FR2767325A1 - EPIL polypeptides encoded by insulin-like gene 4 - Google Patents

Abstract

The following EPIL early placental insulin-like polypeptides encoded by the INSL4 gene insulin-like gene 4 are claimed: (1) EPIL 1, which is expressed in human or animal cells, especially in human placental trophoblasts, and consists of a single chain comprising regions A, B and C with an overall sequence selected from the amino acid sequences between positions 18 and 139, of the 139 bp sequence given in the specification also see WO 9534653, where the overall sequence comprises at least amino acids 24-139, and where EPIL 1 may also comprise a signal peptide; (2) EPIL 2, which is expressed in human or animal cells, especially in human placental trophoblasts, and consists of: (a) an A chain whose amino acid sequence consists of amino acids 115-139 of the 139 amino acid sequence; (b) a B chain whose amino acid sequence is a sequence between positions 18 and 58, inclusive, of the 139 aa sequence, where this sequence comprises at least amino acids 24-53, and where EPIL 2 may also comprise a signal peptide whose amino acid sequence is a sequence between positions 1 and 23, inclusive, of the 139 aa sequence, where the signal peptide comprises at least amino acids 1-17; (3) EPIL 3, which is expressed in human or animal cells, especially in human placental trophoblasts, and consists of a C chain whose amino acid sequence is a sequence between positions 54 and 114, inclusive, of the 139 aa sequence Also claimed are: (4) a biologically active fragment of a polypeptide as above; (5) a nucleic acid sequence encoding a polypeptide or fragment as above; (6) a cloning and/or expression vector containing the nucleic acid sequence of (5); (7) a host cell transformed with the vector of (6); (8) monoclonal and polyclonal antibodies, antibody fragments and chimeric antibodies that specifically recognise a polypeptide or fragment as above; (9) an oligonucleotide probe or primer capable of hybridising to the nucleic acid sequence of (5); (10) a method for cell labelling using one or more antibodies as in (8); (11) a method for obtaining a cell containing a polypeptide or nucleic acid sequence as above, comprising using the method of (10) and isolating the labelled cell; (12) a cell that can be selected by the method of (11); (13) a method for selecting a chemical or biochemical compound capable of modulating the activity of a polypeptide as above, using a polypeptide or fragment as above, a nucleic acid sequence as in (5) or a cell as in (7); (14) a chemical or biochemical compound that can be selected by the method of (13).

Description

IDENTIFICATION AND LOCATION OF EPIL POLYPEPTIDES
EXPRESSES, CODES BY THE INSL4 GENE AND THEIR APPLICATIONS
The present invention relates to the identification and tissue localization of new polypeptides expressed by the insulin-like 4 gene (INSL4), their amino acid and nucleic acid sequence, methods of detection and diagnosis, methods of labeling. , selection and detection of cells expressing said polypeptides or their corresponding nucleic sequence and methods of selecting compounds capable of modulating the activity of said polypeptides. The invention also relates to medicaments for the treatment of tumors, the vascularization of specific tissues and / or the regeneration of tissues.

 Insulin, IGF-1, IGF-2 (Insulin-like growth factors 1 and 2) and relaxin belong to a family of peptide hormones with certain structures and common functions, including their influence on proliferation, development, differentiation and cell metabolism.

 Insulin is well known as a pancreatic endocrine hormone that regulates energy metabolism.

Insulin-IGF-1 growth factors are growth promoter peptides involved in the endocrine, paracrine and autocrine regulation of cell growth and which are expressed in many tissues. IGF-2 has similar properties but is expressed in greater amounts during the prenatal period and is considered to be a factor in fetal growth.

 Relaxin induces a remodeling of the connective tissues in the reproductive tract and inhibits uterine contractions. The relaxin gene is expressed in the corpus luteum, the decidua, the trophoblasts and the prostate (Bogic, L.V. et al., 1995). Its functional role in the brain, where a very important expression has been observed, remains to be elucidated.

We have also added to this family the Ley ILs which are currently cloned in the form of cDNA and whose biological activity is still to be defined. Transcripts
Ley IL are present in Leydig cells such as in the corpus luteum, trophoplasts, fetal membranes and breast tissue (Adham, IM et al., 1993; Tashima, LS et al., 1995). This peptide family has in common structural characteristics defined by the position of different cysteines essential for the formation of a tertiary structure.

 It has been shown that all members of this family bind to cell surface receptors. These receptors were identified by molecular cloning and characterized in detail for insulin and IGFs. They belong to the super-family of tyrosinekinase receptors (RTK's) which includes growth factor receptors and their oncogenic analogs such as cerbB2 / neu (EGF receptor), c-met (hepatocyte growth factor receptor), fms ( CSF-1 receptor) and trk (NGF receptor).

The intracellular signal transduction pathway for these receptors is characterized by tyrosinekinase activity which produces autophosphorylation of tyrosine residues on the receptor followed by a chain of events corresponding to phosphorylations. This notably includes the activation of IRS-1 (in particular for insulin and IGFs), P13K, Shc, GBR2, Sos, Ras,
Raf and the mitogen-activating protein (MAP), a kinase culminating when the phosphorylation cascade affects various cellular processes such as transcription.

 The pleiotropic physiological effects of this cascade of signals in general are the subject of intensive research.

 The member polypeptides of this family are all characterized by the presence of a signal peptide, a B chain, a C junction peptide or C chain and an A chain.

 For these polypeptide hormones, the terms preprohormone, prohormone or mature hormone are defined on the basis of the presence or absence of certain elements and their arrangement in a tertiary structure. Pre-prohormone is distinguished from prohormone by the presence of the signal peptide element. The distinction between prohormone and active mature hormone is more complex and depends on the hormone considered.

 For example, in proinsulin and prorelaxin, the B and A chains are located respectively at the N- and C-terminal ends and are separated by a long C junction peptide which is cleaved during the maturation phase which results in the form active hormone.

 Unlike insulin and relaxin, the C peptide of IGFs is not cleaved during the maturation phase. The mature forms of the IGFs peptides contain, in addition to the domains A, B and C, two terminal carboxyl peptides (domains D and E) which are cleaved after expression.

 Recently, a new gene, called INSL4, from the insulin-like family (related insulins) has been characterized (WO 95 34653, Chassin, D. et al., 1995). This gene was cloned from an early placenta cDNA library and located on chromosome 9p24.

 The INSL4 gene codes for a polypeptide of 139 amino acids called EPIL (insulin-like peptide of early placenta).

 For greater clarity in the present description, the expression EPIL polypeptide means the putative polypeptide of 139 amino acids deduced from the coding nucleic acid sequence and as defined in FIG. 1.

The deduced amino acid sequence has shown, in agreement with the general organization of this family of hormones, certain structural homologies such as the presence of a signal peptide, a B and A chain and a C peptide of junction or C chain, as well as the presence of 6 cysteine residues, which must probably be involved in the formation of 3 disulfide bridges
These documents describe the organization of the INSL4 gene which is composed of two exons and an intron. These documents also show that the mRNA transcripts of the INSL4 gene are present in particular in the placental and uterine tissue.

 Analysis by analogy of the peptide and nucleotide sequence of EPIL, compared with the other sequences of family members, as well as the study of the distribution of transcripts suggests to the authors of these documents that the EPIL polypeptide has a structure closer to insulin, relaxin and LEY-IL than to IGF 1 and 2 (Chassin, D. et al., 1995). The homology analysis of EPIL compared with the other members of the family does not make it possible to deduce the essential characteristics such as for example the identification and the localization of the expressed form of the EPIL polypeptide coded by the INSL4 gene.

 Knowledge of these essential characteristics would indeed make it possible to develop, for example, synthetic peptides corresponding to predetermined forms (pre-prohormone, prohormone or mature hormone) expressed or to particular domains of these predetermined forms (chains A, B or peptide VS) . These synthetic peptides can be used as agonists or antagonists of the corresponding activity. They can also be used as an antigenic peptide and allow the obtaining of specific antibodies directed against particular forms or domains of this hormone. The tissue localization of the form or forms expressed by the INSL4 gene would make it possible to pre-establish the type or types of biological activity in which this new hormone is involved. This is precisely the object of the present invention.

In view of the above, in particular the structural analogy between insulin, relaxin and the EPIL polypeptide, thus suggesting that the expressed and mature form of the EPIL polypeptide is unique and in a form devoid of C chain and taking into account the usual practices of identification and localization of the expressed form of a hormone from the knowledge of its coding sequence, the inventors nevertheless used anti-C chain antibodies, from peptide constructs mimicking the domain C of the putative EPIL polypeptide, in addition to anti-chain antibodies
A and B in their search for identification and localization of the forms expressed.

The inventors were thus able to demonstrate in a surprising manner two expressed forms of the polypeptide
EPIL. A form with the C chain and a form without the C chain.

 The inventors have also, and just as surprisingly, demonstrated that these two expressed forms were localized at different tissue levels.

The present invention therefore relates to a polypeptide
EPIL 1 coded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in human placenta trophoblasts, and in that it consists of a single chain comprising the A regions,
B and C of global amino acid sequence chosen from the amino acid sequences between positions 18 and 139, ends included, of the amino acid sequence defined in FIG. 1, said global sequence comprising at least the sequence amino acids between positions 24 and 139, ends included, said EPIL 1 polypeptide possibly further comprising a signal peptide such that the amino acid sequence of the EPIL 1 polypeptide comprising the signal peptide is the sequence between positions 1 and 139, ends included, of the amino acid sequence defined in FIG. 1.

 In the present description, the term polypeptide also denotes a protein or a peptide.

 It should be understood that the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their natural environment but that they could have been. obtained by purification from natural sources, or else obtained by genetic recombination, or even by chemical synthesis and which can then contain non-natural or modified amino acids.

 The invention also relates to an EPIL 1 polypeptide according to the invention, characterized in that it is preferentially expressed in cytotrophoblasts of human placenta.

 The invention further relates to an EPIL 1 polypeptide according to the invention, characterized in that it is present in endothelial cells, preferably vascular endothelial cells, in particular fetal vascular endothelial cells.

 Preferably, the EPIL 1 polypeptide according to the invention, is characterized in that it is secreted in the amniotic fluid and / or in the fetal circulation.

The invention also relates to an EPIL 2 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in human placenta trophoblasts and in that it consists
a) of a chain A whose amino acid sequence is the sequence between positions 115 and 139, inclusive ends of the amino acid sequence defined in FIG. 1
b) a chain B whose amino acid sequence is chosen from a sequence between positions 18 and 58, ends included, of the amino acid sequence defined in FIG. 1, said sequence comprising at least the sequence between positions 24 and 53 ends inclusive
said EPIL 2 polypeptide possibly further comprising an amino acid sequence signal peptide chosen from a sequence between positions 1 and 23, ends included, of the amino acid sequence defined in FIG. 1, said signal peptide sequence comprising at least the sequence between positions 1 and 17, ends included.

 The invention further comprises an EPIL 2 polypeptide according to the invention, characterized in that it is preferentially expressed in syncytiotrophoblasts of human placenta.

 The invention also includes an EPIL 2 polypeptide according to the invention, characterized in that it is secreted in the maternal circulation.

 The invention also relates to an EPIL 3 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in human placenta trophoblasts and in that it consists of a chain C, the amino acid sequence of which is chosen from a sequence between positions 54 and 114, ends included, of the amino acid sequence defined in FIG. 1, said sequence comprising at least the sequence between positions 59 and 114, ends included.

 The invention relates to polypeptides according to the invention, characterized in that they are involved in the process of differentiation, proliferation and / or regeneration of human and / or animal cells, in particular endothelial cells.

 The polypeptides according to the invention are also characterized in that they are involved in the process of vascularization of embryonic cells and / or in the process of vascularization of tumors.

 The polypeptide fragments according to the invention, characterized in that they are biologically active, also form part of the invention.

 The term “biologically active fragment” is intended to denote in particular a fragment of the amino acid sequence of a polypeptide according to the invention having at least one of the activities of a polypeptide according to the invention in particular - the ability to modulate the differentiation, the regeneration and / or the proliferation of cells, such as in particular endothelial cells, in particular vascular cells - the ability to be recognized by an antibody specific for a polypeptide according to the invention; and / or - the ability to modulate the activity of a polypeptide according to the invention, by neutralizing for example the binding of a polypeptide according to the invention to its specific cellular receptor.

 The term “polypeptide fragment” is intended to denote a polypeptide of amino acid sequence comprising at least 5 amino acids, preferably 10 amino acids and 15 amino acids.

 The invention also includes the homologous polypeptides and variants of the polypeptides according to the invention.

 The term “homologous polypeptides” will be understood to mean the polypeptides having, with respect to the polypeptides according to the invention, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion, and / or a mutation, in particular point. Among the homologous polypeptides, those whose amino acid sequence has at least 80k, preferably 90k, of similarity with the amino acid sequences of the polypeptides according to the invention are preferred.

 The term “polypeptide varying” means all of the mutated polypeptides which may exist, in particular in humans, and which correspond in particular to truncations, substitutions, deletions and / or additions of at least one amino acid residue.

 The invention further comprises the nucleic acid sequences characterized in that they code for a polypeptide or one of its fragments according to the invention.

 The invention also includes the cloning and / or expression vectors containing a nucleic acid sequence according to the invention as well as the host cells transformed by said vectors.

 The vectors according to the invention, characterized in that it comprises the elements allowing the expression and / or the secretion of said sequences in said host cells, also form part of the invention.

 The invention also relates to a method for producing a recombinant polypeptide characterized in that it implements a cell according to the invention. The method for obtaining a polypeptide according to the invention in recombinant form is preferably characterized in that the transformed cells are cultivated under conditions allowing the expression of a recombinant polypeptide, and that said recovery is obtained. recombinant polypeptide.

 The recombinant polypeptides capable of being obtained by said methods also form part of the invention.

 The invention also includes the polypeptides or one of their fragments according to the invention, characterized in that they are obtained by chemical synthesis.

 The polypeptides corresponding to the polypeptides according to the invention obtained by chemical synthesis and which may contain unnatural amino acids, are also included in the invention.

 The recombinant polypeptides obtained as indicated above can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure.

 These polypeptides can be produced from the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.

 An efficient system for producing a recombinant polypeptide requires a vector, for example of plasmid or viral origin, and a compatible host cell.

 The cellular host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells such as CHO cells (Chinese hamster ovary cells) or murine cells or any other system advantageously available. A preferred cellular host for the expression of the proteins of the invention consists of CHO Chinese hamster ovary cells.

 The vector. must include a promoter, translation initiation and termination signals, and appropriate regions for transcription regulation.

It must be able to be maintained stably in the cell and may possibly have particular signals specifying the secretion of the translated protein.

 These different control signals are chosen according to the cellular host used. For this purpose, the nucleic acid sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or integrative vectors of the chosen host.

 Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock.

 The invention further relates to the host cells transformed by the preceding vectors. These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.

 These cells can be used in a method for producing a recombinant polypeptide according to the invention.

 Among the cells which can be used for these purposes, it is of course possible to mention bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cell cultures. mammals (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary cells (CHO), but also insect cells in which methods using baculoviruses can be used for example (Luckow, 1993).

 The methods of purification of recombinant or synthetic polypeptide used are known to those skilled in the art.

The recombinant or synthetic polypeptide can be purified using methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific mono or polyclonal antibodies, etc.

 A preferred variant consists in producing a recombinant polypeptide fused to a carrier protein (conjugated protein). The advantage of this system is that it allows stabilization and a decrease in the proteolysis of the recombinant product, an increase in the solubility during the in vitro renaturation and / or a simplification of the purification when the fusion partner has a affinity for a specific ligand.

 Mono or polyclonal antibodies or their fragments, chimeric antibodies, characterized in that they are capable of specifically recognizing a polypeptide according to the invention, form part of the invention.

 Specific polyclonal antibodies can be obtained from a serum of an animal immunized against, for example - a polypeptide according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures, from a nucleic acid sequence according to the invention.

 Antibodies. Specific monoclonals can be obtained according to the conventional method of culture of hybridones described by Kôhler and Milstein, 1975.

 The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments. According to the invention, they can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.

Furthermore, the invention relates to the use of one or more antibodies for the detection, identification, localization, in particular tissue or cell, and / or the assay of a polypeptide according to the invention, or for the diagnosis of pathologies linked to the abnormal presence of polypeptide according to the invention
Said use is included in the invention.

 It is understood that the expression “several antibodies” in the present description means at least two antibodies according to the invention of different specificity, that is to say capable of recognizing and binding to two different epitopes or domains of polypeptide according to the invention. It is moreover preferred according to the invention to use at least two antibodies of the invention of different specificity for the methods, the methods and the kits of the invention which include said antibodies. In this case, it is possible, for example, to use two different antibody labels or two different development systems.

 They thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of the polypeptide according to the invention on sections of specific tissues, for example by immunofluorescence, gold labeling, enzyme immunoconjugates.

 They make it possible in particular to demonstrate and quantify the normal or abnormal specific presence of a polypeptide according to the invention in tissues or biological samples, which makes them useful for the identification and localization of the expression of polypeptide according to the invention, or for the diagnosis of pathologies linked to the abnormal presence of the polypeptide according to the invention.

 More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a polypeptide according to the invention must be observed qualitatively and / or quantitatively.

 The invention also relates to oligonucleotide probes or primers, characterized in that they consist of a nucleic acid sequence according to the invention or one of its fragments capable of hybridizing specifically to said sequence, preferably the probes can be marked.

 The invention further relates to the use of probe or primer, for the detection, identification, localization, in particular tissue or cell, amplification, and / or assay of nucleic acid sequence according to the invention. invention as well as for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to the invention.

 The oligonucleotide probes or primers will have a minimum size of 10 bases and fragments of 20 bases, and preferably 30 bases, will be preferred.

 Among the nucleic acid fragments of interest according to the invention, mention may be made in particular of antisense oligonucleotides, that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the polypeptides according to the invention, will induce either an inhibition or an activation of this expression. These sense or antisense oligonucleotides also form part of the invention.

 The invention also relates to a method for the detection, identification, localization and / or specific assay of a polypeptide or one of its fragments according to the invention in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of polypeptide according to the invention, characterized in that it comprises the following stages a) bringing the biological sample into contact with one or more antibodies according to the invention; b) highlighting, identification, location and / or determination of the antigen-antibody complex formed.

 The invention also relates to a kit or kit for the detection, identification, localization and / or specific assay of a polypeptide or one of its fragments according to the invention in a biological sample, or for the diagnosis of related pathologies the abnormal presence of polypeptide according to the invention characterized in that it comprises the following elements a) one or more antibodies according to the invention; b) reagents for constituting the medium suitable for the immunological reaction c) reagents for the detection, identification and / or determination of the antigen-antibody complexes produced by the immunological reaction.

 The specific techniques and reagents for detecting and / or assaying the antigen-antibody complexes which can be used in the methods of the invention are well known to those skilled in the art and are, for example, ELISA techniques, RIA or immunofluorescence which can be combined, in the case where the antibodies of the invention are not already immunoconjugated or labeled, with the appropriate reagents such as immunoconjugated, labeled with fluoroscéine or radiolabelled antibodies capable of specifically recognizing the antibodies of the invention, as well as the chromogenic substrates specific for the conjugated enzymes and the control reagents of positive, negative and quantitative control.

 The invention further relates to a method for the detection, identification and / or specific assay of nucleic acid sequence according to the invention in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of sequence of nucleic acid according to the invention, characterized in that it comprises the following stages: a) isolation of the DNA from the biological sample to be analyzed, or production of a cDNA from the RNA of the biological sample b) specific amplification of the nucleic acid sequences according to the invention using primers according to the invention; c) qualitative and / or quantitative analysis of the amplification products.

 The invention also relates to a method for the detection, identification and / or specific assay of nucleic acid sequence according to the invention in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of acid sequence nucleic acid according to the invention, characterized in that it comprises the following steps a) bringing an oligonucleotide probe according to the invention into contact with a biological sample, the DNA contained in the biological sample having, where appropriate, previously made accessible to hybridization, under conditions allowing hybridization of the probe to the DNA contained in the biological sample; b) detection, identification and / or assay of the hybrid formed between the oligonucleotide probe and the DNA of the biological sample.

 The invention also includes a method for the detection, identification and / or specific assay of nucleic acid sequence according to the invention in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of acid sequence nucleic acid according to the invention, characterized in that it comprises the following steps a) contacting an oligonucleotide probe according to the invention immobilized on a support with a biological sample, the DNA of the sample, having the if necessary, been previously amplified using primers according to the invention and / or made accessible to hybridization, under conditions allowing hybridization of the probe to the DNA of said biological sample; b) bringing the hybrid formed into contact with said oligonucleotide probe immobilized on a support and the DNA contained in the biological sample, where appropriate after elimination of the DNA from the biological sample which has not hybridized with the probe, with an oligonucleotide probe, in particular labeled, according to the invention.

 The invention also relates to a kit or kit for the detection, identification and / or specific assay of nucleic acid sequence according to the invention. C) If necessary, a pair of primers according to the invention as well as the reagents necessary for a DNA amplification reaction.

 The invention further comprises a kit or kit for the detection, identification and / or specific determination of nucleic acid sequence in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of acid sequence nucleic acid according to the invention, characterized in that it comprises the following elements a) an oligonucleotide probe, called capture probe, according to the invention which can be supplied pre-immobilized on a support; b) an oligonucleotide probe, known as a revelation probe, in particular labeled, according to the invention; c) Where appropriate, a pair of primers according to the invention as well as the reagents necessary for a DNA amplification reaction.

 The techniques of specific amplification and of qualitative and / or quantitative analysis of nucleic sequence which can be used in the methods of the invention are well known to those skilled in the art and are, for example, those which will be described below.

 The invention therefore also relates to cell labeling methods, characterized in that they use one or more antibodies according to the invention or a probe, in particular labeled, and / or a primer according to the invention.

 The marking methods according to the invention are preferred, characterized in that they implement a method according to the invention.

 The techniques for labeling cells with antibodies specific for the compound of said cell, such as that described in Example 4, are well known to immumocytochemists or immunohistochemists and will not be developed in the present description.

 Among the methods for labeling a cell according to the invention using a probe of the invention, preference is also given to methods comprising the use of a labeled probe according to the invention and / or methods comprising at least one step d amplification called by PCR (polymerase chain reaction) or by PCR-like of the target sequence according to the invention capable of being present with the help of pairs of primers of nucleic sequence according to the invention. The amplified products may be treated with the aid of an appropriate restriction enzyme before detecting or assaying the targeted product, in particular by a method according to the invention.

By PCR-like is meant to denote all the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified. These techniques are of course known. In general it is the amplification of DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently many methods for this amplification, for example the so-called NASBA Nucleic Acid Sequence Based Amplification (Compton 1991), TAS Transcription based Amplification System (Guatelli et al. 1990), LCR Ligase Chain Reaction (Landegren et al. 1988 ), Endo Run Amplification (ERA), Cycling Probe Reaction (CPR), and SDA Strand
Displacement Amplification (Walker et al. 1992), well known to those skilled in the art.

 The invention relates in particular to cell labeling methods, characterized in that the cell is an endothelial cell, in particular a vascular cell.

 The subject of the invention is also a method for obtaining a cell containing a polypeptide according to the invention and / or a nucleic acid sequence according to the invention, characterized in that it implements a labeling method according to the invention, and in that said labeled cell is isolated.

 The invention also relates to a cell in that it is capable of being selected by a method of production according to the invention.

 The invention also relates to a method for detecting tumor cells, characterized in that it implements a labeling method according to the invention.

 Another aspect of the invention relates to methods of selecting a chemical or biochemical compound capable of modulating the activity of a polypeptide according to the invention, characterized in that they use a polypeptide or one of its fragments according to l invention, or a cell according to the invention. Said cell can be a transformed or selected cell according to the invention.

 Said compounds may, for example, be selected on their capacity to bind to one of the polypeptides according to the invention and may thus inhibit or promote the activity of said polypeptides as, for example, agonist or antagonist ligands of a receptor specific for said polypeptides .

 Said compounds can also be selected on their capacity to modulate on said cells in vitro or in vivo, the biological activity of said polypeptides expressed by said cells, such as in particular the differentiation, proliferation or regeneration of cells.

 The invention further relates to selection methods according to the invention, characterized in that said selected compound is capable of modulating the differentiation, proliferation and / or regeneration of human and / or animal cells, in particular of promoting or to inhibit the differentiation, regeneration and / or proliferation of said cells, in particular endothelial cells, preferably vascular endothelial cells.

 The invention also includes chemical or biochemical compounds, which can be selected by a method according to the invention.

The invention further relates to compounds according to the invention as a medicament, preferably chosen from
a) a polypeptide or a fragment thereof according to the invention;
b) an antibody according to the invention;
c) a chemical or biochemical compound, characterized in that it is a ligand of a polypeptide or one of its fragments according to the invention;
d) a vector according to the invention;
e) a vector according to the invention having on its surface a specific marker of the target cell which one seeks to modulate the activity of a polypeptide according to the invention.

The invention relates to compounds according to the invention, intended for the treatment of tumors1 in particular intended for the treatment of tumors of the pancreas, liver, uterus, breast, angiosarcoma, glioblastoma, neuroblastoma, rhabdomyosarcoma or a leiomyosarcoma
We could, for example, refer to the publications of
Clark, R. (1997); Ferrara, N., et al. (1997), or from the journal Scrip NO 2209, pp21, and Scrip NO 2202 concerning the article on the company Genentech, for the biological activities in which IGF1 and EGF (endothelial growth factor) are involved, related hormones to the EPIL polypeptide.

 The invention also includes compounds according to the invention, intended to promote the vascularization of specific tissues, and for the treatment of retinopathy, degeneration of the macula, psoriasis, endometriosis, rheumatoid arthritis, atherosclerosis or hyperthyroidism.

 Finally, the invention comprises compounds according to the invention, intended for regenerating and / or differentiating a tissue, in particular endothelial, following a degenerative disease or a disease of the endocrine glands, in particular the thyroid or the pancreas, or following damage to said tissue caused by trauma.

 The compounds of the invention as active principles of medicament, will preferably be in soluble form, associated with a pharmaceutically acceptable vehicle.

 Such compounds which can be used as medicaments offer a new approach for the treatment of diseases linked to the unregulated differentiation or proliferation of specific cells or tissues, or to the absence or insufficiency of certain cells or tissues, continued for example to trauma.

 Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal route or by the oral route.

 Their optimal methods of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and observed side effects, etc.

 The deficiency or overexpression of polypeptides according to the invention can also be generally remedied by a so-called replacement therapy allowing the amplification or the reduction of the activities of said polypeptides.

 The replacement therapy may be carried out by gene therapy, that is to say by introducing the nucleic acid sequences according to the invention and / or the corresponding genes with the elements which allow their expression in vivo, in the case where one of the genes is insufficiently expressed, for example, or when it is expressed in an abnormal form.

The principles of gene therapy are known. Viral vectors can be used, for example on the basis of adenoviruses (Perricaudet et al., 1992), AAVs,
Adenovirus Associated Virus (Carter, 1993), retroviruses (Temin, 1986), poxviruses or herpes viruses (Epstein et al., 1992). Most of the time, these viruses are used in defective form and, in general, with or without integration into the cell genome. It is also possible to provide non-viral vectors, that is to say synthetic vectors, which mimic viral sequences or which are constituted by naked DNA or RNA according to the technique developed in particular by the company VICAL. .

 In most cases, targeting elements ensuring specific expression will have to be provided so as to be able to limit the areas of expression of the polypeptides. It is even advantageous, in certain cases, to have vectors of transient expression or at least of controlled expression which can be blocked when necessary.

 Other characteristics and advantages of the invention appear in the following description with the examples and the figures, the legends of which are shown below.

Legend of Cracks
Figure 1: Nucleic acid sequence of the gene's cDNA
INSL4 and amino acid sequence of the EPIL polypeptide encoded by this sequence.

Figure 2
A. Schematic representation of the structure of the EPIL polypeptide and localization of the binding sites of the domains of the EPIL polypeptide recognized by the antibodies 1661 and 7381.

 B. Primary structures of the amino-terminal part of the C chain of EPIL AND RLX-H2 (relaxin H2). Identical damino acid residues are denoted by *.

Figure 3: Competitive inhibition curves, obtained by ELISA method, of the binding of antibodies 7381 and 1661, by different competing peptides
A. Inhibition of the binding of the antibody 7381 (diluted to 1/500) directed against the peptide EPIL 59-108-Y adsorbed on a microplate, by the competing peptides EPIL 59-108
Y (+), EPIL 88-108-Y (0), RLX-H2 63-92 (A) and RLX-H2 74-92 (O).

 B. Inhibition of the binding of the antibody 1661 (diluted to 1/300) directed against the peptides EPIL 23-52 and EPIL 115-139 adsorbed on microplate, by the competing peptides EPIL 23-52 (+), EPIL 115- 139 (0), and by a mixture of peptides EPIL 23-52 and EPIL 115-139 (O).

 Figure 4: Competitive inhibition curve for the binding of polyclonal antibody 7383.

The results were obtained by ELISA method on a microplate support with the peptide EPIL 23-52 adsorbed
The percentage inhibition of the binding of the antibody 7383 diluted to 1/500 is calculated as a function of the increasing quantity of inhibitor peptide chain B (23-52), having served as an immunogen, the molar concentration of which is expressed in notation scientific: 1.0 E-10 equivalent to 1.0 x 1010.

 Figure 5: Competitive inhibition curve for the binding of the EPIL 02 monoclonal antibody.

FIG. 5 represents the competitive inhibition analysis of the EPIL 02 antibody for two different dilutions: dilution to 1/2500 (g) and dilution to 1/5000 (+). The results were obtained by ELISA method on microplate support on which the peptide is adsorbed
EPIL (125-137-Y).

 Figure 6: Diagram representing the construction characteristics of the INSL4-pSec TAG CM vector containing the INSL4 insert of 418 bp.

 Figure 7: Immunohistochemical detection of the EPIL 1 and EPIL 2 polypeptides.

Slices of villous chorionic tissue from placenta less than 3 months old (6 to 8 weeks) (photographs A, B,
E and F) or term placenta (photographs C and D) have been marked
- with pre-absorbed anti-EPIL 1 antibodies (photographs A and C) and anti-EPIL 1 antibodies 7381, both diluted 1/500 (photographs B and D);
- with pre-absorbed anti-EPIL 2 antibodies (photograph E) and anti-EPIL 2 antibodies 1161 (photograph F), both diluted 1/300.

Cytotrophoblasts of placenta less than 3 months old (photograph B) and of placenta at term (photograph
D) show a predominant labeling with anti-EPIL 1 antibodies while anti-EPIL 2 antibodies mark both cytotrophoblasts and syncytiotrophoblasts of placenta less than 3 months old (photograph F).

 Magnification: photographs A and B: x 400, photographs C to F: x 1,000.

 Figure 8: Quantitative analysis of the levels of mRNA transcripts of the INSL4 gene in cytotrophoblasts (CT) compared to syncytiotrophoblasts (ST).

The quantification is carried out by competitive RT-PCR using a standard quantitative DNA in internal control (IQS). Electrophoresis is performed on the DNA sequencer
ABI by Perking Elmer and we measure the peak areas obtained. The results are expressed as a percentage of peak area obtained with the target cDNA and the corresponding IQS.

 The experiments were carried out twice independently, the bars representing the difference between the two results obtained.

Figure 9: Analysis by immunoblotting (Western blot) - Figure 9A: Revealing antibody EPIL 02
Line 1: Supernatant obtained after solubilization of proteins from infected Sf9 cell cultures.

The bands corresponding to the recombinant EPIL polypeptides are very little marked and hardly visible taking into account the factor of dilution of the polypeptides in the crude culture supernatant.

Line 2: Effluent after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 3: Eluate after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 4: Fraction 7 after reverse phase chromatography.

Line 5: Fraction 8 after reverse phase chromatography.

Line 6: Fraction 9 after reverse phase chromatography.

Line 7: Fraction 16 after reverse phase chromatography.

Figure 9B: 7381 Developer Antibody
Line 1: Molecular mass standard.

Line 2: Supernatant obtained after solubilization of proteins from infected Sf9 cell cultures.

Line 3: Effluent after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 4: Eluate after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 5: Fraction 7 after reverse phase chromatography.

Line 6: Fraction 8 after reverse phase chromatography.

Line 7: Fraction 9 after reverse phase chromatography.

- Figure 9C: Revealing antibody 7383.

Line 1: Supernatant obtained after solubilization of proteins from infected Sf9 cell cultures.

Line 2: Effluent after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 3: Eluate after affinity chromatography (Sepharose-CNBr-EPIL 02).

Line 4: Fraction 7 after reverse phase chromatography.

Line 5: Fraction 8 after reverse phase chromatography.

Line 6: Fraction 9 after reverse phase chromatography.

Line 7: Fraction 16 after reverse phase chromatography.

EXAMPLE 1
Characterization of the form (s) of EPIL polvreptide expressed in tissues
Production of anti-EPIL antibodies, specific to specific domains of the EPIL polypeptide.

 The strategy for detecting the expressed form or forms of the EPIL polypeptide is based on the production and use of monoclonal and polyclonal antibodies directed against synthetic peptides, coupled to carrier proteins, mimicking different regions of putative protein.

1 Synthesis of peptides and preparation of immunogens.

Synthetic peptides are constructed according to the conventional solid phase method on an automaton
Applied Biosystems model 431A. The peptides synthesized are presented during the description of the antibodies (see 3). These peptides are purified by exclusion chromatography followed by HPLC and then they are analyzed (amino acid composition and peptide sequencing).

They are then conjugated to carrier proteins to prepare immunogens.

2 Production and characterization of polyclonal and monoclonal antibodies.

The production of polyclonal antibodies is carried out by injection of immunogens (peptide-carrier protein conjugates) in female Fauve rabbits.
Burgundy aged around 70 days. The immunogen (150 μg) is injected by intradermal multipoint into the back in the presence of complete Freund's adjuvant in physiological saline. Two subsequent boosters are made on D + 21 and D + 42 respectively by injection of 25 Sg of immunogen in incomplete Freund's adjuvant by subcutaneous route. A third reminder can be made on D + 70. One week after the recall, a blood sample is taken from the ear to test for the presence of antibodies.

Good responder rabbits are bled white by intracardiac puncture.

The production of monoclonal antibodies is carried out by the cell hybridization technique. Briefly, mice of BALB / c strain are immunized by the injection of 50-100 μg of peptide-carrier protein conjugate in complete Freund's adjuvant by subcutaneous route. Recalls by intraperitoneal route (25 Ci d of immunogen in incomplete adjuvant of Freund) are carried out with three months of interval until obtaining an immune response, attested by the presence of detectable antibodies in the serum of animal. Cell fusion is carried out 3 days after iv (intravenous) injection of the immunogen. It consists in removing and then dissociating the spleen from the mouse and, in a second step, fusing with polyethylene glycol (PEG 1000) the lymphocytes thus released with myeloma cells, maintained in continuous culture. The hybridomas are selected in a selective medium containing aminopterin. The production of monoclonal antibodies by hybridomas is analyzed by an immunoenzymatic solid phase method (binding to the immunogenic peptide). The producing hybridomas are cloned twice by limiting dilution and the mass production of antibodies is carried out by injection of the hybridomas intraperitoneally into the Nude mouse. The monoclonal antibodies are then purified from the ascites liquids by an affinity chromatography method on
Sepharose-protein A (Pharmacia) after precipitation of ammonium sulfate proteins.

 Antibody binding characteristics are established by solid phase immunoenzymatic (ELISA) and radioimmunological (RIA) methods. Briefly, the ELISA method is carried out by adsorbing one or more peptides mimicking a sequence of a domain of the putative polypeptide EPIL, diluted in a 0.1 M phosphate buffer, pH 7.4, on a solid support (polystyrene plate COSTAR / EIA).

After an incubation of 18 hours at + 40 ° C., the supports are saturated with an albumin solution and the antibodies to be tested are added for an incubation of 1 hour at 37 ° C.

After washing, the binding of the antibodies is detected by the addition of a secondary antibody labeled with peroxidase (RAM / IgG (H + L) Po, TEBU). The chromogen solution (ophenylene diamine) in the presence of H2O2 reveals the enzymatic reaction. A competitive inhibition method can be performed to specify the location of the sites recognized by the antibodies. For this, the antibodies are previously incubated with synthetic peptides at different concentrations, for example representative of shorter sequences than that of the peptide adsorbed on the support. The mixture is then deposited on the support and the residual antibody activity is measured. The results are expressed as a percentage of binding inhibition. The RIA method is developed in a similar way, except that the peptide is radiolabeled by a 125I isotope according to the so-called Iodogen method. The immune complexes potentially formed with the antibody are precipitated by polyethylene glycol (PEG 6000) and, after washing, the radioactivity is measured. It is also possible to carry out a competitive inhibition experiment as described above.

3 Antibody characteristics.

 The numbers associated with the sequences below correspond to the positions of the amino acids defined in the amino acid sequence of FIG. 1.

 Three polyclonal antibodies and one monoclonal antibody directed against synthetic peptides are described below o A polyclonal antibody, numbered 7381, is obtained by immunization of rabbits with an immunogen comprising a synthetic peptide mimicking the sequence 59-108 Tyrosine (Y) of the C chain putative (Figure 2) coupled by benzidine to keyhole limpet hemocyanin (KLH), carrier protein. This antibody binds to the sequence 59-108 of domain C of the EPIL polypeptide (Figure 3A). I1 does not recognize the analogous sequence in relaxin H2 (RLX-H2), a protein which has the greatest structural similarity (67%) in the C domain. This antibody is capable of detecting the recombinant polypeptide comprising the C domain, for example in an immunoblot method, and to highlight the physiological EPIL 1 or EPIL 3 polypeptide, for example at the level of the placenta (see example 4, concerning immunohistochemistry).

 A polyclonal antibody, numbered 1661, is obtained by immunization of rabbits with an immunogen comprising a mixture of synthetic peptides mimicking the sequences 23-52 of the putative B chain and 115-139 of the putative A chain (Figure 2) coupled with benzidine. with tetanus toxoid, a carrier protein. This polyclonal antibody contains antibodies mainly directed against the region 115-139 of chain A and to a lesser extent against the sequence 23-59 of chain B (FIG. 3B). I1 makes it possible to detect a recombinant polypeptide comprising the A and / or B domains, for example in an immunoblotting method, as well as the native EPIL polypeptide and its molecular forms comprising the A and / or B domains, such as for example the polypeptides EPIL 1 and EPIL 2 (see example 4).

 A polyclonal antibody, numbered 7383, and obtained by immunization of rabbits with an immunogen comprising a mixture of synthetic peptides mimicking the sequences (3149) + (31-50) + (31-51) + (31-52) + (31- 53) of the putative B chain coupled by MBS (m-maleimidobenzoyl-Nhydroxysuccimimide) to keyhole limpet hemocyanin (KLH), carrier protein (Figure 4).

 A monoclonal antibody, called EPIL 02 (IgG2a), is selected after immunization of mice with an immunogen comprising a synthetic peptide mimicking the sequence 125137-Y of the putative A chain coupled by benzidine to tetanus toxoid, carrier protein (FIG. 5 ). This antibody can be used for the preparation of an affinity chromatography column making it possible to separate and purify the polypeptide of recombinant origin (see example 2).

EXAMPLE 2
Production of the extra short form in SF9 cell
The following steps are carried out: 1 Construction in the expression vector pFastBac.

 The extra short form INSL4 (coding sequence) is inserted into the lower eukaryotic (baculovirus) expression vector pFastBacTM (GIBCOBRL) at the Kpnl site. Subsequently, coinfection of the SF9 insect cells with wild-type baculovirus DNA (AcMNPV, GIBCOBRL) and the transfer vector containing the gene of interest is carried out, in order to constitute homologous recombination.

2 Sequencing.

In order to verify the presence and the orientation of the INSL4 coding sequence in the recombinant construct, the analysis of the restriction profile of the recombinant DNA is carried out using different endonucleases (Kpnl, EcoRi) and by automatic sequencing (specific primers of DNA
INSL4 and the multiple cloning site of the bacmid pFastBacTM.

3 Analysis of the expression of the EPIL polypeptide.

 After infection of the ovarian insect cells (Spodoptera frugiperda 9) and amplification of the viral titer, the expression of the recombinant EPIL polypeptide, coded by the INSL4 gene, is analyzed by SDS-PAGE followed by immunoblotting.

4 Purification and characterization of the recombinant EPIL polypeptide.

 Solubilization of proteins.

 About 200 x 106 infected Sf9 cells are centrifuged. The centrifugation pellet is taken up in 1 ml of 0.05 M Na2HPO buffer, pH 8.5 containing 0.1% NP 40 and 5 Ag / ml of leupeptin. After stirring by tilting for 4 h at 40C, the mixture is centrifuged for 10 min at 14,000 rpm at + 40C. The supernatant is recovered for the following steps.

 Affinity chromatography.

At first, a column of Sepharose
CNBr ~ EPIL 02 is prepared as follows. Three grams of gel are swelled in 20 ml of HCl for 4 hours. The gel is then introduced into a column (20 x 1.5 cm) and then rinsed with a coupling solution (0.1 M NaHCO3, 0.5 M NaCl, pH 8.5). After elimination thereof, 1.5 to 4 mg of purified monoclonal antibody EPIL 02, diluted in the coupling solution, are added. After 18 hours at + 40C with stirring, the free sites are blocked with 1 M ethanolamine then the column is washed with a 0.1 M CH3COONa solution, 0.1 M CH3COOH, 0.5 M NaCl, pH 4. Finally , the column is rinsed with 0.05 M Na2HPO4, pH 8.5.

 The supernatant recovered after solubilization is diluted 1/10 in 0.05 M Na2HPO4 buffer, pH 8.5. After dilution, the solution is brought into contact for 18 h at + 40C with the Sepharose-CNBr-EPIL 02 support with slow stirring. Washes are carried out with a 0.05 M NazHPO4 buffer, pH 8.5 in the presence of 0.01 NP40. The procedure is automated on FPLC equipment (Pharmacia). The washing time is conditioned by the return of the optical density to the basic level. The fractions collected constitute the effluent. The elution of the antigen linked to the antibody is carried out with a 2.5 acetic acid buffer. The fractions collected constitute the eluate. The effluent and the eluent are analyzed by an immunoblotting technique with EPIL 02, 7381 and 7383 as revealing antibodies.

 High performance liquid chromatography (HPLC) in reverse phase.

Two successive chromatographies are then carried out on the fractions collected after elution with 2.5 t acetic acid. These fractions are combined and then first deposited on a Lichrosorb RPB C8 7 Am column (250 mm x 4 mm) mounted on a Beckman HPLC system.
GOLD. The loading is carried out continuously by an external valve with a flow rate of 2 ml / min and the fractions are collected by automatic collection every 30 seconds. Two mobile phases are used: phase A
0.1% TFA - 99.9 k H2O and p polyacrylamide (30% w / v acrylamide, 0.8% w / v bis acrylamide; Tris-Hcl 1.5 m, pH 8.8) for 30 minutes under 200 volts. After migration, the proteins are transferred to a support of the PVDF filter paper type under the influence of an electric field. The support is then saturated in blocking buffer and then incubated with the antibodies. After washing, the possible binding of the antibodies is revealed either by a secondary antibody labeled with peroxidase or by the luminol method (kit
ECL, Amersham, NW). The relative molecular weights are calculated from reference standards. Under these conditions, the EPIL 02, 7381 and 7383 antibodies reveal several positive fractions after reverse phase chromatography and correspond to different elution times.

These fractions referenced 7, 8, 9 and 16 contain different populations of proteins varying by their relative molecular mass (Figure 9). These relative masses are between 13 kDa and 32 kDa.

6 Analysis by mass spectrometry by ionization at
electro-spray in positive mode.

This study is carried out on a PlatformII analyzer (Micromas Ltd) using horse myoglobin as standard (Molecular mass: 16,951.50 t 0.17 Da). Fraction 7 obtained after reverse and positive phase chromatography after an immunoblot analysis with the 3 antibodies EPIL 02, 7381 and 7383, is lyophilized and then taken up in a solution of CH3CN / H2O (V / V) and 0.1% formic acid. . Analysis of this sample reveals a mixture of two products with average experimental molecular weights
M1: 13,346.62 t 2.14 Da and M2: 15,491.86 + 3.46 Da.

 The theoretical molecular mass of the EPIL 1 polypeptide (1-139) corresponds to a mass of 15,445.05 Da. The mass difference of 43 Da with respect to the experimental molecular mass M2 could be attributed to an acetyl group (CH3-CO-).

 These results suggest the presence in the sample of the acetylated EPIL 1 molecule in the N-terminal position.

 A theoretical molecular mass equal to 13,347.46 Da is calculated when the first 18 amino acids of the EPIL 1 polypeptide are deleted and when 3 disulphide bridges are made between the 6 cysteines. The experimental mass found (13,346.62 + 2 Da) is close to this calculated theoretical mass. This observation could suggest that the second molecule present mainly in the sample corresponds to the EPIL 1 molecule without the signal peptide 1-18 and with a tertiary structure having 3 disulfide bridges.

EXAMPLE 3
Production of the extra short form in CHO cells
The following steps are carried out 1 Construction.

The coding sequence corresponding to the extra short form INSL4 is cloned into the eukaryotic expression vector pSec Tag C (INVITROGEN) at the BamHI-Xbal site (diagram in FIG. 6). In parallel, a reference vector pSec Tag C, digested with the same restriction endonucleases (pSec Tag C MOCK), is constructed. After transformation of TOP10 bacteria (INVITROGEN), a recombinant clone is selected 1 / by PCR analysis, 2 / by a study of the restriction profile of the recombinant DNA with respect to different endonucleases (BamH1, Xbal, ECOR1) and 3 / by automatic sequencing (primers specific for DNA INSL4 and for the multiple cloning site of the plasmid pSec
Tag Cl '). The functionality of the open reading frame is confirmed by an in vitro transcription / translation study (TNT T7 Quick coupled Transcription / Translation
System, PROMEGA). This experiment highlights the expression of two neosynthesized protein species.

These two species, identified by the antibody 7381, specific for peptide C, and the antibody 7383 directed against the B chain, have molecular masses respectively equal to 15 and 20 kDa.

 In order to obtain a line producing the recombinant EPIL polypeptide in a stable manner, the expression vector having integrated the INSL4 gene is transfected into Chinese hamster ovary cells (CHO line). Two transfection methods can be used: the Calcium Phosphate method and electroporation.

The use of 10 to 20 Mg of plasmid DNA allows genomic integration of the INSL4 recombinant DNA. Thanks to the presence of an antibiotic resistance marker present in the vector, the stable recombinant cell clones, having integrated pSec Tag C INSL4, are selected by culture in a medium containing 600 ssg / ml of ZEOCINTM (INVITROGEN).

2 Northern blot.

 The selected clones are established in culture.

The study of the expression of the pSec TagC INSL4 transcripts is carried out by Northern blot after extraction of the mRNAs. Twenty-four hours after hybridization of the INSL4 probe (BamHI-Xbal insert), the expression of the recombinant transcript pSec Tag
C INSL4 is demonstrated in all transfected cells. The size of the mRNA, 900 bp, corresponds to that expected for the putative transcript (907 bp).

3 RT-PCR.

 The expression of the recombinant INSL4 transcript in CHO cells is also confirmed by an RT-PCR analysis.

Analysis by RT-PCR of the mRNAs extracted from the CHO cells transfected with the CHO-pSec Tag C MOCK construct (empty control vector) as well as those extracted from the CHO parental cells shows the absence of expression of the recombinant INSL4 transcript after 30 amplification cycles.

4 Detection of the recombinant polypeptide.

 The purification of various recombinant placental hormonal forms produced from the media. culture and transfected cells shows, by the immunoblotting method, the presence of protein forms of 16.5 and 20 kDa in the transfected cells, as well as protein species of 6.5, 10 and 16.5 kDa in the medium of pSec Tag C INSL4 transfected CHO cells.

EXAMPLE 4
Detection of the expressed physiological forms of the EPIL polypeptide
The existence of the expressed physiological forms of the EPIL polypeptide in normal human tissues is sought by an immunohistochemical method using the antibodies described above. This technique is performed on the placenta since the INSL4 gene has been cloned from this tissue.

Fabric samples
Placental villi obtained from early (7 weeks) and long-term placenta were supplied by the Obstetrics and Gynecology Department of the Hospital
Antoine Béclère, France, according to the appropriate procedures by the ethics committee.

 The tissues are fixed in a neutral pH buffer containing 10 W of formalin and then included in paraffin.

It also uses cells of villous cytotrophoblasts and syncytiotrophoblasts previously purified from placenta at term. Isolation of cells from. cytotrophoblasts from villous placenta is performed as described by Guillaudeux T. et al. (1995) by enzymatic digestion and centrifugation in Percoll gradient as for the primary cultures. The villous cytotrophoblasts and the differentiated villous syncytiotrophoblasts, formed in vitro from cytotrophoblasts (at 4-5 days of culture), are highly purified (less than 1 k of contaminating cells). For clarity, the cells purified from villous cytotrophoblasts and the cells differentiated in vitro from syncytiotrophoblasts are referred to herein as cytotrophoblasts and syncytiotrophoblasts respectively.

Immunohistochemistry
Placental tissue aged 6 to 12 weeks (n = 6) and term placental tissue (n = 2) are used.

Fabric cuts of 3 µm are made, mounted on SuperFrost / Plus plates and degreased on xylene. The rehydrated sections are microwaved for 15 minutes in the presence of 10 mM citrate buffer, pH 6.0. After several washes in phosphate buffered saline (PBS), the sections are incubated for 20 min in the presence of the antibody given.

 The sections are then washed and then labeled with a biotinylated antibody followed by an alkaline phosphatase coupled to streptavidin. The labeling is then revealed by incubation in the presence of the chromogenic substrate solution (Fuchsine DAKO, New Fuschin substrate system, Carpinteria, CA). The negative control control tissue sections are produced with sections incubated in the presence of non-specific antibodies (preimmune serum) or in the presence of antibodies preabsorbed on an excess of corresponding peptide (FIG. 7).

EXAMPLE 5
Analysis of mRNA transcripts of cytotrophoblasts and syncytiotrophoblasts
INSL4 mRNAs are quantified by the RT-PCR (reverse transcriptase polymerase chain reaction) method as described in Bellet, D. et al. (1997) . Briefly, the TFIID transcripts are quantified and used as endogenous control mRNA. The PCR is carried out over 30 amplification cycles. The nucleic acid sequences of the primers used for PCR amplification are as follows
INSL4-F: F 5'- AACTCCTTAGAGAAGCCTAGCA-3 '
INSL4-R: 5'-TCGTACCTAAGGCTTGTCCATCT-3 '
TFIID-F: F 5'-ACAGGAGCCAAGAGTGAAGAA-3 '
TFIID-R: 5'-CCAGAAACAAAAATAAGGAGA-3 '(F: fluorescein dye)
Results
Two antibodies, designated 7381 and 1661, directed respectively against the polypeptides EPIL 1 and / or EPIL 3, and EPIL 2 were selected.

The antibody 7381 directed against the 59-108-Y domain of the C chain conjugated with a carrier protein has been found to be completely specific for the EPIL 1 polypeptide and / or
EPIL 3. Indeed, competitive inhibition experiments carried out with peptides or sub-peptides analogous to the C chain portion of the putative EPIL polypeptide or the corresponding portion of human relaxin, demonstrate that the antibody 7381 is binds only to domain
C of the putative EPIL polypeptide at its portion 59-88 and does not recognize the C chain of human relaxin (Figure 3A). The hormone relaxin is the only member of the insulin-like superfamily with a significant percentage (37 t) of similarity with the putative EPIL polypeptide in the portion of domain C corresponding to region 59-88 of the EPIL polypeptide.

The antibody 1661 was obtained by immunization carried out with a mixture of two peptides analogous to domain 115-139 of chain A and to domain 23-52 of chain B. These two regions of the putative polypeptide EPIL do not have a significant percentage similarity with corresponding regions of other members of the superfamily (<16%). Competitive inhibition experiments demonstrate that this polyclonal antibody is mainly directed against domain 115-139 of chain A and also contains a subpopulation of antibodies directed against domain 23-52 of chain B (Figure 3B). The parallelism of the antigen competition curves observed with the chain A and chain peptides
B more likely reflects similar affinities of these two antibody subpopulations for their chain A and chain B epitopes, since the primary structure of these two distinct regions is not significantly similar.

* The immunohistochemical methods based on antibodies 7381 and 1661 were used to detect the presence of the polypeptides EPIL 1 and / or EPIL 3, and EPIL 2 respectively in the tissues of early placenta (less than 3 months) and of placenta at term. Figure 7 represents the results obtained from experiments carried out on tissues of at least two pregnant women.

The immunostaining carried out with the antibody 7381 directed against the polypeptide EPIL 1 and / or EPIL 3 is very important in the cytotrophoblasts of early placenta whereas it is light or even absent in the syncytiotrophoblasts (FIG. 7B).

 In the long term placenta, rare cytotrophoblasts show intense coloring while syncytiotrophoblasts do not show appreciable coloring (Figure 7D). In addition, this EPIL 1 antipolypeptide antibody labels vascular endothelial fetal cells, particularly in the early placenta (Figure 7B).

 The results obtained with the anti-EPIL 2 polypeptide 1661 antibody show that both the cytotrophoblasts and the syncytiotrophoblasts of the early placenta are stained (FIG. 7F). In contrast, this antibody does not mark the placental trophoblasts at term (results not shown).

Analysis of mRNA transcripts
The results obtained by the RT-PCR method and the quantitative analysis method by PCR show that the levels of INSL4 mRNA transcripts in differentiated villous syncytiotrophoblasts in vitro obtained from term placenta are ten times higher. (one log unit) higher than those observed in cytotrophoblasts (Figure 8). In contrast, the levels of TFIID transcripts are similar in the two cell types.

 Detection of mRNA does not imply that proteins are synthesized, as for example for HLA class I expression in the placenta. In fact, the villous cytotrophoblasts express the HLA class I mRNAs, but do not synthesize the corresponding proteins, while the extravillous trophoblasts transcribe and translate the HLA class I genes (Hunt, J. S. et al., 1990).

However, the placenta, which transcribes a large number of genes, is also a source of many polypeptides, including insulin-like growth factors, which are known to be essential at the early stage of embryonic development and, after birth, and to participate in the growth and functional activity of almost all the organs of the human body (Le Roith, D.

1997).

These results show for the first time that the trophoblasts not only express the mRNA of INSL4, but contain EPIL 1 and / or EPIL 3 polypeptides, and
EPIL 2 immunoreactives. On the other hand, it is of great interest to observe that the important immunostaining of the trophoblastic tissues of early placenta obtained the EPIL.

2, is similar to those described for several growth factors, including TGFα and EGF (Horowitz, G. M. et al., 1993).

 Another surprising result is the significant expression of EPIL 1 and / or EPIL 3 polypeptides in cytotrophoblasts compared to syncytiotrophoblasts, while both in situ hybridization studies performed on early placenta (results not shown) and l quantitative analysis by competitive PCR performed on term placenta indicating that INSL4 transcripts are more abundant in syncytiotrophoblasts. In addition, EPIL 2 polypeptides are detected in both cytotrophoblasts and early placenta syncytiotrophoblasts with similar labeling intensities in both cell types.

 Taken together, these observations show that, in vivo, the maturation of EPIL differs in cytotrophoblasts in comparison with syncytiotrophoblasts.

 In the insulin-like superfamily, the C junction peptides or C chain are mediators for the formation of the tertiary structure of proteins and in particular for the correct assembly of the three disulfide bridges of the mature hormone.

 These results show that in the cytotrophoblasts, the C peptide of the putative EPIL polypeptide is maintained while the EPIL 2 present in the syncytiotrophoblasts does not present a C peptide junction. Thus, we can deduce from these results and from these observations that the EPIL 1 and / or EPIL 3 polypeptide containing peptide C can be secreted from cytotrophoblasts in the amniotic fluid and that EPIL 2 not containing peptide C can be secreted from syncytiotrophoblasts into the maternal circulation.

EPIL 3, corresponding to peptide C, is by definition also recognized by anti-C chain antibodies, such as antibody 7381. This form of the putative EPIL polypeptide and which corresponds to the cleavage by-product leading to EPIL 2 , can also have a biological activity other than that of participating in the formation of the tertiary structure of EPIL (Ido, Y. et al., 1997,
Steiner, DF et al., 1997). This capacity for selective excretion of different peptides in various biological compartments has already been illustrated by other polypeptide hormones such as for HCG and its free subunits.

On the other hand, it is remarkable to see that the EPIL 1 and / or EPIL 3 polypeptide is (are) also present in vascular endothelial fetal cells. Recent results (Zhou, Y. et al., 1997) have shown that extravillous cytotrophoblasts are plastic and have the capacity to acquire characteristics of endothelial cells. Thus, these results in fact establish that the villous cytotrophoblasts and the endothelial fetal cells must most certainly share common phenotypic characteristics. These results also show that the EPIL polypeptide, in particular EPIL 1, is involved in the process of differentiation of trophoblastic cells, and in general the polypeptide
EPIL is most certainly involved in the process of differentiation, proliferation and regeneration of cells, in particular endothelial cells such as vascular cells.

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Claims (57)

CLAIMS 1. EPIL 1 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in human placenta trophoblasts, and in that it consists of a single chain comprising regions A, B and C of global amino acid sequence chosen from the amino acid sequences between positions 18 and 139, ends included, of the amino acid sequence defined in FIG. 1, said global sequence comprising at least minus the amino acid sequence between positions 24 and 139, ends included, said EPIL 1 polypeptide possibly further comprising a signal peptide such that the amino acid sequence of EPIL 1 polypeptide comprising the signal peptide is the sequence between positions 1 and 139, ends included, of the amino acid sequence defined in FIG. 1. 2. EPIL 1 polypeptide according to claim 1,  characterized in that it is expressed preferentially  in human placenta cytotrophoblasts. 3. EPIL 1 polypeptide according to claim 1,  characterized in that it is present in the cells  endothelial. 4. EPIL 1 polypeptide according to claim 3,  characterized in that the endothelial cells are  vascular endothelial cells. 5. EPIL 1 polypeptide according to claim 4,  characterized in that the endothelial cells  vascular are endothelial cells  vascular. 6. EPIL 1 polypeptide according to one of claims 1 to  5, characterized in that it is secreted in the liquid  amniotic and / or in the fetal circulation. 7. EPIL 2 polypeptide encoded by the INSL4 gene, characterized  in that it is expressed in human cells or  animal, especially in placenta trophoblasts  human and what it is made of  a) of a chain A whose amino acid sequence is  the sequence between positions 115 and 139,  included ends of the amino acid sequence  defined in figure 1  b) a chain B of which the amino acid sequence is  chosen from a sequence between  positions 18 and 58, ends included, of the  amino acid sequence defined in Figure 1, said  sequence comprising at least the sequence included  between positions 24 and 53, ends included  said EPIL 2 polypeptide can also comprise a  signal peptide of selected amino acid sequence  from a sequence between positions 1 and  23, ends included, of the acid sequence  amines defined in Figure 1, said sequence of  signal peptide comprising at least the sequence  between positions 1 and 17, ends  included. 8. EPIL 2 polypeptide according to claim 7,  characterized in that it is expressed preferentially  in human placenta syncytiotrophoblasts. 9. EPIL 2 polypeptide according to one of claims 7 and  8, characterized in that it is secreted in the  maternal circulation. 10. EPIL 3 polypeptide encoded by the INSL4 gene, characterized  in that it is expressed in human cells or  animal, especially in the trophoblast cells of  human placenta and in that it consists of a  chain C of which the amino acid sequence is chosen  from a sequence between positions 54 and  114, ends inclusive, of the acid sequence  amines defined in Figure 1, said sequence  comprising at least the sequence between  positions 59 and 114, ends included. 11. Polypeptide according to one of claims 1 to 10,  characterized in that it is involved in the process  differentiation of human cells and / or  animal. 12. Polypeptide according to one of claims 1 to 10,  characterized in that it is involved in the process  proliferation of human and / or animal cells. 13. Polypeptide according to one of claims 1 to 10,  characterized in that it is involved in the process  regeneration of human and / or animal cells. 14. Polypeptide according to one of claims 11 to 13,  characterized in that human cells and / or  animal cells are endothelial cells. 15. Polypeptide according to one of claims 1 to 10,  characterized in that it is involved in the process  of vascularization of embryonic cells. 16. Polypeptide according to one of claims 1 to 10,  characterized in that it is involved in the process  of vascularisation of tumors. 17. Fragment of a polypeptide according to one of claims  1 to 16, characterized in that it is biologically  active. 18. Nucleic acid sequence characterized in that it  code for a polypeptide or a fragment thereof according to  one of claims 1 to 17. 19. Cloning and / or expression vector containing a  A nucleic acid sequence according to claim 18. 20. Vector according to claim 19, characterized in that  that it contains the elements allowing expression  and / or the secretion of said sequences in a cell  host. 21. Host cell transformed by a vector according to one  of claims 19 and 20. 22. Method for producing a recombinant polypeptide  characterized in that it implements a cell  according to claim 21. 23. Method for obtaining a recombinant polypeptide according to  claim 22, characterized in that one  cultivates transformed cells according to the  claim 21 under conditions allowing  expression of a recombinant polypeptide, and that one  recovers said recombinant polypeptide. 24. Recombinant polypeptide capable of being obtained by  a method according to one of claims 22 and 23. 25. Polypeptide or a fragment thereof according to one of  claims 1 to 17, characterized in that it is  obtained by chemical synthesis. 26. Monoclonal and polyclonal antibodies or their fragments,  chimeric antibodies, characterized in that it is  capable of specifically recognizing a polypeptide  or one of its fragments according to one of claims  1 to 17, 24 and 25. 27. Antibody according to claim 26, characterized in that  that it is immunoconjugated or labeled. 28. Oligonucleotide probe or primer, characterized in  that it is made up of an acid sequence  nucleic acid according to claim 18 or one of its  fragments capable of specifically hybridizing to  said sequence. 29. oligonucleotide probe according to claim 28,  characterized in that it is marked. 30. Use of one or more antibodies according to one  claims 26 and 27, for the detection,  identification, location and / or dosage of a  polypeptide or a fragment thereof according to one of  claims 1 to 17, 24 and 25, or for diagnosis  pathologies related to the abnormal expression of  polypeptide according to one of claims 1 to 16. 31. Use of a probe or primer according to one of the  claims 28 and 29, for detection,  1 identification, localization, amplification,  and / or the nucleic acid sequence assay according to  claim 18, or for the diagnosis of  pathologies linked to the abnormal presence of sequence  nucleic acid according to claim 18. 32. Method for the detection, identification,  location and / or specific dosage of a  polypeptide or a fragment thereof according to one of  claims 1 to 17, 24 and 25 in a sample  biological, or for the diagnosis of related pathologies  the abnormal presence of polypeptide according to one of  claims 1 to 16, characterized in that it  includes the following steps  a) bringing the biological sample into contact with a  or more antibodies according to one of claims  26 and 27;  b) highlighting, identification, location and / or  assay of the antigen-antibody complex formed. 33. Kit or necessary for detection, identification,  the location and / or specific dosage of a  polypeptide or a fragment thereof according to one of  claims 1 to 17, 24 and 25 in a sample  biological, or for the diagnosis of related pathologies  the abnormal presence of polypeptide according to one of  claims 1 to 16, characterized in that it  includes the following  a) one or more antibodies according to one of  claims 26 and 27  b) the reagents for the constitution of the favorable environment  to the immunological reaction  c) reagents for detection,  identification and / or determination of complexes  antigen-antibody produced by the reaction  immunological. 34. Method of detection, identification and / or determination  specific for nucleic acid sequence according to the  claims 18 in a biological sample, or  for the diagnosis of pathologies linked to the presence  abnormal nucleic acid sequence according to the  claims 18, characterized in that it comprises  the following steps:  a) isolation of DNA from the sample  biological to be analyzed, or obtaining a cDNA from  RNA from the biological sample  b) specific amplification of the acid sequences  nucleic acid according to claims 18 using  primers according to claim 28;  c) qualitative and / or quantitative analysis of the products  amplification. 35. Method of detection, identification and / or  specific nucleic acid sequence assay according to  claim 18 in a biological sample,  or for the diagnosis of pathologies linked to  abnormal presence of nucleic acid sequence according to  claim 18, characterized in that it  includes the following steps  a) contacting an oligonucleotide probe  according to one of claims 28 and 29 with a  biological sample, the DNA contained in  the biological sample having, where appropriate,  previously made available for hybridization,  under conditions allowing hybridization of the  DNA probe contained in the biological sample;  b) detection, identification and / or assay of the hybrid  formed between said oligonucleotide probe and  The DNA of the biological sample. 36. Method of detection, identification and / or  specific nucleic acid sequence assay according to  claim 18 in a biological sample,  or for the diagnosis of pathologies linked to  abnormal presence of nucleic acid sequence according to  claim 18, characterized in that it comprises  the following steps:  a) contacting an oligonucleotide probe  according to claim 28 immobilized on a support  with a biological sample, the DNA of  the sample, having, where applicable, been  previously amplified using primers according to the  claim 28 and / or made available to  hybridization, under conditions allowing  hybridization of the DNA probe of said sample  biological;  b) contacting the hybrid formed between said  oligonucleotide probe immobilized on a support and  The DNA contained in the biological sample, if applicable  if necessary after removal of DNA from the sample  biological that has not hybridized with the probe, with a  oligonucleotide probe, in particular labeled, according to  one of claims 28 and 29. 37. Kit or necessary for detection, identification  and / or the specific acid sequence assay  nucleic acid according to claims 18 in a  biological sample or for the diagnosis of  pathologies linked to the abnormal presence of sequence  nucleic acid according to claim 18,  characterized in that it comprises the elements  following:  a) An oligonucleotide probe according to one of  claims 28 and 29;  b) The reagents necessary for the implementation of a  hybridization reaction;  c) If necessary, a pair of primers according to the  claim 28 and the reagents necessary for  a DNA amplification reaction. 38. Kit or necessary for detection, identification  and / or the specific acid sequence assay  nucleic acid according to claims 18 in a  biological sample, or for the diagnosis of  pathologies linked to the abnormal presence of sequence  nucleic acid according to claim 18,  characterized in that it comprises the elements  following:  a) an oligonucleotide probe, called a probe  capture according to claim 28 which can be  supplied pre-immobilized on a support;  b) an oligonucleotide probe, called a probe  revelation, notably marked, according to one of  claims 28 and 29  c) If necessary, a pair of primers according to the  claim 28 and the reagents necessary for  a DNA amplification reaction. 39. Cell labeling method, characterized in  that it uses one or more antibodies according to  one of claims 26 and 27. 40. Marking method according to claim 39,  characterized in that it implements a process  according to claim 32. 41. Cell labeling method, characterized in  that it implements a primer and / or a probe,  in particular marked, according to one of claims 28  and 29. 42. Marking method according to claim 41,  characterized in that it implements a process  according to one of claims 34 to 36. 43. Cell labeling method according to one of the  claims 39 to 42, characterized in that the  cell is an endothelial cell. 44. Cell labeling method according to claim  43, characterized in that the endothelial cell is  a vascular cell. 45. Method for obtaining a cell containing a  polypeptide according to one of claims 1 to 17, 24  and 25 and / or a nucleic acid sequence according to the  claim 18, characterized in that it sets  implements a method according to one of claims 39 to  44, and in that said labeled cell is isolated. 46. Cell in that it is likely to be  selected by a method according to claim  45. 47 Method for detecting tumor cells,  characterized in that it implements a method  according to one of claims 39 to 44 48. Method for selecting a chemical compound or  biochemical capable of modulating the activity of a  polypeptide according to one of claims 1 to 16,  characterized in that it implements a  polypeptide or a fragment thereof according to one of  claims 1 to 17, 24 and 25, or a cell according to  one of claims 21 and 46. 49. Selection method according to claim 48,  characterized in that said selected compound is  able to modulate differentiation, regeneration  and / or proliferation of human cells and / or  animal. 50. Selection method according to claim 49,  characterized in that said selected compound is  capable of promoting differentiation,  cell regeneration and / or proliferation  human and / or animal. 51. Selection method according to claim 49,  characterized in that said selected compound is  capable of inhibiting differentiation, regeneration  and / or proliferation of human cells and / or  animal. 52. Selection method according to one of claims 49  to 51, characterized in that said cells are  endothelial cells. 53. Selection method according to claim 52,  characterized in that the endothelial cells are  vascular cells. 54. Chemical or biochemical compound, in that it is  likely to be selected by a method according to  one of claims 48 to 53. 55. A compound according to claim 54 as  drug. 56. Compound according to claim 55, characterized in that  that he is chosen from  a) a polypeptide or a fragment thereof according to one  of claims 1 to 17, 24 and 25  b) an antibody according to claim 26  c) a chemical or biochemical compound, characterized in  what it is a ligand of a polypeptide or one of its  fragments according to one of claims 1 to 17  d) a vector according to one of claims 19 and  20  e) a vector according to one of claims 19 and 20  presenting on its surface a specific marker of the  target cell whose activity is sought to modulate  of a polypeptide according to one of claims 1 to  16. 57. Compound according to one of claims 55 and 56,  intended for the treatment of tumors. 58. Compound according to claim 57, characterized in that  that the tumor is a tumor of the pancreas, liver,  uterus, breast, angiosarcoma, glioblastoma,  a neuroblastoma, a rhabdomyosarcoma, or a leiomyosarcoma 59. Compound according to one of claims 55 and 56,  intended to favor the vascularisation of tissues  specific. 60. Compound according to one of claims 55 and 56,  for the treatment of retinopathy,  degeneration of the macula, psoriasis,  endometriosis, rheumatoid arthritis,  atherosclerosis or hyperthyroidism. 61. Compound according to one of claims 55 and 56,  intended to regenerate and / or differentiate a tissue,  especially endothelial, following an illness  degenerative or endocrine gland disease,  including the thyroid or pancreas, or following a  damage to said tissue caused by trauma.