognized by hA21. Finally, these authors have shown that the antibody hA21 was able to block the intracellular release of calcium, but with a efficiency 10 times lower than that obtained with the ETA-R peptide antagonist that is "BQ-123", (5-amino acid cyclopeptide of formula D-tryptamine-D-aspartic acid-L-proline-D-valine-L-leucine, CAS: 136553-81-6). US Patent Application 2010/003240 filed on behalf of the University of New York and published January 7, 2010 relates to therapeutic protocols and pharmaceutical mixtures in the context of the treatment and prevention of cancers and especially melanomas. For this purpose and more specifically, the use of ETB-R antagonists is claimed. The examples described for these antagonists relate only to modified forms of endothelin-1, but by extension, the use of ETB-R antagonist antibodies is claimed without any particular example of such antibodies being described. or provided. Finally, the article by Yamaguchi et al., 2004, entitled "Characterization and application of monoclonal antibodies against human endothelin B receptors in insect cells" in Biotechnology Letters, vol. 26, pp. 293-299 concerns the characterization of the binding properties of mouse monoclonal antibodies obtained after protein immunization with recombinant human ETB-R produced in insect cells.

Four of them have a similar affinity (in the nanomolar range) for ETB-R, while the fifth is 10 times less affine. The epitopic analysis of 3 of them (N-6, N-3 and N-1) revealed that they recognize the N-terminal domain of ETB-R and more particularly the sequence corresponding to the amino acids 27- ETB-R for N-6; the sequence corresponding to amino acids 27-41 of ETB-R for N-3 and the sequence corresponding to amino acids 71-85 for N-1. Finally, these 5 antibodies are able to recognize COS cells overexpressing ETB-R. It should be noted that no experiments presenting the ETA-R specificity study are provided in this publication, the only evidence that the monoclonal antibodies described are ETB-specific antibodies. R. These antibodies also recognize ETB-R expressed on the surface of higher eukaryotic cells. However, nothing is described regarding their ability to prevent the binding of natural ligands (endothelins), nor to block the release of intracellular calcium. It is therefore not, as is, ETB-R antagonistic antibodies.

The inventors have therefore set themselves the goal of obtaining antibodies capable of specifically recognizing the subtype B of the endothelin receptor, having effective antagonist properties rendering them useful both in the therapeutic and diagnostic fields. DISCLOSURE OF THE INVENTION The present invention makes it possible to solve the technical problems as defined above and to achieve the goal that the inventors have set themselves. In fact, the work of the inventors made it possible to develop a particular immunization and selection strategy. This strategy consists, first of all, of carrying out several gene immunizations by injection into the tibial muscle of plasmid DNA mice containing the DNA encoding ETB-R. These injections are systematically followed by in vivo electroporation. After 3 gene immunizations, a first immunization boost by injection of higher eukaryotic cells, overexpressing ETB-R in its functional form was performed. The functionality of ETB-R expressed on the surface of the cells used for the immunization boost was verified by various radioactively labeled or fluorescently labeled endothelin-1 binding experiments. This strategy coupled with a procedure for screening hybridomas in ELISA-eye then by flow cytometry privileged the obtention of specific monoclonal antibodies of ETB-R in its native conformation. This approach also has the advantage of not needing the extracted and purified interest receptor, which is still a difficult challenge today. By this strategy, the inventors were able to select a monoclonal antibody antagonist of the pharmacological properties of subtype B of endothelin receptors and in particular of human subtype B, hereinafter referred to as Rendomab-B1. Regarding the antagonistic nature of this antibody, it should be emphasized that, in comparison with the only peptide antagonist specific for ETB-R (BQ-788) available to date, the antibody which is the subject of the present invention is a 10-fold antagonist. more effective (see paragraph III below). In addition, the interest, as compared with BQ-788 for example, is also related to the fact that the antibody according to the present invention is capable, via its Fc domain, of recruiting different cellular effectors such as cytotoxic T lymphocytes, and therefore to induce the lysis of the targeted cells (ADCC), or molecular as the complement. The labeling of the antibody with a radionuclide such as yttrium-90 is also conceivable, as well as its use in imaging in a smaller recombinant form such as a fragment as defined below.

The present invention relates to an endothelin receptor subtype B antagonist antibody, fragment or derivative thereof. Before going into more detail in the description of the invention, we recall or propose the following definitions. The terms "antibody" and "immunoglobulin" are equivalent and may be used interchangeably in the present invention. An antibody is a glycoprotein comprising at least two heavy chains (H) and at least two light chains (L) interconnected by one or more disulfide bridges. Each heavy chain comprises a variable region (or domain) (VH) and a constant region comprising 3 domains, usually designated CH1, CH2 and CH3. Each light chain comprises a variable region (or domain) (VL) and a constant region comprising a single domain, usually designated CL. The variable regions of the heavy and light chains involved in antigen recognition can be further subdivided into 3 hypervariable regions, also called complementarity determining regions (CDRs), framed by 4 more conserved regions, also called framework regions (FR). The organization of each heavy chain (or light chain) variable region is, from the N-terminus to the C-terminus, as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In the context of the present invention, the definition of CDR and FR that has been used is that of the IMGT (the international ImMunoGeneTics database http://imgt.cines.fr:8104). The calculations of the percentages of identity of the CDR sequences mentioned and claimed hereafter are therefore to be taken into account on the basis of this annotation. In addition, the term "antibody" includes, in the context of the present invention, not only complete antibody molecules but also fragments and derivatives thereof. By "antibody fragment" is meant, in the context of the present invention, both a monovalent fragment which has a single antigen-binding site and a divalent fragment which has two antigen-combining sites. . Among these fragments, mention may be made of Fab, F (ab ') 2r Fv fragments, and other fragments which retain the antigen binding site (scFv and diabody). An Fab fragment is a monovalent fragment consisting of the entire light chain and a portion of the heavy chain (Fd) comprising the VH and CHI domains as previously defined. An F (ab ') 2 fragment is a divalent fragment corresponding to the association of two Fab fragments connected by the disulfide bridges present at the hinge region of the immunoglobulins ("Hinge") located between the constant domains CH1 and CH2. A Fv fragment is a monovalent fragment consisting only of the variable regions VL and VH of the light and heavy chains of an antibody. An scFv fragment is a monovalent polypeptide fragment, obtained only by genetic engineering, corresponding to the variable domains linked by a peptide bond. A diabody is a recombinant and divalent antibody molecule consisting of two scFv molecules upside down because of a peptide link that is too short to allow the formation of scFv. The fragments according to the invention also cover the fragments as mentioned above, the half-life of which has been increased by chemical modification, in particular by incorporation into a liposome or by the introduction of a poly (alkylene) glycol such as a polyethylene. glycol (PEG), this technique being called "PEGylation" and giving fragments such as Fab-PEG, F (ab ') 2-PEG or Fv-PEG. Recombinantly, it is also possible to generate single or fused fragments of the antibody according to the present invention, exhibiting more effective and better controlled properties of solid tumor penetration and pharmacokinetics. The antibody fragments useful in the context of the present invention may be natural or recombinant. By "antibody derivative" is meant, in the context of the present invention, fragments of antibodies obtained by genetic engineering such as single chain Fv (scFv) and single domain (dAb) antibodies. The term also includes antibody-like molecules that can be produced using phage display techniques or other random selection techniques for molecules that bind to subtype B of the invention. endothelin receptors or specific regions of this subtype. Thus, the "antibody fragments" and "antibody derivatives" cover all molecules that contain a structure, advantageously peptide, which is part of the recognition site (that is, the part of the antibody which binds to or combines with the epitope or antigen) of an antibody according to the present invention. In addition, the fragments and antibody derivatives according to the present invention show an antagonistic activity towards the endothelin receptor subtype B. By "antagonist" is meant, in the context of the present invention, an antibody antagonist of the pharmacological properties of the subtype B endothelin receptors (ETB-R). More particularly, such an antibody is capable of decreasing, inhibiting or blocking one or more biological activity (s) of ETB-R. An antibody, fragment or derivative thereof, antagonist may act: by directly reducing, altering or completely blocking, directly or indirectly, the interactions between an ETB-R and an endothelin such as ET1, ET2 or ET3; by directly or indirectly interfering with the activation of ETB-R normally mediated by the binding of an endothelin such as ET1, ET2 or ET3; Interfering directly or indirectly with signal transduction normally mediated by the binding of an endothelin such as ET1, ET2 or ET3 to ETB-R; by disrupting or blocking the ETB-R acquisition of a homodimeric and / or oligomeric conformation or organization necessary for the expression of its pharmacological properties, such an antagonistic potential being, in this case, independent of the binding of the natural effectors endothelin ET-1, ET-2 and ET-3; and / or by inducing the internalization of ETB-R ("down-regulation"), which results in a sharp decrease or even disappearance of ETB-R from the surface of the targeted cells, such an effect, leading to the cessation of the effects induced by ETT-R binding of endothelins, in particular the effects responsible for the proliferation of cancer cells. More particularly, the antibodies according to the present invention cover antibodies which bind to ETB-R, which directly or indirectly interfere with the formation of the ETBR / endothelin complex such as ET1, ET2 or ET3. Among the biological activities of ETB-R that the antibody can reduce, inhibit or block, include proliferation, survival and / or dissemination of cells including cancer; the release of nitric oxide (NO); angiogenesis; Release of prostacyclin, VEGF (vascular endothelial growth factor) or MMP 20 13 (metalloproteinases); prevention of apoptosis; vasodilatation and / or vasoconstriction. The antibody of the present invention is also capable of lifting, at least partially, the lack of lymphocyte infiltration by the immune system at the level of the cancer cells.

The present invention relates to an endothelin receptor subtype B antagonist antibody having at least one heavy chain variable region comprising a CDR3 having at least 80% identity with the following amino acid sequence: RNAYDGYFDFW (SEQ ID NO: 2). In the context of the present invention, the amino acid sequences are given in accordance with the international 1-letter code. Advantageously, the antibody according to the present invention has at least one heavy chain variable region whose CDR1, CDR2 and CDR3 respectively have at least 80% identity with the following amino acid sequences: GFSLTTYA (SEQ ID NO: 1) : 4); IWTGGDT (SEQ ID NO: 6); - ARNYDGYFDFW (SEQ ID NO: 2).

The CDR1 of the heavy chain variable region of the antibody according to the invention has at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, and or at least 98% identity with the following amino acid sequence: GFSLTTYA (SEQ ID NO: 4). The CDR2 of the heavy chain variable region of the antibody according to the invention has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 95% identity. identity and / or at least 98% identity with the following amino acid sequence: IWTGGDT (SEQ ID NO: 6). The CDR3 of the heavy chain variable region of the antibody according to the invention has at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, and or at least 98% identity with the following amino acid sequence: RNAYDGYFDFW (SEQ ID NO: 2).

In particular, the antibody according to the invention has at least one heavy chain variable region comprising a CDR1, a CDR2 and a CDR3 respectively having the following amino acid sequences. GFSLTTYA (SEQ ID NO: 4); IWTGGDT (SEQ ID NO: 6); - ARNYDGYFDFW (SEQ ID NO: 2).

More particularly, the antibody according to the invention comprises a heavy chain variable region whose amino acid sequence exhibiting at least 80% identity with the following sequence: QVQLKESGPGLVAPSQSLSLTCTVTGFSLTTYAIVWVRQPPGKGLEW LGVIWTGGDTNYNSDLRSRLRIDKDNSRNQVFLKMSSLQTDDTARYYCARNYDGY FDFWGQGTTLTVSS (SEQ ID NO: 8).

Thus, the antibody according to the invention comprises a heavy chain variable region whose amino acid sequence has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the amino acid sequence SEQ ID NO: 8. More particularly, the antibody according to the invention comprises a heavy chain variable region whose sequence in amino acids corresponds to the amino acid sequence SEQ ID NO: 8.

In addition, the antibody according to the present invention has at least one light chain variable region whose CDR1, CDR2 and CDR3 respectively have at least 80% identity with the following amino acid sequences: - QNLLYSGNQKTY (SEQ ID N0: 10); - WAS (SEQ ID NO: 12;); QQYFIYPRT (SEQ ID NO: 14). Advantageously, the CDR1 of the light-chain variable region of the antibody according to the invention has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 95% identity. identity and / or at least 98% identity with the following amino acid sequence: QNLLYSGNQKTY (SEQ ID NO: 10). CDR2 of the light chain variable region of the antibody according to the invention has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 95% identity. identity and / or at least 98% identity with the following amino acid sequence: WAS (SEQ ID NO: 12). Finally, the CDR3 of the light chain variable region of the antibody according to the invention has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 95 % identity and / or at least 98% identity with the following amino acid sequence: QQYFIYPRT (SEQ ID NO: 14). In particular, the antibody according to the invention has at least one variable light chain region of which CDR1, CDR2 and CDR3 respectively present the following amino acid sequences. QNLLYSGNQKTY (SEQ ID NO: 10); - WAS (SEQ ID NO: 12); QQYFIYPRT (SEQ ID NO: 14).

More particularly, the antibody according to the invention comprises a light chain variable region whose amino acid sequence exhibiting at least 80% identity with the following amino acid sequence: DIVMSQSPSSLIVSVGQKVTMNCKSSQNLLYSGNQKTYLAWYQQKPG QSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCQQYFIYPR TFGGGTKLEIKR (SEQ ID NO: 16).

Thus, the antibody according to the invention comprises a light chain variable region whose amino acid sequence has at least 80% identity and can have at least 85% identity, at least 90% identity, at least 50% identity. at least 95% identity and / or at least 98% identity with the amino acid sequence SEQ ID NO: 16. More particularly, the antibody according to the invention comprises a light chain variable region whose sequence in amino acids corresponds to the amino acid sequence SEQ ID NO: 16.

Advantageously, the antibody according to the invention has a light chain variable region and a heavy chain variable region as previously defined.

The light chain of the antibody according to the invention is typically a kappa light chain. The heavy chain of the antibody according to the invention is in particular a gamma 2b heavy chain. In particular, the antibody according to the present invention is a type G immunoglobulin. More particularly, the antibody according to the present invention is an immunoglobulin of type IgG2b / kappa.

The work of the inventors has made it possible to show that the endothelin receptor subtype B antagonist antibody, which is the subject of the present invention, selectively binds to three extracellular segments of ETB-R, segments respectively consisting of acid residues. Amines 45 to 71, amino acid residues 80 to 94 and amino acid residues 244 to 272 of human endothelin receptor subtype B (SEQ ID NO: 17). The sequences of the human subtype B of the endothelin receptors selectively recognized by the antibody object of the present invention are: 18 - IMTPPTKTLWPKGSNASLARSLAPAEV corresponding to amino acid residues 45 to 71 of the amino acid sequence of subtype B endothelin receptors (SEQ ID NO: 17); SPPRTISPPPCQGPI corresponding to amino acid residues 80 to 94 of the amino acid sequence of endothelin receptor subtype B (SEQ ID NO: 17); - TMDYKGSYLRICLLHPVQKTAFMQFYKTA corresponding to amino acid residues 244 to 272 of the amino acid sequence of subtype B endothelin receptors (SEQ ID NO: 17). These sequences mentioned above, were identified via overlapping peptides corresponding to the set of extracellular domains of the human ETB-R receptor. By "antibody which selectively binds" at least one specified domain or region of ETB-R, in particular human ETB-R, is meant, in the context of the present invention, an antibody which binds the domain (s) specific with greater affinity than any other region of ETB-R. Advantageously, the antibody binds the specified domain (s) of ETB-R with an affinity of at least 2, or at least 5, or at least 10, or at least 50 times greater than that which it is present for any other region of ETB-R. This binding can be determined by methods well known in the art such as flow cytometry, radioimmunoassay (RIA), confocal microscopy, enzymo-immunoassay labeling (EIA) by direct or indirect revelation of the antibody to test (ELISA). The antibody which is the subject of the present invention may be obtained from an animal immunized with endothelin receptor subtype B or against a fragment of this receptor comprising the epitope (s) recognized by the antibody according to the present invention. The immunized animal may be any animal usually used for the production of antibodies such as a mouse, a rat, a rabbit, a goat, a dog, a horse or a camelid such as camel or llama. The antibody thus obtained may be purified on an affinity column on which the endothelin receptor subtype B or one of the sequences recognized specifically by the antibody according to the invention has been immobilized beforehand. This purification may also involve affinity chromatography on protein A. In the context of the present invention, the antibody may be a polyspecific or monospecific polyclonal antibody, or a monoclonal antibody. Advantageously, the antibody of the present invention is monoclonal. A "monoclonal antibody" refers, by definition in immunology, to an antibody obtained from a substantially homogeneous antibody population i.e. a population of identical antibodies, a relatively small amount of which may possibly have a mutation. A monoclonal antibody is obtained from the proliferation of a single cell clone such as a hybridoma. More particularly, the antibody according to the present invention is the monoclonal murine antibody obtained from the hybridoma deposited at the CNCM on September 30, 2010 under the number CNCM I-4370. The present invention also relates to such a hybridoma.

Alternatively, the antibody according to the present invention may be a chimeric antibody ie an antibody which contains variable regions or hypervariable regions of heavy and light chain (s) derived from an antibody of a species given in combination with heavy and light chain constant regions derived from an antibody of another species heterologous to the above.

A first variant of the present invention corresponds to a chimericized antibody and in particular a chimeric monoclonal antibody, that is to say an antibody whose variable domains derived from the murine antibody previously described are associated with constant domains of human origin. . It should be recalled that several therapeutic antibodies used in humans are chimerised antibodies. A second particularly advantageous variant may be a humanized antibody and in particular a humanized monoclonal antibody. Indeed, it is preferable to use a humanized antibody, if the latter is to be administered repeatedly to a human subject. In the case of a humanized monoclonal antibody according to the present invention, the latter may be prepared by insertion of the CDRs of a murine antibody and in particular of the murine antibody derived from the hybridoma deposited on September 30, 2010 at the CNCM under the number CNCM I-4370 within a human antibody, regardless of its isotype (IgG, IgA, IgM). The humanized antibodies can be made using the techniques and approaches described in the article by Verhoeyen et al., 1988, entitled "Reshaping human antibodies: Grafting antilyssozyme activity", in Science, vol. 239, pages 1534-1536 and in US Patent 4,816,567 in the name of Genentech and published March 28, 1989.

The antibodies may also be human antibodies in the sense that they have the amino acid sequence of human anti-ETB-R antibodies via methods of preparation known in the art that do not require vaccination of humans. For example, such antibodies can be obtained by gene immunization / transgenic mouse cell immunoassociations that are available and which in essence contain human immunoglobulin genes (see Vaughan et al., 1998, entitled " Human antibodies by design "in Nature Biotechnol, vol 16, pages 535-539). Alternatively, such antibodies can be obtained by cloning cDNAs encoding human B cells against ETB-R. The present invention also relates to an isolated polynucleotide selected from the following polynucleotides: i) a polynucleotide encoding an antibody as previously defined; ii) a polynucleotide complementary to the polynucleotide as defined in (i); iii) a polynucleotide of at least 18 nucleotides capable of hybridizing under high stringency conditions to the polynucleotides as defined in (i) and (ii). By "polynucleotide" is meant in the context of the present invention a nucleic acid, a nucleic sequence, a nucleic acid sequence, an oligonucleotide, a polynucleotide sequence, a nucleotide sequence, a single-stranded DNA, a DNA double-stranded or RNA. A polynucleotide according to the present invention may comprise natural nucleotides and unnatural nucleotides. The polynucleotide according to the invention does not correspond to a nucleotide sequence in its natural state i.e. in its natural chromosomal environment. On the contrary, the polynucleotide according to the invention has been isolated and optionally purified, and its environment has therefore been modified. The polynucleotide according to the invention can also be obtained by genetic recombination or by chemical synthesis. The conditions of high stringency correspond to temperature and ionic strength conditions which make it possible to maintain hybridization between two complementary nucleotide sequences. Those skilled in the art will be able to determine the most stringent conditions of high stringency, particularly as a function of the size of the nucleotide sequences, and this, referring to the teaching of Sambrook et al. (Molecular cloning, 1989, Noland C. ed., NY: Cold Spring Harbor Laboratory Press).

The polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity. Identity% with the following nucleotide sequence: GCC AGA AAT TAT GAT GGA TAC TT GAC TTC TGG (SEQ ID NO: 1).

Alternatively, the polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the sequence complementary to the following nucleotide sequence: GCC AGA AAT TAT GAT GGA TAC TT GAC TTC TGG (SEQ ID NO: 1).

In an embodiment, the polynucleotide according to the present invention comprises at least three nucleotide sequences having at least 80% identity, at least 85% identity, at least 24 90% identity, at least 95% identity. identity and / or at least 98% identity with respectively the following nucleotide sequences: GGC TTC TCA TTA ACC ACT TAT GCT (SEQ ID NO: 3), ATA TGG ACT GGT GGA GAC ACA (SEQ ID NO: 5 ) and - GCC AGA AAT TAT GAT GGA TAC TT GAC TTC TGG (SEQ ID NO: 1). Advantageously, the polynucleotide according to the present invention comprises at least three nucleotide sequences corresponding respectively to the nucleotide sequences SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 1. It is clear that the three sequences listed above (ie SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 1) must be organized relative to each other so that the polypeptide obtained following the translation of the polynucleotide according to the invention comprises 3 peptide sequences presenting at least 80% identity and advantageously 100% identity with the CDR1, CDR2 and CDR3 of the variable region of the heavy chain as previously defined. In particular, the polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the following nucleotide sequence: CAG GTG CAG CTG GAG TCA GGA CCT GGC CTG GTG GCG CCC TCT CAG AGC CTG TCC CTC ACG TGC ACT GTC ACT GGC TTC TCA TTA ACT ACT TAT GCT ATT GTT TGG GTT CGC CAG CCA 25 CCG GGA AAG GGT CTG GAG TGG CTT GGA GTA ATA TGG ACT GGT GGA GAC ACA AAT TAT AAT TCA GAT CTC AGA TCT AGA TTG CGC ATC GAC AAA GAC AAC TCC AGG AAT CAA GTT TTC TTA AAA ATG AGC AGT CTA CAA ACT GAT GAC ACA GCC CGG TAC TAC TGT GCC AGA AAT TAT GAT GGA TAC TT GAC TTC TGG GGC CAG GGC ACC ACT CTC ACA GTC TCC TCA (SEQ ID NO: 7). More particularly, the polynucleotide according to the present invention comprises at least one nucleotide sequence corresponding to the nucleotide sequence SEQ ID NO: 7.

As a variant of this embodiment, the polynucleotide according to the present invention comprises at least three nucleotide sequences having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity. identity and / or at least 98% identity with respectively the complementary sequences of the nucleotide sequences SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 1.

Advantageously, the polynucleotide according to the present invention comprises at least three nucleotide sequences corresponding respectively to the sequences complementary to the nucleotide sequences SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 1.

In particular, the polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 98% identity with the sequence complementary to the nucleotide sequence SEQ ID NO: 7. More particularly, the polynucleotide according to the present invention comprises at least one nucleotide sequence corresponding to the sequence complementary to the nucleotide sequence SEQ ID NO: 7.

In one embodiment, the polynucleotide according to the present invention comprises at least three nucleotide sequences having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity. identity and / or at least 98% identity with respectively the following nucleotide sequences: CAG AAC CTT TTA TAT AGT GGC AAT CAA AGA ACC TAC (SEQ ID NO: 9), TGG GCG TCC (SEQ ID NO: 11) and CAA CAA TAT TTTATC TAT CCT CGG ACG (SEQ ID NO: 13). Advantageously, the polynucleotide according to the present invention comprises at least three nucleotide sequences corresponding respectively to the nucleotide sequences SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13. It is clear that the three sequences listed above (ie SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13) must be organized relative to each other so that the polypeptide obtained following the translation of the polynucleotide according to the invention comprises 3 peptide sequences presenting at least 80% identity and advantageously 100% identity with the CDR1, CDR2 and CDR3 of the variable region of the light chain as previously defined. In particular, the polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the following nucleotide sequence: GAC ATT GTG ATG TCA CAG TCT CCA TCC CTA ATT GTG TCA GTT GGA CAG AAG GTT ACT ATG AAC TGC AAG TCC AGT CAG AAC CTT TTA TAT AGT GGC AAT CAA AAG ACC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG TCT CCT AAA CTG CTG ATT TAT TGG GCG TCC ACT AGG GAA TCT GGG GTC CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT GTG AAG GCT GAA GAC CTG GCA ATT TAT TAC TGA CAA CAA TAT TTT ATC TAT CCT CGG ACG TTC GGT GGA GGC ACC AGE CTG GAA ATC AAA CGG (SEQ ID NO: 15). More particularly, the polynucleotide according to the present invention comprises at least one nucleotide sequence corresponding to the nucleotide sequence SEQ ID NO: 15.

As a variant of this embodiment, the polynucleotide according to the present invention comprises at least three nucleotide sequences having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the sequences complementary to the nucleotide sequences SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13. Advantageously, the polynucleotide according to the present invention comprises at least three nucleotide sequences corresponding respectively to the sequences complementary to the nucleotide sequences SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13. In particular, the polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity and / or at least 98% identity with the sequence complementary to the SEQ nucleotide sequence ID NO: 15. More particularly, the polynucleotide according to the present invention comprises at least one nucleotide sequence corresponding to the sequence complementary to the nucleotide sequence SEQ ID NO: 15.

In a third embodiment, the polynucleotide according to the present invention comprises at least the nucleotide sequence (s) as defined in the embodiment and at least the (or) the nucleotide sequence (s) as defined in the second embodiment.

By "percentage identity" between two amino acid sequences (or between two nucleotide sequences) is meant, in the context of the present invention, a percentage of identical amino acid residues (or nucleotides) between the two Compared sequences, this percentage being obtained after implementation of the best alignment (optimum alignment) between the two sequences. Those skilled in the art know different techniques for obtaining such a percentage of identity and involving homology algorithms or computer programs such as the BLAST program. The percent identity is statistical and the differences between the two sequences are randomly distributed along these sequences. The differences between the two sequences may consist of different types of sequence modifications: deletions, substitutions or additions of amino acid residues (or nucleotides). In one embodiment, the modifications implemented in the sequences result in equivalent amino acid substitutions i.e. amino acids having structural homologies or not substantially altering the biological activity of the corresponding antibodies. In a second embodiment, the modifications implemented in the sequences result in substitutions by non-equivalent amino acids i.e. amino acids having no structural homology. These modifications are likely to improve the biological properties of the antibody ie improved affinity and / or specificity, expanded recognition spectrum, increased stability, reduced immunogenicity etc. Applied to the two embodiments previously described, these modifications, Insertions or deletions may target a CDR essential or not to the properties of the antibody according to the invention. Applied to the two embodiments previously described, these modifications, insertions or deletions may also target an RF region, knowing that such regions, within the variable domains of the antibodies, may, depending on the antibodies, also play a role in the expression of the properties. of the antibody according to the invention.

The present invention also relates to a cloning and / or expression vector containing at least one polynucleotide according to the present invention. Such a vector is particularly useful for transforming a host organism and expressing in the latter an antibody according to the present invention. The vector according to the present invention further comprises one (or more) element (s) which allows (s) the expression of the polynucleotide according to the present invention and / or the secretion of the product resulting from the translation of the polynucleotide according to the present invention. Among these elements, mention may be made of a constitutive or inducible promoter, a transcription initiation signal or a transcription termination signal, a translation initiation sequence or an end of translation signal. Advantageously, the vector according to the present invention comprises, operably linked, a promoter, a polynucleotide of the invention and a terminator element. The term "operatively linked to each other" according to the invention means elements interconnected so that the operation of one of the elements is affected by that of another. By way of example, a promoter is operably linked to a coding sequence when it is able to affect the expression of the latter. The regulatory elements for the transcription, translation and maturation of the peptides that the vector can comprise are known to those skilled in the art and the latter is capable of selecting them according to the host organism in which the expression or cloning must be done. The vector according to the present invention is advantageously chosen from a plasmid, a cosmid, a bacteriophage and a virus such as a baculovirus. In particular, the vector of the invention is a vector with autonomous replication comprising elements allowing its maintenance and its replication in the host organism as an origin of replication. In addition, the vector may include elements allowing its selection in the host organism such as, for example, an antibiotic resistance gene or a selection gene which ensures complementation with the respective gene deleted at the level of the genome of the host organism. Such cloning and / or expression vectors are well known to those skilled in the art and widely described in the literature.

The invention also relates to a host organism transformed with or comprising a polynucleotide 32 according to the present invention or a vector according to the present invention. By "host organism" is meant any uni- or multicellular organism, inferior or superior, in which a polynucleotide of the invention is introduced for the production of an antibody according to the present invention. Those skilled in the art know different methods for effectively introducing a polynucleotide into a host organism so that in the host organism the antibody encoded by said polynucleotide is produced. By way of example and non-exhaustively, this method can be electroporation, lipofection, a biological transformation of a plant using Agrobacterium tumefasciens, a thermal shock or a chemical process. Advantageously, the host organism is a microorganism such as a yeast, a bacterium or a fungus. The transformation of such microorganisms makes it possible to produce the antibody of the invention on a semi-industrial or industrial scale. Alternatively, the host organism is an animal cell such as a mammalian cell, a plant cell, an insect cell, an animal with the exception of a human, or a plant. Such host organisms can be used to produce an antibody according to the present invention. Indeed, a method for producing an antibody according to the present invention comprises the steps of. A) culturing a host organism according to the present invention and in particular a unicellular host organism in a culture medium and under appropriate conditions; b) recovering said antibody from the culture medium of said cultured host organism or from said cultured host organism.

The antibody according to the present invention is also modifiable in order to a) generate an antibody labeled with a radioactive isotope, a pro-drug, an enzyme or a toxin, and b) modify the binding specificity and / or affinity, and / or the stability, and / or the immunogenicity of said antibody targeting the cells which overexpress ETB-R, in particular cancer cells such as melanomas, glioblastomas, etc. The antibody according to the present invention is also modifiable in order to to couple it chemically or genetically to a peptide molecule; a protein molecule; a nucleic molecule such as DNA, RNA, RNAi, aptamer, PNA or LNA; a lipid molecule; a carbohydrate molecule or a chemical molecule.

The present invention therefore relates to a compound comprising an antibody according to the present invention conjugated to an element selected from the group consisting of a cytotoxic group, an easily detectable group or an effector group. By "cytotoxic group" is meant a directly or indirectly toxic group for the cells targeted by the antibody according to the present invention. By "directly cytotoxic" is meant a group which is in itself cytotoxic. By "indirectly cytotoxic" is meant a group which, although not cytotoxic in itself, can induce cytotoxicity, for example by its action on another molecule or by an additional action on it. In a first embodiment, the cytotoxic group is a cytotoxic chemotherapeutic agent, one skilled in the art knows various cytotoxic chemotherapeutic agents that can be used in the context of the present invention, the activity of these agents can be increased under irradiation. By way of illustrative and nonlimiting examples, mention may be made of alkylating agents such as mechlorethamine or chlorambucil, methotrexate, 5-fluorouracil, vinblastine, gemcitabine, fludarabine, nicotinamide, doxorubicin and mitomycin. L-asparaginase, cisplatin, taxol and its analogues / derivatives In a second form of implementation, the cytotoxic group is a cytotoxic (poly) peptide group such as ricin, abrin, exotoxin. Pseudomonas, TNFOE and interleukin 2. In a third form of implementation, the cytotoxic group is an indirectly cytotoxic chemotherapeutic agent. Also referred to as pro-drug is not or little cytotoxic as such but is able to give, especially following an enzymatic reaction or irradiation, a cytotoxic substance (or drug) especially as defined in the " form of implementation. By way of illustrative and nonlimiting examples, mention may be made of methotrexate-alanine; mitomycin phosphate, 5-fluorocytosine; photofrin and capecitabine.

In a fourth embodiment, the cytotoxic moiety is an indirectly cytotoxic (poly) peptide moiety.An indirectly cytotoxic poly (peptide) moiety is a polypeptide which exhibits enzymatic activity and can convert a relatively unimportant prodrug. Particularly toxic as defined in the third embodiment of a cytotoxic substance, especially as defined in the implementation form, Among such indirectly cytotoxic (poly) peptide groups, there may be mentioned a peptidase such as carboxypeptidase, aminopeptidase or endopeptidase, phosphatase, sulfatase, amidase, kinase, glycosidase, deaminase, reductase, and oxidase, In a fifth embodiment, the cytotoxic moiety is a nucleic acid molecule which is directly or indirectly cytotoxic such as antisense oligonucleotide or aptamer. Those skilled in the art are aware of various techniques for conjugating such groups to an antibody according to the present invention once it has been obtained or produced.

These techniques allow a covalent coupling between an antibody according to the invention and a cytotoxic group by making use of particular chemical groups carried by the antibody according to the invention and by the cytotoxic group. Among these particular chemical groups include a thiol group, an ester group, an amino group, an acid group and any chemical element likely to be implemented in the "click-chemistry". As an alternative and in particular when the cytotoxic group is a group of peptide nature, this conjugation may consist in producing the compound according to the invention in the form of a fusion compound by genetic recombination techniques, in which a polynucleotide comprises respective regions. encoding the antibody of the present invention and the cytotoxic moiety, adjacent to each other, juxtaposed or separated by a region encoding a peptide linker that does not destroy the desired properties of the final hybrid compound.

Whatever the technique used to conjugate an antibody according to the present invention with a cytotoxic group, the only constraint to be respected in the context of this conjugation is that the conjugated antibody retains its binding specificity for ETB-R and its character. antagonist, properties associated with those of the cytotoxic group. By "easily detectable group" is meant, in the context of the present invention, a group which can be detected by use of a suitable noninvasive appropriate detection technique such as microscopy, scintigraphy and imaging. magnetic resonance imaging (MRI). A compound according to the invention comprising such an easily detectable group is particularly suited to the field of imaging and diagnosis. In particular, it makes it possible to identify and locate sites at which ETB-R is overexpressed due to the ETB-R binding specificity of the antibody present in this compound.

In a first embodiment, the easily detectable group can be an enzyme or a molecule capable of generating a detectable and possibly quantifiable signal under particular conditions, such as when placing a suitable substrate in the presence of a suitable substrate. illustrative and nonlimiting examples include biotin, digoxygenine, 5-bromodeoxyuridine, alkaline phosphatase, peroxidase, acetylcholine esterase (AChE), glucose amylase and lysozyme.

In a second form of implementation, the easily detectable group may be a fluorescent, chemofluorescent or bioluminescent label such as fluorescein and its derivatives, rhodamine and its derivatives, GFP (for "Green Fluorescent Protein") and its derivatives. and umbelliferone, luminol, luciferase and luciferin In a third embodiment, the easily detectable moiety may be a radioactive label or isotope such as iodine-123, iodine-125, iodine-126, iodine-133, indium-111, 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. It should be noted that certain radioactive atoms used as easily detectable groups can also constitute cytotoxic groups because of the amount of radioactivity they can deliver. All that has previously been explained for the conjugation of the antibody according to the invention with the cytotoxic groups applies mutatis mutandis to the conjugation of the antibody according to the invention with the easily detectable groups. The conjugation of the antibody according to the invention with the easily detectable groups can also be carried out in relation to nano-objects, in order to densify their concentration, and thus to improve the emitted signal, the contrast or the toxicity. . In the case where this easily detectable group is a radioactive marker, the latter can be introduced into the peptide sequence of the antibody according to the invention. This introduction can take place during the synthesis of the antibody using one or more labeled amino acids. Alternatively, this introduction can take place following this synthesis by fixing the radioactive label on 39 residues of the peptide sequence of the synthesized antibody. For example, yttrium-90 can be attached via a lysine residue. In another variant, the radioactive marker may be indirectly attached to the antibody by known means. For example, EDTA or another chelating agent may be attached to the antibody of the invention and used to bind indium-111. The present invention relates to the use of a compound comprising an antibody and an easily detectable moiety as a highly effective diagnostic, prognostic and in vivo monitoring tool in medical imaging. The antibody format is chosen to generate the best signal-to-noise ratio and the best pharmacokinetics.

In other words, the present invention relates to a method for detecting and quantifying in vivo or in vitro expression or overexpression of endothelin receptor subtype B, comprising: a1) contacting a biological sample with a compound according to the present invention; b1) detecting any complex between said compound and said subtype B endothelin receptors. Such a method can be implemented to detect, diagnose, predict or follow a state in which the subtype B of endothelin receptors is overexpressed and in particular to detect, diagnose, predict or follow a cancerous state (presence, size and evolution cancerous tumors). By "effector group" is meant, in the context of the present invention, a group capable of specifically recognizing a cancer marker, or which allows the recruitment of a) an effector cell of the immune system ie cytotoxic T cells, or (b) the complement system. By "group capable of specifically recognizing a cancer marker" is meant, in the context of the present invention, a ligand of a cancer marker; an antibody identical to or different from an antibody according to the present invention; a protein; a peptide; or a nucleic molecule such as DNA, RNA, RNAi, aptamer, PNA or LNA. By "cancer marker", an ETB-R is considered as well as another membrane marker. In a 1st embodiment, the effector group recognizes a cancer marker, identical to or different from ETB-R, expressed on the surface of the cancer cells, thus ensuring better recognition specificity and therefore increased targeting of the cancer cells. In a second embodiment, the effector moiety has recognition specificity for a marker specifically present on the surface of cytotoxic T cells, such recruitment provides targeted lysis of cancer cells recognized by the antibody of the present invention. In a third embodiment, the effector moiety has recognition specificity for the complement system and, in particular, for the C1 protein or truncated C1q form, which initiates the cascade of molecular events that result in at the death of the targeted cell. Such recruitment provides targeted lysis of the cancer cells recognized by the antibody of the present invention. In a fourth embodiment, the effector group has a recognition specificity for the complement system and, in particular, for the C3 protein or its truncated C3b form, thus ensuring the recruitment of effector cells of the immune system, cells which induce the death of the targeted cell. Such recruitment provides targeted lysis of the cancer cells recognized by the antibody of the present invention.

The present invention relates to an antibody according to the present invention, a polynucleotide according to the present invention or a compound according to the present invention for use as a medicament. Thus, the present invention relates to a pharmaceutical composition comprising, as active ingredient, an antibody according to the present invention, or a polynucleotide according to the present invention or a compound according to the present invention and a pharmaceutically acceptable vehicle. By "pharmaceutically acceptable carrier" is meant according to the present invention, any substance which is added to an antibody, a polynucleotide or a compound according to the present invention to promote its transport, to avoid its substantial degradation in said composition, to increase its half-life. life and / or preserve its antagonistic properties. Advantageously, such a pharmaceutically acceptable vehicle is sterile and pyrogen-free. It is chosen according to the type of application of the pharmaceutical composition of the invention and in particular according to its mode of administration. Thus, the pharmaceutical composition according to the invention consists of at least one antibody, or a polynucleotide or a compound according to the present invention in free form or in the form of an addition salt with a pharmaceutically acceptable acid, in the state pure or in the form of a composition in which it is associated with any other pharmaceutically compatible product. The pharmaceutical compositions according to the invention can be used systemically; parenterally, for example intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular or subcutaneous; topically; oral; rectally; intranasally or by inhalation. As solid compositions for oral administration, it is possible to use tablets, pills, powders, etc. where the antibody, the polynucleotide or the compound according to the invention are mixed with one or more inert diluents conventionally used, and optionally to other substances such as, for example, a lubricant, a dye, a coating etc. As liquid compositions for oral or ocular administration, pharmaceutically acceptable suspensions, solutions, emulsions, syrups may be used. containing inert diluents conventionally used, and possibly other substances such as wetting agents, sweeteners, thickeners, etc. The sterile compositions for parenteral administration may be aqueous solutions or not, suspensions or emulsions. As the solvent or vehicle, water, propylene glycol, vegetable oils or other suitable organic solvents may be employed. These compositions may also contain adjuvants, such as wetting agents, isotonic agents, emulsifiers, etc. The compositions for topical administration may be, for example, creams, lotions, mouthwashes, nasal or eye drops or aerosol. The daily dose level of the antibody, polynucleotide or compound according to the present invention is usually 1 to 1000 mg per adult (i.e., about 0.015 to 15 mg / kg), administered in single or split doses. These doses are given for illustrative purposes only. In any case, the doctor will be able to determine the actual dose that is best for a given individual patient, and this varies according to the patient's age, weight and response.

The present invention relates to an antibody according to the present invention, a polynucleotide according to the present invention, a compound according to the present invention or a pharmaceutical composition according to the present invention for treating and / or preventing a disorder or pathology involving a dysfunction, direct or otherwise. in combination with another physiological pathway, the axis comprising an endothelin and at least one of its receptors such as, in particular, the subtype B endothelin receptors. Advantageously, such a disorder or such a pathology is selected from the group consisting of arterial hypertension, atherosclerosis, coronary heart disease, renal dysfunction, cerebrovascular diseases, Crohn's disease, pulmonary fibrosis, lymphadenopathy, asthma, angiogenesis and cancer. As cancers, there may be mentioned melanoma, colon cancer, Kaposi's sarcoma, glioblastoma, ovarian cancer and bladder cancer. In other words, the present invention relates to the use of an antibody according to the present invention, a polynucleotide according to the present invention, a compound according to the present invention or a pharmaceutical composition according to the present invention for the preparation of a medicament for the treatment, prevention and / or prophylaxis of a disorder or pathology as defined above. In other words, the present invention relates to a method for treating and / or preventing a disorder or pathology involving dysfunction, direct or in combination with another physiological pathway, of the axis comprising an endothelin and at least one its receptors such as, in particular, the endothelin receptor subtype B in a patient suffering or likely to suffer from such a disorder or such pathology. This method comprises administering to said patient an effective amount of an antibody according to the present invention, a polynucleotide according to the present invention, a compound according to the present invention or a pharmaceutical composition according to the present invention.

Finally, the present invention relates to the use of an antibody according to the present invention or a compound according to the present invention as a research tool particularly suitable for the study of signaling pathways associated with the endothelin axis / receptors. endothelin, as well as to progress in understanding the structural and functional characteristics of this family of receptors.

Other features and advantages of the present invention will become apparent to those skilled in the art on reading the examples below given for illustrative and non-limiting, with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the flow cytometry (Guava) studies, the fixation on CHO 46 cells overexpressing ETB-R (diamonds) of the 9 blood samples and containing the polyclonal antibodies, carried out during the treatment process. gene immunization. The samples are written BO to B8, and correspond to the times D0, D28, D42, D52, D59, D76, D83, D104 and D112 days. The responses of control mice immunized with empty plasmid vector (not containing ETB-R DNA) are indicated as dots. Figure 2 shows the isotyping of the Rendomab-B1 monoclonal antibody by ELISA technique. Figure 3 shows the binding curves of Rendomab-B1 on two different sources of CHO cells overexpressing ETB-R: CHOs obtained in the laboratory (large triangles) and commercial cells (points) provided by Lonza. In both cases, a nanomolar affinity was measured. No binding of Rendomab-B1 on cells overexpressing endothelin receptor subtype A (ETA-R) is observed (small triangles). FIG. 4 shows the labeling of CHO-ETB-R cells by Rendomab-B1 (FIG. 4A) and the absence of labeling by Rendomab-B1 of CHO cells that do not express the endothelin receptor subtype B (FIG. Figure 4B). Figure 5 shows the competitive experiments for binding Rendomab-B1 on CHO-ETB-R cells in the presence of increasing concentrations of endothelin 1, 2, 3 and sarafotoxin-c.

An irrelevant peptide of 21 amino acids was tested as a negative control. The IC50 values of the different competing peptides are indicated. FIG. 6 shows images obtained by confocal microscopy on CHO-ETB-R cells in the presence of 2 nM labeled Rendomab-B1 (FIG. 6A) and in the presence of 2 nM of labeled Rendomab-B1 and 20 nM of endothelin 1 (Figure 6B). Figure 7 shows the competition curves of Rendomab-B1 (solid circles) and irrelevant antibody (solid squares) to inhibit fluorescent ET-1 binding to CHO-ETB-R cells. Figure 8 shows the activation curves for measuring the EC50 on ETB-R of Rendomab-B1 (Figure 8A) and BQ-788 (Figure 8B) respectively for a range of Rendomab-B1 from 2 nM to 500 nM final on CHO-ETB-R cells and for a range of BQ-788 from 0.8 nM to 500 nM final on CHO-ETB-R cells. The ETB-R inactivation curves after addition of Rendomab-B1, BQ-788, ET1 or an irrelevant antibody of the same isotype are also presented. FIG. 9 shows the nucleic and deduced amino acid sequences of the variable domains of the light chain (VL) (FIG. 9A) and of the heavy chain (VH) (FIG. 9B) of the specific murine IgG2b / kappa Rendomab-B1 antibody. for the B receptor endothelins. Hypervariable loops (CDRs) are underlined in deduced amino acid sequences. The germinal parts of the different segments of the VL and VH are indicated under each sequence (IMGT annotation). Figure 10 shows the mapping of the epitope recognized by Rendomab-B1, via overlapping dodecapeptides of 1 amino acid and corresponding to all the regions exposed on the surface of the ETB-R receptor. Dark spots indicate the peptides best recognized by Rendomab-B1. The peptides are sequentially arranged from left to right from the N-terminus of the receptor. Positive and negative controls are present at the bottom left of the nitrocellulose membrane. Figure 11 shows the effect of adding different molecules alone or in combination to the growth of human endothelial cells of the umbilical vein (Huvec). Figure 12 shows the effect of the addition of Rendomab-B1 at a concentration of 10 mg / kg on the development of human melanoma tumors implanted in two xenografted nude mice (stars and full triangles), the negative control "vehicle" being performed in the absence of treatment (big round dots). DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS I. Materials and Methods. I.1. Immunization. The immunization strategy implemented consisted of combining DNA injections with protein boosters in the form of injection of cells overexpressing ETB R. Three injections, in the tibial muscle of mice, of 50 μg of plasmid DNA 49 pcDNA3 / ETB-R, were performed with a periodicity of 14 days (Figure 1: D0, D14 and D28). Each DNA injection was followed by electrostimulation according to the following characteristics 8 pulses of 20 ms each, 80 volts, 1 Hz. 4 C57BL6 mice were immunized with empty vector (control), and with the recombinant vector. Three immunization boosts at days 49, 70 and 101 (FIG. 1: D49, D70 and D101, respectively) were then carried out, by intraperitoneal injection of 2.106 COS transiently overexpressing ETB-R cells, or control cells which do not express the receiver. Nine blood samples noted BO to B8 in Figure 1 were made at times 0, 28, 42, 52, 59, 76, 83, 104 and 112 days (Figure 1: D0, D28, D42, D52, D59, D76, D83, D104 and D112, respectively), in order to study the nature of the polyclonal antibody response. Figure 1 illustrates the intensity of this response.

The two best responder mice were sacrificed for cell fusion of lymphoid cells from their spleens with NS-1 murine myeloma.

I.2. Screening of hybridomas. The hybridomas obtained were first screened by ELISA on stably expressing CHO cells ETB-R, with negative control of CHO cells expressing the irrelevant NK1 receptor. The hybridomas retained were then screened in flow cytometry (Guava apparatus, Millipore). A single hybridoma named "Rendomab-B1" was finally retained at the end of these two screens. The secreted antibody was then produced from liquid tumors (ascites) induced in the mouse by intraperitoneal injection of the selected hybridoma.

II. Biochemical characterization. After purification of Rendomab-B1, it was proceeded to the characterization of its biochemical properties. Heavy and light chain isotyping of Rendomab-B1 was performed using the Rapid ELISA Mouse mAb lsotyping kit from Pierce. The result is shown in Figure 2. It is an immunoglobulin type G, isotype 2b for the heavy chain and kappa for the light chain. Rendomab-B1 is therefore an IgG2b / kappa type immunoglobulin.

The binding specificity of Rendomab-B1 was established by flow cytometry and confocal microscopy, by direct labeling of Rendomab-B1, or indirectly by means of a fluorescent anti-mouse secondary antibody.

The affinity (1 nM) and exclusive specificity of Rendomab-B1 for human endothelin receptor subtype B are illustrated in Figure 3. The ability of Rendomab-B1 to recognize ETBR when overexpressed at the surface of CHO cells is illustrated by confocal microscopy in FIG. 4. The specific inhibition of the binding of Rendomab-B1 to ETB-R on the surface of CHO cells by the three human endothelins (ET1, ET2 and ET3) as well as sarafotoxin c, are illustrated in FIGS. 5 and 6. Competitive binding experiments were performed on CHO-ETB-R cells in the presence of radioactively labeled endothelin-1. with iodine 125, or fluorescent (ET-1fam). The results obtained in FIG. 7 show the ability of Rendomab-B1 to inhibit the binding of ET-1fam, with the control of an irrelevant control antibody of the same isotype. III. Antagonistic pharmacological properties. The study of the Rendomab-B1 antagonistic properties was carried out on a Flex 2 station (Molecular Device) using a Fluo4 type calcium flow kit. The antagonistic potential of BQ-788 peptide (N - [(cis-2,6-dimethyl-1-piperidinyl) carbonyl] -4-methyl-L-leucyl-1- (methoxycarbonyl) -D-tryptophyl-D sodium salt CAS RN: 156161-89-6), specific for ETB-R, was carried out in parallel. Peak calcium flux was established in the presence of ET-1 alone or in combination with an irrelevant control antibody of the same isotype as Rendomab-B1. The antagonistic potential of the pharmacological properties of ETB-R (ability to block intracellular calcium release normally induced by endothelin-1) by Rendomab-B1 or BQ-788, a classical peptide inhibitor of ETB-R is shown in Figure 8A and Figure 8B, respectively. In conclusion, Rendomab-B1 is a better ETB-R antagonist than BQ-788.

IV. Molecular cloning. The cloning of the nucleic precursors encoding the heavy chain and the light chain of Rendomab-B1 was carried out using the kits: "Gene-Elute / Total RNA" (Sigma) and "RACE-PCR" (Invitrogen). The nucleic and deduced amino acid sequences of the Rendomab-B1 light chain (VL) and heavy chain (VH) variable domains are shown in Figure 9.

V. Epitopic Mapping. The mapping of the epitope recognized by Rendomab-B1 on the surface of ETB-R was carried out by the "Pep-scan" technique in collaboration and according to the protocols developed within the UMR 3145 "SysDiag" CNRS / BioRad located in Montpellier (Dr. Claude Granier). To do this, 144 peptides of 12 amino acids shifted one amino acid each were used. These peptides correspond to all of the ETB-R sequences exposed on the extra-cytoplasmic side of the membrane. The result of the epitope analysis of Rendomab-B1 is shown in FIG. 10. The three peptides identified in this analysis as being the most recognized by Rendomab-B1 are the peptides of sequence: ## EQU1 ## amino acids 45 to 71 of the amino acid sequence of endothelin receptor subtype B (SEQ ID NO: 1); SPPRTISPPPCQGPI corresponding to amino acid residues 80 to 94 of the amino acid sequence of subtype B of endothelin receptors (SEQ ID NO: 1); - TMDYKGSYLRICLLHPVQKTAFMQFYKTA corresponding to amino acid residues 244 to 272 of the amino acid sequence of subtype B of endothelin receptors (SEQ ID NO: 1).

VI. Antiproliferative potential in vitro. The potential of Rendomab-B1 to slow the proliferation of cells naturally expressing at least ETB-R has been tested on HUVEC cells, which are endothelial cells of the human umbilical vein. After establishing the proliferative effect of ET1 on cell multiplication of HUVECs, a range of Rendomab-B1, a control antibody or BQ-788, was added to the culture medium. The effect of this addition was monitored and measured. The results obtained are shown in FIG. 11. 50% growth inhibition is observed in the presence of Rendomab-B1 with respect to the proliferative action of endothelin-1 (ET1). The simultaneous addition of Rendomab-B1 and Et1 does not alter the effect due to Rendomab-B1 alone. The two negative controls, involving an irrelevant antibody (Ctrl IgG2b) alone or in combination with ET1, well confirm the antiproliferative effect induced by Rendomab-B1. Finally, the addition of another peptide inhibitor of ETB-R (BQ-788) alone or with ET1 induces no effect on the growth of Huvec cells.

VII. Antiproliferative potential in vivo. The potential of Rendomab-B1 to slow the proliferation of human melanoma cancer cells, and naturally expressing at least ETB-R, was tested on tumors implanted in nude mice. Rendomab-B1 is injected intraperitoneally every 3 days at a concentration of 10 mg / kg of mouse, or 0.2 mg of antibody per mouse. The results obtained are presented in FIG. 12. A very marked slowdown in development is observed as early as the sixth day of treatment, volume volume of the tumors in the mice treated with Rendomab-B1, compared to the absence of treatment. This result establishes the potential and the therapeutic interest of the antibody which is the subject of the present application, in particular in the case of melanomas, cancers against which no specific treatment exists to date.

Claims (21)

CLAIMS1) Endothelin receptor subtype B antagonist antibody, said antibody having at least one heavy chain variable region comprising a CDR3 having at least 80% identity with the following amino acid sequence: RNAYDGYFDFW (SEQ ID N0: 2), a fragment or a derivative thereof. 2) Antibody according to claim 1, characterized in that said antibody has at least one heavy chain variable region whose CDR1, CDR2 and CDR3 respectively have at least 80% identity with the following amino acid sequences. GFSLTTYA (SEQ ID NO: 4); IWTGGDT (SEQ ID NO: 6); - ARNYDGYFDFW (SEQ ID NO: 2). 3) Antibody according to claim 1 or 2, characterized in that said antibody comprises a heavy chain variable region whose amino acid sequence having at least 80% identity with the following sequence: QVQLKESGPGLVAPSQSLSLTCTVTGFSLTTYAIVWVRQPPGKGLEW LGVIWTGGDTNYNSDLRSRLRIDKDNSRNQVFLKMSSLQTDDTARYYCARNYDGY FDFWGQGTTLTVSS (SEQ ID NO: 8). 4) An antibody according to any one of the preceding claims, characterized in that said antibody comprises a heavy chain variable region whose amino acid sequence corresponds to the amino acid sequence SEQ ID NO: 8. 5) Antibody according to any one of the preceding claims, characterized in that said antibody has at least one light chain variable region whose CDR1, CDR2 and CDR3 respectively have at least 80% identity with the acid sequences. following amino acids: - QNLLYSGNQKTY (SEQ ID NO: 10); - WAS (SEQ ID NO: 12;); QQYFIYPRT (SEQ ID NO: 14). 6) Antibody according to any one of the preceding claims, characterized in that said antibody comprises a light chain variable region whose amino acid sequence having at least 80% identity with the following amino acid sequence: DIVMSQSPSSLIVSVGQKVTMNCKSSQNLLYSGNQKTYLAWYQQKPG QSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCQQYYYPR TFGGGTKLEIKR (SEQ ID NO: 16). 7) Antibody according to any one of the preceding claims, characterized in that said antibody comprises a light chain variable region whose amino acid sequence corresponds to the amino acid sequence SEQ ID NO: 16.30 57 8) Antibody according to any one of the preceding claims, characterized in that said antibody is an IgG2b / kappa type immunoglobulin. 9) Antibody according to any one of the preceding claims, characterized in that said antibody is monoclonal. 10) Antibody according to any one of the preceding claims, characterized in that said antibody is the murine monoclonal antibody obtained from the hybridoma deposited at the CNCM on September 30, 2010 under number CNCM I-4370. 11) Antibody according to any one of the preceding claims, characterized in that said antibody is a chimeric antibody. 12) Antibody according to any one of the preceding claims, characterized in that said antibody is a humanized antibody. 13) Hybridome deposited at the CNCM on September 30, 2010 under number CNCM I-4370. 14) isolated polynucleotide selected from the following polynucleotides: i) a polynucleotide encoding an antibody as defined in any one of claims 1 to 12; Ii) a polynucleotide complementary to the polynucleotide as defined in (i); iii) a polynucleotide of at least 18 nucleotides, capable of hybridizing under conditions of high stringency to polynucleotides as defined in points (i) and (ii). 15) Cloning and / or expression vector containing at least one polynucleotide according to claim 14. 16) A host organism transformed with or comprising a polynucleotide according to claim 14 or a vector according to claim 15. A compound comprising an antibody according to any one of claims 1 to 12 conjugated with a member selected from the group consisting of a cytotoxic moiety, an easily detectable moiety, or an effector moiety. 18) An antibody according to any one of claims 1 to 12, polynucleotide according to claim 14 or a compound according to claim 17 for use as a medicament. 19) A pharmaceutical composition comprising, as active ingredient, an antibody according to any one of claims 1 to 12, a polynucleotide according to claim 14 or a compound according to claim 17 and a pharmaceutically acceptable carrier. 20) An antibody according to any one of claims 1 to 12, polynucleotide according to claim 14, compound according to claim 17 or pharmaceutical composition according to claim 19, for treating and / or preventing a disorder or pathology involving a dysfunction, direct or in combination with another physiological pathway, the axis comprising an endothelin and at least one of its receptors such as, in particular, the subtype B endothelin receptors. 21) Antibody, polynucleotide, compound or pharmaceutical composition according to claim 20, characterized in that said disorder or said pathology is chosen from the group consisting of arterial hypertension, atherosclerosis, coronary heart disease, renal dysfunction. , cerebrovascular diseases, Crohn's disease, pulmonary fibrosis, asthma, angiogenesis and cancer.