FR2809734A1 - New human tetraspan protein, useful for identifying specific modulators, e.g. for treating or diagnosing neurodegeneration, also related nucleic acid - Google Patents

Abstract

Isolated human tetraspan protein (I) having a 248 amino acid (aa) sequence (2), reproduced, its fragments of at least 5 aa and their derivatives. Independent claims are also included for the following: (1) nucleic acid (II) encoding (I), and its complement; (2) nucleic acid (IIa) containing at least 15 nucleotides (nt) able to hybridize to (II); (3) vector (III) containing (II); (4) cell (IV) containing (III); (5) preparation of (I) by culturing (IV); (6) mono- or poly-clonal antibodies (Ab), or their fragments, specific for (I); (7) (ant)agonists (A) of the activity of (I); (8) pharmaceutical composition containing (I), (II), Ab, (A), (III) or (IV); (9) diagnostic kit containing Ab or (II); and (10) (k) non-human mammal containing (IV).

Description

<Desc / Clms Page number 1>

 The subject of the present invention is a human tetraspane protein and its fragments, in particular its extracellular domain EC2, the corresponding nucleic acids, the vectors containing said nucleic acids, the antibodies directed against said tetraspane or its fragments as well as the pharmaceutical compositions comprising these preceding objects. . The present invention also relates to the use of said tetraspane or of an agonist of the latter in particular for reducing the migration of tumor cells.

 The tetraspanes superfamily (abbreviation TM4SF) groups together glycoproteins with structural and sequence homologies. These tetraspanes have four hydrophobic domains TM1 to TM4 (TM for transmembrane) which can constitute transmembrane domains allowing the formation of two outer loops, one of small size called EC1 (EC for extracellular) located at the N-terminal end and one more large size called EC2 located at the Cterminal end) (Maecker et al., 1997). The strongest homologies between the various members of the tetraspan superfamily are confined to the transmembrane domains. The greatest sequence diversity is found at the level of the extracellular loops and in particular EC2. However, four cysteine residues are conserved at the level of EC2: CCG motif located at approximately 50 amino acids after the TM3 domain, a cysteine residue at approximately 11 amino acids upstream of TM4, and a cysteine residue frequently found in a PXSC motif (X possibly be any of the amino acids) which is placed interchangeably in the EC2 domain.

 The tetraspans superfamily contains around twenty members, some of which are ubiquitous (such as, for example, the molecules CD9, CD63, CD81 and CD82) (Oren et al., 1990; Boucheix et al., 1991; Metzelaar et al., 1991; Gil et al., 1992) or of a more limited expression (such as, for example, the CD37 molecule on mature B lymphocytes, the CD53 molecule on lymphoid and meloid cells or neurospanin on nerve cells) (Classon et al., 1989; Amiot, 1990; and Bixby, 1999). The molecules of the tetraspan superfamily act as facilitators in the interactions of membrane molecules (Maecker et al., 1997).

 Many molecules of the tetraspan superfamily were initially identified as tumor antigens such as TAPA-1 / CD63 (Oren et al., 1990), Motility related protein-1 / CD9 (Boucheix et al., 1991), L6 (Marken et al.,

<Desc / Clms Page number 2>

 1992), Sarcoma Amplified Sequence (SAS) (Jankowski et al, 1994), TI-1 (Kallin et al., 1991) or CO-029 (Szala et al., 1990). Remarkably, it appears that the decrease in expression of certain molecules of the tetraspan superfamily is associated with an increase in the migration properties of tumor cells (tumor invasion process or metastasis). This phenomenon has been widely reported in the literature for the molecules CD9 (Miyake et al., 1991), CD63 (Hotta et al., 1988; Azorsa et al., 1991), and CD82 (Adachi et al., 1996; White et al., 1998). In the same way, the transfection of tumor cells with the cDNAs coding for the molecules CD9 (Ikeyama et al., 1993), CD63 (Hotta et al., 1991; Kondoh et al., 1993; Radford et al., 1995; Radford et al., 1996) or CD82 (Dong et al., 1995) induces a decrease in the mobility and therefore the aggressiveness of the tumor.

 The molecules of the tetraspan superfamily play a co-receptor role by forming oligomeric complexes whose role remains obscure. Among these multimeric structures, let us quote the interaction of the CD19 molecule with the tetaspanes CD9 / CD81 / CD82 (Horvath et al., 1998). The molecules of the major class II histocompatibility complex form oligomeric complexes with the tetaspanes CD37 / CD53 / CD81 / CD82 (Angelisova et al., 1994) or CD9 / CD63 / CD81 / CD82 (Rubinstein et al., 1996). The CD2 molecule interacts with tetraspane CD53 and the CD5 molecule interacts with tetraspane CD9 (Toyo-oka et al., 1999). The CD81 and CD82 molecules associate with the intracellular domains of the CD4 and CD8 molecules (Imai & Yoshie, 1993; Imai et al., 1995).

 The molecules of the tetraspanes superfamily also interact with the integrins, mainly those of the ss1 family, for example the interaction of the CD63 molecule with the integrins [alpha] 3ss1 (VLA-3) and [alpha] 6ss1 (VLA-6 ), of the molecule CD9 with the integrins [alpha] 4ss1 (VLA-4) and VLA-6, of the molecule CD 151 with the integrin [alpha] 5ss1 (Berdichevski et al., 1996; Rubinstein et al., 1994 ; Rubinstein et al., 1996; Mannion et al., 1996; Radford et al., 1996; Sincock et al., 1997) or of the molecule NAG-2 with [alpha] 3ss1 and [alpha] 6ss1 (Tachibana et al ., 1997). The CD9 and CD63 molecules also have the property of interacting with the LFA-1 molecule ([alpha] 1ss2). The role of integrins in cell adhesion and migration mechanisms as well as the capacity of molecules in the tetraspan superfamily to form complexes

<Desc / Clms Page number 3>

 oligomers with integrins make it possible to emphasize the role of the latter in the processes of tumor invasion.

 The association of the molecules of the tetraspan superfamily with costimulation molecules expressed by the cells of the immune system has made it possible to demonstrate their participation in the activation of certain immune cells.

Activation of the CD9 and CD82 molecules using monoclonal antibodies provides a costimulation signal to T lymphocytes stimulated with an anti-CD3 antibody (Lebelbinay et al., 1995; Tai et al., 1996). Anti-CD151 and anti-CD9 antibodies induce platelet aggregation and activation, respectively (Slupsky et al., 1989; Griffith et al., 1991). Cellular activation mediated by the molecules of the tetraspan superfamily is associated with the generation of second signals involved in signal transduction. An anti-CD53 antibody provides a costimulation signal to B cells activated via the immunoglobulin receptor (Rasmussen et al., 1994); this activation signal is mediated in part by protein kinase C (Bosca & Lazo, 1994). An anti-CD9 antibody activates the kinase p72syk (Ozaki et al., 1995) and intracellular signaling is associated with a GTP-binding protein (Seehafer & Shaw, 1991). Immunoprecipitation experiments on the CD63 and CD53 molecules show that they are associated with unidentified phosphatases (Carmo & Wright, 1995).

 The CD81 molecule has been identified as a receptor for the hepatitis C virus E2 molecule (Pileri et al., 1998; Flint et al., 1999). Mice not expressing the CD9 molecule (CD9 knockout) exhibit reduced fertility following an alteration in sperm-egg interactions (Kaji et al, 2000).

 The tetraspans superfamily has recently developed by the identification of seven new cDNAs coding for molecules presenting the structural characteristics of this family (Serru et al., 2000) as well as by the cloning in chicken of a new molecule, called neurospanin (Perron and Bixby, 2000).

 Thus, there remains today a great need to isolate and identify new tetraspanes. Such tetraspanes would indeed be likely to have an important role in modulating the interactions of membrane molecules but also in the process of tumor invasion. There is therefore a need for new pharmaceutical compositions useful in the treatment of diseases linked to alteration of expression

<Desc / Clms Page number 4>

 molecules from the tetraspan superfamily as well as new diagnostics to detect these diseases. This is precisely the object of the present invention.

 The present invention thus relates to a new isolated human tetraspane protein of sequence SEQ ID No. 2, one of its fragments of at least 5 amino acids or one of its derived proteins.

 This new tetraspane according to the invention was surprisingly isolated on the basis in particular of an expressed sequence label EST (EST for Expressed Sequence Tag) chosen from 1856102 EST from the bank db EST, available at the internet address "www. ncbi.nlm.nih.gov/dbEST/ ".

 In addition, as demonstrated in Example 2 below, the expression of this tetaspane is not limited to neurons. Thus, it has been demonstrated that this new teteraspane was also expressed in particular at the level of endothelial cells and T lymphocytes.

 The term “protein” is also intended to denote the terms of peptide or polypeptide, these 3 terms being used interchangeably in the present description.

 In the present description, in particular in the claims, unless specifically mentioned, the term “human tetraspane” will be understood to mean isolated human tetraspane of sequence SEQ ID No. 2.

 By fragment of human tetraspane of sequence SEQ ID No. 2, is meant in the present description any fragment whose sequence comprises at least 5 consecutive amino acids of the sequence SEQ ID No. 2, preferably 8, 10, 15, 20, 25.50 and 100 consecutive amino acids of the sequence SEQ ID No. 2.

 By protein derived from human tetraspane of sequence SEQ ID No. 2, is meant in the present description the proteins comprising a sequence having a percentage of identity after optimal alignment of at least 90%, preferably 95%, and 99 % with the sequence SEQ ID No. 2.

 Among said fragments and said proteins derived from the protein of sequence SEQ ID No. 2, preference is given to those capable of exercising at least partially one of the activities of the tetraspane proteins as mentioned above, or of modulating at least partially one of the activities tetraspane proteins mentioned above.

 Among said fragments and said proteins derived from the protein of sequence SEQ ID No. 2, particular preference is given to those capable of inhibiting or

<Desc / Clms Page number 5>

 to at least partially increase one of the activities of the tetraspane proteins as mentioned above, said fragments and said derived proteins thus being able to be used to modulate, in particular decrease, the biological activity of tetraspane.

 Thus, said derived proteins may comprise one or more substitutions, preferably conservative, deletions or insertions, compared with the wild protein of sequence SEQ ID No. 2.

 The term “conservative substitution” is intended to denote any amino acid substitution which has little or no effect on the total charge, the polarity or the hydrophobicity of the reference protein, said substitution making it possible to increase or decrease the activity sought for tetraspane. This sought-after activity can either be an agonist or an antagonist of tetraspane.

 By percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is intended to denote a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by comparison window to identify and compare the local regions of sequence similarity. The optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol.

48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or even by BLAST N comparison software or BLAST P).

 The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid sequence or

<Desc / Clms Page number 6>

 of amino acids to be compared can include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

 For example, we could use the BLAST program, BLAST 2 sequences, available on the site http://www.ncbi.nlm.nih.gov/BLAST/, the parameters used being those given by default (in particular for the open parameters gap penalty: 5, and extension gap penalty: 2; the chosen matrix being for example the BLOSUM 62 matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

 Among said fragments of the protein of sequence SEQ ID No. 2 according to the invention, preferred is the isolated protein of sequence SEQ ID No. 4 corresponding to the EC2 extracellular domain of human tetraspane, or one of its proteins derived.

 Among said fragments of the protein of sequence SEQ ID No. 2 according to the invention, preference is also given to the isolated protein of sequence SEQ ID No. 6 corresponding to the extracellular domain EC2 of human tetraspan flanked by its two transmembrane domains TM3 and TM4, or one of its derived proteins.

 The term “protein derived from the protein of sequence SEQ ID No. 4 or of sequence SEQ ID No. 6” is intended to denote respectively a protein whose sequence has a percentage of identity after optimal alignment of at least 90%, preferably 95 %, and 99% with the sequence SEQ ID No. 4 or SEQ ID No. 6.

 Preferably, said proteins derived from the protein of sequence SEQ ID No. 4 and of sequence SEQ ID No. 6 are capable of exercising at least partially, respectively, one of the activities of the fragments of sequences SEQ ID No. 4 and SEQ ID No. 6 of the tetraspane protein or at least partially modulating one of said activities, as mentioned above for the fragments and proteins derived from the sequence SEQ ID No. 2.

<Desc / Clms Page number 7>

 In another aspect, the invention includes isolated nucleic acids whose sequence codes for a protein according to the present invention, their nucleic acid of complementary sequence or the sequence of their corresponding RNA.

 The term “nucleic acid, nucleic sequence, polynucleotide sequence or polynucleotide” means a fragment of DNA, single-stranded or double-stranded, or of natural, recombinant or synthetic RNA, comprising or not non-natural nucleotides, designating a precise sequence nucleotides, modified or not, making it possible to define a fragment, a segment or a region.

 The invention comprises in particular a nucleic acid according to the invention, the sequence of which codes for the protein of sequence SEQ ID No. 2 or for one of its derived proteins. Such a nucleic acid is for example and preferably a nucleic acid of sequence SEQ ID No. 1.

 The invention comprises in particular a nucleic acid according to the invention, the sequence of which codes for the protein of sequence SEQ ID No. 4 or for one of its derived proteins. Such a nucleic acid is for example and preferably a nucleic acid of sequence SEQ ID No. 3.

 The invention comprises in particular a nucleic acid according to the invention, the sequence of which codes for the protein of sequence SEQ ID No. 6 or for one of its derived proteins. Such a nucleic acid is for example and preferably a nucleic acid of sequence SEQ ID No. 5.

 The invention also relates to nucleic acids capable of hybridizing with one of the nucleic acid sequences according to the present invention.

 The term “nucleic acid capable of hybridizing specifically with one of the nucleic acids according to the invention” is intended to denote the nucleic acids of at least 15 nucleotides, preferably 18, 20, 25 and 50 nucleotides capable of hybridizing in stringent hybridization conditions with one of said nucleic acids according to the invention, these conditions being well known to those skilled in the art, such as the conditions described in particular by Sambrook et al. (Molecular cloning, A laboratory Manual, Sec. Edition, Cold Spring Harbor Laboratory Press, pages 11. 50 and 11.51, 1989).

 Among said stringent conditions, preference is given to the conditions such as they provide for nucleic acids which specifically hybridize with one of the acids

<Desc / Clms Page number 8>

 nucleic acids according to the invention, a sequence having at least 90%, preferably 95% and 99% identity after alignment with the complementary sequence of one of said nucleic acids according to the invention.

 In another aspect, the invention includes the use of the nucleic acids according to the invention as a primer, in particular for the amplification of nucleic acid by a polymerase chain polymerization technique (PCR or method related to PCR) or as a probe for the detection or identification of nucleic acid.

 The present invention relates to all the probes which can be deduced from the nucleic acids of the invention, in particular sequences capable of hybridizing with them, and which can make it possible to demonstrate said nucleic acids of the invention, in particular of discriminate normal sequences from mutated sequences.

 All of the probes and primers according to the invention can be labeled by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal.

 The invention further comprises the use of the nucleic acids according to the invention, as sense or antisense oligonucleotides.

 The antisense nucleic acids may in particular be used in so-called antisense therapeutic treatments known to those skilled in the art. They mainly consist in combining a naturally produced RNA sequence with an antisense polynucleotide which hybridizes to the latter so as to form a duplex. This duplex then blocks the rest of the transcription or prohibits translation.

 Mention may also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression, and thus be used in other therapeutic treatments requiring the modulation of this expression.

 Another object of the invention is a vector, for cloning and / or expression, which contains a nucleic acid according to the invention, in particular a nucleic acid chosen from:

<Desc / Clms Page number 9>

 a nucleic acid coding for a tetaspane of sequence SEQ ID No. 2 or for a protein whose sequence has a percentage of identity after optimal alignment of at least 90%, preferably 95%, and 99% with the sequence SEQ ID No. 2; - a nucleic acid of sequence SEQ ID No. 1; a nucleic acid which codes for the EC2 extracellular domain of sequence SEQ ID No. 4, or for a protein whose sequence has a percentage of identity after optimal alignment of at least 90%, preferably 95%, and 99% with the sequence SEQ ID No. 4; - a nucleic acid of sequence SEQ ID No. 3; a nucleic acid coding for the EC2 extracellular domain of human tetraspane flanked by its two transmembrane domains TM3 and TM4 of sequence SEQ ID No. 6, or for a protein whose sequence has a percentage identity after optimal alignment of minus 90%, preferably 95%, and 99% with the sequence SEQ ID No. 6; - a nucleic acid of sequence SEQ ID No. 5.

 Among the vectors according to the invention, the vectors characterized in that they comprise the elements allowing the expression of said sequences in a host cell and optionally the secretion of said sequences out of the host cell are preferred. By expression vector is meant both expression vectors with autonomous replication of the plasmid type and systems intended to ensure integration into cells, but these expression vectors may also be expression vectors of the type viral or even, when it is desired to carry out, for example, gene therapy, naked DNA. Among the viral vectors, those derived from adenovirus, from virus associated with adenovirus (AAV), from retroviruses, from lentiviruses, and preferably derivatives from HIV, poxviruses, from herpesvirus are preferred. in eukaryotic system. Among the non-viral vectors, naked polynucleotides such as naked DNA or naked RNA are preferred according to the technique developed by the company VICAL, artificial yeast chromosomes (YAC) for expression in yeast. , mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC, human artificial chromosome) for expression in human cells.

<Desc / Clms Page number 10>

 A cell which contains a vector according to the invention is also an object of the present invention. Said cell is preferably a eukaryotic or prokaryotic cell and more preferably a mammalian cell.

 The cell can thus be chosen from bacterial cells but also from yeast cells, as well as from plant and animal cells; preferably, the cell host is a mammalian cell, but also an insect cell in which methods using baculoviruses can be used, for example. These cells can be obtained by the introduction into host cells of nucleic acid inserted into a vector as defined above, then the cultivation of said cells under conditions allowing the expression of the transfected nucleic acid.

 The invention also relates to a process for the preparation of a tetaspane, of a fragment thereof or of a derivative thereof according to the invention, which comprises the following steps: a) culturing a cell according to the invention, in particular a cell containing a DNA sequence coding for the tetraspan of sequence SEQ ID No. 2 or for its extracellular domain of sequence SEQ ID No. 4 or SEQ ID No. 6, in a culture medium suitable for said cell ; and b) recovering said tetraspane, said fragment or said derived protein, in particular the tetraspane of sequence SEQ ID No. 2 or its extracellular domain of sequence SEQ ID No. 4 or SEQ ID No. 6, from the medium of culture and / or lysate of said cultured cells.

 For the implementation of this process, said cell in step a) is preferably a cell according to the invention as defined above.

 The recombinant tetraspane, its fragments or its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein SEQ ID No. 6 are capable of being obtained according to the preparation process of the invention and according to the techniques for producing recombinant proteins known to those skilled in the art. The present invention therefore also relates to tetraspane, its fragments or its derived proteins which can be obtained by the method presented above. In this case, the nucleic acid used is placed under the control of signals allowing its expression in a

<Desc / Clms Page number 11>

 cell host. An efficient recombinant protein production system requires a vector, for example of plasmid or viral origin, and a compatible host cell. 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 in a stable manner in the cell and may possibly have particular signals specifying the secretion of the proteins translated. These different control signals are chosen according to the cellular host used. To this end, the nucleic acids coding for tetraspane, its fragments or its derived proteins can be inserted into vectors with autonomous replication within the chosen host, or vectors integrative 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, calcium phosphate precipitation transfection, lipofection, electroporation, thermal shock.

 Tetraspane, its fragments or its derived proteins according to the invention obtained as indicated above, can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure.

 Tetraspane, its fragments or its proteins derived according to the invention can be obtained by chemical synthesis and can thus contain unnatural amino acids. Such proteins are also included in the invention.

 The methods for purifying recombinant or natural proteins, such as tetraspane, its fragments or its derived proteins according to the present invention, are known to those skilled in the art. Tetraspane, its fragments or its proteins derived according to the invention, in particular recombinant, can be purified from lysates and cell extracts, from the supernatant of the culture medium, by 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 protein fused to a carrier protein (chimeric protein). The advantage of this system is that it allows stabilization and a decrease in the proteolysis of the recombinant product, an increase in the solubility during the renaturation in

<Desc / Clms Page number 12>

 in vitro and / or a simplification of the purification when the fusion partner has an affinity for a specific ligand.

 In another aspect, the present invention comprises monoclonal, polyclonal antibodies, or a fragment thereof, directed specifically against tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular directed against human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6.

 Preferably, the monoclonal, polyclonal antibody or one of their fragments, is characterized in that it is capable of binding specifically to one of the epitopes or determinant of human tetraspane of sequence SEQ ID No. 2, in particular a epitope carried by one of its extracellular domains, such as the extracellular domain of sequence SEQ ID No. 4, or carried by the protein of sequence SEQ ID No. 6. The epitopic determinants usually consist of groups of molecules having chemically active surfaces such as amino acids or sugar side chains and having a specific three-dimensional structure and / or a characteristic specific charge.

 The monoclonal antibodies can advantageously be prepared from hybridomas according to the technique described, for example, by Kohler and Milstein in 1975 (Kohler and Milstein. Nature, 256: 1975).

 The polyclonal antibodies can be prepared, for example by immunization of an animal, in particular a mouse or a rabbit, with tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, associated with an adjuvant of the immune response, then purification of the specific antibodies contained in the serum of animals immunized on an affinity column on which tetraspane, one of its fragments or one of its proteins derived according to the invention has previously been fixed, has served as an antigen.

 The antibodies of the invention, or their fragments, can also be labeled with an enzymatic, fluorescent or radioactive type of labeling.

 Such an antibody could not only be useful for studying and thus better understanding the role played by human tetraspane according to the invention, in particular in

<Desc / Clms Page number 13>

 level of interactions of membrane molecules; but also for other applications such as therapeutic or diagnostic applications, or for targeting compounds capable of modulating the biological activity of the human tetraspane of the invention.

 The fragments of monoclonal or polyclonal antibody according to the invention comprise any fragment of said monoclonal antibody capable of binding to an epitope of tetaspane, one of its fragments or one of its derived proteins according to the invention on which fixes the monoclonal or polyclonal antibody from which said fragment is derived. Examples of such fragments include in particular single-chain monoclonal antibodies or monovalent fragments Fab or Fab 'and divalent fragments such as F (ab') 2, have the same binding specificity as the monoclonal or polyclonal antibody of which they are issues. A fragment according to the invention may also be a single chain Fv fragment produced by methods known to those skilled in the art and as described for example by Skerra et al. (Skerra et al. Science, 240: 1988) and King et al. (King, et al. Biochemical J., 290: 723-729, 1991).

 According to the present invention, fragments of monoclonal or polyclonal antibodies of the invention can be obtained from the monoclonal or polyclonal antibodies as described above by methods such as digestion with enzymes, such as pepsin or papain and / or by cleavage of the disulfide bridges by chemical reduction. Alternatively, the monoclonal or polyclonal antibody fragments included in the present invention can be synthesized by automatic peptide synthesizers such as those provided by the company Applied Biosystems, etc., or can be prepared manually using techniques known to those skilled in the art and as described for example by Geysen et al.

 (Geysen, et al. J. Immunol. Methods, 102: 1978).

 In general, for the preparation of monoclonal, polyclonal antibodies or their fragments, reference may be made to the techniques which are in particular described in the Antibodies manual (Harlow et al., Antibodies: Laboratory Manual, Cold Spring Harbor Publications pp. 726, 1988) or to the technique of preparation from hybridomas described by Kohler and Milstein in 1975.

<Desc / Clms Page number 14>

 The monoclonal or polyclonal antibodies according to the invention may for example be purified on an affinity column on which human tetraspane has previously been immobilized, an extracellular domain of said tetraspane, or one of its fragments comprising the epitope specifically recognized by said monoclonal or polyclonal antibodies according to the invention.

 The invention further comprises a monoclonal antibody or one of its fragments according to the invention, characterized in that it is chosen from humanized, chimeric or anti-idiotypic antibodies.

The humanized monoclonal antibodies according to the invention or their fragments can be prepared by techniques known to those skilled in the art (Carter et al., PNAS 89: 4285-4289,1992; Mountain, et al. Biotechnol. Genet. Eng. Rev., 10:
1992).

 Such humanized monoclonal antibodies according to the invention are preferred for their use in therapeutic methods.

 The monoclonal antibodies or their chimeric type fragments according to the invention can be produced using the techniques of genetic recombination.

 The invention also comprises a monoclonal, polyclonal antibody or one of their fragments according to the invention, characterized in that it is labeled.

 The monoclonal or polyclonal antibodies according to the invention or their fragments can also, according to the invention, be in the form of labeled antibodies in order to obtain a detectable and / or quantifiable signal.

 The monoclonal antibodies labeled according to the invention or their fragments include, for example, so-called immunoconjugate antibodies which can be conjugated, for example, with enzymes such as peroxidase, alkaline phosphatase, ss-Dgalactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase or by a molecule such as biotin, digoxigenin or 5-bromodesoxyuridine. Fluorescent markers can also be conjugated to the monoclonal antibodies or their fragments of the invention and include in particular fluorescein and its derivatives, rhodamine and its derivatives, GFP (GFP for Green Fluorescent Protein), dansyl, umbelliferone etc. In such conjugates, the monoclonal antibodies of the invention or their fragments can be prepared by

<Desc / Clms Page number 15>

 methods known to those skilled in the art. They can be coupled to enzymes or fluorescent markers directly or through a spacer group or a linking group such as a polyaldehyde, such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of coupling agents such as periodate, etc. The conjugates comprising markers of the fluorescein type can be prepared by reaction with an isothiocyanate.

 Other conjugates can also include chemiluminescent markers such as luminol and dioxetanes or bioluminescent markers such as luciferase and luciferin.

 Among the markers which can be attached to the monoclonal antibody or one of its fragments according to the invention, radioactive markers such as 14C, 36Cl, 57Co, 58Co, 51Cr, 152 Eu, 59Fe, 3H, 125I, 131I are also preferred. 32P, 35S, 75SE and 99mTc which can be detected by known means such as for example the gamma or scintillation counter or by autoradiography.

 The subject of the invention is also an agonist or an antagonist making it possible respectively to increase or decrease the activity of tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular tetraspane human with sequence SEQ ID No. 2, its extracellular domain with sequence SEQ ID No. 4 or the protein with SEQ ID No. 6.

 The invention also relates to the use of a tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, of an antibody according to the invention, of an agonist according to the invention or of an antagonist according to the invention for, or for the preparation of a composition intended to, modulate the interactions of membrane molecules and / or to regulate the immune response.

 Indeed, Example 2 below demonstrates a specific expression (compared to the other immune cells) of this new molecule at the level of T lymphocytes.

 A particularly preferred use is the use of a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain

<Desc / Clms Page number 16>

 of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, or of an agonist according to the invention for, or for the preparation of a composition intended to, decrease, or even suppress, survival, migration, the growth or proliferation of tumor cells.

Indeed, it has been found that the tetraspane protein according to the present invention is expressed at the endothelial cells, which are involved in the angiogenesis process and particularly in tumor neovascularization.

 Another essential object of the invention is a pharmaceutical composition characterized in that it comprises at least one compound chosen from the following compounds: a) a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No.

2, its extracellular domain with sequence SEQ ID No. 4 or the protein of SEQ ID No.

6; b) a nucleic acid according to the invention; c) an antibody according to the invention; d) an agonist according to the invention; e) an antagonist according to the invention; f) a vector according to the invention; and g) a cell according to the invention.

 This pharmaceutical composition can in particular be used for the prevention or treatment of neurodegenerative diseases, for the regulation of the immune system, for the regulation of angiogenesis or for the repair or regeneration of nervous tissue.

 Preferably, it is used for the prevention or treatment of cancer.

 The pharmaceutical compositions comprising a vector according to the invention may in particular be used for the transfer of nucleic acid encoding a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular tetraspane human of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, in a given cell in vivo in the context of a treatment of a neurodegenerative disease by gene therapy . This new technology, the scope of which is vast, makes it possible to envisage the treatment of neurodegenerative diseases for which the

<Desc / Clms Page number 17>

 conventional therapeutic alternatives are ineffective or even non-existent and concern both genetic and acquired diseases. The most practiced approach consists in using a viral vehicle to introduce the therapeutic nucleic acid into the cell to be treated and, in particular, retroviral and adenoviral. In fact, viruses have developed sophisticated mechanisms to cross cell membranes, escape degradation at the level of lysosomes and make their genome penetrate into the nuclei in order to ensure the expression of the therapeutic gene.

 More and more gene transfer methods use non-viral vectors. Other methods of gene transfer can be employed such as for example the delivery of the therapeutic nucleic acid by means of synthetic vectors, such as the cationic lipids which interact spontaneously with the nucleic acid to form positively charged complexes capable of fusing with the anionic cell membranes and penetrate the nucleic acid they transport (see for example Behr, Bioconjugate Chemistry (1994) 5: 382). Finally, an even simpler approach can also be envisaged by direct administration of naked DNA coding for a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain with sequence SEQ ID No. 4 or the protein of SEQ ID No. 6.

 Such pharmaceutical compositions comprising a vector or vector system according to the invention may also be used for the transfer of nucleic acid coding for a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, in a given cell ex vivo as part of a treatment for neurodegenerative disease , in particular for the repair or regeneration of neurons or neuronal tissue, which will then be implanted or reimplanted in the patient to be treated (xenograft or allograft).

 A preferred embodiment according to the invention is a pharmaceutical composition for the prevention or treatment of metastases, which comprises at least one compound chosen from the following compounds:

<Desc / Clms Page number 18>

 a) a tetraspane protein, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No.

2, its extracellular domain with sequence SEQ ID No. 4 or the protein of SEQ ID No.

6; b) a nucleic acid according to the invention; c) an agonist according to the invention; d) a vector according to the invention; and e) a cell according to the invention.

 The amount of compounds included in the pharmaceutical compositions according to the present invention and necessary for effective therapy will depend on various factors such as the mode of administration, the targeted part of the body, the physiological state of the patient, the administration of other drugs, possible side effects, etc. The dosage for such preventions or therapeutic treatments should be carried out in such a way as to optimize its safety and effectiveness. In general, animal models can be used to determine the effective amounts of the compounds entering into the pharmaceutical compositions according to the present invention for the treatment or the prevention of particular pathologies. These methods are in particular discussed in works such as Gilman et al. (Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed. Pergamon Press, 1990) and Remington's Pharmaceutical Sciences (17th ed., 1990) for administration methods such as oral, intravenous, intraperitoneal, intramuscular or transdermal. The pharmaceutically acceptable excipients include in particular water, saline solutions, buffers or any other compound described for example in the Merck Index.

 The invention also relates to a diagnostic kit comprising at least: - a monoclonal, polyclonal antibody according to the invention or one of their fragments; or - a nucleic acid according to the invention making it possible to carry out a PCR in order to quantitatively and / or qualitatively determine the presence or not of tetraspane.

 Thus, also within the scope of the invention, a diagnostic kit or kit comprising an antibody according to the invention, in particular for the assay (including the detection) or the identification of tetraspane, one of its fragments or one of his

<Desc / Clms Page number 19>

 proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, in a biological sample, preferably in vitro.

 In particular, a diagnostic kit characterized in that it comprises the following elements: - a polyclonal, monoclonal antibody or one of their fragments according to the invention, if appropriate labeled; - If necessary, a reagent for the constitution of the medium suitable for carrying out the immunological reaction; - where appropriate, a reagent allowing the detection of antigen-antibody complexes produced by the immunological reaction, this reagent can also carry a marker, or be capable of being recognized in turn by a labeled reagent, more particularly in the case where said antibody monoclonal, polyclonal or one of their fragments is not marked.

 The use of a monoclonal, polyclonal antibody, or a fragment thereof, for the assay or in vitro identification of a tetaspane, one of its fragments or one of its proteins derived according to the invention, in particular Another subject of the invention is human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6.

 The monoclonal, polyclonal antibodies or their fragments according to the invention can thus be used for the identification or the localization, in particular tissue or cell, and / or the assay of tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, said use being included in the invention.

 The monoclonal, polyclonal antibodies or their fragments according to the invention also constitute a means of analysis of the expression of tetaspane, one of its fragments or one of its proteins derived according to the invention, in biological samples (fluids or on specific tissue sections), for example by immunofluorescence, by enzymatic, radioactive or gold labeling. They make it possible in particular to highlight and quantify the normal specific presence or

<Desc / Clms Page number 20>

 abnormal tetraspane protein according to the invention in tissues or biological samples, which makes them useful for the identification and localization of tetraspane protein according to the invention, for the diagnosis of pathologies linked to the abnormal presence of tetraspane protein according to the invention. invention but also for monitoring the evolution of methods of prevention or treatment of pathology requiring said detection or said assay.

 More generally, the monoclonal, polyclonal antibodies or their fragments according to the invention can be advantageously used in any situation where the expression of tetaspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, must be observed qualitatively and / or quantitatively.

 Preferably, the biological sample consists of a biological fluid, such as serum, whole blood, cells, a tissue sample or biopsies of human origin.

 Any conventional procedure or test can be used to carry out such a detection and / or assay. Said test can be a competition or sandwich test, or any test known to those skilled in the art dependent on the formation of an antibody-antigen immune complex. Depending on the applications according to the invention, the monoclonal, polyclonal antibody or one of their fragments can be immobilized or labeled. This immobilization can be carried out on numerous supports known to those skilled in the art.

These supports may in particular include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, or natural or modified celluloses. These supports can be either soluble or insoluble.

 For example, a preferred method involves immunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radioimmunological (RIA) or equivalent.

 Thus, the invention also comprises a method for the detection and / or the determination of tetraspane or of an extracellular domain of tetraspane according to the invention in a biological sample, characterized in that it comprises the following steps:

<Desc / Clms Page number 21>

 a) bringing the biological sample (tissue or biological fluid) into contact with a monoclonal, polyclonal antibody or one of their fragments according to the invention (under conditions allowing an immunological reaction between said antibodies and said tetaspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6; b) evidence of the antigen-antibody complex possibly formed.

 The invention also includes the use of a nucleic acid according to the invention capable of hybridizing with a DNA sequence, and / or corresponding RNA, of the gene coding for tetraspane, one of its fragments or one of its proteins derived according to the invention, in particular human tetraspane of sequence SEQ ID No. 2, its extracellular domain of sequence SEQ ID No. 4 or the protein of SEQ ID No. 6, for the assay or The in vitro identification of such a DNA sequence, and / or of corresponding RNA, in a biological sample is also an embodiment of the invention.

 These different uses may in particular make it possible to predict the severity of a tumor.

 An object of the invention also relates to a mammal, except man, in particular as an animal model, comprising a nucleic acid or a transformed cell according to the invention, in particular characterized in that at least one of the alleles of the gene corresponding to the homolog of the gene coding for human tetraspane of sequence SEQ ID No. 2 of said animal is not functional or is overexpressed.

 Among the mammals according to the invention, animals such as mice, rats or rabbits are preferred, whose gene corresponding to the homolog of the gene coding for human tetraspane of sequence SEQ ID No. 2 carried by one at least of the two alleles of said animal is mutated such that the protein encoded by such a gene is not expressed or is not functional.

 Among the animal models which are more particularly interesting here, there are in particular: - transgenic animals exhibiting a deficit of the protein homologous to human tetraspane of sequence SEQ ID No. 2 of one of the components of LSR. They are obtained for example by homologous recombination on embryonic stem cells,

<Desc / Clms Page number 22>

 transfer of these stem cells to embryos, selection of the affected chimeras in the reproductive lines, and growth of said chimeras; - transgenic mice overexpressing said homologous gene. The mice are obtained by transfection of multiple copies of said gene under the control of a powerful promoter of ubiquitous nature, or selective of a type of tissue; - transgenic animals (preferably mice) rendered deficient for one of said genes carried by the two alleles, by inactivation using the LOXP / CRE recombinase system or any other system for inactivation of the expression of a gene to a specific age of the animal.

 Thus, such an animal model could in particular be used to screen molecules for the prevention or treatment of neurodegenerative diseases, for the repair or regeneration of nervous tissue, for the prevention or treatment of cancer and preferably for the prevention or treatment of metastases.

 Other characteristics and advantages of the invention appear in the following description with the examples and the figure, the legend of which is given below.

Figure Figure: Expression of tetraspane in endothelial cells and T lymphocytes
1% agarose gel electrophoresis demonstrating the expression of mRNA encoding neurospanin in different types of human cells.

 The expression of neurospanin-coding mRNA was analyzed by RTPCR in T lymphocytes, LAK cells, endothelial cells, B cells, monocytes and neutrophils. The integrity and quality control of the cDNA used in the experiments was evaluated by analyzing the expression of the mRNA coding for GAPDH.

Example 1: Cloning of the cDNA Coding for the New Human Tetraspane
In order to identify new molecules expressed selectively on the surface of human cells, sequences which may contain a transmembrane motif have been sought in EST databases (Expressed

<Desc / Clms Page number 23>

 Sequence Tag) human and particularly in the db EST bank, available at the internet address "www.ncbi.nlm.nih.gov/dbEST/". This bank contains 1856102 different ESTs.

 The identification of human ESTs sequences coding for potential transmembrane motifs was carried out using TopPred 2 software (membrane protein prediction software) which is available on the internet (http://www.biokemi.su.se/ TopPred2).

 Among the 3000 sequences with the highest score, EST # AA411274 was selected. The available nucleotide sequence (http: //www.ncbi.nih.nlm.gov) presents an open reading frame of 109 amino acids before a stop codon. Its translation product has a transmembrane motif (position 69-91). The peptide sequence has 50% identity with a protein called neurospanin whose sequence has been cloned in chicken. Neurospanin is a molecule belonging to the tetraspan superfamily. The coding nucleotide sequence of EST # AA411274 has 85% identity with the neurospanin sequence. Based on the structural characteristics of tetraspanes, it appears that the protein domain 69-91 corresponds to the 4th transmembrane domain (EC4) of tetraspans.

 Homologues of the sequence # AA411274 were then searched for in the human EST database. Eight new nucleotide sequences were thus selected on the basis of homology with the sequence # AA411274 (clones # AA132131, AA132070, T85007, AA031888, R60397. 1, AA031808, AA329968 and AA324463). The clones were obtained from Research Genetics (New York, NY).

The bacterial clones were selected using the appropriate antibiotic. The plasmid DNA was purified using a commercial kit (Midiprep DNA extraction kit, Qiagen, Germany) and sequenced using the ABI Prism BigDye Terminator DNA kit (Perkin Elmer Applied Biosystems, Warrington, UK) following supplier recommendations. The sequence was analyzed with an ABI Prism 377 automatic sequencer (Perkin Elmer Applied Biosystems). The sequences were analyzed using Sequencher sequence analysis software (Genecodes, Ann Arbor, MI). After alignment of the different sequences, a DNA fragment of 1345 base pairs having an open reading frame of 747 nucleotides containing a codon

<Desc / Clms Page number 24>

 initiation and a stop codon has been defined. This fragment was amplified by PCR using specific primers corresponding to the 5 'and 3' ends of the aligned sequence. The complete cDNA sequence was obtained by a series of PCR amplification cycles of the 5 'end (5'RACE) using commercial kits. The DNA templates were cDNA prepared from human bone marrow (Marathon-Ready cDNA; Clontech). The prediction of a signal peptide was defined using software available on the web (http://www.cbs.dtu.dk/services/SignaIP).

Results
By analysis of the available human EST databases, a cDNA clone (# AA411274) coding for a molecule exhibiting a transmembrane motif was selected. The cDNA sequence obtained is 1345 base pairs long, see sequence SEQ No 1. The predicted protein sequence contains 248 amino acids and has a molecular weight of 27.5 kDa and an isoelectric point of 4. 4. has 85% homology with chicken neurospanin (belonging to the tetraspan superfamily). The initiation codon is at position nt 281 and the stop codon is located at nucleotide nt 1015. The translation product presents: - 4 predicted transmembrane domains (TM1-4) located at positions aal3- 36, aa50-73, aa83 -107 and aa218-243 of the sequence SEQ ID No. 2; - 2 predicted extracellular domains (EC1 and EC2) located at positions aa37-49 and aal08-217 of the sequence SEQ ID No. 2.

 The greatest diversity is contained in the external EC2 loop (62% identity).

 The signal peptide is in position aal-32 of the sequence SEQ ID No. 2.

EXAMPLE 2 Demonstration of the Expression of Tetraspane in Endothelial Cells and T Lymphocytes

 The expression of the mRNA coding for neurospanin was evaluated by RT-PCR in the following cell types: B lymphocytes, T lymphocytes, monocytes, neutrophils, T cells activated by IL-2 (LAK or Lymphokine Activcated Killer cells) and endothelial cells. Briefly, the cell pellets are taken up in an extraction buffer containing guanidium isothiocyanate (Trizol; Life

<Desc / Clms Page number 25>

 technologies) at a rate of 1 ml for 10 x 106 cells. After two extractions with phenol / chloroform / isoamyl alcohol (25/24/1), the total RNAs are precipitated by adding an equal volume of isopropanol (18 hours at -20 ° C.) and then collected by centrifugation (13,000 rpm) , 30 min, 4 C). The pellet is rinsed with 70% ethanol.

The total RNA is resuspended in water containing an Rnase inhibitor (RNAsin; Promega, Madison, WI), denatured by heating (5 min at 65 ° C.) then quantified by spectrophotometry (# = 260 nm). The complementary DNAs (cDNAs) are synthesized using a commercial kit (First strand cDNA synthesis, Amersham Pharmacia Biotech, Uppsala, Sweden) are retrotranscribed. Reverse transcription (2 g of total RNA) is carried out in 10 mM Tris-HCl buffer pH 8.3 containing 300 μg / ml of oligodT primer (5'-AACTGGAAGAATTCGCGGCCGCAGGAA (T) i8-3 ': SEQ ID No 7), 10 M dithiothreitol (DTT), 5 mM of the 4 nucleotides and 1 U / ml of recombinant reverse transcriptase. The reaction mixture is incubated for 1 hour at 37 ° C. and the reaction is stopped by heating at 65 ° C. for 5 minutes. The reaction mixture for RT-PCR is as follows: 1 l of cDNA (corresponding to 25 ng of total RNA), 25 pmol of primer (5'-CGGGGCCTGCCTGCTGGCCATCGGCATC-3 ': SEQ ID No. 8 and 5' CCCTGGTAGTGCTTGGTGAGCTCC-3 ': SEQ ID No. 9), dNTP 200 M, MgCl2 1.5 mM, Tris-HCl 10 mM pH 8.3, KCI 50 mM and 0.4 units of Taq polymerase (Perkin Elmer). The PCR conditions are: 5 min at 95 ° C., 30 cycles: 1 min at 94 ° C., 1 min at 55 ° C. and 1 min at 72 ° C., a final extension of 5 min at 72 ° C. The samples are taken up in the buffer sample (0.25% bromophenol blue, 40% sucrose) and analyzed by electrophoresis in 1% agarose gel containing ethidium bromide. After migration, the samples are visualized by UV illumination of the gel. The integrity of the RNA is analyzed by evaluating the expression of the cDNA encoding the actin p molecule using the following primers: 5'-TCCACCACCCTGTTGCTGTA-3 ': SEQ ID No.

 10 and 5'-ACCACAGTCCATGCCATCAC-3 ': SEQID No. 11.

 The figure shows that neurospanin is expressed in the following cell types: T lymphocytes, T cells activated by IL-2 (LAK or Lymphokine Activated Killer cells) and endothelial cells.

<Desc / Clms Page number 26>

BIBLIOGRAPHY Adachi M, Taki T, leki Y, Huang CL, Higashiyama M, Miyake M. 1996. Correlation of KAI1 / CD82 gene expression with good prognosis in patients with non-small cell lung cancer. Cancer Res. 56: 1751-1755.

Amiot M. 1990. Identification and analysis of cDNA clones encoding CD53. A panleukocyte antigen related to membrane transport proteins. J. Immunol. 145: 4322-4325.

Angelisova P, Hilgert I, Horejsi V. 1994. Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2 / C33) with MHC class II glycoproteins.

Immunogenetics 39: 249.

Azorsa DO, Hyman JA, Hildreth JE. 1991. CD63 / Pltgp40: a platelet activation antigen identical to the stage-specific, melanoma-associated antigen ME491. Blood 78: 280-284.

Berditchevski F, Tolias KF, Wong K, Carpenter CL, Hemler ME. 1997. A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase. J. Biol. Chem. 272: 2595-2598.

Bosca L, Lazo PA. 1994. Induction of nitric oxide release by MRC OX-44 (anti-CD53) through a protein kinase C-dependent pathway in rat macrophages. J. Exp. Med.

179: 1119-1126.

Boucheix C, Benoit P, Frachet P, Billard M, Worthington RE, Gagnon J, Uzan G. 1991.

Molecular cloning of the CD9 antigen. A new family of cell surface proteins. J. Biol.

Chem. 266: 117-122.

Bradbury LE, Goldmacher VS, Tedder TF. 1993. The CD19 signal transduction complex of B lymphocytes. Deletion of the CD19 cytoplasmic domain alters signal transduction but not complex formation with TAPA-1 and Leu 13. J. Immunol.

151: 2915-2927.

Carmo AM, Wright MD. 1995. Association of the transmembrane 4 superfamily molecule CD53 with a tyrosine phosphatase activity. Eur. J. Immunol. 25: 2090-2095.

Carmo AM, Wright MD. 1995. Association of the transmembrane 4 superfamily molecule CD53 with a tyrosine phosphatase activity. Eur. J. Immunol. 25: 2090-2095.

Classon BJ, Williams AF, Willis AC, Seed B, Stamenkovic I. 1989. The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen. J. Exp. Med. 169: 1497-1502.

<Desc / Clms Page number 27>

Dong JT, Lamb PW, Rinker-Schaeffer CW, Vukanovic J, Ichikawa T, Isaacs JT, Barrett JC. 1995. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome llpll.2. Science 268: 884-886.

Flint M, Thomas JM, Maidens CM, Shotton C, Levy S, Barclay WS, McKeating JA.

1999. Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J.

Virol. 73: 6782-6790.

Gil ML, Vita N, Lebel-Binay S, Miloux B, Chalon P, Kaghad M, Marchiol-Fournigault C, Conjeaud H, Caput D, Ferrara P, et al. 1992. A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line. Cloning and preliminary functional studies. J. Immunol. 148: 2826-2833.

Griffith L, Slupsky J, Seehafer J, Boshkov L, Shaw AR. 1991. Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal. Blood 78: 1753-1759.

Horvath G, Serru V, Clay D, Billard M, Boucheix C, Rubinstein E. 1998. CD19 is linked to the integrin-associated tetraspans CD9, CD81, and CD82. J. Biol. Chem.

273: 30537-30543.

Hotta H, Hara I, Miyamoto H, Homma M. 1991 Overexpression of the human melanoma-associated antigen ME491 partially suppresses in vivo malignant phenotypes of H-ras-transformed NIH3T3 cells in athymic nude mice. Melanoma Res. 1: 125-132.

Hotta H, Ross AH, Huebner K, Isobe M, Wendeborn S, Chao MV, Ricciardi RP, Tsujimoto Y, Croce CM, Koprowski H. 1988. Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res.

48: 2955-2962.

Ikeyama S, Koyama M, Yamaoko M, Sasada R, Miyake M. 1993. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP- 1 / CD9) DNA. J. Exp. Med. 177: 1231-1237.

Imai T, Kakizaki M, Nishimura M, Yoshie O. 1995. Molecular analyzes of the association of CD4 with two members of the transmembrane 4 superfamily, CD81and CD82. J. Immunol. 155: 1229-1239.

<Desc / Clms Page number 28>

 Imai T, Yoshie 0. 1993. C33 antigen and M38 antigen recognized by monoclonal antibodies inhibitory to syncytium formation by human T cell leukemia virus type 1 are both members of the transmembrane 4 superfamily and associate with each other and with CD4 or CD8 in T cells . J. Immunol. 151: 6470-6481.

Jankowski SA, Mitchell DS, Smith SH, Trent JM, Meltzer PS. 1994. SAS, a gene amplified in human sarcomas, encodes a new member of the transmembrane 4 superfamily of proteins. Oncogene 9: 1205-1211.

Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J, Tada N, Miyazaki S, Kudo A. 2000. The gamete fusion process is defective in eggs of Cd9-deficient mice.

Nat. Broom. 24: 279-282.

Kallin B, de Martin R, Etzold T, Sorrentino V, Philipson L. 1991. Cloning of a growth arrest-specific and transforming growth factor beta-regulated gene, TI 1, from an epithelial cell line. Mol. Cell. Biol. 11: 5338-5345.

Kondoh M, Ueda M, Ichihashi M, Mishima Y. 1993. Decreased expression of human melanoma-associated antigen ME491 along the progression of melanoma precanceroses to invasive and metastatic melanomas. Melanoma Res. 3: 241-245.

Lebel-Binay S, Lagaudriere C, Fradelizi D, Conjeaud H. 1995. CD82, member of the tetra-span-transmembrane protein family, is a costimulatory protein for T cell activation. J. Immunol. 155: 101-110.

Maecker HT, Todd SC, Levy S. 1997. The tetraspanin superfamily: facilitators. FASEB J. 11: 428-442.

Mannion BA, Berditchevski F, Kraeft SK, Chen LB, Hemler ME. 1996.

Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d / CD29). J. Immunol.

 157: 2039-2047.

Marken JS, Schieven GL, Hellstrom I, Hellstrom KE, Aruffo A. 1992. Cloning and expression of the tumor-associated antigen L6. Proc. Natl. Acad. Sci. USA. 89: 3503-3507.

Metzelaar MJ, Wijngaard PL, Peters PJ, Sixma JJ, Nieuwenhuis HK, Clevers HC. 1991.

CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J. Biol. Chem. 266: 3239-3245.

<Desc / Clms Page number 29>

 Miyake M, Koyama M, Seno M, Ikeyama S. 1991. Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility. J. Exp. Med. 174: 1347-1754.

Oren R, Takahashi S, Doss C, Levy R, Levy S. 1990. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol. Cell.

Biol. 10: 4007-4015.

Ozaki Y, Satoh K, Kuroda K, Qi R, Yatomi Y, Yanagi S, Sada K, Yamamura H, Yanabu M, Nomura S, et al. 1995. Anti-CD9 monoclonal antibody activates p72syk in human platelets. J. Biol. Chem. 270: 15119-15124.

Perron JC, Bixby JL. 1999. Tetraspanins expressed in the embryonic chick nervous system. FEBS Lett. 461: 86-90.

Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S. 1998. Binding of hepatitis C virus to CD81. Science 282: 938-941.

Radford KJ, Mallesch J, Hersey P. 1995. Suppression of human melanoma cell growth and metastasis by the melanoma-associated antigen CD63 (ME491). Int. J. Cancer.

62: 631-635.

Radford KJ, Thorne RF, Hersey P. 1996. CD63 associates with transmembrane 4 superfamily members, CD9 and CD81, and with beta 1 integrins in human melanoma.

Biochem. Biophys. Res. Common. 222: 13-18.

Radford KJ, Thorne RF, Hersey P. 1997. Regulation of tumor cell motility and migration by CD63 in a human melanoma cell line. J. Immunol. 158: 3353-3358.

Rasmussen AM, Blomhoff HK, Stokke T, Horejsi V, Smeland EB. 1994. Cross-linking of CD53 promotes activation of resting human B lymphocytes. J. Immunol. 153: 4997-45007.

Rubinstein E, Le Naour F, Billard M, Prenant M, Boucheix C. 1994. CD9 antigen is an accessory subunit of the VLA integrin complexes. Eur. J. Immunol. 24: 3005-3013.

Rubinstein E, Le Naour F, Lagaudriere-Gesbert C, Billard M, Conjeaud H, Boucheix C.

 1996. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur. J. Immunol. 26: 2657-2665.

<Desc / Clms Page number 30>

 Seehafer JG, Shaw AR. 1991. Evidence that the signal-initiating membrane protein CD9 is associated with small GTP-binding proteins. Biochem. Biophys. Res. Common.

179: 401-406.

Serru V, Dessen P, Boucheix C, Rubinstein E. 2000. Sequence and expression of seven new tetraspans. Biochim. Biophys. Acta. 1478: 159-163.

Sincock PM, Mayrhofer G, Ashman LK. 1997. Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD 151) in normal human tissues: with CD9, CD63, and alpha5betal integrin. J. Histochem. Cytochem. 45: 515-525.

Slupsky JR, Seehafer JG, Tang SC, Masellis-Smith A, Shaw AR. 1989. Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex. J Biol. Chem. 264: 12289-12293.

Szala S, Kasai Y, Steplewski Z, Rodeck U, Koprowski H, Linnenbach AJ. 1990.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens. Proc. Natl. Acad. Sci. USA 87: 6833- 6837.

Tachibana I, Bodorova J, Berditchevski F, Zutter MM, Hemler ME. 1997. NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins. J. Biol. Chem. 272: 29181-29189.

Tai XG, Yashiro Y, Abe R, Toyooka K, Wood CR, Morris J, Long A, Ono S, Kobayashi M, Hamaoka T, Neben S, Fujiwara H. 1996. A role for CD9 molecules in T cell activation. J. Exp. Med. 184: 753-758.

Testa JE, Brooks PC, Lin JM, Quigley JP. 1999. Eukaryotic expression cloning with an antimetastatic monoclonal antibody identified with tetraspanin (PETA-3 / CD151) as an effector of human tumor cell migration and metastasis. Cancer Res. 59: 3812-3820.

Toyo-oka K, Yashiro-Ohtani Y, Park CS, Tai XG, Miyake K, Hamaoka T, Fujiwara H.

 1999. Association of a tetraspanin CD9 with CD5 on the T cell surface: role of particular transmembrane domains in the association. Int. Immunol. 11: 2043-2052.

Travis GH, Christerson L, Danielson PE, Klisak I, Sparkes RS, Hahn LB, Dryja TP, Sutcliffe JG. 1991. The human retinal degeneration slow (RDS) gene: chromosome assignment and structure of the mRNA. Genomics 10: 733-739.

White A, Lamb PW, Barrett JC. 1998. Frequent downregulation of the KAI1 (CD82) metastasis suppressor protein in human cancer cell lines. Oncogene 16: 3143-3149.

Claims (30)

1. Isolated human tetraspane protein of sequence SEQ ID No. 2, one of its fragments of at least 5 amino acids or one of its derived proteins.  2. Protein according to claim 1 of sequence SEQ ID No. 4, or one of its derived proteins.  3. Protein according to claim 1 of sequence SEQ ID No. 6, or one of its derived proteins.  4. Isolated nucleic acid whose sequence codes for a protein according to one of claims 1 to 3 or its nucleic acid of complementary sequence.  5. Nucleic acid according to claim 4, the sequence of which is chosen from the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, their complementary sequence and the sequence of their corresponding RNA.  6. Nucleic acid of at least 15 nucleotides capable of hybridizing with a nucleic acid according to one of claims 4 and 5.  7. In vitro use of a nucleic acid according to one of claims 4 to 6 as a primer or probe.  8. Use of a nucleic acid according to one of claims 4 to 6 as sense or antisense oligonucleotide for the preparation of a pharmaceutical composition.  9. Vector characterized in that said vector contains a nucleic acid according to one of claims 4 to 6.  10. Vector according to claim 9, characterized in that said vector contains a nucleic acid chosen from: a) a nucleic acid coding for a protein of sequence SEQ ID No. 2 or for a protein whose sequence has a percentage of identity after optimal alignment of at least 90% with the sequence SEQ ID No. 2; b) a nucleic acid of sequence SEQ ID No. 1; c) a nucleic acid coding for a protein of sequence SEQ ID No. 4, or for a protein whose sequence has a percentage identity after optimal alignment of at least 90% with the sequence SEQ ID No. 4; d) a nucleic acid of sequence SEQ ID No. 3; <Desc / Clms Page number 32>  e) a nucleic acid coding for a protein of sequence SEQ ID No. 6, or for a protein whose sequence has a percentage identity after optimal alignment of at least 90% with the sequence SEQ ID No. 6; and f) a nucleic acid of sequence SEQ ID No. 5.  11. Cell, characterized in that it contains a vector according to claim 9 or 10.  12. Cell according to claim 11, characterized in that said cell is a eukaryotic or prokaryotic cell.  13. Cell according to claim 12, characterized in that said cell is a mammalian cell.  14. Method for preparing a protein according to one of claims 1 to 3, characterized in that it comprises the following steps: a) the culture of a cell according to one of claims 11 to 13, in a medium culture suitable for said cell; b) recovering said protein from the culture medium and / or lysate of said cultured cells.  15. Monoclonal, polyclonal antibodies, or a fragment thereof, directed specifically against a protein according to one of claims 1 to 3.  16. Agonist making it possible to increase the activity of a protein according to one of claims 1 to 3.  17. Antagonist making it possible to decrease the activity of a protein according to one of claims 1 to 3.  18. Use of a protein according to one of claims 1 to 3, an antibody as defined in claim 15, an agonist as defined in claim 16 or an antagonist as defined in claim 17 for the preparation of a composition intended to modulate the interactions of membrane molecules and / or to regulate the immune response.  19. Use of a protein according to one of claims 1 to 3 or of an agonist as defined in claim 16 for the preparation of a composition intended to decrease, suppress survival, migration, growth or proliferation of tumor cells. <Desc / Clms Page number 33>  20. Pharmaceutical composition characterized in that it comprises at least one compound chosen from the following compounds: a) a protein according to one of claims 1 to 3; b) a nucleic acid according to one of claims 4 to 6; c) an antibody according to claim 15; d) an agonist according to claim 16; e) an antagonist according to claim 17; f) a vector according to one of claims 9 and 10; g) a cell according to one of claims 11 to 13.  21. Pharmaceutical composition according to claim 20, for the prevention or treatment of neurodegenerative diseases, the regulation of the immune system, the regulation of angiogenesis or the repair or regeneration of nervous tissue.  22. Pharmaceutical composition according to claim 20, for the prevention or treatment of cancer.  23. Pharmaceutical composition for the prevention or treatment of metastases, characterized in that it comprises at least one compound chosen from the following compounds: a) a protein according to one of claims 1 to 3; b) a nucleic acid according to one of claims 4 to 6; c) an agonist according to claim 16; d) an antagonist according to claim 17; e) a vector according to one of claims 9 and 10; and f) a cell according to one of claims 11 to 13.  24. A diagnostic kit comprising at least: - a monoclonal, polyclonal antibody, or one of their fragments according to claim 15; or - a nucleic acid according to one of claims 4 to 6.  25. Use of a monoclonal, polyclonal antibody, or a fragment thereof according to claim 15 for the assay or in vitro identification of a protein according to one of claims 1 to 3 in a biological sample. <Desc / Clms Page number 34>  26. Use of a nucleic acid according to one of claims 4 to 6 for the assay or the identification in vitro of a sequence of the gene coding for a protein according to one of claims 1 to 3 in a biological sample.  27. Use according to claim 25 or 26 for predicting the severity of a tumor.  28. Mammal, except man, characterized in that it contains a cell according to one of claims 11 to 13.  29. A mammal according to claim 28, characterized in that the gene corresponding to the homolog of the gene coding for human tetraspane of sequence SEQ ID No. 2 carried by at least one of the two alleles of said animal is mutated or inactivated by such that the protein encoded by such a gene is not expressed or is not functional.  30. A mammal as an animal model according to claim 28 or 29, characterized in that said mammal is a mouse.