Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to the identification and characterization of a new gene expressed in tumor cells and involved in the regulation of the immune response, the cloning and characterization of its cDNA and the corresponding polypeptides.
• the invention also relates to vectors containing a nucleic acid encoding this gene, cells transformed by such vectors as well as diagnostic methods and methods for selecting a chemical or biochemical compound capable of interacting directly or indirectly with a nucleic acid or polypeptide according to the invention.
• the invention further relates to compounds for the treatment of immune pathologies, including autoimmune diseases and cancer.
• genes coding for proteins translocated in subcellular compartments or secreted outside the cell during or after the translational process are of great interest. Mention may in particular be made of genes coding for factor proteins, such as cytokines, or genes coding for membrane receptors which constitute therapeutic targets of choice.
• proteins such as membrane proteins, present for the most part an N-terminal domain corresponding to a signal peptide, an extracellular domain and a transmembrane domain.
• the present invention relates to a purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) the sequence SEQ ID No. 1; b) the sequence of a fragment of at least 18 consecutive nucleotides, preferably 20, 25, 30, 50, 100 or 200 consecutive nucleotides, of the sequence SEQ ID No. 1 deleted from its terminal polyA fragment, preferably d said fragment of the sequence SEQ ID No. 3.
• the present invention relates to a purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the group of following sequences: a) the sequence SEQ ID No. 1; b) the sequence of a fragment of at least 18 consecutive nucleotides of the sequence SEQ ID No. 1 deleted from its terminal polyA fragment; c) a nucleic sequence having a percentage identity of at least 80% after optimal alignment with a sequence as defined in a) or b); d) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b) or c), with the exception of the sequence SEQ ID No. 11 of document WO 99/54461, of the sequence SEQ ID No. 51 of document WO 99/06548 and of the three sequences described in the documents of the EMBL database under the identification numbers Al 672868, AA 890726 and Al 301140.
• WO 99/54461 is a patent document which relates to nucleic acid sequences expressed by tumor endometrial tissues. This document describes in particular among 635 sequences a nucleic sequence SEQ ID No. 11 and its corresponding peptide sequence SEQ ID No. 173 having an identity close to 100% with respectively a fragment of the sequence SEQ ID No. 1 (nt 1597-3313 ) and of the sequence SEQ ID No. 2 (aa 519-1013) of the present invention. This document indicates, page 153, that the peptide sequence SEQ ID No. 173 does not correspond to any sequence whose function is known.
• WO 99/06548 is a document relating to EST sequences coding for proteins secreted from non-specific tissues.
• this document describes in particular the nucleic and peptide sequence SEQ ID No. 51 whose sequence aa37-113 (150 amino acids) has a very strong homology with the fragment aa1- 150 of the sequence SEQ ID No. 2 according to the invention (identity of approximately 85% for the fragment of 150 amino acids; ⁇ 94% for the first 135 amino acids).
• This document also indicates that the DNA corresponding to this 5 ′ sequence SEQ ID No. 51 originates from human prostate tumor tissue (cf. page 40 of the "original source” sequence list).
• the document "EMBL” Al 672868 describes an mRNA sequence originating from a bank of human colon tissues suffering from Crohn's disease, and exhibiting a high level of identity with the sequence complementary to the cDNA fragment nt 441- 971 of the sequence SEQ ID No. 1 according to the invention.
• the document "EMBL” AA890726 describes an mRNA sequence originating from a human tissue bank of a parathyroid with sporadic adenoma, and exhibiting a high level of identity with the sequence complementary to the cDNA fragment nt 2779-3309 of the sequence SEQ ID No. 1 according to the present invention.
• EBL EML A1 301140 describes an mRNA sequence originating from a human pulmonary carcinoid library of the neuroendocrine type described, and having a high level of identity with the complementary sequence of the cDNA fragment in position nt 2736 -3299 of the sequence SEQ ID No. 1 according to the invention.
• nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs.
• nucleotide sequences in their natural chromosomal environment that is to say in the natural state.
• sequences which have been isolated and / or purified that is to say that they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.
• the length of the nucleic acid sequence according to the present invention will be less than 150 kb, even more preferably less than 100 kb, 50 kb, 25 kb, 10 kb or 5 kb.
• percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or 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.
• the term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. 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 regions. sequence similarity locale.
• 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, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
• the percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid or amino acid sequence to be compared may include additions or deletions compared to the reference sequence for optimal alignment between these two sequences.
• the percentage 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 compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
• nucleic acid sequences having a percentage identity of at least 80%, preferably 90%, 95% or 99% after optimal alignment with a reference sequence, the nucleic acid sequences presenting, with respect to the reference nucleic acid sequence certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion, and / or a substitution, in particular punctual, and whose nucleic sequence has at least 80%, preferably 90%, 95% or 99%, identity after optimal alignment with the reference nucleic sequence.
• They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the sequence SEQ ID No. 1 of the invention.
• the specific hybridization conditions or high stringency will be such that they ensure at least 80%, preferably 90%, 95% or 99% identity after optimal alignment between one of the two sequences and the sequence complementary to the other.
• Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two fragments of complementary DNAs.
• high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.
• DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS.
• the last washing is carried out in 0.1 ⁇ SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides.
• the high stringency hybridization conditions described above for a polynucleotide of defined size will be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al. (Molecular cloning: a laboratory manual., Sec. Ed., Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1989).
• variant nucleic sequences are also preferred. of the nucleic sequence SEQ ID No. 1, or of its fragments, that is to say all of the nucleic sequences corresponding to allelic variants, that is to say individual variations of the nucleic sequence SEQ ID No. 1.
• SEQ ID No. 1 or of its fragments, that is to say all of the nucleic sequences corresponding to allelic variants, that is to say individual variations of the nucleic sequence SEQ ID No. 1.
• These natural mutated sequences correspond to polymorphisms present in mammals, in particular in humans and, in particular, to polymorphisms which can lead to the onset of a pathology.
• the present invention relates to the variant nucleic acid sequences in which the mutations lead to a modification of the amino acid sequence of the polypeptide, or of its fragments, coded by the normal sequence of sequence SEQ ID No. 1.
• variant nucleic sequences any RNA or cDNA resulting from a mutation of a splicing site of the genomic nucleic sequence whose cDNA has the sequence SEQ ID No. 1.
• the invention preferably relates to a purified or isolated nucleic acid according to the present invention, characterized in that it consists of the sequence SEQ ID No. 1, the complementary sequence or the sequence of the corresponding RNA of the sequence SEQ ID No. 1, preferably its fragment coding for the peptide of sequence SEQ ID No. 2, or of sequence SEQ ID No. 2 deleted from the peptide signed aa1-41.
• the invention preferably relates to a purified or isolated nucleic acid according to the invention, characterized in that it comprises the sequences chosen from the following sequences: a) the sequences SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 19; b) the sequence of a fragment of at least 18 nucleotides of the sequence SEQ ID No. 19; c) a nucleic sequence having a percentage identity of at least 80% after optimal alignment with a sequence as defined in a) or b); and d) the complementary sequence or the sequence of the RNA corresponding to a sequence as defined in a), b) or c).
• the invention preferably relates to a purified or isolated nucleic acid according to the invention, characterized in that its sequence is chosen from the following sequences: a) SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19 and SEQ ID No. 21; b) a nucleic sequence having an identity percentage of at least 80% after optimal alignment with a sequence as defined in a); and c) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a) or b).
• the primers or probes characterized in that they comprise a sequence of a nucleic acid according to the invention, also form part of the invention.
• the present invention preferably thus relates to the primers or probes according to the invention which can make it possible in particular to demonstrate or discriminate the variant nucleic sequences, or to identify the genomic sequence of the gene whose cDNA is represented by the sequence SEQ ID No. 1, in particular using an amplification method such as the PCR method, or a related method.
• the invention also relates to the use of a nucleic acid sequence according to the invention as probe or primer, for the detection, identification, assay or amplification of nucleic acid sequence.
• the polynucleotides which can be used as probe or as primer in methods of detection, identification, assay or amplification of nucleic sequence will have a minimum size of 15 bases, preferably 20 bases, or better from 25 to 30 bases.
• All the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal.
• the unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.
• the sequences are generally marked to obtain sequences which can be used for numerous applications.
• the labeling of the primers or probes according to the invention is carried out with radioactive elements or with non-radioactive molecules.
• radioactive isotopes used, mention may be made of 32 P, ⁇ P, ⁇ S, 3 H or 125 l.
• Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.
• the polynucleotides according to the invention can thus be used as a primer and / or probe in methods using in particular the technique of PCR (polymerase chain reaction) (Erlich, HA, New York: Stockton Press, 1989; Innis, MA, et al., Académie Press, 1990; and Rolfs, A., et al., Berlin: Springer-Verlag, 1991).
• PCR polymerase chain reaction
• This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified.
• the amplified fragments can be identified, for example after electrophoresis in agarose or polyacrylamide gel, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced.
• the specificity of the amplification can be controlled by using, as primer, the nucleotide sequences of polynucleotides of the invention as template, plasmids containing these sequences or else the derived amplification products.
• the amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
• the invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.
• Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.
• PCR-like will be understood to mean all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or else in which the labeling systems have been amplified, these techniques are of course known, in general these are amplification of DNA with a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand.
• thermostable ligase which uses a thermostable ligase, the RCR technique (Repair Chain Reaction) described by Segev, D. in 1992 (Kessler C. Springer Verlag, Berlin, New York, 197-205), the CPR (Cycling Probe Reaction) technique described by Duck, P., et al. in 1990 (Biotechniques, 9: 142-147), the Q-beta-replicase amplification technique described by Miele, EA, et al. in 1983 (J. Mol. biol., 171: 281-295) and improved in particular by Chu, BCF, et al.
• RCR Repair Chain Reaction
• CPR Cycling Probe Reaction
• the target polynucleotide to be detected is an mRNA
• an enzyme of reverse transcriptase type in order to obtain a cDNA from the mRNA contained in the biological sample.
• the cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention.
• the probe hybridization technique can be carried out in various ways
• the most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).
• a support such as nitrocellulose, nylon, polystyrene
• the latter can be used as a capture probe.
• a probe called a “capture probe”
• a probe is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected.
• a second probe called a “detection probe”, marked by an easily detectable element.
• the invention comprises a method for the screening of cDNA or genomic DNA libraries, or for the cloning of isolated cDNA or genomic, characterized in that it implements a nucleic sequence according to l 'invention.
• these methods according to the invention can be used for the identification and thus the obtaining of cDNAs of nucleic variants.
• screening and / or cloning methods will in particular comprise a step of hybridization of a nucleic acid according to the invention with a nucleic acid contained in a genomic or cDNA library.
• the invention also includes a method for identifying the nucleic acid sequences which promote and / or regulate the expression of the gene whose cDNA has the sequence SEQ ID No. 1, characterized in that it uses a nucleic acid according to the invention.
• the computer tools available to those skilled in the art allow him easily to identify, from the genomic nucleic sequences according to the invention, the promoter regulatory boxes necessary and sufficient for the control of gene expression, in particular the TATA, CCAAT boxes, GC, as well as the stimulatory (“enhancer”) or inhibitory (“silent”) regulatory sequences which control in CIS the expression of the genes according to the invention; among these regulatory sequences, mention should be made of TIRE, MRE, CRE.
• the invention also relates to methods for the identification of mutations carried by the gene coding for the polypeptide of sequence SEQ ID No. 2, in particular responsible mutations, characterized in that they implement a nucleic sequence according to the invention .
• the invention relates particularly to a method for identifying a nucleic acid of at least 18 consecutive nucleotides of the sequence of the gene coding for the polypeptide of sequence SEQ ID No. 2, said nucleic acid comprising at least one mutation, of preferably responsible for the abnormal expression of said gene, characterized in that it implements a nucleic sequence according to the invention.
• These methods of identifying these mutations will include in particular the following steps: (i) isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the mRNA of the biological sample ; (ii) specific amplification of the target DNA capable of presenting a mutation using primers according to the invention; (iii) analysis of the amplification products, in particular the size and / or the sequence of the amplification products, with respect to a reference sequence.
• Also part of the invention are the promoter and / or regulatory sequences of said gene according to the invention having mutations capable of modifying the expression of the corresponding protein.
• Nucleic acids characterized in that they are capable of being obtained by one of the preceding methods according to the invention, or nucleic acids capable of hybridizing under conditions of high stringency (percentage identity of at least 80 % between one of the two sequences and the complementary sequence of the other) with said nucleic acids, are part of the invention, in particular the nucleic acids of allelic variants of the gene whose cDNA has the sequence SEQ ID No. 1, as well as the genomic sequences of the homologous genes of other mammals such as the mouse.
• nucleic acid sequence according to the invention as probe or primer for the screening of genomic library or cDNA is of course part of the object of the present invention.
• the invention comprises a purified or isolated polypeptide encoded by a nucleic acid according to the invention.
• polypeptide will be used to also denote a protein or a peptide.
• the present invention relates to a purified or isolated polypeptide, characterized in that it comprises an amino acid sequence chosen from the following group: a) the sequence SEQ ID No. 2; b) the sequence of a fragment of at least 6, 10, 15, 30 or 50 consecutive amino acids of the sequence SEQ ID No. 2, preferably of a said fragment of the sequence SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8 or SEQ ID No. 10; c) an amino acid sequence having a percentage identity of at least
• the subject of the invention is a polypeptide of amino acid sequence SEQ ID No. 2, of sequence SEQ ID No. 2 deleted from the fragment aa1-41, or an amino acid sequence having a percentage d identity of at least 80%, preferably 90%, 95% or 99%, after optimal alignment with the sequence SEQ ID No. 2.
• Protein sequence analysis predicts that the polypeptide of sequence SEQ ID No. 2 has a signal peptide whose sequence is between positions aa 1-41, an extracellular domain whose sequence is between positions aa 42-911 , a transmembrane domain whose sequence is between the positions aa 912-930 and an intracellular domain whose sequence is between the positions aa 931-1013 on the sequence SEQ ID No. 2.
• polypeptides according to the invention the polypeptide whose sequence is between the positions aa 42-911of the sequence SEQ ID No. 2 corresponding to the extramembrane domain or any fragment of at least 6, 10, 15, 30 is also preferred. or 50 consecutive amino acids of said sequence aa 42-911, preferably consecutive of the sequence SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8 or SEQ ID No. 10.
• the invention preferably relates to a purified or isolated polypeptide, characterized in that it comprises an amino acid sequence chosen from the following group: a) the sequence SEQ ID No. 2; b) the sequence of a fragment of at least 6 amino acids of the sequence SEQ ID No. 22, preferably of the sequence SEQ ID No. 20; and c) an amino acid sequence having a percentage identity of at least 80% after optimal alignment with a sequence as defined in a) or b).
• the invention preferably relates to a polypeptide according to the invention, characterized in that its sequence is chosen from the sequences SEQ ID No. 2, SEQ ID No. 4,
• SEQ ID No. 20 SEQ ID No. 22 or a sequence having an identity percentage of at least 80% after optimal alignment with these sequences.
• the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their environment.
• the invention relates to peptides obtained by purification from natural sources, or else obtained by genetic recombination, or even by chemical synthesis and which can then comprise non-natural amino acids.
• the production of a recombinant polypeptide, which can be carried out using one of the nucleotide sequences according to the invention is particularly advantageous since it makes it possible to obtain an increased level of purity of the desired polypeptide.
• polypeptide whose amino acid sequence exhibiting a percentage identity of at least 80%, preferably 90%, 95% or 99%, after optimal alignment with a reference sequence is intended to denote the polypeptides exhibiting certain modifications. relative to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and / or one or more substitutions.
• polypeptides whose amino acid sequence having a percentage identity of at least 80%, preferably 90%, 95% or 99% after optimal alignment with the sequence SEQ ID No.
• the variant polypeptides encoded by the variant nucleic sequences as defined above are preferred, in particular the polypeptides whose amino acid sequence has at least one mutation corresponding in particular to truncation, deletion, substitution and / or addition of at least one amino acid residue with respect to the sequence SEQ ID No. 2 or with one of its fragments, more preferably the variant polypeptides having a mutation linked to a pathology.
• the invention also includes the cloning and / or expression vectors containing a nucleic acid sequence according to the invention.
• the vectors according to the invention characterized in that they comprise the elements allowing the expression and / or the secretion of said sequences in a host cell, or even a cell addressing sequence, also form part of the invention.
• Said vectors will preferably comprise a promoter, translation initiation and termination signals, as well as suitable regions for regulating transcription. They must be able to be maintained stably in the cell and may possibly have specific signals specifying the secretion of the translated protein.
• control signals are chosen according to the cellular host used.
• the nucleic acid sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or vectors integrative of the chosen host.
• plasmid or viral type systems will preferably be used depending on the host cell, the viral vectors possibly being in particular adenoviruses (Perricaudet, M., et al., La Diego, 23: 471 -473, 1992), retroviruses, lentiviruses, poxviruses or herpesviruses (Epstein, A., Medicine / Sciences, 8: 902-911, 1992).
• adenoviruses Perricaudet, M., et al., La Diego, 23: 471 -473, 1992
• retroviruses retroviruses
• lentiviruses lentiviruses
• poxviruses poxviruses
• herpesviruses Epstein, A., Medicine / Sciences, 8: 902-911, 1992.
• viruses When it is desired to integrate the sequence into the chromosomes of the host cell, use may be made, for example, of systems of the plasmid or viral type; such viruses will be, for example, retroviruses (Temin, H. M., Retrovirus vectors for gene transfer, In Kucherlapati R., éd. Gène Transfer, New York, Plénum Press,
• 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.
• YAC artificial yeast chromosome
• MAC mouse artificial chromosomes
• HAC human artificial chromosome
• Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock.
• the invention further comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed by the vectors according to the invention as well as the transgenic animals, except humans, comprising one of said cells transformed according to the invention.
• a preferred cellular host for the expression of the proteins of the invention consists of COS cells.
• mice, rats or rabbits are preferred, expressing a polypeptide according to the invention.
• animals such as mice, rats or rabbits are also preferred, characterized in that the gene coding for the protein with sequence SEQ ID No. 2, or whose sequence is coded by homologous gene for these animals, is not functional, is invalidated or has at least one mutation.
• transgenic animals are obtained for example by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras.
• transgenic animals may overexpress the gene coding for the protein according to the invention, or their homologous gene, or express said gene into which a mutation may be introduced.
• These transgenic animals are obtained for example by transfection of a copy of this gene under the control of a strong promoter of ubiquitous nature, or selective for a type of tissue, or after viral transcription.
• These transgenic animals may be made deficient for the gene coding for the polypeptide of sequence SEQ ID No.
• the cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can also be used as an analysis model.
• the transformed cells or mammals as described above can thus be used as models in order to study the interactions between the polypeptides according to the invention, and the chemical or protein compounds, involved directly or indirectly in the activities of the polypeptides according to the invention, this in order to study the different mechanisms and interactions involved.
• polypeptides according to the invention can be used for the selection of products interacting with the polypeptides according to the invention, in particular the protein of sequence SEQ ID No. 2 or its variants according to the invention, as cofactor, or inhibitor, in particular competitive , or also having an agonist or antagonist activity of the activity of the polypeptides according to the invention.
• said transformed cells or transgenic animals will be used as a model, in particular for the selection of products making it possible to combat pathologies linked to an abnormal expression of this gene.
• the invention also relates to the use of cell, mammal or polypeptide according to the invention for the screening of chemical or biochemical compound which can interact directly or indirectly with the polypeptides according to the invention, and / or capable of modulating the expression or the activity of these polypeptides.
• the invention also relates to the use of a nucleic acid sequence according to the invention for the synthesis of recombinant polypeptides.
• the method for producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultivated in conditions allowing the expression of a recombinant polypeptide encoded by a nucleic acid sequence according to the invention, and that said recombinant polypeptide is recovered.
• Recombinant polypeptides characterized in that they are capable of being obtained by said production method, also form part of 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.
• sequences of the recombinant polypeptides can also be modified in order to improve their solubility, in particular in aqueous solvents. Such modifications are known to those skilled in the art, such as the deletion of hydrophobic domains or the substitution of hydrophobic amino acids with hydrophilic amino acids.
• 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.
• the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.
• An efficient system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.
• 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.
• the recombinant polypeptide purification methods used are known to those skilled in the art.
• the recombinant polypeptide can be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using '' specific monoclonal or polyclonal antibodies, etc.
• polypeptides according to the present invention can be obtained by chemical synthesis and this using one of the many known peptide syntheses, for example the techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a synthesis in conventional solution.
• polyclonal antibodies can be obtained from a serum of an animal immunized against the polypeptides according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures.
• Mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are capable of specifically recognizing the extramembrane domain of the polypeptide of sequence SEQ ID No. 2 are particularly preferred.
• the specific monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by K ⁇ hler and Milstein (Nature, 256: 495-497, 1975).
• the antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.
• the invention also relates to methods for the detection and / or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention.
• the invention further comprises purified polypeptides, characterized in that they are obtained by a method according to the invention.
• the antibodies of the invention in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample.
• polypeptides according to the invention in particular the polypeptide of sequence SEQ ID No. 2 or one of its variants, on sections of specific tissues, by example by immunofluorescence, gold labeling, enzyme immunoconjugates.
• the antibodies of the invention can be advantageously used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed.
• Also forming part of the invention are the methods for determining an allelic variability, a mutation, a deletion, a loss of heterozygosity or any genetic anomaly of the gene according to the invention, characterized in that that they use a nucleic acid sequence or an antibody according to the invention.
• the invention relates to a method for diagnosing a disease associated with the presence of at least one mutation in a gene sequence according to the invention from a biological sample from a patient, characterized in that it comprises the following steps: a) if necessary, isolation of the genomic DNA from the biological sample to be analyzed, or obtaining cDNA from the RNA of the biological sample; b) specific amplification of said DNA sequence of the gene capable of containing a mutation using primers according to the invention; c) analysis of the amplification products obtained and comparison of their sequence with the normal sequence.
• the invention also includes a method for diagnosing a disease associated with an abnormal expression of a polypeptide according to the invention, in particular the polypeptide of sequence SEQ ID No. 2 or one of its variants, characterized in that one brings one or more antibodies according to the invention into contact with the biological material to be tested, under conditions allowing the possible formation of specific immunological complexes between said polypeptide and said antibody or antibodies, and in that one detects and / or quantifies the immunological complexes possibly formed.
• These diagnostic methods are intended, for example, to diagnose diseases associated with an abnormal expression of the gene coding for the polypeptide of sequence SEQ ID No. 2, such as overexpression or repression, by determining from a biological sample. of the patient the presence in abnormal quantity of a sequence according to the invention.
• the nucleic acid sequences analyzed may as well be genomic DNA, cDNA, or mRNA.
• the methods which make it possible to demonstrate a mutation in a gene relative to the wild-type gene are, of course, very numerous. They can basically be divided into two main categories.
• the first type of method is that in which the presence of a mutation is detected by comparison of the mutated sequence with the corresponding wild-type sequence
• the second type is one in which the presence of the mutation is detected indirectly, for example by evidence of mismatches due to the presence of the mutation.
• probes and primers of the present invention can use the probes and primers of the present invention. They are generally purified hybridization nucleic sequences comprising at least 15 nucleotides, preferably 20, 25 or 30 to 200 nucleotides, characterized in that they can hybridize specifically with a nucleic sequence according to the invention.
• the specific hybridization conditions are such as those defined above or in the examples.
• the methods for determining an allelic variability, a mutation, a deletion, or a genetic abnormality preference is given to methods comprising at least one amplification step called by PCR (polymerase chain reaction ) or by PCR-like of the target sequence according to the invention capable of presenting an anomaly using a pair of primers of nucleotide sequences according to the invention.
• the amplified products may be treated with the appropriate restriction enzyme before detecting or assaying the targeted product.
• the mutations of the gene according to the invention can be responsible for different modifications of its translation product, modifications which can be used for a diagnostic approach. Indeed, the modifications of antigenicity linked to these mutations can allow the development of specific antibodies. Discrimination of the mutated gene product can be achieved by these methods. All these modifications can be used in a diagnostic approach using several well-known methods based on the use of mono- or polyclonal antibodies recognizing the normal polypeptide or mutated variants, such as for example by RIA or by ELISA.
• the invention comprises a method of selecting chemical or biochemical compounds capable of interacting directly or indirectly with the polypeptides according to the invention, in particular the polypeptide of sequence SEQ ID No. 2, or with the nucleic acids according to the invention, and / or allowing the expression or activity of the polypeptides according to the invention to be modulated.
• the invention comprises a method of selecting chemical or biochemical compounds capable of interacting in vitro or in vivo with a nucleic acid sequence according to the invention, characterized in that it comprises contacting a cell expressing normally or abnormally the gene coding for the polypeptide of sequence SEQ ID No. 2 with a candidate compound, and the detection direct or indirect modification of the expression or activity of the polypeptide of sequence SEQ ID No. 2.
• the screening can be used to test compounds capable of modifying the level and / or the specificity of expression of the polypeptides according to the invention, in particular the polypeptide of sequence SEQ ID No. 2, either by binding competitively to the binding sites of the transcription factors located in the promoter of the gene coding for the polypeptide of sequence SEQ ID No. 2, either by directly binding to the transcription factors.
• the level of expression of the gene coding for the polypeptide of sequence SEQ ID No. 2 and its location can be analyzed by hybridization in solution with large probes as indicated in PCT patent WO 97/05277.
• a quantitative analysis of the expression of the gene coding for the polypeptide of sequence SEQ ID No. 2 can also be carried out using DNA templates, which may include all or part of the cDNA sequence of sequence SEQ ID No. 1 of the present invention, and having a sufficient length to allow specific detection of the expression of mRNAs capable of hybridizing to it.
• the fragments comprise at least 15, 25, 50, 100 or 500 consecutive nucleotides of the nucleic sequences from which they are derived as described in Schena, et al. (Science, 270: 467-470, 1995).
• DNAs according to the invention can also be carried out with DNAs according to the invention on DNA matrices according to the description of Pietu et al. (1996).
• the DNAs according to the invention or their fragments are amplified by PCR and fixed on membranes.
• MRNAs from different tissues or cells are labeled with radioactive nucleotides. After hybridization and washing under controlled conditions, the hybrid mRNAs are detected with a Phosphor Imager or by autoradiography. The experiments are carried out in duplicate and a quantitative analysis of the differentially expressed mRNAs can be carried out.
• Another aspect of the present invention relates to methods of identifying molecules capable of binding with the polypeptides according to the invention, in particular the polypeptide of sequence SEQ ID No. 2.
• Such molecules can be used to modulate the biological activity of these polypeptides.
• such molecules can be used to stimulate or reduce the activity of these polypeptides.
• the invention comprises a method of selecting chemical or biochemical compounds capable of modulating the activity of the polypeptide of sequence SEQ ID No. 2, characterized in that it comprises contacting a polypeptide according to the invention or of a cell according to the invention, with a candidate compound and, the detection of a modification of the activity of the polypeptide of sequence SEQ ID No. 2.
• the invention also relates to a method of selecting chemical compounds or biochemical in vivo or in vitro capable of binding with the polypeptide of sequence SEQ ID No. 2, characterized in that it comprises contacting a polypeptide according to the invention or a cell according to the invention , with a candidate compound, and detecting the formation of a complex between the candidate compound and said polypeptide.
• the peptides or small molecules capable of interacting with the polypeptide of sequence SEQ ID No. 2 according to the invention can be linked to detectable markers such as radioactive, fluorescent or enzymatic markers. These labeled molecules are brought into contact with said immobilized polypeptide according to the invention, under conditions allowing a specific interaction. After elimination of the molecules which are not specifically fixed, the linked molecules are detected by appropriate means.
• B1ACORE technology can also be used to screen for compounds capable of interacting with the polypeptide of sequence SEQ ID No. 2. This technology, described in Szabo, A., et al. (Curr. Opin. Struct.
• Biol., 5: 699-705, 1995 makes it possible to detect interactions between molecules in real time without the use of labeling. It is based on the phenomenon of SPR (surface plasmon resonance).
• SPR surface plasmon resonance
• One of the main advantages of this method is that it allows the determination of the association constants between the polypeptide of sequence SEQ ID No. 2 and the interacting molecules. Thus, it is possible to specifically select the molecules interacting with strong or weak association constants.
• Proteins or other molecules interacting with the polypeptide of sequence SEQ ID No. 2 according to the invention can be identified using affinity columns which contain said polypeptide or one of its fragments according to the invention.
• Said polypeptide of sequence SEQ ID No. 2, or a fragment thereof can be attached to the column using conventional techniques including chemical coupling to an appropriate column matrix such as agarose, Affi Gel, or d other matrices known to those skilled in the art.
• the affinity column can contain chimeric proteins in which said polypeptide of sequence SEQ ID No. 2, or one of its fragments, would be fused for example with gluthation S-transferase.
• the test molecules described above are then deposited on the column.
• the molecules interacting with said polypeptide of sequence SEQ ID No. 2 are retained by the column and can be isolated by elution.
• the invention also relates to a method of screening and / or of selection of compounds interacting with the promoter and / or regulatory sequences of the gene coding for the polypeptide of sequence SEQ ID No. 2 according to the invention.
• nucleic acids coding for proteins interacting with the promoter and / or regulatory sequences of the gene coding for the polypeptide of sequence SEQ ID No. 2 according to the invention can be screened and / or selected using a single hybrid system such as the one described in the manual accompanying the Clontech One-Hybrid System Matchmaker kit (Catalog N ° K1603).
• the selection of compounds capable of modifying the expression of the gene coding for the polypeptide of sequence SEQ ID No. 2 according to the invention by binding to its regulatory and / or promoter sequences can also be carried out using “reporter” genes "Such as alkaline phosphatase, ⁇ -galactosidase, or GFP (green fluorescent protein).
• reporter genes "Such as alkaline phosphatase, ⁇ -galactosidase, or GFP (green fluorescent protein).
• the effect of candidate compounds on the expression resulting from the regulatory and / or promoter sequences of the gene coding for the polypeptide of sequence SEQ ID No. 2 according to the invention can thus be evaluated.
• the invention comprises the use of nucleic acid or polypeptide according to the invention, of a cell according to the invention, or of a mammal according to the invention, for the study of the expression or activity of the polypeptide of sequence SEQ ID No. 2 according to the invention, and / or interactions, direct or indirect, between said polypeptide of sequence SEQ ID No. 2, and chemical or biochemical compounds which may be involved in its activity.
• the chemical or biochemical compounds characterized in that they are selected by said methods defined above also form part of the invention.
• compounds intended to modulate the expression or the activity of the polypeptide of sequence SEQ ID No. 2 according to the invention is intended to denote the compounds which make it possible in particular to reduce, stabilize or increase the quantity or the specificity of the polypeptide of sequence SEQ ID No. 2 according to the invention, or of promoting or inhibiting the overall activity or the activity of one of the specific domains of said polypeptide or else of restoring the normal expression of said gene coding for the polypeptide of sequence SEQ ID No. 2 according to the invention in the case, for example, where a genetic abnormality is observed.
• These compounds may for example interact as specific ligands of said polypeptide of sequence SEQ ID No. 2 according to the invention or of one of its domains as a cofactor, or an inhibitor, in particular a competitive one, or else having an agonist activity. or antagonist of the activity of said polypeptide of sequence SEQ ID No. 2 according to the invention.
• These compounds may also interact either by neutralizing the specific natural ligands of said polypeptide of sequence SEQ ID No. 2 according to the invention and thereby inhibiting the activity of said polypeptide induced by these ligands, or by neutralizing the binding site of these natural ligands .
• the invention comprises the compounds according to the invention, characterized in that they are agonists or antagonists of the activity of the polypeptide of sequence SEQ ID No. 2 according to the invention.
• the invention also comprises compounds according to the invention, characterized in that they are capable of binding to a polypeptide of sequence SEQ ID No. 2 according to the invention.
• polypeptide according to the invention including the polypeptides of sequence SEQ ID No. 173 and SEQ ID No. 51 respectively described in documents WO 99/54461 and WO 99/06458, - a polypeptide according to the invention, characterized in that that it corresponds to a fragment or to the soluble fraction of the polypeptide of sequence SEQ ID No. 2 according to the invention,
• nucleic acid according to the invention including the nucleic acid of sequence SEQ ID No. 11 from document WO 99/54461, of sequence ID No. 51 of document WO 99/06548, of sequence Al 672868, AA 890726 or Al 30140 EMBL documents with corresponding identification number, meaning or antisense, and
• the invention comprises compounds according to the invention, as a medicament for the prevention and / or treatment of pathologies linked to an abnormality in the expression and / or the activity of the polypeptide of sequence SEQ ID No. 2 according to the invention.
• the invention comprises compounds according to the invention, as a medicament for the prevention and / or treatment of immune pathologies, in particular for the prevention and / or treatment of autoimmune diseases or cancers.
• the invention finally comprises compounds according to the invention, as a medicament for the prevention and / or treatment of infections of the viral, fungal, bacterial or parasitic type.
• the compounds of the invention as active principles of medicament will preferably be in soluble form, associated with a pharmaceutically acceptable vehicle.
• 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 modes of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and observed side effects, etc.
• RNA coding for the peptide with sequence SEQ ID No. 2 was analyzed by Northern Blotting using commercial membranes (Multiple Tissue Northern; Clontech, Palo Alto, CA). Representative results obtained with two different membranes are presented. 1. fetal liver, 2. bone marrow, 3. mononuclear cells, 4. thymus, 5. peripheral nodes, 6. spleen, 7. mononuclear cells, 8. lung, 9. placenta, 10. small intestine, 11. liver, 12. kidney, 13. spleen, 14. thymus, 15. colon, 16. skeletal muscle, 17. heart, 18. brain.
• RNA encoding the polypeptide of sequence SEQ ID No. 2 was evaluated by PCR in the different subpopulations of unstimulated mononuclear cells: T lymphocytes (line 1), B lymphocytes (line 2), NK cells ( lane 3) and monocytes (lane 4) as well as in dendritic cells generated in vitro (lane 5).
• T lymphocytes line 1
• B lymphocytes line 2
• NK cells lane 3
• monocytes lane 4
• the integrity of the RNA as well as the quality of the synthesized cDNA are evaluated by amplifying the cDNA encoding the GAPDH molecule.
• the mRNA encoding the polypeptide of sequence SEQ ID No. 2 in human tumor lines was analyzed by PCR.
• the cell lines analyzed are line 1: A549, line 2: THP1, line 3: HTB10, line 4: T24, line 5: Daudi, line 6: A427, line 7: U373, line 8: HEP2, line 9: HT29, line 10: BT20.
• the mRNA coding for the polypeptide of sequence SEQ ID No. 2 is expressed constitutively by different human tumor lines.
• COS cells were transfected with the vector pCDNA3.1 containing the complete cDNA coding for the polypeptide of sequence SEQ ID No. 2.
• Cytoplamic and non-cytoplasmic fractions of the cells were obtained three days after transfection by freezing / thawing .
• the protein extracts were separated by SDS-PAGE electrophoresis; the expression of the recombinant protein was evaluated by Western Blotting using an anti-c-myc Ab.
• Line 1 non-cytoplasmic fraction
• line 2 cytoplasmic fraction.
• polypeptide of sequence SEQ ID No. 2 is expressed in the non-cytoplasmic fraction of the transfected cells.
• sequences which may contain a transmembrane motif have been searched in the databases of expressed EST (Expressed Sequence Tag) sequence labels.
• EST # AI301140 based on the fact that the translation product has a transmembrane motif (position 14-34 with a prediction score of 1.868) and that the nucleotide sequence is not homologous to any other sequence known.
• the nucleotide sequence available http://www.ncbi.nih.nlm.gov) presents an open reading frame of 116 amino acids before a stop codon.
• the complete cDNA sequence was obtained by a series of PCR amplification cycles of the 5 'end (5'RACE) (Frohman, MA, et al., Proc. Natl. Acad. Sci. USA, 85 : 8998-9002, 1988) using commercial kits.
• the DNA templates were cDNA prepared from the human brain and bone marrow (Marathon-Ready cDNA; Clontech).
• the prediction of a signal peptide has been defined using software available on the web (http://www.cbs.dtu.dk/services/SignalP).
• the molecule has a predicted molecular weight of 113 kDa, a pI of 6.5 as well as 3 possible glycosylation sites (Threonine and Serine residues).
• a cDNA clone (# AI301140) encoding a molecule exhibiting a transmembrane motif was selected.
• the sequence SEQ ID No. 1 of cDNA obtained is 3331 base pairs long and does not show any homology with the molecules present in the databases. It presents an open reading frame of 3042 base pairs encoding a putative protein of 1013 amino acids.
• the initiation codon is located in position nt 45-47 and the stop codon is located in position nt 3084-3086 of the sequence SEQ ID No. 1.
• sequence SEQ ID No 2 presents a signal peptide of sequence aa 1-41, an extracellular domain of sequence aa 42-911, a transmembrane domain of sequence aa 912-930 and an intracellular domain of sequence aa 931-1013.
• sequences SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9 correspond to fragments of the sequence SEQ ID No. 1 not homologous to the EST sequences selected in Example 1.
• tissue expression of the polypeptide of sequence SEQ ID No. 2 is analyzed by Northern Blotting using commercial membranes which make it possible to evaluate the expression of an mRNA in different tissues (Multiple Tissue Northern or MTN, Clontech).
• a cDNA fragment corresponding to fragment nt 385-nt 1137 is generated by PCR using specific primers whose sequence is deduced from the sequence SEQ ID No. 1. After purification, 25 ⁇ g of this fragment are radioactively labeled using a commercial kit (Multiprime DNA labeling kit, Amersham, Amrsham, UK) and the radioactive nucleotide [ ⁇ -32P] -dATP.
• the labeled probe is purified by chromatography.
• the membranes are preincubated in the SSC 2X solution (300 mM sodium citrate pH 7 - 3M NaCl - 0.1% sodium dodecyl sulfate) for 2 hours at 50 ° C. with stirring. The membrane is then incubated overnight at 50 ° C with shaking with the radiolabelled probe which will have been denatured by heat (10 minutes at 95 ° C). After three washes (15 minutes at 55 ° C with stirring) in a 0.1% SSC 2X-SDS solution. The membranes are exposed to an autoradiographic film (Kodak, Rochester, NY) for 24 hours. Results
• the Northern Blotting membranes shown in Figures 1A and 1B, show a strong hybridization signal in peripheral blood mononuclear cells and the small intestine; the mRNA encoding the polypeptide of sequence SEQ ID No. 2 is also expressed in the thymus, the spleen, the brain and the lung.
• the mRNA encoding the polypeptide of sequence SEQ ID No. 2 is preferentially expressed in human lymphoid tissues.
• EXAMPLE 3 Expression of the mRNA Coding for the Polypeptide of Sequence ID No. 2 in the Different Populations of Circulating Blood iWéf ⁇ oco / og / e
• CFN Mononuclear cells
• the CMNs are resuspended at a concentration of 200 x 10 6 cells / ml and mixed with 1 ml of a 50% solution of sheep erythrocytes (BioMérieux, Marcy l'Etoile, France). The cell suspension is incubated at 4 ° C overnight. After a gentle resuspension, the T cells are isolated by centrifugation on a Ficoll-Hypaque gradient (1500 rpm for 30 minutes at room temperature). The sheep erythrocyte-T lymphocyte complexes are collected at the bottom of the tube. The red blood cells are lysed by two successive hypotonic shocks.
• the purity of the T cells thus isolated, evaluated by labeling with an anti-CD3 FITC antibody is> 95%.
• the cells are washed in FACS buffer (10 mM phosphate buffer, pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) before being distributed in wells of a plate of culture 96 wells with conical bottom (Nunc, Roskilde, Denmark) at a rate of 2 ⁇ 10 5 cells in a volume of 50 ⁇ l of FACS buffer.
• FACS buffer 10 mM phosphate buffer, pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide
• the cells After 20 minutes of incubation at 4 ° C, the cells are washed twice with 200 ⁇ l of FACS buffer and then are resuspended in 200 ⁇ l of this same buffer.
• the expression of CD3 on the surface of T lymphocytes is analyzed by FACS.
• the monocytes are purified by a positive selection technique using magnetic beads coated with an anti-human CD14 antibody (MACS TM system; Miltenyi Biotex, Bergish Gladbach, Germany). Briefly, the mononuclear cells (300 x 10 6 / ml) are incubated with 60 ml of a suspension of beads (Miltenyi) at 4 ° C for 15 minutes in PBS / EDTA buffer 2 mM / bovine serum albumin 0.5% (PBS / EDTA / BSA). After washing, the cells are placed on a column subjected to a magnetic field. After three washes with the PBS / EDTA / BSA solution, the column is removed from the magnetic field and the monocytes are collected by gravity. The purity of the monocytes, evaluated by cytometry on the basis of the size / grain size parameters, is greater than 95%.
• Immature human dendritic cells are generated in vitro from the purified monocytes as described above.
• the monocytes are cultured at a concentration of 10 6 cells / ml in the following medium (hereinafter called complete culture medium): RPMI 1640 medium supplemented with 10% fetal calf serum (heating at 56 ° C. for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ⁇ g / ml of streptomycin (Life technologies) in 6-well culture plates (Nunc, Roskilde, Denmark) at a rate of 5 ml of medium per well .
• complete culture medium hereinafter called complete culture medium: RPMI 1640 medium supplemented with 10% fetal calf serum (heating at 56 ° C. for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ⁇ g / ml of streptomycin (Life technologies) in 6-well culture plates (N
• the cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom). After 6 days of culture (37 ° C, 5% CO 2 in a humid atmosphere), the phenotype of the cells is defined by cytofluorometry by analyzing the expression of the CD1a and CD83 molecules using a labeled anti-human CD1a antibody fluorescein is an anti-human CD83 antibody (Becton Dickinson) which is detected by an anti-mouse immunoglobulin antibody labeled with fluorescein (Silenus, Hauworth, Australia). Immature dendritic cells are characterized by an expression of the CD1a molecule (average fluorescence intensity (IMF)> 100) and the absence of expression of the CD83 molecule.
• IMF average fluorescence intensity
• NK cells (CD56 + / CD3-) are isolated from CMN by cytofluorometry (positive selection) after labeling with an anti-CD56 antibody labeled with fluorescein (Becton Dickinson) and an anti-CD3 antibody labeled with phycoerythrin. Labeling of the cells with the antibody is carried out as described above. The cells are sorted on a FACS Vantage (Becton Dickinson); the purity is> 98%.
• B lymphocytes are isolated from tonsils collected after surgical excision. Briefly, the tonsils are mechanically ground and then passed through a sieve of porosity 50 ⁇ m. Mononuclear cells are isolated by centrifugation on a Ficoll-Hypaque gradient as described above. T cells are removed from the CMN suspension by the so-called rosette technique, as described above. The purity of the B cell population is evaluated by cytofluorometry using an anti-CD20 antibody labeled FITC; the purity is> 95%. PCR technique The expression of the RNA coding for the polypeptide of sequence SEQ ID No. 2 was analyzed by PCR in the different CMN subpopulations.
• the cells are washed twice in PBS and then lysed in the extraction medium for Trizol nucleic acids (Life technologies).
• the proteins are removed by two successive extractions with Phenol / chloroform / isoamylic acid (25: 24: 1) (Life technologies).
• An equivalent volume of isopropyl alcohol is added to the aqueous phase to precipitate the nucleic acids (overnight at -20 ° C).
• the nucleic acids are collected by centrifugation (12,000 rpm, 30 minutes at 4 ° C).
• the pellet is washed once with an ethanol solution at 70 ° C.
• RNA After denaturation (65 ° C, 10 min), 2 ⁇ g of total RNA are transcribed inversely in using an oligo-dT primer using a commercial kit (Promega, Madison, Ml) according to the manufacturer's recommendations.
• the cDNA coding for the polypeptide of sequence SEQ ID No. 2 is amplified by PCR using the following primers: 5'-TGACTCGGAATCACCTCCCAGCT-3 '(SEQ ID No. 11) and 5'-CAAGACGGTGAGCAGGATGGCAGTACAG-3' (SEQ ID No. 12).
• RNA which was used to prepare the cDNAs is analyzed by evaluating the expression of the cDNA encoding the GAPDH molecule using the following primers:
• 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 sample buffer (0.25% bromophenol blue, 40% sucrose).
• the samples are analyzed by electrophoresis in 1% agarose gel which contains ethidium bromide. After migration, the samples are visualized by UV illumination of the gel. Results
• the mRNA encoding the polypeptide of sequence SEQ ID No. 2 is detected by
• RNA coding for the polypeptide of sequence SEQ ID No. 2 is selectively expressed by the T lymphocytes of the circulating blood.
• Example 4 The RNA coding for the polypeptide of sequence SEQ ID No. 2 is expressed in different tumor cell lines. Methodology We evaluated the expression of the mRNA coding for the polypeptide of sequence SEQ ID No. 2 in different lines tumor cells. These cell lines were obtained from ATCC and cultured according to the instructions provided. The extraction of total RNA as well as the synthesis of cDNA were carried out as described above. The expression of the RNA coding for the polypeptide of sequence SEQ ID No. 2 and the GAPDH molecule were analyzed by PCR as described above.
• the PCR experiments reveal that the following human cell lines constitutively express the mRNA coding for the polypeptide of sequence SEQ ID No. 2: lung adenocarcinoma (line A549), monocyte leukemia (line THP1), neuroblastoma (line HTB10), bladder carcinoma (T24), burkitt lymphoma (Daudi line), lung carcinoma (line A427), glioblastoma (line U373), larynx carcinoma (line HEP2), colon carcinoma (line HT29) and carcinoma of the breast (line BT20).
• lung adenocarcinoma line A549
• monocyte leukemia line THP1
• neuroblastoma line HTB10
• bladder carcinoma T24
• burkitt lymphoma Duudi line
• lung carcinoma line A427)
• glioblastoma line U373
• larynx carcinoma line HEP2
• colon carcinoma line HT29
• carcinoma of the breast line BT20.
• RNA coding for the polypeptide of sequence SEQ ID No. 2 is expressed by a number of tumor lines.
• Example 5 The recombinant polypeptide of sequence SEQ ID No. 2 is predominantly expressed in the membrane fraction of the transfected COS cells.
• COS cells are transfected with the vector pCDNA3.1 (Invitrogen) containing the insert coding for the polypeptide with sequence SEQ ID No. 2.
• This system makes it possible to add a c-myc epitope to the recombinant molecule.
• the transfection is carried out by the lipofection technique (Fugene; Boehringer Mannheim, Germany). Three days after transfection, the cells are collected and washed three times with PBS. The cells are then subjected to three rapid freezing / thawing series and then centrifuged at 12,000 revolutions per minute (rpm) for 10 min at 4 ° C. The supernatant corresponds to the cytoplasmic fraction and the pellet to the membrane part plus nucleus plus organelles.
• rpm revolutions per minute
• the pellet is resuspended in a 10 mM phosphate buffer, pH 7.4 containing 0.5% of Nonidet P40 and a cocktail of protease inhibitors (Boehringer Mannheim, Germany) for 20 minutes at 4 ° C.
• the cell lysates are then centrifuged at 12,000 rpm for 10 minutes at 4 ° C to remove the nuclei.
• the supernatants corresponding to the membrane and cytoplasmic fractions are taken up in an equivalent volume of 10 mM phosphate buffer containing 0.1% bromophenol blue, 5% glycerol and 0.1% ⁇ -mercaptoethanol.
• the proteins are then separated according to their molecular weight by polyacrylamide gel electrophoresis (5% concentration gel - 10% separation gel).
• a quantity of proteins corresponding to 5 x 10 6 cells is deposited in each line. After migration, the proteins are transferred by Western blotting onto a nitrocellulose membrane. After saturation in a phosphate solution containing 10% skim milk, the membranes are incubated overnight at 4 ° C with shaking with an anti-c-myc antibody (clone 9E10; ATCC, Manassas, VA) at a concentration of 10 ⁇ g / ml. After washing with a phosphate buffer containing 10% skimmed milk, the membranes are incubated with an anti-mouse immunoglobulin antibody labeled with peroxidase (Amersham) for 2 hours at room temperature with shaking. The membranes are washed in phosphate buffer.
• an anti-c-myc antibody clone 9E10; ATCC, Manassas, VA
• the attached antibodies are detected by chemiluminescence using the ECL system from Amersham.
• Results The Western Blotting presented in FIG. 4 indicates that the recombinant protein is mainly expressed in the insoluble fraction of the cellular extracts (cytoplasmic membrane plus intracytoplasmic organelles). This result as well as the prediction of a potential transmembrane domain suggests that the recombinant polypeptide is expressed on the surface of the transfected cells.
• the recombinant polypeptide of sequence SEQ ID No. 2 is expressed in the membrane (non-cytoplasmic) fraction of the transfected COS cells.
• Example 6 The gene coding for the polypeptide of sequence SEQ ID No.
• a genomic DNA sequence with 98% homology to EST # AI301140 was found on a fragment of chromosome 11q at cluster 33-34. Many tumor susceptibility genes have been located in the 11q23 genomic region -40 (Koreth, J., et al., J. Pathol., 187: 28-38, 1999).
• the gene coding for the polypeptide of sequence SEQ ID No. 2 is located in chromosome 11 region q33-q34. This result makes it possible to confirm that the polypeptide of sequence SEQ ID No. 2 is implicated in the regulation of the immune response and that an alteration of its expression could be associated with immunological disorders capable of inducing a tumor character in cells presenting a abnormal expression of the gene coding for the polypeptide of sequence SEQ ID No. 2.

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

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• 1999
  • 1999-10-29 FR FR9913629A patent/FR2800388A1/fr active Pending
• 2000
  • 2000-10-30 CN CN00816059A patent/CN1391608A/zh active Pending
  • 2000-10-30 AU AU12848/01A patent/AU1284801A/en not_active Abandoned
  • 2000-10-30 BR BR0015161-0A patent/BR0015161A/pt not_active Application Discontinuation
  • 2000-10-30 EP EP00974612A patent/EP1226248A1/fr not_active Withdrawn
  • 2000-10-30 CA CA002389522A patent/CA2389522A1/fr not_active Abandoned
  • 2000-10-30 MX MXPA02004297A patent/MXPA02004297A/es unknown
  • 2000-10-30 WO PCT/FR2000/003032 patent/WO2001031003A1/fr not_active Application Discontinuation
  • 2000-10-30 JP JP2001533984A patent/JP2003516126A/ja active Pending
• 2002
  • 2002-04-26 ZA ZA200203340A patent/ZA200203340B/en unknown

Non-Patent Citations (1)

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