Antibodies to endothelin receptor beta subtypes

Abstract

The present disclosure relates to an endothelin receptor (EBTR) subtype B antagonist antibody, fragment, or derivative thereof, designated "rendomab-B49." Therapeutic and diagnostic uses of the claimed antibody are suitable for patients suffering from glioblastoma. Experimental results demonstrate the strong affinity of rendomab-B49 for glioblastoma.

Description

[Technical field]

[0001] The present invention relates to the technical field of antibodies, their therapeutic and diagnostic uses, and their use as research tools.

[0002] More specifically, the present invention provides antibodies, preferably monoclonal antibodies, specific to the native and functional conformation of endothelin receptor subtype B and in particular the human endothelin receptor expressed on the surface of cancer cells such as glioblastoma cells.

[0003] The present invention relates to the use of these antibodies for therapeutic and diagnostic purposes, as well as for research purposes. [Background technology]

[0004] The receptors for the different endothelins (called ET1, ET2 and ET3 in humans) belong to a family of receptors with seven transmembrane domains, also called GPCRs for "G Protein Coupled Receptors". Endothelin receptors have two main subtypes in humans: subtype A (ETA-R) and subtype B (ETB-R). The fact that these receptors are classified into the GPCR family gives them a complex three-dimensional structure. This feature partly explains the difficulty in obtaining antibodies that recognize the native structure of these receptors expressed in the cell membrane. In fact, the difficulty in obtaining monoclonal antibodies specific for GPCRs is a consequence of the problems associated with obtaining these receptors in their native and functional form, outside of their association with the membrane.

[0005] The endothelin axis and its receptors are involved in several physiopathological functions and dysfunctions, including, but not limited to, arterial hypertension, atherosclerosis, coronary artery disease, liver dysfunction, cerebrovascular disease, Crohn's disease, pulmonary fibrosis, asthma, and others (see review by R. Shah, 2007, "Endothelins in health and disease", Eur. J. Int. Med., Vol. 18, pp. 272-282).

[0006] Furthermore, endothelin receptors have also been found to be associated with the progression of many cancers by promoting the proliferation, survival and dissemination of cancer cells as well as angiogenesis (Bagnato & Rosano, 2008, "The endothelin axis in cancer", Int. J. of Biochem. & Cell Biology, vol. 40, pp. 1443-1451). As for endothelin receptor subtype B, the latter has altered expression levels, especially in melanoma, colon cancer, Kaposi's sarcoma, glioblastoma (brain tumor), and bladder cancer.

[0007] It has also been established that endothelin receptor subtype B is involved in the lack of recognition of some cancer cells, especially ovarian cancer cells, by the immune system by inducing a strong reduction in lymphocyte infiltration (Buckanovich et al., 2008, "Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy", Nature Medicine, vol. 14, pp. 28-36).

[0008] Therefore, targeting ETB-R conformers expressed on the surface of cancer cells, especially glioblastoma cells, seems particularly relevant in human clinical biology, not only for tracking the progression of these tumors, especially these brain tumors, and their recurrence after surgery, but also for diagnostic tools for therapeutic applications targeting these tumor cells. Moreover, in the passive immunotherapy arsenal for cancer using monoclonal antibodies (40 antibodies approved to date), none of them targets GPCRs.

[0009] Few antibodies targeting endothelin receptors in general, and the subtype ETB-R in particular, have been described so far.

[0010] Kondoh et al., 1990 ("Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization", BBRC, Vol. 172, pp. 503-510) describe the binding properties of four monoclonal antibodies (A2, G9, E7 and G10) against solubilized complexes of endothelin receptors present on the surface of rat lung membranes. Antibodies G9 and G10 are G-type isotype 2a immunoglobulins (IgG2a), whereas antibodies A2 and E7 are IgG1 immunoglobulins. If these four antibodies are indeed specific for solubilized endothelin receptors, Kondoh et al. provide no information on the fine specificity of these antibodies (ETA-R and / or ETB-R), neither with regard to the recognition of receptors of human origin, nor with regard to possible antagonist properties.

[0011] Yamaguchi et al., 2004 ("Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells", Biotechnology Letters, vol. 26, pp. 293-299) concern the characterization of the binding properties of five murine monoclonal antibodies obtained after protein immunization with recombinant human ETB-R produced in insect cells. Four of them have similar affinities (nanomolar range) for ETB-R, whereas the fifth has a ten-fold lower affinity. Epitope analysis of three of them (N-6, N-3 and N-1) revealed that they recognize the N-terminal domain of ETB-R, more specifically the sequence corresponding to amino acids 27-35 of ETB-R in N-6, the sequence corresponding to amino acids 27-41 of ETB-R in N-3 and the sequence corresponding to amino acids 71-85 of N-1. Finally, these five antibodies are able to recognize COS cells overexpressing ETB-R.

[0012] Patent application US2010 / 003240, filed on behalf of New York University and published on January 7, 2010, relates to therapeutic protocols and pharmaceutical mixtures within the scope of the treatment and prevention of cancer, particularly melanoma. For this purpose, and more specifically, the use of ETB-R antagonists is claimed. The examples described for these antagonists relate only to modified endothelin-1 type, but by extension, the use of ETB-R antagonist antibodies is claimed, although no specific examples of such antibodies are described or provided.

[0013] Patent application No. 2012-111706, filed on behalf of Seikisui Chemical Co Ltd and published on June 14, 2012, relates to a monoclonal antibody called hB07, which is an IgG2a / lambda isotype mouse immunoglobulin specific for the endothelin receptor human subtype B. The authors report that antibody hB07 has a cytotoxicity of 1.7×10 -7 M and the calculated efficiency (IC 50 ) can competitively block endothelin-1 binding. Thus, this antibody, whose sequence has not been described, has antagonistic properties.

[0014] Similarly, a monoclonal antibody antagonist against endothelin receptor subtype B, specifically human subtype B, called Rendomab-B1, was described in international application WO2012 / 045776, filed on behalf of the CEA and published on April 12, 2012.

[0015] Finally, international application WO2013 / 063001, filed on behalf of Genentech and Hoffmann-LaRoche and published on May 2, 2013, describes a therapeutic antibody conjugated to a cytotoxic molecule for the treatment of melanoma, without reference to the conformer preferentially expressed by tumor cells. This antibody, called 5E9, targets the human ETB-R, which is overexpressed on the surface of melanoma. It should be noted that the antibody 5E9 cross-reacts with rodent ETB-R as well as with non-human primate receptors, as described in Asundi et al., 2011 ("An antibody-drug conjugate targeting the endothelin B receptor for the treatment of melanoma", Clinical Cancer Research, Vol. 17, pp. 965-975). [Prior art documents] [Patent documents]

[0016] [Patent Document 1] US2010 / 003240 [Patent Document 2] Japanese Patent Application No. 2012-111706 [Patent Document 3] WO2012 / 045776 [Patent Document 4] WO2013 / 063001 [Patent Document 5] U.S. Patent No. 4,816,567 [Non-Patent Document]

[0017] [Non-Patent Document 1] R. Shah, 2007, "Endothelins in health and disease", Eur. J. Int. Med., Vol. 18, pp. 272 - 282 [Non-Patent Document 2] Bagnato & Rosano, 2008, "The endothelin axis in cancer", Int. J. of Biochem. & Cell Biology, Vol. 40, pp. 1443 - 1451 [Non-Patent Document 3] Buckanovich et al., 2008, "Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy", Nature Medecine, Vol. 14, pp. 28 - 36 [Non-Patent Document 4] Kondoh et al., 1990, "Isolation of anti - endothelin receptor monoclonal antibodies for use in receptor characterization", BBRC, Vol. 172, pp. 503 - 510 [Non-Patent Document 5] Yamaguchi et al., 2004, "Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells", Biotechnology Letters, vol. 26, pp. 293-299. [Non-Patent Document 6] Asundi et al., 2011, “An antibody-drug conjugate targeting the endothelin B receptor for the treatment of melanoma”, Clinical Cancer Research, vol. 17, pp. 965–975. [Non-Patent Document 7] Allard et al., 2011, “Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor”, DNA and Cell Biology, vol. 30, pp. 727–737. [Non-Patent Document 8] International ImMunoGeneTics Database http: / / imgt.cines.fr:8104 [Non-Patent Document 9] Verhoeyen et al., 1988, "Reshaping human antibodies: Grafting an antilysozyme activity", Science, vol. 239, pp. 1534-1536. [Non-Patent Document 10] Vaughan et al., 1998, "Human antibodies by design", Nature Biotechnol., vol. 16, pp. 535-539 [Non-Patent Document 11] Sambrook et al., 1989, Molecular cloning, edited by Noland C., New York: Cold Spring Harbor Laboratory Press. Summary of the Invention [Problem to be solved by the invention]

[0018] Therefore, the present inventors aimed to obtain antibodies capable of targeting specific conformers of endothelin receptor subtype B expressed on the surface of cancer cells. [Means for solving the problem]

[0019] The present invention makes it possible to solve the technical problems such as those defined above and to achieve the objectives set out by the inventors.

[0020] In fact, the inventors developed and used a specific selective immunization strategy already published in Allard et al., 2011 ("Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor", DNA and Cell Biology, vol. 30, pp. 727-737). This strategy, coupled with a hybridoma screening technique in ELISA cells and then by flow cytometry, favors the acquisition of monoclonal antibodies specific for ETB-R in its native conformation. This method further has the advantage that it does not require the receptor of interest to be extracted and purified, which is particularly challenging even today.

[0021] This strategy allowed the inventors to select different monoclonal antibodies against endothelin receptor subtype B, in particular against human subtype B, hereafter referred to as Rendomab-B49, Rendomab-B41 and Rendomab-B36. These antibodies are close to each other not only in terms of their nucleotide and peptide sequences but also in terms of their properties. Thus, none of these antibodies are antagonists against the pharmacological properties of endothelin receptor subtype B (ETB-R). That is, the antibodies according to the invention are not able to inhibit or block the binding of endothelin ligands, in particular the ligands ET1, ET2 and ET3 on ETB-R. On the contrary, the antibodies according to the invention are able to recognize a specific conformational isomer of endothelin receptor subtype B expressed on the surface of cancer cells, in particular glioblastoma cells.

[0022] The present invention relates to antibodies against endothelin receptor subtype B, fragments or derivatives thereof.

[0023] Before describing the invention in more detail, the following definitions are reviewed or proposed.

[0024] The terms "antibody" and "immunoglobulin" are equivalent and can be used interchangeably in the present invention.

[0025] An antibody is a glycoprotein that comprises at least two heavy chains (H) and at least two light chains (L) linked together by one or more disulfide bridges. Each heavy chain comprises a variable region (or domain) (VH) and a constant region that comprises three domains, usually called CH1, CH2 and CH3. Each light chain comprises a variable region (or domain) (VL) and a constant region that comprises a single domain, usually called CL. The variable regions of the heavy and light chains involved in antigen recognition can be further subdivided into three hypervariable regions, also called "complementarity determining regions" (CDRs), surrounded by four more conserved regions, also called framework regions (FRs). The composition of each heavy (or light) chain variable region, from N-terminus to C-terminus, is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Within the scope of the present invention, the definitions of CDR and FR used are those of IMGT (International ImMunoGeneTics database http: / / imgt.cines.fr:8104), and therefore the calculations of the percent identity of the CDR sequences mentioned and claimed below shall be considered based on this annotation.

[0026] Furthermore, the term "antibody" includes within the scope of the present invention not only complete antibody molecules but also fragments and derivatives thereof.

[0027] By "antibody fragment" is meant within the scope of the present invention both monovalent fragments with a single antigen-binding site as well as bivalent fragments with two antigen-binding sites. Thus, a fragment according to the invention has at least one antigen-binding site. Among these fragments, fragments Fab, F(ab')2, Fv and other fragments that preserve the antigen-binding site (scFv and bispecific antibodies) are mentioned. The fragment Fab is a monovalent fragment consisting of the complete light chain and part of the heavy chain (Fd) including the domains VH and CH1 as defined above. The fragment F(ab')2 is a bivalent fragment corresponding to the association of two fragments Fab linked by a disulfide bridge present in the hinge region of the immunoglobulin located between the constant domains CH1 and CH2. The fragment Fv is a monovalent fragment consisting only of the variable regions VL and VH of the light and heavy chains of an antibody. The fragment scFv is a monovalent polypeptide fragment corresponding to the variable domains linked by peptide bonds, obtainable exclusively by genetic engineering. Bispecific antibodies are recombinant bivalent antibody molecules consisting of two head-to-tail scFv molecules, since the peptide bonds are too short to allow the formation of scFv. The fragments according to the invention also encompass the aforementioned fragments, whose half-life has been extended, in particular by incorporation into liposomes or by chemical modification by the introduction of polyalkylene glycols such as polyethylene glycol (PEG), a technique called "PEGylation", generating fragments such as Fab-PEG, F(ab')2-PEG or Fv-PEG. By recombinant routes it is also possible to generate single or fusion fragments of the antibodies according to the invention, with more efficient and better controlled properties of solid tumor penetration and pharmacokinetics. Antibody fragments useful within the scope of the invention can be natural or recombinant.

[0028] By "antibody derivatives" is meant within the scope of the present invention antibody fragments obtained by genetic engineering, such as single chain Fv (scFv) and single domain antibody (dAb) molecules. This term also includes antibody type molecules which can be derived using phage display techniques or other random selection methods for molecules that bind to endothelin receptor subtype B or to regions specific for this subtype.

[0029] Thus, "antibody fragments" and "antibody derivatives" encompass all molecules that contain an advantageous peptide structure that is part of the recognition site of the antibody according to the invention (i.e., the part of the antibody that binds to or associates with the epitope or antigen). Furthermore, the antibody fragments and derivatives according to the invention are capable of recognizing a specific conformer of the endothelin receptor subtype B expressed on the surface of cancer cells, in particular glioblastoma cells.

[0030] The present invention relates to an antibody against endothelin receptor subtype B, - CDR1 (hereinafter referred to as CDR1 H ), CDR2 (hereinafter referred to as CDR2 H ) and CDR3 (hereinafter referred to as CDR3 H A heavy chain variable region comprising CDR1 H , CDR2 H and CDR3 H a heavy chain variable region, the ordered arrangement of which shows at least 80% identity with the following amino acid sequence: GYTFISYWIDPDSGGTAREGDYAWFAY (SEQ ID NO: 1); - CDR1 (hereinafter referred to as CDR1 L ), CDR2 (hereinafter referred to as CDR2 L ) and CDR3 (hereinafter referred to as CDR3 L A light chain variable region comprising CDR1 L , CDR2 L and CDR3 L a light chain variable region, wherein the ordered arrangement formed by the amino acid sequence of: The present invention relates to an antibody comprising the compound

[0031] Within the scope of the present invention, amino acid sequences are given according to the international one-letter code.

[0032] "CDR1 H , CDR2 H and CDR3 H (or CDR1 L , CDR2 L and CDR3 L The "ordered arrangement formed by the amino acid sequence of CDR1" is an arrangement of the following formula: H aa sequence + CDR2 H aa sequence + CDR3 H aa sequence (or CDR1 L aa sequence + CDR2 L aa sequence + CDR3 L It means an artificial amino acid (aa) sequence having the aa sequence (aa sequence of the above).

[0033] Generally, CDR1 H , CDR2 H and CDR3 H The ordered arrangement formed by the amino acid sequence of SEQ ID NO: 1 has at least 81% identity with the amino acid sequence of SEQ ID NO: 1. L , CDR2 L and CDR3 L has at least 85% identity with the amino acid sequence of SEQ ID NO:2.

[0034] Advantageously, the antibody according to the invention comprises a heavy chain variable region, the CDR1 H has the following consensus sequence: GYTFX1SYW (SEQ ID NO: 3), where X1 represents any amino acid. In a particular embodiment, X1 is either I or T, and the CDR1 of the heavy chain variable region of the antibody according to the invention H The amino acid sequence of is either GYTFISYW (SEQ ID NO: 5) or GYTFTSYW (SEQ ID NO: 7).

[0035] More preferably, the antibody according to the invention comprises a heavy chain variable region, the CDR2 H has the following consensus sequence: IDPX2SGGT (SEQ ID NO: 8), where X2 represents any amino acid. In a particular embodiment, X2 is either D or N, and the CDR2 of the heavy chain variable region of the antibody according to the invention H is either IDPDSGGT (SEQ ID NO: 10) or IDPNSGGT (SEQ ID NO: 12).

[0036] More preferably, the antibody according to the invention comprises a heavy chain variable region, H has the following consensus sequence: X3REGX4X5AWFX6Y (SEQ ID NO: 13), where X3, X4, X5 and X6 are the same or different and represent any amino acid. In a particular embodiment, X3, X4, X5 and X6 are the same or different and are selected from the group consisting of A, D, Y, E, F, V and W. In a more particular embodiment, X3 is either A or V, X4 is selected from the group consisting of D, W and E, X5 is selected from the group consisting of Y, D and F, and X6 is either A or V. In an even more particular embodiment, the CDR3 of the heavy chain variable region of the antibody according to the invention is H is selected from the group consisting of AREGDYAWFAY (SEQ ID NO: 15), VREGWDAWFVY (SEQ ID NO: 17) and AREGEFAWFAY (SEQ ID NO: 19).

[0037] Advantageously, the antibody according to the invention comprises a light chain variable region, the CDR1 L has the following consensus sequence: QX7IVHSNGX8TY (SEQ ID NO: 20), where X7 and X8 are the same or different and represent any amino acid. In a particular embodiment, X7 and X8 are the same or different and are selected from the group consisting of N, Y and S. In a more particular embodiment, X7 is either N or S and X8 is either N or Y. In an even more particular embodiment, CDR1 of the light chain variable region of the antibody according to the invention is Lis either QSIVHSNGNTY (SEQ ID NO: 22) or QNIVHSNGYTY (SEQ ID NO: 24).

[0038] More preferably, the antibody according to the invention comprises a light chain variable region, the CDR2 of which is L has the following consensus sequence: KVX9, where X9 represents any amino acid. In a particular embodiment, X9 is either S or F, and the CDR2 of the light chain variable region of the antibody according to the invention L The amino acid sequence of is either KVS or KVF.

[0039] More preferably, the antibody according to the invention comprises a light chain variable region, the CDR3 L The consensus sequence is: FQGSHVPX 10 T (SEQ ID NO: 25) (wherein X 10 In certain embodiments, X 10 is either W or L, and the CDR3 of the light chain variable region of the antibody according to the present invention L The amino acid sequence of is either FQGSHVPWT (SEQ ID NO: 27) or FQGSHVPLT (SEQ ID NO: 29).

[0040] Advantageously, the antibody according to the invention comprises i1) - CDR1, the amino acid sequence of which is GYTFISYW (SEQ ID NO: 5) H , - CDR2 having the amino acid sequence IDPDSGGT (SEQ ID NO: 10) H , and - CDR3 having the amino acid sequence AREGDYAWFAY (SEQ ID NO: 15) H a heavy chain variable region comprising or ii1) - CDR1, the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7) H , - CDR2 having the amino acid sequence IDPDSGGT (SEQ ID NO: 10) H , and - CDR3 having the amino acid sequence VREGWDAWFVY (SEQ ID NO: 17) H a heavy chain variable region comprising or iii1) - CDR1, the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7) H , - CDR2 having the amino acid sequence IDPNSGGT (SEQ ID NO: 12) H , and - CDR3 having the amino acid sequence AREGEFAWFAY (SEQ ID NO: 19) H A heavy chain variable region comprising Includes.

[0041] More particularly, the antibody according to the invention comprises a heavy chain variable region, the amino acid sequence of which is as follows: QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVPVSA (SEQ ID NO: 31) It shows at least 80% identity with.

[0042] Thus, an antibody according to the invention comprises a heavy chain variable region whose amino acid sequence exhibits at least 80% identity with the amino acid sequence of SEQ ID NO: 31, and may exhibit at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or even at least 90% identity.

[0043] Even more particularly, the antibody according to the invention comprises a heavy chain variable region, the amino acid sequence of which corresponds to, i.e. consists of, the amino acid sequence of SEQ ID NO: 31 (for Rendomab-B49).

[0044] Alternatively (in the case of Rendomab-B41), the antibody according to the invention comprises a heavy chain variable region, the amino acid sequence of which is the following amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATLTVDKPSNTANMQLSSLTSEDSAVYYCVREGWDAWFVYWGQGTLLTVSA (SEQ ID NO: 33) corresponds to, i.e. consists of,

[0045] Alternatively (in the case of Rendomab-B36), the antibody according to the invention comprises a heavy chain variable region, the amino acid sequence of which is the following amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWIHWVNQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKTSSTAYMQFSSLTSEDSAVYYCAREGEFAWFAYWGQGTLVTVSA (SEQ ID NO: 35) corresponds to, i.e. consists of,

[0046] Advantageously, the antibody according to the invention comprises i2) - CDR1, the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22) L , - CDR2 whose amino acid sequence is KVS L , - CDR3 having the amino acid sequence FQGSHVPWT (SEQ ID NO: 27) L a light chain variable region comprising or ii2) - CDR1, the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22) L , - CDR2 with the amino acid sequence KVF L , - CDR3 having the amino acid sequence FQGSHVPLT (SEQ ID NO: 29) L a light chain variable region comprising or iii2) - CDR1, the amino acid sequence of which is QNIVHSNGYTY (SEQ ID NO: 24) L , - CDR2 whose amino acid sequence is KVS L , - CDR3 having the amino acid sequence FQGSHVPLT (SEQ ID NO: 29) L A light chain variable region comprising Includes.

[0047] More particularly, the antibody according to the invention comprises a light chain variable region, the amino acid sequence of which is as follows: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 37) It shows at least 80% identity with.

[0048] Thus, an antibody according to the invention comprises a light chain variable region whose amino acid sequence exhibits at least 80% identity with the amino acid sequence of SEQ ID NO: 37, and may exhibit at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or even at least 95% identity.

[0049] Even more particularly, the antibody according to the invention comprises a light chain variable region, the amino acid sequence of which corresponds to, i.e. consists of, the amino acid sequence of SEQ ID NO: 37 (for Rendomab-B49).

[0050] Alternatively (in the case of Rendomab-B41), the antibody according to the invention comprises a light chain variable region, the amino acid sequence of which is the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKR (SEQ ID NO: 39) corresponds to, i.e. consists of,

[0051] Alternatively (in the case of Rendomab-B36), the antibody according to the invention comprises a light chain variable region, the amino acid sequence of which is the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGYTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGSGTKLEIKR (SEQ ID NO: 41) corresponds to, i.e. consists of,

[0052] Advantageously, an antibody according to the invention comprises a light chain variable region and a heavy chain variable region as defined above.

[0053] The light chain of the antibody according to the invention is generally a kappa light chain.

[0054] The heavy chain of the antibody according to the invention is in particular a gamma1 heavy chain or a gamma3 heavy chain.

[0055] In particular, the antibody according to the invention is a G-type immunoglobulin.

[0056] More particularly, the antibodies according to the invention are IgG1 / kappa or IgG3 kappa immunoglobulins.

[0057] The antibody against endothelin receptor subtype B, which is the subject of the present invention, selectively binds to the extracellular segment of ETB-R. By "antibody selectively binding" to at least one specific domain or region of ETB-R, in particular human ETB-R, is meant within the scope of the present invention an antibody that binds to a specific domain with a higher affinity than any other region of ETB-R. Advantageously, the antibody binds to a specific domain of ETB-R with an affinity at least 2-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold higher than it exhibits for any other region of ETB-R. This binding can be determined by direct or indirect characterization of the antibody to be tested by methods well known in the art, such as flow cytometry, radioimmunoassay (RIA), confocal microscopy, enzyme immunoassay (EIA) labeling (ELISA).

[0058] The antibodies that are the subject of the present invention can be obtained from animals immunized against endothelin receptor subtype B or against a fragment of this receptor containing the epitope recognized by the antibody according to the invention. The immunized animal can be any animal normally used to produce antibodies, such as a mouse, rat, rabbit, goat, dog, horse or a camelid, such as a camel or llama.

[0059] The antibodies that are the subject of the present invention can also be obtained from naive recombinant (scFv, Fab, ...) libraries expressed on the surface of viruses, phages, bacteria, yeast or other eukaryotic cells, these libraries being constructed from the immunoglobulins of different immunized animals as disclosed above.

[0060] The antibodies thus obtained can be purified on an affinity column on which endothelin receptor subtype B or one of the sequences specifically recognized by the antibody according to the invention has been previously immobilized, which purification can also involve protein A affinity chromatography.

[0061] Within the scope of the present invention, antibodies can be polyclonal multispecific or monospecific antibodies, or monoclonal antibodies.

[0062] Advantageously, the antibodies of the invention are monoclonal. A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. identical antibodies, according to the usual definition in immunology, with a relatively small number possibly having mutations. Monoclonal antibodies are obtained from the propagation of a single clone of cells, such as a hybridoma.

[0063] More particularly, the antibody according to the invention is a mouse monoclonal antibody obtained from a hybridoma selected from the hybridoma deposited at the CNCM under the accession number CNCM I-5084 on May 19, 2016 (Rendomab-B49), the hybridoma deposited at the CNCM under the accession number CNCM I-5104 on June 7, 2016 (Rendomab-B41), and the hybridoma deposited at the CNCM under the accession number CNCM I-5103 on June 7, 2016 (Rendomab-B36). The invention also relates to such hybridomas.

[0064] Alternatively, an antibody according to the invention may be a chimeric antibody, i.e. an antibody comprising heavy and light chain variable or hypervariable regions derived from an antibody of a given species combined with heavy and light chain constant regions derived from an antibody of another species, heterologous to the former.

[0065] A first alternative of the present invention corresponds to chimerized antibodies, in particular chimerized monoclonal antibodies, i.e. antibodies in which the aforementioned variable domains derived from mouse antibodies are associated with constant domains of human origin. It should be noted that some therapeutic antibodies used in humans are chimerized antibodies.

[0066] A second particularly interesting alternative would be humanized antibodies, and in particular humanized monoclonal antibodies. Indeed, when the latter require repeated administration to human subjects, it is preferable to use humanized antibodies.

[0067] In the case of humanized monoclonal antibodies according to the invention, the latter can be prepared by inserting into a human antibody the CDRs of a murine antibody, regardless of isotype (IgG, IgA, IgM), in particular from a hybridoma selected from the hybridoma deposited at the CNCM on May 19, 2016 under the accession number CNCM I-5084 (Rendomab-B49), the hybridoma deposited at the CNCM on June 7, 2016 under the accession number CNCM I-5104 (Rendomab-B41) and the hybridoma deposited at the CNCM on June 7, 2016 under the accession number CNCM I-5103 (Rendomab-B36). Humanized antibodies can be made using the techniques and methods described in Verhoeyen et al., 1988 ("Reshaping human antibodies: Grafting an antilysozyme activity", Science, Vol. 239, pp. 1534-1536) and U.S. Pat. No. 4,816,567, issued March 28, 1989 on behalf of Genentech.

[0068] The antibodies can also be human antibodies in that they have the amino acid sequence of anti-ETB-R human antibodies through preparation methods known in the art that do not require human vaccination. For example, such antibodies can be obtained by genetic immunization / cellular immunization boosting of transgenic mice that are available and contain essentially human immunoglobulin genes (see Vaughan et al., 1998, "Human antibodies by design", Nature Biotechnol., vol. 16, pp. 535-539). Alternatively, such antibodies can be obtained by cloning CDNAs that code for human B lymphocytes against ETB-R.

[0069] The present invention relates to the following different polynucleotides: α) a polynucleotide encoding an antibody as defined above, β) a polynucleotide complementary to the polynucleotide defined in (α); γ) a polynucleotide of at least 18 nucleotides capable of hybridizing to the polynucleotides defined in (α) and (β) under high stringency conditions; The present invention also relates to an isolated polynucleotide selected from:

[0070] By "polynucleotide" is meant within the scope of the present invention a nucleic acid, a nucleic acid sequence, a nucleic acid sequence, an oligonucleotide, a polynucleotide sequence, a nucleotide sequence, a single-stranded DNA, a double-stranded DNA or an RNA. A polynucleotide according to the present invention can include naturally occurring and non-naturally occurring nucleotides.

[0071] A polynucleotide according to the invention does not correspond to the nucleotide sequence in its natural state, i.e. in its natural chromosomal environment. On the contrary, a polynucleotide according to the invention has been isolated and possibly purified, the environment of which has been altered as a result. A polynucleotide according to the invention can also be obtained by genetic recombination or chemical synthesis.

[0072] High stringency conditions correspond to the temperature and ionic strength conditions that allow the hybridization between two complementary nucleotide sequences to be maintained. Those skilled in the art will be able to determine the most appropriate high stringency conditions depending on the size of the nucleotide sequence, in particular by referring to the teachings of Sambrook et al., 1989 (Molecular cloning, Noland C., ed., New York: Cold Spring Harbor Laboratory Press).

[0073] The polynucleotide according to the invention has the following nucleotide sequence: GGC TAC ACC TTC ATC AGC TAC TGG (SEQ ID NO: 4) and GGC TAC ACC TTC ACC AGC TAC TGG (SEQ ID NO: 6) CDR1 selected from H The present invention comprises at least one nucleotide sequence encoding

[0074] Advantageously, the polynucleotide according to the invention has the following nucleotide sequence: ATT GAT CCT GAT AGN1GGT GGT ACT (wherein N1 represents either C or T) (SEQ ID NO: 9) and ATT GAT CCT AAT AGT GGT GGC ACT (SEQ ID NO: 11) CDR2 selected from H The present invention comprises at least one nucleotide sequence encoding

[0075] More preferably, the polynucleotide according to the invention has the following nucleotide sequence: GCA AGA GAA GGG GAT TAC GCC TGG TTT GCT TAC (SEQ ID NO: 14); GTA AGA GAA GGG TGG GAC GCC TGG TTT GTT TAC (SEQ ID NO: 16) and GCA AGA GAG GGG GAA TTC GCC TGG TTT GCT TAC (SEQ ID NO: 18) A CDR3 selected from H The present invention comprises at least one nucleotide sequence encoding

[0076] Generally, a polynucleotide according to the invention has the following nucleotide sequence: CAG AGC ATT GTA CAT AGT AAT GGA AAC ACC TAT (SEQ ID NO: 21) and CAG AAC ATT GTC CAT AGT AAT GGA TAC ACC TAT (SEQ ID NO: 23) CDR1 selected from L The present invention comprises at least one nucleotide sequence encoding

[0077] In particular, the polynucleotide according to the invention has the following nucleotide sequence: AAA GTT TCC and AAA GTT TTC CDR2 selected fromL The present invention comprises at least one nucleotide sequence encoding

[0078] More particularly, the polynucleotide according to the invention has the following nucleotide sequence: TTT CAA GGT TCA CAT GTT CCG TGG ACG (SEQ ID NO: 26) and TTT CAA GGT TCA CAT GTT CCN2CTC ACG (wherein N2 represents either G or T) (SEQ ID NO: 28) A CDR3 selected from L The present invention comprises at least one nucleotide sequence encoding

[0079] Advantageously, the polynucleotide according to the invention is selected from the group: (i1') the nucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 9 (wherein N1 represents C), and SEQ ID NO: 14; (ii1') the nucleotide sequences of SEQ ID NO:6, SEQ ID NO:9 (wherein N1 represents T), and SEQ ID NO:16, and (iii1') The nucleotide sequences of SEQ ID NO:6, SEQ ID NO:11 (wherein N1 represents T), and SEQ ID NO:18. The present invention comprises at least three nucleotide sequences corresponding to one of the following:

[0080] For each group, the polypeptide obtained at the end of translation of the polynucleotide according to the present invention is CDR1 H , CDR2 H and CDR3 H It is clear that the three sequences listed above need to be structured with respect to each other to include three peptide sequences corresponding to:

[0081] More particularly, the polynucleotide according to the invention has the following nucleotide sequence: CAGGTCCAACTGCAGCAGCCTGGGGCTGCGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCATCAGCTACTGGATGCTCTGGGTGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTGATAGCGGTGGTACTAAG TACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGAGAAGGGGATTACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCCCTGTCTCTGCA (Sequence number 30) The nucleic acid sequence of the present invention comprises at least one nucleotide sequence having at least 80% identity to the nucleic acid sequence of the present invention.

[0082] Thus, a polynucleotide according to the present invention comprises at least one nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 30, and may exhibit at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or even at least 90% identity.

[0083] Even more particularly, the polynucleotide according to the invention comprises a nucleotide sequence that corresponds to the nucleotide sequence of SEQ ID NO: 30 (in the case of Rendomab-B49). Thus, the nucleotide sequence encoding the heavy chain variable region of Rendomab-B49 comprises or consists of the nucleotide sequence of SEQ ID NO: 30.

[0084] Alternatively (in the case of Rendomab-B41), the polynucleotide according to the invention has the following nucleotide sequence: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTGATAGTGGTGGTACTAAA TACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAACCCTCCAACACAGCCAACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCGGTCTATTATTGTGTAAGAGAAGGGTGGGACGCCTGGTTTGTTTACTGGGGCCAAGGGACTCTGCTCACTGTCTCTGCA (Sequence number 32) Includes.

[0085] Thus, the nucleotide sequence encoding the heavy chain variable region of Rendomab-B41 comprises or consists of the nucleotide sequence of SEQ ID NO:32.

[0086] Alternatively (in the case of Rendomab-B36), the polynucleotide according to the invention may have the following nucleotide sequence: CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATACACTGGGTAAATCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGTGGCACTAAG TACAATGAGAAGTTCAAGAGTAAGGCCACACTGACTGTAGACAAAACCTCCAGCACAGCCTACATGCAGTTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGAGAGGGGGAATTCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (Sequence number 34) Includes.

[0087] Thus, the nucleotide sequence encoding the heavy chain variable region of Rendomab-B36 comprises or consists of the nucleotide sequence of SEQ ID NO:34.

[0088] Advantageously, the polynucleotide according to the invention is selected from the group: (i2') the nucleotide sequences of SEQ ID NO:21, AAAGTTTCC, and SEQ ID NO:26; (ii2') the nucleotide sequences of SEQ ID NO: 21, AAAGTTTTC, and SEQ ID NO: 28 (wherein N2 represents G); and (iii2') the nucleotide sequences of SEQ ID NO: 23, AAAGTTTCC, and SEQ ID NO: 28 (wherein N2 represents T); The present invention comprises at least three nucleotide sequences corresponding to one of the following:

[0089] For each group, the polypeptide obtained at the end of translation of the polynucleotide according to the present invention is CDR1 L , CDR2 L and CDR3 L It is clear that the three sequences listed above need to be structured with respect to each other to include three peptide sequences corresponding to:

[0090] More particularly, the polynucleotide according to the invention has the following nucleotide sequence: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (Sequence number 36) The nucleic acid sequence of the present invention comprises at least one nucleotide sequence having at least 80% identity to the nucleic acid sequence of the present invention.

[0091] Thus, a polynucleotide according to the invention comprises at least one nucleotide sequence exhibiting at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or even at least 95% identity to the nucleotide sequence of SEQ ID NO:36.

[0092] Even more particularly, the polynucleotide according to the invention comprises a nucleotide sequence that corresponds to the nucleotide sequence of SEQ ID NO: 36 (in the case of Rendomab-B49). Thus, the nucleotide sequence encoding the light chain variable region of Rendomab-B49 comprises or consists of the nucleotide sequence of SEQ ID NO: 36.

[0093] Alternatively (in the case of Rendomab-B41), the polynucleotide according to the invention has the following nucleotide sequence: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACTTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT TCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (Sequence number 38) Includes.

[0094] Thus, the nucleotide sequence encoding the light chain variable region of Rendomab-B41 comprises or consists of the nucleotide sequence of SEQ ID NO:38.

[0095] Alternatively (in the case of Rendomab-B36), the polynucleotide according to the invention may have the following nucleotide sequence: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTCCATAGTAATGGATACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT CCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG (Sequence number 40) Includes.

[0096] Thus, the nucleotide sequence encoding the light chain variable region of Rendomab-B36 comprises or consists of the nucleotide sequence of SEQ ID NO:40.

[0097] By "percent identity" between two amino acid sequences (or between two nucleotide sequences) is meant within the scope of the present invention the percentage of identical amino acid (or nucleotide) residues between the two sequences being compared, this percentage being obtained after carrying out the best alignment (optimal alignment) between both sequences. The skilled person knows different techniques that make it possible to obtain such a percentage identity, including homology algorithms or computer programs such as the program BLAST.

[0098] The percent identity is a statistical measure and the differences between both sequences are randomly distributed along these sequences. The differences between both sequences can consist of different modified forms of the sequences: deletions, substitutions or additions of amino acid (or nucleotide) residues.

[0099] In a first embodiment, modifications made to the sequence result in substitutions between equivalent amino acids, ie amino acids that have structural homology or do not substantially modify the biological activity of the corresponding antibody.

[0100] In a second embodiment, the modifications made to the sequence result in the replacement of non-equivalent amino acids, i.e. amino acids with no structural homology, which are likely to improve the biological properties of the antibody, i.e. increase affinity and / or specificity, broaden the recognition range, increase stability, reduce immunogenicity, etc.

[0101] When applied to both of the aforementioned embodiments, these modifications, insertions or deletions, whether essential or not, can be targeted to the CDRs for the properties of the antibodies according to the invention.

[0102] When applied to both of the aforementioned embodiments, these modifications, insertions or deletions can also be targeted to the FR region, given that such regions within the variable domain of the antibody can play a role in the expression by the antibody of the properties of the antibody according to the invention.

[0103] The present invention also relates to a cloning and / or expression vector containing at least one polynucleotide according to the invention. Such vectors are particularly useful for transforming host organisms and for expressing the antibodies according to the invention in the latter.

[0104] A vector according to the invention further comprises one (or more) elements that allow a polynucleotide according to the invention to be expressed and / or a product resulting from translation of a polynucleotide according to the invention to be secreted, including a constitutive or inducible promoter, transcription initiation or termination signals, translation initiation or termination signals.

[0105] Advantageously, the vector according to the invention comprises a promoter, a polynucleotide of the invention and a terminator element operably linked to each other. "Operatively linked to each other" according to the invention means elements that are linked to each other such that the function of one element is influenced by the function of another element. By way of example, a promoter is operably linked to a coding sequence if it is capable of influencing its expression. The peptide transcription, translation and maturation regulatory elements that the vector may contain are known to those skilled in the art who can select them depending on the host organism in which expression or cloning is to be carried out.

[0106] The vector according to the invention is advantageously selected from plasmids, cosmids, bacteriophages and viruses such as baculoviruses. In particular, the vector according to the invention is an autonomously replicating vector which comprises an element that allows it to be maintained and replicated in the host organism as an origin of replication. Furthermore, the vector may contain an element that allows it to be selected in the host organism, for example as an antibiotic resistance gene or a selection gene that ensures complementation with the respective gene deleted in the genome of the host organism. Such cloning and / or expression vectors are well known to the person skilled in the art and have been extensively described in the literature.

[0107] The present invention also relates to host organisms transformed by or containing a polynucleotide according to the invention or a vector according to the invention.

[0108] By "host organism" is meant an isolated unicellular or multicellular lower or higher organism into which a polynucleotide of the invention has been introduced to produce an antibody according to the invention.

[0109] The skilled artisan knows different methods to efficiently introduce a polynucleotide into a host organism in order to produce the antibody encoded by said polynucleotide in the host organism. By way of example and in a non-exhaustive manner, these methods may be electroporation, lipofection, biological transformation of plants using Agrobacterium tumefasciens, heat shock or chemical methods.

[0110] 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 antibodies of the invention on a semi-industrial or industrial scale.

[0111] Alternatively, the host organism is an animal cell, such as a mammalian cell, a plant cell, an insect cell, a non-human animal or a plant.

[0112] Such a host organism can be used to produce an antibody according to the invention. Indeed, a method for producing an antibody according to the invention comprises the following steps: a) culturing a host organism according to the invention, in particular a unicellular host organism, in a culture medium under suitable conditions; b) recovering the antibody from the culture medium of the cultured host organism or from the cultured host organism. Includes.

[0113] The antibodies according to the invention can also be modified to i) generate antibodies labelled with radioisotopes, prodrugs, enzymes or toxins, and ii) modify the binding specificity and / or affinity, and / or stability, and / or immunogenicity of the antibodies to specifically target cells that overexpress ETB-R, in particular cancer cells such as melanoma, glioblastoma, etc.

[0114] The antibody according to the present invention can also be modified to chemically or genetically link it to a peptide molecule; a protein molecule; a nuclear molecule such as DNA, RNA, RNAi, aptamer, PNA or LNA; a lipid molecule; a carbohydrate molecule or a chemical molecule.

[0115] The present invention therefore relates to a compound comprising an antibody according to the invention linked to a member selected from the group consisting of a cytotoxic group, a readily detectable group, or an effector group.

[0116] By "cytotoxic group" is meant a group that is directly or indirectly toxic to cells targeted by an antibody according to the invention. By "directly cytotoxic" is meant a group that is cytotoxic in itself. By "indirectly cytotoxic" is meant a group that is not cytotoxic in itself but which is capable of inducing cytotoxicity, for example through its action on other molecules or through a further action on itself.

[0117] In a first embodiment, the cytotoxic group is a cytotoxic chemotherapeutic agent. The skilled artisan knows different cytotoxic chemotherapeutic agents that can be used within the scope of the present invention. The activity of these agents can be increased under irradiation. Illustrative and non-limiting examples include alkylating agents such as mechlorethamine or chlorambucil; methotrexate; 5-fluoro-uracil; vinblastine; gemcitabine; fludarabine; nicotinamide; doxorubicin; mitomycin; L-asparaginase; cisplatin; taxol and its analogs / derivatives.

[0118] In a second embodiment the cytotoxic group is a cytotoxic (poly)peptide group such as ricin, abrin, Pseudomonas exotoxin, TNFα and interleukin-2.

[0119] In a third embodiment, the cytotoxic group is an indirectly cytotoxic chemotherapeutic agent. Such agents, also called prodrugs, have little or no cytotoxicity themselves, but can provide a cytotoxic substance (or drug), especially as defined in the first embodiment, especially after enzymatic reaction or irradiation. Illustrative and non-limiting examples include methotrexate-alanine; mitomycin phosphate, 5-fluorocytosine; photofrin and capecitabine.

[0120] In a fourth embodiment, the cytotoxic group is an indirectly cytotoxic (poly)peptide group. By indirectly cytotoxic polypeptide group is meant a peptide or polypeptide which exhibits enzymatic activity and is capable of converting a relatively non-toxic prodrug, in particular as defined in the third embodiment, into a cytotoxic substance, in particular as defined in the first embodiment. Among such indirectly cytotoxic (poly)peptide groups, mention may be made of peptidases, such as carboxypeptidases, aminopeptidases or endopeptidases; phosphatases; sulfatases; amidases; kinases; glycosidases; deaminases; reductases; and oxidases.

[0121] In a fifth embodiment, the cytotoxic agent is a directly or indirectly cytotoxic nucleic acid molecule, such as an antisense oligonucleotide or an aptamer.

[0122] The person skilled in the art is aware of the different techniques making it possible to bind such groups to the antibodies according to the invention, once the latter are obtained or produced.

[0123] These techniques allow the covalent linkage between the antibody according to the invention and the cytotoxic group by taking advantage of specific chemical groups carried by the antibody according to the invention and among which thiol groups, ester groups, amino groups, acid groups and any chemical element that may be implemented in "click chemistry" may be mentioned.

[0124] Alternatively, and particularly, when the cytotoxic group is a peptidic group, the conjugation may consist of producing the compound according to the invention as a fusion compound by recombinant genetic techniques, in which the polynucleotide comprises the respective regions encoding the antibody according to the invention and the cytotoxic group 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.

[0125] Regardless of the technique used to conjugate an antibody according to the invention with a cytotoxic group, the only requirement to be met within this conjugation is that the conjugated antibody properly preserves its ETB-R binding specificity and its absence of an antagonist, these properties being related to those of the cytotoxic group.

[0126] By "easily detectable group" is meant within the scope of the present invention a group that can be detected by carrying out suitable detection techniques, advantageously non-invasive, such as microscopy, scintigraphy, positron emission tomography (TEP) and magnetic resonance imaging (MRI). Compounds according to the invention that contain such easily detectable groups are particularly suitable for the field of imaging and diagnostics. Due to the ETB-R binding specificity of the antibody according to the invention present in this compound, it makes it possible to identify and localize in particular the sites where ETB-R is overexpressed.

[0127] In a first embodiment, the readily detectable group may be an enzyme or molecule capable of generating a detectable and possibly quantifiable signal under certain conditions, such as when contacted with a compatible substrate. Illustrative and non-limiting examples include biotin, digoxigenin, 5-bromodeoxyuridine, alkaline phosphatase, peroxidase, acetylcholinesterase (AChE), glucose amylase and lysozyme.

[0128] In a second embodiment, the readily detectable group may be a fluorescent, chemiluminescent or bioluminescent label, such as fluorescein and its derivatives, rhodamine and its derivatives, GFP (green fluorescent protein) and its derivatives, as well as umbelliferone; luminol; luciferase and luciferin.

[0129] In a third embodiment, the readily detectable group 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 some radioactive atoms used as readily detectable groups may also be cytotoxic groups due to the amount of radioactivity they can deliver.

[0130] Everything already explained for the conjugation of the antibodies according to the invention with cytotoxic groups is applicable mutatis mutandis to the conjugation of the antibodies according to the invention with easily detectable groups, which can also be carried out in connection with nano objects in order to increase their concentration and thus improve the emitted signal, contrast or toxicity.

[0131] If this easily detectable group is a radioactive label, the latter can be introduced into the peptide sequence of the antibody according to the invention. This can be done during the synthesis of the antibody by using one or more labeled amino acids. Alternatively, this can be done after the synthesis by binding the radioactive label to a residue of the peptide sequence of the synthesized antibody. For example, yttrium-90 can be bound via a lysine residue. Alternatively, the radioactive label can be indirectly bound to the antibody by known means. For example, EDTA or other chelating agents can be bound to the antibody according to the invention and used to bind indium-111.

[0132] The present invention relates to the use of antibodies and compounds containing easily detectable groups as highly efficient diagnostic, prognostic and in vivo follow-up tools for medical imaging. The antibody type is selected to give the best signal to noise ratio and the best pharmacokinetics.

[0133] Briefly, the present invention provides a method for detecting and quantifying expression or overexpression of endothelin receptor subtype B in vivo or in vitro, comprising the steps of: a1) contacting a biological sample with a compound according to the invention; b1) detecting a possible complex between said compound and said endothelin receptor subtype B; This relates to a method comprising:

[0134] Such methods can be carried out to detect, diagnose, prognose or follow up conditions in which endothelin receptor subtype B is overexpressed, in particular to detect, diagnose, prognose or follow up cancer conditions (presence, size and progression of cancer tumors). In the case of methods for diagnosing cancers such as glioblastoma, the latter can be a1') contacting a biological sample from a subject with a compound according to the invention; b1') detecting the signal emitted by the easily detectable group; and c1') determining the presence or absence of cancer in the subject based on the signal detected in step (b1'). Includes.

[0135] In a particular embodiment, the diagnostic method according to the present invention is an in vitro method, in which a biological sample, such as a biopsy, is taken from the subject before carrying out step (a1'). Alternatively, this method can correspond to an in vivo imaging method, in which an effective amount of a compound according to the present invention is administered to the subject beforehand. By "effective amount" is meant an amount of a compound according to the present invention sufficient for imaging cancer. This amount varies depending on the mode of administration, the formulation administered, the excipients and the cancer to be diagnosed. However, it is a routine task for the person skilled in the art to determine this effective amount.

[0136] By "effector group" is meant within the scope of the present invention a group capable of specifically recognizing a cancer marker or capable of recruiting (i) effector cells of the immune system, i.e. NK cells, cytotoxic T cells, macrophages or (ii) the complement system. By "group capable of specifically recognizing a cancer marker" is meant within the scope of the present invention a ligand of a cancer marker; an antibody identical or different to the antibody according to the present invention; a protein; a peptide; or a nuclear molecule such as DNA, RNA, RNAi, aptamer, PNA or LNA. By "cancer marker" both ETB-R and other membrane markers are contemplated.

[0137] In a first embodiment, the effector group recognises a cancer marker that is the same as or different from ETB-R, expressed on the surface of cancer cells, thus ensuring greater recognition specificity and thereby increasing the targeting of cancer cells.

[0138] In a second embodiment, the effector group exhibits recognition specificity for markers specifically present on the surface of effector cells of the immune system, i.e. NK cells, macrophages or cytotoxic T cells, such recruitment ensuring the targeted lysis of cancer cells recognized by the antibody of the invention.

[0139] In a third embodiment, the effector group has recognition specificity for the complement system, in particular for protein C1 or its truncated form C1q, which initiates a cascade of molecular events that leads to the death of the target cell, such recruitment ensuring the targeted lysis of the cancer cells recognized by the antibodies of the invention.

[0140] In a fourth embodiment, the effector group exhibits recognition specificity for the complement system, in particular for protein C3 or its truncated form C3b, thus ensuring the recruitment of effector cells of the immune system which induce the death of target cells, such recruitment ensuring the targeted lysis of cancer cells recognised by the antibody of the invention.

[0141] The present invention relates to an antibody according to the invention, a polynucleotide according to the invention or a compound according to the invention for use as a drug or medicament.

[0142] The present invention therefore relates to a pharmaceutical composition which comprises as active ingredient an antibody according to the invention, or a polynucleotide according to the invention or a compound according to the invention, and a pharma- ceutically acceptable vehicle.

[0143] By "pharmaceutically acceptable vehicle" is meant according to the invention any substance added to an antibody, polynucleotide or compound according to the invention to facilitate its transport, to avoid its substantial degradation in said composition and / or to increase its half-life. Advantageously, such a pharmaceutically acceptable vehicle is sterile and non-pyrogenic. It is selected depending on the type of application of the pharmaceutical composition of the invention, in particular depending on its mode of administration.

[0144] Thus, the pharmaceutical composition according to the invention consists of at least one antibody, or polynucleotide or compound according to the invention, in free form or in the form of an addition salt with a pharma- ceutically acceptable acid, in the pure state or in the form of a composition in which it is associated with any other pharma- ceutical compatible product. The pharmaceutical composition according to the invention can be used by the systemic route; by the parenteral route, for example the intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular or subcutaneous route; by the topical route; by the oral route; by the rectal route; by the intranasal route or by inhalation.

[0145] As solid compositions for oral administration, the antibodies, polynucleotides or compounds according to the invention can be used in the form of tablets, pills, powders, etc. when mixed with one or more conventionally used inert diluents, and possibly other substances such as lubricants, dyes, coatings, etc.

[0146] Pharmaceutically acceptable liquid compositions for oral or ocular administration may be suspensions, solutions, emulsions, syrups containing conventionally used inert diluents, and possibly other substances such as wetting agents, sweeteners, thickeners, etc.

[0147] Sterile compositions for parenteral administration may be aqueous or non-aqueous solutions, suspensions or emulsions. As solvents or vehicles, water, propylene glycol, vegetable oils or other suitable organic solvents may be used. These compositions may also contain adjuvants such as wetting agents, isotonicity agents, emulsifiers, etc.

[0148] Compositions for topical application can be, for example, creams, lotions, oral sprays, nasal or eye drops or aerosols.

[0149] The daily dosage levels of the antibodies, polynucleotides or compounds according to the invention are typically 1-1000 mg per adult (i.e., about 0.015-15 mg / kg), administered in single or divided doses. These dosages are given for illustrative purposes only. In any event, the physician can determine the actual dosage most suitable for the individual patient to be administered, which will vary with the age, weight and response of the patient.

[0150] The present invention relates to an antibody according to the invention, a polynucleotide according to the invention, a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of disorders or conditions involving dysfunction associated with a direct or alternative physiological pathway of an axis involving endothelin and at least one of its receptors, such as in particular endothelin receptor subtype B.

[0151] Advantageously, such disorder or such condition is cancer. Cancer may include melanoma, colon cancer, Kaposi's sarcoma, glioblastoma, ovarian cancer and bladder cancer. Typically, the disorder is melanoma or glioblastoma.

[0152] Furthermore, the present invention thus relates to a method for the treatment and / or prevention of a disorder or condition involving a malfunction associated with a direct or alternative physiological pathway of an axis involving endothelin and in particular at least one of its receptors, such as endothelin receptor subtype B, in a patient having or likely to have such a disorder or condition, which method comprises administering to said patient an effective amount of an antibody according to the invention, a polynucleotide according to the invention, a compound according to the invention or a pharmaceutical composition according to the invention.

[0153] Finally, the present invention relates to the use of the antibodies according to the invention or the compounds according to the invention as research tools particularly suitable for studying signal transduction pathways related to the endothelin / endothelin receptor axis and for advancing the understanding of the structural and functional properties of these receptor families.

[0154] Further characteristics and advantages of the present invention will become more apparent to those skilled in the art on reading the examples given below, given for illustrative and non-limiting purposes, with reference to the attached figures, in which: [Brief description of the drawings]

[0155] [Figure 1] Binding curves of Rendomab-B49 (FIG. 1A), Rendomab-B41 (FIG. 1B) and Rendomab-B36 (FIG. 1C) to CHO cells overexpressing ETB-R with (squares) or without (dots) preincubation of the cells in the presence of 300 nM endothelin 1. In both cases, affinities of the order of 1 nanomolar were measured. [Diagram 2] Confocal microscopy images of neurospheres from a biopsy of a patient with high-grade glioblastoma in the presence of 1 μg / mL of labeled Rendomab-B1 (FIG. 2A) or 1 μg / mL of labeled Rendomab-B49 (FIG. 2B). Only nuclei of DAPI-labeled cells are seen in FIG. 2A. [Diagram 3]3A-3B show images obtained by confocal microscopy of tumor cells from a biopsy of a patient with low-grade glioblastoma in the presence of DAPI (FIG. 3A) or 1 μg / mL Rendomab-B49 (FIG. 3B). [Figure 4] FIG. 1 shows images obtained by confocal microscopy of tumor cells from a biopsy of a patient with glioblastoma in the presence of 1 μg / mL Rendomab-B36. [Diagram 5] FIG. 5 shows the core sequences deduced from the amino acids of the variable domains of the light chain (VL) (FIG. 5A) and heavy chain (VH) (FIG. 5B) of the endothelin receptor B-specific IgG1 / kappa murine antibody Rendomab-B49. [Figure 6] 6A and 6B show the core sequences deduced from the amino acids of the variable domains of the light chain (VL) (FIG. 6A) and heavy chain (VH) (FIG. 6B) of the endothelin receptor B-specific IgG1 / kappa murine antibody Rendomab-B41. [Figure 7] FIG. 7 shows the core sequences deduced from the amino acids of the variable domains of the light chain (VL) (FIG. 7A) and heavy chain (VH) (FIG. 7B) of the endothelin receptor B-specific IgG3 / kappa murine antibody Rendomab-B36. [Figure 8] A. Epitope mapping. Revealed "PepScan" membrane. B. Epitope mapping. Sequences of peptides recognized with high intensity by Rendomab-B49 (C5, C6, C7, C8, C9 and C10 peptides). C. Epitope mapping. Location of epitopes recognized by Rendomab-B49 in the sequence of human endothelin receptor subtype B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0156] I. Materials and Methods I.1.Immunization The so-called "genetic immunization" strategy developed in our laboratory consists of combining DNA injection with a protein boost 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, 30, 727-737).

[0157] Briefly, mouse tibialis muscles were injected with 50 μg of plasmid DNA pcDNA3 / ETB-R three times over a 14-day period. Each DNA injection was followed by electrical stimulation with the following characteristics: 8 pulses of 20 ms, 80 volts, 1 Hz each. Then, mice were injected with 2.10 transiently overexpressing ETB-R by intraperitoneal route. 6 Three booster immunizations were performed by injecting 100 COS cells each.

[0158] The highest responding mice were sacrificed for cell fusion of their splenic lymphocyte cells with the mouse myeloma NS-1.

[0159] I.2. Hybridoma Screening The resulting hybridomas were initially screened by ELISA against CHO cells stably expressing ETB-R, together with CHO cells expressing the irrelevant receptor NK1 (CHO-WT) as a negative control.

[0160] The retained hybridomas were then screened by flow cytometry (instrument, Guava, Millipore). Three hybridomas, designated "Rendomab-B49", "Rendomab-B41" and "Rendomab-B36", were finally retained at the end of both these screens. Secreted antibodies were then produced from liquid tumors (ascites) induced in mice by intraperitoneal injection of the selected hybridomas.

[0161] II. Biochemical characterization After purification of Rendomab-B49, Rendomab-B41 and Rendomab-B36, their biochemical properties were characterized.

[0162] Isotype identification of the heavy and light chains of Rendomab-B49 was performed using the "Rapid ELISA Mouse mAb Isotyping" kit from Piercell. The heavy chain is an immunoglobulin of isotype 1, G, and the light chain is an immunoglobulin of kappa. Rendomab-B49 is therefore an IgG1 / kappa immunoglobulin. Rendomab-B41 and Rendomab-B36 are IgG1 / kappa and IgG3 / kappa immunoglobulins, respectively.

[0163] After purification of Rendomab-B49, Rendomab-B41 and Rendomab-B36, their binding specificity was established by flow cytometry (Facs Calibur, Becton Dickinson BD Bioscience) by indirect labeling with a commercially available fluorescent anti-mouse secondary antibody (Life Technologies: AlexaFluor® 488 F(ab')2 fragment of goat anti-mouse IgG(H+L)*2mg / mL).

[0164] CHO-ETBR cells were divided into two groups: one group was pre-incubated in culture medium in the presence of endothelin-1 at a final concentration of 300 nM for 2 h at 37°C to internalize the ETBR receptor, in order to demonstrate the recognition specificity of the three Rendomab antibodies against ETB-R.

[0165] The cells were then aliquoted into 15 ml Falcon tubes (300000 cells / tube) in the presence of increasing concentrations of the antibody Rendomab-B49 (concentration range 0.04 nM to 800 nM, 2 x 2). After 2 h of incubation at 4°C, the cells were washed 3 times with PBS buffer and then incubated with secondary antibody at a final concentration of 4 μg / mL for 1 h at 4°C. The cells were then washed 3 times with PBS buffer and then 30000 cells were counted and analyzed on a FACs Calibur.

[0166] III. Binding properties to glioblastoma cells III.1. Protocol Cells are cultured as neurospheres (suspension culture) or adherent to poly-D-lysine / laminin coated glass slides. Cells are then bound with 4% paraformaldehyde solution at room temperature for 15 minutes and then washed twice with PBS (SigmaAldrich). Non-specific sites are blocked and cells are permeabilized with PBS solution + 5% donkey serum + 0.1% Triton for 30 minutes.

[0167] The primary antibody Rendomab-B49, diluted to 1 μg / mL in the previous solution, is placed in contact with the cells overnight at 8° C. After rinsing twice with PBS, the cells are incubated for 2 hours with a commercial secondary antibody coupled with either Alexa 488 (Life Technologies: Alexa Fluor® 488 F(ab′)2 fragment of goat anti-mouse IgG(H+L)*2 mg / mL*) or Alexa 680 (Life Technologies: Alexa Fluor® 680 F(ab′)2 fragment of goat anti-mouse IgG(H+L)*2 mg / mL*) at a final concentration of 4 μg / ml according to the supplier's recommendations, then rinsed twice with PBS.

[0168] To observe the nuclei, the cells are briefly incubated with 1 µg / ml DAPI (4',6-diamidino-2-phenylindole) solution, then washed with PBS, after which a coverslip is mounted between the slide and the slide using ad-hoc assembly medium for observation. Pictures are taken with a Zeiss microscope equipped with an apotome module at 400x magnification.

[0169] III.2. Results The near nanomolar range affinities of Rendomab-B49, Rendomab-B41 and Rendomab-B36, and their exclusive specificity for human endothelin receptor subtype B, are shown in FIG.

[0170] The binding curves observed for Rendomab-B49 (Figure 1A), Rendomab-B41 (Figure 1B) and Rendomab-B36 (Figure 1C) are characteristic of an antibody binding to its target with a saturation plateau.

[0171] Furthermore, preincubation of CHO-ETBR cells in the presence of 300 nM endothelin-1 for 2 h at 37°C led to internalization of ETB-R and reduced the binding of Rendomab-B49, Rendomab-B41 and Rendomab-B36 under these conditions by more than 50%, thus demonstrating the binding specificity of these antibodies for ETB-R. The apparent dissociation constants K D is determined by taking the value of the concentration that gives an MFI equal to 50% of the value of the MFI at the plateau. This constant is close to 1 nM for the three antibodies.

[0172] Second, the lack of binding of Rendomab-B49, Rendomab-B41 and Rendomab-B36 on CHO-WT cells not transfected with ETBR indicates that the binding observed on CHO-ETBR cells is not due to membrane proteins of CHO cells.

[0173] Binding experiments on tumor cells isolated from glioblastoma patient biopsies.

[0174] In Figure 2, there is no labeling of neurospheres isolated from high-grade glioblastoma tumors with the antibody Rendomab-B1 (Figure 2A), whereas in the same cells, very strong fluorescent labeling is observed with the antibody Rendomab-B49 (Figure 2B).

[0175] Similarly, Figure 3 shows strong fluorescent labeling of tumor cells isolated from a low-grade glioblastoma tumor with the antibody Rendomab-B49 (Figure 3B), and these cells are also labeled with DAPI (Figure 3A).

[0176] Comparable results were obtained with the antibodies Rendomab-B41 and Rendomab-B36, to which end Figure 4 shows the intense fluorescent labeling by the antibody Rendomab-B36 of tumor cells isolated from a glioblastoma tumor.

[0177] IV. Molecular Cloning Cloning of the nuclear precursors encoding the heavy and light chains of Rendomab-B1 was carried out using the kit "Gene-Elute / Total RNA" (Sigma) and "RACE-PCR" (Invitrogen).

[0178] The core sequences deduced from the amino acids of the variable domains of the light chain (VL) and heavy chain (VH) of Rendomab-B49, Rendomab-B41, and Rendomab-B36 are shown in Figures 5 to 7, respectively.

[0179] V. Epitope Mapping V.1. Materials and Methods The mapping of the epitopes recognized by Rendomab-B49, Rendomab-B41 and Rendomab-B36 on the surface of ETB-R was carried out by the "Pep-Scan" technique in conjunction with and according to the protocol developed within the company "SysDiag" CNRS / BioRad (Dr. Claude Granier) UMR3145, Montpellier. The sequence of the human endothelin receptor subtype B has the following amino acid sequence: MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPTKTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTVVSCL VFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLHIVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRYRAVASWSRIKGIGVPKWTAVE IVLIWVVSVVLAVPEAIGFDIITMDYKGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEMLRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLYNQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS (SEQ ID NO:42) Shows.

[0180] For this purpose, 144 peptides of 12 amino acids each supplemented by one amino acid were used, which correspond to all ETB-R sequences that are expressed on the cytoplasmic side of the membrane.

[0181] To reveal the epitope peptide recognized by Rendomab-B49, the "Pep-Scan" membrane was treated according to the following protocol: - Wet the membrane in an ethanol bath. - Wash 3 times in 25 ml of TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4) for 10 minutes at room temperature with agitation. - Saturate the membrane with 25 ml of saturation buffer (TBS, 5% skimmed milk powder, 0.1% Tween 20) for 30 minutes at room temperature with stirring. - Incubate with 25 ml of saturation buffer containing Rendomab-B49 antibody at a final concentration of 1 μg / ml overnight at 4° C. with agitation. - 3 short washes (30 seconds) with TBS buffer, then 3 washes with 25 ml of TBST buffer (TBST=TBS+0.1% Tween 20) for 10 minutes with agitation. - Incubate with 25 ml of saturation buffer containing goat anti-mouse secondary antibody diluted 1 / 5,000 for 30 minutes at room temperature with agitation. - 3 short washes (30 seconds) with TBS buffer, then 3 washes with 25 ml of TBST buffer for 10 minutes with agitation, - The membrane is revealed by immersion for 5 minutes in the revealing solution from Pierce (Pierce ECL Plus Western Blotting Ref:32132) and signal acquisition in automatic mode with the BioRad system ChemiDoc™.

[0182] V.2.Results The results of the epitope analysis of Rendomab-B49 are shown in Figure 8. The peptide sequences hybridizing with high intensity are C5, C6, C7, C8, C9 and C10 peptides (Figure 8A).

[0183] Their alignment makes it possible to identify the epitope mainly recognized by Rendomab-B49, the latter being EVPKGDR, corresponding to the sequence from amino acid 70 to amino acid 76 of SEQ ID NO: 42 (FIG. 8B). As soon as glutamic acid E70 is deleted in peptide C11 of sequence VPKGDRTAGSPP, corresponding to the sequence from amino acid 71 to amino acid 82 of SEQ ID NO: 42, the binding of the antibody is significantly reduced (FIG. 8A).

[0184] The location of the epitope peptide in the sequence of human endothelin receptor subtype B is shown in Figure 8C. This sequence is at the N-terminus of the receptor.

Claims

1. An antibody, fragment or derivative thereof against endothelin receptor subtype B, i) - CDR1 (hereinafter CDR1) having the amino acid sequence GYTFISYW (SEQ ID NO: 5) H That is, - CDR2 (hereinafter CDR2) having the amino acid sequence IDPDSGGT (SEQ ID NO: 10) H (That is, - CDR3 having the amino acid sequence AREGDYAWFAY (SEQ ID NO: 15) (hereinafter CDR3 H (That is, and a heavy chain variable region comprising - CDR1 (hereinafter CDR1) having the amino acid sequence QSIVHSNGNTY (SEQ ID NO: 22) L That is, - CDR2 whose amino acid sequence is KVS (hereinafter referred to as CDR2 L (That is, - CDR3 (hereinafter CDR3) having the amino acid sequence FQGSHVPWT (SEQ ID NO: 27) L (That is, A light chain variable region comprising Including, or ii) - CDR1 H , the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7), - CDR2 H , the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10), and - CDR3 H, the amino acid sequence of which is VREGWDAWFVY (SEQ ID NO: 17) and a heavy chain variable region comprising - CDR1 L , the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22), - a CDR2 L whose amino acid sequence is KVF, - CDR3 L, the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29) A light chain variable region comprising Including, or iii) - CDR1 H , the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7), - CDR2 H , the amino acid sequence of which is IDPNSGGT (SEQ ID NO: 12), and - CDR3 H, the amino acid sequence of which is AREGEFAWFAY (SEQ ID NO: 19) and a heavy chain variable region comprising - CDR1 L , the amino acid sequence of which is QNIVHSNGYTY (SEQ ID NO: 24), - a CDR2 L whose amino acid sequence is KVS, - CDR3 L, the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29) A light chain variable region comprising Including, the fragment has at least one antigen-binding site; the derivative is a single chain Fv, It can recognize a specific conformational isomer of endothelin receptor subtype B expressed on the surface of glioblastoma cells. An antibody, a fragment or derivative thereof.

2. The amino acid sequence is: QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGRIDPDSGGTKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREGDYAWFAYWGQGTLVPVSA (SEQ ID NO: 31) The antibody of claim 1, comprising a heavy chain variable region that exhibits at least 80% identity to

3. The amino acid sequence is: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 37) The antibody of claim 1 or 2, comprising a light chain variable region that exhibits at least 80% identity with

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

5. 5. The antibody of claim 1, which is monoclonal.

6. 6. The antibody according to any one of claims 1 to 5, which is a monoclonal mouse antibody obtained from a hybridoma selected from the hybridoma deposited at the CNCM on May 19, 2016 under the accession number CNCM I-5084, the hybridoma deposited at the CNCM on June 7, 2016 under the accession number CNCM I-5104 and the hybridoma deposited at the CNCM on June 7, 2016 under the accession number CNCM I-5103.

7. A hybridoma selected from the hybridoma deposited at the CNCM on May 19, 2016 under accession number CNCM I-5084, the hybridoma deposited at the CNCM on June 7, 2016 under accession number CNCM I-5104 and the hybridoma deposited at the CNCM on June 7, 2016 under accession number CNCM I-5103.

8. The following distinct polynucleotides: α) a polynucleotide encoding an antibody as defined in any one of claims 1 to 6, and β) a polynucleotide that is 100% complementary to the entire length of the polynucleotide defined in (α); An isolated polynucleotide selected from:

9. A cloning and / or expression vector containing at least one polynucleotide according to claim 8.

10. A non-human host organism transformed by or containing a polynucleotide according to claim 8 or a vector according to claim 9.

11. 7. A compound comprising an antibody according to any one of claims 1 to 6 conjugated to a member selected from the group consisting of a cytotoxic group, a readily detectable group, or an effector group, wherein the readily detectable group is a group that can be detected by carrying out a suitable non-invasive detection technique selected from microscopy, scintigraphy, positron emission tomography (TEP) and magnetic resonance imaging (MRI).

12. 12. An antibody according to any one of claims 1 to 6, a polynucleotide according to claim 8 or a compound according to claim 11 for use in medicine.

13. A pharmaceutical composition comprising as an active ingredient an antibody according to any one of claims 1 to 6, a polynucleotide according to claim 8 or a compound according to claim 11, and a pharma- ceutically acceptable vehicle.

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