ANTIBODY AGAINST THE ENDOTHELIN RECEPTOR SUBTYPE A, AND USES THEREOF

Abstract

The present invention relates to antibodies against the endothelin receptor subtype A, in particular monoclonal antibodies, fragments or derivatives thereof. The present invention also relates to the therapeutic or diagnostic use of this antibody or as a research tool in the field of cancers, in particular glioblastoma.

Claims

1. Antibody directed against the endothelin receptor subtype A having: i) at least one light-chain variable region, wherein the amino acid sequence: of the CDR1.sub.L has at least 60% identity with the following amino acid sequence: SQSIVYSNGKIYL (SEQ ID NO: 2); of the CDR2.sub.L has at least 60% identity with the following amino acid sequence: KVS; of the CDR3.sub.L has at least 60% identity with the following amino acid sequence: FQGSHLPLT (SEQ ID NO: 4); and ii) at least one heavy-chain variable region wherein the amino acid sequence: of the CDR1.sub.H has at least 60% identity with the following amino acid sequence: GFTFNIYA (SEQ ID NO: 6); of the CDR2.sub.H has at least 60% identity with the following amino acid sequence: IRSKSNNYAT (SEQ ID NO: 8); of the CDR3.sub.H has at least 60% identity with the following amino acid sequence: VSSYYSGSFFAY (SEQ ID NO: 10); a fragment or a derivative thereof.

2. The antibody according to claim 1, wherein said antibody comprises at least one light-chain variable region wherein the amino acid sequence: of the CDR1.sub.L is SQSIVYSNGKIYL (SEQ ID NO: 2); of the CDR2.sub.L is KVS; and of the CDR3.sub.L is FQGSHLPLT (SEQ ID NO: 4).

3. The antibody according to claim 1, wherein said antibody comprises at least one light-chain variable region wherein the amino acid sequence has at least 60% identity with the following sequence: TABLE-US-00011 (SEQ ID NO: 12) DVLMTQTPLSLPVSLGDQASISCRSSQSIVYSNGKIYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPLT FGAGTKLELKR.

4. The antibody according to claim 1, wherein said antibody comprises at least one heavy-chain variable region wherein the amino acid sequence: of the CDR1.sub.H is GFTFNIYA (SEQ ID NO: 6); of the CDR2.sub.H is IRSKSNNYAT (SEQ ID NO: 8); and of the CDR3.sub.H is VSSYYSGSFFAY (SEQ ID NO: 10).

5. The antibody according to claim 1, wherein said antibody comprises at least one heavy-chain variable region wherein the amino acid sequence has at least 60% identity with the following sequence: TABLE-US-00012 (SEQ ID NO: 14) EVQLVESGGGLVQPKGSLKLSCAASGFTFNIYAMNWIRQAPGKGLEWIARI RSKSNNYATYYADSVKDRFTISRDDSQNMVYLQMNNLKTEDTAMYYCVSSY YSGSFFAYWGQGTLVTVSA.

6. The antibody according to claim 1, wherein said antibody is an immunoglobulin of type IgG2b/kappa.

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 the hybridoma filed at the CNCM on 18 Oct. 2017 under the number CNCM 1-5250.

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

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

11. Hybridoma filed at the CNCM on 18 Oct. 2017 under the number CNCM 1-5250.

12. Isolated polynucleotide chosen from the following various polynucleotides: a) a polynucleotide coding an antibody as defined in claim 1; β) a polynucleotide that is complementary to the polynucleotide as defined at point (α); and γ) a polynucleotide of at least 18 nucleotides, capable of hybridising under high-stringency conditions to the polynucleotides as defined at points (α) and (β).

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

14. Host organism transformed by or comprising a polynucleotide according to claim 12 or a vector according to claim 13.

15. 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. Antibody according to claim 1, polynucleotide according to claim 12 or compound according to claim 15 for use as a drug.

17. Pharmaceutical composition comprising, as active principle, an antibody according to claim 1 and a pharmaceutically acceptable vehicle.

18. Antibody according to claim 1, for use in the treatment of and/or prevention of an illness or a pathology involving a malfunctioning, direct or in association with another physiological route, of the axis comprising an endothelin and at least one of the receptors thereof such as in particular the endothelin receptor subtype A.

19. Antibody, polynucleotide, compound or pharmaceutical composition for use according to claim 18, characterised in that said illness or said pathology is a cancer.

20. Method for diagnosing a cancer such as a glioblastoma in vitro comprising the steps of: a.sub.1′) putting a biological sample of the subject in contact with a compound according to claim 15; b.sub.1′) detecting a signal emitted by the easily detectable group, and c.sub.1′) determining a presence or absence of a cancer in said subject on the basis of the signal detected at step (b.sub.1′) optionally compared with a control signal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0187] FIG. 1 presents the curves for bonding of Rendomab-A63 to CHO cells overexpressing ETA-R (CHO-ETAR) or CHO not expressing ETA-R (CHO-WT) in the presence or not of endothelin 1 (ET1).

[0188] FIG. 2 presents the binding curves of Rendomab-A63 to glioblastoma strain cells, GLI-7, overexpressing ETA-R.

[0189] FIG. 3 presents the difference in the level of expression of the EDNRA transcript coding for the endothelin receptor A in glioblastoma cells in patients (denoted GBM) compared with non-tumoral neurone cells (denoted non-tumour).

[0190] FIG. 4 presents results of immunofluorescence on glioblastoma GLI-7 strain cells, in the presence of 1 μM of ET1-FAM (FIG. 4A), in the presence of 30 nM of Rendomab-A63 (RA63) (FIG. 4B) or in the presence of 30 nM of a control antibody (NC) (FIG. 4C). The nuclei are coloured with DRAQ5.

[0191] FIG. 5 presents the curve for bonding of the Rendomab Axx antibody, which does not form part of the invention, to CHO cells transfected with the endothelin type A receptor (CHO ETAR) (continuous black line) and in the presence of 100 nM of endothelin 1 (ET1) (broken grey line).

[0192] FIG. 6 presents the marking by immunofluorescence on the glioblastoma GLI-7 strain cells in the presence of 30 nM of Rendomab-Axx (RAxx) revealed by a secondary antibody labeled with Alexa fluor 488 (Raxx-AF488). The nuclei are coloured with DRAQ5. The two images are merged (Fusion).

[0193] FIG. 7 presents the results of in vivo fluorescence imaging on a preclinical nude mouse model xenografted in the orthotopical position with GLI-7 cells. FIG. 7A presents the positive control for detection of Rendomab A63-AF680 and of Control-AF750. FIG. 7B presents the detection of the in vivo fluorescence in the grafted mouse and the control mouse. FIG. 7C presents the detection of the ex vivo fluorescence in the grafted brain and the control brain.

[0194] FIG. 8 presents the nucleotide sequences and the amino acid sequences deduced from the variable domains of the light chain and of the heavy chain of Rendomab-A63.

DETAILED DISCLOSURE OF PARTICULAR EMBODIMENTS

[0195] I. Equipment and Methods

[0196] The strategy for immunisation and screening of the hybridomas used in the present invention is identical to that used in the international applications WO 2012/045776 [14] and WO 2017/220739 [15].

[0197] II. Biochemical Characterisation

[0198] After purification of the Rendomab-A63 on Protein A—PropSep high capacity (Millipore), characterisation of the biochemical properties thereof was carried out.

[0199] The isotyping of the heavy and light chains of the Rendomab-A63 was carried out using the “Rapid ELISA Mouse mAb Isotyping” kit from Piercell. This is type G immunoglobulin, of isotype 2b for the heavy chain and kappa for the light chain. Rendomab-A63 is therefore an immunoglobulin of type IgG2b/kappa.

[0200] Recognition of the ETA-R in its cell context (CHO-ETAR and Gli-7 cells) by Rendomab-A63 was established by flow cytometry and immunofluorescence cell marking.

[0201] The binding curves for Rendomab-A63 were produced

[0202] i) on the CHO (standing for “Chinese Hamster Ovary”) cell line, not expressing ETA-R (CHO-WT),

[0203] ii) on the CHO-ETAR line, CHO cells, transfected in a stable manner, to allow strong expression of ETA-R, and

[0204] iii) on the line of glioblastoma cell strains, Gli-7, established by Dr Jean-Philippe Hugnot from a biopsy following exeresis of a glioblastoma in a patient.

[0205] The fluorescence is quantified by flow cytometry on an FACSCalibur™ (BD Bioscience). A range of concentrations of antibodies lying between, at a maximum, 1 μM and, at a minimum, 5 μM was incubated for 2 hours at 4° C. in the presence or not of 100 nM ET1 (preincubated for 30 minutes at 4° C.). At 90% confluence, the cells are detached in the presence of versene and then aliquoted in tubes (300 μl/300,000 cells) in the presence of a saturation buffer (PBS-SNC 5%-BSA 0.1%) stored at 4° C. After three washes in PBS buffer, 300 μl of secondary antibody labeled with Alexa Fluor™ 488 (AF-488) diluted to 1/400 was added and incubated for 60 minutes at 4° C. (Goat anti-mouse IgG, Invitrogen-ref A10684). For each antibody concentration point, after three washes in PBS, 10,000 cells were counted with flow cytometry.

[0206] The data were analysed in the GraphPad Prism software and the curves modelled according to the parameter: a specific binding site. The apparent dissociation constants calculated gave the values of Kd close to 0.5 nM on the CHO-ETAR line in the presence or not of ET1 (FIG. 1). The binding of RA63 was therefore not modified in the presence of ET1. In addition, the absence of binding of Rendomab-A63 on the CHO-WT cells was noted, thus demonstrating the binding specificity for ETA-R.

[0207] As illustrated in FIG. 2, the affinity of RA63 for ETA-R expressed on the surface of the Gli-7 cells is around one nanomolar (15 nM).

[0208] III. Immunofluorescence on Glioblastoma Gli-7 Strain Cells

[0209] III.1. Preliminary Remarks

[0210] Glioblastoma tumour cells are known for overexpressing ET-1 [20] and the endothelin type A receptor (ETAR) as shown by the transcriptomic data of the Gliovis public base (http://gliovis.bioinfo.cnio.es/) presented in FIG. 3. These data indicate clearly an overexpression of the EDNRA transcript in the glioblastoma cells of the patients.

[0211] III.2. Results

[0212] FIG. 4 presents results of immunofluorescence on the glioblastoma Gli-7 strain cells under the following operating conditions: [0213] in the presence of 1 μM of ET1-FAM (Phoenix Pharmaceuticals FG-023-01A) (FIG. 4A); [0214] in the presence of 30 nM of Rendomab-A63 (FIG. 4B); and [0215] in the presence of 30 nM of a control antibody (NC) and of the same secondary antibody also diluted to 1/400 (FIG. 4C).

[0216] The nuclei are coloured with DRAQ5 (Abcam ab108410) following the protocol of the supplier. The Gli-7 cells were incubated for 12 hours at 4° C. with either 1 μM of ET1FAM, or 30 nM final of RA63, or 30 nM final of NC. After three washes with PBS, for the labeling with the antibodies, the secondary antibody is added, diluted to 1/400, coupled with AF488 (Invitrogen—ref A10684) to the cells for 2 hours at 4° C. After three washes with PBS, the cells were mounted in an aqueous medium, Aquatex® (VWR 1 08562 0050) on plates, in order to be observed under confocal microscope (ZEISS).

[0217] FIG. 4A makes it possible to ensure the presence of the endothelin receptors capable of binding endothelin 1 on the Gli-7 cells. FIG. 4B shows a very strong immune-labeling of the Gli-7 cells with the RA63 antibody recognising the endothelin receptor A, whereas a negative control antibody (NC), of the same isotype as RA63 (IgG2b Kappa), does not generate any signal on the Gli-7 cells (FIG. 4C). All these results make it possible to conclude that there is a strong expression of ETA-R on the surface of the Gli-7 cells revealed by the binding of the specific RA63 antibody of the ETA-R. This binding is well mediated by the variable region of the RA63 antibody, as shown by the absence of a signal when the control antibody is used.

[0218] III.3. Comparative Results

[0219] In the immunisation process that made it possible to generate the RA63 antibody according to the invention, a plurality of monoclonal antibodies were obtained.

[0220] In particular, the Rendomab-Axx antibody directed against ETAR having an nM affinity but the binding of which on the endothelin receptor A is shifted by a concentration of 100 nm of ET1 (FIG. 5). This antibody therefore has properties comparable to those of the antibodies described in the patent application CA 2 971 491 [13].

[0221] This same antibody Raxx is not capable of binding to glioblastoma cells as shown by the immunofluorescence experiment presented in FIG. 6. This experiment demonstrates the existence of a conformation of the ETAR receptor present on the surface of the glioblastoma cells not recognised by the Raxx antibody but recognised by the RA63 antibody.

[0222] IV. In Vivo Imaging by Fluorescence Tomography on a Preclinical Animal Model

[0223] FIG. 7 presents the results of in vivo fluorescence imaging on a preclinical nude mouse model xenografted in the orthotopical position with Gli-7 cells.

[0224] For FIG. 7A, a range of quantities of RA-AF680 and NC-AF750 tracers were imaged on a solid FMT 1500 support (Perkin Elmer) in order to demonstrate the specificity of the filters used for detecting fluorescence, either i) at 680 nm where only the RA63-AF680 emits a detectable fluorescence signal, or ii) at 750 nm where only the NC-AF750 emits a detectable fluorescence signal.

[0225] For FIG. 7B, the mice are imaged at D0+3 months after xenografting. A co-injection of the RA63-AF680 tracers (14 nmols of RA63 coupled with 8 nmols of AF680) and NC-AF750 (17 nmols of control antibody coupled with 7 nmols of AF750) were injected by intraperitoneal route (IP) in the xenografted nude mouse and in the normal nude mouse (control mouse).

[0226] Ten days after IP injection, necessary for eliminating the tracers from the blood circulation, the mice were imaged with FMT 1500 (Perkin Elmer). For each mouse, a simultaneous acquisition at 680 nm and 750 nm was made. A strong fluorescence at 680 nm at the head of the xenografted mouse was observed, whereas, on this same mouse, the NC-AF750 tracer does not generate any fluorescence at 750 nm. Thus only the RA63-AF680 tracer was detected on the xenografted living mouse with the GLI-7 line. The absence of detection of the RA63-AF680 and NC-AF750 injected into the normal mouse was also noted.

[0227] For FIG. 7C, the mice were sacrificed, their brain removed and fixed with a 4% paraformaldehyde solution (PFA 4%) for 1 hour at 4° C. The brains were imaged as before. Once again a strong fluorescence with the RA63-AF680 tracer was observed in the brain invaded by the tumour cells, whereas no signal is detected with the NC-AF750 tracer. The absence of fluorescence is also found with the two tracers RA63-AF680 and NC-AF750 in the brain of the control mouse.

[0228] In conclusion, the RA63 antibody can be used as a tracer (RA63-AF680), for diagnosing the presence of glioblastoma tumour cells (Gli-7) for imaging applications.

[0229] V. Molecular Cloning

[0230] Cloning of the nucleic transcripts coding the heavy chain and the light chain of Rendomab-A63, was performed using the kits: “GenElute/Total RNA” (Sigma-Aldrich) and “RACE-PCR” (Invitrogen).

[0231] The deduced nucleic amino acid sequences of the variable domains of the light chain (VL) and of the heavy chain (VH) of Rendomab-A63 are presented in FIG. 8.

BIBLIOGRAPHIC REFERENCES

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