Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
• • 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
• • 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
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • 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 present invention relates to new nucleotide sequences coding for variable regions of ⁇ chains of T cell receptors, the corresponding peptide segments and diagnostic and therapeutic applications.
• T cell receptors the receptors recognizing the antigens on the surface of mature T lymphocytes (hereinafter referred to as T cell receptors) have a structure having a certain analogy with those of immunoglobulins. Thus, they include heterodimeric structures comprising _ and ⁇ glycoprotein chains or ⁇ and S glycoprotein chains (see Meuer et al. (1), Moingeon et al. (2), Brenner et al. (3), Bank et al . (4)).
• the repertoire of T cell receptors must be able to cope with the immense diversity of antigenic determinants. This is obtained by genetic recombination of the different discontinuous segments of the genes which code for the different structural regions of the T-cell receptors.
• the genes include V segments (variable segments), possibly D segments (diversity segments), segments J (junction segments) and C segments (constant segments).
• V segments variable segments
• D segments diversity segments
• segments J junction segments
• C segments constant segments.
• specific genes are created by recombination of the V, D and J segments for the ⁇ and ⁇ loci and of the V and J segments for the ⁇ and ⁇ loci. These specific combinations as well as the pairing of the two chains create combinatorial diversity.
• This diversity is greatly amplified by two additional mechanisms, namely the imprecise joining of the V-D-J or V-J segments and the addition of nucleotides corresponding to the N region (Davis et al. (5)).
• V gene segments have been grouped into subfamilies according to the similarity of the sequences. By definition, the segments which have more than 75% similarity in the nucleotide sequence have been considered to be members of the same subfamily (Crews et al. 6)).
• V ⁇ gene segments are known (Wilson et al. (7), Robinson (8), Sense et al. (9), Reynolds (10), Li et al. (11)) which have been classified into 20 subfamilies including 7 with a single member (see Wilson et al. Already cited).
• WO 90/06758 has recently described monoclonal antibodies directed against specific segments of the variable parts of the T cell receptors, in particular ⁇ and chains. S. These monoclonal antibodies are useful not only as diagnostic tools but also as therapeutic tools, for example with respect to rheumatoid arthritis.
• the present invention aims to enrich the repertoire of gene segments coding for variable regions of the ⁇ chains of T cell receptors by providing new V ⁇ gene segments belonging to new subfamilies or belonging to subfamilies of which at least one is already known. a member.
• the present invention thus relates to nucleotide sequences coding for variable regions of ⁇ chains of receptors for human T lymphocytes, corresponding to cDNAs comprising nucleotide sequences chosen from any of the V ⁇ segments corresponding to one of the sequences SEQ ID No 2 to 19, and the sequences which differ from it by one or more nucleotides.
• the present invention relates more particularly to sequences coding for variable regions of the ⁇ chains of human T lymphocyte receptors, corresponding to cDNAs comprising nucleotide sequences chosen from any of the V ⁇ segments corresponding to one of the sequences SEQ ID No 2 to 5, and the sequences which differ from it by one or more nucleotides.
• nucleotides By the expression “and the sequences which differ from it by one or more nucleotides”, we include alleles which have up to 8 nucleotides of difference, but most often 1 or 2 nucleotides of difference, or which can differ by deletion or the addition of one or two codons.
• a more particular subject of the present invention is also:
• nucleotide sequences coding for variable regions of ⁇ chains of human T lymphocyte receptors corresponding to cDNAs corresponding to all or part of the nucleotide sequences chosen from any one of the V ⁇ segments corresponding to one of the sequences
• nucleotide sequences coding for variable regions of ⁇ chains of human T lymphocyte receptors corresponding to cDNAs corresponding to one of the nucleotide sequences chosen from any one of the V ⁇ segments corresponding to one of the sequences SEQ ID No 6 to 15, the sequences which differ by one or two nucleotides and the fragments thereof, in particular the fragments of the sequences which correspond to all or part of the nucleotide sequences chosen from any one of the V ⁇ segments corresponding to one of the sequences
• nucleotide sequences coding for variable regions of ⁇ chains of human T lymphocyte receptors corresponding to cDNAs corresponding to all or part of the nucleotide sequences chosen from any one of the V ⁇ segments corresponding to one of the sequences
• nucleotide sequences corresponding to cDNAs corresponding to all or part of the nucleotide sequences denotes both the complete sequences and fragments of these sequences, including short fragments which find applications as probes (gene - actually comprising at least 10 nucleotides) or as a primer (generally comprising at least 15 nucleotides).
• the present invention generally encompasses all of the new oligonucleotides which are fragments of the V ⁇ sequences according to the invention.
• sequences which differ by one or two nucleotides correspond to variations which have been observed experimentally during the determination of the nucleotide sequence of several cDNAs.
• the subject of the present invention is also the peptides encoded by nucleotide sequences according to the invention as well as the alleles and the derivatives thereof which have the same function.
• the present invention encompasses the peptides constituted by or comprising a peptide sequence encoded by the nucleotide sequences according to the invention as well as the fragments of these peptides. It also includes peptides which differ from these by one or more
• allelic variation 29
• the present invention can be reproduced in particular by bispecific gene amplification (polymerase chain reaction or PCR) starting from peripheral lymphocytes expressing the mRNAs including the variable or junctional ⁇ segments corresponding to the sequences ID No. 2 to 19 of the invention or alternatively by applying this PCR technique to genomic DNA of any somatic cell of an individual taken at random.
• the invention can also be reproduced by preparing the above gene sequences by chemical synthesis of oligonucleotides.
• the peptides according to the invention can be obtained by conventional peptide synthesis. They can also be obtained by application of known techniques of genetic engineering comprising the insertion of a DNA sequence coding for a peptide according to the invention in an expression vector such as a plasmid and the transformation of cells with this expression vector.
• the present invention therefore also relates to plasmids and expression vectors comprising a sequence DNA encoding a peptide according to the invention as well as hosts transformed with this vector.
• the present invention also relates to antibodies and in particular monoclonal antibodies, directed against an antigenic determinant belonging to or comprising a peptide according to the invention.
• Monoclonal antibodies can be obtained by any technique which allows the production of antibody molecules from cell line culture. These techniques include the various techniques using hybridomas.
• the production of antibodies can be obtained in animals by immunization of animals by injection of the peptides or fragments according to the invention, whether they are natural, recombinant or synthetic, optionally after coupling with an immunogenic agent such as toxoid tetanus, or by injection of human T lymphocytes expressing the corresponding sequences on their surface, including recombinant cells transfected with the corresponding coding sequences.
• an immunogenic agent such as toxoid tetanus
• the present invention also relates to hybridomas producing monoclonal antibodies directed against the polypeptides according to the invention.
• the present invention also encompasses the fragments and derivatives of monoclonal antibodies according to the invention which are reactive with defined variable regions of T cell receptors.
• These fragments are in particular the F (ab ′) 2 fragments which can be obtained by cleavage enzymatic antibody molecules with pepsin, the Fab 'fragments which can be obtained by reduction of the disulfide bridges of the F (ab') 2 fragments and the Fab fragments which can be obtained by enzymatic cleavage of the antibody molecules with papain in the presence of a reducing agent.
• the fragments can also be obtained by genetic engineering.
• Monoclonal antibody derivatives are by:
• Monoclonal antibody derivatives are also antibodies or fragments of these antibodies to which therapeutically active molecules are linked, in particular cytotoxic compounds.
• the products of the invention find several types of application in the field of diagnosis and in the field of therapy.
• the oligonucleotides contained in the nucleotide sequences according to the invention can be used to constitute detection probes (generally at least 10 nucleotides) capable of hybridizing to a variable region of a ⁇ chain or primers for amplification DNA (generally comprising at least 15 nucleotides and preferably at least 17 nucleotides) capable of binding to a sequence to be amplified.
• detection probes generally at least 10 nucleotides
• primers for amplification DNA generally comprising at least 15 nucleotides and preferably at least 17 nucleotides
• the oligonucleotides thus find an application in the diagnosis of immune disorders by detecting the presence of nucleic acid sequences homologous to a gene coding for variable regions of ⁇ chains of T cell receptors in the mRNA of a sample of a patient.
• T cell genes Different methods can be used to link the expression of T cell genes to a disease. These methods include:
• the primers find application in PCR reactions in a method such as that defined under c.
• Monoclonal antibodies, fragments or derivatives of these antibodies according to the invention can be used to study type T immune responses, for example in the field of autoimmune diseases of oncology, allergy, transplantation and infectious diseases.
• the repertoire of the different variable ⁇ segments of the T receptor can be studied, whether they are blood T cells or tissues.
• the techniques used can be in vitro or in vivo methods.
• the samples used can be samples of body fluids or samples of tissue.
• the techniques used can notably include flow cytofluorimetry to analyze T lymphocytes in the blood or immunoperoxidase labeling on anatomopathological section to study lymphocytes infiltrating tissue.
• the antibodies, their fragments or their derivatives are administered by the usual routes, for example by the intravenous route, and the immunospecific bonds are detected. This can be obtained for example in the case where an antibody labeled with a radioisotope is used.
• the oligonucleotides contained in the nucleotide sequences according to the invention can be used in therapy as antisense oligonucleotides. It is known in fact that it is possible in vitro to inhibit the expression of a gene transcribed in human lymphocytes by incubating these lymphocytes with an antisense olignoculeotide specific for the gene in question (30). These antisense oligonucleotides generally comprise at least 10 and preferably at least 16 nucleotides. These antisense oligonucleotides can in particular be the inverted and complemented sequences corresponding to the 20 nucleotides upstream from the translation initiation site (ATG).
• ATG translation initiation site
• the advantage of using in vitro antisense oligonucleotides specific for a V ⁇ gene segment is to abolish (or reduce strongly) the expression of a T receptor comprising this V ⁇ segment and therefore of obtaining a clonal deletion phenomenon at the level of the specific reactivity of T lymphocytes.
• the antisense oligonucleotides can not only be used in vitro on human T lymphocytes which are then reinjected, but also in vivo by local or systemic injection preferably after modification to increase the stability in vivo and the penetration inside the lymphocytes of these oligonucleotides.
• the monoclonal antibodies according to the invention can be used to modulate the immune system. This is how antibodies can be administered to block the interaction of effector T cells with their specific antigen. It is also possible to administer anti-T receptor antibodies linked for example to a cytotoxic molecule or to a radioisotope so as to obtain a clonal deletion, by virtue of the specific binding to a ⁇ chain of a T cell receptor.
• the monoclonal antibodies according to the invention can be used therapeutically at low mitogenic concentrations so as to specifically activate certain subsets of T cells or can be used at much higher concentrations to bind to the receptors concerned and thus mark these subsets for elimination by the reticuloendothelial system.
• An important criterion for the treatment of a disease is the ability to modulate subsets of T cells linked to a disease.
• the exact nature of this therapeutic modulation namely blocking or suppressing a particular subset of T cells or, on the contrary, stimulating and activating a
• This type of treatment has an advantage compared to current treatments using antibodies such as treatment with anti CD3 antibodies in patients who have undergone a renal transplant and who have a rejection problem since, thanks to the invention, there is no will not modulate the entire T cell population but only the subset of T cells expressing the specific ⁇ subfamily of T cell receptors
• T cell response is often oligoclonal, it is generally advisable to use in therapy "cocktails" of several antibodies.
• anti-V ⁇ antibodies can be used to select T cells in vitro, for example by passing through a column containing beads carrying the antibody. This separation of certain T lymphocytes can be used with a view to culturing these lymphocytes before reinjecting them into the patient.
• nucleotide sequences according to the invention or the fragments of these sequences (generally comprising at least 8 to 10 amino acids).
• sequences or fragments administered to humans or animals can act as a decoy, that is to say that they will bind to the epitope carried by the harmful antigen and prevent the reaction of normal T cells with the antigen, thereby preventing the development of an aggressive disease against self-determinants. They can also be used as immunogens in the manufacture of vaccines (possibly after coupling with protein carriers).
• Figs. 1 to 6 give in alignment both known V ⁇ sequences and partial sequences of the new sequences according to the invention (SEQ ID Nos. 6 to 19), denoted IGRa 08 to IGRa 20 belong to known V ⁇ subfamilies.
• the numbering of the nucleotides begins at the initiation ATG codon (which is underlined). The dots indicate the identical nucleotides. Sequences that are assumed to be leader sequences are highlighted.
• Fig. 7 gives the Southern blot analyzes of the genomic DNA treated with a restriction enzyme using probes specific for the V ⁇ subfamilies.
• the restriction enzymes used are EcoR I (column R), Hind III
• - Fig. 8 represents the detection by autoradiography of the amplified transcripts of the ⁇ chains of the TCR expressed by the peripheral lymphocytes of a healthy individual, and of the co-amplified ⁇ -actin control.
• Fig. 9 represents the analysis by cytofluorimetry of the reactivity of the monoclonal antibody RO-73 respectively with respect to the immunizing clone 3025 (9A), the clone 12410 (9B) and the circulating lymphocytes (9C).
• the control reactivity of the NKTa or OKT 3 antibodies is given respectively for each type of cell.
• the number of cells counted (linear scale) is given as a function of the intensity of the fluorescence (logarithmic scale).
• FIG. 10 represents the cytofluorimetry analysis of the reactivity of the monoclonal antibody JU-74 (FIGS. 10A, 10B, 10C: same conditions as for FIGS. 9A, 9B, 9C).
• Fig. 11 shows the cytofluorimetry analysis of the co-modulation with the CD3 molecule of the TCR structure of clone 3025 recognized by the monoclonal antibody R0-73 respectively in the absence (Fig. 11A) or in the presence of anti-CD3 antibodies (Fig. 11B).
• the co-control is given respectively with the monoclonal antibodies NKTa, OKT3 and anti-CD2.
• Fig. 12 represents the cytofluorimetric analysis of the co-modulation with the CD3 molecule of the TCR structure of clone 3025 recognized by the monoclonal antibody JU-73, respectively in the absence (FIG. 12A) or in the presence of anti-CD3 antibodies (Fig. 12B).
• FIG. 13 represents the detection by autoradiography of the amplified transcripts of the a chains (FIG. 13A) and of the ⁇ chains (FIG. 13B) of the TCR expressed by the RO-73 + cells.
• RNA was prepared according to the guanidinium isothiocyanate and cesium chloride method (Chirgwin (12)) or the one-step method by extraction with guanidinium isothiocyanate, phenol and chloroform (Chomczynski (13)).
• the first strand of cDNA was synthesized in a final volume of 50 microliters at a temperature of 42o C for 1 hour using 5 micrograms of total RNA, reverse transcriptase and a primer A specific for the C ⁇ region constituted by the sequence 5'-TATCTGGAGTCA TTGAGGGCGGGC (SEQ ID No 20). This material was then purified by extraction with phenol / chloroform and precipitation with ammonium acetate.
• the first strand of G-terminated cDNA was precipitated with ethanol and subjected to amplification using the PCR (Polymerase Chain Reaction technique described by Saiki et al. (14)) in a final volume of 100 microliters containing 50 mM KCl, 10 mM Tris-Ci pH 8.3, 1.5 mM MgCl 2 , 0.1%
• the two primers used are, on the one hand, a poly-C primer (5'-GCATGCGCGCGGCC GCGGAGG-14C) (SEQ ID No 21) described by Loh et al. (15) as well as a primer B specific for the C ⁇ region (5'-TGTGGCCAGGCATGCCAGTGTGGCC) (SEQ ID No. 22).
• each cycle comprises a denaturation step at 92 ° C for 1 minute, a hybridization step at 55 ° C for 2 min and an elongation period at 72 ° C for 4 min.
• the amplified products are then precipitated with ethanol, resuspended in 30 mM sodium acetate pH5, 50 mM NaCl, 1 mM ZnCl 2 , glycerol 5% by volume, and 1/10 of this material is purified according to the size on an agarose gel with low melting point 1%.
• a second amplification phase is then carried out directly on approximately 10% of the band containing the agarose following the same conditions as above, except that the primer C specific for the C ⁇ region is used the primer 5'-GGTGTGGGAGAA TTCTGCTTCTGA (SEQ ID No 23).
• the reaction mixture is then precipitated with ethanol and resuspended in 60 ⁇ l HUO.
• 1/3 of the product of the second amplification is digested with Sac II, separated on 1% agarose gel and purified by adsorption on glass beads.
• the material is inserted into the Bluescript SK + vector (Stratagene, La Jolla, USA) and the recombinants obtained are used to transform XLl-blue strains of E. Coli (Stratagene).
• a test is carried out on the white colonies using a "dot blot" technique and as a probe a third oligonucleotide specific for the C ⁇ region
• the DNA was extracted from the human erythroleucic cell line K562 and digested with one of the following restriction enzymes: Eco RI, BamH I or Hind III.
• the DNA (15 micrograms) was subjected to 0.7% agarose electrophoresis and was transferred to nylon membranes as described by Triebel et al. (20). Hybridizations were carried out at 65 ° C. with 6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denhardt's and 100 micrograms of denatured salmon sperm DNA within 16 hours. The membranes were washed at 65 ° C with 2 ⁇ SSC, 0.2% SDS.
• V ⁇ probes As the specific V ⁇ probes, probes obtained by amplification of VJC cDNA were used, using the primer poly-C and primer C. The probes were purified on 1% agarose gel. DNA probes labeled with 3 2 P were prepared from the fragments purified on agarose by the method of Feinberg (21). IV - Results
• V ⁇ sequences of the invention appear in the list of sequences under SEQ ID N "2 to 19.
• SEQ ID N 3 to 5 correspond to 3 new sub-families while the SEQ ID No. 2 and 6 to 19 correspond to
• This subfamily was identified by the clone IGR b02 (SEQ ID No. 2).
• the segment SEQ ID No. 3 was defined as a consensus sequence from 23 distinct cDNA clones. We observed in position 322 a C instead of a T and in position 350 an A takes the place of a G.
• the segment ID H ′ 4 was defined as a consensus sequence from 4 distinct clones. We observed in position 154 a G instead of an A and in position 160 an A instead of a G. I has a homology of 75.7% with the sequence VB12A1 (Leiden already cited) but has a lower homology 75% with the other members of the V ⁇ 5 subfamily (shown in Fig. 1). It is therefore not part of the V ⁇ 5 subfamily.
• Subfamily V ⁇ W24 (SEQ ID No 5)
• the SEQ ID No 5 segment was defined from 2 distinct cDNA clones.
• the sizes of the restriction fragments of the germinal DNA are as follows:
• V ⁇ W21 ECOR I 1.7-, 3- and 6.5 kb, Hind III 2.5-, 7.2-, 11.7-, 14- and 18 kb, BamH I 5.5-, 16, 5- and 23 kb; V ⁇ W22; EcoR I 2.8 kb, Hind III 8.8 kb, BamH I 5.3 kb;
• V ⁇ W23 EcoR I 3.2- and 4.4 kb, Hind III 7.4-, 15.5- and 16.5 kb, BamH I 2.5- and 5.7 kb;
• V ⁇ w24 EcoR I 8 kb, Hind III 20 kb and 7.3 kb, BamH I 11, - and 22 kb.
• V ⁇ 5 subfamily (Fig. 1):
• sequences have a homology of 79 to 86 I and 76 to 70 1 respectively with the 4 previously known segments VB12A1 (Leiden already cited), HBP51 (Kimura (23)), PH24 (Tillinghast already cited) and PL25 (Concannon (24 )) and represent new members.
• V ⁇ 6 subfamily (Fig. 2):
• This sequence corresponds to an extension on the 5 ′ side of the HBP25 clone (Kimura, already cited).
• This sequence which represents a new member has a homology of the nucleotides of 94% with PH 16 (Tillinghast, already cited), GPPA (Li, already cited) and HT45 (Kimura (25)).
• V ⁇ 12 subfamily (Fig. 3): SEQ ID N ° 12 (IGR b13)
• This sequence which represents a new member corresponds to more than 85% homology with the PH27 sequences
• V ⁇ 13 subfamily (Fig. 4):
• SEQ ID N ° 13, 14 and 15 (IGR bl4, IGR bl5 and IGR b16)
• the sequences SEQ ID N "13 and 14 which represent new members have homology of 78 to 91% and 77 to 79% respectively with the other known sequences
• sequence SEQ ID No 15 has a 94% homology with HBVP34. It should be noted that the sequence SEQ ID No. 15 has an intron (represented in lowercase characters) in the leader region.
• the sequence SEQ ID No. 15 is a consensus sequence. At position 231 we observed a C instead of a T and at position 259 an A instead of a G.
• V ⁇ 7 subfamily (Fig. 5):
• sequence PL4.9 Concannon, already cited
• V ⁇ 9 subfamily (Fig. 6): SEQ ID No 19 (IGR b20)
• This sequence extends the PL2.6 sequence (Concannon, already cited) on the 5 'side. There is a difference between the two sequences at positions 98 and 100 corresponding to different amino acids.
• the present invention also aims to provide specific oligonucleotides of the different V ⁇ subfamilies, which can be used as primers for the amplification of DNA. corresponding to these different V ⁇ subfamilies, with a view for example to a study of the expression of certain V ⁇ subfamilies in a patient and finally to a diagnosis of immune disorders, as indicated above.
• V ⁇ subfamilies The predominant expression of certain V ⁇ subfamilies has already been studied using an incomplete range of oligonucleotides.
• Sottini et al. (33) have demonstrated, using a range of oligonucleotides, a predominant expression of certain V ⁇ in patients with rheumatoid arthritis.
• the present invention aims to provide a full range of oligonucleotides allowing the study of both
• V ⁇ subfamilies and new V ⁇ subfamilies of the invention which are entirely specific to each subfamily.
• the oligonucleotides were therefore chosen and synthesized for this purpose and, if necessary, modifications of one or two nucleotides were introduced compared to the
• the present invention thus also relates to oligonucleotides, which can be used as primers for the amplification of DNA corresponding to variable regions of ⁇ chains of T cell receptors, chosen from the sequences SEQ ID N ° 25 to 48.
• the subject of the present invention is also the use, as primers for the amplification of DNA corresponding to variable regions of chains of T cell receptors, of oligonucleotides chosen from the sequences SEQ ID Nos. 25 to 48.
• the present invention also relates to a method of detecting nucleotide sequences coding for V ⁇ segments of T receptors or of cDNA corresponding to the transcription products thereof, in a biological sample, characterized in that it comprises: a) amplification of the DNA with at least one pair of primers formed by one of the oligonucleotides defined above and an oligonucleotide belonging to the C ⁇ segment, and
• the oligonucleotide belonging to a C ⁇ segment used for the amplification can in particular be chosen from the sequences SEQ ID Nos. 49 and 50.
• the procedure is advantageously carried out in the presence of a pair of control primers and the corresponding control sequence amplified is detected using a corresponding control probe.
• This pair of control primers can correspond to two C ⁇ segments, for example the primers C ⁇ E and C ⁇ J corresponding to the sequences SEQ ID N ° 55 and 56.
• a C ⁇ detection probe is then used (corresponding for example to the sequence SEQ ID No 57 ).
• this pair of primers is advantageously constituted by two primers belonging to ⁇ -actin, in particular those corresponding to sequences SEQ ID No. 52 and 53. O then uses a detection probe corresponding to a sequence of 0-actin, such the sequence SEQ ID N ° 54.
• the present invention also relates to a diagnostic kit for implementing the method defined above, which comprises:
• oligonucleotide chosen from the sequences SEQ ID N ° 25 to 48,
• This kit can notably include:
• sequences SEQ ID No. 25 to 45 correspond to sequences belonging to clones of the known subfamilies V ⁇ 1 to V ⁇ 20 (accessible in the database of EMBL data) or to sequences which differ by one or two nucleotides.
• sequences SEQ ID No 45, 46, 47 and 48 correspond to sequences belonging to clones of new subfamilies of the invention, correspond to subfamilies provisionally named V ⁇ w21, V ⁇ w22, V ⁇ w23 and V ⁇ w24 ( w indicating that the designation is awaiting final designation).
• sequences SEQ ID No. 49 and 50 are two examples of C ⁇ oligonucleotides which can be used as primers for amplification.
• sequence SEQ ID No. 51 is the sequence of a C ⁇ probe which can be used for the detection of the amplified DNAs.
• sequences SEQ ID No. 52, 53 and 54 are respectively the sequences of a pair of oligonucleotides belonging to the sequence of 0-actin which can be used for the control of the amplification and the sequence of a probe to detect corresponding amplified DNAs.
• the position indicated is the position of the 5 ′ end from the predictive initiation site for ATG translation.
• the position is given relative to the first nucleotide of the sequence.
• the underlined nucleotides correspond to the mismatches introduced with respect to the natural sequence.
• oligonucleotides were synthesized on an Applied Biosystems 381 A automated DNA synthesizer using the ⁇ -cyanoethylphosphoramidite method (Sinha N. et al.
• oligonucleotides were detritylated in the apparatus, cleaved from the support and deprotected with ammonia (at 60 ° C. for 5 hours).
• the crude products were purified by high-pressure reverse phase chromatography on a column of ⁇ -bondapak C18 using a gradient of acetonitrile (9 to 15%) in a 0.01 M triethylammonium acetate buffer at pH 5.5.
• the amplification carried out using the primers according to the invention can in particular be the amplification technique by PCR (Polymerase Chain Reaction) as described by Saiki et al. (14) and patents US-A-4,683,195, 4,683,202,
• PCR it is possible to use a double stranded DNA that denatures or a cDNA obtained from RNA using reverse transcriptase as mentioned above.
• the polymerization agent is a DNA polymerase, in particular Taq polymerase.
• the amplification cycle is repeated 25 to 4 times.
• amplified sequences can be obtained by labeling oligonucleotides with a radioactive isotope, which leads to detection by autoradiography, or by coupling with an enzyme such as peroxidase (ECL Amersham system), alkaline phosphatase or ⁇ -galactosidase (system Tropix Ozyme), which leads to chemiluminescence detection.
• ECL Amersham system peroxidase
• alkaline phosphatase alkaline phosphatase
• ⁇ -galactosidase system Tropix Ozyme
• RNA was extracted according to the one-step method by extraction with guanidium isothiocyanate, phenol and chloroform (Chomczynski, 13).
• the complementary DNA was synthesized in a final volume of 20 ⁇ l at 42 ° C for 1 hour using 1 to 5 ⁇ g of total RNA, the reverse transcrip tase and the primer C ⁇ B (1.25 M).
• the material obtained was then heated at 95 ° C for 3 minutes before being subjected to an amplification according to the PCR technique using in parallel each of the specific V ⁇ primers corresponding to the sequences SEQ ID No 25 to 48 and the primer C ⁇ B specific for the C ⁇ region (SEQ ID No. 50).
• This amplification was carried out in a final volume of 10 ⁇ l per tube containing 50 mM KCl, 10 mM tris-HCl pH 8.3, 1.5 mM MgCl2 0.1% (weight / volume) of gelatin, 200 M of dNTP, 0.25 units of Taq polymerase and 0.25 M of each primer.
• a control amplification was carried out starting from 25 mM of a DNA fragment of ⁇ -actin of 877 base pairs prepared by PCR and of the primers Act 1 and Act 2 (SEQ ID N ° 52 and 53 ) specific for actin.
• 30 amplification cycles were carried out followed by a final elongation step of 5 minutes at 72 ° C.
• Each cycle included a denaturation step at 94 ° C for 1 minute, a hybridization step at 65 ° C for 1 minute and an elongation period at 72 ° C for 1 minute.
• the products obtained were separated by electrophoresis on a 2% agarose gel, transferred to nylon membranes in an alkaline buffer and hybridized simultaneously with the oligonucleotide probes C ⁇ C (SEQ ID No. 51) and Act 3 (SEQ ID N ° 54) labeled with 32 P by the enzyme polynucleotidyl T4 kinase.
• Hybridization was performed at 42 ° C for 16 hours in a buffer containing 6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denhart's, 0.05% PO4H2Na and 100 ⁇ g / ml of salmon sperm DNA
• the membranes were then washed with 6 ⁇ SSC, 20mM PO4H2Na, twice at room temperature for 5 minutes and once at 50 ° C for 30 minutes and then autoradiographed.
• Actin control (band of 877 base pairs) makes it possible to verify the amplification in all the wells. A specific signal appears below this band, the size of which corresponds to the size of the corresponding amplified fragments, each fragment having a length corresponding to the distance between the location of the specific oligonucleotide V ⁇ and the primer C ⁇ .
• Clone T 3025 (Moebius et al. (35)) was cultured in complete medium comprising DMEM (Seromed), 8% human AB serum, IL-2 and TCGF as described by Hercend et al. (36). Periodic restimulations were carried out on allogenic cells in the presence of IL-2.
• mice 6 week Biozzi mice (Institut Curie, Paris, France) were immunized with the whole T cells of clone 3025. After a first intraperitoneal injection of ⁇ 10 6 cells with complete Freund's adjuvant, the mice received three intraperitoneal injections. 5 ⁇ 10 6 cells with incomplete Freund's adjuvant three weeks apart Two weeks after the last intraperitoneal injection, the mice received 2 ⁇ 10 6 viable cells by intravenous injection. The mice were sacrificed three days later and the spleen was removed. 3) Merger
• NS-1 cells (Kohler and Milstein (39) were cultured in medium comprising DMEM (Seromed), 8 azaguanine (Sigma, Saint Louis, MI), 10% horse serum (Seromed, lot no. 5Z04), penicillin and streptomycin (Eurobio), glutamine (Seromed, 200 mM) and sodium pyruvate (Gibco, 100 mM).
• the spenocytes were fused with the NS-1 cells by polyethylene glycol (PEG 1000, Merck) with a ratio of 4 splenic cells to a myeloma cell. After fusion, the cells were cultured at 3 ⁇ 10 6 cells per ml in 96-well plates (Nunc) in a HAT selection medium containing DMEM, 10% horse serum, 10% fetal calf serum ( Seromed, lot no. 219195), aminopterin (Gibco), hypoxanthine and thymidine (Gibco), penicillin and streptomycin, glutamine, sodium pyruvate and NCTC 109 (Eurobio ). Fresh medium was added to the wells 2 days (50 ⁇ l per well) and 9 days after the fusion (100 ⁇ l per well). The culture was carried out at 37 ° C., in an incubator containing 10% CO 2 . 4) Screening of hybridomas
• the supernatant of the hybridomas obtained was collected 15 days after the fusion and tested for its reactivity with the immunizing cell by indirect immunofluorescence and analysis by flow cytometry. Briefly, the T3025 cells were incubated at 4 ° C. for 30 minutes with the hybridoma supernatant (100 ⁇ l per 300,000 cells), washed and labeled with a goat anti-mouse immunoglobulin antibody conjugated to fluorescein (Coulter Electronics, Hialeah, FL). The cells were then analyzed by cytofluorometry (Coulter Profile). As shown in FIGS.
• the supernatants of the hybridomas R0-73 and JU-74 allow the labeling of 100% of the cells of the immunizing clone 3025.
• An anti-CD3 antibody (OKT3 Ortho-Co) and the anti- NKTa clonotype (IgG1, Hercend et al. (40)) served as positive and negative controls respectively in this experiment.
• the anti-T receptor specificity of the monoclonal antibodies was analyzed according to the following criteria:
• TCR T cell receptor
• the supernatants of the RO-73 and JU-74 hybridomas react with 100% of the cells of the immunizing clone 3025 (FIG. 9A and 10A), less than% of the cells of clone 12410 (FIG. 9B and 10B) and 1 to 3% of the PBL (Fig. 9C and 10C).
• the cells of clone 3025 (10 6 cells per ml) were incubated in medium alone or in the presence of anti-CD3 antibody (OKT3) in 24-well culture plates. After 24 hours of incubation, the cells were harvested and labeled with the hybridoma supernatant RO-73 or JU-74, the anti-CD3 monoclonal antibody or a anti-CD2 control monoclonal antibody (Coultronics Co.) and then analyzed. by cytofluorimetry. As shown in FIGS. 11 and 12, the cytofluorimetry analysis of the cells incubated in the presence of anti-CD3 monoclonal antibody (FIGS.
• FIG. 11B and 12B shows a decrease in the fluorescence intensity for the anti-CD3 monoclonal antibody as well as for RO-73 and JU-74, while the intensity of labeling with the anti-CD2 monoclonal antibody increases in comparison with the intensity obtained respectively in the absence of anti-CD3 antibody (FIG. 11A and FIG. 11B).
• the cells of the initial hybridomas were distributed in culture plates at the rate of 0.5 cell per well in complete HAT medium, on irradiated syngenic splenic cells. 3 subclones were selected for each of the RO-73 and JU-74 hybridomas. These cells produce monoclonal antibodies whose reactivity is identical to that of the initial hybridomas (results not shown).
• the subclones were cultured in a non-selective medium containing DMEM, 10% fetal calf serum, 10% horse serum, hypoxanthine, thymidine, penicillin and streptomycin, glutamine , sodium pyruvate and NCTC 109.
• the hybridoma or subclone cells were frozen in fetal calf serum containing 10% dimethyl sulfoxide (DMSO, Merck) and stored in liquid nitrogen.
• DMSO dimethyl sulfoxide
• the isotypes were determined by immunodiffusion on solid support using an "INNO-LIA mouse mAb kit.
• Ascites have been produced in nude mice.
• the ascites liquid obtained was filtered through cotton to remove the fibrin and precipitated by sodium sulfate (18%).
• the pellet obtained was suspended in PBS buffer, diluted 1/3 in buffer (4.5 M NaCl, 2.25 M glycine, pH 8.8) and loaded onto a column of Protein A-Sepharose 4 Fast Balanced flow in the loading buffer (3M NaCl, 1.5 M glycine, pH 8.8).
• a majority peak of immunoglobulins was eluted at pH 6 using successive elution buffers of decreasing pH. This majority peak was purified on an ion exchange column (Q Sepharose Fast Flow) in 50 mM Tris buffer, pH 8 and eluted with a NaCl gradient.
• the purity of the preparation was checked by electrophoresis in a PHAST system (Pharmacia LKB, Uppsala, Sweden) and the purified immunoglobulins were tested by indirect immunofluorescence on cell 3025, as indicated previously.
• Sepharose Fast Flow 32 mg of purified immunoglobulins.
• the percentages of circulating lymphocytes recognized respectively by the RO-73 and JU-74 monoclonal antibodies were determined for 10 different healthy donors. The results are shown in Table 1.
• the monoclonal antibody JU-74 recognizes less than 0.5% to 2.1% of the PBLs (average 1.08%) and the monoclonal antibody RO-73 recognizes from 0, 5% to 2.2% of PBL depending on the individual (average 1.09%).
• the RO-73 and JU-74 monoclonal antibodies recognize approximately the same percentages of circulating lymphocytes, respectively.
• the PBLs recognized respectively by the RO-73 and JU-74 monoclonal antibodies were purified from a normal donor using a positive selection process using magnetic beads (Dynabeads, Dynal). Briefly, 1 to 4 ⁇ 10 9 PBLs were labeled with one or other of the above purified monoclonal antibodies and incubated with the ready-to-use Dynabeads M-450 beads covered with goat serum anti-mouse IgG, in the proportion of 3 beads for a labeled cell. The positive cells were then separated using a magnet.
• the selected positive cells were cultured in a microplate in the presence of IL-2 on irradiated allogenic cells and then again purified by magnetic beads after approximately one week of culture in order to obtain a preparation of purity greater than 95 %.
• oligonucleotides specific for the V ⁇ 1 to V ⁇ 24 type segments described above (SEQ ID No 25 to No 48) was used as specific primers to analyze the ⁇ TCR transcripts expressed in RO-73 + and JU-74 cells +.
• the procedure used is identical to that described in the example above for the peripheral lymphocytes of a healthy individual.
• the complementary DNA was synthesized using the reverse transcriptase and the primer C ⁇ (SEQ ID No. 50).
• the material obtained was subjected to 30 amplification cycles according to the PCR technique using in parallel each of the specific V ⁇ primers corresponding to the sequences SEQ ID N ° 25 to 48 and the primer C ⁇ B specific for the C ⁇ region (SEQ ID No 50) as described above.
• the amplified products obtained were separated by electrophoresis on a 2% agarose gel, transferred to nylon membranes and hybridized with the C ⁇ C oligonucleotide probe (SEQ ID No. 51) labeled with 32 P. The membranes were then washed as described above and then autoradiographed.
• the sequencing of the transcripts of the ⁇ chain of the TCRs was carried out by following the cloning and sequencing method described above for the cDNA.
• the material amplified by the specific oligonucleotide of the V ⁇ 13 subfamily (SEQ ID No. 37) was digested with the enzyme SacII and purified by electrophoresis on agarose gel.
• the material obtained was introduced into the vector pBS SK + [as described above for the A-PCR technique] and used to transfect the E. Coli XL-1-blue bacteria.
• the transformed colonies obtained were tested by dot-blot hybridization using the oligonucleotide probe C ⁇ C (SEQ ID No. 51) labeled with 32P.
• the plasmid DNA was sequenced as previously described.
• Figure 13 shows the results obtained for the analysis of the transcripts of the ⁇ chains (Fig. 13 A) and the ⁇ chains (Fig. 13B) of the TCR expressed by the RO-73 + cells recognized by the monoclonal antibody RO-73 . It can be seen that many different Va segments are expressed in these cells (FIG. 13A). On the other hand, only the oligonucleotide specific for the sequences of the V ⁇ 13 subfamily allows amplification of the cDNA (FIG. 13B).
• the monoclonal antibodies JU-74 and RO-73 have the same specificity and exclusively recognize the product of the new V / 313 IGRbl6 gene segment of the invention (SEQ ID No. 15 indicated above).
• TITLE OF THE INVENTION Nucleotide sequences coding for variable regions of the ⁇ chains of human T lymphocyte receptors, corresponding peptide segments and diagnostic and therapeutic applications.
• TYPE nucleotide and its corresponding protein
• GGT ACC AGG CTC CTC TGC CGG GTG GCC TTC TGT CTC CTG GTG GAA GAA 96 Gly Thr Arg Leu Leu Cys Arg Val Ala Phe Cys Leu Leu Val Glu Glu
• AAA AAG CAG CCT GTG GCT TTT TGG TGC AAT CCT ATT TCT GGC CAC AAT 192 Lys Lys Gln Pro Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His Asn
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• GAG GGT ACC ACT GAC AAA GGA GAA GTC CCC AAT GGC TAC AAT GTC TCC 288 Glu Gly Thr Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr Asn Val Ser
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• TYPE nucleotide and its corresponding protein
• GAG AAA CTC TCT ATA AAT GAA AGT GTG CCA AGT CGC TTC TCA CCT GAA 287 Glu Lys Leu Ser Ile Asn Glu Ser Val Pro Ser Arg Phe Ser Pro Glu
• TYPE nucleotide and its corresponding protein

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