WO2019063580A1

WO2019063580A1 - Human soluble cd146 proteins, preparation and uses thereof

Info

Publication number: WO2019063580A1
Application number: PCT/EP2018/076046
Authority: WO
Inventors: Marcel Blot-Chabaud; Marie NOLLET; Richard Bachelier; Nathalie Bardin; Françoise DIGNAT-GEORGE
Original assignee: Universite D'aix-Marseille; Assistance Publique Hôpitaux De Marseille; INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2017-09-26
Filing date: 2018-09-26
Publication date: 2019-04-04

Abstract

The present invention relates to compositions and methods for modulating angiogenesis in vivo, ex vivo or in vitro. More particularly, the invention relates to new soluble CD146 proteins usable in the context of human therapy, as well as to corresponding antibodies. The invention also relates to compositions comprising such compounds, particularly pharmaceutical or diagnostic compositions, including kits and the like, as well as methods of therapy or diagnosis using said compounds, compositions and cells.

Description

HUMAN SOLUBLE CD146 PROTEINS, PREPARATION AND USES THEREOF.

FIELD OF THE INVENTION The present invention relates to compositions and methods for modulating angiogenesis in vivo, ex vivo or in vitro.

More particularly, the invention relates to new soluble CD 146 proteins usable in the context of human therapy, as well as to corresponding antibodies. Particular forms of CD 146, herein described, may be used to mobilize, in vivo or ex vivo, both mature and immature endothelial cells, as well as to increase their influence on angiogenesis. They can further be used to prepare compositions, in particular pharmaceutical, diagnostic or cosmetic compositions, and corresponding kits.

The invention further relates to methods of prophylaxis, therapy or diagnosis, and to cosmetic treatments, using the previously mentioned compounds, compositions and cells.

BACKGROUND OF THE INVENTION

The formation of new blood vessels either from differentiating endothelial cells during embryonic development (vasculogenesis) or from pre-existing vessels during adult life (angiogenesis) is an essential feature of organ development, reproduction, and wound healing in higher organisms.

Therapeutic angiogenesis is an effective means to treat patients suffering from a disease or a disorder leading to, resulting from or associated to (tissue) ischemia.

Treatment of ischemia, using non-surgical therapy, has become possible with the discovery of angiogenic factors favouring formation of new blood vessels. Several candidate angiogenic factors have been described so far which were the object of clinical trials.

Enthusiasm has however been hampered by series of negative clinical outcomes. Regarding VEGF for example, despite the potent angiogenic effects of this factor, its expression did not efficiently improve muscle blood flow in patients. This was explained by the formation of leaky vascular lacunae and arteriovenous shunts interfering with the downstream microcirculation.

Endothelial progenitor cells (EPCs) have been identified in adult human peripheral blood, in bone marrow and in cord blood (Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T,Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997 275:964-7). Circulating EPCs participate in postnatal neovascularization after mobilization from the bone marrow. Transplantation of culture- expanded EPCs, obtained either from blood or from autologous bone marrow mononuclear cells, was found to be able to augment ischemia-induced neovascularization in vivo.

The use of cultured cells as a therapeutic approach in patients is however considerably limited by the small proportion of EPCs in the peripheral blood, the necessity of harvesting a large amount of bone marrow to isolate a sufficient number of EPCs, and the heterogeneity of the recovered EPCs.

Despite their drawbacks, the use of angiogenic growth factors thus remains to date the primary strategy of therapeutic angiogenesis for the treatment of patients, such as patients presenting with severe peripheral arterial disease (also called peripheral vascular disease) or ischemic heart disease.

CD 146, also known as MCAM, MUC18, or Mel-CAM, is a glycoprotein essentially expressed on the vascular system which belongs to the immunoglobulin superfamily (Bardin N, Anfosso F, Masse JM, Cramer E, Sabatier F, Le Bivic A, Sampol J, Dignat-George F. Identification of CD 146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001 ; 98:3677-84). As a member of such a family, it consists in five Ig domains, a transmembrane domain, and a cytoplasmic region.

CD 146 is mainly known to occur in two distinct forms differing by the length of their cytoplasmic domain: a long isoform (herein identified as "long CD 146") and a short isoform (herein identified as "short CD 146"), both present in the membrane of cells, mainly endothelial cells. In endothelial cells, the long isoform is a component of the endothelial junction primarily involved in the control of cell-cell cohesion, vascular permeability and tissue architecture (Kebir et al., 2010). In contrast, the short isoform is rather expressed at the apical membrane and displays angiogenic functions (Kebir et al., 2010).

CD 146 is involved in the control of cell and tissue architecture, as demonstrated by the regulation of its expression during endothelium monolayer formation, its involvement in the control of paracellular permeability (Bardin N, Anfosso F, Masse JM, Cramer E, Sabatier F, Le Bivic A, Sampol J,Dignat-George F. Identification of CD 146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001 ; 98:3677-84) and its colocalization with the actin cytoskeleton (Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F. Outside- in signaling pathway linked to CD 146 engagement in human endothelial cells. J Biol Chem. 2001; 276:1564-9).

Membranous CD 146 has been reported to promote tumor growth, angiogenesis, and metastasis in human melanoma. Membranous CD 146 expression levels and distribution are closely associated with tumor progression and onset of metastasis in human malignant melanoma. Anti- membranous CD 146 antibodies have been described as capable of significantly inhibiting the growth and metastasic properties of human melanoma cells in nude mice (Mills L, Tellez C, Huang S, Baker C, McCarty M, Green L, Gudas JM, Feng X, Bar-Eli M. Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res. 2002; 62:5106-14.). Membranous CD146 has been shown to display angiogenic properties, both in an in vitro model of human umbilical vein endothelial cells (HUVEC) (Kang Y, Wang F, Feng J, Yang D, Yang X, Yan X. Knockdown of CD 146 reduces the migration and proliferation of human endothelial cells. Cell Res. 2006; 16(3):313-8) and in in vivo models of chicken chorioallantoic membrane (CAM) assays and tumor growth in mice (Yan X, Lin Y, Yang D, Shen Y, Yuan M, Zhang Z, Li P, Xia H, Li L, Luo D, Liu Q, Mann K, Bader BL. A novel anti-CD 146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood. 2003;102: 184-91). mAb AA98 has been shown by Yan et al. to display a remarkably restricted immunoreactivity against intratumoral vasculature compared with blood vessels of normal tissues.

Inventors also showed that CD 146 was involved in the regulation of monocytes transendothelial migration (CD 146 and its soluble form regulate monocytes transendothelial migration. Arteriosclerosis, thrombosis and Vascular Biology, 2009; 29: 746-53).

Different localisations and functional differences have been identified in the literature for the two membranous isoforms of chicken CD 146. In one study, authors analyzed chicken CD 146 targeting in polarized epithelial Madin-Darby canine kidney (MDCK) cells using CD146-GFP chimeras, to identify the respective role of each isoform. They showed by confocal microscopy that short CD 146 and long CD 146 were addressed to the apical and basolateral membranes, respectively (Guezguez B, Vigneron P, Alais S, Jaffredo T, Gavard J, Mege RM, Dunon D. A dileucine motif targets MCAM-1 cell adhesion molecule to the basolateral membrane in MDCK cells. FEBS Lett. 2006; 580:3649-56). In another study, the same group showed that long CD 146 promoted rolling via microvilli induction in lymphocytes and displayed adhesion receptor activity, suggesting its involvement in the recruitment of activated T cells to inflammation sites (Guezguez B, Vigneron P, Lamerant N, Kieda C, Jaffredo T, Dunon D. Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction: MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptor. J Immunol. 2007; 179:6673-85).

A breakthrough results from the inventors' discovery (Bardin et al., 2003) that biologically active forms of human CD 146 exist not only as membrane-bound forms but also as a soluble form present in the human serum. Soluble CD 146 is detectable in the human serum and its level is modulated in different pathologies, such as inflammatory bowel diseases (Bardin et al., 2006), pathological pregnancies (Pasquier et al., 2005), chronic renal failure (Bardin et al., 2003) and cancer (Stalin et al., 2016).

Inventors described, for the first time, the structure of soluble forms of CD 146 in WO2010/086405. A soluble form generated by alternate splicing with retention of intron 11 has also been described. This isoform is only constituted by the extracellular part of the molecule and differs from the membrane forms at the C terminal part (Vainio et al., 1996).

Inventors also demonstrated the therapeutic properties of human soluble CD 146, in particular the angiogenic properties thereof. Indeed, it displays chemotactic effects on vascular cells and it is able to stimulate endothelial cell proliferation, migration and ability to generate vascular structures in matrigel. In addition, inventors have shown that the soluble form was able to increase neo-vascularization and to restore blood flow in an animal model of hindlimb ischemia (Harhouri et al., 2010).

In the literature, different soluble receptors, as soluble EphB4 or soluble Notchl, have been shown to act as endogenous inhibitors of angiogenesis, acting as traps for their ligand. This is also the case for the soluble form of VEGFR2 which blocks the angiogenic effect of VEGF (Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002; 99: 11393-8.). In contrast, other soluble molecules have been shown to act as activators of angiogenesis, such as the soluble N-cadherin fragment (Derycke L, Morbidelli L, Ziche M, et al. Soluble N-cadherin fragment promotes angiogenesis. Clin Exp Metastasis. 2006; 23: 187-201) or the soluble CD40 ligand (Melter M, Reinders ME, Sho M, et al. Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo. Blood. 2000; 96: 3801-8.). The reason for the observed opposite effects of soluble molecules, inhibitor or activator, is unknown but may result from distinct signalling pathways. Thus, one can hypothesize that soluble forms of receptor molecules may trap the ligand and inhibit the effect. In contrast, other soluble molecules, such as soluble CD 146, resulting from a membrane protein shedding, could serve as a ligand that activates its receptor.

Inventors now herein identify new soluble CD 146 proteins and describe advantageous uses thereof.

Inventors in particular herein provide new tools, using the newly identified soluble forms of CD 146, improving the treatment of tissue ischemia while reducing deleterious side effects observed with classically used therapies. They herein demonstrate that these new soluble forms of CD 146 fulfill key functions in the neovascularisation process.

Inventors herein characterize new human soluble forms of CD 146 (herein generally identified as "soluble CD 146s", "sCD146s" or "soluble CD 146 proteins") and describe for the first time nucleic acid and amino acid sequences thereof usable in the context of a prevention and treatment methods. Inventors in particular describe their advantageous chemotactic and angiogenic effects on endothelial cells, in particular on endothelial progenitor cells ("EPC", also herein identified as "Endothelial Colony Forming Cells" or "ECFC"). These human soluble forms of CD 146 are able to promote a therapeutic vasculogenesis and/or angiogenesis in a subject, typically in a mammal, in particular in a human subject.

Other advantages of the products and compositions herein described are further indicated below. SUMMARY OF THE INVENTION

Inventors herein demonstrate for the first time that the short and long membrane isoforms of CD 146 are shed through the Tace and AD AMI 0 proteinases, respectively. In addition, RNA sequencing experiments revealed the existence of two alternative sCD146 mRNA spliced variants, herein identified as "I5-13-sCD146" and "I10-sCD146". The proteins generated from these mRNA splice variants promote angiogenesis in vitro and in vivo, in a mouse model of limb ischemia. Of interest, spliced variants of sCD146 are differentially expressed in organs and differentially regulated. Thus, I5-13-sCD146 is increased by WNT5a and TNF and is increased in the sera of patients with systemic sclerosis (SSc). In contrast, I10-sCD146 is decreased by VEGF, netrin and WNT3a, and is decreased in SSc.

Herein described human soluble CD 146s induce the migration ability or mobilisation (chemotactic activity) and activation of endothelial cells, in particular endothelial progenitor cells, of smooth muscle cells and of hematopoietic cells, and demonstrate that these molecules are able to promote vasculogenesis and/or angiogenesis in vivo. Any of these molecules which may be administered either alone or in combination with a distinct angiogenic factor and/or with a mature or immature endothelial cell, is an advantageous tool for therapeutic angiogenesis in patients presenting with tissue ischemia or at risk of developing such a tissue ischemia.

The present invention provides novel proteins corresponding to human soluble CD 146 proteins. These proteins, which are biologically active and naturally present in the human serum, have been isolated by inventors and are herein described for the first time.

Inventors herein describe a human soluble CD 146 protein, typically an isolated human soluble CD 146 protein, comprising or consisting in a sequence selected from SEQ ID NO:l and SEQ ID NO:2. In a particular aspect, any of these human soluble CD 146 proteins, comprising or consisting in an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, is usable in the context of a treatment, in particular of a human treatment, as herein described, typically as a drug or medicament, or for preparing such a drug or medicament.

In another aspect, inventors provides a composition comprising at least one soluble CD 146 protein as herein described, possibly two distinct soluble CD 146 protein as herein described, typically selected from SEQ ID NO: 1 and SEQ ID NO: 2, and a pharmaceutically acceptable carrier. The composition is typically for use as a drug or medicament.

In another aspect, this composition further comprises a mature or immature endothelial cell, in particular an endothelial progenitor cell, and/or another angiogenic factor.

A pharmaceutical composition as herein described may also comprise, as the only biologically active agent, a mature or immature endothelial cell which has been contacted with at least one human soluble CD146 protein as herein described and/or which has been genetically modified to express a human soluble CD146 protein as herein described and/or a human short CD146 protein.

Further herein disclosed is the use of a protein as herein described to prepare, in vitro or ex vivo, a mature or immature endothelial cell capable of stimulating angiogenesis in a human body. In a further aspect, the invention relates to a soluble CD 146 protein as herein described for use (as a biomarker) in the diagnosis of a disease, disorder or dysfunctional state characterized by dysregulated angiogenesis, undesirable excessive (neo)vascularization or undesirable vascular permeability, by an overexpression or excessive activation of a soluble form of CD 146 and/or of a receptor for CD 146, in particular soluble CD 146, or by an excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR in a mammal, compared to standard expression. In this context, the terms "dysregulated angiogenesis" mean, in particular, (abnormally) increased angiogenesis, typically excessive angiogenesis, when compared to angiogenesis of a healthy subject.

Such a disease, disorder or dysfunctional state can be a cancer, systemic sclerosis (SSc), an ischemic disease, a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia, or age-related macular degeneration (AMD).

In another aspect, the invention relates to a soluble CD 146 protein as herein described or a composition comprising such a soluble CD 146 protein for use in the prevention, in the treatment or (as a biomarker) in the diagnosis of tissue ischemia or of a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia or characterized by a decreased expression or activation of a receptor for CD 146, or by a decreased expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to standard expression.

An antibody, preferably a monoclonal antibody, which selectively binds a human soluble CD 146 protein as herein described, typically a human soluble CD 146 protein comprising or consisting in an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, is further herein provided. This antibody preferably also neutralizes a biological activity of the human soluble CD 146 protein of the invention. Preferably, the antibody decreases or inhibits neovascularization vascular permeability and/or vascular endothelial cell growth in a mammal, preferably in a human being. Further herein described is the use of such an antibody as a drug or medicament, or for preparing such a drug or medicament.

The antibody, or a pharmaceutical composition comprising such an antibody and a pharmaceutically acceptable carrier, herein disclosed, can be used in a mammal, preferably in a human being, for preventing, treating or diagnosing a disease, disorder or dysfunctional state characterized by dysregulated angiogenesis, undesirable excessive (neo)vascularization or undesirable vascular permeability, by an overexpression or excessive activation of a soluble form of CD 146 and/or of a receptor for CD 146, in particular soluble CD 146, or by an excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to standard expression.

In a particular embodiment, the invention provides a method of diagnosing, preventing or treating a disease, a disorder or a dysfunctional state in a subject, typically in a mammal, preferably in a human being, as herein identified, in particular a method of diagnosing cancer, systemic sclerosis (SSc), an ischemic disease or a disease or disorder leading to, resulting from or associated to (tissue) ischemia, or a method of preventing or treating an ischemic disease, typically a tissue ischemia, a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia, or age-related macular degeneration (AMD).

The method of diagnosing cancer, systemic sclerosis (SSc), age-related macular degeneration (AMD), an ischemic disease or a disease or disorder leading to, resulting from or associated to (tissue) ischemia preferably comprises a step of dosing, in the subject's serum, the amount of a soluble CD 146 protein as herein described. Such a method typically involves the use of an antibody binding a human soluble CD 146 protein as herein described. The method of preventing or treating a tissue ischemia, or a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia, preferably comprises a step of administering to the subject, an effective amount of a soluble CD 146 protein as herein described or composition comprising such a soluble CD 146 protein.

In a further aspect, the invention relates to the use of a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, to improve the aesthetic appearance of a scar or, in prevention, to facilitate the cicatrization or healing of a wound, a cut or an incision.

An object of the present invention is a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD146 protein, for use in the cicatrization of a subject's, typically mammal's epithelium, in particular a human epithelium, in particular following a wound, a cut or an incision or in the context of a skin graft.

Another object of the present invention is a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the prevention or treatment of an eschar or a bedsore in a subject, typically in a mammal, in particular in a human being.

A further object of the present invention is a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the context of a skin graft in a subject, typically in a mammal, in particular in a human being.

In a further embodiment, the invention provides isolated nucleic acid molecules encoding a human soluble CD 146 as herein described, optionally together with the human short form of CD 146, or recombinant forms thereof.

The nucleic acid molecule can be provided in a replicable vector comprising the nucleic acid molecule operably linked to control sequences recognized by a host cell transfected or transformed with the vector, in particular a mature or immature endothelial cell or a progenitor cell, preferably an endothelial progenitor cell. The invention further provides such an host cell comprising the nucleic acid molecule or the vector. In particular, the invention relates to the use of a product as herein described, typically of protein, nucleic acid, antibody or cell as herein described, to prepare a composition for diagnosing, preventing or treating a disease, a disorder or a dysfunctional state as herein identified. In another aspect, the present disclosure provides kits comprising any one or more of the herein- described protein, antibody, cell or composition. Typically, the kit also comprises instructions for using the protein, antibody, cell or composition according to the disclosed methods.

LEGEND TO THE FIGURES

Figure 1: Characterization of I10-sCD146 and I5-13-sCD146 isoforms

A: Schematic representation of the I10-sCD146 and I5-13-sCD146 isoforms as compared to the shed form. The differences in amino acids are indicated.

B: The mRNA expression of I10-sCD146 and I5-13-sCD146 isoforms was analyzed in ECFC and HUVEC and compared to mRNA expression of the short and long isoforms of CD 146. Results are the mean values +/- SEM of 3 experiments.

C: The expression of I10-sCD146 and I5-13-sCD146 isoforms was analyzed by immunofluorescence in ECFC. Nuclei were labelled with Dapi. The picture is representative of 3 different experiments.

D: The protein expression of I10-sCD146 and 15-13-sCD 146 isoforms was visualized by western-blot in ECFC supernatants. The picture is representative of 3 different experiments. E: The mRNA expression of I10-sCD146 and I5-13-sCD146 isoforms was analyzed by qPCR in ECFC after treatment of the cells for 24h with the recombinant shed soluble CD 146 50 ng/ml, VEGF 20 ng/ml, TNF 20 ng/ml, TGF beta 5 ng/ml, and netrin 50 ng/ml. Cells were also treated for 15 min with WNT5a 200 ng/ml and lh with WNT3a 50 ng/ml. Results are the mean values +/- SEM of 3 experiments.

*: P<0.05, experimental vs Control.

Figure 2: Effect of I5-13-sCD146 and I10-sCD146 on angiogenesis

A: Effect of siRNA targeting I10-sCD146 and I5-13-sCD146 on proliferation of ECFC. Experiments were performed in the presence of GM6001 10 μΜ. Results are mean values of 4 different experiments and representative pictures of the cells in one experiment are given.

B: Effect of over-expression of I10-sCD146 (p-I10-sCD146), I5-13-sCD146 (p-I5-13-sCD146) and shed sCD146 (p-shed sCD146) performed by plasmid transfection on proliferation of

ECFC. Experiments were performed in the presence of GM6001 10 μΜ.

C: Effect of recombinant proteins I10-sCD146, I5-13-sCD146 and shed sCD146 50 ng/ml on the proliferation of ECFC. Experiments were performed in the presence of GM6001 10 μΜ.

Results are mean values of 4 different experiments.

D: Yolk sac membrane assays were performed in the presence of 25 ng/ml or 50 ng/ml 15-13- sCD146 and I10-sCD146 for 48h and compared to control. Representative pictures of 4 different experiments are given. E: Effect of recombinant proteins I10-sCD146, I5-13-sCD146 and shed sCD146 50 ng/ml on the number of sprouts, branched points and cumulative sprout length in spheroid experiments performed with ECFC. Results were compared to VEGF 20 ng/ml and complete EGM2-MV medium. Average of the experiments and representative pictures are given. Results are mean values of 4 different experiments.

*: P<0.05, ***: P<0,001, experimental vs Control.

Figure 3: Effect of local injection of recombinant I5-13-sCD146 and I10-sCD146 in a mouse ischemic hind limb model

A: Ischemic mice were subjected to local injection of either PBS or 2 μg of rh-sCD146/ rh-I10- sCD146/ rh-I5-13-sCD146 twice a week for 28 days. Blood perfusion rate was monitored by laser-Doppler as a function of time in all animals. Results are mean values of 6 different animals in each group and expressed as % of the control leg. *: P<0.05, 110-sCD146 vs. PBS; $: P<0.05, I5-13-sCD146 vs. PBS; #: P<0.05, shed sCD146 vs. PBS.

B: Representative pictures of laser-Doppler experiments are given at day 0 (DO) and at day 28 (D28) after treatment with shed sCD146, I5-13-sCD146 and I10-sCD146, in comparison with PBS.

C: Blood vessels examination in hind limb muscle sections from control (PBS) and shed sCD146/ I10-sCD146/ rh-I5-13-sCD146 treated animals 28 days after surgery. Vessels are labelled with isolectin B4. Pictures are representative of different 3 different experiments in 6 animals.

D: Angiographic pictures of control (PBS) and shed sCD146/ I10-sCD146/ I5-13-sCD146 treated animals at D28. Pictures are representative of the 6 animals in each group.

Figure 4: Expression of I5-13-sCD146 and I10-sCD146 isoforms in normal tissues and in systemic sclerosis

A: mRNA expression of I10-sCD146 and I5-13-sCD146 was examined in various organs by tissue array. Results were normalized as a function of the expression in lymph node.

B: I5-13-sCD146 and I10-sCD146 concentration was determined in 24 patients with systemic sclerosis as compared to matched control.

*: P<0.05 **: P<0.01, experimental vs. Control.

Figure 5: Characterization of recombinant I10-sCD146 and I5-13-sCD146

Recombinant I10-sCD146 and I5-13-sCD146 proteins were generated in CHO cells after stable transfection of the plasmids. The mRNA expression of the molecules was examined by qPCR (A) and proteins were characterized by western-blot using specific polyclonal antibodies generated against the molecules (B). Pictures are representative of 3 different experiments. Figure 6: Yolk sac membrane assay quantification and histology A: Quantification of the YSM assay performed with I5-13-sCD146 and I10-sCD146 is given. The number of junctions, nodes and branches is given.

B: Histological analysis of the YSM assay performed with I5-13-sCD146 and I10-sCD146 is shown. Arrows indicate with areas with angiogenesis.

Figure 7: Yolk sac membrane assay with shed sCD146

Experiments were performed in the presence of 25 ng/ml or 50 ng/ml recombinant shed sCD146 for 48h and compared to control. Representative pictures of 4 different experiments are given.

DETAILED DESCRIPTION OF THE INVENTION

In the below description of the invention, the following terms will be employed and are intended to be defined as indicated below.

"Human long CD 146 protein" or "long CD 146" refers to a human protein, peptide or amino acid molecule, mainly present in the membrane of endothelial cells and having an amino acid sequence corresponding to the following SEQ ID NO: 3:

MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLS

HVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLC

QGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYK NGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNH MKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTRE AEEETTNDNGVLVLEPARKEHSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPA APERQEGS SLTLTCEAES SQDLEFQ WLREETDQVLERGPVLQLHDLKREAGGGYRCVA SVPSIPGLNRTQLVKLAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTISWNVN GTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSN TTTGLSTSTASPHTRANSTSTERKLPEPESRGWIVAVIVCILVLAVLGAVLYFLYKKGK LPCRRSGKQEITLPPSRKTELWEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRH

"Human short CD 146 protein" or "short CD 146" refers to a human protein, peptide or amino acid molecule mainly present in the membrane of endothelial cells and having an amino acid sequence corresponding to the following SEQ ID NO: 4:

MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLS HVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLC QGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYK NGRPLKEEKNRVHIQ S S QTVES S GLYTLQ SILKAQLVKEDKD AQF YCELNYRLPS GNH MKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTRE AEEETTNDNGVLVLEPARKEHSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPA APERQEGS SLTLTCEAES SQDLEFQ WLREETDQVLERGPVLQLHDLKREAGGGYRCVA SVPSIPGLNRTQLVKLAIFGPPWMAFKERKV KENMVLNLSCEASGHPRPTISWNVN GTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSN TTTGLSTSTASPHTRANSTSTERKLPEPESRGWIVAVIVCILVLAVLGAVLYFLYKKGK LPCRRSGKQEMERNTSI

"Human soluble CD 146 protein" or "soluble CD 146" typically refers to a human protein, peptide or amino acid molecule herein described for the first time.

A particular human soluble CD 146 protein according to the present invention comprises or consists in the amino acid sequence SEQ ID NO: 1 :

MVYIVRQFLLYNVSGSVYLDQLIVLLTAKFSILRIAGSRVHHSPFSGHLDGCSFLSLQHS LHTSLDMSRHENVFLGLTLSSKSAGLKGFQLAFVPGLLQGTGGYLDGPLPTPVDNPRV GLEVGLRLSLPPLPPCPGVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRL PSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQ NPSTREAEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISLLSEPQELLVNYVSDV RVSPAAPERQEGSSLTLTCEAESSQDLEFQWLREETGQVLERGPVLQLHDLKREAGGG YRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTI SWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTT LTPDSNTTTGLSTSTASPHTRANSTSTGKPGLAREQGCARASFLPCPSPESPVQKGE

This sequence corresponds to a preferred human soluble CD 146 protein usable in the context of a mammal treatment, in particular in a human being treatment, as herein described. Another human soluble CD 146 protein usable in the context of a mammal treatment has an amino acid sequence comprising or consisting in SEQ ID NO: 2:

MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLS HVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLC QGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYK NGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNH MKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTRE AEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISLLSEPQELLVNYVSDVRVSPA APERQEGS SLTLTCEAES SQDLEFQ WLREETGQVLERGPVLQLHDLKREAGGGYRCVA SVPSIPGLNRTQLVNVAIFGEALPLGRDQVTPSGWFKLFDKKPPAALGSSGAEGEAG Any of the herein above described preferred soluble CD146 protein of SEQ ID NO: l and 2 according to the invention can be used, in a particular aspect of the invention, together with the below described SEQ ID NO:5, in any one of the herein described compositions and methods. SEQ ID NO: 5:

MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLS HVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQPvLSLQDRGATLALTQVTPQDERIFLC QGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYK NGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNH MKESREVTVPVF YPTEKVWLEVEP VGMLKEGDRVEIRCLAD GNPPPHF SI SKQNP STRE AEEETTNDNGVLVLEPARKEHSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPA APERQEGS SLTLTCEAES SQDLEFQ WLREETDQVLERGPVLQLHDLKREAGGGYRCVA SVPSIPGLNRTQLVKLAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTISWNVN GTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSN TTTGLSTSTASPHTRANSTSTERKLPEPESR

The soluble CD 146 proteins of the invention are present in the human serum and extracted therefrom or artificially reproduced. In a preferred embodiment, the soluble CD 146 comprises the amino acid sequence consisting in SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the herein disclosed human soluble CD146 is a biologically active human soluble CD146, i.e., it initiates, promotes, increases or stimulates vasculogenesis and/or angiogenesis in vitro, ex vivo or in vivo. Preferably the soluble CD 146 displays a chemotactic activity, i.e., the soluble CD 146 is able to induce mobilization or migration of endogeneous or exogeneous cells to the site wherein vasculogenesis and/or angiogenesis is to occurred, preferably cells of endothelial origin (KDR+ and/or CD31+ cells), preferably selected from immature endothelial cells (in particular CD117+ cells), mature endothelial cells (in particular KDR+ cells), endothelial progenitor cells (EPC), such as stem cell (typically bone-marrow derived stem cells), and mixtures thereof, and/or allows or favors the organization of such cells into vascular-like structures. A biologically active human soluble CD146 is also able to activate endothelial cells as defined previously, i.e., to increase their ability to proliferate and/or promote pseudo-capillaries genesis.

The herein disclosed human soluble CD 146 is further preferably capable of interacting with the short isoform of CD 146 ("short CD 146"), a receptor of CD 146, in particular a receptor of soluble CD146, and/or to a complex comprising such a short isoform of CD146 and receptor of soluble CD 146, on a cell preferably selected from the above identified cells. A typical human soluble CD 146 protein according to the present invention is, as explained previously, a protein usable in the context of a treatment (a therapeutic or prophylactic protein) or in the context of diagnostic, and compatible with an administration to a human, in particular by way of injection in the bloodstream, and/or by way of subcutaneous and/or intramuscular administration.

Preferably, the human soluble CD 146 protein according to the present invention is for use as a drug or medicament, or for preparing such a drug or medicament.

The term "Treatment" refers to both therapeutic and prophylactic or preventive treatment or measures able to alleviate or cure a disease, disorder or dysfunctional state. Such a treatment is intended for a mammal subject, preferably a human subject in need thereof. Are considered as such, the subjects suffering from a disease, disorder or dysfunctional state leading to, resulting from or associated with tissue ischemia, or those considered "at risk of developing" such a disease, disorder or dysfunctional state, in which this has to be prevented.

Disease, disorder or dysfunctional state leading to, resulting from or associated with tissue ischemia are disease, disorder or dysfunctional state leading to abnormal vasculogenesis and/or angiogenesis, in particular disease, disorder or dysfunctional state leading to, resulting from or associated with an undesirable excessive (abnormally increased) neovascularization easily determinable (easy to assess) by the skilled person in the art, vascular permeability (alteration of the intercellular junctions of endothelial cells) and/or vascular endothelial cell growth. Examples of such diseases include cancer; diabetes; age-related macular degeneration ("AMD"); rheumatoid arthritis; psoriasis; systemic sclerosis (SSc); an ischemic disease, typically a tissue ischemia, or a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia; any known vascular diseases including atherosclerotic vascular disease, cardiovascular disease such as coronary artery disease, ischemic heart disease and stroke, cerebrovascular ischemia, peripheral vascular disease such as peripheral artery occlusive disease.

In the context of the present invention, the cancer can be a cancer whose cells express membranous CD 146 such as pancreatic cancer, melanoma, renal cancer or breast cancer for example, or a cancer whose cells do not express membranous CD146 but express soluble form(s) thereof.

The cancer is for example selected from breast cancer, pancreatic cancer, renal cancer, melanoma and colon cancer. In these conditions leading to an undesirable neovascularization, new blood vessels feed diseased tissues, destroy normal tissues, and in the case of cancer, the new vessels allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases).

Disorders may be the consequence of a disease as described above or of a trauma. Typical disorders are for example inflammation, oedema or necrosis.

Examples of dysfunctional states are characterized by a lack of or, on the contrary, an excessive expression of at least one particular form of CD 146 or of a receptor for CD 146, in particular for soluble CD 146, compared to a standard expression (typically the corresponding expression in a healthy subject). Other examples of dysfunctional states are characterized by a decreased or excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to a standard expression.

Dysfunctional states characterized by a lack of expression are advantageously treated by a human soluble CD 146, or a therapeutic composition comprising such a soluble CD 146, or a cell (as described below) according to the present invention.

Dysfunctional states characterized by an excessive expression, such as cancer, are advantageously treated by an antibody directed against a human soluble CD 146, or any other antagonist directed against such a human soluble CD 146, as herein described. In a particular embodiment, a dysfunctional state such as a cancer, may be treated by an antibody directed against a human soluble CD 146 together with an antibody directed against an angiogenic factor such as VEGF.

In a particular aspect, an antibody or any other antagonist directed against I5-13-sCD146 (SEQ ID NO: 1), or a composition comprising such an antagonist, is advantageously usable for treating dysregulated or in other words abnormally increased (compared to healthy subjects) angiogenesis associated to SSc in SSc subjects.

The present description therefore identifies isolated human soluble CD 146 proteins respectively comprising or consisting in SEQ ID NO: 1 and SEQ ID NO: 2. "Isolated" means identified and separated or recovered from a component of its natural source or environment in a human subject, in particular from bone marrow, blood, serum or plasma of said subject.

A preferred human soluble CD146 protein comprises or consists in SEQ ID NO: 1 or SEQ ID NO: 2, and corresponds to a protein usable in the context of a mammal treatment, in particular a human treatment, as herein described. The present description further provides nucleic acid molecules which respectively encode the herein described proteins of the invention.

Such nucleic acid molecules are RNA or DNA that preferably each encode a biologically active human CD 146, in particular a human soluble CD 146 protein of the invention, the human short form of CD 146, and recombinant forms thereof.

Examples of nucleic acid sequences are provided below:

SEQ ID NO: 6 (encoding short CD146 of SEQ ID NO: 4)

rGGGGCTTCCCAGGCTGGTCTGCGCCTTCTTGCTCGCCGCCTGCTGCTGCTGTCCTC GCGTCGCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCCTGAGCTGGTGGAGGT GGAAGTGGGCAGCACAGCCCTTCTGAAGTGCGGCCTCTCCCAGTCCCAAGGCAACC TCAGCCATGTCGACTGGTTTTCTGTCCACAAGGAGAAGCGGACGCTCATCTTCCGTG TGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGGCTCAGCCT CCAGGACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCA TCTTCTTGTGCCAGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGC GTCTACAAAGCTCCGGAGGAGCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGT GAACAGTAAGGAGCCTGAGGAGGTCGCTACCTGTGTAGGGAGGAACGGGTACCCC ATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCCTCTGAAGGAGGAGAAGAACCG GGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTTGTACACCTTGCAGA GTATTCTGAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTACTGTGAG CTCAACTACCGGCTGCCCAGTGGGAACCACATGAAGGAGTCCAGGGAAGTCACCGT CCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGGAAGTGGAGCCCGTGGGAATGC TGAAGGAAGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCCTCCACCA CACTTCAGCATCAGCAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAA CCAACGACAACGGGGTCCTGGTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCG CTATGAATGTCAGGGCCTGGACTTGGACACCATGATATCGCTGCTGAGTGAACCAC AGGAACTACTGGTGAACTATGTGTCTGACGTCCGAGTGAGTCCCGCAGCCCCTGAG AGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAGAGTAGCCAGGACC TCGAGTTCCAGTGGCTGAGAGAAGAGACAGGCCAGGTGCTGGAAAGGGGGCCTGT GCTTCAGTTGCATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGT

GGCCCCCCTTGGATGGCATTCAAGGAGAGGAAGGTGTGGGTGAAAGAGAATATGG TGTTGAATCTGTCTTGTGAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGAAC GTCAACGGCACGGCAAGTGAACAAGACCAAGATCCACAGCGAGTCCTGAGCACCC TGAATGTCCTCGTGACCCCGGAGCTGTTGGAGACAGGTGTTGAATGCACGGCCTCC AACGACCTGGGCAAAAACACCAGCATCCTCTTCCTGGAGCTGGTCAATTTAACCAC CCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTTCCACTGCCAGTCCTC ATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGAGCCG GGGCGTGGTCA TCGTGGCTGTGA TTGTGTGCA TCCTGGTCCTGGCGGTGCTGGGCGCT GTCCTCTA TTTCCTCTA 7AAGAAGGGCAAGCTGCCGTGCAGGAGCTCAGGGAAGCAG GAGATGGAGAGAAATACATCGATCTGA

SEQ ID NO: 7 (encoding soluble CD146 of SEQ ID NO: 1):

ATGGTATACATTGTTAGACAATTTCTTTTATACAATGTTTCTGGGTCAGTCTATTTAG ATCAACTGATCGTTTTGCTTACTGCCAAGTTTTCCATACTACGCATAGCAGGTAGTC GAGTTCACCATTCCCCATTTAGTGGACATCTAGACGGCTGCTCGTTTTTATCATTGC

TGACACTGAGCAGTAAAAGTGCTGGGTTGAAGGGTTTCCAGCTTGCATTTGTACCT GGCCTTCTACAGGGGACAGGGGGCTATTTAGATGGTCCCCTGCCAACCCCAGTGGA CAACCCTAGGGTGGGGCTGGAGGTGGGGCTGAGGCTGAGTCTTCCTCCCCTTCCTC CCTGCCCAGGGGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTTGTAC ACCTTGCAGAGTATTCTGAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTT TTACTGTGAGCTCAACTACCGGCTGCCCAGTGGGAACCACATGAAGGAGTCCAGGG AAGTCACCGTCCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGGAAGTGGAGCCC GTGGGAATGCTGAAGGAAGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCA ACCCTCCACCACACTTCAGCATCAGCAAGCAGAACCCCAGCACCAGGGAGGCAGA GGAAGAGACAACCAACGACAACGGGGTCCTGGTGCTGGAGCCTGCCCGGAAGGAA CACAGTGGGCGCTATGAATGTCAGGGCCTGGACTTGGACACCATGATATCGCTGCT GAGTGAACCACAGGAACTACTGGTGAACTATGTGTCTGACGTCCGAGTGAGTCCCG CAGCCCCTGAGAGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAGAG TAGCCAGGACCTCGAGTTCCAGTGGCTGAGAGAAGAGACAGGCCAGGTGCTGGAA AGGGGGCCTGTGCTTCAGTTGCATGACCTGAAACGGGAGGCAGGAGGCGGCTATC GCTGCGTGGCGTCTGTGCCCAGCATACCCGGCCTGAACCGCACACAGCTGGTCAAC GTGGCCATTTTTGGCCCCCCTTGGATGGCATTCAAGGAGAGGAAGGTGTGGGTGAA AGAGAATATGGTGTTGAATCTGTCTTGTGAAGCGTCAGGGCACCCCCGGCCCACCA TCTCCTGGAACGTCAACGGCACGGCAAGTGAACAAGACCAAGATCCACAGCGAGT CCTGAGCACCCTGAATGTCCTCGTGACCCCGGAGCTGTTGGAGACAGGTGTTGAAT GCACGGCCTCCAACGACCTGGGCAAAAACACCAGCATCCTCTTCCTGGAGCTGGTC AATTTAACCACCCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTTCCAC TGCCAGTCCTCATACCAGAGCCAACAGCACCTCCACAGGTAAGCCAGGCCTGGCAA GAGAACAGGGCTGTGCCAGGGCATCCTTTCTGCCCTGTCCCTCCCCAGAGAGCCCT GTCCAGAAAGGTGAGTAG

SEQ ID NO: 8 (encoding soluble CD 146 of SEQ ID NO: 2):

ATGGGGCTTCCCAGGCTGGTCTGCGCCTTCTTGCTCGCCGCCTGCTGCTGCTGTCCT CGCGTCGCGGGTGTGCCCGGAGAGGCTGAGCAGCCTGCGCCTGAGCTGGTGGAGGT GGAAGTGGGCAGCACAGCCCTTCTGAAGTGCGGCCTCTCCCAGTCCCAAGGCAACC TCAGCCATGTCGACTGGTTTTCTGTCCACAAGGAGAAGCGGACGCTCATCTTCCGTG TGCGCCAGGGCCAGGGCCAGAGCGAACCTGGGGAGTACGAGCAGCGGCTCAGCCT CCAGGACAGAGGGGCTACTCTGGCCCTGACTCAAGTCACCCCCCAAGACGAGCGCA TCTTCTTGTGCCAGGGCAAGCGCCCTCGGTCCCAGGAGTACCGCATCCAGCTCCGC GTCTACAAAGCTCCGGAGGAGCCAAACATCCAGGTCAACCCCCTGGGCATCCCTGT GAACAGTAAGGAGCCTGAGGAGGTCGCTACCTGTGTAGGGAGGAACGGGTACCCC ATTCCTCAAGTCATCTGGTACAAGAATGGCCGGCCTCTGAAGGAGGAGAAGAACCG GGTCCACATTCAGTCGTCCCAGACTGTGGAGTCGAGTGGTTTGTACACCTTGCAGA GTATTCTGAAGGCACAGCTGGTTAAAGAAGACAAAGATGCCCAGTTTTACTGTGAG CTCAACTACCGGCTGCCCAGTGGGAACCACATGAAGGAGTCCAGGGAAGTCACCGT CCCTGTTTTCTACCCGACAGAAAAAGTGTGGCTGGAAGTGGAGCCCGTGGGAATGC TGAAGGAAGGGGACCGCGTGGAAATCAGGTGTTTGGCTGATGGCAACCCTCCACCA CACTTCAGCATCAGCAAGCAGAACCCCAGCACCAGGGAGGCAGAGGAAGAGACAA CCAACGACAACGGGGTCCTGGTGCTGGAGCCTGCCCGGAAGGAACACAGTGGGCG CTATGAATGTCAGGGCCTGGACTTGGACACCATGATATCGCTGCTGAGTGAACCAC AGGAACTACTGGTGAACTATGTGTCTGACGTCCGAGTGAGTCCCGCAGCCCCTGAG AGACAGGAAGGCAGCAGCCTCACCCTGACCTGTGAGGCAGAGAGTAGCCAGGACC TCGAGTTCCAGTGGCTGAGAGAAGAGACAGGCCAGGTGCTGGAAAGGGGGCCTGT GCTTCAGTTGCATGACCTGAAACGGGAGGCAGGAGGCGGCTATCGCTGCGTGGCGT

GGTGAGGCCCTCCCTCTGGGTAGAGACCAGGTCACCCCAAGTGGGTGGTTTTTAAG CTCTTTGACAAAAAGCCACCTGCTGCCCTGGGGAGCTCTGGTGCGGAGGGGGAGGC AGGCTAG

SEQ ID NO: 9 (encoding soluble CD146 of SEQ ID NO: 5):

GGCCCCCCTTGGATGGCATTCAAGGAGAGGAAGGTGTGGGTGAAAGAGAATATGG TGTTGAATCTGTCTTGTGAAGCGTCAGGGCACCCCCGGCCCACCATCTCCTGGAAC GTCAACGGCACGGCAAGTGAACAAGACCAAGATCCACAGCGAGTCCTGAGCACCC TGAATGTCCTCGTGACCCCGGAGCTGTTGGAGACAGGTGTTGAATGCACGGCCTCC AACGACCTGGGCAAAAACACCAGCATCCTCTTCCTGGAGCTGGTCAATTTAACCAC CCTCACACCAGACTCCAACACAACCACTGGCCTCAGCACTTCCACTGCCAGTCCTC ATACCAGAGCCAACAGCACCTCCACAGAGAGAAAGCTGCCGGAGCCGGAGAGCCG G

The natural (non recombinant) molecules may be isolated, for example from a nucleic acid library prepared from a tissue known to express the desired protein, e.g., blood, in particular serum or bone marrow, the library being screened with an appropriate probe, or from a tissue sample (bone marrow, blood, serum, etc.), preferably a sample of the subject to be treated (see for example Hoskins RA, Stapleton M, George RA, Yu C, Wan KH, Carlson JW, Celniker SE. Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP). Nucleic Acids Research 2005; 33: 185-197).

The nucleic acid molecules may otherwise be artificially produced, for example by oligonucleotides synthesis (see for example Michaels ML, Hsiao HM, Miller JH. Using PCR to extend the limit of oligonucleotide synthesis. Biotechniques. 1992; 12:44- 48).

A preferred human soluble CD 146 protein according to the present invention may be obtained using a method comprising the following steps of transfecting a mammalian cell with an appropriate vector expressing the human soluble CD 146 protein, typically the human soluble CD 146 protein of SEQ ID NO: 1 or 2, and isolating the expressed human CD 146 protein.

Herein described is a recombinant human soluble CD 146 (rh-sCD146) protein fused to another polypeptide, such as a tag polypeptide sequence (see the c-myc tagged human soluble CD 146 in the experimental part wherein the sequence of the soluble CD146 is SEQ ID NO: 1 or 2).

The nucleic acid molecule can be provided in a replicable vector comprising the nucleic acid molecule operably linked to control sequences recognized by a host cell transfected or transformed with the vector, in particular a mature or immature endothelial cell or a progenitor cell, preferably an endothelial progenitor cell (also herein identified as EPC or ECFC), typically a cell as previously herein described. The invention further provides such an host cell comprising the vector or the nucleic acid molecule as further explained below.

The amino acid molecules of the present invention can be designed to improve their performance and/or biocompatibility with a diagnostic, therapeutic or prophylactic use in a subject, typically in a mammal, preferably in a human being. They can be, for example, glycosylated, methylated, acetylated, phosphorylated or fused to another polypeptide, preferably methylated, acetylated, phosphorylated or fused to another polypeptide, for targeting different types of tissues, in particular a pathological tissue such as, typically, an ischemic tissue, preferably in a human being.

Suitable host cells for the expression of a glycosylated soluble human CD 146 may be selected from mammalian cell lines, for example CHO cells.

In another embodiment, the invention provides a composition, in particular a pharmaceutical composition useful for promotion of vascular cell growth, typically endothelial cell growth, comprising, preferably in a therapeutically effective amount, at least one soluble CD 146 protein as herein described, in a pharmaceutically acceptable carrier or excipient. The composition is typically for use as a drug or medicament.

A "therapeutically affective amount" of a soluble CD 146 protein is an amount allowing the treatment, as previously defined, of a subject, typically of a mammal, preferably of a human being.

A pharmaceutically acceptable excipient, vehicle or carrier, usable in the context of the present invention, is for example a saline, isotonic, buffered solution such as Mannitol 20 %, optionally combined with stabilizing agents such as isogenic albumin or any other stabilizing protein, glycerol, etc., and also adjuvants such as polybrene or DEAE dextrans, etc..

In a particular aspect, the herein described compositions comprising at least one soluble CD 146 protein as herein described, possibly at least two soluble CD 146 proteins selected from SEQ ID NO: 1, 2 and 5, preferably SEQ ID NO: 1 and 2, and may further comprise at least one other angiogenic factor.

In the context of the present invention, an angiogenic factor is a factor which favors blood vessel development.

Angiogenic factors usable in the context of the present invention may be selected from angiogenin, angiopoietin-1, Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), follistatin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), hepatocyte growth factor (HGF) /scatter factor (SF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), erytropoietin (EPO), endothelial nitric oxyd synthase (e-NOS), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), Angiopoietin-1 (Angl), plasminogen activator urokinase (PLAU/u-Pa), the matrix metallopeptidase MMP-2, the VEGF receptor 2 (KDR), stromal-cell-derived- factor- 1 (SDF-1), etc., and a mixture thereof.

Preferred angiogenic factors may be selected from vascular endothelial growth factor, stromal- cell-derived- factor- 1 (SDF-1), basic fibroblast growth factors (bFGF), erytropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-8 (IL-8) and a mixture thereof. In another particular aspect, the herein described compositions comprising an antibody directed against a soluble CD 146 may further comprise at least one other antibody directed against one of the previously identified angiogenic factor. In another particular aspect, the herein described compositions comprising a human soluble CD 146 may in addition comprise a mature or immature endothelial cell or a progenitor cell, typically of human origin, preferably in vzYro-expanded progenitor cells, in particular stem cells or endothelial progenitor cells, typically cells derived from blood or bone marrow, for example selected from cells expressing CD34, CD133, CD31, VE-cadherin, VEGFR2, c-Kit, CD45 and/or Tie-2. Preferably, such a cell has been contacted with at least one human soluble CD146 protein as herein described.

Compositions comprising cells as previously mentioned contacted with a soluble CD 146 as herein defined before being optionally incorporated in a pharmaceutically acceptable excipient are an embodiment of the present invention as further explained below.

A particular composition as previously described does not comprise added soluble CD 146 protein as herein described.

In a preferred embodiment, the progenitor cell is a recombinant progenitor cell. Such a recombinant cell may be genetically modified using an appropriate vector comprising or consisting in a genetic or nucleic acid construct expressing, preferably enabling an overexpression of, a particular biologically active form of CD 146, preferably of the human soluble form of CD146 as herein described and/or of the human short form of CD146.

In a particular aspect, the nucleic acid construct expresses at least one human soluble protein as herein described and optionally the human short CD 146 protein of SEQ ID NO: 5.

Many vectors are available. Preferred vectors may be selected from a plasmid, a retrovirus, a lentivirus and an adenovirus. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, easily selectable by the man of the art (see for example Liu JW, Pernod G, Dunoyer-Geindre S, Fish RJ, Yang H, Bounameaux H, Kruithof EK. Promoter dependence of transgene expression by lentivirus-transduced human blood-derived endothelial progenitor cells. Stem Cells. 2006; 24:199-208). Such a progenitor cell may be transfected with the appropriate vector, preferably with a plasmid as described previously, according to known protocols, preferably a protocol appropriate to endothelial progenitor cells such as electroporation or use of liposomes, and then cultured in any known suitable media (a medium comprising EGM-2 for example), optionally supplemented with one or more of the following: an appropriate hormone, growth factor, buffer, etc. The progenitor cells may then be expanded in vitro using any method known by the man of the art (see for example Delorme B et al. Presence of endothelial progenitor cells, distinct from mature endothelial cells, within human CD146+ blood cells. Thromb Haemost. 2005 ;94:1270-9). Also incorporated in the present invention is the use of a human soluble CD 146 protein as herein described for the first time to prepare, ex vivo, a mature or immature endothelial cell or a progenitor cell, as previously defined, exhibiting therapeutic or prophylactic properties, in particular capable of stimulating angiogenesis in a human body. Inventors have indeed discovered that such cells contacted, "pre-treated" or "primed" with a human soluble CD 146 protein according to the invention are able by themselves, once administered to a subject, to induce or stimulate vasculogenesis and/or angiogenesis. The "pre-treated" or "primed" cells have been contacted with, or cultured in the presence of, a human soluble CD 146 protein according to the invention before being optionally incorporated into a pharmaceutically acceptable support.

Such cells are further objects of the present invention as well as the pharmaceutical compositions comprising said cells, preferably in a pharmaceutically acceptable support. While preparing such pharmaceutical compositions further adding human soluble CD 146 proteins as herein described is optional.

Preferred "pre-treated" or "primed" cells are cells which have been genetically modified, as explained previously, to overexpress anyone of the herein described soluble CD 146 proteins or both, and optionally the short form of the CD 146 protein.

In a further aspect, inventors herein disclose a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the prevention, in the treatment or (as a biomarker) in the diagnosis of a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia or characterized by a decreased expression or activation of a receptor for CD 146, in particular soluble CD 146, or by a decreased expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to standard expression values. The invention relates to a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the prevention or treatment of ischemia.

In particular, the invention relates to the use of a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, to prepare a composition for preventing or treating a disease, a disorder or a dysfunctional state as herein previously identified.

In another particular embodiment, the invention provides a method of preventing or treating a disease, disorder or dysfunctional state, for example tissue ischemia, in a subject, typically in a mammal, preferably in a human being, as herein identified, wherein the method comprises a step of administering to the subject (an effective amount of) at least one soluble CD 146 protein as herein described, or (of) a composition comprising said at least one soluble CD 146 protein. Also herein described is a soluble CD 146 protein as herein described for use as a biomarker in the diagnosis of a disease, disorder or dysfunctional state characterized by dysregulated angiogenesis, undesirable excessive (neo)vascularization or undesirable vascular permeability, by an overexpression or excessive activation of a soluble form of CD 146 and/or of a receptor for CD 146, in particular soluble CD 146, or by an excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR in a mammal, compared to standard expression.

Such a disease, disorder or dysfunctional state is typically a cancer, systemic sclerosis (SSc), age-related macular degeneration (ASD), an ischemic disease or a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia.

An object of the present invention is a soluble CD146 protein as herein described, or a composition comprising such a soluble CD146 protein, for use in the cicatrization of a subject's, typically mammal's epithelium, in particular a human epithelium, in particular following a wound, a cut or an incision or in the context of a skin graft. Another object of the present invention is a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the prevention or treatment of an eschar or a bedsore in a subject, typically in a mammal, in particular in a human being.

A further object of the present invention is a soluble CD 146 protein as herein described, or a composition comprising such a soluble CD 146 protein, for use in the context of a skin graft in a mammal, in particular in a human being.

Preferred compositions for use in the context of cicatrisation will be advantageously formulated for topic application according to methods known by the man of the art.

In another embodiment, the present invention further provide an antibody, in particular a monoclonal antibody, which selectively binds to a human soluble CD 146 protein according to the present invention and herein described for the first time, preferably to the protein comprising or consisting in SEQ ID NO: 1 or SEQ NO: 2.

This antibody preferably also neutralizes a biological activity of the targeted human soluble CD 146 protein of the invention. Preferably, the monoclonal antibody decreases or inhibits neovascularization vascular permeability and/or vascular endothelial cell growth in a subject as herein defined, typically a mammal, preferably a human being.

The monoclonal antibody is also preferably able to reduce or suppress an excessive expression (compared to a standard expression) of a soluble CD 146 receptor, or an excessive expression (compared to a standard expression) of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR.

Antibodies binding both a human soluble CD 146 protein as herein described for the first time of SEQ ID NO: 1 or 2 and a CD 146 or soluble CD 146 protein receptor (or a CD 146 protein receptor subunit) are also within the scope of the present invention. Methods of making such antibodies are known in the art (See for example Despoix N, Walzer T, Jouve N, Blot-Chabaud M, Bardin N, Paul P, Lyonnet L, Vivier E, Dignat-George F, Vely F. Mouse CD146/MCAM is a marker of natural killer cell maturation. Eur J Immunol. 2008;38: 2855-64).

Further herein described is the use of an antibody as herein described as a drug or medicament, or for preparing such a drug or medicament.

Preferred antibodies selected by inventors in the context of the present invention, capable of selectively binding a soluble CD 146 protein as herein described for the first time of SEQ ID NO: 1 or 2 (versus membrane CD 146 or a distinct known soluble CD 146), are herein disclosed and identified respectively as I5-13-sCD146 antibody (I5-13-sCD146 Ab) for the sCD146 protein comprising or consisting of SEQ ID NO:l and I10-sCD146 antibody (I10-sCD146 Ab) for the sCD146 protein comprising or consisting of SEQ ID NO:2.

Typically, I5-13-sCD146 Ab recognizes the amino acid sequence of SEQ ID NO: 29 and/or of SEQ ID NO: 31 of the I5-13-sCD146 protein, or at least a fragment of SEQ ID NO: 29 and/or of SEQ ID NO: 31, and I10-sCD146 Ab recognizes the amino acid sequence of SEQ ID NO: 33 of I10-SCD146, or at least a fragment of SEQ ID NO: 33.

SEQ ID NO: 28 is the nucleic acid sequence encoding SEQ ID NO: 29.

SEQ ID NO: 30 is the nucleic acid sequence encoding SEQ ID NO: 31.

SEQ ID NO: 32 is the nucleic acid sequence encoding SEQ ID NO: 33.

The herein described antibodies may be incorporated into a composition further comprising a pharmaceutically acceptable carrier, in respective appropriate dosages.

In a particular aspect, the herein described compositions comprising an antibody as herein described for the first time, preferably an antibody which selectively binds (is directed against) a human soluble CD 146 protein as herein described, typically a human soluble CD 146 protein comprising or consisting in an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, may further comprise at least one other anti-angiogenic factor. In the context of the present invention an anti-angiogenic factor is a factor which inhibits or interferes with blood vessel development.

Anti-angiogenic factors usable in the context of the present invention may be selected from an antibody directed against an angiogenic factor as previously defined, angioarrestin, angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage- derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro- beta, an heparinase, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, Interferon inducible protein (IP- 10), interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-Methoxyestradiol, placental ribonuc lease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin 16kD fragment, proliferin-related protein (PRP), a retinoid, tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growth factor-beta (TGF-b), vasculostatin, vasostatin (calreticulin fragment), etc., and a mixture thereof. The antibody, or a pharmaceutical composition comprising at least said antibody and a pharmaceutically acceptable carrier, herein disclosed, can be used in a subject, typically in a mammal, preferably in a human being, for preventing or treating a disease, disorder or dysfunctional state characterized by a dysregulated angiogenesis, an undesirable excessive (neo)vascularization or undesirable vascular permeability, by an overexpression or excessive activation of a soluble form of CD146 and/or of a receptor for CD146, in particular soluble CD 146, or by an excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to standard expression in a reference subject.

In a particular aspect, the description relates to an antibody which binds (is directed against), preferably selectively binds, I5-13-sCD146 (SEQ ID NO: 1), or a composition comprising such an antibody, for use in the prevention or treatment of dysregulated (abnormally increased) angiogenesis.

Also herein disclosed is an antibody as herein disclosed, for use in a subject, typically in a mammal, preferably in a human being, for diagnosing a disease, disorder or dysfunctional state characterized by a dysregulated angiogenesis, an undesirable excessive (neo)vascularization or undesirable vascular permeability, by an overexpression or excessive activation of a soluble form of CD 146 and/or of a receptor for CD 146, in particular soluble CD 146, or by an excessive expression of a gene selected from the gene encoding e-NOS, uPa, MMP-2 and KDR, compared to standard expression in a reference subject.

In a particular aspect, the disease is selected from a cancer, systemic sclerosis, an ischemic disease or a disease, disorder, dysfunctional state leading to, resulting from or associated to (tissue) ischemia or age-related macular degeneration (AMD). Inventors also herein disclose a method of diagnosing such a disease, disorder or dysfunctional state, for example tissue ischemia, in a subject, typically in a mammal, preferably in a human being, as herein identified, wherein the method comprises a step of administering to the subject, (an effective amount of) an antibody as herein described, or (of) a composition comprising such an antibody.

In a particular aspect where the disease to be diagnosed is systemic sclerosis and the antibody is an antibody selectively binding a soluble CD 146 protein comprising or consisting in SEQ ID NO: 1, an increased level of said soluble CD 146 protein in the serum of the tested subject when compared to a reference level is indicative of systemic sclerosis or AMD.

In another particular aspect where the disease to be diagnosed is systemic sclerosis and the antibody is an antibody selectively binding a soluble CD 146 protein comprising or consisting in SEQ ID NO: 2, a decreased level of said soluble CD146 protein in the serum of the tested subject when compared to a reference level is indicative of systemic sclerosis or AMD.

The doses of the diagnostic or pharmaceutical composition may be adjusted by the skilled person depending on the treated subject, the route of administration, the targeted tissue, the biologically active compound (as herein disclosed), etc.

Various protocols may be used for the administration, such as simultaneous or sequential administration of the human soluble CD 146 protein(s) and of any other compound as defined previously ("pre-treated", "primed", and/or genetically modified cells as described previously for example), single or repeated administration, etc., which may be adjusted by the skilled person.

The pharmaceutical composition containing a product according to the invention may be administered to a patient for example systemically, subcutaneously, intraspinally or intracerebrally, given the targeted pathological tissue or area. Preferred modes of injection are systemical injections, in particular intra-venous or intra-arterial injections, or subcutaneous injections. The molecules of the present invention may further be used in methods of diagnostic. The term diagnostic designates any in vivo, ex vivo or in vitro diagnosis, including molecule detection, monitoring, quantification, comparison, etc. In particular, any of the herein described human soluble CD 146 proteins may be used as a biomarker providing an indication of the presence of a disease in a subject, typically in a mammal, preferably in a human being, in particular of an ischemia, a disease, disorder or dysfunctional state leading to, resulting from or associated to (tissue) ischemia, a systemic sclerosis, AMD, a cancer, of metastasis of a tumor, or of the evolution of such a diseased state. In particular, the serum concentration of the considered human soluble CD 146 protein may be an indication of high value in this regards. The measured value may be indeed compared to standard values associated to a healthy status of a subject. An overexpression of the considered soluble form of CD146 may be, in particular, indicative of the presence of a cancer.

The term diagnosis also includes the use of the molecules to screen compounds or treatments that cause or increase apoptosis of a cell, in vitro, ex vivo or in vivo. Also herein provided is a kit comprising at least one biologically active product as herein described, such as typically a human soluble CD 146 protein as herein described for the first time, an antibody, in particular a monoclonal antibody, directed against one of said human soluble CD 146 protein, a "pre-treated" or "primed" (stimulated by such a human soluble CD 146) and/or genetically modified (to overexpress at least one of the herein described soluble CD 146 and/or the short form of the CD 146 protein) cell, and optionally (ii) a leaflet providing guidelines.

Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting. All references cited in the present application are herein incorporated by reference.

EXAMPLES EXAMPLE 1 : Two novel pro-angiogenic spliced variants of soluble CD146 are differentially expressed and regulated in pathology.

Materials and Methods

Cells

Endothelial Colony Forming Cells (ECFC) were cultured in endothelial EGM-2 MV medium

Clonetics, Walkersville, USA), as previously described (Delorme et al., 2005).

HUVEC were obtained as previously described and were cultured in EGM2 medium.

For stimulation experiments, cells were maintained for 3 hours in basal medium and then stimulated.

Cell proliferation assay

Experiments were performed as previously described (Stalin et al., 2013). Results were expressed as arbitrary units. Experiments were performed in triplicates.

Endothelial cell tube formation in spheroids

Formation of spheroid experiments was performed as previously described (Korff et al., 2011). RNA isolation, reverse transcription and real time PCR

Total cellular RNA was isolated from cells, reverse transcribed into cDNA and the resulting cDNA was subjected to qPCR as previously described (Stalin et al., 2013).

Forward and reverse specific primer sequences are identified herein below:

GAPDH:

Forward sequence: GGTGGTCTCCTGACTTCAACA (SEQ ID NO: 10), Reverse sequence: GTTGCTGTAGCCAATTCGTTGT (SEQ ID NO: 11)

CD146sh:

Forward sequence: CCACTGGCCTCAGCACTTCC (SEQ ID NO: 12),

Reverse sequence: CTACTCACCTTTCTGGACAG (SEQ ID NO: 13)

CD1461g:

Forward sequence: TGGTTTGTACACCTTGCAGAGTATTC (SEQ ID NO: 14),

Reverse sequence: TGGGCAGCCGGTAGTTG (SEQ ID NO: 15)

I10-SCD146:

Forward sequence: GGCAGAGGAAGAGACAACCA (SEQ ID NO: 16),

Reverse sequence: TTGGGGTGACCTGGTCTCTA (SEQ ID NO: 17)

I5-13-SCD146:

Forward sequence: GGACATCTAGACGGTGCTC (SEQ ID NO: 18),

Reverse sequence: ACAAATGCAAGCTGGAAACC (SEQ ID NO: 19).

The values given refer to the number of transcript copies.

Co-immunoprecipitation experiments

Experiments were performed as previously described (Stalin et al., 2016).

Western-blot analysis

Western-blot analysis was performed as previously described⁸. Membranes were probed with specific primary antibodies (7A4 diluted 1/3000, actine diluted 1/5000, Tace and AD AMI 0 diluted 1/1000, I10-sCD146 and I5-13-sCD146 Ab diluted 1/250) followed by secondary antibodies coupled to peroxidase. Blots were revealed with the ECL substrate (Pierce). Membranes probed with various antibodies were stripped between antibodies.

Yolk sac membrane assay

The yolk sac membrane (YSM) assay was performed using a modified protocol as previously described (Beckers et al., 1997).

Lipid raft preparation

Lipid rafts were isolated as previously described (Tellier et al., 2006) by sucrose density gradient centrifugation of cells treated with non-ionic detergents.

Immunofluorescence experiment

The localization of the different isoforms of CD 146 and ADAMIO/Tace was examined in cells seeded on coverslip. Cells were fixed 10 minutes with paraformaldehyde at room temperature, then permeabilized with saponin (0.2%) then incubated during 30 minutes at room temperature with the anti-CD146 antibodies (1/100) and anti-Tace (1/200) or anti-ADAMlO (1/100) antibodies. After 3 rinses in PBS, cells were incubated with a secondary antibody associated to a fluorescent probe. In some experiments, the nucleus was stained with DAPI. In sections of muscles from ischemic mice, vascularisation was visualized with isolectin B4 (Lifetechnologies, reference 121411) (1/100). Cells and tissue sections were examined by fluorescence microscopy (Leica sp5, Leica, Nanterre, France).

Induction of hind limb ischemia in mice

Female mice were subjected to unilateral hind limb ischemia by complete resection of the entire left femoral artery. Laser-doppler tissue imaging showed that obstruction of the left common femoral artery decreased blood perfusion by about 50% at day 1. After surgery, animals were split in four treatment groups: one control group injected in ischemic adductor muscles twice a week with PBS for 28 days; three experimental groups treated as the control groups with the three different recombinant human soluble CD146 (rh-sCD146, rh-I5-13-sCD146, rh-I10- sCD146) 2 μ_δ.

Histological Analysis

The YSM was surgically removed and fixed in 10% buffered formaldehyde for 10 hours, dehydrated in graded alcohol, cleared in xylene and embedded in paraffin. 5μιη thick sections were cut in a plane parallel to the surface of the YSM and stained by hematoxylin-eosin which was observed under a light photomicroscope.

Plasmid transfection experiments and clone selection

Plasmids encoding for the different proteins were introduced in CHO cells using Fugen kit as described by the manufacturer (Promega). Stable clones were then selected with Geneticin and FACS and western blot or RT-PCR analysis was performed to identify the clone with the highest expression.

siRNA experiments

SiRNA designed to silence the different isoforms of sCD146 were generated. A control siRNA was also used in each experiment. siRNAs were introduced in endothelial cells using the Silencemag magnetofection kit as described by the manufacturer (OZ Biosciences). Silencing resulted in about 80-95%> of protein expression as a function of the experiments.

Sens and antisens sequences of the different siRNAs are herein below identified:

sishCD146:

sens: 5 ' CAGGAGAUGGAGAGAAAUACAUCGA (SEQ ID NO: 20),

antisens: 5 'UCGAUGUAUUUCUCUCCAUCUCCUG (SEQ ID NO: 21)

silgCD146:

sens: 5'CCCGUCUCGUAAGACCGAACUUGUA (SEQ ID NO: 22),

antisens: 5'UACAAGUUCGGUCUUACGAGACGGG (SEQ ID NO: 23)

siI10-sCD146:

sens: CCCUCCCUCUGGGUAGAGACCAGGU (SEQ ID NO: 24), antisens: ACCUGGUCUCUACCCAGAGGGAGGG (SEQ ID NO: 25) siI5-13-sCD146:

sens: UGGACAUCUAGACGGCUGCUCGUUU (SEQ ID NO: 26),

antisens: AAAC G AGC AGC CGUCUAG AUGUC C A (SEQ ID NO: 27).

Quantitative flow cytometry

The level of membrane expression of the different isoforms of CD 146 was determined by labeling cells with the antibody or the isotype matched control antibody coupled to fluorescent dye (10 μg/ml) for lh at 4°C. After washing, samples were analyzed by flow cytometry (GalliosTM Flow Cytometer, Beckman Coulter, Villepinte). The results were then analyzed using Kaluza software (Kaluza® Analysis Software, Beckman Coulter).

Tissue array

TissueScan Human Normal Tissue qPCR Arrays (OriGeneTechnologies, Rockville MD) were used to screen for I5-13-sCD146 and I10-sCD146 expression in 48 different tissues and following manufacturer's instructions.

ELISA

For detecting the two spliced isoforms of sCD146 (I5-13-sCD146 and I10-sCD146), a sandwich immunoassay was used (Biocytex). The protocol was modified for the second antibody (detection antibody) that was replaced by the anti-I5-13-sCD146 or anti-I10-sCD146 antibodies coupled to HRP.

Laser Doppler blood flow analysis

The ratio of the ischemic versus normal hind limb blood flow was measured using a laser doppler blood flow analyzer. At different time points post-surgery (days 1, 3, 7, 14, 21 and 28), animals were subjected to 3 consecutive laser scannings over the regions of interest (leg and feet). Blood flow was expressed as the ischemic versus normal hind limb ratio.

Angiographic CT imaging studies

The animals were deeply anesthetised with mix of Ketamine Xylasine (lOOmg/kg and lOmg/kg respectively) and perfused intracardially with 2ml of lipiodole. CT 3D images were obtained with a microPET/microCT rodent model scanner (nanoPET/CT®, Mediso) under following conditions: 70 kVp energy, exposure time of 300 ms and 720 projections.

Subjects

Sera from 24 systemic sclerosis (SSc) patients admitted to internal medicine departments in Marseille (France) were analyzed. All patients fulfilled the 2013 ACR/EULAR Classification Criteria for Scleroderma and were then sub classified according to LeRoy et al. criteria. As controls, we studied the sera of 24 age and sex matched blood donors. All samples came from a declared Biobank (DC 2012-1704) with respect of ethical directives and were stored to - 80°C prior to use.

Ethics committee approval

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; n°13328).

The experiments on human samples were performed retrospectively on plasmas from patients with systemic sclerosis. They were performed in accordance with the Helsinki declaration of 1975, revised in 1983, approved by relevant institutional review board and informed consent of the patients was obtained.

Peptides, plasmids, antibodies, and inhibitors

The recombinant human soluble form of CD 146 corresponding to the shed sCD146 (rh- sCD146) was obtained from Biocytex. The rh-I5-13-sCD146 and rh-I10-sCD146 arise from the purification on the column of protein tagged with HA peptide (Trial Kit 3320A, MBL).

The plasmid encoding for Tace was from Dr F. Peiretti, UMR-S1062, Marseille, France.

The plasmid encoding for ADAM10 was a kind gift from Dr B. Charreau, UMR-S1064, Nantes, France.

Polyclonal antibodies against I5-13-sCD146 and I10-sCD146 were generated by Covalab company by injecting peptides corresponding to the specific parts of the two proteins. Antibodies against ADAM- 17 (ab2051), ADAM- 10 (EPR5622), CD 146 (clone 7A4) and actine were respectively purchase from Abeam, Millipore, Biocytex and Cell signaling.

GM6001 was from Selleck chemicals.

Statistical analysis

Data were expressed as mean ± SEM of the indicated number of experiments. Statistical analysis was performed with the Prism software (GraphPad Software Inc., San Diego, CA). Significant differences were determined using non parametric Mann Whitney test. A P-value < 0.05 was considered as significant. Results

Additional forms of soluble CD146 are generated by alternative splicing

Since a fraction of the sCD146 secretion is GM6001 -insensitive, inventors hypothesized that this fraction could be due to the generation of additional forms of sCD146 generated by alternative splicing. To test this hypothesis, they performed RNA seq on ECFC cells. Results showed the existence of at least two additional isoforms of sCD146. One of these isoforms contained the intron 10 of the molecule (this isoform is herein identified as "I10-sCD146 isoform" and consists in SEQ ID NO: 2). Another one contained both the introns 5 and 13 of the molecule (this isoform is herein identified as "I5-13-sCD146 isoform" and consists in SEQ ID NO: 1). Figure 1A gives a schematic representation of these two isoforms in comparison with the shed form. Inventors verified the expression of I10-sCD146 and I5-13-sCD146 transcripts in ECFC and in other endothelial cells, HUVEC. Results show that both transcripts are present (figure IB) in ECFC and HUVEC and that they are expressed as protein as demonstrated by immunofluorescence on ECFC (figure 1C) and by western-blot in ECFC supernatants (figure ID). Inventors tested the effect of different pro-angiogenic molecules on the expression of the two variants. Of interest, they are differentially modulated. Indeed, I5-13-sCD146 was up-regulated at the mRNA level by TNF and WNT5a whereas it was not modified by the shed form of CD 146, VEGF, TGFb, netrin and WNT3a. In contrast, 110- sCD146 was not up-regulated at the mRNA level by these different factors. The mRNA level of I10-sCD146 variant was even down-regulated by VEGF, netrin and WNT3a (figure IE).

I5-13-sCD146 and I10-sCD146 isoforms of sCD146 display pro-angiogenic effects in vitro and in vivo

Since ECFC constitute a useful model for ex vivo angiogenesis studies, inventors evaluated the effect of the two spliced sCD146 isoforms (110 and 15-13) on ECFC proliferation. Experiments were performed in the presence of GM6001 in order to prevent membrane CD 146 shedding. Results showed that both siRNA targeting I10-sCD146 and I5-13-sCD146 inhibited the proliferation of ECFC (figure 2A). The efficiency of both siRNA was verified. Plasmids encoding for both isoforms were also transfected into ECFC to examine their effect on proliferation. Results (figure 2B) show that both plasmids significantly increased the proliferation. The angiogenic potential of these two sCD146 isoforms was also estimated after generation of the recombinant proteins. Recombinant I10-sCD146 and I5-13-sCD146 proteins (rh-I10-sCD146 and rh-I5-13-sCD146, respectively) were produced and characterized (Figure 5). Their effect was tested on the proliferation of ECFC and compared to that of the recombinant shed sCD146 form (rh-sCD146) (figure 2C). Results show that rh-I10-sCD146, rh- I5-13-sCD146 and rh-sCD146 increased ECFC proliferation. They also increased vascularisation in a yolk sac membrane assay (figure 2D, Figure 6 and Figure 7). Inventors also performed experiments of capillary- like formation in 3D (spheroid experiments) with the three recombinant proteins. Results show that the three recombinant proteins increased the ability to generate pseudo-capillaries, as measured by the number of sprouts, branch points and cumulative sprout length. Pro-angiogenic properties of the different forms of sCD146 led them to investigate their angiogenic effect in a mouse model of hind limb ischemia. Ischemic mice were locally treated twice a week with 2μg of rh-sCD146, rh-I10-sCD146 and rh-I5-13-sCD146 for 28 days and blood flow was estimated by laser-Doppler. Results show that the three forms were able to increase blood flow as compared to animals treated with PBS (figure 3 A and B). Labelling of muscle with isolectin B4 showed that vascularisation was increased in animals treated with rh- I10-sCD146, rh-I5-13-sCD146 and rh-sCD146, as compared to control animals treated with PBS (figure 3C). Finally, angiography pictures showed also an increase in the number of vessels in the legs treated with rh-I10-sCD146, rh-I5-13-sCD146 and rh-sCD146, as compared to legs treated with PBS (figure 3D).

I5-13-sCD146 and I10-sCD146 are differentially expressed and modulated in systemic sclerosis

Inventors analyzed the mRNA levels of the newly identified sCD146 variants using tissue array. Results are given in figure 4A. Whereas many organs did not express I5-13-sCD146 or 110- sCD146, lung, lymph node, or rectum, highly expressed the two isoforms of sCD146. Of interest, peripheral blood lymphocytes expressed high levels of I5-13-sCD146 but did not express I10-sCD146.

Since I5-13-sCD146 and I10-sCD146 are highly expressed in lung and differentially modulated by TNF and WNT5a/3a, inventors decided to focus on the expression of these isoforms in pathological conditions and in particular in systemic sclerosis (SSc) which is an autoimmune disorder characterized by dysregulated angiogenesis among the most serious complications. They thus analyzed the expression of I5-13-sCD146 and I10-sCD146 in the sera of patients with SSc, as compared to control patients (Figure 4B). Results show that I5-13-sCD146 is significantly increased in patients with SSc whereas I10-sCD146 is decreased.

Analyses

Inventors demonstrated that multiple isoforms of sCD146 are found in the soluble compartment and determined their mechanisms of generation. They herein identify for the first time the proteases involved in the shedding of both membrane CD 146 isoforms and report the existence of two supplementary isoforms generated by alternative splicing and encoding for new sCD146 proteins. These isoforms are secreted by endothelial cells and also display angiogenic properties. The fact that different sCD146 isoforms exist with the same physiological function highlights the importance of the molecule, as reported for VEGF, and indicates that they could be differentially regulated in physiology and/or pathology. Concerning the shed form of CD 146, inventors have demonstrated that it can be generated by two different ADAMs as a function of the membrane CD 146 isoform. Indeed, the long form is shed by AD AMI 0 whereas the short form is shed by Tace. This indicates that the two shed forms of CD 146 might probably differ by a couple of amino acids since ADAM 10 and Tace do not cleave the same sequences of amino acids and that these shedding might be differentially regulated. Inventors have recently shown that the short isoform of CD 146 displayed a complete proteolytic processing with the shedding of the extracellular part followed by the shedding of the intracellular part through presenilin-1, generating an intracellular domain (shCD146-ICD). In contrast, the long isoform displayed only an extracellular shedding (Stalin et al., 2016). They showed that the short CD 146 isoform could thus display direct transcriptional effects through shCD146-ICD translocation into the nucleus. In the present study, they show that the ADAM involved in shCD146 shedding is Tace. Of interest Tace and presenilin-1 are often associated to generate sequential shedding of proteins. Thus, notch is also processed through these two molecules, leading to the generation of NICD with transcriptional effects (Gudey et al., 2014). In addition to the shed form of CD 146, inventors' experiments show that endothelial cells are able to secrete at least two other forms of sCD146. These two isoforms are generated by alternative splicing and differ from the shed form at the C- terminal part for I10-sCD146 and at both the N- and C- terminal parts for I5-13-sCD146. These isoforms are also able to stimulate angiogenesis both in vitro and in vivo. Different angiogenic factors have been shown to exist as different isoforms. Thus, the major angiogenic molecule VEGF displays many different isoforms (Guyot et al., 2015). These isoforms display different receptors, VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin that can be expressed in distinct cells and affect angiogenesis or lymphangiogenesis (Jussila et al., 2002). Of interest, different isoforms can also bind the same receptor and induce different signal transduction and trafficking that will elicit diverse cellular outcome (Fearnley et al., 2016). Recently, both pro-angiogenic and anti-angiogenic isoforms of VEGF have been described. This is of major interest since in tumors both types of molecules are expressed. However, they are both inhibited by anti-VEGF molecules as bevacizumab, reducing the effect of the therapy (Biselli-Chicote et al., 2012). In the context of the present invention, only pro-angiogenic molecules have been found. Inventors have shown that the shed form of sCD146 binds the p80 isoform of angiomotin to generate its effects (Stalin et al., 2013). For VEGFs, at least four different receptors have been identified, VEGFR1-2-3 and neuropilin- 1. These receptors are involved in angiogenesis but also in lymphangiogenesis. Finally, inventors believe that these isoforms are involved in other physiological processes. Along this line, they have shown that the shed form of sCD146 is also involved in inflammation. Indeed, it is able to bind monocytes and to increase their transendothelial migration. These new isoforms of sCD146 thus also display roles in inflammation, or in immunity, as described for the VEGF 164 (Ishida et al., 2003).

Of interest, inventors have shown that the regulation of the two new splice variants of sCD146 is different since I5-13-sCD146 is up-regulated at the mRNA level by WNT5a and TNF whereas I10-sCD146 is not. In addition, I5-13-sCD146 is expressed in lymphocytes whereas 110 is not. This is in favor of different roles and regulatory pathways for each isoform. The effect of WNT5a on I5-13-sCD146 and its high expression in lung and lymphocytes prompted inventors to analyze its expression in the sera of patients with systemic sclerosis (SSc). Indeed, dysregulated angiogenesis plays a major role in the development of this pathology and abnormalities in many factors regulating angiogenesis have been reported (Hummers et al., 2009). Thus, VEGF, PDGF, FGF-2 and P1GF are increased in patients with SSc whereas the pro-angiogenic HGF is decreased. In addition, it was reported that Thl7 lymphocytes express CD 146 and that their number was increased in human autoimmune diseases (Dagur et al., 2015). In the present invention, inventors showed that I5-13-sCD146 splice variant is increased by WNT5a and TNF, and that the protein is significantly enhanced in SSc patients whereas 110- sCD146 is decreased by WNT3A and VEGF, and is decreased in SSc. The observed regulation of the two isoforms in the sera of patients is in agreement with the described increase of TNF, Wnt3a and VEGF in SSc patients. This result is important since it confirms the involvement of CD146/sCD146 in SSc and shows that different pro-angiogenic forms of sCD146 are involved in the development of the pathology.

Inventors have also shown that lymphocytes express a high level of mRNA encoding for the 15- 13-sCD146 isoform.

Taken together, inventors' results identified the two proteinases involved in the shedding of the two membrane isoforms of CD 146 and identified two new pro-angiogenic isoforms of sCD146 generated by alternative splicing (Figure 7). One of these isoforms, I5-13-sCD146, appears to be specifically increased in systemic sclerosis.

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Claims

A human soluble CD 146 protein consisting in a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 1.

A composition comprising at least one protein as described in claim 1 and a pharmaceutically acceptable carrier.

The composition according to claim 2, further comprising an angiogenic factor selected from the group consisting of vascular endothelial growth factor (VEGF), stromal-cell- derived- factor- 1 (SDF-1), basic fibroblast growth factors (bFGF), erytropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-8 (IL-8) and a mixture thereof.

The composition according to claim 3, further comprising an endothelial progenitor cell (EPC), preferably a cell which has been contacted with at least one protein as described in claim 1.

The composition according to claim 4, wherein the endothelial progenitor cell is a recombinant cell comprising a nucleic acid construct expressing at least one protein as described in claim 1 and optionally the human short CD 146 protein of SEQ ID NO: 5.

The composition according to claim 5, wherein the nucleic acid construct is a vector, for example a plasmid.

An antibody which selectively binds the protein of claim 1.

A composition comprising the antibody of claim 7 and a pharmaceutically acceptable carrier.

A protein as described in claim 1 or a composition as described in anyone of claims 2 to 6, for use in the prevention or treatment of a disease or disorder leading to, resulting from or associated to tissue ischemia in a mammal.

10. Use of a protein as described in claim 1 to prepare, ex vivo, a mature or immature endothelial cell capable of stimulating angiogenesis in a human body.

11. A protein as described in claim 1 for use as a biomarker in the diagnostic of a disease or disorder characterized by dysregulated angiogenesis, undesirable excessive

(neo)vascularization, undesirable vascular permeability and/or excessive activation of a receptor for CD 146 in a mammal.

12. An antibody as described in claim 7 or a composition as described in claim 8 for use in the treatment of a disease or disorder characterized by dysregulated angiogenesis, undesirable excessive (neo)vascularization, undesirable vascular permeability and/or excessive activation of a receptor for CD 146 in a mammal.

13. An antibody as described in claim 7 for use in the diagnostic of a disease or disorder characterized by dysregulated angiogenesis, undesirable excessive (neo)vascularization, undesirable vascular permeability and/or excessive activation of a receptor for CD 146 in a mammal.

14. A protein for use according to claim 11, an antibody or a composition for use according to claim 12 or an antibody for use according to claim 13, wherein the disease is a cancer, an ischemic disease, a disease or disorder leading to, resulting from or associated to tissue ischemia, or age-related macular degeneration (AMD).

15. A nucleic acid molecule encoding a protein as described in claim 1