WO1997004093A1 - Nucleotide and peptide sequences for treating myasthenia - Google Patents

Abstract

The use of nucleotide or peptide sequences for preparing drugs suitable for treating or preventing acquired auto-immune myasthenia is disclosed. Said nucleotide sequences are selected from sequences capable of coding for a peptide sequence that is in turn capable of causing the formation of antibodies that recognise and form an immunological complex with all or part of the amino acid sequence of human T cell receptor segment Vβ5.1; or sequences capable of coding for a peptide sequence that itself represents an antibody capable of recognising and forming an immunological complex with all or part of the amino acid sequence of said segment Vβ5.1; sequences capable of coding for an anti-sense RNA that is in turn capable of hybridising with all or part of the mRNA coded for by the DNA sequence coding for the amino acid sequence of said segment Vβ5.1; or sequences capable of hybridising with all or part of the DNA sequence coding for the amino acid sequence of said segment Vβ5.1, or with all or part of the mRNA coded for by said DNA sequence. Said peptide sequences are selected from sequences capable of causing the formation of antibodies that recognise and form an immunological complex with human T cell receptor segment Vβ5.1; or sequences corresponding to antibodies capable of recognising and forming an immunological complex with all or part of the amino acid sequence of T cell receptor segment Vβ5.1.

Description

NUCLEOTIDE AND PEPTIDE SEQUENCES FOR THE TREATMENT OF MYASTHENIA.

The present invention relates to the use of nucleotide and peptide sequences for obtaining drugs intended for the prevention or treatment of myasthenia gravis autoimmune acquired. The invention also relates to pharmaceutical compositions and vaccines comprising the above nucleotide and peptide sequences.

Myasthenia gravis (Myasthenia Gravis) is an autoimmune disease which is characterized by fatigue and muscular weakness. This disease is caused by autoantibodies that recognize the nicotinic acetylcholine receptor (RACh) on the muscle, thereby hindering neuromuscular transmission (1). These anti-RACh autoantibodies are found in the sera of 85% of patients (2) and at least some of them are pathogenic (3, 4). The presence and activation of T helper cells participate in the pathogenesis of myasthenia gravis (5-9). The thymus is supposed to play a direct role in the physiology of this pathology, as confirmed by the favorable effect of thymectomy on clinical evolution (10,11) and its frequent morphological anomalies (50-70% of hyperplasia, 10 -15% thy dreary) (12-14).

Several experimental results strongly support the role of the thymus in primary self-sensitization and / or in the triggering and perpetuation of the autoimmune process: hyperactivity of thymic T and B cells (15-18), self-reactivity towards the receptor for l acetylcholine in B and T cells (18-23), presence of the autoantigen (24). functional abnormalities of the epithelial cells and in particular a disruption of the production of IL-6 (25).

In short, the production of the above-mentioned anti-RACh autoantibodies is under the control of CD4 + helper T cells restricted to MHC (Major Histocompatibility Complex) class II. The peptides resulting from the degradation of the autoantigen, namely RACh, in macrophages or other cells presenting the antigen, are coupled to the MHC class II molecule, HLA DR. T cells are stimulated by recognizing this peptide-HLA DR complex, via a receptor, also known as a T cell receptor, the latter thus stimulated being able in turn to stimulate B cells which will produce antibodies recognizing RACh .

The immunogenetic aspect of the pathology was studied by HLA typing on a large number of patients. The HLA DR3 or DR5 haplotypes have been serologically defined in correlation with the pathology (26-28). A strong association with HLA DR3 has been found in young patients with thymic hyperplasia (27). Molecular typing confirmed these results (29) and identified the HLA-DQ alleles (DQA1 * 0501 and DQB1 * 0201) as being frequently expressed in patients with thymic hyperplasia (30). As well as the autoantigen RACh involved in myasthenia gravis is well characterized at the molecular and functional levels (31), and that numerous monoclonal antibodies have been produced (32). immunodominant B epitopes have been identified on the α subunit (MIR. HIR) (33-35). T cell epitopes have also been identified using recombinant fragments and synthetic peptides (36-40), thus revealing the multiepitopic nature of the autoantigen.

The aim of the present invention is to provide medicaments intended for the treatment or prevention of autoimmune myasthenia gravis, these medicaments comprising molecules capable of specifically binding either to the segment of the T cell receptor capable of recognizing and binding to peptide-HLA DR complex mentioned above, either to the gene or to the RNA. in particular the messenger RNA (mRNA), coding for this segment, and this in a sufficiently stable (or in a sufficiently durable equilibrium) so that either the abovementioned complex can no longer bind to said segment, or the abovementioned gene or mRNA cannot no longer be translated into a peptide sequence corresponding to said segment.

Another object of the present invention is to provide medicaments capable of causing the production in the organism of molecules capable of specifically binding to the segment of the T cell receptor or to the gene or to the RNA coding for this segment.

Another object of the present invention is to provide medicaments which can be used as vaccines against myasthenia gravis, these vaccines comprising either molecules capable of generating the production in the organism of antibodies capable of specifically binding to the receptor segment of the T cells, ie vectors containing nucleotide sequences coding for molecules capable of generating the production in the body of antibodies as described above.

However, it should be noted that the T receptor is very polymorphic. It consists of two α and β subunits, each encoded by the combination of several genes (V, D, J, C). 24 families of V genes have been described for the β chain.

No correlation between the particular families of genes coding for the different regions of the α and β subunits on the one hand, and myasthenia gravis ^{on the} other hand, has been demonstrated to date, so that no approach Therapeutics by blocking a specific segment of the T cell receptor or a gene coding for such a specific segment has not been possible so far.

The present invention stems from the discovery made by the Inventors that the Vβ5.1 segment of the T cell receptor is closely linked to myasthenia gravis, and this more particularly in patients of the HLA DR3 haplotype. namely in patients expressing molecules of the class II histocompatibility complex (HLA DR) of the HLA DR3 type.

It should be noted that myasthenia gravis is understood in the foregoing and what follows, myasthenia gravis autoimmune acquired.

Reference will be made in the remainder of this text to the sequences SEQ ID NO 1 to SEQ ID NO 5 described in the list of sequences below, these sequences being the following:

- the DNA sequence represented by SEQ ID NO 1 code for the Vβ5.1 segment of the T cell receptor;

- the amino acid sequence represented by SEQ ID NO 2 comprises the aforementioned Vβ5.1 segment, said segment being delimited by the amino acids located at positions 20 and 113, and being preceded by a signal peptide delimited by the amino acids located at positions 1 and 19; - the mRNA sequence represented by SEQ ID NO 3 codes for the above-mentioned SEQ ID NO 2 sequence, in particular for the aforementioned Vβ5.1 segment;

- the antisense DNA sequence represented by SEQ ID NO 4 is the sequence complementary to the sequence represented by SEQ ID NO 1; - the antisense RNA sequence represented by SEQ ID NO 5 is the sequence complementary to the sequence represented by SEQ ID NO 3.

The subject of the present invention is the use of nucleotide or peptide sequences, for obtaining medicaments capable of being used in the context of the prevention or treatment of myasthenia gravis, said nucleotide sequences being chosen from:

- those capable of coding for a peptide sequence, itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the cell receptor Human T, or - those capable of coding for a peptide sequence which itself represents an antibody capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment, - those capable of coding for an antisense RNA, itself capable of hybridizing with all or part of the mRNA coded by the DNA sequence coding for the amino acid sequence of said segment Vβ5.1. or

- those capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of said segment Vβ5.1, or with all or part of the mRNA coded by this DNA sequence, said peptide sequences being chosen from:

- those capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with the Vβ5.1 segment of the human T cell receptor, or

- those corresponding to antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the T cell receptor

A more particular subject of the invention is the use of nucleoditic or peptide sequences as described above, for obtaining medicaments capable of being used in the context of the prevention or treatment of myasthenia gravis in patients with HLA DR3 haplotype.

Preferably, patients in whom the above-mentioned drugs are likely to be used to prevent or treat myasthenia gravis are patients:

- generally starting the disease before having reached about forty years,

- presenting with a generalized myasthenia gravis, most often of severe form: grade IIB as defined according to the classification of Osserman (41), - presenting a hyperplastic thymus, thymic hyperplasia being defined at the same time by a significant thymic mass and the presence of lymphoid follicles (12-14),

- having a level of antibodies directed against the RACh receptor greater than approximately 10 nM (and capable of being measured by the method described in the reference (42).

According to a preferred embodiment of the invention, the nucleoditic sequences used are those capable of coding for a peptide sequence, itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor. Advantageously, such nucleotide sequences are chosen from:

the DNA sequence represented by SEQ ID N0 1, comprising in particular the nucleotide sequence coding for the peptide sequence of Vβ5.1 segment of the human T cell receptor, said peptide sequence consisting of the sequence of 94 amino acids delimited by the amino acids located at positions 20 and 113 of the sequence represented by SEQ ID NO 2, - any fragment of DNA of at least approximately 15 contiguous nucleotides of the DNA sequence represented by SEQ ID NO 1, capable of coding for a peptide fragment of at least approximately 5 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, said peptide fragment itself being capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ 5.1 segment,

- any DNA sequence derived, for example by mutation, from the DNA sequence represented by SEQ ID NO 1, or from a DNA fragment as defined above, in particular by substitution, deletion or -addition d '' one (or more) nucleotide (s) of the DNA sequence represented by SEQ ID NO 1 or of the DNA fragment mentioned above, said derived DNA sequence being capable of coding either for a peptide sequence identical to the peptide sequence represented by SEQ ID NO 2, or identical to a peptide fragment as defined above, either for a peptide sequence derived from that represented by SEQ ID N0 2, or derived from a peptide fragment as defined above, in particular by substitution, deletion or addition of one (or more) amino acid (s) of the peptide sequence represented by SEQ ID N0 2, or of a peptide fragment as defined above, said derivative peptide sequence being herself susceptible to age ndering the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ segment 5.1.

It goes without saying that by peptide sequence capable of causing the formation of antibodies, is meant in what precedes and what follows, any peptide sequence capable, in particular when it is in the blood circulation of an individual, of elicit an immune response in this individual by the production of antibodies by the latter's immune system, these antibodies being capable of specifically recognizing and forming a stable immunological complex with the above-mentioned peptide sequence or with the amino acid sequence of the Vβ5 segment .1 above. Advantageously, the abovementioned DNA fragments comprise approximately 15 nucleotides to approximately 408 contiguous nucleotides, preferably from approximately 60 nucleotides to approximately 300 contiguous nucleotides of the DNA sequence represented by SEQ ID NO 1, capable of respectively coding for a fragment peptide from about 5 amino acids to about 136 acids contiguous amines. preferably from about 20 amino acids to about 100 amino acids of the peptide sequence represented by SEQ ID NO 2.

Preferred DNA fragments capable of being used in the context of the present invention are those comprising all or part: of the nucleotide sequence delimited by the nucleotides located at positions 1 and 339 of the sequence represented by SEQ ID NO 1 and coding for the polypeptide comprising a signal peptide delimited by the amino acids located in positions 1 and 19 of the sequence represented by SEQ ID NO 2. this signal peptide being followed by the Vβ5.1 segment delimited by the amino acids located in positions 20 and 113 of the sequence represented by SEQ ID NO 2.

- the nucleotide sequence delimited by the nucleotides located at positions 58 and 339 of the sequence represented by SEQ ID NO 1 and coding for the Vβ5 segment. 1 delimited by the amino acids located in positions 20 and 1 13 of the sequence represented by SEQ ID NO 2, - the nucleotide sequence delimited by the nucleotides located in positions 1 and 57 of the sequence represented by SEQ ID NO 1 and coding for the aforementioned signal peptide delimited by the amino acids located in positions 1 and 19 of the sequence represented by SEQ ID NO 2.

Preferred DNA fragments capable of being used in the context of the present invention are those mentioned above from approximately 60 nucleotides to approximately 300 nucleotides comprising all or part of the nucleotide sequence coding for the CDR1 region of the Vβ5.1 segment and / or all or part of the nucleotide sequence coding for the CDR2 region of the Vβ5.1 segment.

The aforementioned CDRl and CDR2 regions of the Vβ5 segment. 1 could be determined from the following articles:

- Kronenberg et al. , Ann. Rev. Immunol. , 4: 529-591 (1986),

- Novotny et al. , Proc. Natl. Acad. Sci. , USA, 83: 742-746 (1986),

- Chothia et al. , The EMBO Journal, 7, n ° 12, 3745-3755 (1988),

- Claverie et al. , Immunology Today, vol. 10, 10-14 (1989), - Davis et al. , Nature, 334, 395-402 (1988),

- Jorgensen et al. , Ann. Rev. Immunol. , 10, 835-873 (1992),

- Bellio et al. , J. Exp. Med. , 129, 1087-1097 (1994),

- Prochnika-Chalufour, International Immunology, vol. 3, No. 9, 853-864 (1991). - Chien and Davis, Immunology Today, vol. 14, No. 12, 597-602 (1993).

Particularly preferred DNA fragments mentioned above are those comprising:

- all or part of the nucleotide sequence delimited by the nucleotides located at positions 133 and 150 of the sequence represented by SEQ ID NO 1. and coding for the CDR1 region of 6 amino acids delimited by the amino acids located at positions 45 and 50 of the sequence represented by SEQ ID NO 2. and / or

- all or part of the nucleotide sequence delimited by the nucleotides located in positions 199 and 243 of the sequence represented by SEQ ID NO 1, and coding for the CDR2 region of 15 amino acids delimited by the amino acids located in positions 67 and 81 of the sequence represented by SEQ ID NO 2.

Advantageously, the percentage of homology between the nucleotides constituting the abovementioned derived DNA sequences, and those constituting the DNA sequence represented by SEQ ID NO 1, or the abovementioned DNA fragments, is at least approximately 60% . preferably about 90%.

Advantageously still, the percentage of homology between the amino acids constituting the above-mentioned derived peptide sequences, and those constituting the peptide sequence represented by SEQ ID NO 2. or the above-mentioned peptide fragments, is at least about 60%. preferably about 90%.

According to another preferred embodiment of the invention, the nucleotide sequences used are chosen from those capable of coding for an antisense RNA, this antisense RNA itself being capable of hybridizing with all or part of the mRNA represented by SEQ ID N0 3, said mRNA being coded by the DNA sequence coding for the amino acid sequence of said segment

Vβ5.1, namely by the DNA sequence represented by SEQ ID N0 1. Advantageously, such nucleotide sequences are chosen from:

the antisense DNA sequence represented by SEQ ID N0 4, said DNA sequence coding for the antisense RNA represented by SEQ ID N0 5. capable of hybridizing with the mRNA represented by SEQ ID NO 3,

- any antisense DNA fragment of at least about 12 contiguous nucleotides of the antisense DNA sequence represented by SEQ ID N0 4. capable of coding for an antisense RNA fragment of at least about 15 contiguous nucleotides of the Antisense RNA represented by SEQ ID N0 5. said antisense RNA fragment being capable of hybridizing with all or part of the mRNA defined above,

- any antisense DNA sequence derived, for example by mutation, from the antisense DNA sequence represented by SEQ ID NO 4, or from an antisense DNA fragment as defined above, in particular by substitution. deletion or addition of one (or more) nucleotide (s) from the antisense DNA sequence of the aforementioned antisense DNA fragment, said antisense DNA sequence being capable of coding either for an antisense RNA identical to that represented by SEQ ID NO 5, or identical to an antisense RNA fragment as defined above, or for an antisense RNA derived from that represented by SEQ ID NO 5, or derivative of an antisense RNA fragment as defined above, in particular by substitution, deletion or addition of one (or more) nucleotide (s) of the antisense RNA represented by SEQ ID NO 5, or of an antisense RNA fragment as defined above, said derived antisense RNA itself being capable of hybridizing with all or part of the mRNA defined above.

It goes without saying that the nucleotide sequences of the invention represented by SEQ ID NO 1. SEQ ID NO 3. SEQ ID NO 4 and SEQ ID NO 5, the derivative sequences and the sequence fragments of the invention mentioned above , must be taken into consideration as being represented in the 5 '→ 3' direction.

Thus, the first nucleotide of a complementary sequence in the 5 '- »3' direction as described above, is the complement of the last nucleotide of the sequence in the 5 '-> 3' direction coding for the peptide sequence Vβ5 .1 (or by a fragment of this peptide sequence or a derived peptide), the second nucleotide of this complementary sequence is the complement of the penultimate nucleotide of the sequence coding for the peptide sequence Vβ5.1, and so on , up to the last nucleotide of said complementary sequence which is the complement of the first nucleotide of the sequence coding for the peptide sequence Vβ5.1. Antisense RNA encoded by the complementary sequence represented by

SEQ ID NO 4 mentioned above is such that, when this antisense RNA is represented in the 5 '→ 3' direction, its first nucleotide corresponds to the last nucleotide of the DNA sequence represented by SEQ ID NO 1 coding for the peptide sequence Vβ5. 1, and therefore hybridizes with the last nucleotide of the mRNA represented by SEQ ID NO 3 encoded by the latter, while its last nucleotide corresponds to the first nucleotide of the DNA sequence coding for the peptide sequence Vβ5.1, and therefore hybridizes with the first nucleotide of the mRNA encoded by the latter.

Thus, by antisense RNA is meant in the above and what follows, any RNA coded by the above-mentioned complementary sequence and represented in the reverse direction (3 '- »5') from the direction in which the mRNA coded by the sequence coding for the Vβ5.1 peptide sequence (or fragment or derived peptide), the latter mRNA being also designated sense mRNA (5 '→ 3').

The term antisense RNA is therefore intended for an RNA sequence complementary to the base sequence of messenger RNA, the complementary term to be understood in the sense that each base (or a majority of bases) of the sequence antisense (read in the direction 3 '→ - 5') is capable of pairing with the corresponding bases (G with C, A with U) of the RNA messenger (sequence read in the 5 '→ 3' direction). Antisense RNA is an RNA produced by the transcription of the non-coding DNA strand (nonsense strand).

This antisense strategy is more particularly described in European patent n ° 240 208, or in "Antisense strategies" Armais of the New York Academy of Sciences, Volume 660 (1992), Editors: Renato Baserga, David T.,

Denhardt. or in Medicine / Sciences, 10: 250-281 (1994), or in Grooke S. T., Lebleu B.: Antisens Research and Applications, New York: CRC Press Inc., 1993.

It is believed that the inhibition of the synthesis of a protein according to the antisense strategy, in this case of the peptide sequence Vβ5.1 in the present case, is the consequence of the formation of a duplex between the two complementary RNAs ( sense and antisense) thus preventing the production of the protein. The mechanism, however, remains unclear. The RNA-RNA complex can interfere either with subsequent transcription, with maturation, transport or translation, or even lead to degradation of the mRNA.

Advantageously, the abovementioned antisense DNA fragments comprise from approximately 12 nucleotides to approximately 30 contiguous nucleotides. preferably from approximately 15 nucleotides to approximately 20 contiguous nucleotides of the DNA sequence represented by SEQ ID NO 4, capable of respectively coding for an antisense RNA fragment of approximately 12 to approximately 30 nucleotides, preferably approximately 15 nucleotides to approximately 20 nucleotides of the antisense RNA sequence represented by SEQ ID NO 5.

Preferred antisense DNA fragments capable of being used in the context of the present invention are those comprising all or part: of the nucleotide sequence delimited by the nucleotides located at positions 73 and 41 1 of the sequence represented by SEQ ID NO 4 and coding for the antisense RNA delimited by the nucleotides located at positions 73 and 411 of the sequence represented by SEQ ID NO 5,

- the nucleotide sequence delimited by the nucleotides located in positions 73 and 354 of the sequence represented by SEQ ID NO 4 and coding for the antisense RNA delimited by the nucleotides located in positions 73 and 354 of the sequence represented by SEQ ID NO 5 ,

- the nucleotide sequence delimited by the nucleotides located at positions 355 and 41 1 of the sequence represented by SEQ ID NO 4 and coding for the antisense RNA delimited by the nucleotides located at positions 355 and 41 1 of the sequence represented by SEQ ID NO 5.

Advantageously, the percentage of homology between the nucleotides constituting the abovementioned derivative antisense DNA sequences, and those constituting the antisense DNA sequence represented by SEQ ID NO 4. or the The above-mentioned antisense DNA fragments is at least about 60%, preferably about 90%.

Advantageously still, the percentage of homology between the nucleotides constituting the abovementioned derivative antisense RNA sequences, and those constituting the antisense RNA sequence represented by SEQ ID NO 5, is at least about 60%, preferably of about 90%.

Advantageously, the nucleotide sequences chosen from those capable of coding for a peptide sequence or for an antisense RNA as described above, and used in the context of the present invention. are contained in recombinant nucleotide sequences comprising the elements necessary to allow transcription of the abovementioned nucleotide sequences, in particular a promoter and a transcription terminator.

As such, the subject of the present invention is any recombinant nucleotide sequence containing one (or more) nucleotide sequence (s) of the invention coding for a peptide sequence or for an antisense RNA as described above, as well as the elements necessary for the transcription of these sequences, in particular a promoter or a transcription terminator.

Where appropriate, the recombinant nucleotide sequences of the invention comprise a nucleotide sequence coding for an amino acid sequence, said nucleotide sequence being located upstream and / or downstream of the nucleotide sequence of the invention, said recombinant nucleotide sequences coding thus for a hybrid protein containing a peptide sequence as described above of the invention preceded and / or followed by an amino acid sequence, in particular an amino acid sequence capable of increasing the immunogenic character of the hybrid protein compared to to the aforementioned single peptide sequence of the invention.

Advantageously, the abovementioned recombinant nucleotide sequences of the invention are inserted into vectors, in particular into plasmids, capable of allowing the expression of said recombinant nucleotide sequences in the organism.

As such, the subject of the present invention is any vector, in particular any plasmid, containing one (or more) recombinant nucleotide sequence (s) as defined above. A more particular subject of the invention is the use of vectors as described above, in particular of plasmids. containing recombinant nucleotide sequences according to the invention, the latter themselves containing at least one nucleotide sequence according to the invention coding for a peptide sequence, as described above, capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, for obtaining vaccines against myasthenia gravis, which may in particular be used in prophylaxis or therapy as part of the treatment of the patients described above, these vaccines still being designated naked DNA vaccines.

By way of illustration, mention may be made of the general and more specific reviews of the following naked DNA vaccines: Ladenheim, Biofutur, May 1995, pp. 15-21; Xiang et al. Virology, 199, 132-140 (1994); Xu and Liew. Vaccine, vol. 12, No. 16, 1534-1536. (1994); Cichutek. Vaccine, vol. 12. No. 16,

1520-1525, (1994); Danko and Wolff, Vaccine, vol. 12, No. 16, 1499-1502 (1994); Davis et al., Vaccine, vol. 12. No. 16, 1503-1508 (1994); Spooner et al. Gene Therapy, 2, 173-180 (1995); Manthorpe et al., Human Gene Therapy, 4, 419-431 (1993); Whalen and Davis. Clinical Immunology and Immunopathology, vol. 75, n ° 1, April, pp. 1-12 (1995); Ulmer et al. Science.

259, 1745-1749 (1993).

According to another embodiment of the invention, the nucleotide sequences used are those capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of the Vβ5.1 segment of the human T cell receptor . Advantageously, such nucleotide sequences are chosen from those complementary to all or part of one of the strands of the DNA sequence represented by SEQ ID NO 1, in particular from nucleotide sequences comprising at least approximately 12 contiguous nucleotides up to the number maximum of nucleotides of the DNA sequence represented by SEQ ID NO 4 complementary to the DNA sequence represented by SEQ ID NO 1, or among antisense DNA fragments as described above, or also among sequences of 'DNA derived from all or part of the DNA sequence represented by SEQ ID NO 4 or from the above-mentioned antisense DNA fragments, in particular by substitution, deletion and / or addition of one (or more) nucleotide (s).

According to another embodiment of the invention, the nucleotide sequences used are those which hybridize with all or part of the mRNA coded by the DNA sequence coding for the amino acid sequence of the Vβ5.1 segment of the receptor for human T cells, these nucleotide sequences themselves represent an antisense RNA, this antisense RNA advantageously consisting of at least about 12 contiguous nucleotides complementary to nucleotides of the abovementioned mRNA. Advantageously, such nucleotide sequences are chosen from:

- the antisense RNA sequence represented by SEQ ID N0 5, any fragment of at least approximately 12 contiguous nucleotides of the antisense RNA sequence represented by SEQ ID NO 5, said fragment being capable of hybridizing with all or part of the mRNA represented by SEQ ID NO 3 defined above above, - any antisense RNA sequence derived, for example by mutation, from the antisense RNA sequence represented by SEQ ID NO 5, or from a fragment as defined above, in particular by substitution, deletion or addition of one (or more) nucleotide (s) of the above-mentioned antisense RNA sequence or fragment, said derived antisense RNA sequence being capable of hybridizing with all or part of the mRNA defined above.

Advantageously, the abovementioned antisense RNA fragments comprise approximately 12 nucleotides to approximately 30 contiguous nucleotides, preferably from approximately 15 nucleotides to approximately 20 contiguous nucleotides of the RNA sequence represented by SEQ ID NO 5. capable of hybridizing with the mRNA represented by SEQ ID NO 3 defined above.

Preferred antisense DNA fragments capable of being used in the context of the present invention are those comprising all or part:

- the nucleotide sequence delimited by the nucleotides located at positions 73 and 411 of the sequence represented by SEQ ID NO 5, - the nucleotide sequence delimited by the nucleotides located at positions 73 and 354 of the sequence represented by SEQ ID NO 5,

- the nucleotide sequence delimited by the nucleotides located at positions 355 and 411 of the sequence represented by SEQ ID NO 5.

Advantageously, the percentage of homology between the nucleotides constituting the abovementioned derivative antisense RNA sequences and those constituting the RNA sequence represented by SEQ ID NO 5, or the abovementioned antisense RNA fragments, is at least approximately 60 %, preferably around 90%.

According to another embodiment of the invention, the peptide sequences used are those capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with the Vβ5.1 segment of the receptor for human T cells. Advantageously, the above-mentioned peptide sequences are chosen from:

- the peptide sequence represented by SEQ ID N0 2, - any peptide fragment of at least about 5 contiguous amino acids of the peptide sequence represented by SEQ ID N0 2, said fragment being capable of generating the production of antibodies in a patient , these antibodies being able to recognize and form an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment mentioned above,

- any peptide sequence derived, in particular by substitution, deletion or addition of one (or more) amino acid (s) of the peptide sequence represented by SEQ ID NO 2 or of a peptide fragment as defined above , said peptide sequence being capable of generating the production of antibodies in a patient, these antibodies being capable of recognizing and of forming an immunological complex with all or part of the amino acid sequence of said segment Vβ5.1. Advantageously, the abovementioned peptide fragments comprise approximately 5 amino acids to approximately 136 contiguous amino acids preferably, from approximately 20 amino acids to approximately 100 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, capable of causing the formation of 'antibodies as defined above. Preferred peptide fragments are those comprising all or part:

- the peptide sequence of 114 amino acids delimited by the amino acids located in positions 1 and 113 of the sequence represented by SEQ ID NO 2, this peptide sequence consisting successively on the one hand by the 19 amino acids delimited by the amino acids located at positions 1 and 19 of the sequence represented by SEQ ID NO 2 and corresponding to the signal peptide, and on the other hand, by the 94 amino acids delimited by the amino acids located at positions 20 and 113 of the sequence represented by SEQ ID NO 2,

- the peptide sequence of 94 amino acids delimited by the amino acids located in positions 20 and 113 of the sequence represented by

SEQ ID NO 2,

- the peptide sequence of 19 amino acids delimited by the amino acids located in positions 1 and 19 of the sequence represented by SEQ ID NO 2. Preferred peptide fragments are those of approximately 20 amino acids to approximately 100 amino acids comprising all or part of the CDR1 region and / or the CDR2 region mentioned above of the Vβ5.1 segment.

Particularly preferred peptide fragments mentioned above are those comprising: - all or part of the peptide of 6 amino acids delimited by the amino acids located at positions 45 and 50 of the sequence represented by

SEQ ID NO 2, and corresponding to the CDR1 region. and or - All or part of the 15 amino acid peptide delimited by the amino acids located at positions 67 and 81 of the sequence represented by SEQ ID NO 2, and corresponding to the CDR2 region.

Advantageously still, the percentage of homology between the amino acids constituting the above-mentioned derived peptide sequences, and those constituting the peptide sequence represented by SEQ ID NO 2, or the above-mentioned peptide fragments, is at least about 60%, preferably d 'about 90%.

According to another embodiment of the invention, the peptide sequences used as antibodies are those capable of recognizing and forming an immunological complex with the amino acid sequence of the Vβ5.1 segment of the human T cell receptor. Advantageously, said antibodies are polyclonal or monoclonal antibodies as obtained by immunization of an animal with peptide sequences chosen from: - the peptide sequence represented by SEQ ID N0 2,

- any fragment of approximately 5 to approximately 136 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, in particular any peptide fragment as described above, said fragment being capable of allowing the production by the immune system of a animal, of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment,

- any peptide sequence derived, in particular by substitution, deletion or addition of one (or more) amino acid (s), of the peptide sequence represented by SEQ ID N0 2 or of a peptide fragment as defined above above, said derived peptide sequence being capable of allowing the production by the immune system of an animal, of antibodies capable of recognizing and of forming an immunological complex with all or part of the amino acid sequence of said segment Vβ5.1.

A subject of the invention is also pharmaceutical compositions comprising, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) as described above, coding for a sequence peptide itself representing an antibody capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, said nucleotide sequence (s) ) being advantageously contained in recombinant nucleotide sequences as defined above, the latter themselves being inserted into vectors as defined above. The invention also relates to pharmaceutical compositions comprising, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) as described above, coding for an antisense RNA, itself capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of the Vβ5 segment. 1 of the human T cell receptor, said nucleotide sequence (s) being advantageously contained in recombinant nucleotide sequences as defined above, the latter themselves being inserted into vectors as defined above -above. The invention also relates to pharmaceutical compositions comprising, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) as described above, capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, or with all or part of the mRNA encoded by this DNA sequence.

A subject of the invention is also pharmaceutical compositions comprising, in association with a pharmaceutically acceptable vehicle, one (or more) peptide sequence (s) as described above, corresponding to antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor.

The invention also relates to the vaccines, also known as naked DNA vaccines as described above, and comprising, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) as described (s) ) above, capable of coding for a peptide sequence, itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5 segment .1 the human T cell receptor, said nucleotide sequence (s) being advantageously contained in recombinant nucleotide sequences as defined above, the latter themselves being inserted into vectors as defined above.

A subject of the invention is also the vaccines comprising, in association with a pharmaceutically acceptable vehicle, one (or more) peptide sequence (s) as described above, capable of '' generate the formation of antibodies capable of recognizing and forming an immunological complex with the amino acid sequence of the Vβ5 segment. 1 of the human T cell receptor. Advantageously, the pharmaceutical compositions and vaccines according to the invention are in the form of injectable preparations, in particular by the intravenous route, preferably by the intramuscular route, or by the subcutaneous route. The invention also relates to the complexes formed between the abovementioned nucleotide sequences used in the context of the present invention, and the DNA or RNA sequence coding for said Vβ5.1 segment.

As such, the subject of the invention is more particularly:

- the complexes formed between the complementary DNA sequence represented by SEQ ID NO 4, or a fragment or a sequence derived from the latter, as defined above, with all or part of the DNA sequence coding for said segment Vβ5.1;

- the complexes formed between the antisense RNA sequence represented by SEQ ID NO 5, or a fragment or a sequence derived from the latter as defined above, with all or part of the mRNA sequence coding for said Vβ5 segment .1.

The invention also relates to the complexes formed between the above-mentioned peptide sequences used in the context of the present invention, and the amino acid sequence of said segment Vβ5.1. As such, the invention more particularly relates to the complexes formed on the one hand between the antibodies, or directly administered to a patient. either generated by the introduction into the body, or by the production in the body (case of the naked DNA vaccine), of the peptide sequence represented by SEQ ID NO 2, or of a fragment or of a derived sequence of the latter as defined above, and on the other hand all or part of the amino acid sequence of the Vβ5.1 segment.

The nucleotide sequences used in the context of the present invention are obtained by any conventional method well known to those skilled in the art of cloning or nucleotide synthesis. As regards the DNA sequence represented by SEQ ID NO 1. the latter was cloned by Kimura et al. , J. Exp. Med. , 184, 739-750 (1988); its access number in Gene bank is X04927.

The peptide sequences used in the context of the present invention are obtained by any conventional method well known to those skilled in the art of peptide synthesis in liquid or solid phase.

A more particular subject of the invention is the recombinant polypeptides, and in particular the peptide sequence corresponding to the segment.

Vβ5.1 of the human T cell receptor, namely the peptide sequence represented by SEQ ID NO 2, or the fragments or the peptide sequences aforementioned derivatives, as obtained by transformation of cells (such as bacteria, in particular E. coli. yeasts, insect cells infected with a Baculovirus, eukaryotic cells such as Cos or CHO cells) with a nucleotide sequence recombinant as defined above, containing a DNA sequence according to the invention, in particular using a vector as described above.

The expression “recombinant polypeptides” should be understood to mean any molecule having a polypeptide chain capable of being produced by genetic engineering, via a phase of transcription of the DNA of the corresponding gene, which leads to the obtaining RNA which is subsequently transformed into mRNA (by suppression of introns), the latter then being translated by ribosomes, in the form of proteins, the whole being carried out under the control of appropriate regulatory elements inside ^a host cell. Consequently, the expression "recombinant polypeptides" used does not exclude the possibility that said polypeptides include other groups, such as glycosylated groups.

Of course, the term "recombinant" indicates that the polypeptide was produced by genetic engineering, because it results from the expression, in an appropriate cellular host, of the corresponding nucleotide sequence which was previously introduced into an expression vector used to transform said cell host.

The invention also relates to the antibodies as described above directed against the peptide sequences used in the context of the invention, and more particularly those directed against the aforementioned recombinant polypeptides, in particular those directed against the peptide sequence represented by SEQ ID NO 2, or a fragment or a sequence derived from the latter as defined above.

Such antibodies can be obtained by immunizing an animal with these polypeptides, followed by the recovery of the antibodies formed. It goes without saying that this production is not limited to polyclonal antibodies.

It also applies to any monoclonal antibody produced by any hybridoma capable of being formed, by conventional methods, from the spleen cells of an animal, in particular mouse or rat. immunized against one of the purified polypeptides of the invention on the one hand, and cells of an appropriate myeloma on the other hand, and to be selected, by its capacity to produce monoclonal antibodies recognizing the above-mentioned polypeptide initially put used for animal immunization. As such, the subject of the invention is more particularly humanized or chimeric antibodies as obtained from DNA encoding the above-mentioned antibodies according to the conventional methods for obtaining these antibodies, these methods being described and commented on in particular in: Winter G., Harris WJ, Immunology Today, vol. 14, No. 6, 243-246 (1993); Bach JF et al. , Immunology Today, vol. 14, No. 9, 421-425 (1993); Bach JF. The Immunologist, 214, 135-137 (1994).

These humanized antibodies are more particularly characterized in that their specific part recognizing the Vβ5.1 fragment consists of an amino acid sequence of animal origin, in particular of mice, while their non-specific part does not recognize the Vβ5 segment. 1 consists of an amino acid sequence of human origin.

The invention also relates to pharmaceutical compositions comprising the above-mentioned humanized antibodies in association with a pharmaceutically acceptable vehicle.

The subject of the invention is also any method of treatment and / or prevention of myasthenia gravis by administration of one or more pharmaceutical compositions, and / or of one or more vaccines described above according to the invention, to a patient myasthenia gravis, and more particularly to a patient with an HLA-DR3 haplotype.

The invention will be further illustrated in the following detailed description of the demonstration of the involvement of the Vβ5.1 segment of the T cell receptor in autoimmune myasthenia gravis in patients of the HLA DR3 haplotype.

Material and methods

Patients

The patients were followed up at Marie Lannelongue hospital. The diagnosis of myasthenia gravis was made on the basis of clinical features, electromyographic decrement and the positive effect of anticholinesterase drugs. Seven patients were selected in this study, based on specific criteria. They are young, almost all female, with generalized disease, a hyperplastic thymus and a high titer of anti-RACh antibodies and have preferably recently developed the pathology. The only treatment was the use of anticholinasterase therapy. Patients undergoing corticotherapy were excluded, since this treatment considerably modifies thymic populations (14). HLA typing was determined by oligotyping according to the method described by (reference 29). Hyperplasia is limited to the thymus characterized by the presence of germinal centers, as described by Rosaï and Levine (13). Osserman's modified classification was used to grade the severity of the condition (41). All patients have a generalized form of pathology.

Patients with an IIA degree of severity exhibit moderately disordered functional activity and moderate weakness of the limb or eye muscles. Patients with a degree of severity IIB have a large dysregulation of functional activity and significant signs of weakness of the muscles of the limbs and bulbar. The anti-RACh were titrated using the RACh of human muscle complexed with the iodinated α-bungarotoxin as described in (42).

Isolation of thymic T cells and peripheral blood

The thymuses were obtained from myasthenic patients who underwent a thymectomy after therapeutic prescription of the latter at Marie Lannelongue hospital (Le Plessis-Robinson, France). All the thymuses of the seven patients were hyperplastic and contained lymphoid follicles as described in (13, 18).

The thymuses were rinsed in HBSS (Hank's Balanced Sait Solution) and cell suspensions were obtained by gentle grinding of the thymic tissue, followed by passage through sterile gauze, washing and freezing in liquid nitrogen until use. After thawing, a loss of approximately 15-20% of immature cortical thymocytes CD1 + (CD4 + CD8 +) was observed, resulting in a proportional increase in the percentage of mature medullary thymocytes CD4 + CD8- and CD4-CD8 +. As this study was mainly focused on mature CD4 + thymic cells (thymocytes without CD8), this was not detrimental. The peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation of venous blood collected on EDTA. The isolated cells were washed in HBSS and frozen ^until use. No alteration of PBMC subpopulations was observed after thawing, and viability confirmed by exclusion of trypan blue staining. is greater than 95% at the time of use. Peptide synthesis

The synthesis and purification of the peptides corresponding to positions 169-181 and 351-368 of the α subunit of RACh was carried out by the solid phase method (43). The amino acid composition of the peptides was checked by amino acid analysis. The amino acid sequences of these peptides are described in reference 36. The two peptides correspond to selected domains of the α subunit of RACh. Peptide 169-181 is extra cytoplasmic and corresponds to a sequence bordering on the binding site of α-bungarotoxin on the α subunit. It contains a motif of 5 residues (sequence

175-179) which is generally found in the known antigenic sites of T cells (44). The peptide 351-368 is intracytoplasmic and corresponds to a highly immunogenic region designated HIR (35). Its sequence is in a helical conformation in the intact protein and therefore presages an antigenic site according to Delisi et al. (45). In addition, the two peptides contain one to two anchoring residues in a position suitable for binding to the HLA-DR3 molecule which, moreover, represents a HLA class II allele associated with myasthenia gravis, according to one or the other of the link patterns described (46-50).

Recombinant RACh α fragments

The cDNA of the α subunit of human RACh contains exon P3A (51). The larger fragment extracytoplasmic corresponding to amino acids 1-210 was cloned as a fusion protein in a commercially available vector (pMAL ^® -C2, New England Biolabs, Beverly, MA) downstream of the wrong E gene that encodes a maltose binding protein (MBP). and has been strongly expressed in E. coli.

A stop codon was placed in position 211, in order to ensure the termination of the translation. The majority (90%) of the fusion protein was insoluble and found as an inclusion body.

After extraction in 8M urea of the washed inclusion bodies, then step-by-step dialysis in buffers with decreasing concentrations of urea for renaturation, the fusion protein was 70% pure. The fusion protein was then purified in one step using the affinity of MBP for maltose. then split using coagulation factor Xa according to the manufacturer's recommendations. The ri -210 fragment was purified by preparative electrophoresis with continuous elution with a high yield (80-90%) and a high level of purity. Purity is a critical factor as it minimizes the risk of observing false positive protein reactions recombinant due to contamination with bacterial proteins. The αr 1 -120 fragment was sterilized by filtration through 0.22 μm membranes and stored at -20 ° C. until used in proliferation experiments. It leads to the appearance of proliferative responses from PBMCs and thymocytes from many MG patients included or not in this study.

Depletion experiences of CD8 and Vβ subpopulations

The subpopulations of PBMC and thymic lymphocytes expressing CD8 (as well as the doubly positive CD4-r-CD8 + cells of thymus) were depleted by positive selection of CD8 -K cells using paramagnetic beads coupled to anti-CD8 + (IObeads Immunotech, Marseille. France), following the manufacturer's instructions. For the depletion experiments of the Vβ subpopulation, paramagnetic beads coupled to mouse immunoglobulins linked to anti-Vβ5.1 or anti-Vβ8 were used according to the manufacturer's recommendations (Immunotech) immediately after depletion CD8, and the culture was carried out according to the method described above.

Cell culture for the analysis of the expression of the Vβ segment of T cell receptors (TCR)

The PBMCs or thymic T cells depleted in CD8 were cultured for 6 days in 48-well plates (Falcon. Becton Dickinson) in a medium (RPMI 1640) supplemented with 5% human AB serum, 2 mM glutamine, penicillin 100 IU / ml, streptomycin 100 μg / ml. 1% MEM non-essential amino acids, 1 mM sodium pyruvate, 10 mM hepes buffer (all from Gibco. Detroit, MI), 5 x 10 ^"5 M β-mercaptoethanol with or without a selected synthetic peptide (5 μg / ml) or the ri -210 fragment (5 μg / ml), then purified natural IL-2 (10 U / ml) (Boehringer Mannheim.

GmbH, Mannheim, Germany) is added for 2 days. At least 10 wells (up to 15 wells) were prepared for each of the culture conditions and were pooled for the fluorescent labeling process. In the inhibition experiments using the specific anti-DR3 alloantisera, the serum was added at the start of the culture at a concentration varying from 0.1% to 10%. Immunofluorescence and flow cytometry

Two-color flow cytometry analyzes were performed 8 days after stimulation with IL-2 alone, or peptide + IL-2, using specific anti-CD4 and anti-TCR-Vβ monoclonal antibodies.

The following monoclonal antibodies (mAbs), directed against the V region of the TCR were used: anti-Vβ5a (recognizing Vβ5-2 and 5.3), anti-Vβ5b (recognizing Vβ5-3 only), anti-Vβ5c (recognizing Vβ5.1 only), anti-Vβόa (recognizing an allotypic allele, Vβ6.7), anti-Vβ8a (recognizing the Vβ8 family), anti-Vβl2a (recognizing the Vβ l2 family), all commercially available from T Cell Sciences, Cambridge, MA. All antibodies were coupled to FITC. Direct immunofluorescence was performed according to the manufacturer 's instructions. For double labeling, after 3 washes, an additional step consists in staining with an anti-CD4 conjugated with phycoerythrin (Immunotech) for 30 minutes at 4 ° C. After washing, the cells were incubated for 20 minutes with 2 μg / ml of propidium iodide (PI, Becton Dickinson, Mountain View, CA) (52). Dead cells were excluded during the analysis by positive staining with propidium iodide.

Proliferation and depletion experience of the Vβ subpopulation

The peripheral or thymic T cells depleted in CD8 cells were cultured in microtiter plate wells (Costar) (0.2 × 10 ⁵ cells / well) in the medium described above with or without 5 μg / ml of peptide .

After 6 days, rIL-2 (10 U / ml) is added and 2 days after 1 μCi of tritiated thymidine (specific activity 6.7 Ci / mmole: New England Nuclear. Boston, MA) is added to each well for 18 hours . The cells are then harvested and the incorporation of tritiated thymidine detected by counting the radioactivity on glass fiber filters in a scintillation liquid counter (LKB, Bromma, Sweden). Six identical wells are prepared for each culture condition. The results are expressed in stimulation index defined by: cpm with peptide for 10 ^ cells / cpm without peptide for 10 cells. When indicated, an anti-DR3 alloantiserum has been added to inhibit cell proliferation as described above. Results

Experimental strategy.

To determine the repertoire of T cells involved in the response to peptides of the α subunit of RACh. CD8 thymocytes or PBMCs from myasthenic patients were stimulated with a peptide and then with IL-2 to allow the regeneration of potentially modulated receptors (53, 54). The cells were then harvested and analyzed by two-color immunofluorescence and flow cytometry for the expression of

CD4 and Vβ on the one hand on small cells (at rest) and on the other hand on large cells (activated). Depletion of CD8 + cells has been very effective in blood lymphocytes as well as in thymic lymphocytes. Analysis by flow cytometry gives less than 0.5% of CD8 + cells or CD4 + CD8 + cells after depletion. In addition, no cell expansion

CD8 + was only observed after nine days of culture with the peptide and IL-2.

The in vitro activation of thymic CD4 + T cells leads to blastogenesis which can be easily identified by size (FSC) and granulosity (SSC) parameters. Activated blast cells appear larger and more granular than non-blast cells, the latter having a size and particle size typical of lymphocytes taken ex-vivo unstimulated. Activated cells represent approximately 5% to 12% of the total population in culture, based on the expression of DR and CD25. Analysis of the expression of Vβ by CD4 + cells after stimulation with the peptides, was carried out with antibodies against members of the Vβ5 families.

Vβ8, Vβ6 and Vβl2. It is important to note that, in all cytometric analyzes, dead cells were excluded by staining with propidium iodide. Furthermore, the specificity of the binding of the antibody to the Vβ region of the TCRs was confirmed by the use of control antibodies of the same isotype which stain less than 0.5% of T cells in the cultures.

An increase in the proportion of blast cells, as well as variations in the distribution of Vβ segments within blast cells. are considered to represent a significant response to the peptide. When the analysis is carried out on all of the living cells, no measurable difference is observed in the cultures stimulated by the RACh peptides in comparison with the non-stimulated control cultures. A measurable increase in selective Vβ subpopulations can only be observed in activated blast cells. This is why the cytotluorographic analysis was carried out on an accumulation of events (several thousand) corresponding to the living activated population, thus allowing a more precise determination of CD4 + cells expressing a given Vβ.

Specific expansion of cells expressing a given Vβ in mature thymic CD4 + T cells from MG patients in response to synthetic peptides.

The experiments described above were carried out to analyze the expression of the Vβ segment in response to antigens derived from RACh in 7 MG patients. Cytofluorographic analysis of the use of the Vβ region in response to peptides 169-181 and 351-368 and to the recombinant fragment rαl-210 of RACh. leads to the following observations. In a patient taken as an example (patient # 2). stimulation of thymic CD4 + cells with peptide 169-181 leads to a specific and measurable increase in Vβ5 cells. 1 + (from 5.3 to 14.4%) and Vβ8 + cells (from 7.4 to 10.4%) whereas this same peptide does not induce any increase in the other Vβ families tested. The peptide 351-368 also induces a significant increase in Vβ5.1 + in the CD4 + thymocytes of this patient. The recombinant fragment rαl-210 was used initially as a source of peptides capable of being able to be naturally generated. However, the engagement of numerous Vβ gene segments in the response of thymocytes and PBMCs to this fragment has led the inventors to use another strategy by using selected peptides to study the repertoire of T cells involved in the response to the autoantigen. in MG patients. Anyway, the experiments using the ri -210 fragment confirm the multidetermining nature of the autoantigen.

RACh and especially that of the α chain which has been intensively studied (36-40). In order to visualize the specific stimulation of the Vβ region by the peptides in all patients as well as to compare appropriately among different patients the subpopulations of T cells carrying a given Vβ segment after peptide stimulation, the results below are expressed as differences calculated between the percentage of CD4 + blast T cells carrying a particular Vβ segment after stimulation and the percentage of CD4 + T cell carrying this Vβ segment in the control cultures (without peptide). In terms of variation in the repertoire of T cells in response to a peptide, it is reasonable to consider that a difference of 2% is significant. Therefore differences greater than 2% constitute significant expansions. This also takes into account the fact that the frequency of cells responding to the peptide in myasthenia gravis is between 1 / 100,000 to 1/200 000 (55, 56) and that the variations due to errors in analysis by FACS ^® are usually evaluated at 0.5%. Therefore, it is very significant that T cells carrying Vβ5.1 and, to a lesser degree Vβ8. are enriched in large cells after activation by the peptides. Of the 6 patients studied. 5 (all of HLA-DR3 haplotype) exhibit this expansion of the Vβ5 region. 1 in thymocytes. The sixth patient who does not exhibit expansion has clinical and biological characteristics which are specified below, and in particular does not have the HLA-DR3 haplotype. The negative differences observed in other Vβ subpopulations are probably due to compensation due to enrichment in Vβ5 cells. 1 + (and Vβ8 +). The expansion of Vβ5. 1 varies approximately from 2 to 10% in thymocytes stimulated with p 169-181. and from 2 to 4% in thymocytes stimulated with p351-368.

Use of Vβ segments in the response to synthetic peptides in PBMC

As for the study of thymocytes, the expansions could only be detected among the activated cells, so that only the results on the activated cells are presented. The prevalence of expansion of cells expressing Vβ5.1 in response to peptide 169-181 is very clear in PBMC and concerns 6 of the 7 patients studied, although lower levels of expansion are observed in comparison with thymocytes.

Furthermore, the restriction to Vβ5.1 is more marked; the other families or other members of the Vβ5 family are used less frequently, with the exception of Vβ8 which is increased in 3 out of 7 patients. Few significant expansions have been observed with the peptide 351-368; they only involved the Vβ5.1 family (out of 1 patient out of the 7 studied, MG). Again, the expansion of cells expressing Vβ5.1 was only observed in CD4 + cells activated in response to the peptides.

Specific proliferation in response to peptides is suppressed in CD4 + thymic cells after depletion in Vβ5.1

Depletion experiments were carried out to further study the specificity and selectivity of the involvement of Vβ5.1 in the response to synthetic peptides. The thymic CD4 + T cells depleted in cells expressing Vβ5.1 were compared with the total CD4 + thymic T cells from the point of view of their proliferative capacity in response to the peptides. Over 86% depletion of cells expressing Vβ5. 1 in two patients suppress proliferative responses to peptides and to the recombinant fragment rαl-210.

Analysis of the results in all the patients studied

A summary of the analysis of the use of Vβ fragments by CD4 + T cells from myasthenic patients in response to peptides 169-181 and 351-368. is shown in Table 2.

The differences in expression of Vβ calculated as indicated above, and representing the stimulation of a population expressing a given Vβ, reveal the prevalence of the expansion of cells expressing Vβ5.1 in response to the peptide 169-181 in the thymus and the periphery. The implication of Vβ5.1 is less clear in response to peptide 351-368. It appears in the thymocytes or PBMC from patients in whom the disease has recently appeared. In all cases, and at least with regard to thymocytes, the use of Vβ5.1 in response to this peptide is observed exclusively in severely affected patients.

The DR3 haplotype is strongly associated with the use of Vβ5. 1 in response to peptide 169-181

All but one patient expressed the DR3 haplotype (patient 7). In this patient, the percentage of Vβ5.1 + CD4 + cells (thymocytes or PBMC) is not increased in response to peptides 169-181 or peptide 351-368. In addition, when the specific anti-DR3 alloantisera are added to the cultures, both the proliferation consecutive to the peptide 169-181 and the expansion of the cells expressing Vβ5.1 were inhibited, which indicates that the DR3 molecule plays a major role in the presentation of the peptide.

In conclusion:

T cells recognize a complex formed by a peptide antigen linked to MHC molecules via a specific receptor as the third component of a trimolecular complex. This work provides for the first time results showing an association between the expression of a TCR gene and the specificity for a particular combination of self peptide / MHC in human autoimmune myasthenia gravis.

These results show the recurrent use of a TCR gene segment also called Vβ5.1. in response to synthetic peptide 169-181 (and to a lesser degree to peptide p351-368) by thymic CD4 + T cells and peripheral blood in a subgroup of myasthenic patients sharing the DR3 haplotype. Consequently, in vivo interactions involving the peptide 169-181 of the α subunit of RACh, the T cell receptors made up of the Vβ5.1 segment. and the HLA DR3 molecule are highly probable, which allows potential immunotherapy in a subgroup of young myasthenic patients.

This study on a selected homogeneous group of myasthenic patients with common clinical signs such as a hyperplastic thymus, a generalized disease and a high antibody titer, demonstrates a shared Vβ selectivity between different patients in the cellular immune response to the synthetic peptide 169- 181, (and to a lesser degree peptide 351-368), of the α subunit of RACh, which is strongly associated with the HLA DR3 haplotype. The specificity of this original, measurable expansion in response to the peptide is supported by several experimental arguments. First of all, it is observed exclusively in the population of activated blast cells, indicating a real effect of the peptide on cell expansion, and not only a selection from a population already existing in culture. Second, the artifacts in the analysis of the results were carefully avoided by excluding dead cells from cultures using the incorporation of propidium iodide. In addition, the fluorescent labeling experiments were carried out with antibodies directly coupled to fluorochrome, and always included negative controls. Third, the expansion of Vβ5.1 is included in the expansion of the entire CD4 + population. Fourth, the patient's T cells respond to peptides by proliferation, and the depletion of cells expressing Vβ5.1 inhibited this proliferation. Fifth, an anti-DR3 alloantiserum not only inhibits proliferation of peptide 369-391, but also the expansion of cells expressing Vβ5.1 in response to this peptide.

It can be appreciated that the specific increase in CD4 + T cells expressing Vβ5.1 after peptide stimulation is greater in the thymus. It is possible that the T cells responding to the peptide originate from the thymus at the stage of initiation of the disease and migrate towards the periphery where they are in the active state in patients with recent onset of the disease.

RACh is a multi-epitopic autoantigen as indicated by the large number of T cell epitopes, found by different research groups on the α subunit as well as on the other subunits (36-40). The inventors' approach is based on the selection of two peptides of the α subunit of RACh for which the proliferative memory T response in patients is correlated with clinical data (antibody titer, disease severity). These Peptides are therefore potentially pathogenic and involved in the disease (36) when the following arguments are taken into account:

1) the role of these peptides in the in vitro proliferative response of memory T cells in myasthenic patients has also been confirmed by two independent studies carried out on Italian (39) and Israeli (40) patients;

2) other studies aimed at defining T cell epitopes in myasthenic patients. did not take into account the variation of the clinical forms of the disease and the patient subpopulations, and provided data on few patients including elderly patients with low antibody levels, representing a minority of the total population myasthenic patients (37, 57-59);

3) unlike several epitopes described in the literature, the aforementioned peptides generally do not generate a response in the controls (22, 40, 60). The normal immune repertoire contains autoreactive T cells for RACh and other autoantigens (56, 61. 62,63), and it may be admitted that the specificities of the most frequent autoreactive T cells in the normal immune repertoire are less involved. in the disease and vice versa:

4) the follow-up, after thymectomy, of the PBMC reaction of three myasthenic patients (not included in this study) responding to the peptide p 169-181 before the intervention, does not reveal any proliferative response one to two years after the surgical intervention ;

5) the expansion of CD4 cells in response to the peptide involves the cells expressing Vβ5.1 in all patients sharing the HLA-DRB 1 * 03 allele. The sharing of T cell specificity by different myasthenic patients expressing a common HLA DR B1 allele (HLA-DR3), which is also associated with the disease, demonstrates that the cells which have proliferated in response to the peptide are characteristic of the disease.

All these points taken together indicate that the two peptides selected in this study, exhibiting reactivity associated with clinical data, are likely to be involved in the pathogenesis of this disease.

Myasthenia gravis is associated with an increase in the frequency of the DR3 allele (26-30). this association is more evident in women, in young patients and in patients with thymic hyperplasia (27). In addition, thymic hyperplasia is associated with a high antibody titer and a generalized form of the disease (1, 10, 14). This would explain the importance of the expression of the HLA-DR3 haplotype in patients selected solely on the basis of the above clinical criteria. It should be noted that the only patient not expressing the HLA DRB1 * 03 haplotype, does not exhibit expansion of the cells expressing Vβ5.1, either ex vivo or in culture after stimulation. with the peptide. All HLA-DR3 patients also express HLA DQA1 501 and HLA DQB1 201, which are strongly linked with HLA DRB1 * 03.

Several arguments are in favor of the role of HLA-DR3 in the presentation of the p169-181 peptide. First of all, all cell lines and clones derived from myasthenic patients for whom the HLA restriction has been examined, are restricted to the DR molecule, but not DQ or SB (61, 64, 65). Second, several cell lines or clones directed against the entire RACh molecule, or more importantly at the extracytoplasmic fragments of the α subunit of RACh, have been found to be restricted to DR3 (60, 63, 66, 67). However, no data were available on the use of the T cell receptor by such cell lines. Third, the two peptides selected in this study contain the key residues proposed and arranged according to the consensus motifs defined for the peptides associated with HLA-DR3 (46-50). Finally, the direct demonstration of the implication of HLA-DR3 in the presentation of the peptide 169-181. is provided by our experiences of inhibiting the expansion of CD4 + Vβ5.1 + cells and proliferation in response to the peptide by a specific anti DR3 alloantiserum in two patients tested. Therefore, the peptides used in this study are important components in the ternary complex to direct the observed Vβ5.1 expansion.

Finally, this study showed that peptides potentially implicated in the disease, presented by the HLA DR3 molecule which also represents a dominant allele in the subpopulation of myasthenic patients of this study, are responsible in vitro for the specific expansion and recurrent cells expressing Vβ5.1. This may be related to the fact that the expression of Vβ5.1 by cells of myasthenic patients is generally increased in the CD4 + thymic T populations analyzed ex vivo. In addition, this increase in the thymus may be the result of an active selection mechanism leading to the escape of self-reactive cells during the step of differentiating the population of double positive CD4 + CD8 + cells expressing weak CD3 towards those expressing strongly CD3. These results underline the importance of the molecular interaction in the ternary complex represented by the HLA-DR3 molecule. peptide 169-181 and T cell receptors using Vβ5. 1, in the disease, and open perspectives for new therapeutic means within the framework of the treatment of myasthenia gravis, at least in the subpopulation of patients described above. The current therapeutic means of myasthenia gravis are thymectomy. the use of anticholinesterase compounds in combination or not with corticosteroids. These therapeutic means have never been shown to reduce the process of spread of this disease, or lead to remissions. Only thymectomy is well accepted for young patients with a hyperplastic thymus, and can act by suppressing the source of potentially self-reactive T and B cells, as well as the potential site for reactivation of the autoimmune process (25). Clinical improvement after thymectomy is apparent after only one or two years. The use of anticholinesterase compounds is only a symptomatic treatment which is difficult to balance and for which the risk of overdose is permanent. As for corticosteroid treatment, this is accompanied by serious side effects.

Suppressing the immune response to RACh in myasthenic patients with more specific means would improve the management of these patients. The disease-specific T cell fraction is probably very small in myasthenics, and could include cells carrying Vβ5. 1 in HLA-DR3 patients. This could explain the reason why global experimental approaches on the Vβ repertoire in the context of human autoimmune diseases have had very limited success.

The interest of our study was to define the implication in the pathogenesis of a small population of T cells expressing Vβ5.1 and representing at most 5% of CD4 + T cells as a potential target for selective and specific therapy of myasthenia gravis in a clinically and biologically homogeneous subgroup of patients expressing the HLA-DR3 haplotype.

Table 1 v

β

Clinical data of myasthenic patients O • t-

Patient (a) Sex Age (b) Beginning of the Title in Histology of the severity from there Tyi HLA page (f) disease (c) anti-RACh Thymus (d) disease (s) DRB 1 * DQA 1 "DQB 1 -

1 F 32 1 month 752 hyperplasia (2) MB 0301- 1501 501-102 201-601

2 M 20 6 months 21.8 hyperplasia (4) MB 0301-04 501-301 201-301

3 F 31 8 months 12.6 hyperplasia (3) II Λ 03- 13 501 - 102 201 -604

4 F 20 9 months 6.8 hyperplasia (2) I1B 03- 1 101 501-501 201-301

5 F 31 12 months> 10.5 hyperplasia (2) IIΛ 0301 -O iOl 501- 101 201 -501

6 F 22 13 months 54.9 hyperplasia (4) 1IB 0301-04 501 -301 201 -302

7 F 22 10 years> 30 hyperplasia (4) IIB 0103-0101 501 - 101 501-301

(a) All patients received anticholinesterase therapy. Patients treated with coiticoids were excluded from the study

(b) At the time of the thymectomy

(c) Time interval between the onset of the first signs of myasthenia gravis and thymectomy

(d) Degree of hyperplasia defined by the size and number of lymphoid follicles. 20 (e) Graduation according to Osserman's classification.

(f) Oligotyping by PCR.

O

H

S. Sβ

W

Table 2

Summary of data in all patients tested.

Vβ family thymocytes

5.1 5-2.3J 5J 6.7 I 8 π

MG1

MG2

MG3 MG4- MG5 nd J 6d

MG6

MG7

The filled boxes represent a significant difference (Δ> 3%) in percentage of cells expressing Vβ between the control cultures without peptide and the cultures with peptide.

Note the recurrence of the use of the Vβ5.1 segment in all the HLA DR3 patients tested (and not in the MG 7 patient, not DR3), in response to the peptide 169-181 both at the level of the thymocytes and of the PBMCs. REFERENCES

1. Engel, A. G. 1986. Acquired autoimmune Myasthenia Gravis. In "Miology" (A. G. Engel and B.Q. Banker, Eds. Pp. 1925-1954. Mac Graw-Hill.

5 New York.

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3. Patrick. J., J. Lindstrom. 1973. Autoimmune response to acetylcholine H) receptor. Science 180: 871-872.

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(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: NATIONAL CENTER FOR SCIENTIFIC RESEARCH

(B) STREET: 3, rue Michel-Ange

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75016

(ii) TITLE OF THE INVENTION: NUCLEOTIDE AND PEPTIDE SEQUENCES FOR THE TREATMENT OF MYASTHENIA

(iii) NUMBER OF SEQUENCES: 5

(iv) COMPUTER-READABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPO)

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ATG GGC TCC AGG CTG CTC TGT TGG GTG CTG CTT TGT CTC CTG GGA GCA 48 Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala 1 5 10 15

GGC CCA GTA AAG GCT GGA GTC ACT CAA ACT CCA AGA TAT CTG ATC AAA 96 Gly Pro Val Lys Ala Gly Val Thr Gin Thr Pro Arg Tyr Leu Ile Lys 20 25 30

ACG AGA GGA CAG CAA GTG ACA CTG AGC TGC TCC CCT ATC TCT GGG CAT 144 Thr Arg Gly Gin Gin Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His 35 40 45 AGG AGT GTA TCC TGG TAC CAA CAG ACC CCA GGA CAG GGC CTT CAG TTC 192 Arg Ser Val Ser Trp Tyr Gin Gin Thr Pro Gly Gin Gly Leu Gin Phe 50 55 60

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GTG AGC ACC TTG GAG CTG GGG GAC TCG GCC CTT TAT CTT TGC GCC AGC 336 Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110

AGC TTG TCC GTC TCC GGG GAG CTG TTT TTT GGA GAA GGC TCT AGG CTG 384 Ser Leu Ser Val Ser Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu 115 120 125

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GGUCGAUUCU CAGGGCGCCA GUUCUCUAAC UCUCGCUCUG AGAUGAAUGU GAGCACCUUG 300

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CACATTCATC TCAGAGCGAG AGTTAGAGAA CTGGCGCCCT GAGAATCGAC CAGGGAAGTT 180

TCCTTTGTTT CTCTGTGTCT CACTGAAGTA TTCAAAGAGG AACTGAAGGC CCTGTCCTGG 240 GGTCTGTTGG TACCAGGATA CACTCCTATG CCCAGAGATA GGGGAGCAGC TCAGTGTCAC 300 TTGCTGTCCT CTCGTTTTGA TCAGATATCT TGGAGTTTGA GTGACTCCAG CCTTTACTGG 360 GCCTGCTCCC AGGAGACAAA GCAGCACCCA ACAGAGCAGCAGA

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CACAUUCAUC UCAGAGCGAG AGUUAGAGAA CUGGCGCCCU GAGAAUCGAC CAGGGAAGUU 180

UCCUUUGUUU CUCUGUGUCU CACUGAAGUA UUCAAAGAGG AACUGAAGGC CCUGUCCUGG 240

GGUCUGUUGG UACCAGGAUA CACUCCUAUG CCCAGAGAUA GGGGAGCAGC UCAGUGUCAC 300

UUGCUGUCCU CUCGUUUUGA UCAGAUAUCU UGGAGUUUGA GUGACUCCAG CCUUUACUGG 360

GCCUGCUCCC AGGAGACAAA GCAGCACCCA ACAGAGCAGC CUGGAGCCCA U 411

Claims

1. Use of nucleotide or peptide sequences, for obtaining drugs which can be used in the context of prevention or

5 treatment of myasthenia gravis autoimmune acquired. said nucleotide sequences being chosen from:

- those capable of coding for a peptide sequence, itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the l acides amino acid sequence of the Vβ5.1 segment of the receptor for human T cells, or

- those capable of coding for a peptide sequence itself representing an antibody capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5 segment. 1.

15 - those capable of coding for an antisense RNA, itself capable of hybridizing with all or part of the mRNA coded by the DNA sequence coding for the amino acid sequence of said Vβ5 segment. 1, or

- those capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of said segment Vβ5.1. or with

All or part of the mRNA encoded by this DNA sequence, said peptide sequences being chosen from:

- those capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with the Vβ5.1 segment of the human T cell receptor, or

25 - those corresponding to antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the T cell receptor

2. Use of nucleoditic or peptide sequences according to claim 1, for obtaining medicaments capable of being used in the context of the prevention or treatment of myasthenia gravis in patients of the HLA DR3 haplotype, namely in patients expressing HLA DR3 class II histocompatibility complex (HLA DR) molecules.

35

3. Use according to claim 1 or claim 2, characterized in that the nucleoditic sequences capable of coding for a peptide sequence, itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with any or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, are chosen from:

the DNA sequence represented by SEQ ID NO 1. comprising in particular the nucleotide sequence coding for the peptide sequence of the Vβ5.1 segment of the human T cell receptor, said peptide sequence consisting of the chain of 94 amino acids delimited by the amino acids located at positions 20 and 113 of the sequence represented by SEQ ID NO 2

- any DNA fragment of at least approximately 15 contiguous nucleotides of the DNA sequence represented by SEQ ID NO 1, capable of coding for a peptide fragment of at least approximately 5 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, said peptide fragment being itself capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment,

- any DNA sequence derived, for example by mutation, from the DNA sequence represented by SEQ ID NO 1, or from a DNA fragment as defined above, in particular by substitution, deletion or addition of one (or more) nucleotide (s) of the DNA sequence represented by SEQ ID NO 1 or of the DNA fragment mentioned above, said derived DNA sequence being capable of coding either for a peptide sequence identical to the peptide sequence represented by SEQ ID NO 2, or identical to a peptide fragment as defined above, either for a peptide sequence derived from that represented by SEQ ID NO 2, or derived from a peptide fragment as defined above, in particular by substitution, deletion or addition of one (or more) amino acid (s) of the peptide sequence represented by SEQ ID NO 2, or of a peptide fragment as defined above, said derivative peptide sequence being - even susceptible to engen drer the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment.

4. Use according to claim 1 or claim 2, characterized in that the nucleotide sequences chosen from those capable of coding for an antisense RNA, this antisense RNA itself being capable of hybridizing with all or part of the mRNA represented by SEQ ID NO 3 and coded by the DNA sequence represented by SEQ ID NO 1, are chosen from:

the antisense DNA sequence represented by SEQ ID NO 4, said DNA sequence coding for the antisense RNA represented by SEQ ID NO 5. - any antisense DNA fragment of at least about 12 contiguous nucleotides of the antisense DNA sequence represented by SEQ ID NO 4, capable of coding for an antisense RNA fragment of at least about 12 nucleotides of RNA antisense represented by SEQ ID NO 5, said antisense RNA fragment being capable of hybridizing with all or part of the mRNA defined above,

any antisense DNA sequence derived, for example by mutation, from the antisense DNA sequence represented by SEQ ID NO 4, or from an antisense DNA fragment as defined above, in particular by substitution, deletion or addition of one (or more) nucleotide (s) of the antisense DNA sequence of the abovementioned antisense DNA fragment, said antisense DNA sequence being capable of coding either for an antisense RNA identical to that represented by SEQ ID NO 5, or identical to an antisense RNA fragment as defined above, either for an antisense RNA derived from that represented by SEQ ID NO 5, or derived from an antisense RNA fragment as defined above, in particular by substitution, deletion or addition of one (or more) nucleotide (s) of the antisense RNA represented by SEQ ID NO 5, or of an antisense RNA fragment as defined above, said antisense RNA derived being itself capable of hybridizing with all or part of the mRNA challenge neither above.

5. Use according to one of claims 1 to 4, characterized in that the nucleotide sequences chosen from those capable of coding for a peptide sequence or for an antisense RNA, are contained in recombinant nucleotide sequences comprising the elements necessary to allow the transcription of the abovementioned nucleotide sequences, in particular a promoter and a transcription terminator.

6. Use according to claim 5, characterized in that the recombinant nucleotide sequences are inserted into vectors, in particular into plasmids, capable of allowing the expression of said recombinant nucleotide sequences in the organism.

7. Use according to claim 1 or claim 2. characterized in that the nucleotide sequences capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of the Vβ5.1 region of the receptor for human T cells, are chosen from those complementary to all or part of one strand of the ^DNA sequence represented by SEQ ID N0 1, in particular from the nucleotide sequences comprising at least about 12 contiguous nucleotides up to the number maximum of nucleotides of the DNA sequence represented by SEQ ID NO 4, complementary to the DNA sequence represented by SEQ ID NO 1, or also among DNA sequences derived from all or part of the DNA sequence represented by SEQ ID NO 4, in particular by substitution, deletion and / or addition of one (or more) nucleotide (s).

8. Use according to claim 1 or claim 2, characterized in that the nucleotide sequences hybridizing with all or part of the mRNA coded by the DNA sequence coding for the amino acid sequence of the Vβ5.1 region of the receptor for human T cells, themselves represent an antisense RNA, this antisense RNA advantageously consisting of at least about 12 contiguous nucleotides complementary to all or part of the aforementioned mRNA, said nucleotide sequences being chosen from: - the sequence antisense RNA represented by SEQ ID NO 5,

- any fragment of at least about 12 contiguous nucleotides of the antisense RNA sequence represented by SEQ ID NO 5, said fragment being capable of hybridizing with all or part of the mRNA defined above,

- any antisense RNA sequence derived, for example by mutation, from the antisense RNA sequence represented by SEQ ID NO 5, or from a fragment as defined above, in particular by substitution, deletion or addition of a (or more) nucleotide (s) of the abovementioned antisense RNA sequence or fragment, said derived antisense RNA sequence being capable of hybridizing with all or part of the mRNA defined above.

9. Use according to claim 1 or claim 2, of peptide sequences capable of generating the formation of antibodies capable of recognizing and of forming an immunological complex with the Vβ5.1 segment of the human T cell receptor, said peptide sequences being chosen from:

- the peptide sequence represented by SEQ ID NO 2,

- any peptide fragment of approximately 5 to approximately 136 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, said fragment being capable of generating the production of antibodies in a patient, these antibodies being capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment mentioned above,

- any peptide sequence derived, in particular by substitution, deletion or addition of one (or more) amino acid (s) of the sequence peptide represented by SEQ ID NO 2 or of a peptide fragment as defined above, said peptide sequence being capable of generating the production of antibodies in a patient, these antibodies being capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment.

10. Use according to claim 1 or claim 2, of peptide sequences as antibodies capable of recognizing and of forming an immunological complex with the amino acid sequence of said Vβ5.1 segment of the human T cell receptor, said antibodies being polyclonal or monoclonal antibodies, where appropriate, humanized, as obtained by immunization of an animal with peptide sequences chosen from:

- the peptide sequence represented by SEQ ID NO 2.

- any fragment of approximately 5 to approximately 136 contiguous amino acids of the peptide sequence represented by SEQ ID NO 2, said fragment being capable of allowing the production, by the immune system of an animal, of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of said segment Vβ5.1, - any peptide sequence derived, in particular by substitution, deletion or addition of one (or more) amino acid (s), of the peptide sequence represented by SEQ ID NO 2 or of a peptide fragment as defined above, said derived peptide sequence being capable of allowing the production, by the immune system of an animal, of antibodies capable of recognizing and forming a immunological complex with all or part of the amino acid sequence of said Vβ5.1 segment.

11. Pharmaceutical compositions characterized in that they comprise, in association with a pharmaceutically acceptable vehicle. one (or more) nucleotide sequence (s) coding for a peptide sequence itself representing an antibody capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the receptor human T cells, said nucleotide sequence (s) being advantageously contained in recombinant nucleotide sequences as defined in claim

5, the latter being themselves inserted into vectors as defined in claim 6.

12. Pharmaceutical compositions characterized in that they comprise, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) coding for an antisense RNA, itself capable of hybridizing with all or part of the DNA sequence coding for the Vβ5.1 segment of the human T cell receptor, and more particularly one (or more) nucleotide sequence (s) as defined in claim 4 , said nucleotide sequence (s) being advantageously contained in recombinant nucleotide sequences as defined in claim 5, the latter being themselves inserted into vectors as defined in claim 6.

13. Pharmaceutical compositions characterized in that they comprise, in association with a pharmaceutically acceptable vehicle. one (or more) nucleotide sequence (s) capable of hybridizing with all or part of the DNA sequence coding for the amino acid sequence of said Vβ5.1 segment, or with all or part of the mRNA encoded by this DNA sequence, and more particularly one (or more) nucleotide sequence (s) as defined in claims 7 and 8.

14. Pharmaceutical compositions characterized in that they comprise, in association with a pharmaceutically acceptable vehicle, one (or more) peptide sequence (s) corresponding to antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, and more particularly one (or more) peptide sequence (s) as defined in claim 10.

15. Vaccines characterized in that they comprise, in association with a pharmaceutically acceptable vehicle, one (or more) nucleotide sequence (s) capable of coding for a peptide sequence, itself capable of causing the formation of antibodies capable of recognizing and forming an immunological complex with all or part of the amino acid sequence of the Vβ5.1 segment of the human T cell receptor, and more particularly one (or more) nucleotide sequence (s) such (s) as defined in claim 3, said nucleotide sequence (s) being advantageously contained in recombinant nucleotide sequences as defined in claim 5. these the latter being themselves inserted into vectors as defined in claim 6.

16. Vaccines characterized in that they comprise, in association with a pharmaceutically acceptable vehicle, one (or more) peptide sequence (s) capable of generating the formation of antibodies capable of recognizing and forming an immunological complex with the Vβ5.1 segment of the human T cell receptor, and more particularly one (or more) peptide sequence (s) as defined in claim 9.