Anti-CD146 antibodies and uses thereof

Abstract

The present invention relates to the field of diagnostic and treatment of cancer, particularly melanoma, pancreatic cancer, kidney cancer and colon cancer. In particular, the invention relates to an antibody directed specifically to CD146-positive tumors and its applications, particularly for use as a medicament for the prevention and/or treatment of cancer, for use in a method of diagnostic or prognostic of a cancer, or for use as a radiotracer when labelled with a radioactive element.

Claims

1. An antibody or a fragment thereof which is directed against a CD146 protein, wherein said antibody or fragment comprises light chain variable region (VL) CDR polypeptide sequences comprising SEQ ID NO: 1, sequence NAN and SEQ ID NO: 2, and heavy chain variable region (VH) CDR polypeptide sequences comprising SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

2. The antibody or fragment thereof according to claim 1, wherein the sequence of the light chain variable region (VL) comprises the sequence of SEQ ID NO: 6 and wherein the sequence of the heavy chain variable region (VH) comprises the sequence of SEQ ID NO: 7.

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

4. The antibody according to claim 3, wherein said antibody is produced by the hybridoma deposited on Apr. 10, 2017 at the Collection Nationale de Cultures de Microorganismes (CNCM) under the number CNCM I-5246.

5. A hybridoma deposited on Apr. 10, 2017 at the Collection Nationale de Cultures de Microorganismes (CNCM) under the number CNCM I-5246.

6. A nucleic acid molecule encoding an antibody or a fragment thereof according to claim 1.

7. A vector comprising a nucleic acid molecule according to claim 6.

8. A host cell comprising a nucleic acid molecule according to claim 6 or a vector comprising said nucleic acid molecule.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: an antibody and/or a fragment thereof according to claim 1; a nucleic acid encoding said an antibody and/or a fragment thereof; a vector comprising said nucleic acid; and a host cell comprising said nucleic acid.

10. A method of treating cancer comprising administering an antibody and/or a fragment thereof according to claim 1, or a pharmaceutical composition thereof, to a subject having cancer.

11. The method according to claim 10, wherein the cancer is selected from melanoma, kidney cancer, pancreatic cancer and colon cancer.

12. An in vitro diagnostic and/or prognostic method of detecting cancer in a subject wherein said method comprises a step of determining CD146 protein expression and/or level of expression in a biological sample of said subject using at least one antibody and/or a fragment thereof according to claim 1, the detection of CD146 protein expression or levels indicating the presence of cancer in said subject.

13. An in vitro method for monitoring the response to an anticancer treatment of a subject suffering from cancer comprising determining CD146 protein level of expression in a biological sample of said subject using at least one antibody and/or a fragment thereof according to claim 1 at two or more time points during said anticancer treatment, wherein an equal or higher CD146 protein level of expression in a biological sample of the subject at a later time point, compared to a reference value obtained in a biological sample of the subject at an earlier time point, is indicative of a resistance of the subject to said anticancer treatment whereas a lower CD146 protein level is indicative of a response of the subject to said anticancer treatment.

14. A kit comprising at least one antibody and/or a fragment thereof as defined in claim 1 and, at least one reagent for detecting said at least one antibody and/or fragment thereof.

15. A method of imaging a subject comprising administering to the subject a radiolabeled antibody or a fragment thereof according to claim 1, performing an imaging method on said subject, and determining or analyzing the presence and/or amount of said radiolabeled antibody or fragment thereof present in the subject.

16. The method according to claim 15, wherein the imaging comprises Single Photon Emission Computed Tomography (SPECT-CT) or Positron Emission Tomography-Computed Tomography (PET-CT).

17. The antibody or fragment thereof according to claim 1, wherein said antibody or fragment thereof is conjugated to a drug.

18. The antibody or fragment thereof according to claim 17, wherein the drug is Monomethyl Auristatin E (MMAE).

19. The antibody or fragment thereof according to claim 1, wherein said antibody or fragment thereof is human or humanized.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1C: Effect of TsCD146 mAb antibody on cell proliferation and CD146 expression

(2) FIG. 1A. Analysis of the proliferation of HUVEC, HMEC-1, UACC-1273, C81-61 and Panc-1 cells after 72 h in the presence of IgG or TsCD146 mAb (5 μg/ml). The results are expressed as mean values+/−standard deviation of 3 experiments.

(3) FIG. 1B. Total CD146 expression was determined by Elisa on lysates of UACC, Panc-1, HUVEC and HMEC-1 cells after 72 h of treatment with control IgG or TsCD146 mAb. Mean values of three different experiments are given.

(4) FIG. 1C. Membrane expression of CD146 was determined by flow cytometry experiments with S-endo1 antibody in the different cell lines after treatment with TsCD146 mAb (5 μg/ml) or IgG (5 μg/ml) for 72 hours. The results are expressed as mean values+/−standard deviation of 3 experiments.

(5) * p<0.05, ** p<0.01, *** p<0.001, experimental versus control.

(6) FIGS. 2A-2C: Effect of TsCD146 mAb on growth of C81-61 cells in an animal model of xenograft

(7) FIG. 2A. 8 NOD/SCID mice xenografted with C8161 cells were treated for 46 days with control IgG or the TsCD146 mAb. Tumor volume was determined twice a week with caliper.

(8) FIG. 2B. Tumor weight was determined in IgG and TsCD146 mAb treated animals after the sacrifice of the animals.

(9) FIG. 2C. Tumors from IgG and TsCD146 mAb treated animals were photographed after sacrifice of the animals.

(10) FIG. 3: Effect of TsCD146 mAb on growth of Panc-1 cells in an animal model of xenograft

(11) 8 NOD/SCID mice xenografted with Panc-1 cells were treated for 46 days with control IgG or the TsCD146 mAb. Tumor volume was determined twice a week with caliper.

(12) *: p<0.05, experimental versus control.

(13) FIG. 4 relates to a deposited microorganism or other related biological material.

EXPERIMENTAL PART

(14) Inventors have generated antibodies recognizing the CD146 expressed in cancer cells but not CD146 expressed in other cell types. Of interest, one antibody (TsCD146 mAb) displayed these properties. This antibody as well as fragments thereof were thus further characterized in order to evaluate their interest for diagnostic and/or therapeutic applications.

(15) Materials and Methods

(16) Cell Culture

(17) HUVEC (Human Umbilical Vein Endothelial Cells) were grown in Endothelial Cell Growth Media (EGM-2 Bulletkit™) (Lonza, Amboise, France). The HMEC-1 (Human Microvascular Endothelial Cell Line) cell line was grown in Endothelial Basal Medium (EBM) (PAA, Velizy-Villacoublay, France) supplemented with fetal calf serum (FCS 10%), penicillin and streptomycin, (1%), L-glutamine (1%), epidermal growth factor (10 ng/mL) and hydrocortisone (50 μg/mL). HUA-SMC (Smooth Muscle Cells) were grown in Dulbecco's Modified Eagles medium (DMEM) added with 10% SVF.

(18) Tumor cell lines PANC-1 (pancreatic cancer) and LOVO (colon cancer) were cultured in DMEM (Life Technologies, Saint Aubin, France) supplemented with FCS (10%), PS (1%), L-glutamine (1%) and sodium pyruvate (1%). The tumor cell lines UACC-1273 (melanoma), C8161 (melanoma) and SW620 (colon cancer) were cultured in RPMI 1640 Glutamax™ (life technologies) supplemented with FCS (10%) and PS (1%). The cells were grown in a humidified atmosphere with 5% CO.sub.2 at 37° C.

(19) Generation of Antibodies Against CD146

(20) Antibodies against CD146 were generated by the platform of monoclonal antibodies (Mi-mAbs; Marseille-Luminy, France) by injection in rat of a recombinant protein, generated in mouse myeloma, corresponding to the extracellular part of CD146. After obtaining the hybridoma, anti-CD146 antibodies were purified by affinity chromatography on a HiTrap protein G column (GE Healthcare). Such an extracellular part of CD146 typically comprises or consists of an amino acid sequence comprising or consisting of a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, preferably SEQ ID NO: 14.

(21) qPCR

(22) Cell RNA was extracted using the RNeasy Mini Kit (Qiagen, Hamburg, Germany), according to the supplier's instructions. The extracted RNA was assayed using NanoDrop at 260 nm (Thermo scientific). The reverse transcription (RT) was then carried out with 1 μg of RNA to which is added 10 μL of a mixture (RT buffer, DNX 25×100 mM, RT random primer 10×, multiscribe reverse T, H.sub.2O (Applied Biosystems)). The RT was performed in a thermocycler at 25° C. for 10 minutes, 37° C. for 2 hours, 85° C. for 5 minutes and then at 4° C. 50 ng/μl of cDNA was then taken and placed with 24 μl of a mixture comprising sybergreen (Agilent, Stratagene), right and left primers and H.sub.2O. The PCR (Polymerase Chain Reaction) was then carried out using the Step One Plus real-time PCR system (Life Technologies) according to the following profile: 1 cycle of 10 minutes at 95° C. and then 40 cycles of 30 seconds at 95° C. and 1 minute at 60° C. The results were then analyzed using the Step One software. Primers were: GAPDH: R: 5′-GGTGGTCTCCTGACTTCAACA (SEQ ID NO: 15); F: 5′-GTTGCTGTAGCCAATTCGTTGT (SEQ ID NO: 16); CD146: R: 5′-GGCTAATGCCTCAGATCGATG (SEQ ID NO: 17); F: 5′-AATATGGTGTTGAATCTGTCTTG (SEQ ID NO: 18).

(23) Flow Cytometry

(24) After detachment of the cells with trypsin, cells were plated with 0.1% PBS-BSA. Primary antibody (S-endo1, mouse IgG, TsCD146 mAb, rat IgG) was then added to the cells for 45 minutes at 4° C. After rinsing with 0.1% PBS-BSA, the secondary antibody was added for 45 minutes at 4° C. in the dark. After rinsing with 0.1% PBS-BSA, the cells were taken up in 0.1% PBS-BSA and analyzed by flow cytometer (Gallios™ Flow Cytometer, Beckman Coulter, Villepinte). The results were then analyzed using Kaluza software (Kaluza® Analysis Software, Beckman Coulter).

(25) UACC derived microparticles (MP) were first generated by serial centrifugations (3 centrifugations at 70,000 g for 1 h30) of UACC supernatant medium. MP were then labeled with 10 μl of AnnexinV-FITC (Tau Technologies, Netherlands) and 10 μl of various concentration of PE-TsCD146 mAb during 30 minutes at room temperature, without light. 500 μl of AnnexinV binding buffer were finally added and tube was analysed on flow cytometer. Titration curve was therefore established, by determination of the maximum percentage of double positive AnnexinV/Ts CD146 microparticles among AnnexinV-positive MP (data unshown). The TsCD146 antibody suspension on vesicles was also adjusted equivalently with IgG isotypic control to confirm staining specificity.

(26) The same staining was performed on plasmatic MP derived from healthy patients or patients with melanoma. Briefly, Platelet Free Plasma (PFP) were prepared from EDTA blood collection tubes using a first centrifugation on ficoll sucrose gradient 30 min at 800 g. Then the part above PBMC ring was collected. Then 30 μl of PFP were thawed and stained with AnnexinV FITC and TsCD146 PE Ab or IgG isotypic control during 30 minutes at room temperature, without light. After addition of 500 μl of AnnexinV binding buffer+hirudine (Cryopep, Montpellier, France), 30 μl of MP count beads (Biocytex, Marseille, France) were added to evaluate the tumoral MP concentration.

(27) MP analyses were performed on Gallios Flow Cytometer using the Megamix+FSC strategy previously published by inventors' team (Robert et al., 2012). Cancer MP were defined as AnnexinV/Ts CD146-positive events.

(28) Confocal Microscopy

(29) The cells were seeded on glass plates at 50000 cells per well. At confluence, they were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then rinsed 3 times with PBS Immunofluorescence experiments were then carried out on these cells with the primary anti-CD146 antibodies S-endo1 (Biocytex) and TsCD146 mAb (dilution 1/200), for 1 hour in a humid chamber in the dark, then with the secondary anti-mouse and anti-rat (dilution 1/200) antibodies coupled to Alexa 488 diluted in PBS-saponin (0.2%) —FCS (10%) for 30 minutes. The lamellae were then mounted with DAPI (Prolong® gold antifade reagent with DAPI, Invitrogen) mounting liquid and observed with the confocal microscope (Leica SP5, Leica, Nanterre, France).

(30) For experiments on biopsies, organs were fixed in 4% formalin and cut using a cryostat. The slides were then pretreated in a pH 6 citrate buffer at 96° C. for 30 minutes. They were then preincubated for 30 minutes in PBS-BSA 0.5%-Triton 10×0.1% Immunofluorescence experiments were then carried out on these sections with primary antibodies S-endo1 and TsCD146 mAb (dilution 1/100) overnight in a humidity chamber and anti-mouse and anti-rat secondary antibodies (dilution 1/200) coupled to Alexa diluted in PBS-BSA 0.5%-Triton 10×0.1% for 1 hour. The lamellae were then mounted with mounting fluid (Prolong, Invitrogen) and observed with the confocal microscope (Leica SP5).

(31) For experiments with rab11, cells were first treated for 72 hours with TsCD146 mAb. Then cells were fixed with PFA 4% and incubated for 5 minutes in PBS-Triton 10×0.2% followed by 30 minutes incubation with PBS-BSA 0.5%. They were then incubated with rab11 antibody (Abcam, Ab3612) for 1 hour and secondary antibodies (anti-rat alexa 647 for TsCD146 mAb and anti-rabbit alexa 488 for rab 11) for 1 hour. Colocalization of both proteins was visualized with confocal microscope.

(32) CD146 Elisa

(33) Total CD146 expression was determined on the different cell lines using the CD146 Elisa kit (Biocytex), as recommended by the manufacturer.

(34) Immunoprecipitation and Western-Blot Experiments

(35) Immunoprecipitation of CD146 was performed by incubating 5 μg of TsCD146 or S-Endo1 antibodies overnight in cell lysates (Lysis buffer was: 120 mM NaCl, 10 mM Tris HCl pH 7.5, 2 mM EDTA, 1% Triton, 25 mM KCl). Protein A sepharose was then added in the lysate before centrifugation, and denaturation with NuPAGE sample-reducing agent (Invitrogen). Samples were then subjected to NuPAGE using 4-12% Novex Bis-Tris gels (Invitrogen) and separated proteins were transferred onto nitrocellulose membranes (Invitrogen). Membranes were probed with COM 7A4 (Biocytex) anti-CD146 diluted 1/1000 followed by secondary antibodies coupled to peroxidase. Blots were revealed with the ECL substrate (Pierce).

(36) Proliferation Experiments

(37) The different cell lines were seeded in 96-well plates at 10,000 cells per well. 12 hours later, the rat control IgG or TsCD146 mAb was added at the indicated concentration for 72 hours. After 48 hours, BrdU (BromodeoxyUridine) was added for 24 h. The proliferation was analyzed using the “Cell proliferation ELISA, BrdU” kit (Roche Diagnostics, Meylan, France), according to the supplier's instructions. The reaction was then stopped with a solution of 1M H.sub.2SO.sub.4. The absorbance was read at 450 nm using a microplate photometer.

(38) Internalization Experiments with Protonex Red 600 SE

(39) Cells were seeded in 96-well plates, 10,000 cells per well, in complete cell culture medium containing 2.5-30 μg/ml Ts CD146 mAb conjugated to Protonex red 600 SE (AAT Bioquest, CA). Protonex is a pH-sensitive dye that is non-fluorescent at basic pH (extracellular: culture medium) and fluorescent at acidic pH (intracellular: endosomes, lysosomes). Serially-diluted antibodies were added and plates were incubated at 37° C. for 3, 6 and 9 hours. Experiments were also performed at 4° C. as a control in order to block the internalization processes. Mean fluorescent intensities (MFI) of intracellular Protonex were measured per well using Glomax (Promega).

(40) Analysis of Tumor Growth on Tumor Xenografted Animals

(41) Xenografts of human tumor cell lines were produced by injecting tumor cells (5×10.sup.6 cells resuspended in PBS) subcutaneously into the back of NOD/SCID mice. When tumors reached 20 mm.sup.3, peri-tumoral administration of purified TsCD146 mAb or rat control IgG were performed at a dose of 10 μg, twice a week, for 46 days. Tumor size was measured twice a week with caliper and tumor volume was determined according to the equation:

(42) (length*width*thickness)*0.5236. Tumor weight was also evaluated at the end of the experiment after the sacrifice of the animal.

(43) NODAGA Conjugation and Radiolabeling

(44) TsCD146 Fab′2 were generated and purified with Amicon Ultra-0.5 3 KDa Centrifugal Filter 500 μL (Millipore Corporation) and added 10 equivalents of p-NCS-benzyl-NODAGA in 0.2M bicarbonate buffered. The mixture was left at 37° C. for 3 h. The conjugate was then transferred to an Amicon Ultra-0.5 3 KDa Centrifugal Filter Devices 500 μL, washed 3 times with PBS to eliminate small molecules which did not react, concentrated in distilled water (0.5 mg/mL) and stored at −80° C.

(45) Radiochemistry

(46) Gallium was obtained in .sup.68GaCl.sub.3 form using a commercial TiO.sub.2-based .sup.68Ge/.sup.68Ga generator (Obninsk). .sup.68GaCl.sub.3 (200.69±40.97 MBq/0.5 mL) was eluted from a .sup.68Ge-.sup.68Ga generator using 0.1 N HCl, after which 4M ammonium acetate buffer (pH 7.4) was added. This solution was then added to NODAGA-TsCD.sub.146 Fab′2 (15 μg); final pH of the mixture was 6.0. The reaction mixture was stirred at room temperature for 15 min with manual shaking.

(47) Determination of radiochemical purity was done by radio-thin-layer chromatography (ITLC_SG) and was performed using a Ray-test miniGITA radio-TLC scanner detector (Straubenhardt, Ge) (eluents, 1:1 [v/v] mixture of 1M aqueous ammonium acetate solution and methanol, RF .sup.68Ga/.sup.68Ga-NODAGA-TsCD146 Fab′2: 0/1).

(48) MicroTEP Imaging

(49) Mice xenografted with C8161 cells (n=6) were injected IV at day 36 post implementation with 5-10 MBq of .sup.68Ga-NODAGA-TsCD146 Fab′2 under 2% isoflurane anesthesia. PET images were acquired 1 h30 and 3H00 after .sup.68Ga-NODAGA-TsCD146 Fab′2 IV injection with a MedisoNanoPET/CT under 2% isoflurane anesthesia.

(50) Ethics Committee Approval.

(51) The animal experiments conformed to the directive 2010/63/EU of the European Parliament and were approved by the Institution's Animal care and Use Committee (Aix-Marseille University). The procedures described above were conducted under an institutional approved animal use protocol (Marseille Ethical Committee) and under the supervision of an authorized researcher (B. Guillet; no 13328).

(52) Statistical Analysis and Expression of Results

(53) The data were expressed as mean values±SEM of the indicated number of experiments. Statistical analysis was performed with the Prism software (GraphPad Software Inc., San Diego, Calif., USA). The variance between the different groups to be compared was estimated before statistical analysis. When comparing more than two groups, a non-parametric one-way ANOVA followed by a Dunn's multiple comparison test was used. Significant differences between two groups were determined using the unpaired student's t test or Mann-Whitney test. A value of P≤0.05 was considered to be significant.

(54) The sample size was chosen for each type of experiment using an a priori power analysis. No exclusion criteria were taken into account; all the values obtained during the experiments were used for both in vitro and in vivo studies. In animal studies, the groups' sizes were chosen using an a priori power analysis, the investigator was blinded to the group allocation and mice were distributed at random in the different groups.

(55) Results

(56) Generation of a Newly Developed Monoclonal Anti-CD146 Antibody Specifically Targeting Tumor CD146.

(57) The recombinant extracellular part of CD146 was produced in mouse myeloma cells and used as immunogen to generate rat monoclonal antibodies. Hybridomas were screened for clones producing antibodies that i/bind the CD146 expressed by cancer cells, and ii/do not bind to the CD146 expressed on the surface of endothelial and smooth muscle cells. The hybridoma clone TsCD146 mAb (IgG1 subtype) was selected on these criteria and was further characterized.

(58) Characterization of TsCD146 mAb

(59) Since it has been shown that many tumors express CD146, inventors examined the expression of the molecule in five cancer lines with reference to two types of micro- and macrovascular endothelial cells, and smooth muscle cells. CD146 expression was evaluated in these different cells at the mRNA level by RT-PCR with primers directed against the extracellular portion of CD146 and at the protein level by Elisa assay on cell lysates. UACC-1273 and C8161, two metastatic melanoma cell lines; Panc-1, a pancreatic cancer line; SW620 and Lovo, two colon cancer lines were used. In these different cancer cell lines, only Lovo do not express CD146. For endothelial cells, HUVECs which are macroendothelial primary cells and the HMEC-1 line which is a microendothelial cell line were used. Finally, the HUA-SMC smooth muscle cells were also used. In all the cells, except Lovo, inventors observed CD146 expression both at the mRNA and protein levels. TsCD146 mAb was characterized in comparison to the commercially available S-Endo1 antibody on its ability to bind cancer cells, endothelial cells and smooth muscle cells. To this end, both flow cytometry and immunofluorescence experiments were performed. Results show that the TsCD146 mAb was able to bind UACC-1273, Panc-1, C8161, SW620 cancer cells and not Lovo cells which do not express CD146. In contrast, it was not able to bind HUVEC, HMEC-1 and HUA-SMC cells. As a control, the S-Endo1 antibody was able to bind all cells, except Lovo cells. In addition, immunoprecipitation experiments were performed on the different cell lines with the TsCD146 mAb and CD146 was revealed by western-blot. Results show that TsCD146 mAb immunoprecipitates CD146 only in UACC and Panc-1 cell lines, but not in HUVEC and HMEC-1 cells, and that the molecule is evidenced around 110 kDa.

(60) Use of TsCD146 mAb for Immunodetection of Cancer Cells in Biopsies of Human Tissues

(61) In order to demonstrate that TsCD146 mAb detects tumor but not vascular cells, inventors carried out immunofluorescence experiments on human biopsies of melanoma, renal carcinoma and colon adenocarcinoma. Sections of tissues were also labeled with an antibody directed against the vascular endothelium (anti-CD31 antibody). Binding of TsCD146 mAb was evidenced on tumor cells but not on endothelial cells which were labeled with CD31 mAb. In contrast, S-Endo1 antibody was able to bind both tumor and endothelial cells in renal carcinoma, as demonstrated by its colocalization with CD31 antibody in vessels.

(62) Use of Radiolabeled TsCD146 Fab′2 for Immunodetection of Melanoma Cells by PET Imaging

(63) Inventors investigated whether TsCD146 mAb was able to detect human melanoma cells in vivo using a xenograft model. To this end, the inventors used Fab′2 fragment from TsCD146 mAb coupled to .sup.68Ga (see Material and Methods). Nude mice were injected with C8161 cells and 36 days after, .sup.68Ga Fab′2-TsCD146 was injected in animals. Results obtained by PET imaging showed that the tumor was visualized by the radiolabeled Fab′2. Of interest, only the external part of the tumor was labeled, without labeling in the central part. To further analyze this result, inventors performed immunofluorescence and histological experiments on these tumors after the sacrifice of the animal. Results confirmed the results obtained in vivo. Indeed, only the external part of the tumor was labeled with the TsCD146 mAb and the internal part corresponded to necrotic tissues.

(64) TsCD146 mAb Decreases Cancer Cell Proliferation and CD146 Expression in the Membrane Cancer Cells by Internalizing the Molecule

(65) The inventors tested the effect of TsCD146 mAb on cancer cell proliferation as compared to proliferation of endothelial cells. After a 72-hour treatment with the antibody at 5 μg/ml, the inventors observed a decrease in proliferation of UACC-1273, C8161 and Panc-1 cells whereas there was no effect on HUVEC and HMEC-1 cells (FIG. 1A).

(66) Since an anti-proliferative effect of TsCD146 mAb was observed on cancer cells and since CD146 is involved in cell proliferation, the inventors investigated whether this effect could be due to a decrease in the expression of CD146. In a first series of experiments, they analyzed the total expression of CD146 by Elisa on whole cell lysates. Results (FIG. 1B) showed that total CD146 was reduced in cancer cells but not in endothelial cells after 72 h of treatment with TsCD146 mAb. In a second series of experiments, inventors then investigated whether this could be due to a decrease in the membrane expression of CD146. The membrane CD146 expression was thus evaluated by flow cytometry in cancer cells after 72 hours of treatment with the TsCD146 mAb or the control IgG. A significant decrease in membrane expression of CD146 was observed in the different cancer cell lines whereas there was no effect in endothelial cells (FIG. 1C).

(67) In view of these results, inventors performed experiments to determine whether this down-regulation of membranous CD146 was due to membranous CD146 internalization and degradation. To this end, they used a probe that was fluorescent in acidic subcellular compartments as endosomes or lysosomes. Results show that after treatment of C81-61 cells with the TsCD146 mAb, CD146 was directed towards intracellular acidic compartments of the cells at 37° C. but not at 4° C., this last condition preventing the internalization of proteins from the membrane to the intracellular compartments. In addition, time-course experiments showed that the phenomenon started rapidly since it could be observed as soon as three hours after the beginning of treatment with the TsCD146 mAb. Similar results were obtained with Panc-1 and UACC cells. This result was confirmed by confocal microscopy experiments. Indeed, inventors showed that, after 72 h of treatment with TsCD146 mAb, CD146 was colocalized with rab11 in intracellular compartments in C81-61 cells.

(68) TsCD146 mAb Decreases Tumor Growth in Animal Models of Xenograft

(69) The effect of TsCD146 mAb was tested on tumor growth in C8161 melanoma cells orthotopically xenografted in NOD/SCID mice. When tumors reached about 20 mm.sup.3, TsCD146 mAb or an isotype control IgG (rat IgG1 mAb) were injected, twice a week, for 46 days. Tumor growth was monitored by determination of relative tumor volume by caliper. Caliper measurement revealed a significant decrease of tumor size in the group of mice injected with the TsCD146 mAb as compared to the rat IgG control group after 32 days of treatment (FIG. 2A). Tumor weight was also determined at day 46 on isolated tumors after the sacrifice of animals and revealed that tumor weight was significantly reduced in the TsCD146 mAb treated group as compared to the rat IgG control group (FIG. 2B). Finally, tumors were photographed at day 46 after the sacrifice of the animals and confirmed the inhibitory effect of TsCD146 mAb (FIG. 2C).

(70) These results were confirmed in another model of CD146-positive tumors subcutaneously xenografted in nude mice, the Panc-1 cells. Using the same experiments, inventors showed that tumor volume measured with caliper was significantly reduced in the TsCD146 mAb treated tumor group as compared to the control group from day 43 after the beginning of the treatment (FIG. 3).

(71) Discussion

(72) Up to now, it was impossible to specifically target CD146 expressed by cancer cells or their derivatives as circulating cancer cells or microparticles, leading to major problems for diagnosis and/or targeted therapy. Thanks to the present invention, specifically targeting the tumor form of CD146 with an antibody or a fragment thereof is possible and allows detecting and targeting tumor cells without affecting vascular or immune integrity and functions.

(73) Concerning the interest of TsCD146 mAb and fragment F(ab′)2 for diagnosis, the inventors have shown that it is able to detect CD146 in human biopsies of different tumors and in vivo in xenografted tumors by PET imaging. Concerning this last technique, it will be useful for the specific detection of CD146-positive tumors before targeted therapy. Today, tumor cells are detected by imaging with common markers as .sup.18FDG which do not permit to distinguish between tumors (Singnurkar et al., 2017). There is a need for new biomarkers able to discriminate between tumors thereby allowing personalized medicine. The TsCD146 mAb and its F(ab′)2 fragment constitute such biomarkers.

(74) In addition to the diagnostic interest, inventors show that TsCD146 mAb is of great interest for therapeutic purposes. Indeed, results demonstrate that treatment with the antibody induces a decrease in the expression of the tumor CD146 molecule. This is due in part to an internalization of the membranous molecule after treatment with the antibody, as demonstrated by the use of a pH-dependent fluorescent dye and the colocalization with rab11, which is involved in vesicle transport between membrane and intracellular compartments (Welz et al., 2014). This mechanism has been described for other antibodies, as trastuzumab. This antibody is currently used for the treatment of breast cancer and internalizes the HER2 receptor, a marker of bad prognosis as CD146 (Zum et al., 2002). Thus, the tumor CD146 molecule can be removed from the plasma membrane and then be directed from the endosomal to the lysosomal compartments where it is degraded. Moreover, in xenografted models of cancer cells, inventor's experiments showed that TsCD146 mAb administration leads to a significant decrease in tumor growth. In view of its mechanism of action, TsCD146 mAb is of therapeutic interest, administrated either alone or coupled to toxins or radioactive atoms, in order to fight cancer cells.

(75) Conclusion

(76) In conclusion, the herein provided data demonstrate that it is possible to specifically target the CD146 molecule expressed by tumor cells (also herein identified as “tumor CD146” or “tumor CD146 protein”). For the first time, inventors have successfully generated a monoclonal antibody, herein referred to as TsCD146 mAb, as well as fragments thereof, in particular a F(ab′)2 fragment, that specifically bind and internalize tumor membranous CD146. This antibody is of interest in diagnosis since it is able to recognize CD146-positive tumors on biopsies and in vivo by PET imaging. In addition, because of its mechanism of action, this antibody is of interest in therapy since it is able to reduce the growth of CD146-positive tumors xenografted in nude mice.

REFERENCES

(77) Bardin N, Anfosso F, Masse J M et al. Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood 2001; 98(13): 3677-84. Bardin N, Blot-Chabaud M, Despoix N et al. CD146 and its soluble form regulate monocyte transendothelial migration. Arterioscler Thromb Vasc Biol. 2009; 29(5): 746-53. Bardin N, Frances V, Lesaule G et al. Identification of the S-Endo 1 endothelial-associated antigen. Biochem Biophys Res Commun. 1996; 218(1): 210-6. Bardin N, Moal V, Anfosso F et al. Soluble CD146, a novel endothelial marker, is increased in physiopathological settings linked to endothelial junctional alteration. Thromb Haemost. 2003; 90(5): 915-20. De Souza P S, Faccion R S, Bernardo P S et al. Membrane microparticles: shedding new light into cancer cell communication. J Cancer Res Clin Oncol. 2016; 142(7): 1395-406. Dignat-George F, Boulanger C M. The many faces of endothelial microparticles. Arterioscler Thromb Vasc Biol. 2011; 31(1):27-33. Duan H, Xing S, Luo Y et al. Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS. Sci Rep. 2013; 3: 1687. Imbert AM1, Garulli C, Choquet E, Koubi M, Aurrand-Lions M, Chabannon C. CD146 expression in human breast cancer cell lines induces phenotypic and functional changes observed in Epithelial to Mesenchymal Transition. PLoS One. 2012; 7(8):e43752. Kebir A, Harhouri K, Guillet B et al. CD146 short isoform increases the proangiogenic potential of endothelial progenitor cells in vitro and in vivo. Circ Res. 2010; 107(1): 66-75. Lehmann J M, Riethmüller G and Johnson J P. MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc Natl Acad Sci USA. 1989; 86(24): 9891-5. Lin Y, Wu X, Shen Y et al. A novel antibody AA98 V(H)/L directed against CD146 efficiently inhibits angiogenesis. Anticancer Res. 2007; 27(6B): 4219-24. Maitra A, Hansel D E, Argani P et al. Global expression analysis of well-differentiated pancreatic endocrine neoplasms using oligonucleotide microarrays. Clin Cancer Res. 2003; 9: 5988-95. Mills L, Tellez C, Huang S et al. Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res. 2002; 62(17): 5106-14. Pickl W F, Majdic O, Fischer G F, Petzelbauer P, Faé I, Waclavicek M, Stöckl J, Scheinecker C, Vidicki T, Aschauer H, Johnson J P, Knapp W. MUC18/MCAM (CD146), an activation antigen of human T lymphocytes. J Immunol 1997 158(5): 2107-15. Robert S, Lacroix R, Poncelet P et al. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles. Arterioscler Thromb Vasc Biol. 2012; 32(4): 1054-8. Sers C, Kirsch K, Rothbächer U et al. Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains. Proc Natl Acad Sci USA. 1993; 90(18): 8514-8. Shih I M. The role of CD146 (Mel-CAM) in biology and pathology. J Pathol. 1999; 189(1):4-11. Singnurkar A, Poon R, Metser U. Comparison of 18F-FDG-PET/CT and 18F-FDG-PET/MR imaging in oncology: a systematic review. Ann Nucl Med. 2017; 28: 1-13. Stalin J, Nollet M, Garigue P et al. Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors. Oncogene. 2016; 35(42): 5489-5500. Ueno K, Hirata H, Majid S et al. IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma. Int J Cancer. 2011; 129(10): 2360-9. Wang Z, Yan X. CD146, a multi-functional molecule beyond adhesion. Cancer Lett. 2013; 330(2): 150-62. Welz T, Wellbourne-Wood J, Kerkhoff E Orchestration of cell surface proteins by Rab11. Trends Cell Biol. 2014; 24(7):407-15. Wu G J, Fu P, Chiang C F et al. Increased expression of MUC18 correlates with the metastatic progression of mouse prostate adenocarcinoma in the TRAMP model. J Urol. 2005; 173(5): 1778-83. Zeng G F, Cai S X and Wu G J. Up-regulation of METCAM/MUC18 promotes motility, invasion, and tumorigenesis of human breast cancer cells. BMC Cancer. 2011; 11:113-126. Zum Büschenfelde CM, Hermann C, Schmidt B et al. Antihuman epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab enhances cytolytic activity of class I-restricted HER2-specific T lymphocytes against HER2-overexpressing tumor cells. Cancer Res. 2002; 62(8): 2244-7.