ANTIBODY DIRECTED AGAINST THE ENDOTHELIN RECEPTOR BETA SUB-TYPE

Abstract

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

Claims

1) An antibody directed against the endothelin receptor sub-type B comprising: a heavy chain variable region comprising a CDR1 (hereinafter designated CDR1.sub.H), a CDR2 (hereinafter designated CDR2.sub.H) and a CDR3 (hereinafter designated CDR3.sub.H) such that the ordered juxtaposition formed by the amino acid sequences of the CDR1.sub.H, CDR2.sub.H and CDR3.sub.H exhibits at least 80% identity with the following amino acid sequence: GYTFISYWIDPDSGGTAREGDYAWFAY (SEQ ID NO: 1) and a light chain variable region comprising a CDR1 (hereinafter designated CDR1.sub.L), a CDR2 (hereinafter designated CDR2.sub.L) and a CDR3 (hereinafter designated CDR3.sub.L) such that the ordered juxtaposition formed by the amino acid sequences of the CDR1.sub.L, CDR2.sub.L and CDR3.sub.L exhibits at least 80% identity with the following amino acid sequence: TABLE-US-00020 (SEQIDNO:2) QSIVHSNGNTYKVSFQGSHVPWT, a fragment or derivative thereof.

2) The antibody according to claim 1, wherein said antibody comprises: i.sub.1) a heavy chain variable region comprising: a CDR1.sub.H the amino acid sequence of which is GYTFISYW (SEQ ID NO: 5); a CDR2.sub.H the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3.sub.H the amino acid sequence of which is AREGDYAWFAY (SEQ ID NO: 15); or ii.sub.1) a heavy chain variable region comprising: a CDR1.sub.H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2.sub.H the amino acid sequence of which is IDPDSGGT (SEQ ID NO: 10); and a CDR3.sub.H the amino acid sequence of which is VREGWDAWFVY (SEQ ID NO: 17); or iii.sub.1) a heavy chain variable region comprising: a CDR1.sub.H the amino acid sequence of which is GYTFTSYW (SEQ ID NO: 7); a CDR2.sub.H the amino acid sequence of which is IDPNSGGT (SEQ ID NO: 12); and a CDR3.sub.H the amino acid sequence of which is AREGEFAWFAY (SEQ ID NO: 19).

3) The antibody according to claim 1, wherein said antibody comprises a heavy chain variable region the amino acid sequence of which exhibits at least 80% identity with the following sequence: TABLE-US-00021 (SEQIDNO:31) QVQLQQPGAALVKPGASVKLSCKASGYTFISYWMLWVKQRPGRGLEWIGR IDPDSGGTKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREG DYAWFAYWGQGTLVPVSA.

4) The antibody according to claim 1, wherein said antibody comprises: i.sub.2) a light chain variable region comprising: a CDR1.sub.L the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2.sub.L the amino acid sequence of which is KVS; a CDR3.sub.L the amino acid sequence of which is FQGSHVPWT (SEQ ID NO: 27); or ii.sub.2) a light chain variable region comprising: a CDR1.sub.L the amino acid sequence of which is QSIVHSNGNTY (SEQ ID NO: 22); a CDR2.sub.L the amino acid sequence of which is KVF; a CDR3.sub.L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29); or iii.sub.2) a light chain variable region comprising: a CDR1.sub.L the amino acid sequence of which is QNIVHSNGYTY (SEQ ID NO: 24); a CDR2.sub.L the amino acid sequence of which is KVS; a CDR3.sub.L the amino acid sequence of which is FQGSHVPLT (SEQ ID NO: 29).

5) The antibody according to claim 1, wherein said antibody comprises a light chain variable region the amino acid sequence of which exhibits at least 80% identity with the following sequence: TABLE-US-00022 (SEQIDNO:37) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKLEIK.

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

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

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

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

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

11) A hybridoma chosen from the hybridoma deposited with the CNCM on the 19 May 2016 under the accession number CNCM I-5084, the hybridoma deposited with the CNCM on the 7 Jun. 2016 under the accession number CNCM I-5104 and the hybridoma deposited with the CNCM on the 7 Jun. 2016 under the accession number CNCM I-5103.

12) An isolated polynucleotide chosen from the different polynucleotides hereinafter: ) a polynucleotide encoding an antibody as defined in claim 1; ) a polynucleotide complementary to the polynucleotide as defined in (); ) a polynucleotide of at least 18 nucleotides, capable of hybridizing under high stringency conditions with the polynucleotides as defined in () and ().

13) A cloning and/or expression vector containing at least one polynucleotide according to claim 12.

14) A host organism transformed by or comprising a polynucleotide according to claim 12.

15) A compound comprising an antibody according to claim 1 conjugated with an element chosen from the group consisting of a cytotoxic group, an easily detectable group, or an effector group.

16) The antibody according to claim 1 for use in medicine.

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

18) The antibody according to claim 1 for use in the treatment and/or prevention of a disorder or a condition involving a dysfunction, being direct or in association with another physiological route, of the axis comprising an endothelin and at least one of its receptors such as, for example, the endothelin receptor sub-type B.

19) The antibody, the polynucleotide, the compound or the pharmaceutical composition for use according to claim 18, wherein said disorder or said condition is a cancer.

20) A process for diagnosing a cancer such as a glioblastoma in vitro comprising the steps of: a.sub.1) contacting a biological sample taken from a subject with a compound according to claim 15; b.sub.1) detecting the signal emitted by the easily detectable group and c.sub.1) determining the presence or absence of a cancer in said subject based on the signal detected in step (b.sub.1).

21) A host organism transformed by or comprising a vector according to claim 13.

22) The polynucleotide according to claim 12 for use in medicine.

23) The compound according to claim 15 for use in medicine.

24) A pharmaceutical composition comprising, as an active ingredient, a polynucleotide according to claim 12 and a pharmaceutically acceptable vehicle.

25) A pharmaceutical composition comprising, as an active ingredient, a compound according to claim 15 and a pharmaceutically acceptable vehicle.

26) The polynucleotide according to claim 12 for use in the treatment and/or prevention of a disorder or a condition involving a dysfunction, being direct or in association with another physiological route, of the axis comprising an endothelin and at least one of its receptors such as, for example, the endothelin receptor sub-type B.

27) The compound according to claim 15 for use in the treatment and/or prevention of a disorder or a condition involving a dysfunction, being direct or in association with another physiological route, of the axis comprising an endothelin and at least one of its receptors such as, for example, the endothelin receptor sub-type B.

28) The pharmaceutical composition according to claim 17, for use in the treatment and/or prevention of a disorder or a condition involving a dysfunction, being direct or in association with another physiological route, of the axis comprising an endothelin and at least one of its receptors such as, for example, the endothelin receptor sub-type B.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0199] FIG. 1 shows binding curves of Rendomab-B49 (FIG. 1A), Rendomab-B41 (FIG. 1B) and Rendomab-B36 (FIG. 1C) on CHO cells overexpressing ETB-R with (squares) or without (dots) pre-incubation of the cells in the presence of 300 nM of endothelin 1. In both cases, an affinity in the order of one nanomole has been measured.

[0200] FIG. 2 shows images obtained in confocal microscopy on neurospheres from a biopsy of a patient having a high grade glioblastoma in the presence of 1 g/mL of labelled Rendomab-B1 (FIG. 2A) or 1 g/mL of labelled Rendomab-B49 (FIG. 2B). Only the nuclei of DAPI labelled cells are viewed in FIG. 2A.

[0201] FIG. 3 shows images obtained in confocal microscopy on tumor cells from a biopsy of a patient having a low grade glioblastoma in the presence of DAPI (FIG. 3A) or 1 g/mL of Rendomab-B49 (FIG. 3B).

[0202] FIG. 4 shows images obtained in confocal microscopy on tumor cells from a biopsy of a patient having a glioblastoma in the presence of 1 g/mL Rendomab-B36.

[0203] FIG. 5 shows the nucleic sequences deduced in amino acids from the variable domains of the light chain (VL) (FIG. 5A) and the heavy chain (VH) (FIG. 5B) of the IgG1/kappa murine antibody Rendomab-B49 specific to the endothelin receptor B.

[0204] FIG. 6 shows the nucleic sequences deduced in amino acids from the variable domains of the light chain (VL) (FIG. 6A) and the heavy chain (VH) (FIG. 6B) of the IgG1/kappa murine antibody Rendomab-B41 specific to the endothelin receptor B.

[0205] FIG. 7 shows the nucleic sequences deduced in amino acids of the variable domains of the light chain (VL) (FIG. 7A) and the heavy chain (VH) (FIG. 7B) of the IgG3/kappa murine antibody Rendomab-B36 specific to the endothelin receptor B.

[0206] FIG. 8 shows the epitopic mapping. FIG. 8A presents the revealed Pep scan membrane. FIG. 8B presents the sequences of the peptides recognized by Rendomab-B49 with high intensity (C5, C6, C7, C8, C9 and C10 peptides). FIG. 8C presents the location of the epitope recognized by Rendomab-B49 in the sequence of the human endothelin receptor sub-type B.

DETAILED DISCLOSURE OF PARTICULAR EMBODIMENTS

[0207] I. Materials and Methods

[0208] I.1. Immunisation

[0209] The so-called gene immunisation strategy developed in the laboratory of the inventors consists in combining DNA injections with protein boosts as an injection of cells overexpressing ETB-R (Allard et al, 2011, Electroporation-aided DNA immunisation generates polyclonal antibodies against the native conformation of human endothelin B receptor, DNA and Cell Biology, vol. 30, pages 727-737).

[0210] Briefly, three injections, in a mouse tibial muscle, of 50 g of plasmid DNA pcDNA3/ETB-R, were made with a periodicity of 14 days. Each DNA injection was followed by an electrostimulation according to the following characteristics: 8 pulses each of 20 ms, 80 Volts, 1 Hz. Three immunisation boosts were then made, by injection by the intra-peritoneal route of 2.10.sup.6 COS cells transiently overexpressing ETB-R.

[0211] The best responder mice were sacrificed in order to conduct cell fusion of the lymphoid cells of their spleens with the murine myeloma NS-1.

[0212] I.2. Screening Hybridomas

[0213] The hybridomas obtained were first screened by ELISA on CHO cells stably expressing ETB-R, with as a negative control, CHO cells expressing the irrelevant receptor NK1 (CHO-WT).

[0214] The hybridomas retained were then screened in flow cytometry (apparatus Guava, Millipore). Three hybridomas called Rendomab-B49, Rendomab-B41 and Rendomab-B36 were finally retained at the end of both these screens. The antibodies secreted were then produced from liquid tumors (ascites) induced in the mouse by injection by the intra-peritoneal route of the selected hybridomas.

[0215] II. Biochemical Characterisation

[0216] After purifying Rendomab-B49, Rendomab-B41 and Rendomab-B36, the characterisation of their biochemical properties was conducted.

[0217] The isotyping of the heavy and light chains of Rendomab-B49 was made using the Rapid ELISA Mouse mAb Isotyping kit from Piercell. It is an immunoglobulin of isotype 1, G type, for the heavy chain and kappa for the light chain. Rendomab-B49 is thus an IgG1/kappa type immunoglobulin. Rendomab-B41 and Rendomab-B36 are, an IgG1/kappa type immunoglobulin and an IgG3/kappa type immunoglobulin, respectively.

[0218] After purifying Rendomab-B49, Rendomab-B41 and Rendomab-B36, their binding specificity was established by flow cytometry (Facs Calibur, Becton Dickinson BD Bioscience), by indirect labelling using a commercial fluorescent anti-mouse secondary antibody (Life Technologie: Alexa Fluor 488 F(ab)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*).

[0219] CHO-ETBR cells were separated into 2 groups. One of the groups was incubated beforehand in a culture medium for 2 h at 37 C. in the presence of endothelin 1 at a final concentration of 300 nM so as to internalise the ETBR receptor for the purpose of demonstrating the recognition specificity of the 3 Rendomab antibodies to ETB-R.

[0220] Then, the cells were aliquoted in a 15 ml falcon tube (300 000 cells/tube) in the presence of an increasing concentration (concentration range from 0.04 nM to 800 nM two by two) of antibody Rendomab-B49. After 2 h incubation at 4 C., the cells are washed three times in a PBS buffer and then incubated for 1 h at 4 C. with the secondary antibody at a final concentration of 4 g/mL. The cells are then washed 3 times with a PBS buffer and then analyzed with FACs Calibur after counting 30 000 cells.

[0221] III. Binding Properties on Glioblastoma Cells

[0222] III.1. Protocol

[0223] The cells are cultured either in neurospheres (suspended culture) or in adherence on glass slides covered with poly D Lysine/Laminine. The cells are then bound by a 4% paraformaldehyde solution for 15 min at room temperature, and then washed twice with PBS (SigmaAldrich). The non-specific sites are blocked and the cells are permeabilized by a PBS solution+5% donkey serum+0.1% Triton, for 30 min.

[0224] The primary antibody Rendomab-B49, diluted in the previous solution at 1 g/mL is contacted with the cells overnight at 8 C. After 2 rinses with PBS, the cells are incubated with a commercial secondary antibody at a final concentration of 4 g/ml either coupled with Alexa 488 (Life Technologie: Alexa Fluor 488 F(ab)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*) or coupled with Alexa 680 (Life Technologie Alexa Fluor 680 F(ab)2 fragment of goat anti-mouse IgG (H+L)*2 mg/mL*) for 2 h according to the recommendations of the provider, and then rinsed twice with PBS.

[0225] The cells are briefly incubated with a 1 g/ml DAPI (4,6-diamidino-2-phenylindole) solution to view the nuclei, and then after washing with PBS, the coverslips are mounted between slip and slide with an ad hoc assembling medium for observation. The photographs are taken with a Zeiss microscope provided with a 400 magnification apotome module.

[0226] III.2. Results

[0227] The affinity close to the nanomolar range for Rendomab-B49, Rendomab-B41 and Rendomab-B36 and their exclusive specificity for the human endothelin receptor sub-type B are illustrated in FIG. 1.

[0228] The binding curve observed for Rendomab-B49 (FIG. 1A), Rendomab-B41 (FIG. 1B) and Rendomab-B36 (FIG. 1C) is characteristic of the binding of an antibody to its target with a saturation plateau.

[0229] In addition, the pre-incubation of CHO-ETBR cells for 2 h at 37 C. in the presence of 300 nM of endothelin 1 causes the internalisation of ETB-R, which results in a drop of more than 50% of the Rendomab-B49, Rendomab-B41 and Rendomab-B36 binding under these conditions thus demonstrating the binding specificity of these antibodies for ETB-R. The apparent dissociation constant K.sub.D of the antibodies is determined by taking the value of the concentration giving a MFI equal to 50% the value of the MFI at the plateau. This constant is close to 1 nM for the 3 antibodies.

[0230] Then, the absence of binding of Rendomab-B49, Rendomab-B41 and Rendomab-B36 on CHO-WT cells non transfected by ETBR is noted showing that the binding observed on CHO-ETBR cells is not due to a membrane protein of the CHO cells.

[0231] Binding experiments on tumor cells isolated from biopsies of patients with a glioblastoma.

[0232] In FIG. 2, there is an absence of labelling of neurospheres isolated from a high grade glioblastoma tumor with the antibody Rendomab-B1 (FIG. 2A) whereas it is observed, on the same cells, a very strong fluorescent labelling with the antibody Rendomab-B49 (FIG. 2B).

[0233] Likewise, FIG. 3 shows a strong fluorescent labelling by the antibody Rendomab-B49 (FIG. 3B) of tumor cells isolated from a low grade glioblastoma tumor, these cells being also labelled with DAPI (FIG. 3A).

[0234] Comparable results are obtained with the antibodies Rendomab-B41 and Rendomab-B36. To that end, FIG. 4 shows a strong fluorescent labelling with the antibody Rendomab-B36 on tumor cells isolated from a glioblastoma tumor.

[0235] IV. Molecular Cloning

[0236] The cloning of nucleic precursors coding the heavy chain and the light chain of Rendomab-B1 was made using the kits: Gene-Elute/total RNA (Sigma) and RACE-PCR (Invitrogen).

[0237] The nucleic sequences deduced in amino acids of the variable domains of the light chain (VL) and the heavy chain (VH) of Rendomab-B49, Rendomab-B41 and Rendomab-B36 are given in FIGS. 5 to 7 respectively.

[0238] V. Epitopic Mapping

[0239] V.1. Materials and Methods

[0240] The mapping of the epitope recognised by Rendomab-B49, Rendomab-B41 and Rendomab-B36 at the ETB-R surface was made by a Pep-scan technique in collaboration with and according to the protocols developed within UMR 3145 SysDiag CNRS/BioRad located in Montpellier (Dr. Claude Granier). The sequence of the human endothelin receptor sub-type B exhibits the following amino acid sequence:

TABLE-US-00019 (SEQIDNO:42) MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPT KTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETF KYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLH IVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRY RAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGS YLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEM LRKKSGMQIALNDHLKORREVAKTVFCLVLVFALCWLPLHLSRILKLTLY NQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSC LCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS.

[0241] For this, 144 peptides of the 12 amino acids each offset by one amino acid were used. These peptides correspond to all the ETB-R sequences displayed on the extra-cytoplasmic side of the membrane.

[0242] In order to reveal the epitopic peptide(s) recognized by Rendomab-B49, the Pep-scan membrane was treated according to the following protocol: [0243] humidification of the membrane in an ethanol bath; [0244] 3 washes in 25 ml of TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4) for 10 min under agitation at ambient temperature; [0245] saturation of the membrane with 25 ml of the saturation buffer (TBS, 5% skimmed milk powder, 0.1% Tween 20) for 30 min under agitation at ambient temperature; [0246] incubation with 25 ml of saturation buffer containing the Rendomab-B49 antibody at a final concentration of 1 g/ml overnight at 4 C. under agitation; [0247] 3 short washes (30 seconds) with TBS buffer then 3 washes of 10 min under agitation with 25 ml of TBST buffer (TBST=TBS+0.1% Tween 20); [0248] incubation with 25 ml of saturation buffer containing the goat anti-mouse secondary antibody diluted at 1/5,000 for 30 min at ambient temperature under agitation; [0249] 3 short washes (30 seconds) with TBS buffer then 3 washes of 10 min under agitation with 25 ml of TBST buffer; [0250] revelation of the membrane by immersing it in the revelation solution from Pierce (Pierce ECL Plus Western Blotting Ref: 32132) for 5 min and by the signal acquisition in automatic mode by the system ChemiDoc of BioRad).

[0251] V.2. Results

[0252] The results of the epitopic analysis of Rendomab-B49 are presented at FIG. 8. The peptide sequences hybridizing with high intensity are C5, C6, C7, C8, C9 and C10 peptides (FIG. 8A).

[0253] Their alignment makes it possible to identify the epitope predominantly recognized by Rendomab-B49: the latter is EVPKGDR corresponding to the sequence from amino acid 70 to amino acid 76 in SEQ ID NO: 42 (FIG. 8B). As soon as the glutamic acid E70 is lacking in peptide C11 of sequence VPKGDRTAGSPP corresponding to the sequence from amino acid 71 to amino acid 82 in SEQ ID NO: 42, the antibody binding decreases significantly (FIG. 8A).

[0254] The location of the epitopic peptide in the sequence of the human endothelin receptor sub-type B is presented at FIG. 8C. This sequence is at the N-terminal end of the receptor.