WO1999009172A1

WO1999009172A1 - Method for identifying and locating expressed epil peptides, coded by the insl4 gene and their uses

Info

Publication number: WO1999009172A1
Application number: PCT/FR1998/001799
Authority: WO
Inventors: Dominique Bellet; Frédéric TROALEN; Jean-Michel Bidart; Pascal Mock
Original assignee: Institut Gustave-Roussy
Priority date: 1997-08-14
Filing date: 1998-08-12
Publication date: 1999-02-25
Also published as: FR2767326A1; FR2767326B1; AU9075798A; EP1003866A1; CA2301154A1

Abstract

The invention concerns the identification and localisation in the tissue of novel polypeptides expressed by the insulin-like 4 gene (INSL4), their amino acid and nucleic acid sequence, detection and diagnosis methods, methods for marking, selecting and detecting cells expressing said polypeptides or their corresponding nucleic sequence and methods for selecting compounds capable of modulating the activity of said polypeptides. The invention also concerns medicines for treating tumours, such as Kaposi's Sarcoma, for the vascularization of specific tissues, tissue differentiation and/or regeneration, modulating embryonary implantation and treating pathologies related to insulin-like activity such as diabetes and its complications.

Description

IDENTIFICATION AND LOCATION OF EXPRESSED EPIL POLYPEPTIDES, CODED 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 intended for the treatment of tumors, in particular angioproliferative tumors such as Kaposi's sarcoma, for vascularization of specific tissues and / or for regeneration of tissues.

Insulin, IGF-1, IGF-2 ("Insulin-like growth factors 1 and 2" and relaxin like insulin 1 and 2) and relaxin belong to a family of peptide hormones with certain structures and functions in common, 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 trophoblast and the prostate (Bogie, LV 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 yet to be defined. Ley IL transcripts are present in Leydig cells and in other tissues such as the corpus luteu, the trophoblast, the fetal membranes and the 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 tyrosine kinase receptors (RTK's) which includes growth factor receptors and their oncogenic analogs such as c- erbB2 / 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 tyrosine kinase 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 protein activating mitogenesis (MAP), kinase culminating when the cascade of phosphorylation affects different 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 pre-prohormone, prohormone or hormone mature are defined from 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, of the family of insulin-like (related insulins) was 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 showed, 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 RNA 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 analysis by homology 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 C). These synthetic peptides can be used as agonists or antagonists of the corresponding activity. They can also be used as antigenic peptide and make it possible to obtain specific antibodies directed against particular forms or domains of this hormone. tissue form or forms expressed by the INSL4 gene would 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 antibodies C-antichain, from peptide constructs mimicking the C domain of the polypeptide

Putative EPIL, in addition to the antichain A and B antibodies in their search for identification and localization of the expressed forms.

The inventors were thus able to demonstrate, surprisingly, two expressed forms of the EPIL polypeptide. A form provided with the C chain and a form devoid of 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 an EPIL 1 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in the human placenta triphoblasts, and in that it consists of a single chain comprising the regions A, B and C of overall amino acid sequence chosen from the amino acid sequences comprised between positions 18 and 139, ends included, of the amino acid sequence defined in FIG. 1, said overall sequence comprising at least 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 signal peptide is the sequence between positions 1 and 139, ends included, of the amino acid sequence defined in Figure 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 include non-natural or modified amino acids.

The invention also relates to an EPIL 2 polypeptide 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) 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 included; 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 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 is consisting 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 included between positions 59 and 114, ends included.

The invention also relates to an EPIL 1 or EPIL 3 polypeptide according to the invention, characterized in that it is preferentially expressed in the cytotrophoblasts of human placenta.

The invention further relates to an EPIL 1 or

EPIL 3 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 or EPIL 3 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 1 or EPIL 3 polypeptide according to the invention, characterized in that it is present in smooth muscle cells, in particular vascular smooth muscle cells constituting the wall of vessels such as embryonic vessels or umbilical cord. The invention also includes an EPIL 1 or EPIL 3 polypeptide according to the invention, characterized in that it is present in the stromal cells, in particular the amnion and / or the embryonic vestige cells, preferably the yolk sac and the 'allantoid located at the level of the umbilical cord.

The invention further comprises an EPIL 1 or EPIL 3 polypeptide according to the invention, characterized in that it is present in tumor cells, preferably in tumor cells of angioproliferative tumor such as for example tumor cells associated with Kaposi's sarcoma. Also preferred according to the invention are intra-tumor vessel tumor cells and Malpighi epithelial cells.

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

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

Another aspect of 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 , smooth muscle cells, preferably of the vascular type, epithelial cells or stromal cells, in particular of the decidue, or cells of the glandular epithelium. 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, preferably angioproliferative tumors such as for example Kaposi's sarcoma.

The polypeptides according to the invention are also characterized in that they are involved in the process of decidualization and / or implantation of the embryo, in a process of the insulin-like type, in particular paracrine and / or autocrine, preferably a process of response to hypoxic stress and / or hypoglycemic stress, in particular a process promoting the use of glucose as a source of energy.

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 capacity to modulate the differentiation, the regeneration and / or the proliferation of cells, such as in particular endothelial or smooth muscle cells, in particular vascular cells; the ability to be recognized by an antibody specific for a polypeptide according to the invention; and / or the capacity to modulate the activity of a polypeptide according to the invention, by neutralizing for example the binding of a polypeptide according to the invention on 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” is intended to denote 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 a point mutation. Among the homologous polypeptides, those whose amino acid sequence has at least 80%, preferably 90%, 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 complementary sequences or the RNA sequences corresponding to said nucleic acid sequences according to the invention also form part of 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 they comprise 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 sequence of nucleic acid 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 is constituted by Chinese hamster ovary CHO cells, and by 3AsubE cells of human placental origin.

The vector must include a promoter, translation initiation and termination signals, as well as 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, thermal 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 (CHO) cells, human cells such as 3AsubE cells of placental origin, but also insect cells in which methods can be used using baculoviruses 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 solubility during in vitro renaturation and / or a simplification of the purification when the fusion partner has an 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 or an amino acid sequence of a polypeptide according to the invention.

The specific monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas 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 epitopes or different 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, methods and kits or kits of the invention which include said antibodies, in which case, for example, two different antibody labels or two different development systems may be used. They thus constitute a means of immuno-cytochemical or immunohistochemical analysis of the expression of 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 a fragment thereof, or of a nucleic acid sequence capable of specifically hybridize to said sequence according to the invention, preferably the probes may be labeled. Specific hybridization means that the hybridization is carried out under stringency conditions, in particular under temperature and ionic strength conditions such that they allow hybridization to be maintained between two globally complementary DNA fragments.

As an illustration, high stringency conditions of the hybridization step for the purpose of defining the nucleotide sequences described above are advantageously as follows: The hybridization is carried out at a preferential temperature of 65 ° C., in the presence of buffer 6 x SSC, 5 x Denhardt's solution, 0.5% SDS and 100 μg / ml of salmon sperm DNA.

1 x SSC corresponds to 0.15 M NaCl and 0.05M of sodium citrate and a solution of 1 x Denhardt corresponds to 0.02% Ficoll, 0.02% of polyvinylpyrrolidone and 0.02% of bovine serum albumin.

The washing steps can, for example, be carried out for 5 to 30 min. at 65 ° C, in 2 x SSC or 1 x SSC buffer and 0.1% SDS. The high stringency hybridization conditions described above for a polynucleotide of defined size will be adapted by those skilled in the art for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al., 1989. The invention also 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, 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, and in general the nucleic acid sequences according to the invention, will have a minimum size of 10 bases and fragments of 20 bases will be preferred, and preferably

30 bases.

Among the nucleic acid fragments of interest according to the invention, there may be mentioned in particular the antisense oligonucleotides, that is to say the structure of which ensures, by hybridization with the target sequence, an inhibition of the expression of the 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 oligonucleotides or antisense are also part of the probes according to 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) detection, identification, location and / or determination of the antigen-antibody complex formed.

Preferably, the biological sample consists of a fluid, for example a human or animal serum, blood, amniotic fluid or biopsies.

Preferably, in the preceding method according to the invention, the antibody used in step a) is fixed on a solid support and the demonstration, identification, localization and / or assay of the antigen-antibody complex formed from step b) is carried out using an immunoconjugate or labeled antibody according to the invention.

The solid supports which can be used, such as polystyrene beads, microtiter plates, are well known in the field of immunoassays and will not be developed in the present description.

The invention also relates to a kit, or necessary, 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, in particular in the amniotic fluid. , or for the diagnosis of pathologies linked to 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, in particular being able to be fixed on a solid support; b) where appropriate, the reagents for constituting the medium suitable for the immunological reaction; c) where appropriate, the reagents for the detection, identification and / or determination of the antigen-antibody complexes produced by the immunological reaction, in particular an immunoconjugated or labeled antibody according to the invention.

Any conventional procedure can be implemented to carry out such detection, identification, localization and / or assay of the antigen-antibody complex possibly formed.

The specific techniques and reagents enabling the detection, identification, localization and / or assay of 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, RIA or immunofluorescence techniques 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 antibodies, labeled with fluoroscein or radiolabelled capable of specifically recognizing the antibodies of the invention or the forms of EPIL polypeptide or their fragments, 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, where appropriate, has 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 between said oligonucleotide probe immobilized on a support and the DNA contained in the biological sample, if 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 in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of sequence nucleic acid according to the invention, characterized in that it comprises the following elements: a) an oligonucleotide probe according to the invention; b) the reagents necessary for carrying out a hybridization reaction; c) where appropriate, 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 methods or kits according to the invention for the diagnosis of pathologies linked to the presence of tumor cells, in particular angioproliferative cells and in particular for the diagnosis of Kaposi's sarcoma, advantageously form part of the invention. In the present invention, the term “diagnosis of pathologies linked to the abnormal presence of polypeptide or nucleic sequence according to the invention” is also understood to mean “diagnosis of pathologies linked to the presence of tumor cells”, not only the actual diagnosis of the pathology, but also the diagnosis or prognosis of the evolution of said pathology, following for example a therapeutic treatment. Thus, according to the invention, said methods or kits can be used to evaluate the effectiveness of a therapeutic treatment of said pathology.

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. 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 using 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 will be understood to mean all the methods implementing 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. Also known to those skilled in the art, the nucleic acid quantification techniques in which, for example, the nucleic acid to be quantified is amplified by a PCR type method and in the presence of a standard nucleic acid of the same size and quantity. known and capable of hybridizing to the same primers as the target nucleic acid.

The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography. The specificity of the amplification can be checked for example by molecular hybridization using as probes the probes of the invention, in particular labeled.

The invention relates in particular to cell labeling methods, characterized in that the cell is an endothelial cell, in particular a vascular cell, a smooth muscle cell, a stromal cell, in particular of the decidue, a cell of the glandular epithelium, in particular the endometrial epithelium of the uterus, or a tumor cell, in particular angioproliferative and those of Sarcoma from Kaposi in particular.

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 subject of the invention is also a method for detecting tumor cells, in particular tumor cells from kaposi sarcoma, 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 the invention. invention, a nucleic acid sequence according to the 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 ability to bind to a nucleic acid according to the invention and can thus inhibit or promote expression of the gene encoding said polypeptides as an inducer or repressor.

Said compounds can also be selected on their ability to modulate on said cells according to the invention 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, smooth muscle cells, in particular vascular cells, epithelial cells, stromal cells, in particular of the deciduous tissue, and / or the cells of the glandular epithelium.

The invention also has the aspect of selection methods according to the invention, characterized in that said selected compound is capable of modulating the process of decidualization and / or implantation of the embryo, in particular of promoting or inhibiting the decidualization and / or implantation process.

Another aspect of the invention also relates to selection methods according to the invention, characterized in that said selected compound is capable of modulating insulin-like activity, preferably of modulating the response of the cell to hypoxic and / or hypoglycemic stress. , including promoting the use of glucose as an energy source.

The term insulin-like activity will be understood to mean in the present description, the well-known activities of insulin, in particular those which will be described in the examples below. For example, the invention comprises a method for selecting compounds capable of binding to a polypeptide or one of its fragments according to the invention or capable of binding to a nucleotide sequence or one of its fragments according to the invention, characterized in that it comprises the following stages: a) bringing said compound into contact with said polypeptide or said nucleotide sequence; b) determining the capacity of said compound to bind with said polypeptide or said nucleotide sequence.

The cells transformed or selected according to the invention and expressing said polypeptide or said nucleotide sequence can also be used in said selection methods. Methods for selecting compounds based on the affinity of a compound for another compound (search for specific ligands) with agonist or antagonist effect on a specific desired activity (such as cell-type biochemical activity) are well known and will not be described.

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

The compounds capable of being selected may be organic compounds such as polypeptides or hydrates of ^carbon or other compounds already known, or new organic compounds produced from molecular modeling techniques and obtained by chemical synthesis or biochemical, these techniques being known to one skilled in the art.

The invention further relates to compounds according to the invention as a medicament, preferably chosen from: a) a polypeptide or one of its fragments 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; f) a probe according to the invention, such as for example the sense or antisense nucleic acid sequences as defined above.

The invention relates to compounds according to the invention, intended for the treatment of tumors, in particular angioproliferative tumors such as for example Kaposi's sarcoma. Among said tumors, tumors of the pancreas, liver, uterus, breast, angiosarco e, glioblastoma, neuroblastoma, rhabdomyosarcoma or very particularly leiomyosarcoma, are also preferred.

We can, for example, refer to the publications of Clark, R. (1997); Ferrara, N., et al. (1997), or from the review Scrip N ° 2209, pp.21, and Scrip N ° 2202 concerning the article on the company Genentech, for the biological activities in which IGF-1 and EGF are involved

(endothelial growth factor), hormones related to the EPIL polypeptide.

The invention also includes compounds according to the invention, intended to promote the vascularization of specific tissues.

The invention also includes compounds according to the invention, intended for the treatment of retinopathy, degeneration of the macula, psoriasis, endometriosis, rheumatoid arthritis, atherosclerosis or hyperthyroidism. Treatments for lesions due to atherosclerosis as well as restenosis after angioplasty are among the preferred treatments.

Indeed (Owens et al., 1996 and Murry et al., 1997), the factors regulating cell growth smooth muscles, especially those promoting this growth, play an essential role in the formation of atherosclerotic plaques which are made up of a monoclonal population of smooth muscle cells. Thus, among the compounds of the invention, those making it possible to modulate, in particular to inhibit, the growth or proliferation of smooth muscle cells, could be used for the treatment of lesions induced by the formation of these plaques, in particular for the treatment of restenosis after angioplasty, as part of the treatment of cardiovascular diseases.

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

The invention also includes compounds according to the invention, intended to promote or inhibit embryonic implantation.

Finally, the invention comprises compounds according to the invention, intended for preventing and / or treating pathologies directly or indirectly linked to an activity of the insulin-like type, preferably linked to a dysfunction of the carbohydrate metabolism, such as hypoglycemia or hyperglycemia. Among said pathologies, diabetes, such as gestational diabetes, and complications linked to diabetes, in particular cardiovascular complications, are particularly preferred.

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

It is also generally possible to compensate for the deficiency or overexpression of polypeptides according to the invention 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 Viruses (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. he 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. The compounds according to the invention which can be used as medicaments offer a new approach for the treatment of diseases linked to the differentiation or unregulated proliferation of specific cells or tissues, or to the absence or insufficiency of certain cells or tissue, for example from trauma.

Among the diseases linked to the unregulated differentiation or proliferation of specific cells or tissues, such as in particular the diseases linked to the presence of tumors, the inventors have demonstrated, for example, the abnormal presence of a quantity of polypeptides according to the invention in specific tissues in patients with Kaposi's sarcoma, in comparison with the corresponding healthy tissues. Kaposi's sarcoma (SK) can be defined as an angioproliferative disease characterized by a proliferation of spindle-shaped cells probably of heterogeneous origin predominantly endothelial, but also smooth, dendritic and / or monocytic muscle, a neoangiogenesis process, inflammatory cell infiltration as well as edema.

SK affects the skin and / or mucous membranes. It can spread to the soft tissues, bones, and lymph nodes and affect visceral organs such as the lungs, the gastrointestinal tract (intestine, liver and spleen), the genitals (prostate, seminal vesicle, testes, bladder, penis, cervix and vulva), brain, kidneys and adrenal glands. The etiology of SK remains an enigma today. However, four different clinical entities are recognized on the epidemiological level (Tappero et al., 1993): 1. Classic, affecting men (male / female ratio 10: 1) between 50 and 80 years of Mediterranean origin or Eastern European Jews, skin localization, chronic course, with secondary cancer developing in 35% of cases.

2. Endemic, affecting the black African man of around 35 years (male / female ratio 13: 1), of benign or fatal evolution in 5-8 years, affecting the young child of around 3 years, of evolution fatal in 2-3 years. SK represents between 9% and 12.8% of all neoplasias in Zaire.

3. Iatrogenic, affecting recipients of an immunosuppressed transplanted organ, usually with remission if the immunosuppressive therapy is stopped.

4. Epidemic, associated with AIDS, with approximately 30% of AIDS patients developing SK. It is the most common tumor associated with HIV-1 infection. The risk of developing SK is different according to the mode of transmission of the HIV virus with a greater risk in homosexual men and women infected by heterosexual contact with a bisexual man compared to hemophiliacs and intravenous drug users. It is of multicentric and symmetrical localization, with an unforeseeable evolution, the progression of SK being most often correlated with the general condition of the patient. Thus, the stage criteria developed by the AIDS Clinical Trials Group (ACTG) ^" take into account immunological status (CD4) and opportunistic infections.

Most studies dealing with the pathophysiology of SK are based on primary cultures of SK cells associated with AIDS. The in vitro survival of SK cells in culture seems to depend on growth factors such as OSM, VEGF, FGF-b, PDGF, IL-6 in particular. In addition, the Tat protein of the HIV-1 virus seems to play a synergistic inducing role with FGF-b in the formation of SK, which could partly explain the more aggressive clinical evolution of SK associated with AIDS (Ensoli et al., 1994). Indeed, in the early stages of the disease, the SK associated with AIDS seems to be a disease strictly linked to cytokines where the inflammatory and angiogenic cytokines and the tat protein HIV-1 have an influence on the induction and progression of SK. In addition, it is interesting to note that Barillari et al. in 1993 demonstrated that inflammatory cytokines have the capacity to increase the expression of integrin fixing the RGD (Arg-Gly-Asp) sequence in SK cells and normal endothelial cells. This sequence corresponds to the regions of the proteins of the extracellular matrix (collagen, fibronectin, laminin, tenascin and thrombospondin) which bind to the membrane integrins of cells, thus modulating cell attachment, therefore, participating in the growth and differentiation process and therefore to tumorigenesis. While the tat protein of the HIV virus contains RGD sequences, it has been demonstrated that the inflammatory cytokines (IL-1 beta, TNF, IFN gamma) can moreover increase the binding of the tat protein to the SK cell. The progression of the disease is characterized by the proliferation of spindle cells in both classical SK and in SK associated with AIDS. Recently, it has been shown that the proto-oncogene Bcl-2, anti-apoptosis gene, seems to be overexpressed in spindle cells of endothelial phenotype (factor VIII RA) of lesions of classic SK and associated with AIDS with a higher level of marked cells. at the nodular stage of the disease (Bohan Morris et al., 1996). Thus, the appearance of this anti-apoptotic process seems to be an important mechanism in the pathogenesis of SK and could be a marker of disease progression.

Histologically, classic SK and SK associated with

AIDS is indistinguishable (Tappero et al., 1993). The origin of SK lesion cells remains unclear. The cells are probably mesenchymal cells of

1 lymphatic or vascular endothelium, of dendrocytes of the dermis expressing factor XlIIa or vascular smooth muscle cells.

The 3 histological stages patch, plaque and nodule are correlated with the clinic and the progression of the lesion. 1. Patch: clinically, macular lesion, histologically, proliferation of small, irregular spaces carpeted by an endothelium surrounding normal dermal vessels. Whether or not associated with variable inflammatory lymphocyte infiltration. 2. Plaque: clinically, small palpable lesion (papule), histologically, expansion of spindle cells in the dermis dispersed among the collagen of the dermis with the formation of irregular vascular channels. 3. Nodule: clinically, large palpable nodule, histologically, range of spindle cells with discrete to moderate cytological atypia, erythrocytes in an extensive network of slit-like vascular spaces. Recently, SK associated with AIDS seems to respond to antiprotease treatment, in particular by triple therapy (4 international conference on retroviruses and opportunistic infections in February 1997 in Washington). When the tumor escapes treatment, treatment with chemotherapy can be started (Bleomycin, Vincristine). Briefly, the treatment of SK in general can be local (mainly cryotherapy and radiotherapy) or systemic (interferon, chemotherapy). The first is usually the one you start with because having fewer side effects provided it is a mild form. The cutaneous and mucosal localizations are the major indications for their accessibility. Cryotherapy with liquid nitrogen and the laser offer an acceptable cosmetic result. However, SK remains in the deep dermis and the incidence of recurrence after 6 months increases. Intralesional cytotoxic chemotherapy with vinblastine or vincristine allows a partial or complete response in 60 to 88% of cases with a 40% recurrence rate for patients with SK associated with AIDS in 4 to 6 months. In addition, there is the frequent problem of cutaneous sequelae such as post-inflammatory hyperpigmentation. The intralesional injection of interferon alpha as well as TNF alpha is still at the experimental stage.

Patients who have not responded or have responded poorly to local treatment or who have a more severe form, for example with an ulcer or extensive form, may benefit from systemic treatment with in general a superior clinical response. The most commonly associated chemotherapeutic agents are Bleomycin and Vincristine. However, the fact remains that these treatments are potentially immunosuppressive because they are relatively aplastic. The development of a therapy based on anti-angiogenic agents such as IFN-alpha inhibiting the production of FGF-b and IL-8 in SK cells, the human platelet factor-4 or the development of inhibitors growth factor (cytokines) and / or receptor specific for SK cells remains relevant. Recently, Masood et al. (1997) demonstrated the action of VEGF antisense oligonucleotide on tumor growth in an in vivo model (nude mouse). During development, we usually cite analogues of TNP-450, cis-trans-retinoic acid and more recently HAF or hCG associated factor, whose ^" mechanism of action seems to be linked to the process of apoptosis ( Lunardi Iskandar 1997) Furthermore, since Huang et al. (1996) have recently been able to demonstrate by PCR the presence of DNA of a new virus of the Herpes family, HHV-8 at the level of SK lesion of the 3 types, SK associated with AIDS, classic SK and endemic African SK, an anti-viral therapy is being developed.

For Kaposi's sarcoma and in general for most diseases linked to the presence of tumors, there is today, on the one hand, a great need for new molecular tools to better understand the mechanism. physiological of these diseases, but also new therapeutic treatments to combat them.

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 Figures

Figure 1: Nucleic acid sequence of the cDNA of the INSL4 gene 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 of RLX-H2 (relaxin H2). Identical amino 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 microplate, by the competing peptides EPIL 59-108-

Y (), EPIL ^" 88-108-Y (D), RLX-H2 63-92 (Δ) and RLX-H2 74-92 (O).

B. Inhibition of the binding of 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 (D), and by a mixture of peptides EPIL 23-52 and EPIL 115-139 (0).

Figure 4: Competitive inhibition curve of the binding of polyclonal antibody 7383. The results were obtained by ELISA method on 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 immunogen, the molar concentration of which is expressed in notation scientific: 1.0 E-10 equivalent to 1.0 x 10 ^"10 .

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: 1/2500 dilution (D) and 1/5000 dilution

(*). The results were obtained by ELISA method on microplate support on which the EPIL peptide (125-137-Y) is adsorbed. Figure 6: Diagram representing the characteristics of the construction of the INSL4-pSec TAG C ™ 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 (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. The cytotrophoblasts of placenta less than 3 months old (photograph B) and of the full-term placenta (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 ABI DNA sequencer from Perking Elmer and the peak areas obtained are measured. 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 monoclonal antibody EPIL 02 (antichain A)

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: Revealing polyclonal antibody 7381 (antichain C)

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 polyclonal antibody 7383 (antichain B)

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.

Figure 10: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Labeling of tumor cells ("Spindle cells") with the 7381 immuniser (1/500 dilution). Magnification: X 100. Figure 11: Classic Kaposi's sarcoma (N ° 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Absence of labeling of tumor cells ("Spindle cells") after inhibition of immunserum 7381 (Dilution 1/500) by peptide 59-108Y (18 hours of incubation). Magnification: X 100.

Figure 12: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB 2). Marking of tumor cells ("Spindle cells") by immunserum 7381 (Dilution 1/500). Magnification: X 200.

Figure 13: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Absence of labeling of tumor cells ("Spindle cells") after inhibition of immunserum 7381 (1/500 dilution) by peptide 59-108Y (18 hours of incubation). Magnification: X 200.

Figure 14: Classic Kaposi's sarcoma (N ° 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Labeling of tumor cells (“Spindle cells”) by immunserum 7381 (Dilution 1/500). Magnification: X 1000.

Figure 15: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Absence of labeling of tumor cells ("Spindle cells") after inhibition of immunserum 7381 (Dilution 1/500) by peptide 59-108Y (18 hours of incubation). Magnification: X 1000.

Figure 16: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Marking of tumor cells ("Spindle cells") by immunserum 7381 (Dilution 1/500). Magnification: X 400.

Figure 17: Classic Kaposi's sarcoma (No. 320818) Immunohistochemistry (alkaline phosphatase, Dako LSAB2). Absence of labeling of tumor cells ("Spindle cells") after inhibition of immunserum 7381 (Dilution 1/500) by peptide 59-108Y (18 hours of incubation). Magnification: X 1000.

^Fig ure 18: Inhibition curves obtained with the monoclonal antibody EPIL 08 for different peptides of the 59-108 portion of the chain C EPIL putative polypeptide.

^Fig ure 19: Calibration curve obtained with the synthetic peptide corresponding to the 76-139 portion of EPIL putative polypeptide by the method of radioimmunoassay in two sites and using the monoclonal antibody EPIL 08 as capture antibody and antibody monoclonal EPIL 02 marked with ¹²⁵ I as a tracer.

Figure 20: Comparative assay curves of EPIL and 1 hCG in amniotic fluid for a panel of pregnant women obtained with the radioimmunoassay method at 2 sites for the assay of EPIL; other curves were obtained on other panels showing higher concentrations.

Figure 21: Marking of the vessels inside the trophoblastic villi by the antibody 7381 directed against the C region of EPIL on trophoblasts aged

8 weeks (X 200).

Figure 22: Marking of smooth muscle cells of cord vessels by the antibody 7381 directed against the C region of EPIL (X 200). Figure 23: Labeling of cells of the endometrial epithelium ^" of the uterus by the antibody EPIL 08 directed against the C region of EPIL on trophoblasts aged

9 weeks (X 200).

EXAMPLE 1

Characterization of the form (s) of EPIL polypeptide 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 the peptides and preparation of the immunogens Synthetic peptides are constructed according to the conventional solid phase method on an Applied Biosystems model 431A automaton. 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 rabbits "Grand Fauve de

Bourgogne ”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. The following two booster shots are given on D + 21 and D + 42 respectively by injection of 25 μg 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. The rabbits "good responders" 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 as a complete Freund's adjuvant subcutaneously. Reminders by intraperitoneal route (25 μg of immunogen in incomplete Freund's adjuvant) are carried out three months apart until obtaining an immune response, evidenced by the presence of detectable antibodies in the animal's serum. Cell fusion is carried out 3 days after iv (intravenous) injection of the immunogen. It consists of removing and 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 1 aminopterin. The production of monoclonal antibodies by hybridomas is analyzed by an immuno-enzymatic 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 + 4 ° 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 (o-phenylene diamine) in the presence of H ₂ 0 ₂ makes it possible to reveal the enzymatic reaction. An inhibition method competitive can be performed to specify the location of sites recognized by 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 for the fact that the peptide is radiolabelled with a ¹²⁵ ι isotope according to the so-called Iodogen process. 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 two monoclonal antibodies directed against synthetic peptides are described below:

• A polyclonal antibody, numbered 7381, is obtained by immunization of rabbits with an immunogen comprising a synthetic peptide mimicking the putative C chain 59-108 Tyrosine (Y) sequence (Figure 2) coupled by benzidine to the keyhole limpet hemocyanin (KLH), carrier protein. This antibody binds to the sequence 59-108 of domain C of the EPIL polypeptide (Figure 3A). It 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 demonstrate the physiological EPIL 1 or EPIL 3 polypeptide, by example at the level of the placenta (see example 4, relating to 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 by benzidine to tetanus toxoid, 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). It 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 (31-49) + (31-50) + (31-51) + (31-52) + (31-53) of the putative B chain coupled by MBS (m-maleimidobenzoyl-N-hydroxy-succimimide) 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 125- 137-Y sequence of the putative A chain coupled by benzidine to tetanus toxoid, carrier protein (figure 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).

• A monoclonal antibody, called EPIL 08 (IgG2a), is selected after immunization of mice with an immunogen comprising a synthetic peptide mimicking the sequence 59- 108 -Y of the putative C chain coupled to the carrier protein. This antibody recognizes a long sequence of 13 amino acid residues comprising the 88-100 portion of the C chain as shown by the results obtained by competitive inhibition (see FIG. 18).

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 pFastBac ™ (GIBCOBRL) at the Kpnl site. Subsequently, coinfection of the SF9 insect cells with DNA of wild baculovirus (AcMNPV, GIBCOBRL) and of the transfer vector containing the gene of interest is carried out, in order to constitute a 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, EcoRl) and by automatic sequencing (specific primers of DNA

INSL4 and the multiple cloning site of the bacmid pFastBac ™.

3. Analysis of the expression of the EPIL polypeptide

After infection of insect ovarian 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 10 ₆ infected Sf9 cells are centrifuged. The centrifugation residue is taken up in 1 ml of 0.05 M Na ₂ HP0 ₄ buffer, pH 8.5 containing 0.1% NP 40 and 5 μg / ml of leupeptin. After stirring by tilting for 4 h at 4 ° C., the mixture is centrifuged for 10 min at 14,000 rpm at + 4 ° C. The supernatant is recovered for the following steps.

• Affinity chromatography.

First, a Sepharose-CNBr-EPIL 02 column 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 NaHCO ₃ , 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 + 4 ° C. with stirring, the free sites are blocked with 1 M ethanolamine then the column is washed with 0.1 M CH ₃ COONa solution, 0.1 M CH ₃ COOH, NaCl 0 , 5 M, pH 4. Finally, the column is rinsed with 0.05 M Na ₂ HP0 ₄ , pH 8.5.

The supernatant recovered after solubilization is diluted 1/10 in 0.05 M Na ₂ HP0 ₄ buffer, pH 8.5. After dilution, the solution is brought into contact for 18 h at + 4 ° C with the Sepharose-CNBr-EPIL 02 support with slow stirring. Washings are carried out with a 0.05 M Na ₂ HP0 ₄ 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 collected fractions 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% acetic acid. These fractions are combined and then deposited first on a Lichrosorb RPB C8 7 μm 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: TFA 0.1% - 99.9% H ₂ 0 and phase B: TFA 0.08 - 4.92% H ₂ 0 -

95% CH ₃ CN. An analysis of the fractions collected after C8 reverse phase chromatography is carried out by electrophoresis and SDS-PAGE / immunoblotting using the antibodies EPIL 02, 7381 and 7383. 5. Analysis by immunoblotting.

The presence as well as the relative molecular weights (M _r ) of the molecular forms of the recombinant EPIL polypeptide are determined by electrophoresis followed by immunoblotting. Briefly, the method consists in migrating the protein preparations to be tested in a polyacrylamide gel (30% w / v acrylamide, 0.8% w / v bis acrylamide; Tris-Hcl 1.5 m, pH 8.8) for 30 minutes at 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 (ECL kit, 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. , 9 and 16 contain different protein populations varying by their mass relative molecular (Figure 9). These relative masses are between 13 kDa and 32 kDa.

6. Analysis by mass spectrometry by electro-spray ionization in positive mode This study is carried out on a PlatformII analyzer (Micromas Ltd) using horse myoglobin as standard (Molecular mass: 16,951.50 ± 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 CH ₃ CN / H20 (V / V) and formic acid 0, 1%. Analysis of this sample reveals a mixture of two products with average experimental molecular weights: MI: 13,346.62 ± 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 disulfide 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 (BamHI, Xbal, ECOR1) and 3 / by automatic sequencing (primers specific for INSL4 DNA and the multiple cloning site of the plas ide pSec

Tag C ™). The functionality of the open reading frame is confirmed by a study of transcription / translation in vi tro (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 μg of plasmid DNA allows genomic integration of the INSL4 recombinant DNA. Thanks to the presence of an antibiotic resistance marker present in the vector, cell clones stable recombinants, having integrated pSec Tag C INSL4, are selected by culture in a medium containing 600 μg / ml of ZEOCIN ™ (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 pSec Tag C INSL4 recombinant transcript is demonstrated in all the 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.

RT-PCR analysis of mRNAs extracted from CHO cells transfected with the CHO-pSec Tag C MOCK construct

(empty control vector) as well as those extracted from the parental CHO cells shows the absence of expression of the recombinant INSL4 transcript after 30 amplification cycles.

4. Detection of the recombinant polypeptide

The purification of the various recombinant placental hormonal forms produced from the culture media and the 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, on umbilical cord vascular tissue and on tissue specific to patients with Kaposi's sarcoma. A) Tissue samples 1) Placental tissue samples

Placental villi obtained from early (7 weeks) and long-term placenta were provided by the Hospital's Obstetrics and Gynecology Department

Antoine Béclère, France, according to the appropriate procedures by the ethics committee.

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

It also uses cells of villous cytotrophoblasts and syncytiotrophoblasts previously purified from placenta at term. The isolation of cytotrophoblast cells from the villous placenta is carried out 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 differentiated villous syncytiotrophoblasts, formed in vitro from cytotrophoblasts (4-5 days in culture), are highly purified (less than 1% of contaminating cells). For clarity, the cells purified from villous cytotrophoblasts and the cells in vitro differentiated from syncytiotrophoblasts are referred to herein as cytotrophoblasts and syncytiotrophoblasts respectively. 2) Tissue of embryonic vessels

The immunohistochemical analysis of the embryonic vessels was carried out both on trophoblastic villus sections of 10 SA (Antoine Béclère hospital) and of an umbilical cord of a human embryo of 11 SA (Robert Debré hospital, France) 2 samples obtained as part of the voluntary termination of pregnancy.

3) Specific tissue of patients with Kaposi's sarcoma

28 skin biopsies of classical SK and associated with AIDS were searched in the archives of the Gustave-Roussy Institute and used for immunohistochemistry.

These are tissues fixed in a Bouin solution, included in paraffin. The tissue sections (3 μm) were mounted on SuperFrost / Plus slides and dewaxed with Xylene. The sections, once rehydrated, were heated by microwaves (3 x 5 min.) In a solution of citrate buffer (10 mM, pH 6). B) Immunohistochemistry

1) Samples of placental tissue

Placental tissue aged 6 to 12 weeks (n = 6) and term placental tissue (n = 2) are used. Tissue sections of 3 μm are produced, mounted on SuperFrost / Plus plates and degreased on xylene. The rehydrated sections are passed over a microwave 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 made with sections incubated in the presence of non-specific antibodies (pre-immune serum) or in the presence of antibodies pre-absorbed on an excess of corresponding peptide (FIG. 7).

2) Tissue of embryonic vessels and specific tissue of patients with Kaposi's sarcoma

After washing with a TBS buffer (Dako), the sections were incubated with the given antiserum for 20 min., Then washed and revealed using a biotinylated antibody complex followed by an alkaline phosphatase coupled to streptavidin (PA-streptavidin) . The labeling is completed after incubation with a chromogenic solution of the substrate (DAKO New Fuchsin substrate System, Carpinteria, CA).

The polyclonal antibody 7381 (immune serum diluted with

1: 500) was used as the primary antibody. It is an antibody recognizing EPIL 1 or EPIL 3 since recognizing the region 59-108 of the polypeptide chain

EPIL.

The negative controls used are the preimmune serum (Jo) and the 7381 immunserum inhibited by the corresponding peptide (59-108Y) with an incubation of 18 hours. The other primary antibodies (Ab) were chosen as positive controls making it possible to confirm the diagnosis of SK: Antifactor VIII Ab (endothelial phenotype) and alpha vascular smooth muscle antiactin Ab (smooth muscle phenotype).

The conditions for inhibition of Ac 7381 are carried out by incubation for 18 hours in the presence of:

150 μl Ac 7381 diluted to 1/250 and 150 μl of corresponding peptide (59-108Y) (629bBis) at 2 10 ^"4 M equivalent to 300 μl Ac 7381 diluted to 1/500 for 10 ^{~ 4} M of corresponding peptide (59 -108Y) (629bBis).

For the immunostaining of the following samples: - trophoblastic villus n ° X991 formalin (12SA), 8785 (umbilical cord) 320818 (2) (SK), 293905-7 (SK), 240444 (SK), 293626-1 (SK) (3 μm-microtome sections), the following antibodies (Ab) were used:

- 7381 imun anti chain C serum (59-108Y) diluted to 1/500;

- 7381 OJ (preimmun) diluted to 1/500; - Anti-factor VIII antibody diluted 1/30;

- Anti-actin mouse anti-actin mouse from prediluted human smooth muscle alpha (Dako, code N 1584) (1A4).

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 the 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

1) Placental tissue

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 and / or EPIL 3 polypeptide. Indeed, the 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 antibody 7381 binds only to the C domain of the putative EPIL polypeptide at its 59-88 portion 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%) of similarity with the putative EPIL polypeptide in the portion of domain C corresponding to region 59-88 of the EPIL polypeptide.

The 1661 antibody 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 B peptides 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 regions are not significantly similar.

The munohistochemical methods based on the 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 immunolabeling carried out with the antibody 7381 directed against the polypeptide EPIL 1 and / or EPIL 3 is very important in cytotrophoblasts of early placenta whereas it is light or even absent in 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 anti-EPIL 1 polypeptide 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 the differentiated villous syncytiotrophoblasts in vi tro 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, whereas the extravillous trophoblasts transcribe and translate the HLA class I genes (Hunt, JS 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 INSR4 mRNA, but contain immunoreactive EPIL 1 and / or EPIL 3 polypeptides, and EPIL 2. On the other hand, it is of great interest to observe that the important immunostaining of the trophoblastic tissues of the early placenta obtained with EPIL 2, is similar to those described for several growth factors, including TGFα and 1 EGF. (Horowitz, GM et al., 1993). Another surprising result is the significant expression of EPIL 1 and / or EPIL 3 polypeptides in cytotrophoblasts compared to syncytiotrophoblasts, whereas both hybridization studies in if performed on early placenta (results not shown) and 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 syncytio-trophoblasts.

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 peptide C of the putative EPIL polypeptide is maintained whereas the EPIL 2 present in the syncytiotrophoblasts does not present a peptide C junction. Thus, we can deduce from these results and these observations that the EPIL 1 and / or EPIL 3 polypeptide containing the peptide C can be secreted from the cytotrophoblasts in the liquid amniotic and that EPIL 2 not containing peptide C can be secreted from syncytiotrophoblasts in the maternal circulation. EPIL 3, corresponding to peptide C, is by definition also recognized by antichain C 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 EPIL polypeptide is most certainly involved in the process of differentiation, proliferation and regeneration. cells, especially endothelial cells like vascular cells.

2) Tissue of embryonic vessels

The objective of the immunohistochemistry experiment series is to study more precisely the intravillository embryonic vessels as well as the umbilical cord of the 1st, 2nd and 3rd trimesters. Using the 7381 antiserum at a dilution of 1: 500, the immunohistochemistry experiments confirm the presence of immunostaining at the level of the embryonic vessels of the floating villi, in particular at the level of the endothelial cells in a case of 12 SA trophoblastic sampling. . However, it is noted for the first time that the embryonic vessels of primary villi exhibit immunolabelling of the cells constituting the vessel wall, the smooth muscle cells, while the endothelial cells are almost no longer marked.

Similarly, this phenomenon seems to be amplified at the level of the vessels of the umbilical cord (see Figure 22) with, at the level of the vein and the 2 arteries a marking of the cells of the media therefore of the smooth muscle cells, with a complete disappearance of the labeling of endothelial cells (see Tables 1 and 2 below).

Table 1

Smooth Endothelial Muscle Cells

Embryonic vx

1. floating + ++ villosity

2. primary + ++ or secondary trunk

(villus)

3. umbilical cord - ++

Legend of Table 1: Immunohistochemical detection (PA, Dako, LSAB2) of the EPIL 1 and / or EPIL 3 polypeptide by the immune serum 7381 (1/500 dilution) in the embryonic vessels of trophoblastic villi (12 SA) and of an umbilical cord (AA SA) from the ^1st trimester of pregnancy

(3μ cut). We observe both at the level of the trophoblastic villi of the primary trunks (see Figure 21) and at the level of the umbilical cord, a stronger immunostaining of smooth muscle cells (media) (see Figure 22) compared to endothelial cells. The intensity of the marking cells is represented as follows: (-) absence of labeling; (+) low intensity marking; (++) high intensity marking.

Table 2

Cytotrophoblast

Syncytio- too oblast

Vx embryonic (primary trunk) endothelium + + Media ++ ++

Umbilical cord (11 SA) Media + NT * Endoth + Media + Vx (3 vx) (3 vx) (3 vx)

Amnion + NT NT

Remains embryo. Vitellin bag ++ Allantoide ++ not tested.

Legend of Table 2: Immunohistochemical analysis (AP,

Dako, LSAB2) with the immune serum 7381 anti-peptide C

(dilution 1/500), the polyconal anti-factor VIII antibody

(Dako, Ref. A0082) and anti-actin α smooth muscle monoclonal (Dako, Ref. M1584) on sections (3 μ) of trophoblastic villi (primary trunk) and of an umbilical cord from the ^1st trimester of pregnancy. A labeling similarity is observed between the immune serum 7381. Furthermore, there is an absence of labeling by the pre-immune polyclonal antibody 7381 and the immune serum 7381 inhibited by the peptide 59-108Y. The intensity of labeling of the cells is represented as follows: (-) absence of labeling; (+) low intensity marking; (++) high intensity marking. Remains embryo. : embryonic remains; Endoth. : endothelium; Vx: vessels; NT: not tested; Im. Ser. : immune serum; OJ: preimmune serum.

Reading the slides corresponding to the umbilical cord (see Table 2 and Figure 22)) also highlights the endodermal embryonic vestiges, in particular the yolk sac (yolk sac) and the allantois. These structures are visible alongside the three vessels and are marked by the immune serum 7381 antichaine C. It is interesting to note that the allantois, as well as the yolk sac (yolk sac) are embryonic structures (of endodermal origin) very important in terms of vasculogenesis. Indeed, these structures are the seat of the process of differentiation of hemangioblastic cells (derived from the mesoderm) into angioblastic and hematopoietic cells. Moreover, it is interesting to recall that this differentiation process corresponding to the first stages of vasculogenesis (from the ^3rd week of gestation) and appears to be induced VEGF not synthesized by the endoderm (Risau, W., 1997).

Role of the EPIL polypeptide in the process of decidualization and embryonic implantation The decidualization process of the endometrium is essential for implantation of the embryo to be possible. This process is dependent on the ovarian steroid hormones, especially progesterone. One of the modifications during decidualization concerns the stromal cells of the endometrium. Indeed, the stromal cells undergo in the vicinity of the implantation site morphological (polygonal, cytoplasm of enlarged size, appearance of epitheloid cells) and functional modifications (rich in glycogen and lipid, autocrine secretion and / or paracrine of prolactin, relaxin, renin, IGF and IGFBP) (Speroff, L. et al., 1995; Edwards, RG, 1995). More recently, it has been shown that growth factors of the EGF family (EGF, TGF-alpha, HB-EGF, amphiregulin, heregulin, betacellulin, epiregulin) could play an important role in the mechanisms of initiation of implantation. , including attachment. Indeed, in murine models, the expression of the EGF receptor (EGF-R) regulated by progesterone and E2 has been demonstrated both in the blastocyst and in uterine stromal cells, which makes molecules of this large family of EGF of potential ligands with modulating activity of implantation (SK Das, International Symposium on Embryo Implantation, October 1997). It is interesting to note that in the human species, a study has demonstrated the localization of transforming growth factor-alpha (TGF-alpha) in the endometrium, the decidue and the trophoblast (Horowitz, GM et al., 1993). In addition, decidualization of stromal cells is accompanied by changes in cell motility which involves a change in the extracellular matrix (secretion of laminin by stromal cells, for example). It appears in rats that the expression of basic bFGF or fibroblast growth factor at the level of uterine stromal cells, is dependent on progesterone and could be a growth factor co-regulating stromal cells and therefore be involved in the differentiation process. (decidualization). Decidualized stromal cells synthesize and secrete prolactin (PRL). An in vitro study of stromal endometrial cells has shown that the production of PRL is probably regulated by the combined effect of steroid hormones (estrogen and projesterone) and a peptide, relaxin (Huang, JR et al., 1987) . More recently, while the source of endogenous relaxin is the corpus luteum, the presence of relaxin in the glandular epithelium and endometrial stromal cells has been demonstrated as well as its autocrine / paracrine role on the secretion of PRL by the endometrium in vitro (Bryant-Greewood et al., 1993; Zhu, HH et al., 1990; Rosenberg, M. et al., 1991).

When we consider the expression of EPIL 1 or 3, we have demonstrated by immunohistochemistry with the immune antichain C serum a marking of the decidualized stromal cells, as well as of the endometrial glandular epithelium of the deciduous or deciduous parietal and basal 1 ^st trimester pregnancies (see Figure 23). It is interesting to note that in a similar way to the growth factors of the family of EGF, in particular TGF-alpha, the localization of EPIL 1 or 3 is found both at the level of the trophoblast and at the level of the deciduous

(especially in the endometrial glands and stromal cells). Thus, the EPIL polypeptide should be involved in the implantation process at the time of attachment, like TGF-alpha. In addition, the EPIL polypeptide should also be involved in the differentiation of internal stromal cells which constitute one of the cellular changes in decidualization. Indeed, like relaxin, the EPIL polypeptide should have a stimulating role on the synthesis of PRL by stromal cells and thus participate in the decidualization process. Furthermore, due to a labeling of trophoblastic cells of non-invasive phenotype (intravillous cytotrophoblast), it is reasonable to think that the EPIL polypeptide secreted by the stromal cells and by the cells of the glandular epithelium of the endometrium should play a role in controlling the invasion of extravillous cytotrophoblasts in the same way as TGF-beta (Giudice, L., 1994).

3) Determination of the different forms of the EPIL polypeptide, application to the assay of EPIL in the ammiotic liquid a) Assay method

For example, a sandwich assay by 2-site immuno-radiometry (IRMA) is used for the detection and assay of the forms of EPIL polypeptide obtained obtained by recombinant route, in different fractions in the chromatography procedures or in the biological samples where these forms are likely to be present. This assay can be carried out for example by using a solid phase, such as polystyrene beads (Precision Plastic Bail, Chicago, MN), coated with monoclonal antibody EPIL 08 as capture antibody, and by using the monoclonal antibody EPIL 02 marked with ¹²⁵ I as a radiolabelled indicator. The beads coated with EPIL 08 monoclonal antibody are for example incubated for 2 hours in the presence of the test sample diluted in calf serum. One can for example use as controls synthetic peptides such as the peptide corresponding to the portion 76-139 of the EPIL, in a range of concentrations such as a range going from 5 ng / ml to 2000 ng / ml. The sensitivity of the assay obtained, approximately 50 ng / ml with a 2 hour incubation of the sample to be analyzed, can be improved for example by proceeding with the 2-stage incubation of 6 and 18 hour incubations, approximately 10 ng / mL (see Figure 19). b) Application to the dosage of EPIL in amniotic fluid

The dosage of EPIL in the amniotic fluid of pregnant women is carried out by immunoradiometric assay according to the method described above. Concentrations of EPIL polypeptides ranging from 10 to several hundred ng / mL are detected in pregnant women. Figure 20 shows the results obtained on a panel of women, other results have shown that the concentrations can reach several hundred ng / mL. c) Application to the assay of EPIL in the serum The assay of EPIL in the serum is carried out by immunoradiometric assay according to the method described above. Concentrations of EPIL polypeptides ranging from 10 to 100 ng / mL are detected. 4) Kaposi's sarcoma

The objective of the immunohistochemistry experiment series is to study the expression of the EPIL 1 or EPIL 3 polypeptide at the level of SK biopsies. The various immunohistochemistry performed with the polyclonal antichain C antiserum directed against EPIL 1 and / or EPIL 3 (ac 7381 immunserum diluted 1: 500) made it possible to detect the polypeptides EPIL 1 and / or EPIL 3 at the level of '' about 50% of the 28 cases of SK. The immunostaining is localized at the level of the fusiform tumor cells or spindle cells as well as at the level of intratumoral vessels and / or vessels of the "slit-like" type (without endothelium) and most often in late lesions of the nodular type. The intensity of the immunostaining is moderate (+ to ++) in the majority of cases, with however in 8 cases the observation of an intense immunostaining (+++). Out of the 4 cases of SK associated with AIDS, only one case has weak to moderate labeling in tumor cells, the others being all negative. The experiment, the results of which are shown in Table 3 (cf. Table 3 below), has confirmed an intense immunostaining, noted (3) on the table, at the level of 4 classic skin nodular stage SKs. Immunolabelling is relatively heterogeneous localized in the cytoplasm of fusiform tumor cells (SC). In ^'addition, all cases of SK show immunostaining by 1' 7381 immune serum of the squamous epithelium (skin) and the sebaceous glands. In 1 case, we can note a marking gradient effect with a greater marking intensity of the skin in the region overlying the tumor. Skin marking rather concerns the basal cells of the squamous epithelium with cytoplasmic and nuclear labeling. It should be noted that normal (healthy) skin did not exhibit immunostaining with the immune serum 7381 during experiments carried out in parallel. The negative controls both the pre-immune serum (7381 OJ) and the immune serum 7381 pre-absorbed by the peptide 59-108Y show no labeling.

Figures 10, 12, 14 and 16 (X 100, X 200, X 1000, X 400, respectively) correspond to the results obtained for a case of classic cutaneous Kaposi's sarcoma after immunohistochemistry (PA, Dako LSAB2) with 1 ' immune serum

7381 antichaine C (dilution 1: 500).

Relatively homogeneous labeling of tumor cells (SC) of marked intensity (++) is observed. On the other hand, the fibrous partitions are not marked (Figures 10 and 14).

FIGS. 11, 13, 15 and 17 (X 100, X 200, X 1000 and X 1000, respectively) correspond to the negative control after inhibition of the immune serum 7381 by the peptide 59-108 Y (18 hours of incubation) using the same immunohistochemistry technique. There is an absence of complete labeling.

Table 3

7381 im. 7381 7381 Anti nti actin

Ser. Marking OJ im. ser, A factor alpha- / Tissue 1/500 1/500 inhibited

1/500 VIII smooth muscle

Sarcoma of

E.M. + + (noy> cyt) nb + +

Vx qq + + (media and (Endoth.) Fibrous septum)

Sarcoma of

Kaposi N ° 2

S.C. ++ + qq ++ (<5%)

E.M. ++ (nooy> cyt)

Vx qq ++ + (Endoth.) (Media)

Sarcoma of

Kaposi N ° 3

S.C. ++

E.M. ++

(noy≈cy.

Basal layer ++)

Vx ++ (media) (10%)

Sarcoma of

Kaposi N ° 4

S.C. ++ ++ (30%)

E.M.

(nucleus-cyt.

Basal layer ++)

Vx ++ (media)

SC: Spindle cells; Vx: Vessels EM: Squamous epithelium; Noy: Core; nb: many qq: some; Cyt: Cytoplasm Legend of Table 3: Immunohistochemical analysis (Pa, Dako, LSAB2) with the immune serum 7381 antichain C (dilution (1/500), the polyclonal anti-factor VIII antibody (Dako,

Ref. A0082) and anti-actin α smooth muscle monoclonal (Dako, Ref. M1584) on sections (3 μ) of skin biopsy of Kaposi's sarcoma (classic type) at the nodular histological stage. It is observed that the immune serum 7381 marks the tumor cells "spindle cells" of the SK in a more marked and more extensive manner than the polyclonal antibody antifactor VIII and monoclonal anti-actin α smooth muscle. In addition, there is a marking of the squamous epithelium of the skin by the immune serum 7381 both nuclear and cytoplasmic with a predominance at the level of the basal layer. There is an absence of labeling in the four cases of SK when the pre-immune polyclonal antibody 7381 and the immune serum 7381 inhibited by the peptide 59-108Y are used. The intensity of labeling of the cells is represented as follows: (-) absence of labeling; (+) low intensity marking; (++) high intensity marking. Demonstrated by immunohistochemistry on biopsies and by serum assay, SK cells seem to secrete cytokines, some of which are angiogenic factors (VEGF, FGF-b, PDGF). Recently, by IH and HIS, epidermal keratinocytes bordering an ulcerated region or in a hyperplastic region seem to be one of the sources of VEGF. In addition, expression of the KDR and flt-1 receptor mRNA, two tyrosine kinase receptors of the endothelial cell, seems to be increased in the SK cells themselves, which would be in favor of a double action of paracrine. (Keratinocytes and macrophages) and direct autocrine of VEGF on the SK cell (Brown et al., 1996). More recently, Masood et al. (1997) demonstrated that VEGF is an autocrine growth factor for SK associated with AIDS, making it probably the most important angiogenic factor in this type of tumor. These results show that the expression of the INSL4 gene belonging to the insulin family is localized in particular at the level of the placenta, the umbilical cord and at the level of tumor cells such as those of Kaposi's sarcoma. Monoclonal and polyclonal antibodies have been produced against different synthetic peptides corresponding to the different chains A, C and B of native putative polypeptide.

Polyclonal rabbit antibodies recognizing the C chain (7381 immune serum) and the A and B chain (1661 immune serum) made it possible to demonstrate the polypeptide EPIL 1 and / or EPIL 3, and EPIL 2 respectively at the placenta with expression of the EPIL 1 and / or EPIL 3 polypeptide at the level of cytotrophoblastic cells. In addition, we had observed a surprising labeling of intravillary embryonic endothelial cells by polyclonal Ab 7381. Recently, Zhou et al. (1997) showed how important the differentiation of extravillous cytotrophoblasts into cells with endothelial phenotype was important in the physiology of placental development. Our observation, on the other hand, objectifying the immunolabeling of the EPIL 1 and / EPIL 3 polypeptide at the cytotrophoblastic level and of the intravillository embryonic vessels allows us to deduce therefrom that the EPIL polypeptide would play an essential role in embryonic vasculogenesis ^" as has been recently demonstrated for VEGF, an angiogenic cytokine inducing the proliferation of endothelial cells and an increase in vascular permeability (Risau et al., 1997) .In fact, it is interesting to note that Clark et al. in 1996 demonstrated by immunohistochemistry the presence of the VEGF protein and its Flt-1 receptor at the level of the intravillous cytotrophoblast and the endothelium of the intravillary embryonic vessels. These homologies of immunolabeling with VEGF and other cytokines which we have demonstrated by the presence of EPIL polypeptide at tumor level Angioproliferative, Kaposi's sarcoma, also show that the EPIL polypeptide is implicated as a growth factor for tumor cells like VEGF and other cytokines whose role is well known. Furthermore, it appears that IGF-I, a member of the insulin family, induces the expression of VEGF in colorectal cancer as well as in an in vitro model of osteoblasts (Goad et al., 1996) .

Thus these results show that the EPIL polypeptide is most certainly linked to the process of embryonic vasculogenesis and tumor angiogenesis.

Insulin-like activity of the EPIL polypeptide (EPIL 1, 2 and / or 3)

Reminder of the physiological role of insulin Carbohydrates (HC) or carbohydrates are the main source of energy in the body. Regulatory systems ensure a constant adjustment between HC requirements and glucose formation. The organs involved in this regulation are the pancreas and the liver. The beta cells of the islets of Langerhans of the pancreas are the place of synthesis and secretion of insulin, but also of glucagon and somatostatin. The liver has a function of storing glucose by glycogenesis (like adipocytes) and of synthesis of glucose by neoglucogenesis. Its vital function is to make glucose available by glycogenolysis if necessary (hypoglycemia).

Proinsulin is made up of two chains A and B with a chain C called the connecting peptide. Insulin is biologically active after having undergone a cleavage of its C peptide. It acts on a transmembrane receptor of the tyrosine kinase family which via a phosphorylation cascade intervenes on the regulation of enzymes of the different metabolic pathways ( carbohydrate, fat and protein). The main role of insulin is to allow the use of glucose by the cell as well as the synthesis of lipids and proteins (anabolism).

• The use of glucose is facilitated, for example, in the muscle / adipocyte by a stimulating action of glucose and insulin on its transporter (GLUT-4). Insulin stimulates or inhibits the expression of a series of enzymes involved in the metabolic glucose pathway of E den-Meyerhof such as glucokinase (GK), PFK2 and LPK (insulin stimulating action), PEPCK and F-1,6-P2ase (insulin inhibiting action). Through the action of insulin, glucose is then metabolized to pyruvate and actate, then to C0 ₂ , H ₂ 0 and ATP after passage through the Krebs cycle or citric acid cycle (at the level of mitochodria by oxidative phosphorylation). It should be noted that another way of degrading G6P (glucose 6 phosphate) is possible, the pathway of pentose phosphate, also stimulated by insulin and responsible for the formation NADPH +, coenzyme essential for the biosynthesis of lipids. In addition, the pentose pathway is the source of essential ribose for nucleic acid synthesis.

• The synthesis of lipids is also facilitated by insulin and is carried out from acetyl (CoA). There is also stimulation of protein synthesis by insulin.

Pathophysiology of diabetes

There are two types of diabetes: type I or juvenile diabetes. It is an autoimmune disease characterized by destruction of the islets of Langerhans, therefore not an insulin deficiency; type II or adult diabetes, on the other hand, is characterized by insulin resistance.

Gestational diabetes: its diagnosis is made during pregnancy (fasting hyperglycemia) and generally disappears after childbirth. During the second trimester, the insulin level increases considerably in part by the increase in hormonal anti-insulin activity. Indeed, the placenta produces diabetogenic hormones such as human lactogenic placental hormone (hPL), estrogen and progesterone. In addition, pituitary prolactin and cortisol are antagonists of

1 insulin. The pathophysiological mechanisms are probably multiple. There has been evidence, for example, of an increase in the breakdown of insulin during pregnancy. This degradation is caused by placental enzymes comparable to hepatic insulinases (Freinkel, N. et al., 1991). By a phenomenon combining on the one hand resistance to insulin of hormonal origin

(hPL, PRL) and the increase in the breakdown of insulin, the level of insulin available decreases and a mechanism of pancreatic hypersecretion regulator is then set in motion. If the pancreas has a dysfunction, it will not be able to respond to this new physiological state of pregnancy and glucose intolerance or even gestational diabetes may result (Becker,

K.L. et al. , 1990) . Complications of diabetes

The major complication is cardiovascular. The mechanisms underlying this diabetic complication are those of atherosclerosis and constitute diabetic macro-angiopathy. Briefly, in diabetes, endothelial cells of the vessels undergoes various aggressions ^"Physical and chemical such as hypertension, hyperglycemia, and 1 hypercholesterolemia. These repeated lesions 1 vascular endothelium lead through macrophages, platelets and growth factors to the progressive accumulation of smooth muscle cells, extracellular matrix and lipids in the intima and the media. These are the lesions that contribute to the narrowing of the vascular lumen. of the EPIL polypeptide

• EPIL is an ORP (oxygen-related protein) or GRP (glucose-related protein) type like VEGF. From the third trimester, the placenta becomes the organ through which fetal-maternal exchanges take place, in particular via a considerably developed embryonic vascular network. It is interesting to recall that recently certain studies have demonstrated the role of VEGF in embryonic vasculogenesis with an increased expression from the 3 ^rd week of gestation in the endoderm, responsible for the differentiation of the overlying mesenchyme into hemangioblasts (Risau, W . , 1997) . In addition, VEGF has been shown to be an oxygen-regulated protein (ORP) and glucose (GRP). Indeed, Stein et al. in 1995 demonstrates that hypoxia and hypoglycemia seem to increase the expression of VEGF, in particular by a mechanism for stabilizing its messenger RNA (Stein, I. et al., 1995). Similarly, the glucose transporter GLUT-1 is stimulated by hypoglycemia and allows the cell in a hostile environment to use the maximum amount of glucose as an energy source (Stein, I. et al., 1995). The EPIL polypeptide should be involved in these stress response processes, either hypoxic or hypoglycemic through an insulin-like effect. Indeed, in addition to the structural homology of the EPIL polypeptide and insulin as described above, we clearly demonstrated by immunohistochemistry a similar cellular localization between EPIL and VEGF with labeling of the cytotrophoblast, smooth muscle cells of embryonic vessels as well as embryonic vestiges at the level of the umbilical cord. The coexpression of the EPIL and VEGF polypeptide in identical cells and tissues makes it possible to establish several possible mechanisms. As we said above, insulin allows the use of intracellular glucose and therefore allows the cell to produce ATP, ribose (pentose pathway). Insulin is an anabolic hormone which also allows the biosynthesis of lipids and proteins. Both in placental that at the level of a neoplastic tissue, the rate of cell division is important and the energy need more important. If the neovascularization process is important for the survival of a tissue under stress (VEGF), it is likely that the EPIL polypeptide by an insulin-like para / autocrine effect intervenes in this process by promoting the use of glucose as energy source.

• Insulin-like effect of EPIL and the placenta. The placenta is until the end of the first trimester in an environment of relative hypoxia. Similarly, we can think that in terms of nutrients, in particular glucose, it is most certainly in a hypoglycemic state. However, it is a highly mitogenic tissue of pseudotumoral type, therefore in increased energy need. Also, producing an insulin-like molecule with improved glucose use would allow the placenta to continue its development and growth. In addition, it should be remembered that the placenta is responsible for the breakdown of insulin and that it is the source of diabetogenic proteins such as hPL, estrogens and progesterone. The EPIL polypeptide should thus play the role of insulin by an insulin-like auto / paracrine effect.

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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 the 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 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 included between positions 1 and 139, ends included, of the amino acid sequence defined in FIG. 1.

2. 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 of: a) an A chain, the 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 included; said EPIL 2 polypeptide may also comprise 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 sequence of the signal peptide comprising at least the sequence between positions 1 and 17, ends included.

3. EPIL 3 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in human or animal cells, in particular in human placenta trophoblast cells and in that it consists of a C chain whose sequence of amino acids 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 .

4. Polypeptide according to one of claims 1 and 3, characterized in that it is preferentially expressed in cytotrophoblasts of human placenta.

5. Polypeptide according to one of claims 1 and 3, characterized in that it is present in endothelial cells.

6. Polypeptide according to claim 5, characterized in that the endothelial cells are vascular endothelial cells.

7. Polypeptide according to claim 6, characterized in that the vascular endothelial cells are fetal vascular endothelial cells.

8. Polypeptide according to one of claims 1, and 3 to 7, characterized in that it is secreted in the amniotic fluid and / or in the fetal circulation.

9. Polypeptide according to one of claims 1 and 3, characterized in that it is present in smooth muscle cells.

10. Polypeptide according to claim 9, characterized in that the smooth muscle cells are vascular smooth muscle cells constituting the vessel wall.

11. Polypeptide according to claim 10, characterized in that the vascular smooth muscle cells are cells constituting the wall of the embryonic vessel or of the umbilical cord.

12. Polypeptide according to one of claims 1 and 3, characterized in that it is expressed in stromal cells.

13. Polypeptide according to claim 12, characterized in that the stromal cells are cells of

1 amnion and / or embryonic vestige, preferably the yolk sac and the allantois located at the level of the umbilical cord.

14. Polypeptide according to one of claims 1 and 3, characterized in that it is present in tumor cells.

15. Polypeptide according to claim 14, characterized in that the tumor cells are tumor cells of angioproliferative tumor.

16. Polypeptide according to one of claims 14 and 15, characterized in that the tumor cells are tumor cells associated with Kaposi's sarcoma.

17. Polypeptide according to one of claims 14 to 16, characterized in that the tumor cells are intra-tumor vessel cells.

18. Polypeptide according to claim 16, characterized in that the tumor cells are Malpighi epithelial cells.

19. A polypeptide according to claim 2, characterized in that it is preferentially expressed in syncytiotrophoblasts of human placenta.

20. Polypeptide according to one of claims 2 and 19, characterized in that it is secreted in the maternal circulation.

21. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of differentiation of human and / or animal cells.

22. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of proliferation of human and / or animal cells.

23. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of regeneration of human and / or animal cells.

24. Polypeptide according to one of claims 21 to 23, characterized in that the human and / or animal cells are endothelial cells.

25. Polypeptide according to one of claims 21 to 23, characterized in that the human and / or animal cells are smooth muscle cells, in particular vascular cells.

26. Polypeptide according to one of claims 21 to 23, characterized in that the human and / or animal cells are epithelial cells.

27. Polypeptide according to one of claims 21 to 23, characterized in that the human and / or animal cells are stromal cells, in particular of the decidue, or of the glandular epithelium.

28. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of vascularization of embryonic cells.

29. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of vascularization of tumors.

30. A polypeptide according to claim 29, characterized in that the tumors are angioproliferative tumors.

31. Polypeptide according to one of claims 29 and 30, characterized in that the tumors are tumors associated with Kaposi's sarcoma.

32. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in the process of decidualization and / or implantation of the embryo.

33. Polypeptide according to one of claims 1 to 20, characterized in that it is involved in an insulin-like process, in particular paracrine and / or autocrine.

34. Polypeptide according to claim 33, characterized in that the insulin-like process is a response process to hypoxic stress and / or hypoglycemic stress.

35. Polypeptide according to one of claims 33 and 34, characterized in that it is involved in a process promoting the use of glucose as an energy source.

36. A polypeptide fragment according to one of claims 1 to 35, characterized in that it is biologically active.

37. Nucleic acid sequence characterized in that it codes for a polypeptide or one of its fragments according to one of claims 1 to 36, its complementary sequence or the sequence of its corresponding RNA.

38. A cloning and / or expression vector containing a nucleic acid sequence according to claim 37.

39. Vector according to claim 38, characterized in that it comprises the elements allowing the expression and / or the secretion of said sequences in a host cell.

40. Host cell transformed by a vector according to one of claims 38 and 39.

41. Method for producing a recombinant polypeptide characterized in that it implements a cell according to claim 40.

42. Method for producing a recombinant polypeptide according to claim 41, characterized in that the transformed cells according to claim 40 are cultured under conditions allowing the expression of a recombinant polypeptide, and that said recombinant polypeptide is recovered.

43. A recombinant polypeptide ^"obtainable by a method according to one of claims 41 and 42.

44. Polypeptide or one of its fragments according to one of claims 1 to 36, characterized in that it is obtained by chemical synthesis.

45. Monoclonal and polyclonal antibody 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 36, 43 and 44.

46. Antibody according to claim 45, characterized in that it is immunoconjugated or labeled.

47. Probe or primer oligonucleotide, characterized in that it consists of a nucleic acid sequence according to claim 37 or one of its fragments, or of a nucleic acid sequence capable of hybridizing specifically to a A nucleic acid sequence according to claim 37.

48. Oligonucleotide probe according to claim 47, characterized in that it is labeled.

49. Use of one or more antibodies according to one of claims 45 and 46, for the detection, identification, localization and / or assay of a polypeptide or of a fragment thereof according to one of claims 1 to 36, 43 and 44, or for the diagnosis of pathologies linked to the abnormal expression of the polypeptide according to one of claims 1 to 36.

50. Use of a probe or primer according to one of claims 47 and 48, for the detection, identification, localization, amplification and / or assay of nucleic acid sequence according to claim 37, or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37.

51. Method for the detection, identification, localization and / or specific assay of a polypeptide or one of its fragments according to one of claims 1 to 36, 43 and 44, in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of polypeptide according to one of claims 1 to 36, characterized in that it comprises the following stages: a) bringing the biological sample into contact with one or more antibodies according to one claims 45 and 46; b) highlighting, identification, location and / or determination of the antigen-antibody complex formed.

52. The method of claim 51, characterized in that the antibody used in step a) is fixed on a solid support and in that the detection, identification, localization and / or assay of the antigen complex -antibody formed from step b) is produced using an antibody according to claim 46.

53. The method of claim 51 or 52, for the determination of one of the forms of the EPIL polypeptide in the amniotic fluid and / or the serum.

54. Kit or kit for the detection, identification, localization and / or specific assay of a polypeptide or one of its fragments according to one of the claims 1 to 36, 43 and 44, in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of polypeptide according to one of claims 1 to 36, characterized in that it comprises the following elements: a) one or more antibodies according to one of claims 45 and 46; b) where appropriate, the reagents for constituting the medium suitable for the immunological reaction; c) where appropriate, the reagents allowing the detection, identification and / or determination of the antigen-antibody complexes produced by the immunological reaction.

55. Method for the detection, identification and / or specific assay of nucleic acid sequence according to claim 37 in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37, characterized in that it comprises the following stages: a) isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specific amplification of the nucleic acid sequences according to claim 37 using primers according to claim 47; c) qualitative and / or quantitative analysis of the amplification products.

56. Method for the detection, identification and / or specific assay of nucleic acid sequence according to claim 37 in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37, characterized in that it comprises the following stages: a) bringing an oligonucleotide probe according to one of claims 47 and 48 into contact with a biological sample, the DNA contained in the biological sample having, if necessary, previously been made accessible for hybridization, in 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 said oligonucleotide probe and the DNA of the biological sample.

57. Method for the detection, identification and / or specific assay of nucleic acid sequence according to claim 37 in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37, characterized in that it comprises the following steps: a) contacting an oligonucleotide probe according to claim 47 immobilized on a support with a biological sample, the DNA of the sample, having, where appropriate, has been previously amplified using primers according to claim 47 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 one of claims 47 and 48.

58. Kit or kit for the detection, identification and / or specific assay of acid sequence nucleic acid according to claim 37 in a biological sample or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37, characterized in that it comprises the following elements: a) an oligonucleotide probe according to the one of claims 47 and 48; b) the reagents necessary for carrying out a hybridization reaction; c) where appropriate, a pair of primers according to claim 47 as well as the reagents necessary for a DNA amplification reaction.

59. Kit or kit for the detection, identification and / or specific determination of nucleic acid sequence according to claim 37 in a biological sample, or for the diagnosis of pathologies linked to the abnormal presence of nucleic acid sequence according to claim 37, characterized in that it comprises the following elements: a) an oligonucleotide probe, called capture probe, according to claim 47 which can be supplied pre-immobilized on a support; b) an oligonucleotide probe, said revelation probe, in particular labeled, according to one of claims 47 and 48; c) where appropriate, a pair of primers according to claim 47 as well as the reagents necessary for a DNA amplification reaction.

60. Method or kit according to one of claims 51 to 59, for the diagnosis of pathologies linked to the presence of tumor cells.

61. Method or kit according to claim 60, for the diagnosis of Kaposi's sarcoma, in particular angioproliferative.

62. Cell labeling method, characterized in that it uses one or more antibodies according to one of claims 45 and 46.

63. Method for labeling a cell, characterized in that it uses a primer and / or a probe, in particular labeled, according to one of claims 47 and 48.

64. Marking method according to one of claims 62 and 63, characterized in that it implements a method according to one of claims 51 and 56.

65. A cell labeling method according to one of claims 62 to 64, characterized in that the cell is an endothelial cell.

66. A cell labeling method according to claim 65, characterized in that the endothelial cell is a vascular cell.

67. Method ^" of cell marking according to one of claims 62 to 64, characterized in that the cell is a smooth muscle cell.

68. Method of labeling a cell according to one of claims 62 to 64, characterized in that the cell is a stromal cell, in particular of the deciduous, or of the glandular epithelium, in particular the endometrial epithelium of the uterus.

69. Method for labeling a cell according to one of claims 62 to 64, characterized in that the cell is a tumor cell, in particular an angioproliferative cell.

70. Cell labeling method according to claim

69, characterized in that the cell is a tumor cell of Kaposi's sarcoma.

71. Method for obtaining a cell containing a polypeptide according to one of claims 1 to 36, 43 and 44 and / or a nucleic acid sequence according to claim 37, characterized in that it implements a method according to one of claims 62 to

70, and in that said labeled cell is isolated.

72. Cell in that it is capable of being selected by a method according to claim 71.

73. A method of detecting tumor cells, characterized in that it implements a method according to one of claims 62 to 70.

74. The method of claim 73, for the detection of ^cells' tumor associated Kaposi's sarcoma.

75. Method for selecting a chemical or biochemical compound capable of modulating the activity of a polypeptide according to one of claims 1 to 35, characterized in that it uses a polypeptide or one of its fragments according to one from claims 1 to 36, 43 and 44, a nucleic acid sequence according to claim 47 or a cell according to one of claims 40 and 72.

76. Selection method according to claim 75, characterized in that said selected compound is capable of modulating the differentiation, regeneration and / or proliferation of human and / or animal cells.

77. Selection method according to claim 76, characterized in that said selected compound is capable of promoting the differentiation, regeneration and / or proliferation of human and / or animal cells.

78. Selection method according to claim 76, characterized in that said selected compound is capable of inhibiting the differentiation, the regeneration and / or the proliferation of human and / or animal cells.

79. Selection method according to one of claims 76 to 78, characterized in that said cells are endothelial cells.

80. Selection method according to claim 79, characterized in that the endothelial cells are vascular cells.

81. Selection method according to one of claims 76 to 78, characterized in that said cells are smooth muscle cells.

82. Selection method according to claim 81, characterized in that said cells are vascular smooth muscle cells.

83. Selection method according to one of claims 76 to 78, characterized in that said cells are epithelial cells.

84. Selection method according to one of claims 76 to 78, characterized in that said cells are stromal cells, in particular of the deciduous, or of the glandular epithelium, in particular the endometrial epithelium of the uterus.

85. Selection method according to claim 75, characterized in that said selected compound is capable of modulating the process of decidualization and / or implantation of the embryo.

86. Selection method according to claim 85, characterized in that said selected compound is capable of promoting the process of decidualization and / or implantation of the embryo.

87. Selection method according to claim 85, characterized in that said selected compound is capable of inhibiting the process of decidualization and / or implantation of the embryo.

88. Selection method according to claim 75, characterized in that said selected compound is capable of modulating insulin-like activity.

89. Selection method according to claim 88, characterized in that said selected compound is capable of modulating the response of the cell to hypoxic and / or hypoglycemic stress.

90. Selection method according to claim 89, characterized in that said selected compound is capable of promoting the use of glucose as an energy source.

91. Chemical or biochemical compound, in that it is capable of being selected by a method according to one of claims 75 to 90.

92. A compound according to claim 91, as a medicament.

93. Compound according to one of claims 91 and 92, characterized in that it is chosen from: a) a polypeptide or one of its fragments according to one of claims 1 to 36, 43 and 44; b) an antibody according to claim 45; a chemical or biochemical compound, characterized in that it is a ligand of a polypeptide or one of its fragments according to one of claims 1 to 36; c) a vector according to one of claims 38 and 39; d) a vector according to one of claims 38 and 39 having on its surface a specific marker of the target cell whose one seeks to modulate the activity of a polypeptide according to one of claims 1 to 36; and e) a probe according to claim 47.

94. Compound according to one of claims 92 and 93, intended for the treatment of tumors.

95. Compound according to claim 94, intended for the treatment of angioproliferative tumors.

96. Compound according to claim 95, intended for the treatment of Kaposi's sarcoma.

97. Compound according to one of claims 94 and 95, characterized in that the tumor is a tumor of the pancreas, liver, uterus, breast, angiosarcoma, glioblastoma, neuroblastoma, rhabdomyosarcoma, or a leiomyosarcoma.

98. Compound according to claim 97, characterized in that the tumor is a leiomyosarcoma.

99. Compound according to one of claims 92 and 93, intended to promote the vascularization of specific tissues.

100. Compound according to one of claims 92 and 93, intended for the treatment of retinopathy, degeneration of the macula, psoriasis,

Endometriosis, rheumatoid arthritis, atherosclerosis or hyperthyroidism.

101. Compound according to claim 100, intended for the treatment of atherosclerosis, in particular lesions due to restenosis after angioplasty.

102. Compound according to one of claims 92 and 93, intended for regenerating and / or differentiating tissue, in particular endothelial, smooth or epithelial muscle, following a degenerative disease or a disease of the endocrine glands, in particular the thyroid or the pancreas, or as a result of damage to said tissue caused by trauma.

103. Compound according to one of claims 92 and 93, intended to promote or inhibit embryonic implantation.

104. Compound according to one of claims 92 and 93, intended to prevent and / or treat pathologies directly or indirectly linked to an activity of the insulin-like type.

105. Compound according to one of claim 104 intended to prevent and / or treat pathologies directly or indirectly linked to a dysfunction of the carbohydrate metabolism, in particular to hypoglycemia or hyperglycemia.

106. Compound according to either of claims 104 and 105, characterized in that the pathology is chosen from diabetes, in particular gestational diabetes, and complications linked to diabetes, in particular complications of a cardiovascular nature.