CA2616704A1 - Use of a polypeptide for detecting, preventing or treating a pathological condition associated with a degenerative, neurological or autoimmune disease - Google Patents

Abstract

Use of at least one polypeptide comprising at least one fragment of a protein for obtaining a diagnostic, prognostic, prophylactic or therapeutic composition intended for detecting, preventing or treating a pathological condition associated with a degenerative and / or neurological and / or auto disease -immune, said protein being chosen from proteins whose peptide sequence in the native state corresponds to SEQ ID N ~ 1, SEQ ID N ~ 2, SE Q ID N ~ 3, SEQ ID N ~ 4, SEQ ID N ~ 5, SEQ ID N ~ 6, SEQ ID N ~ 7, SEQ ID N ~ 8, SEQ ID N ~ 1 0, SEQ ID N ~ 11, SEQ ID N ~ 12, SEQ ID N ~ 13, SEQ ID N ~ 14 , SEQ ID N ~ 15, SEQ ID N- 16, SEQ ID N ~ 17, SEQ ID N ~ 18, SEQ ID N ~ 19, SEQ ID N ~ 20, SEQ ID N ~ 21, SE Q ID N ~ 22, SEQ ID N ~ 23, SEQ ID N ~ 24, SEQ ID N ~ 25, SEQ ID N ~ 26, SEQ ID N ~ 27, SEQ ID N ~ 28 and SEQ ID N ~ 29, and the peptide sequences which present to me ns 70% identity, preferably at least 80% identity and advantageously at least 98% identity with one we have peptide sequences SEQ ID N ~ 1 to SEQ ID N ~ 8 and SEQ ID N ~ 10 to SEQ ID N ~ 29, and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, precursor of plasma retinol binding protein, ganglioside activator GM2 precursor, calgranulin B and saposin B.

Description

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USE OF A POLYPEPTIDE TO DETECT, PREVENT OR TREAT
A PATHOLOGICAL CONDITION ASSOCIATED WITH A DEGENERATIVE DISEASE,.
NEUROLOGICAL OR AUTOIMMUNE
The present invention relates in particular to the use of at least one polypeptide, to obtain a diagnostic, prognostic composition, prophylactic or therapy intended to detect, prevent or treat a pathological condition associated with a degenerative and / or autoimmune and / or neurological disease.
According to the invention, the term degenerative disease means a disease in which a process of cell death or cell destruction is associated with troubles physiological and / or clinical. Alzheimer's disease, lateral sclerosis amyotrophic, the Parkinson's disease is classified as a neurodegenerative disease. We means by autoimmune disease, hyperreactivity of the immune system to a or from several autoantigen (s). Multiple sclerosis (MS), arthritis rheumatoid (RA) and the lupus erythematosus are classified as autoimmune diseases.
Multiple sclerosis is a chronic disease of the central nervous system of man, evolving by succession of remission and pushing phases or according to a regular progression, the anatomopathological characteristic of which consists of the formation of well-defined demyelination zones in the white matter of the brain and marrow spinal.
Histologically, these areas present at the early stage of the process injury, degradation of the periaxonal myelin associated with damage cells glial responsible for this demyelination. Macrophagic activation inflammatory involving microglial cells (tissue macrophages resident in the the nervous system central), as well as probably macrophages from monocytes infiltrated blood, is associated with this demyelination process and contributes to the destruction leaflets myelinated. In the center of the demyelinated zone, a relative depletion in glial cells is found when a proliferation of astrocytes develops at the periphery and can invade the demyelinated plaque to generate a fibrous or gliotic plaque. These structures sclerotics are at the origin of the name given to the disease.

2 Another characteristic of these plates is their almost association systematic with a vascular element around which they develop.
Histologically, there is a frequent alteration of the barrier hematoencephalic (BBB) constituted by the capillary endothelium. One of elements determinants in the maintenance of the BBB is constituted by the presence underlying cytoplasmic extensions of astrocytes, called astrocytic feet.
Presumably, astrocytic feet induce the formation or allow the maintenance of tight junction structures which ensure the cohesion of the fence endothelial capillary materializing the BBB. Different models pathological do to the state of the BBB alteration and depletion of the astrocytic feet.
In addition, in the lesional process of MS, the alteration of the BBB
contributes to amplifying the associated inflammatory response, by the influx of cells lymphoids from the bloodstream. The contribution of inflammation associated with immune cells is important in MS and participates in process -15 injury.
The etiology of MS is the source of a current debate because the disease could have diverse origins. Assumptions have been made on a origin bacterial and / or viral. Furthermore, as described in the patent application WO 95/21859, H. Perron et al. were led to seek one or more agents effectors 2o of the pathogenic process leading to the typical formation of plaques of demyelination and astrocytic gliosis. In this study, they put in evidence of the presence in cerebrospinal fluid (CSF) and serum of patients MS of at least one factor which exhibits toxic activity towards cells human or animal astrocytic and oligodendrocytic. This activity poisonous 25 characterized by a cytomorphological disorganization of the filaments intermediates and / or degradation of the proteins of said filaments and / or a dead cells by apoptosis of glial cells. They established a correlation significant between in vitro detection of this toxic activity in samples of LCR and serum from MS patients and multiple sclerosis by a colorimetric assay 30 quantitative methyltetrazolium bromide (MTT) from living cells, as described in patent application WO 95/21859. Furthermore, C. Malcus-Vocanson and al. have shown that urine is a very favorable biological fluid for detection of

3 activity of this toxic factor and developed a process using the flow cytometry for detect and / or quantify dead adherent glial cells by apoptosis.
All the information concerning this process is described in the patent application WO
98/11439.
Tests have been carried out using a protein fraction of LCR and urine from MS patients to try to identify this toxic factor. Protein content of each fraction was separated on 12% SDS-PAGE gel and observed after gel coloring to money.
Among the proteins observed, a protein fraction centered on a weight apparent molecular approximately 21 kD was found to be minority associated with toxic activity detected in vitro and a fraction centered on an apparent molecular weight of about 17 kD has been found mainly associated with this toxic activity.
Injection of the fraction from CSF from MS patients into the brain rat rat and a post-mortem histological observation of brain sections rats a allowed to observe, three months after the injection, apoptosis of the population astrocytic and the formation of demyelination plates. All information is contained in the WO 97/33466. These observations are consistent with those which could be performed on brain sections of MS patients after biopsy (N. Benjelloun et al.
Cell. Mol. Biol., 1998, 44 (4), 579-583).
The present inventors have now identified and analyzed the proteins associated with this toxic activity with respect to glial cells in biological samples MS patients, especially in the urine, cerebrospinal fluid and the serum.
After purification of the proteins and separation on SDS-TRICINE gel, the inventors have highlighted the presence of four bands of interest of different weights apparent molecular, respectively 8, .14, 18 and 20 kD corresponding to at least five families of different proteins.
The proteins of these families were then analyzed by spectrometry of mass and / or sequencing and homology search in databases (NCBI http : /ww.ncbi.nim.nih.gov, Basic Blast Search, Protein Blastp, protein sequences entered in FASTA format in the base of data nr, the algorithm used is Matrix BLOSUM62, the identity called

4 "Identities" corresponds to the number of identical amino acids given in percentage and the positivity "Positive" corresponds to the amino acids presenting a equivalence biological according to the above parameters of the software given in percentage). These proteins belong to the protein families of Perlecan, the precursor of the plasma protein binding to retinol, protein, activator of GM2, of the calgranulin and saposin B. Specifically, the proteins are (i) for the band by 20 kD the C-terminal fragment of Perlecan which begins at amino acid 3464 and this ends with amino acid 3707 (Murdoch AD et al. J Biol Chem, 1992, April 25; 267 (12): 8544-47), and referenced in the sequence identifier SEQ ID N 2 (the protein l0 whole Perlecan being referenced in SEQ ID N 1), (ii) for the band of 20 kD on precursor of plasma retinol binding protein (Monaco HL et al., Science, 1995, 268 (5213): 1039-1041) whose sequence is given in SEQ ID N 4, (iii) for the 18 kD band of ganglioside activator protein GM2 (Furst W et al., Euro J
Biochem, 1990, Sep 24; 193 (3): 709-14) identified in SEQ ID N 8, (iv) for the bandaged 14 kD calgranulin B (Lagasse E et al., Mol Cell Biol, 1988, Jun; 8 (6) : 2402-10) identified in SEQ ID N 17 and (v) for the 8 kD band saposin B (Kleinschmidt T
et al., Biol Chem Hoppe Seyler, 1988, Dec; 369 (12): 1361-5) shown in SEQ
ID N
24. They also highlighted the presence of sequences variants to said reference sequences, in particular for the 18 kD band a sequence variant of GM2 activating protein referenced SEQ ID N 9. These sequences protein variants are the product of mutations in the coding genes for said proteins or are the results = of splicing phenomena. He is at rate by example that calprotectin is a variant of calgranulin B.
The C-terminal fragment of the Perlecan protein (SEQ ID N 2) is encoded for example by the DNA nucleotide sequence SEQ ID N 69, taking into account of genetic code. The precursor protein of the plasma binding protein with retinol (SEQ ID N 4) is coded for example by the DNA nucleotide sequence SEQ ID N

70, taking into account the genetic code. GM2 activator protein (SEQ ID
N 8) is coded for example by the DNA nucleotide sequence SEQ ID N 31, in holding account for the genetic code. The peptides FSWDNCFEGK DPAVIR and YSLPKSEFAV PDLELP
derived from the mutated GM2 activator polypeptide (SEQ ID N 9) are encoded by DNA nucleotide sequences SEQ ID N 66 and SEQ ID N 67 respectively, in taking into account the genetic code. The protein calgranulin B (SEQ ID N 17) is coded for example by the DNA nucleotide sequence SEQ ID N 42, taking into account of genetic code. The protein saposin B (SEQ ID N 24) is coded for example over there

5 DNA nucleotide sequence SEQ ID N 53, taking into account the genetic code.
By protein family is meant all of the proteins encoded from of the same DNA gene and which result from differential multi-splicing of the gene and or of a different reading frame. The DNA gene is transcribed with phenomena alternative splicing which leads to the translation of different sequences primary protein. All these proteins belong to the same protein family.
We also includes in the term "protein family", proteins which present to at least 70% identity, preferably at least 80% identity and advantageously at minus 98% identity with a family reference protein sequence.
By multi-splicing is meant splicing occurring at least once in the nucleotide region of interest.
For example, by protein family precursor of plasma protein binding to retinol, denotes the family of proteins comprising at least the proteins or protein fragments of sequence SEQ ID N 4, SEQ IDN 5, SEQ ID
N 6, SEQ ID N 7, and the proteins encoded by the corresponding gene according to different reading frames.
For example, by family of activating protein of GM2, one indicates the family of proteins comprising at least the proteins or fragments of protein sequence SEQ ID N 8, SEQ ID N 9, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID N 14, SEQ ID N 15, SEQ ID N 16, and proteins coded by the corresponding gene according to different reading frames, which result of a multi differential splicing of the gene and / or a different reading frame.
For example, by family of protein calgranulin B, we denote the family of proteins comprising at least the proteins or protein fragments of sequence SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID N 20, SEQ ID N 21, SEQ ID
N 22, SEQ ID N 23, and the proteins encoded by the corresponding gene according to different reading frames, which result from differential multi-splicing of the gene and / or a different reading frame. Proteins MRP14 (SEQ ID N 17) and MRP8 (SEQ ID
NOT

6 18) have a different protein sequence while being coded by the same uncomfortable ; they belong to the same protein family.
For example, by family of protein saposin B, we denote the family of proteins comprising at least the proteins or protein fragments of sequence SEQ ID N 24, SEQ ID N 25, SEQ ID N 26, SEQ ID N 27, SEQ ID N 28, SEQ
IDN 29, and the proteins encoded by the corresponding gene according to different frames of reading, which result from differential multi-splicing of the gene and / or frame of different reading.
By family of nucleic acids encoding a protein is meant to all of the cDNA and / or RNA nucleic sequences transcribed from a even DNA gene and, which result from differential multi-splicing. The DNA gene is transcribed with differential splicing phenomena and leads to the synthesis of different nucleic acids (cDNA, RNA) of different sequences. All these sequences CDNA
and mRNA are considered to belong to the same family of acids - nucleic acids.
For example, by family of nucleic acids encoding the family of precursor protein of the retinol-binding plasma protein, means the family of nucleic acids comprising at least the nucleic acids or fragments of sequence SEQ ID N 30.
For example, by family of nucleic acids encoding the family of GM2 activating protein, the family of nucleic acids including at minus the nucleic acids or fragments of sequences SEQ ID N 3 1, SEQ ID N
32, SEQ ID N 33, SEQ ID.N 34, SEQ ID N 35, SEQ ID N 36, SEQ ID N 37, SEQ ID
N 38, SEQ ID N 39, SEQ ID N 40, SEQ ID N 41 which result from a multi-splicing differential of the gene and / or a different reading frame.
For example, by family of nucleic acids encoding the family of protein calgranulin B, denotes the family of nucleic acids comprising at minus the nucleic acids or fragments of sequences SEQ ID N 42, SEQ ID N
43, SEQ ID N 44, SEQ ID N 45, SEQ ID N 46, SEQ ID N 47, SEQ ID N 48, SEQ ID
3o N 49, SEQ ID N 50, SEQ ID N 51, SEQ ID N 52 which result from a multi-splicing differential of the gene and / or a different reading frame.

7 For example, by family of nucleic acids encoding the family of protein saposin B, denotes the family of nucleic acids comprising at least the acids nucleic acids or sequence fragment SEQ ID N 53, SEQ ID N 54, SEQ ID N 55 who result from a differential multi-splicing of the gene and / or a reading frame different.
By splicing is meant a mechanism for excision of introns and splicing exons during maturation of transcripts and by "splicing differential "we hear the existence of several splicing patterns of a primary transcript leading to training of different messenger RNAs and, which can give rise to the synthesis of several protein different (Kaplan and Delpech, Molecular Biology and Medicine, 1993, 2 ""' editing, Medicine and Sciences, Flammarion, pages 73-77). This phenomenon is largely described in the scientific literature. As an example, we can cite the gene model who code for heavy and light chains of immunoglobulins, the gene model for dystrophin, the model of the alpha amylase gene, the myelin gene, etc.
It is known that eukaryotic genes, in particular, include regions (exons) which code for fragments of the protein encoded by said gene and other regions (introns) that have no protein equivalent. This is due to. makes them genes are first transcribed into a "primary" RNA which is then cut by enzymes splicing at level specific nucleotide sites (splicing sites). These enzymes butt up then the regions coding for the protein, thus reconstituting a "secondary" RNA
whose regions intronics have been eliminated. Furthermore, according to cell phenotypes (and therefore the fabrics or differentiation) these enzymes are not all expressed and, therefore, a even RNA can be spliced differently in the cells of the same individual, thus generating proteins with sequence differences. However, these phenomena can also apply to nucleotide regions which are fully coding (exons), but which, according to different possible splices will generate several different proteins from the same region nucleotide, by differential splicing phenomenon between the different protein products.
In addition, it is known that nucleotide regions can have several reading frames according to the three potential frames of the code genetic. So,

8 the presence of several translation initiating codons in several reading and / or primary RNA splicing splicing the nucleotic sequences present in phases different readings on DNA, allows the same DNA region to generate products protein without relationship between them, from the point of view of the peptide sequence.
Finally, the genetic polymorphism existing between individuals of the same species and / or individual mutations can create or delete sites splicing in a given DNA region and thus modify the sequence and structure of the or some products proteins normally produced by this region.
Thus, the combination of these different phenomena can allow the same nucleotide sequence corresponding to a segment of DNA, identified as determining a genetic region of interest in a given study, includes information necessary and sufficient to define a whole family of RNAs spliced according to diagrams differentials and alternative, in various reading frames and, by that, obviously, of protein and polypeptides having “mosaic” sequences according to a reading frame or even according to the three potential frameworks and mutations possibly linked to polymorphism genetic.
An example of this phenomenon can be represented by the nucleotide region of the HIV-1 retrovirus env gene. Indeed, several different proteins are encoded by segments of the same sequence: for example, the envelope glycoprotein, and the proteins regulators TAT, REV, NEF, VIF.
It is also known that proteins can result from the assembly of Identical (homodimers, homomultimers) or different subunits (heterodimers, heteromultimers). Thus, the various protein products coded by a same DNA region can also join together to form protein entities complex multimerics. This phenomenon is added to the previous ones and, when a protein is identified by a peptide fragment, we can logically identify all the others building blocks of this complex protein and the spliced DNA and RNA segments that encode them, as well as all members of the protein product family and their assemblies.
Another example is provided by the region of human DNA encoding the family.
protein MRP14 or calgranulin B, MRP8, calprotectin, psoriasin etc.
Also, the subject of the present invention is the use of at least one polypeptide comprising at least one fragment of a protein to obtain a

9 diagnostic, prognostic, prophylactic or therapeutic composition intended to detect, predict, prevent or treat a medical condition associated with a disease degenerative and / or autoimmune, said protein being chosen from proteins whose peptide sequence to the native state corresponds to SEQ ID N 1, SEQ ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N
5, SEQ ID N 6, SEQ ID N 7, SEQ ID N 8, SEQ ID N 10, SEQ ID N 11, SEQ ID N
12, SEQ ID N 13, SEQ ID N 14, SEQ ID N 15, SEQ ID N 16, SEQ ID N 17, SEQ ID N
18, SEQ ID N 19, SEQ ID N 20, SEQ ID N 21, SEQ ID N 22, SEQ ID N 23, SEQ ID N
24, SEQ ID N 25, SEQ ID N 26, SEQ ID N 27, SEQ ID N 28 and SEQ ID N 29 and the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98% identity with any of the sequences peptide mentioned above, and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of plasma protein binding to retinol, GM2 activator protein, calgranulin B
and saposin B. In particular embodiments at least two polypeptides above are used in combination to obtain a diagnostic, prognostic composition, prophylactic or therapy to detect, predict, prevent or treat a condition associated pathology degenerative and / or autoimmune disease.
The invention also relates to the use of at least one polypeptide comprising at least one fragment of a protein to obtain a composition diagnostic, prognostic, prophylactic or therapeutic intended to detect, predict, prevent or treat a medical condition associated with a degenerative and / or self-immune, said protein being chosen from proteins whose peptide sequence to native state matches to SEQ ID N 2, SEQ ID N 4, SEQ ID N 8, SEQ ID N 17 and SEQ ID N 24 and the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98% identity with any of the sequences peptide mentioned above. Advantageously, the five polypeptides which meet the definition previous are used in combination.

Preferably, the peptide sequence of said polypeptide comprises, or consists in a sequence chosen from any one of SEQ ID N 2, SEQ ID N 4, SEQ ID N
8, SEQ ID N 17 and SEQ ID N 24.
The invention also relates to the use of at least a fragment of one of the 5 aforementioned polypeptides for the preparation of an immunogenic peptide, said peptide comprising all or part of at least one of the sequences referenced SEQ ID N s 58 to 65 and being used for the production of monoclonal antibodies.
Another subject of the invention is the use of at least one fragment nucleotide, to obtain a diagnostic, prognostic composition, prophylactic or

10 therapeutic intended to detect, predict, prevent or treat a associated medical condition degenerative and / or autoimmune disease, whereby said fragment nucleotide is chosen from fragments which code for at least one fragment of a protein said protein being chosen from proteins whose peptide sequence to native state matches at SEQ ID N 1, SEQ ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N 5, SEQ ID N
6, SEQ
ID N 7, SEQ ID N 8, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID
N 14, SEQ ID N 15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID
N 20, SEQ ID N 21, SEQ ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID
N 26, SEQ ID N 27, SEQ ID N 28 AND SEQ ID N 29 and the peptide sequences who have at least 70% identity, preferably at least 80% and advantageously at minus 98% identity with any of the peptide sequences ci on it, and fragments complementary to said fragments, and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the plasma retinol binding protein precursor, protein activator of GM2, of calgranulin B and saposin B. It is within the reach of those skilled in the art to determine the nucleic acid sequences of the nucleotide fragments from the sequences peptide and genetic code, this being part of his general knowledge.
Preferably, said nucleotide fragment codes for a protein which the state native consists of a sequence chosen from any one of SEQ ID N s 1 at 8 and SEQ
ID N s 10 to 29 above, and among the peptide sequences or the fragments said sequences belonging to the same family of selected proteins

11 among perlecan, the precursor of the plasma binding protein retinol, protein activator of GM2, calgranulin B and saposin B.
Another object of the invention is the use of at least one fragment nucleotide to obtain a diagnostic, prognostic composition, prophylactic or therapy to detect, predict, prevent or treat a condition associated pathology degenerative and / or neurological and / or autoimmune disease whereby said fragment is a fragment of a nucleic sequence chosen from any one of SEQ ID N 30, SEQ ID N 31, SEQ ID N 32, SEQ ID N 33, SEQ ID N 34, SEQ ID N 35, SEQ ID N
36, SEQ ID N 37, SEQ ID N 38, SEQ ID N 39, SEQ ID N 40, SEQ ID N 41, SEQ ID N
42, SEQ ID N 43, SEQ ID N 44, SEQ ID N 45, SEQ ID N 46 and SEQ ID N 47, SEQ ID
N 48, SEQ ID N 49 and SEQ ID N 50, SEQ ID N 51, SEQ ID N 52, SEQ ID N 53, SEQ ID
N 54, SEQ ID N 55, SEQ ID N 56, SEQ ID N 57, SEQ ID N 67, SEQ ID N 66, SEQ ID N
69, SEQ ID N 70 and SEQ ID N 71 and their complementary sequences.
The invention also relates to the use of a specific ligand of a polypeptide or nucleotide fragment as defined above for get a diagnostic, prognostic, prophylactic or therapeutic composition intended to detect, predict, prevent or treat a medical condition associated with a disease degenerative and / or autoimmune.
By ligand is meant any molecule capable of associating with the polypeptide, Phone that a monoclonal antibody, a polyclonal antibody, a receptor, a activity substrate enzymatic, an enzyme of which said polypeptide is a cofactor. The production antibody polyclonal and monoclonal is part of general human knowledge of career. We may cite by reference Kbhler G. and Milstein C. (1975): Continuous culture of fused cells secreting antibody of predefined specificity, Nature 256 495-497 and Galfre G. et al.
(1977) Nature, 266: 522-550 for the production of monoclonal antibodies and Roda A., Bolelli GF: Production of high-titer antibody to bile acids, Journal of Steroid Biochemistry, Vol. 13, pp. 449-454 (1980) for the production of polyclonal antibodies.
By ligand is also meant any molecule capable of associating with a nucleotide fragment, such as a partially nucleotide fragment or

12 fully complementary, a complementary polynucleotide, an anti-acids nucleic acids. Production of nucleotide fragments or polynucleotides is one of general knowledge of a person skilled in the art. We can notably cite use restriction enzymes, and chemical synthesis on a synthesizer automatic, for example on synthesizers marketed by the company Applied Biosystem. Through elsewhere, we know techniques for the production of anti-nucleic acid antibodies. We can quote as of examples Philippe Cros et al., Nucleic Acides Researc, 1994, Vol. 22, N.
15, 2951-2957;
Anderson, WF et al. (1988) Bioessays, 8 (2), 69-74; Lee, JS et al. (1984) FEBS Lett., 168, 303-306; Malfoy, B. et al. (1982) Biochemistry, 21 (22), 5463-5467; Stollar, BD et al., JJ
(eds) Methods in Enzymology, Academic Press, pp. 70-85; Traincard, F. et al.
(1989) J.
Immunol. Meth., 123, 83-91 and Traincard, F. et al. (1989) Mol. Cell. Probes, 3, 27-38).
The invention also relates to a method for detecting at least one protein.
associated with a degenerative and / or autoimmune disease, in a sample organic in which the biological sample is brought into contact with at least one ligand specific of at at least one polypeptide, said polypeptide comprising at least one fragment of a protein and said protein being chosen from proteins whose peptide sequence native corresponds to SEQ ID N 1, SEQ ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N 5, SEQ ID
N 6, SEQ ID N 7, SEQ ID N 8, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ
ID N

13, SEQ ID N 14, SEQ ID N 15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ
ID N
19, SEQ ID N 20, SEQ ID N 21, SEQ ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ
ID N
25, SEQ ID N 26, SEQ ID N 27, SEQ ID N 28 and SEQ IDN 29 and the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98% identity with any of the sequences peptide SEQ ID N 1 to SEQ ID N 8 and SEQ ID N 10 to 29, and the peptide sequences where the fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of the plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B, then we detect the formation of a complex between said polypeptide and said ligand. Said ligand is advantageously an antibody monoclonal, a polyclonal antibody, a receptor, an enzyme activity substrate or an enzyme of which said polypeptide is a cofactor.
Similarly, the invention relates to a method for detecting at least one ligand associated with a degenerative and / or autoimmune disease, in a sample organic, characterized in that the biological sample is brought into contact with minus one polypeptide comprising at least one fragment of a protein, said protein being chosen among proteins whose native peptide sequence corresponds to SEQ ID N 1, SEQ
ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N 5, SEQ ID N 6, SEQ ID N 7, SEQ
ID N 8, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID N 14, SEQ ID N
15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID N 20, SEQ ID N
21, SEQ ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID N 26, SEQ ID N
27, SEQ ID N 28 and SEQ ID N 29 and the peptide sequences which exhibit minus 70%
identity, preferably at least 80% identity and preferably at least 98%
identity with any of the peptide sequences SEQ ID N 1 to SEQ
ID N 8 and SEQ ID N s 10 to SEQ ID N 29, and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B, then we detect the formation of a complex between said polypeptide and said ligand. The ligand is any molecule that responds to conditions previously described.
Preferably, in the methods described above the sequence of the polypeptide includes or consists of a peptide sequence chosen from any one SEQ IDs N 1 to 8 and SEQ ID N 10 to 29 above and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B.
The invention also relates to a novel polypeptide which comprises at least a fragment of a protein whose peptide sequence corresponds to SEQ ID N
9, said fragment having at least one mutation, in particular at least two mutations compared to the reference sequence SEQ ID N 8. The polypeptide is

14 advantageously chosen from polypeptides which include the sequence in acids amino acids FSWDNCFEGKDPAVIR, referenced SEQ ID N 68 and the acid sequence amines YSLPKSEFAVPDLELP, referenced SEQ ID N 72.
In particular, said polypeptide comprises or consists of SEQ ID N 9.
This polypeptide is used to obtain a diagnostic composition, prognosis, prophylactic or therapeutic intended to detect, predict, prevent or treat a pathological condition associated with a degenerative and / or autoimmune disease, alone or in mixture with at least one polypeptide as defined above.
One of the subject of the invention is also a nucleotide fragment which code for the fragment of the protein whose peptide sequence corresponds to SEQ ID
N 9, said fragment of said protein having at least one mutation, in particular two mutations compared to the reference sequence SEQ ID N 8. Said fragment nucleotide, in particular, comprises or consists of a fragment which codes for SEQ
ID N 9. This fragment is used to obtain a diagnostic composition, prognostic, prophylactic or therapeutic intended to detect, prevent, or treat a pathological condition associated with a degenerative and / or autoimmune disease, alone or in mixing with at least one nucleotide fragment as defined above.
The invention also relates to a method for detecting at least one ligand associated with a degenerative and / or autoimmune disease, in a sample biological, whereby the biological sample is brought into contact with minus the polypeptide which comprises or consists of SEQ ID N 9 or a mixture of polypeptides comprising this polypepide and at least one polypeptide as described above, then we detects the formation of a complex or complexes between the one or more polypeptides and the or the corresponding ligands; it being understood that by ligand is meant a molecule which meets the above conditions.
The invention also relates to a method for detecting at least the reference polypeptide SEQ ID N 9 or a fragment of said polypeptide, this fragment comprising at least one and preferably two mutations with respect to the sequence of reference SEQ ID N 8, in a biological sample according to which contact the biological sample with at least one ligand specific for said polypeptide, then we detects the formation of a complex between said polypeptide and said ligand. The definition of ligand corresponds to that defined above. It can be among others of a monolonal antibody, polyclonal antibody, activity substrate enzymatic, or an enzyme in which said polypeptide is a cofactor, a receptor.
The biological sample can also be brought into contact with a ligand specific for the reference polypeptide SEQ ID N 9 and at least one ligand 5 specific for at least one other polypeptide as defined above, then we detects the formation of complexes between said polypeptides and said ligands specific for said polypeptides; it being understood that by ligand is meant a molecule which meets the conditions described above.
Another subject of the invention is a nucleotide fragment coding for to all or part of the polypeptide SEQ ID N 9, and its use for obtaining a diagnostic, prognostic, prophylactic or therapeutic composition destined to detect, predict, prevent or treat a medical condition associated with sickness degenerative and / or autoimmune, possibly in combination with at least one nucleotide fragment as defined above, and the fragments complementary

15 of said fragments.
By polypeptide fragment is meant at least all or part of the peptide sequence of a protein, in particular a polypeptide fragment who contains approximately between 5 and 15 amino acids and more precisely approximately between 5 and 10 amino acids and 6 and 15 amino acids. And by nucleotide fragment, we hear at less all or part of a nucleotide sequence, it being understood that by sequence nucleotide, the DNA and RNA sequences are covered.
In particular, by polypeptide or nucleotide fragment is meant either fragments associated with the same molecular unit, or fragments in one molecular complex comprising several homologous subunits or heterologists obtained in a natural or artificial way, in particular by multi-splicing differential or by selective synthesis.
The invention also relates to a method for detecting at least one polypeptide as defined above, according to which a sample of a biological fluid of a patient with a medical condition associated with sickness 3o degenerative and / or neurological and / or autoimmune and possibly after purification of said biological fluid sample, it is analyzed by spectrometry of

16 mass the mass profile obtained from the biological fluid and we compare to a profile of reference mass.
The present invention also relates to the use of at least one polypeptide of the invention to define effective agents therapeutically, and the use of these agents to prevent and / or treat auto-immune disease and / or neurological and / or degenerative, especially multiple sclerosis.
Thus, other objects of the invention are the following:
- Use of at least one polypeptide comprising at least one fragment of a protein to test the effectiveness of a therapeutic agent, said protein being chosen from among proteins whose peptide sequence in the native state corresponds to SEQ ID N 1, SEQ ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N 5, SEQ ID N 6, SEQ ID N 7, SEQ ID N 8, SEQ ID
N 9, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID N 14, SEQ ID N
15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID N 20, SEQ
ID N
21, SEQ ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID N 26, SEQ
ID N
27, SEQ ID N 28 and SEQ ID N 29, the peptide sequences which exhibit minus 70%
identity preferably at least 80% identity and preferably at least 98 identity with any of the peptide sequences SEQ ID N 1 to 29, and the sequences peptides or the fragments of said sequences belonging to the same family protein chosen from perlecan, the precursor of the plasma binding protein retinol, activator protein of GM2, calgranulin B and saposin B;
- Use of at least one polypeptide comprising at least one fragment of a protein to define a biological material for the preparation of a composition pharmaceutical intended for the treatment of a degenerative disease and / or neurological and / or autoimmune, such as multiple sclerosis, said protein being selected among proteins whose peptide sequence in the native state corresponds to SEQ ID N 1, SEQ IDN
2, SEQ IDN
3, SEQ ID N 4, SEQ ID N 5, SEQ ID N 6, SEQ ID N 7, SEQ ID N 8, SEQ ID N
9, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID N 14, SEQ ID N
15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID N 20, SEQ ID N

21, SEQ 10 N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID N 26, SEQ
ID
N 27, SEQ ID N 28 and SEQ

17 ID N 29, the peptide sequences which have at least 70% identity of preference to at least 80% identity and advantageously at least 98 identity with one any of peptide sequences SEQ ID N 1 to 29, and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlacan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin and saposin;
According to an advantageous variant of one of the preceding uses, the polypeptide is chosen from SEQ ID N 2, 4,8, 9, 17, 24;
- Use of at least one nucleotide fragment, to test the effectiveness of a therapeutic agent for a pathological condition associated with a disease degenerative and / or neurological and / or auto-immune, whereby said nucleotide fragment is chosen from the fragments which code for at least one fragment of a protein, said protein being chosen from proteins whose peptide sequence in the native state corresponds to SEQ ID N
1, SEQ ID N 2, SEQ ID N 3, SEQ ID N 4, SEQ ID N 5, SEQ ID N 6, SEQ ID N
7, SEQ
ID N 8, SEQ IDN 9, SEQ ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID
N 14, SEQ ID N 15, SEQ ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID
N 20, SEQ ID N 21, SEQ ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID
N 26, SEQ ID N 27, SEQ ID N 28 and SEQ ID N 29, the peptide sequences who have at least 70% identity, preferably at least 80% and advantageously at minus 98% identity with any of the peptide sequences SEQ ID N
1 to 29, and the complementary fragments of said fragments and the fragments which code for the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to retinol, GM2 activator protein, calgranulin B and saposin B.
- Use to test the effectiveness of a therapeutic agent for a condition pathological associated with a degenerative and / or neurological and / or auto-immune, from recombinant proteins and / or encoded by all or part of the fragments defined nucleotides in the previous paragraph;

18 - Use of at least one nucleotide fragment for the preparation of a pharmaceutical composition intended for the treatment degenerative and / or neurological and / or autoimmune disease, such as sclerosis in plates, whereby said nucleotide fragment is selected from fragments that code for at least one fragment of a protein, said protein being chosen from the proteins whose peptide sequence in the native state corresponds to SEQ ID N 1, SEQ ID N
2, SEQ ID N
3, SEQ ID N 4, SEQ ID N 5, SEQ ID N 6, SEQ ID N 7, SEQ ID N 8, SEQ IDN
9, SEQ
ID N 10, SEQ ID N 11, SEQ ID N 12, SEQ ID N 13, SEQ ID N 14, SEQ ID N
15, SEQ
ID N 16, SEQ ID N 17, SEQ ID N 18, SEQ ID N 19, SEQ ID N 20, SEQ ID N
21, SEQ
ID N 22, SEQ ID N 23, SEQ ID N 24, SEQ ID N 25, SEQ ID N 26, SEQ ID N
27, SEQ
ID N 28 and SEQ ID N 29, the peptide sequences which have at least 70 % identity, preferably at least 80% and advantageously at least 98% identity with Moon any of the peptide sequences SEQ ID N 1 to 29, and the fragments complementary of said fragments and the fragments which code for the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B;
- Use for the preparation of a pharmaceutical composition intended for treatment of a degenerative and / or neurological and / or autoimmune disease, such as the multiple sclerosis, recombinant proteins and / or coded by all or part of the fragments nucleotides defined in the previous paragraph.
Advantageously, said nucleotide fragment used codes for said protein.
Preferably, the peptide sequence of said protein in the native state consists in a sequence chosen from any one of SEQ ID N 1 to 29, the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98 of identity with any one of the sequences peptide SEQ ID N 1 to 29, and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma protein binding to retinol,

19 the activating protein of GM2, calgranulin B and saposin B.
polypeptides are preferably chosen from SEQ ID N 2, 4, 8, 9, 17, 24.
- Use of at least one nucleotide fragment, to test the effectiveness of a therapeutic agent for a pathological condition associated with a disease degenerative and / or neurological and / or autoimmune according to which said fragment is a fragment of a sequence nucleic acid chosen from any one of SEQ ID N 30, SEQ ID N 31, SEQ
ID N 32, SEQ ID N 33, SEQ ID N 34, SEQ ID N 35, SEQ ID N 36, SEQ ID N 37, SEQ ID N
38, SEQ ID N 39, SEQ ID N 40, SEQ ID N 41, SEQ ID N 42, SEQ ID N 43, SEQ ID N
44, SEQ ID N 45, SEQ ID N 46 and SEQ ID N 47, SEQ ID N 48, SEQ ID N 49 and SEQ
ID N
50, SEQ ID N 51, SEQ ID N 52, SEQ ID N 53, SEQ ID N 54, SEQ ID N 55, SEQ
ID N
56, SEQ ID N 57, SEQ ID N 66, SEQ ID N 67, SEQ ID N 69, SEQ ID N 70, SEQ
ID N
71, and their complementary sequences.
- Use of at least one nucleotide fragment for the. preparation of a pharmaceutical composition for treatment degenerative and / or neurological and / or autoimmune disease, such as sclerosis in plates according to which said fragment is a fragment of a nucleic sequence chosen from any of SEQ ID N 30, SEQ ID N 31, SEQ ID N 32, SEQ ID N 33, SEQ ID
N 34, SEQ ID N 35, SEQ ID N 36, SEQ ID N 37, SEQ ID N 38, SEQ ID N 39, SEQ ID
N 40, SEQ ID N 41, SEQ ID N 42, SEQ ID N 43, SEQ ID N 44, SEQ ID N 45, SEQ ID
N 46 and SEQ ID N 47, SEQ ID N 48, SEQ ID N 49 and SEQ ID N 50, SEQ ID N
51, SEQ
ID N 52, SEQ ID N 53, SEQ ID N 54, SEQ ID N 55, SEQ ID N 56, SEQ ID N
57, SEQ
ID N 66, SEQ ID N 67, SEQ ID N 68, SEQ ID N 69, SEQ ID N 70, SEQ ID N 71, and their complementary sequences.
The nucleic sequence is preferably chosen from SEQ ID N 30, 31, 42, 53.
- Use of lycorine for the preparation of a composition for the prevention and / or treatment of degenerative and / or neurological disease and / or autoimmune.
By therapeutic efficacy is meant the clinical and biological benefit acquired after administration of a therapeutic agent for improvement, or even a cure for the disease. This benefit translates among other things by a decrease in clinical, biological, and pathological effects of the disease after a clinical analysis by the doctor and / or biological analyzes, such as imagery by magnetic resonance, analysis of the oligoclonal bands in the cephalo- liquid 5 spinal analysis of evoked potentials and the detection test gliotoxicity called bioassay, the principle of which is described in patent application WO 98/11439 previously cited. This decrease in clinical signs and effects pathological must benefit the patient (Schwartz and Lazar, 1995, Elements of statistical medical and biological, eds Flammarion; Lazar and Schwartz, 1995, Elements of to medical and biological statistics, eds Flammarion). The studied disease of preference is multiple sclerosis.
The term “composition for prophylactic and / or therapeutic use” means any composition which includes a therapeutically effective agent. These agents therapeutic - are able (i) to influence qualitatively and / or quantitative The biological activity and / or the function of the proteins of interest identified in this invention, preferably the gliotoxic activity and / or (ii) of modulating and / or to inhibit the expression of these proteins and / or (iii) to decrease the concentration of these protein in an extracellular and / or intracellular compartment, and / or substitute a form non-pathogenic to a pathogenic, for example mutated, form of one of these proteins and or

20 to modulate their attachment to at least one of their ligands; said ligand being a molecule which meets the criteria described above. Different therapeutic agents are products following the classic approaches widely described in the literature. The different groups of therapeutic agents defined from proteins of interest identified in this present invention are described below. Their activity or prophylactic and / or therapeutic efficacy is evaluated in vitro and / or in vivo.
Evaluation of the efficacy of a therapeutic agent in vitro:
urine samples from healthy individuals and patients with sclerosis in plate, of preferably in the active phase, are tested for their gliotoxic activity in vitro following the bioassay protocol described in patent application WO 98/11439, previously cited. The experiment is carried out in parallel by adding or not in the urine samples tested for the therapeutic agent whose efficacy is test. Tests are carried out at different concentrations of this agent, and after different time

21 incubation with the sample, at a temperature of about 37 C or temperature ambient, for each concentration of agent tested, before carrying out the bioassay test.
Gliotoxic activity is determined for each raw or purified sample urine control and patient in the presence or absence of the therapeutic agent tested.
An agent prophylactic and / or therapeutic for multiple sclerosis is an agent which allows a decrease or inhibition of gliotoxic activity in a fluid organic patients, especially in the urine. This decrease or inhibition is assessed by relation to the gliotoxic activity detected in the biological fluid of MS patients in absence of the agent tested which fixes the upper bound and in relation to the activity lo gliotoxic detected in the urine of a healthy individual which determines the terminal lower (Schwartz and Lazar, 1995, Elements of medical and biological statistics, eds .
Flammarion; Lazar and Schwartz, 1995, Elements of medical statistics and organic, eds Flammarion). The therapeutic efficacy of several agents can be assessed in combination in one trial.
Evaluation of the efficacy of a therapeutic agent using a model animal: an animal is injected with purified urine fractions and / or minus one polypeptide of the invention and / or at least one protein obtained by recombination genetics which corresponds to at least one polypeptide of the invention and / or to the minus one synthetic polypeptide whose amino acid sequence corresponds to the sequence 2o of at least one polypeptide of the invention. The injections are carried out, at different established concentrations, in mammalian animals, such as mice or rats, of preferably a Lewis rat according to the protocol described in the patent application W097 / 33466 cited above. A series of animals are injected, by way intradermal, intravenous, intrathecal, intracerebral, intramuscular, or others, different concentrations of a raw or purified urine fraction or at least minus one polypeptide and / or protein, as defined above. Control negative is performed in parallel. The prophylactic and / or therapeutic agent to be assessed and then injected at different concentrations and by different routes of administration has a mammalian animal, preferably a mouse or a rat. The injections are carried out in a single dose or in repeated doses, with different interval times between each administration. A few hours to a few weeks after administration, of

22 biological samples, preferably blood, serum, cephalo-spinal, urine is collected. These samples are made:
(i) a measurement of gliotoxic activity by the bioassay, and / or (ii) a measurement of the activity of the polypeptides and / or proteins of interest of the invention, alone or in combination as described at least in: Li et al., 1983, Am J Hum Genet 35: 629-634; Li et al., '1988 J Biol Chem 263: 6588-6591; Li et al., 1981 J Biol Chem 256: 6234-6240; Li et al., 1976 J Biol Chem 251: 1159; Kase et al., 1996, FebsLetters 393: 74-76; Kishimoto et al., 1992, J Lipid Res 33: 1255-1267;
O'Brien et al., 1991 Fasèb J 5: 301-308; Murthy et a1., 1993 J Immunol 151: 6291-6301 to Murao et al., 1990 Cell growth Differ 1: 447-454, and / or (iii) an assay of the polypeptides and / or proteins of interest, alone or in combination combination, by ELISA (Enzyme Linked-Immunosorbant Assay) and / or Western Blot, using antibodies or antibody fragments capable of binding to at least one of polypeptides and / or proteins of the invention, or their fragment, and / or iv) an assay of antibodies specific for the polypeptides and / or proteins of interest or their fragments, alone or in combination or the determination of at least one ligand capable to attach to the polypeptides and / or proteins of interest or their fragments, and / or (v) an assay of the helper and / or cytotoxic cellular immune response induced against the polypeptides and proteins of interest or their fragments and all peptide 2o immunogen derived from these polypeptides, proteins and fragments, in realizing, by example, an in vitro activation test of helper T lymphocyte cells specific administered antigen; by quantifying the cytotoxic T lymphocytes according to the technique called ELISPOT described by Scheibenbogen et al., 1997 Clinical Cancer Research 3: 221-226. Such a determination is particularly advantageous when we want to evaluate the effectiveness of a vaccine approach for the implementation at a given patient or to diagnose and / or predict a medical condition potential by seeking to highlight a naturally developed immune response through the patient against the antigen, polypeptides, proteins of interest or fragments immunogens derived from these proteins.
By ligand capable of binding to a protein, is meant any molecule able to recognize the protein or a part of the protein. It may be checked by example in vitro by Elisa and / or Western blot tests.

23 The term “polypeptides and / or proteins of interest of the invention” denotes the C-terminal fragment of perlecan (SEQ ID N 2), the precursor of the protein plasma binding to retinol (SEQ ID N 4), the activator protein of GM2 (SEQ
ID N 8), the mutated GM2 activator protein (SEQ ID N 9), the calgranulin B
(SEQ ID N 17), saposin B (SEQ ID N 24), proteins or fragments belonging to the family of plasma retinol binding protein precursor (for example SEQ ID N 5 to 7), proteins or fragments belonging to the family of protein activator of GM2 (for example SEQ ID N 10 to 16), proteins or fragments belonging to the family of the protein calgranulin B (for example SEQ ID N
18 to lo 23), proteins or fragments belonging to the protein family saposin B (by example SEQ ID N 25 to 29) and the peptide sequences which exhibit minus 70 % identity preferably at least 80% identity and preferably at minus 98 % identity with any of the peptide sequences SEQ ID N 1 to 29.
The animal is then sacrificed and histological sections of different t5 tissues are produced, preferably brain sections. Different studies and observations are made to detect and / or quantify the effects characteristics of active polypeptides and / or proteins. associated with the gliotoxic fraction, that is to say a apoptosis of glial cells, and / or the opening of the blood-brain barrier encephalic, and / or demyelination. The presence or expression of the polypeptides and / or protein 2o of identified interest is also observed and / or quantified in these fabrics:
(i) by standard immunohistology analyzes using ligands of polypeptides and / or proteins of interest and / or their fragments and / or antibody monoclonal or polyclonal or fragments of said which bind to polypeptides and / or proteins of interest, or their fragments, and / or (Ii) by conventional in situ hybridization techniques using fragments of nucleic acids or oligonucleotides defined from the sequences polypeptides and / or proteins of interest; and or (iii) by PCR and / or RT-PCR amplification techniques in situ in using fragments of nucleic acids or primers defined from the sequences 30 polypeptides and / or proteins of interest.
By antibody capable of binding to a polypeptide, to a protein or to their fragments, we mean any monoclonal or polyclonal antibody and any fragment

24 said antibodies capable of recognizing the polypeptide, the protein or their fragments.
The capacity of antibodies to i-recognize said polypeptides, proteins or their fragments is verified in vitro, for example by ELISA and / or Western Blot. A
antibody capable of binding to the protein saposin B (SEQ ID N 24) or to all fragment of this protein is described by Misasi et al. 1998, J. NeuroChem. 71 : 2313 and Klein et al. 1994, BBRC 200: 1440-1448 or can be produced using methods conventional, for example those referenced previously for the production monoclonal and polyclonal antibodies, by immunization from the protein natural, a recombinant protein, a synthetic polypeptide or their t0 fragments. Immunogenic peptides for the production of antibodies monoclonals anti-saposin B are the peptides corresponding to the sequences SEQ ID N 61 and SEQ
ID N 62.
For example, an antibody capable of binding to the activator protein of GM2 (SEQ ID N 8) or any fragment of this protein is illustrated by Yuziuk et al., 1998 J Biol Chem 273: 66-72 or can be produced using the methods known to those skilled in the art. This antibody can be by example produced after injection into mice or rabbit of the natural protein or any fragment, and / or the recombinant protein or any fragment, and / or defined peptides and synthesized from the protein sequence of the protein. Peptides immunogens used for the production of anti-GM2 monoclonal antibodies are the peptides references SEQ ID N 58, SEQ ID N 59 and SEQ ID N 60. An antibody capable of is attach to the protein Galgranuline B (SEQ ID N 17) or to any fragment of this protein is described by Saintigny et al., 1992 J Invest Dermatol 99: 639-644 and Goebeler et al 1994 J Leukoc Biol 55: 259-261, or can be produced using methods conventional. Immunogenic peptides for the production of antibodies anti-calgranulin B monoclonals are the peptides corresponding to the sequences SEQ
ID N 63, SEQ ID N 64 and SEQ ID N 65. An antibody capable of binding to the mutated GM2 activator protein (SEQ ID N 9) or any fragment thereof protein can be produced using conventional methods defined below above.
By natural protein and fragment is meant any isolated, purified protein wholly or partially obtained from human or animal samples and all fragment obtained from this protein. For example, we get the natural protein corresponding to saposin B (SEQ ID N 24) following the technique described through Waring et al. 1998 Mol Genet Metab 63: 14-25; natural protein corresponding to the GM2 activating protein (SEQ ID N 8) following the technique described through DeGasperi et al., 1989 Biochem J 260: 777-783, Vogel et al., 1987 Arch Biochem 5 Biophys 259: 627-638, Mitsuyama, 1983 Hokkaido Igaku Zasshi 58: 502-512;
Hirabayashi et al 1983 J Neurochem 40: 168-175, Conzelmann et al, 1979 Hoppe Seylers Z Physiol Chem 360: 1837-1849, Li et al., 1976 J Biol Chem 251: 1159-1163.
The natural protein corresponding to calgranulin B (SEQ ID N 17) is obtained in according to the technique described by Hitomi et al. 1996 J Cell Sci 109: 805-815, Van den 10 Bos et al. 1998 Protein Expr Purif 13: 313-318 and Raftery et al. 1996 Biochem J 316:
285-293.
By recombinant protein or fragment of a recombinant protein, we refers to any protein or protein fragment produced in a cell prokaryotic or eukaryotic from a nucleotide sequence encoding the protein 15 or its fragment and transfected into the cell, this protein or its fragment being then purified. Generally, any cell from an organism prokaryote or eukaryotic can be used in the context of the present invention, but cells from eukaryotic organisms are preferred. We can cite as an example the CHO cells, COS cells; Semliki cells. For the purpose of this invention, Said cell can be wild or mutant. For example, protein recombinant corresponding to saposin B (SEQ ID N 24) can be obtained by following the techniques described by Zaitash et al. 1998 Bebbs letter 423: 1-4 and Qi and al. 1994 J Biol Chem 269: 16746-16753. At least one such recombinant protein is available from Kase et al. 1996 Febs Lett 393: 74-76. Recombinant protein

25 corresponding to the GM2 activating protein (SEQ ID N 8) can be produced by the techniques described by Yuziuk et al. 1998 J Biol Chem 273: 66-72 and Bierfreund and al., 1999 Neurochem Res 24: 295-300. The recombinant protein corresponding to the calgranulin B (SEQ ID N 17) can be obtained according to the Longbottom and al. 1992 Biochim Biophys Acta 1120: 215-222, Raftery et al. 1999 Protein Expr Purif 15: 228-235. Such a recombinant protein is available at least from Klempt et al. 1997 Febs Letter 408: 81-84.

26 By DNA nucleotide sequence or DNA nucleotide fragment coding for all or part of the protein saposin B (SEQ ID N 24), is meant the nucleic acid sequence SEQ ID N 53 or a fragment of this sequence.
Through nucleotide sequence or fragment RNA coding for all or part of the protein saposin B (SEQ ID N 24) means any sequence deduced from the sequence DNA
SEQ ID N 53, taking into account the genetic code and splicing phenomena.
By DNA nucleotide sequence or DNA nucleotide fragment encoding all or part of the GM2 activating protein (SEQ ID N 8), we hears the nucleic acid sequence SEQ ID N 31 or a fragment of this sequence.
Through nucleotide sequence or fragment of RNA encoding all or part of the protein activator of GM2 (SEQ ID N 8), we mean any sequence deduced from the sequence DNA SEQ ID N 31, taking into account the genetic code and the phenomena splicing.
By DNA nucleotide sequence or DNA nucleotide fragment coding for all or part of the protein calgranulin B (SEQ ID N 17), we hear the nucleic acid sequence SEQ ID N 42 or a fragment of this sequence.
Through nucleotide sequence or fragment of RNA encoding all or part of the protein calgranulin B (SEQ ID N 17) means any sequence deduced from the sequence DNA
SEQ ID N 42, taking into account the genetic code and splicing phenomena.
, By nucleotide sequence or fragment encoding all or part of the mutated protein (SEQ ID N 9) means the nucleic acid sequence deducted from the sequence SEQ ID N 9, taking into account the genetic code. By sequence or fragment nucleotide RNA coding for all or part of this mutated protein B (SEQ ID
NOT
9) means any sequence deduced from the DNA sequence, taking into account the coded genetics and splicing phenomena.
By protein activity is meant a characteristic biological function protein. This protein activity can be demonstrated by ~ techniques known to those skilled in the art. For example, the activity of saposin B (SEQ ID
N 24) and proteins of the saposin B family (for example SEQ ID N 25 to 29), can be detected by implementing the protocols described by Li et al., 1983, Am J Hum Genet 35: 629-634 ,; Li et al., 1988 J Biol Chem 263: 6588-6591, Li et al., 1981 J Biol Chem 256: 6234-6240 and Li et al., 1976 J Biol Chem 251: 1159. By activity of the

27 GM2 activating protein (SEQ ID N 8) and proteins of the same family (through example SEQ ID N 10 to 16), we mean at least the activity detected by the setting in implementation of the protocols described for example by Kase et al., 1996, Febs Letters 393: 74-76, Kishimoto et al., 1992, J Lipid Res 33: 1255-1267 and O'Brien et al., 1991 Faseb J
5: 301-308. By activity of calgranulin B (SEQ ID N 17) and proteins of the same family of calgranulin b (for example SEQ ID N 18 to 23) and all, we hears at least the activity detected by the implementation of the protocols described by example by Murthy et al., 1993 J Immunol 151: 6291-6301 and Murao et al., 1990 Cell growth Differ 1: 447-454.
Obtaining a transgenic animal model, preferably murine, for human pathology is technically feasible. Briefly, the animal transgenic is produced using corivalent techniques described and has integrated into its genome the nucleic acids encoding proteins or their fragments.
Evaluation of the effectiveness of a therapeutic agent and therapeutic monitoring ex vivo, in humans:
therapeutic agents to be tested for therapeutic activity and / or for therapeutic monitoring are administered by different routes to humans, as intradermal, intravenous, intramuscular, intracerebral routes, oral, or others.
Different doses are administered to humans. The clinical record of patient at time of first administration is well known. One or more administrations can be performed with different interval times Between each administration can range from a few days to a few years. Of biological samples are taken at specified time intervals after administration of the therapeutic agent, preferably blood, serum, liquid cerebrospinal and urine. Different analyzes are carried out from of these samples. Just before the first administration of the therapeutic agent, these samples and these same analyzes are also carried out. A clinical examination and classic biological (MRI, oligoclonal bands in the cephalo-spinal, evoked potentials) is also carried out in parallel with the analyzes additional who are described below, at different times of the analysis. Analyses carried out are :

28 (i) a measurement of gliotoxic activity by the bioassay from serum samples, CSF and urine; and or (ii) a measure of activity of the proteins of interest identified in the present invention alone or in combination as described for example by: Li et al., 1983, Am J Hum Genet 35: 629-634; Li et al., 1988 J Biol Chèm 263: 6588-6591; Li et al., 1981 J Biol Chem 256: 6234-6240; Li et al., 1976 J Biol Chem 251: 1159; Kase et al., 1996, FebsLetters 393: 74-76;
Kishimoto et al., 1992, J Lipid Res 33: 1255-1267; O'Brien et al., 1991 Faseb J 5: 301-308;
Murthy et al., 1993 J Immunol 151: 6291-6301; Murao et al., 1990 Cell growth Differ 1: 447-454, and / or (iii) an assay of the proteins of interest or their fragments, alone or in combination combination, in blood / serum, CSF, ELISA and / or urine samples Western Blot, using antibodies or antibody fragments capable of binding to at least one of proteins or a fragment thereof, and / or (iv) an assay of antibodies specific for the proteins of interest or their fragments in blood / serum, CSF, urine, ELISA and / or Western blot in using a natural protein or a fragment of the natural protein and / or a protein recombinant or a fragment of this recombinant protein, alone or in combination combination. Of even an assay of ligands capable of binding to the proteins of interest identified, alone or in combination, can be achieved, and / or (v) an assay of the helper and / or cytotoxic cellular immune response induced against proteins of interest and any immunogenic peptide derived from these proteins, for example in performing an in vitro activation test of specific T lymphocyte cells antigen administered. For example by performing an in vitro activation test of cells T cells specific helper of the administered antigen; For example by quantifying the T cells cytotoxic according to the so-called ELISPOT technique described by Scheibenbogen and al., 1997 Clinical Cancer Research 3: 221-226. Such a determination is particularly advantageous when we wish to evaluate the effectiveness of a vaccine approach implemented in a patient given or to diagnose a potential medical condition in a patient in seeking to highlight an immune response naturally developed by said patient against the antigen the proteins of interest or any immunogenic fragment derived from these proteins, alone or in combination, and / or (vi) detection of DNA and / or RNA fragments coding for the proteins or a fragment of the proteins of interest by nucleotide hybridization according to the good techniques

29 known to those skilled in the art (Southern blot, Northern blot, ELOSA "Enzyme-Linked Oligosorbent Assay "(Katz JB et al., Am: J. Vet. Res., 1993 Dec; 54 (12) : 2021-6 and François Mallet et al., Journal of Clinical Microbiology, June 1993, p1444-1449)) and / or by DNA and / or RNA amplification method, for example by PCR, RT-PCR, using nucleic acid fragments encoding the sequence of protein of interest, and / or (vii) by tissue biopsy, preferably from the brain, and observation of effects characteristics of the active proteins associated with the gliotoxic fraction, that is to say apoptosis of glial cells and / or opening of the blood-brain barrier encephalic t0 and / or the observation of demyelination phenomena, and / or (viii) by tissue biopsy or on circulating cells (blood, CSF), observing the presence of the proteins of interest and the estimation of their expression by observation inununohistological on histological sections made from fabrics, in using ligands and / or antibodies or fragments thereof capable of fix to proteins of interest, and / or (ix) by tissue biopsy or on circulating cells (blood, CSF), observation of the expression of the proteins of interest by in situ hybridization of RNA molecules encoding proteins of interest using acids nucleic defined from the sequences of the proteins of interest, and / or (x) by tissue biopsy or on circulating cells (blood, CSF), the determination of the expression of the proteins of interest by amplification of these RNA by conventional techniques, such as RT-PCR, using acids nucleic acids defined from the sequences of the proteins of interest.
The term “polypeptides and / or proteins of interest of the invention” denotes the C-terminal fragment of perlecan (SEQ ID N 2), the precursor of the protein plasma binding to retinol (SEQ ID N 4), the activator protein of GM2 (SEQ
ID N 8), the mutated GM2 activator protein (SEQ ID N 9), the caigranulin B
(SEQ ID N 17), saposin B (SEQ ID N 24), proteins or fragments belonging to the family of plasma retinol binding protein precursor (for example SEQ ID N 5 to 7), proteins or fragments belonging to the family of protein activator of GM2 (for example SEQ ID N 10 to 16), proteins or fragments belonging to the family of the protein calgranulin B (for example SEQ ID N
18 to 23), proteins or fragments belonging to the protein family saposin B (by exeniple SEQ ID N 25 to 29), and the peptide sequences which exhibit minus 70 % identity preferably at least 80% identity and preferably at minus 98 of identity with any of the peptide sequences SEQ ID N 1 to 29.
5 The term nucleic acid sequence denotes DNA or fragments encoding for the 'polypeptides and / or proteins of interest of the invention' the sequence acids nucleic acid coding for the C-terminal fragment of perlecan (SEQ ID N 2), the sequence of nucleic acids encoding the precursor of the plasma protein of link to retinol (SEQ ID N 4), the nucleic acid sequence (SEQ ID N 31) encoding for the 1o GM2 activator protein (SEQ ID N 8), the nucleic acid sequence coding for the mutated GM2 activator protein (SEQ ID N 9), the sequence acids nucleic acid (SEQ ID N 42) coding for calgranulin B (SEQ ID N 17), the sequence nucleic acids (SEQ ID N 53) coding for saposin B (SEQ ID N 24), the DNA and / or RNA nucleic acid sequences (SEQ ID N 30 to 57) coding for the 15 proteins or fragments belonging to the family of the precursor of protein plasma binding to retinol (for example SEQ ID N 5 to 7), proteins or fragments belonging to the family of the GM2 activator protein (by SEQ example ID N 10 to 16), proteins or fragments belonging to the family of protein calgranulin B (for example SEQ ID N 18 to 23), proteins or fragments 20 belonging to the saposin B protein family (for example SEQ ID N
25 to 29).
A protein or a variant of a protein chosen more particularly among the sequences defined in the identifiers SEQ ID N s 2, 4, 8, 9, 17 and 24 or their fragments, or among the sequences corresponding to the proteins of families of these said sequences (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ
25 ID N 10 to 16, SEQ ID N 18 to 24, SEQ ID N 25 to 29), and the sequences peptide who have at least 70% identity, preferably at least 80% identity and advantageously at least 98 of identity with any of the sequences SEQ ID N 1 to 29 peptides, independently or in combination, has a effect toxic directly or indirectly, towards cells, in particular with respect to

30 glial cells, which is highlighted by the aforementioned bioassay. The autoantibody produced in response to the presence of this protein or these proteins are associated with autoimmune process. Thus, the target of the therapeutic agent (s) is through

31 example (i) natural protein or natural proteins or their variants for the purpose of regulate their expression and / or their intracellular concentration and / or their concentration in the circulation, (ii) an antibody specific for at least one such protein.
The therapeutic agent or the defined therapeutic agents eliminate the target directly, by induction of a response specific immune system and / or neutralize it.
The present invention therefore relates to a biological material for the preparation of a pharmaceutical composition intended for the treatment of manunifers suffering from Degenerative and / or autoimmune and / or neurological pathologies, preferably sclerosis in plates, said composition comprising:
(i) either at least one natural protein and / or a recombinant protein, or their fragments whose sequence corresponds to all or part of the sequences referenced SEQ IDN
2, 4, 8, 9, 17 and 24 and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, calgranulin B and saposin B (for example SEQ IDN 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences who present at least 70% identity preferably at least 80% identity and advantageously at least 98 identity with any of the peptide sequences SEQ ID N there 29 ,.
independently or in combination, (ii) either at least one specific ligand of at least one of said proteins or their fragments whose sequence corresponds to all or part of the sequences referenced SEQ ID N
2, 4, 8, 9, 17 and 24, and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, calgranulin B and saposin B (for example SEQ IDN 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences who present at least 70% identity preferably at least 80% identity and advantageously at least 98 of identity with any of the peptide sequences SEQ ID N 1 to 29, independently or in combination,

32 (iii) either at least one polyclonal or monoclonal antibody specific for at least less one of said proteins or their fragments whose sequence corresponds to any or part of sequences referenced SEQ ID N 2, 4, 8, 9, 17 and 24, and the sequences peptide or fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of the plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ
ID N 1, SEQ
ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences which have at least 70% identity in preference to minus 80%
identity and advantageously at least 98 identity with any of the sequences peptides SEQ ID N 1 to 29, independently or in combination, (iv) either at least one nucleic acid sequence comprising at least one gene of therapeutic interest whose nucleic sequence is deduced from the sequences DNA and RNA
coding for all or part of the proteins whose sequences are referenced SEQ iD N 2, 4, 8, 9, 17 and 24, and the DNA and / or RNA sequences (for example SEQ ID N 30 to 57) coding for all or part of the proteins belonging to the same family of proteins chosen from the perlecan, the precursor of the plasma retinol binding protein, of, protein activator of GM2, calgranulin B and saposin B, in combination with elements ensuring the expression of said gene of therapeutic interest in vivo in target cells '' intended to be genetically modified by the nucleic sequence of the gene of interest therapeutic, (v) either at least one mammalian cell which does not naturally produce the protein of interest or proteins of interest or any fragment of this or these protein (s) or antibodies specific for at least one of said proteins or of its fragments said cell mammal being genetically modified in vitro by 'at least one sequence acid nucleic acid or a fragment of a nucleic acid sequence or association of sequences of nucleic acids corresponding to nucleic acid fragments from of the same gene or of different genes, the said nucleic sequence (s) being deduced (s) sequences of DNA and RNA coding for the proteins referenced SEQ ID N 2, 4, 8, 9, 17 and 24, and DNA and / or RNA sequences (for example SEQ ID N 30 to 57) coding for all or part of protein

33 belonging to the same family of proteins chosen from perlecan, precursor plasma retinol binding protein, protein activating GM2, from calgranulin B and saposin B, said gene of therapeutic interest coding for everything or part of the protein of interest, a fragment of the protein of interest or of a antibody specific for the protein of interest which will be expressed on the surface of said mammalian cell (Toes et al., 1997, PNAS 94: 14660-14665). The composition pharmaceutical may contain a therapeutic agent alone directed against a target only or agents taken in combination directed against several targets.

Based on knowledge of the amino acid sequences of proteins of interest identified in the present invention, it is within the scope of the man of the art of define and use the molecules described above and / or any molecule able to attach to said molecules, and / or any molecule capable of inhibiting said molecules.
Thus the present invention relates to the use of natural proteins and or recombinant and / or synthetic polypeptides and their fragments, ligand capable to bind to said proteins or to their fragment (s), for example antibodies; of proteins that inhibit the function and / or expression and / or fixing of said proteins.

34 Use of natural protein (s) and / or peptide (s) and / or protein (s) recombinant (s) and / or synthetic polypeptide (s) corresponding to proteins of interest identified in the present invention.
The present invention relates to biological material for the preparation of pharmaceutical compositions intended for the treatment of mammals suffering from self disease immune, preferably multiple sclerosis, comprising:
(i) either at least one natural protein and / or a recombinant protein and / or a synthetic polypeptide chosen from proteins whose sequences in amino acids are referenced SEQ ID N 2, 4, 8, 9, 17 and 24, and the peptide sequences or the fra.gments said sequences belonging to the same family of proteins chosen parrni the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, to 7, SEQ ID N 10_ to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences who have at least 70% identity, preferably at least 80% identity and advantageously at least 98 of identity with any of the sequences peptide SEQ ID N 1 to 29, alone or in combination, (ii) either at least one natural and / or synthetic fragment of these proteins of interest, for example an immunogenic fragment capable of inducing a response inunune against a target polypeptide, (iii) either at least one mimotope peptide defined from the sequences of reference SEQ ID N 2, 4, 8, 9, 17 and 24 and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences who have at least 70% identity, preferably at least 80% identity and advantageously at least 98 of identity with any of the sequences peptide SEQ ID N 1 to 29, or a combination of mimotopes, capable of inducing immune response against the target polypeptide, (iv) at least any protein or peptide capable of regulating in vivo the transcription and / or translation of the proteins of interest (SEQ ID N 2, 4, 8, 9, 17 and 24) and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to 5 retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 % identity preferably at least 80% identity and advantageously at least 98 identity with one any of the peptide sequences SEQ ID N 1 to 29. The administration of these protein 10 and / or peptides alone or in combination can restore the concentration of a protein of interest in the body.
The immune response directed against a specific antigen can be divided into two distinct categories, one involving antibodies (immune response Of type humoral), the other cytotoxic effector cells such as by example macrophages, 15 cytotoxic lymphocytes (CTL) or killer cells (NK) as well as T cells helper, in particular CD4 + T lymphocytes (immune response of the type cellular). More in particular, the two types of response are distinguished in that the antibodies recognize antigens in their three-dimensional form whereas T lymphocytes, for example, recognize peptide portions of said antigens, associated with encoded glycoproteins 20 by the genes of the major histocompatibility complex (MHC), in particular the genes major type I histocompatibility complex that are expressed so ubiquitous to the cell surface or genes of the major histocompatibility complex of type II which are expressed specifically on the surface of cells involved in presentation of antigens (APC). 1) According to a first aspect, the type immune response cell is 25 characterized in that - CD4 + type T cells (helper T cells), next to a well-known activation phenomenon (for a review see Alberola-lia 1997, Annu Rev Immunol 15, 125-154) produce cytokines which in turn induce proliferation APC cells capable of producing said cytokines, differentiation cell of B cells capable of producing antibodies specific to the antigen, and the 30 stimulation of cytotoxic T lymphocytes (CTL). 2) According to a second aspect of the cellular immune response, cells cytotoxic effector such as for example CD8 + lymphocytes (CTL) are activated a) after interaction with antigenic peptides attached to and presented by the glycoproteins worn by ubiquitous cells and encoded by genes belonging to the CMHI system, and b) possibly by cytokines produced by CD4 +.
The present invention relates to the administration of a protein or a peptide.
derived from the proteins of interest (SEQ ID N 2, 4, 8, 9, 17 and 24) or from their (s) fragment (s), and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29), and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, alone or in combination combination, for prophylaxis and / or therapy of an autoimmune disease, such as multiple sclerosis.
These proteins and peptides administered are characterized in that they must to have lost their toxic activity, for example their gliotoxic activity, or having lost their ability to attach to a ligand, and can significantly induce an immune response mediated by the T cells and / or antibodies to this protein are used.
Such proteins are said to be 'modified', however their immunogenicity is retained. Such molecules modified immunogens are obtained by a number of treatments conventional, by example chemical or heat denaturation, truncation or mutation with deletion, insertion or location of amino acids. An example of truncation consists of the truncation amino acids at the carboxy-terminal end of up to 5-30 amino acids. The modified molecules can be obtained by synthetic techniques or and recombinant or by chemical or physical treatment of molecules natural.
The proteins of natural and / or recombinant interest identified in the present invention (SEQ ID N 2, 4, 8, 9, 17 and 25), and the sequences peptide or fragments of said sequences belonging to the same family of proteins chosen among perlecan, the precursor of the plasma binding protein retinol, protein activator of GM2, calgranulin B and saposin B (for example SEQ
ID N 1, SEQ
ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 'to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29), and the peptide sequences which have at least 70% identity in preference to minus 80%
identity and advantageously at least 98 identity with any of the sequences SEQ ID N 1 to 29 peptides, or their fragment (s), are used in vaccination prophylactic and therapeutic against autoimmune diseases, preferably MS. A
vaccine includes an effective immunogenic amount of the immunogenic protein in association with a pharmaceutically acceptable vehicle and optionally an adjuvant and / or a diluent. Pharmaceutical vehicles, adjuvants and diluents acceptable are well known to those skilled in the art. We can cite by reference the Remington's Pharmaceutical Sciences. The use of vaccine compositions is particularly advantageous in combination with early diagnosis of the disease. The immunogenic protein is used in the preparation of medicament for vaccination prophylactic or therapeutic. Proteins of interest can be eliminated from the body without inducing unwanted side effects. Identification of such proteins or peptides vaccine is carried out as follows: the candidate molecules modified as described previously (natural, recombinant proteins, peptides) are analyzed in a test functional for check that they have lost their toxicity, for example their activity gliotoxic using the test called bioassay, and to check their immunogenicity (i) by performing an in vitro test of proliferation of CD4 + T lymphocytes specific for the administered antigen (T
cell assay) or a in vitro cytotoxicity test for antigen-specific CD8 + lymphocytes administered and (ii) by measuring, among other things, the level of circulating antibodies directed against natural protein. These modified forms are used to immunize men through procedures standard with suitable adjuvants.
The vaccines prepared are injectable, that is to say in liquid solution or in suspension. Optionally, the preparation can also be emulsified. The molecule antigenic can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of favorable excipients are water, a saline, the dextrose, glycerol, ethanol or equivalents and their combinations. If desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, agents who buffer pH or adjuvants like aluminum hydroxide, dipeptide muramyl or their variations. In the case of peptides, their coupling to a larger molecule (KLH, tetanus toxin) sometimes increases immunity. The vaccines are administered conventionally by injection, for example subcutaneous or intramuscular.
Of additional favorable formulations with other modes of administration include suppositories and sometimes oral formulations.
In general the concentration of the polynucleotide in the composition used for in vivo administration is from 0.lg / ml up to 20 mg / ml. The polynucleotide can be homolog or heterolog of the target cell in which it is going to be introduced.
The present invention also relates to the use of vaccines including nucleic acid molecules that code for proteins of interest or peptides immunogens or their fragment (s), not active, corresponding to proteins of interest (SEQ ID
N 2, 4, 8, 9, 17 and 24) and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences who have at least 70% identity, preferably at least 80% identity, and advantageously at least 98% identity with any of the sequences peptides SEQ ID N 1 to 29. Nucleic acid vaccines, in particular the DNA vaccines, are usually given in combination with a vehicle pharmaceutically acceptable for intramuscular injection.
From the amino acid sequence of the proteins of interest described (SEQ
ID N 2, 4, 8, 9, 17 and 24) and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, of the calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ
ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70%
identity preferably at least 80% identity and preferably at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, of peptides or fragments corresponding to all or part of the sequence primary of these proteins can be synthesized by conventional synthesis methods peptide or obtained by genetic recombination.
Recombinant proteins corresponding to the proteins of interest, produced in a prokaryotic or eukaryotic cell system, are available nearby from different teams and are described in the literature. They can also be lo produced by a person skilled in the art from knowledge from sequences of genes correspondents described in the literature and taking into account the degeneration of genetic code. All protein sequences identified herein invention are thus likely to be obtained by genetic recombination. The genes are cloned into suitable vectors. Different vectors are used for transform prokaryotic cells (e.g. E. coli) and eukaryotic cells (e.g.
example COS, CHO cells and Simliki cells). Recombinant proteins corresponding to proteins of interest or to fragments of proteins of interest can to be so produced in prokaryotic and / or encaryotic cell systems. In the cells E. coli, recombinant proteins are produced with a poly- tail histidine. The insoluble protein fraction is dissolved in 8M urea.
The enrichment of product was made on nickel chelated resin (Qiagen). Column a been washed with decreasing concentrations of urea. The elution was made with imidazole in the absence of urea. The complete sequence of proteins of interest can be also cloned into a suitable plasmid and then transferred to the vaccinia virus to get a recombinant virus.
Use of ligands capable of binding to proteins of interest identified in the present invention.
The present invention relates to biological material for the preparation of pharmaceutical compositions intended for the treatment of affected mammals of autoimmune disease, preferably multiple sclerosis, comprising:
(i) either at least one ligand capable of binding to proteins and / or fragments of the proteins chosen from the target proteins SEQ ID N 2, 4, 8, 9, 14 and 24 and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of plasma protein binding to retinol, GM2 activating protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ IDN 3, SEQ IDN 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 5 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, the ligand being capable or not to inhibit protein activity, (ii) either at least one polyclonal or monoclonal antibody capable of binding to at 10 minus a protein or a fragment thereof chosen from proteins targets SEQ ID N 2, 4, 8, 9, 14 and 24 and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, the precursor of protein retinol binding plasma, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ IDN 3, SEQ ID N 5 to 7, SEQ IDN 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and preferably at minus 98%
of identity with any of the peptide sequences SEQ ID N 1 to 29.
This antibody may or may not be neutralizing, i.e. capable or not of inhibiting protein activity of interest. The ligand can be chosen from any molecule or fragment molecule capable of 20 attach to target proteins, for example receptors for this protein, cofactors of these proteins, polyclonal or monoclonal antibodies capable of binding to protein or whatever -fragment of these proteins.
These antibodies are very useful in particular for enabling the implementation of therapeutic compositions because they lead for example to reactions immune, directed 25 specifically against immunodominant epitopes or against antigens with great variability. The patient is administered either soluble antibodies neutralizers to inhibit their function, either specific soluble antibodies to eliminate the peptide by formation of-immune complexes. The invention describes the use of antibodies capable of specifically recognize at least one protein described in the present invention for the treatment and / or for follow-up therapy of degenerative and / or neurological and / or autoimmune disease, preferably the multiple sclerosis. These antibodies are polyclonal and preferably monoclonals. Of preferably these antibodies recognize the active site of the protein and fixing, inhibits the protein function. The ability of the antibody to specifically bind to the protein is analyzed by conventional techniques described, such as for example by ELISA tests or Western blot using the natural immunogenic protein or peptide or synthetic.
The titer of the antibody is determined. The ability of the antibody to neutralize the function of the protein can be analyzed by different means, for example by determining the decrease of the activity of the immunogenic protein or peptide in the presence of the antibody, preferably determining the decrease in gliotoxic activity using the test in vitro bioassay.
For example, monoclonal antibodies against the protein target or part of this protein is produced by techniques used to produce antibodies against antigens of surface Mice or rabbits are immunized (i) with either the protein natural or recombinant of interest, (ii) either with any immunogenic peptide of this protein of interest, (iii) either with murine cells which express the protein or the peptide of interest and the CMHII molecules. The Balb / c murine line is the most frequently used. The immunogen is also a peptide chosen from peptides defined from the primary sequences of the proteins of interest. Through example, the following immunogen was prepared: peptides SEQ ID N s 58, 59, from the sequence of the activator protein of GM2, the peptides SEQ ID N
s 61, 62 derived from the saposin B sequence and the peptides SEQ ID N s 63, 64, 65 from of calgranulin B were coupled to hemocyanin from Lymphet Keyhole, in abbreviated peptide-KLH, as a support for its use in immunization, or coupled with human serum albumin, abbreviated peptide-HSA. Animals were injected with peptide-KLH or peptide-HSA using of Freund's complete adjuvant (IFA). Serums and culture supernatants hybridoma from animals immunized with each peptide were analyzed for the presence of anti-protein antibodies by an ELISA test using the initial proteins. The spleen cells of these mice therefore summer recovered and fused with myeloma cells. Polyethylene glycol (PEG) is the most frequently used fusion agent. Hybridomas producing the the most specific and sensitive antibodies are selected. The antibody monoclonals can be produced in vitro by cell culture of hybridomas produced or by recovery of murine ascites fluid after intraperitoneal injection hybridomas in the mouse. Whatever the mode of production by supernatant or ascites, it then imports from purify the monoclonal antibody. The purification methods used are basically the filtration on ion exchange gel or by exclusion chromatography, or even immunoprecipitation. For each antibody it is necessary to choose the method which will get the best performance. A sufficient number of anti-protein antibodies are screened in functional tests to identify the best performing antibodies for fix the protein of interest and / or to block the activity of the protein of interest. The monoclonal antibodies selected are humanized by standard CDR grafting methods (protocol carried out by many companies, as a service). These humanized antibodies can be clinically tested in the patient. The effectiveness of these antibodies can be followed by clinical parameters.
In vitro production of antibodies, antibody fragments or derivatives antibodies, such as chimeric antibodies, produced by genetic engineering, in cells eukaryotes has been described (EP 120 694 or EP 125 023) and is also applicable to the current invention.
Use of inhibitory molecules for proteins of interest identified in the present invention.
The present invention relates to biological material for the preparation of pharmaceutical compositions intended for the treatment of mammals suffering from sickness degenerative and / or neurological and / or autoimmune, preferably sclerosis in plates, said composition comprising (i) either at least one molecule inhibiting the function of minus one protein selected from the proteins identified herein invention (SEQ ID
N 2, 4, 8, 9, 17, 24) and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences who present at less 70% identity preferably at least 80% identity and advantageously at least 98 % identity with any of the peptide sequences SEQ ID N 1 to 29, for example inhibitor of gliotoxic activity, (ii) at least one molecule expression regulator at least one protein chosen from the proteins SEQ ID N 2, 4, 8, 9, 17, 24 and them peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 % identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, for example for block the transcription or translation, (iii) either at least one regulatory molecule at metabolism minus one protein chosen from the proteins SEQ ID N 2, 4, 8, 9, 17, 24 and the sequences peptides or the fragments of said sequences belonging to the same family protein chosen from perlecan, the precursor of the plasma protein of binding to retinol, activator protein of GM2, calgranulin B and saposin B (by example SEQ ID
N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ
ID N 25 to 29) and the peptide sequences which exhibit at least 70% identity of preference to at least 80% identity and advantageously at least 98% identity with one any peptide sequences SEQ ID N 1 to 29, (iv) or at least one molecule regulator the expression and / or metabolism of a ligand of at least one protein chosen from proteins SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98% identity with any of the sequences SEQ ID N 1 to SEQ ID N 8 and SEQ ID N 10 to 29 peptides and the sequences peptide who present to less 70% identity preferably at least 80% identity and advantageously at least 98 % identity with any of the peptide sequences SEQ ID N 1 to 29, for example a receiver or a cofactor. You might think that these proteins in the body human can be inhibited without side effects.
Another important aspect of the invention relates to the identification and evaluation therapeutic efficacy of natural and / or synthetic substances (i) able to block and / or inhibit the activity of the proteins of interest of the invention and / or their fragment: SEQ ID
N 2, 4, 8, 9, 17, 24 and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, calgranulin B
and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 at 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which present to less 70% identity preferably at least 80% identity and advantageously at least 98 % identity with any of the peptide sequences SEQ ID N 1 to 29 and / or (ii) able to inhibit their metabolism such as metabolism inhibitors correspondent, the inhibitors of enzymes activated by coenzymes, (iii) capable of regulating the expression of proteins of interest (SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the plasma retinol binding protein precursor, protein activator of GM2, of calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptide who have at least 70% identity, preferably at least 80% identity and advantageously at least 98% identity with any of the sequences peptide SEQ ID N 1 to 29, (iv) capable of inhibiting the function and / or expression of ligands proteins of interest SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the plasma retinol binding protein precursor, protein activator of GM2, of calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptides which have at least 70% identity, preferably at least 80 % identity and advantageously at least 98% identity with any of the sequences peptide SEQ ID N 1 to 29, such as for example receivers. These substances can be used in prophylactic and therapeutic treatments of the disease.
The invention relates to Also methods for treating and preventing an autoimmune disease, for example example the MS, by administering effective amounts of these substances. The substances can be proteins,. antibodies, small synthetic molecules or natural, derivatives of proteins identified in this invention, lipids, glycolipids etc ... The little ones molecules can be screened and identified in large quantities using bookstores 10 chemical combinatorics. The invention also relates to compositions pharmaceutical including these substances in association with physiological carriers acceptable, and methods for the preparation of drugs for use in therapy or prevention of autoimmune diseases including MS using these substances.
To identify low molecular weight inhibitory molecules like of 15 candidate drugs for degenerative and / or neurological diseases and / or autoimmune, such as multiple sclerosis, the tests and protocols described are used previously and in the patent applications referenced above, using samples taken from untreated or treated patient, untreated or treated animal model, or model fabrics untreated or treated animal. This aspect of the invention also includes a process for 20 identify substances capable of blocking or inhibiting the activity of proteins of interest, including the introduction of these substances in an in vitro test or in a animal model in vivo. The selected molecules are tested at different concentrations.
These inhibitors are also tested in toxicity and pharmacokinetics tests to find out if they can represent valid candidate drugs. The substances tested for inhibition or 25 blocking protein activities or protein expression in these procedures screening can be proteins, antibodies, antibody fragments, small synthetic or natural molecules, derivatives of proteins of interest, etc. The little molecules can be screened and identified in large quantities using bookstores chemical combinatorics.
By way of example, the following may be mentioned as inhibiting substances:

The protein inhibitors identified in the present invention (SEQ ID N

2, 4, 8, 9, 17, 24), the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of protein retinol binding plasma, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 at 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity. and advantageously at minus 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, and the fragment protein inhibitors. These inhibitors can be included in a prophylactic and therapeutic composition, especially for treatment sclerosis in plates. For example, lycorine, an alkaloid extracted from Amaryllidaceae (ex:
Crinum Asiaticum) is used in vitro at a concentration between 0.1 and 0.5 gg / ml and in vivo at a concentration between 0.1 and 1 mg / kg / day. For example, Rolipram (last name commercial) and Ibudilast (trade name), which are two molecules of the same family of phosphodiesterase 4 (PDE4) inhibitors are used in vitro at concentrations between 1 and 10 M / 1 and in vivo at concentrations between around 10 mg / kg / day.
From the amino acid sequences of the proteins SEQ ID N 2, 4, 8, 9, 17, 24 and peptide sequences or fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the protein plasma binding to retinol, GM2 activating protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) it is obvious that the sequences can be deduced nucleotide DNA
and RNA (SEQ ID N 30, 31, 42, 53) corresponding to the proteins of interest and the sequences coding for proteins of the family of these proteins of interest (by example SEQ ID N 32 to 41, SEQ ID N 43 to 52, SEQ ID N 54 to 57, SEQ ID N 66 to 67), taking into account code genetics and its degeneration. Thus the present invention relates the use of these nucleotide sequences in the form:
- anti-sense sequences, - sequences coding for a therapeutic gene, - of sequences which can be contained in a vector for the realization of cell transformation ex vitro and / or in vivo (gene therapy).
Use of nucleic acids deduced from the amino acid sequences of proteins of interest identified in the present invention; nucleic acids antisense and / or coding for a therapeutic gene.
The present invention relates to biological material for the preparation of pharmaceutical compositions intended for the treatment of mammals suffering from sickness degenerative and / or neurological and / or autoimmune, in particular sclerosis in plates, the composition comprising (i) either at least one nucleic acid sequence able to hybridize to a nucleic acid sequence encoding proteins of interest (SEQ ID N
2, 4, 8, 9, 17, 24) and the peptide sequences or fragments thereof belonging sequences to the same family of proteins chosen from perlecan, the precursor of protein plasma binding to retinol, GM2 activator protein, calgranulin B and saposin B (for example SEQ IDN 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and preferably at minus 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, or their (s) fragment (s), (ii) either at least one nucleic acid sequence comprising at least minus one gene of therapeutic interest coding for proteins or a protein fragment (SEQ ID N 2, 4, 8, 9, 17, 24), the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of plasma protein binding to retinol, GM2 activator protein, calgranulin B and saposin B
(for example SEQ IDN 1, SEQ ID N 3, SEQ iD N 5 to 7, SEQ ID N 10 to 16, SEQ

to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, and elements ensuring expression of said gene in vivo in target cells intended to be genetically modified by said nucleic sequence.
By nucleic acid sequence is meant a DNA and / or RNA fragment, double strand or single strand, linear or circular, natural and isolated or synthesis, designating a precise sequence of nucleotides, modified or not, allowing to define a fragment or a region of a nucleic acid chosen from the group consisting of a CDNA; genomic DNA; plasmid DNA; a messenger RNA. These sequences of nucleic acids are deduced from the amino acid sequence of proteins SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, the precursor of protein plasma binding to retinol, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and preferably at minus 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, using the genetic code. Due to the degeneracy of the genetic code the invention includes also equivalent or homologous sequences. These defined sequences allow those skilled in the art define themselves the suitable nucleic acids.
Also, the present invention relates to biological material for the preparation pharmaceutical compositions comprising at least one acid sequence nucleic capable of hybridizing to a nucleic acid sequence coding for proteins of interest or their fragment (s) (SEQ ID N 2, 4, 8, 9, 17, 24) and the sequences peptide or fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of the plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ
IDN 1, SEQ
ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and peptide sequences which have at least 70% identity in preference to minus 80%
identity and preferably at least 98.% identity with any one sequences peptides SEQ ID N 1 to 29.
The invention consists in defining and using nucleic acid molecules complementary to DNA and / or RNA sequences encoding proteins of interest or their (s) fragment (s). These fragments correspond to antisense molecules or ribozyme and can be synthesized using automatic synthesizers, such as those sold by Applied Biosystem. The invention describes the use of these nucleic acids capable of hybridizing under stringent conditions to DNA and / or RNA
encoding the proteins of the invention or their leu (s) fragment (s). Of conditions of Characteristic stringency are those that correspond to a combination of temperature and the salt concentration chosen approximately between 12 to 20 C below the Tm (melting temperature) of the hybrid under study. Such molecules are synthesized and can be marked using conventional marking methods used to respondents molecular, or can be used as primers in reactions amplification. The sequences that have at least 90% homology to a sequence reference are also part of the invention, as are the fragments of these sequences which have at least 20 nucleotides and preferably 30 contiguous nucleotides counterparts by compared to a reference sequence. In order to reduce the proportion of peptides natural or variants, it is possible to envisage an antisense and / or ribozyme approach.
Such an approach is widely described in the literature. Of course, such molecules antisense can constitute as such vectors. You can also use vectors which include a nucleic acid sequence that codes for antisense.
The present invention relates to biological material for the preparation of pharmaceutical compositions intended for the treatment of mammals suffering from sickness degenerative and / or neurological and / or autoimmune, such as sclerosis in plates, said composition comprising at least one nucleic acid sequence containing at least minus one gene of therapeutic interest and of elements ensuring the expression of said gene in vivo in target cells intended to be genetically modified by said sequence nucleic acid.
These nucleic acid and / or vector sequences (antisense or coding for a protein or a fragment of a protein) can target cells in which the peptide is expressed, such as macrophage cells: (i) either by the use of a targeting molecule introduced onto the vector, (ii) either by the use of a property particular of these cells.
Use of vectors comprising a gene of therapeutic interest corresponding to the genes of the proteins of interest identified herein invention.

The present invention relates to biological material for the preparation of pharmaceutical compositions for the prevention and treatment of diseases degenerative and / or neurological and / or autoimmune, such as sclerosis in plates, the composition comprising a nucleic acid sequence comprising a gene of interest 5 therapeutic and elements of expression of said gene of interest. The genes can be no transferred or transferred. They can also consist of nucleic acids modified so that it is not possible for them to integrate into the genome of the cell target or acids nucleic acids stabilized using agents, such as spermine.
Such a gene of therapeutic interest codes in particular:
10 (i) or at least for a protein chosen from the proteins identified in the present invention (SEQ ID N 2, 4, 8, 9, 17, 24) and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen by parnii the perlecan, the precursor of plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, 15 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ IDN 25 to 29) and the peptide sequences who have at least 70% identity, preferably at least 80% identity and advantageously at least 98% identity with any of the sequences peptide SEQ ID N 1 to 29, or their fragment (s), (ii) at least for all or part of a polyclonal or monoclonal antibody 20 capable of binding to at least one protein or protein fragment chosen from proteins identified in the present invention (SEQ IDN 2, 4, 8, 9, 17, 24) and the sequences peptides or the fragments of said sequences belonging to the same family protein chosen from perlecan, the precursor of the plasma protein of binding to retinol, activator protein of GM2, calgranulin B and saposin B (by example SEQ ID
25 N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity of preference to at least 80% identity and advantageously at least 98% identity with one any of peptide sequences SEQ ID N 1 to 29. They may in particular be antibodies native transmembrane, or fragment or derivative of such an antibody, for as much as said antibody, fragment or derivative of antibody is expressed on the surface of the target cell of the genetically modified mammal and be able to attach to a polypeptide present at the surface of a cytotoxic effector cell or a T helper lymphocyte involved in the method of activating such a cell, (iii) either at least for an inhibitor molecule of at least one protein or of its fragments, said protein being chosen from the proteins identified (SEQ ID N 2, 4, 8, 9, 17, 24) and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of plasma protein binding to retinol, GM2 activator protein, calgranulin B
and saposin B
(for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ

to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29; the proteins inhibitors function and / or metabolism and / or binding of proteins SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the protein plasma binding retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 % identity preferably at least 80% identity and advantageously at least 98%
identity with any one of the peptide sequences SEQ ID N 1 to 29, (iv) at least for a ligand or any part of a ligand capable of attach to at least one protein or a protein fragment chosen from proteins identified (SEQ ID N 2, 4, 8, 9,17, 24) and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, precursor of the plasma retinol binding protein, GM2 activator protein, of the calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ

at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptides which have at least 70% identity, preferably at least 80 %
identity and preferably at least 98%

identity with any of the peptide sequences SEQ ID N 1 to 29, and / or inhibit his function.
More particularly, by antibody fragment is meant the fragments F (ab) 2, Fab ', Fab, sFv (Blazar et al., 1997, Journal of Immunology 159: 5821-5833;
Bird et al., 1988 Science 242: 423-426) of a native antibody and by derivative is understood, by example, a derivative chimeric of such an antibody (see for example the chimeras of the antibodies antiCD3 Mouse / Man in Arakawa et al., 1996 J Biochem 120: 657-662 or immunotoxins such as sFv-toxin de Chaudary et al 1989, Nature 339: 394-397). Through antibody transmembrane means an antibody including at least the functional region able to recognize and attach to its specific antigen is expressed on the surface target cells to allow said recognition and fixation. More specifically, antibody according to present invention consist of fusion polypeptides comprising amino acids defining said functional region and an amino acid sequence (polypeptide transmembrane) allowing anchoring within the lipid double layer membrane of the target cell or the external surface of this bi-layer. The sequences nucleic encoding for many transmembrane polypeptides are described in the literature. According to a entirely advantageous case, the nucleic acid sequence encoding the heavy chain of the antibody is fused with the nucleic acid sequence encoding undit polypeptide transmembrane.
By elements ensuring the expression of said gene in vivo we make in particular reference to the elements necessary to ensure the expression of said gene after his transfer in a target cell. These are, in particular, promoter sequences and / or sequences of efficient regulation in said cell, and possibly the sequences required for allow expression on the surface of target cells of said polypeptide. The promoter used can be a viral, ubiquitous or tissue specific promoter or a promoter synthetic. By way of example, mention will be made of promoters such as promoters of RSV virus (Rous Sarcoma Virus), MPSV, SV40 (Simian Virus), CMV
(Cytomegalovirus) or vaccinia virus, promoters of the gene encoding creatine kinase muscle, for actin. It is also possible to choose a promoter sequence specific of a cell type given, or activable under defined conditions. The literature provides a large amount of information i-elative to such promoter sequences.
Furthermore, said nucleic acid can comprise at least two identical or different sequences exhibiting promoter activity transcriptional and / or at least two identical or different genes located one by relation to the other in a coiltiguë, distant, in the same direction or in the meaning inverse, insofar as the function of transcriptional promoter or the transcription of said genes is not affected.
Similarly in this type of nucleic acid construction, it is possible t0 to introduce neutral nucleic sequences or introns which do no harm not at the transcription and are spliced before the translation step. Such sequences and their uses are described in the literature (reference: patent application PCT WO
94/29471).
Said nucleic acid may also include required sequences for intracellular transport, for replication and / or for integration, for the transcription and / or translation. Such sequences are well known from the man of art.
Furthermore, the nucleic acids which can be used according to the present invention can also be modified nucleic acids so that they don't is not possible to integrate into the genome of the target cell or acids nucleic stabilized with agents, such as for example spermine, which as that such have no effect on the efficiency of transfection.
According to one embodiment of the invention, the nucleic acid sequence is a naked DNA or RNA sequence, i.e. free of any compound facilitating his introduction into cells (nucleic acid sequence transfer).
However, according to a second embodiment of the invention, in order to promote its introduction in the target cells and in order to get the cells genetically modified from the invention, this nucleic acid sequence may be in the form of a vector, and more particularly in the form of a viral vector, such as for example a adenoviral vector, retroviral, a vector derived from a poxvirus, in particular derived from Vaccinia virus or Modified Virus Ankara (MVA) or a non-vector viral such that, for example, a vector consisting of at least one so-called acid sequence nucleic acid complexed or conjugated with at least one molecule or carrier substance selected from the group consisting of a cationic amphiphile, in particular a lipid cationic, a cationic or neutral polymer, a practical polar compound notably chosen among propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or their derivatives, and a polar aprotic compound in particular chosen among the dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethyl sulfone, the sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or their derivatives. The literature provides a large number of examples of such viral vectors and not viral.
Such vectors may further and preferably include elements of targeting that can direct the acid sequence transfer nucleic towards some cell types or certain specific tissues such as cells cytotoxic and cells presenters of the antigen. They can also be used to direct the transfer of a active substance towards certain preferred intracellular compartments such as the nucleus, the mitochondria or peroxisomes, for example. It can also be elements facilitating the penetration into the cell or lysis of compartments intracellular. Such targeting elements are widely described in the literature. He can by example be all or part of lectins, peptides, in particular the peptide JTS-1 (see request of PCT patent WO 94/40958), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands likely to react with an anti-ligand, fusogenic peptides, localization peptides nuclear, or of a composition of such compounds.
Use of cells transformed in vivo after injection of vectors containing at least one gene of therapeutic interest defined from protein of interest identified in the present invention (SEQ ID N 2, 4, 8, 9, 17, 24) and peptide sequences or the fragments of said sequences belonging to a even family of proteins chosen from perlecan, the precursor of the protein plasma binding to retinol, GM2 activator protein, calgranulin B and of the saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 at 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which present at least 70% identity preferably at least 80%

identity and preferably at least 98% identity with any one sequences peptides SEQ ID N 1 to 29.
The present invention relates to biological material for the preparation of pharmaceutical compositions for the prevention and treatment of affected mammals 5 of degenerative and / or neurological and / or autoimmune diseases, preferably sclerosis in plates, the composition comprising at least one vector containing a gene therapeutic as described below, capable of being introduced into a target cell in vivo and express the gene of therapeutic interest in vivo. The advantage of this invention is on the possibility to maintain over the long term a basal level of molecules expressed in the patient treated.
10 Vectors or nucleic acids encoding genes of interest therapeutic are injected.
These vectors and nucleic acids must be transported to cells targets and transfect these cells in which they are to be expressed in vivo.
The invention relates to the in vivo expression of nucleotide sequences and / or vectors as designated in the previous paragraph, i.e.
sequences 15 corresponding to genes of therapeutic interest coding in particular:
(i) at least for a protein chosen from the proteins identified in the present invention (SEQ ID N 2, 4, 8, 9, 17, 24) and the peptide sequences or the fragments said sequences belonging to the same family of proteins chosen from the perlecan, the plasma retinol binding protein precursor, protein activator of GM2, of 20 calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptide who have at least 70% identity, preferably at least 80% identity and advantageously at least 98% identity with any of the sequences peptide SEQ ID N 1 to 29, or their fragment (s), (I) either at least for all or part of a polyclonal antibody or monoclonal capable of binding to at least one protein chosen from protein identified in the present invention (SEQ ID N 2, 4, 8, 9, 17, 24) and the sequences peptides or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the plasma protein link Retinol, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ
ID N
at 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the sequences peptides which have at least 70% identity, preferably at least 80 %
identity and preferably at least 98% identity with any one of 5 peptide sequences SEQ ID N 1 to 29. They may be antibodies transmembrane native, or fragment or derivative of such an antibody, provided that said antibody, antibody fragment or derivative is expressed on the surface of the target cell of genetically modified mammal and in that said antibody is capable of attach to a polypeptide present on the surface of a cytotoxic effector cell or of a T helper lymphocyte and involved in the activation process of such cell. he can they are fragments of antibodies expressed by cells capable of secreting said antibodies in the bloodstream of a mammal or patient with cells genetically modified by the gene encoding the antibody, (ii) either at least for an inhibitor molecule of at least one protein chosen from the proteins identified (SEQ ID N 2, 4, 8, 9, 17, 24) and the sequences peptides or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the plasma protein link retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which exhibit minus 70%
identity preferably at least 80% identity and preferably at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29;
protein inhibitor of the function and / or metabolism and / or SEQ proteins ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or fragments thereof sequences belonging to the same family of proteins chosen from perlecan, precursor retinol-binding plasma protein, activator protein of GM2, of calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity in preference to less 80% identity and advantageously at least 98% identity with any of the SEQ peptide sequences ID N 1 to 29, (i) at least for a ligand or any part of the ligand capable of binding sure at least one protein chosen from the proteins identified (SEQ ID N 2, 4, 8, 9, 17, 24) and the peptide sequences or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the protein plasma binding retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ IDN 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ IDN 25 to 29) and the peptide sequences which have at least 70 % identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ IDN 1 to 29, and / or to inhibit its function.
According to a particular embodiment, it involves using therapy gene from way of directing the immune response against the protein, peptide or molecule of interest target, i.e. against any protein chosen from the proteins SEQ ID N
2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments of said sequences belonging to one family of proteins chosen from perlecan, the precursor of the protein plasma binding to retinol, the GM2 activator protein, calgranulin B and saposin B
(for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ

to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ IDN 1 to 29, their (s) fragment (s) and / or against any molecule that inhibits the function and / or the expressibn and / or the metabolism of said proteins of interest, and / or ligands of said proteins such as for example receptors.
For this it is obvious that the cells to be targeted for transformation with a vector are cells belonging to the immune system, i.e. lymphocyte-like cells (CD4 / CD8), or antigen presenting cells (dendritic cells, macrophages, ...).
According to a particular embodiment, genetically modified, in particular in vivo, the antigen presenting cells (APC). CPA like macrophages, dendritic cells, microgliocytes, astrocytes play a role in initiation of the immune response. They are the first components cell phones that capture the antigen, prime it in the cell and express molecules CMHI and CMHII
transmembrane involved in the presentation of the immunogen to cells T CD4 + and CD8 +, they produce specific accessory proteins which participate in activation of T cells (Debrick et al ;, 1991, J. Inununol 147: 2846; Reis et al., 1993, J
Ep Med 178:
509; Kovacsovics-bankowski et al., 1993, PNAS 90: 4942; Kovacsovics-bankowski et al., 1995 Science 267: 243; Svensson et al., 1997, J Immunol 158: 4229; Norbury et al .; 1997, Eur J Immunol 27: 280). For a vaccination, it may be advantageous to have a gene therapy system that can target gene transfer in such APC cells, that is to say a gene which codes for a polypeptide which can, after its intracellular production and its processing, be presented to CD8 + and / or CD4 + cells by molecules of CMHI and CMHII complexes respectively on the surface of these cells.
We choose to express all or part of the surface of CPA cells in vivo of a antibody and / or a ligand such as for example a receptor, capable of reacting with the protein or target peptide chosen from proteins SEQ ID N 2, 4, 8, 9, 17, 24 and peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to __retino e activator protein eanglioside GM2, calgranulin B
and saposin B
(for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ
ID. N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29. Such cells then go specifically phagocyte said protein or peptide, process it so that fragments of this peptide are presented on the surface of cells presenters of the antigen.
The literature offers a large number of examples of genes coding for antibodies capable of reacting with polypeptides or receptors. He is at the protected from those skilled in the art of obtaining the nucleic acid sequences encoding for such antibodies.
Let us quote for example the genes coding for the light and heavy chains of the antibody YTH 12.5 (anti-CD3) (Routledge et al. 1991, Eur J Immunol 21:
2717-2725), of anti-CD3 according to Arakawa et al; 1996, J. Biochem. 120: 657-662. The sequences acid nucleic acid such antibodies are easily identifiable from the bases of data commonly used by the profession. It is also possible to from hybridomas available from ATCC to clone nucleic acid sequences coding for heavy and / or light chains of these different antibodies by methods amplification such than RT-PCR using specific oligonucleotides or techniques implementing cDNA banks (Maniatis et al., 1982, Molecular cloning. A laboratory manual CSH
Laboratory, Cold Spring Harbor, New York). The sequences thus cloned are so available for cloning into vectors. According to a preferred case of the invention, the nucleic acid sequence encoding the heavy chain of the antibody is merged by homologous recombination with the nucleic acid sequence coding for a polypeptide transmembrane such as rabies glycoprotein or gpl60 (Polydefkis and al., 1990, J Exp Med 171: 875-887). These molecular biology techniques have been perfectly well described.
We choose to express fragments on the surface of CPA cells in vivo immunogens corresponding to at least one protein chosen from proteins SEQ ID N
2, 4, 8, 9, 17, 24 and the peptide sequences or fragments thereof belonging sequences to the same family of proteins chosen from perlecan, the precursor of protein plasma binding to retinol, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N
10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and preferably at minus 98%
of identity with any of the peptide sequences SEQ ID N 1 to 29.
For that, we can choose to have the vector express either the complete polypeptide or preferably polypeptides selected to react with ligands and / or receptors specific. The immunogenic peptide encoded by the polynucleotide introduced into the cell of the vertebrate in vivo can be produced and / or secreted, prepared and then presented to a cell presenter of antigen (APC) in the context of MHC molecules. APCs as well transferred in vivo induce an immune response directed against the immunogen expressed in vivo. CPAs have different mechanisms for capture them antigens: (a) capture of antigens by membrane receptors such as receivers immunoglobulins (Fc) or for the complement available on the surface of granulocytes, monocytes or macrophages allowing efficient delivery of the antigen in the 5 intracellular compartments after phagocytosis mediated by receptors.
(b) entry into APCs by pinocytosis in the fluid phase, involving different mechanisms:
micropinocytosis i.e. the capture of small vesicles (0.1 m) by the covered wells of clathrin and the macropinocytosis, i.e. the capture of larger vesicles (with a size varying ente 0.5 m and about 6 m) (Sallusto et al. 1995, J Exp Med 182: 389-400). While that the 10 micropinocytosis exists constitutively in all cells, the macropinocytosis is limited to cell types, such as macrophages, dendritic cells, astrocytes, epithelial cells stimulated by growth (Racoosin et al., J Cell Sci 1992, 102: 867-880). In this invention, by cells is meant able to macropinocytosis, the cells that can carry out the events described above above and them 15 cells which can capture macromolecules preferably between 0.5 pm and about 6 m in the cytoplasm.
According to a particular embodiment, genetically modified in particular in vivo, cytotoxic effector cells or helper T cells from so that they express on their surface a polypeptide corresponding to the proteins SEQ ID N
2, 4, 8, 9, 17, 20 24 and the peptide sequences or fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the protein plasma binding to retinol, the GM2 activator protein, calgranulin B and saposin B
(for example SEQ ID N 1, SEQ IDN 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID

to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity Preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, to ligands said proteins, naturally not expressed by these cells, and capable of inducing the process activation of such cells, by introducing into these cells acid sequences nucleic acid containing the gene encoding such a polypeptide. In accordance with the present 30 invention, it is also possible to select a nucleic acid sequence containing a gene of therapeutic interest encoding for everything or part of an antibody directed against a protein chosen from proteins SEQ ID N 2, 4, 8, 9, 17, 24 and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of plasma protein binding to retinol, GM2 activator protein, calgranulin B
and saposin B
(for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ

to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70% identity preferably at least 80% identity and advantageously at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, capable of to be expressed at the surface of the target cells of the patient to be treated, said antibody being able to attach to a polypeptide naturally not expressed by these effector cells cytotoxic or T helper lymphocytes.
By cytotoxic effector cells is meant the macrophages, the astrocytes, cytotoxic T lymphocytes (TCL) and killer cells (NK) as well as their derivatives such as for example LAK (Versteeg 1992 Immunology today 13: 244-247; Brittende et al 1996, Cancer 77: T226-1243). By 'helper T cells' we hear designate in particular the CD4, which allow after activation the secretion of factors activation of effector cells of the immune response. The polypeptides and in particular the receptors expressed on the surface of these cells and which are involved in activating such cells consist in particular of all or part of the TCR complex or CD3, all or part of CD8, CD4, CD28, LFA-1, 4-1BB complexes (Melero et al., 1998, Eur J Immunol 28:

1121), CD47, CD2, CDl, CD9, CD45, CD30, CD40, all or part of the receptors for cytolcines (Finke et al., 1998, Gene therapy 5: 31-39), such as IL-7, IL-4, IL-2, IL-15 or GM-CSF, all or part of the NK cell receptor complex such as for example NKAR, Nkp46, ..;
(Kawano et al., 1998 Immunology 95: 5690-5693; Pessino et al., 1998 J Exp Med188: 953-960), Nkp44, all or part of the macrophage receptors such as for example the receiver Fc (Deo et al., 1997, Immunology Today 18: 127-135).
Many tools have been developed to introduce different genes heterologous and / or vectors in cells, in particular cells of mammals. These techniques can be divided into two categories: the first category involves physical techniques like micro-injection, electroporation or bombardment of particles. The second category is based on the use of techniques in molecular and cellular biology with which the gene is transferred with a biological or synthetic vector which facilitates the introduction of material in the cell in vivo. Today, the most effective vectors are the viral vectors, in particular adenovirals and retrovirals. These viruses have natural properties to cross plasma membranes, avoid degradation of their genetic material and introduce their genome into the nucleus of the cell.
These viruses have been widely studied and some are already used experimentally in human applications in vaccination, immunotherapy, or to compensate for genetic deficiencies. However, this viral approach has limitations especially due to the limited cloning capacity in these viral genomes, the risk of disseminate viral particles produced in the body and the environment, risk of artefactual mutagenesis by insertion into the host cell in the case of retrovirus, and the possibility of inducing a strong inflammatory immune response in vivo during the treatment, which limits the number of possible injections (Mc Coy et al.
11995, Human Gene Therapy 6: 1553-1560; Yang et al., 1996 Immunity 1: 433-422). Others Alternative systems to these viral vectors exist. The use of methods no viral such as co-precipitation with calcium phosphate, the use of receptors that mimic viral systems (for a summary see Cotten and Wagner 1993, Current Opinion in Biotechnology, 4: 705-710), or the use of polymers such as polyamidoamines (Haensler and Szoka 1993, Bioconjugate Chem 4: 372-379). Other non-viral techniques are based on the use of liposomes whose effectiveness for the introduction of biological macromolecules such as DNA, the RNA of proteins or active pharmaceutical substances has been widely described in scientific literature. In this area, teams have proposed use cationic lipids before a strong affinity for cell membranes and / or nucleic acids. In fact, it has been shown that a nucleic acid molecule she-even could cross the plasma membrane of certain cells in vivo (WO
, 0 90/11092), the efficiency being dependent in particular on the nature polvanionic of nucleic acid. From 1989 (Felgner et al., Nature 337: 387-388) lipids cationic have been proposed to facilitate the introduction of large molecules anionics, which neutralizes the negative charges of these molecules and promotes their introduction into cells. Different teams have developed such cationic lipids:
DOTMA (Felgner et al., 1987, PNAS 84: 7413-7417), DOGS or TransfectamTM
(Behr and al., 1989, PNAS 86: 6982-6986), DMRIE and DORIE (Felgner et al., 1993 methods 5: 67-75), DC-CHOL (Gao and Huang 1991, BBRC 179: 280-285), DOTAPTM (McLachlan and al., 1995, Gene therapy 2: 674-622) or LipofectamineTM, and others molecules described in patents W09116024, W09514651, W09405624. Other groups have developed cationic polymers which facilitate the transfer of macromolecules into particular of anionic macromolecules in cells. Patent W095 / 24221 describes the use of dendritic polymers, document W096 / 02655 describes the use of polyethyleneimine or polypropyleneimine and the documents US-A-5595897.and FR 2719316.1 conjugates polylysine.
Since it is desired to obtain a targeted transformation in vivo towards a given cell type, it is obvious that the vector used must be able to be himself targeted, as described above.
Use of cells transformed in vitro or ex vivo with vectors containing a gene of therapeutic interest defined in relation to proteins of interest identified in the present invention (SEQ ID N 2, 4, 8, 9, 17, 24) and the sequences peptide or fragments of said sequences belonging to the same family of proteins chosen from the perlecan, the precursor of the plasma retinol binding protein, protein activator of GM2, calgranulin B and saposin B (for example SEQ
ID N 1, SEQ
ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and peptide sequences which have at least 70% identity in preference to minus 80%
identity and preferably at least 98% identity with any one sequences peptides SEQ ID N 1 to 29.
The present invention relates to biological material for the preparation of pharmaceutical compositions for the prevention and treatment of diseases degenerative and / or neurological and / or autoimmune, preferably sclerosis in plates, the composition comprising at least one cell, in particular a cell not not producing naturally antibodies, in a form allowing their administration in the organism of a mammal, human or animal, as well that eventually their prior culture, said cell being genetically modified in vitro by at least one nucleic acid sequence containing at least one gene coding in vivo for:
(i) at least one protein chosen from the proteins SEQ ID N 2, 4, 8, 9, 17, 24, and the peptide sequences or the fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of protein plasma binding to retinol, GM2 activator protein, calgranulin B and saposin B (for example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID
# 10 to 16, SEQ ID N 18 to 23, SEQ ID N 25 to 29) and the peptide sequences which present at least 70% identity preferably at least 80% identity and advantageously at minus 98% identity with any of the peptide sequences SEQ ID N
1 to 29 and the peptide sequences which have at least 70% identity of preference to at least 80% identity and advantageously at least 98% identity with one any peptide sequences SEQ ID N 1 to 29, and any fragment (ü) at least one peptide defined from the primary sequence of at least a protein chosen from the proteins SEQ ID N 2, 4, 8, 9, 17, 24, and the sequences peptides or the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 %
identity preferably at least 80% identity and preferably at least 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, (iii) at least any molecule that inhibits function and / or binding and / or the expression of these proteins, (iv) at least one peptide derived from the primary sequence of a selected protein among the proteins SEQ ID N 2, 4, 8, 9, 17, 24, and the peptide sequences where the fragments of said sequences belonging to the same family of proteins chosen from perlecan, the precursor of the plasma protein of connection to retinol, GM2 activator protein, calgranulin B and saposin B (by EXAMPLE SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 %
5 identity preferably at least 80% identity and preferably at minus 98%
identity with any of the peptide sequences SEQ ID N 1 to 29, and capable to bind to at least one CMHI glycoprotein, (v) at least any antibody and any part of antibody capable of binding to at least one protein chosen from proteins SEQ ID N 2, 4, 8, 9, 17, 24, and 10 peptide sequences or fragments of said sequences belonging to a same family of proteins chosen from perlecan, the precursor of the protein plasma binding retinol, GM2 activator protein, calgranulin B and saposin B (by example SEQ ID N 1, SEQ ID N 3, SEQ ID N 5 to 7, SEQ ID N 10 to 16, SEQ ID N
18 to 23, SEQ ID N 25 to 29) and the peptide sequences which have at least 70 %
15 identity preferably at least 80% identity and preferably at minus 98%
of identity with any of the peptide sequences SEQ ID N 1 to 29.
More particularly, said target cell comes either from the mammal to treat, or from a mammal other than the one to be treated. In the latter case, it should be noted that said target cell will have undergone a treatment making it compatible with the mammal to be treated.
By mammal is preferably meant a human mammal. These cells are established in cell lines and are preferably CMHII + or CMHII + -inducible like the lyrnphocytes, monocytes, astrocytes, oligodendrocytes.
The invention also relates to the modified cells and a method of preparation of a. cell as described above characterized in that we introduce in 25 a naturally occurring antibody-free mammalian cell, for example any means appropriate, at least one nucleic acid sequence containing at least one gene of interest therapeutic and elements ensuring the expression of said gene in said cell, said gene of therapeutic interest containing a nucleic acid sequence coding for a molecule or a fragment of a molecule in vivo, as described just above. More particularly, it relates to prokaryotic cells , cells yeast and animal cells, especially manuniferous cells transformed by at minus a nucleotide sequence and / or a vector as described above.
According to a particular embodiment, the cells (dendritic cells, macrophages, astrocytes, CD4 + T lymphocytes, CD8 + T lymphocytes) of the patient or allogenic are placed in contact with a purified preparation of the polypeptide target this one being internalized, primed and presented on the cell surface associated with CMHI molecules and / or CMHII and thus induce a specific immune response against the peptide.
Cells activated are then administered to the patient in whom they will induce answer immune specific for antigens (we use a natural response pathway inunune, but we controls what the antigen presenting cell will present) According to a particular embodiment, the presenting cells antigen (dendritic cell, macrophage, astrocytes) are modified in vitro to express them antigens in the transformed cell that will associate with the molecules of the CMHI and / or CMHII
and be presented on the surface of cells to induce in the patient where we administers the modified cell a perfectly targeted immune reaction.
Not all vaccine approaches are always satisfactory and drive for example to limited immune reactions directed only against epitopes immunodominant or against antigens with high variability. Of even the incorrect presentation of antigens by glycoproteins of the MHC system at the surface of does not allow the patient treated to develop anti-immunity protein of interest suitable. In order to overcome these problems, some authors have proposed in the framework of such vaccine procedures, to select minimal antigenic fragments corresponding to portions of peptide likely to be specifically recognized by T cells cytotoxic, to express them in cells so that they associate with CMHI molecules and be presented on the surface of cells to induce in the patient treated a reaction perfectly targeted immune system (Toes et al. 1997, PNAS 94: 14660-14665). More in particular, it has been shown that very small epitopes (varying from 7 to about 13 amino acids) which are expressed from introduced minigens in one vaccinia virus, could induce cell-type immunization. he a by elsewhere has been shown that several minigens could be expressed together with from the same vector (this particular construction is called string of beads). The advantage of such a construction is that it induces a reaction immune from synergistic CTL type (Whitton et al;, 1993 J. of Virology 67: 348-352).
Protocol for bringing the cells and the antigenic fragment into contact:
Presentation of antigenic fragments by CMHI molecules is based on an identified intracellular process (see Groettrup et al., 1996 Immunology 1 o Today 17: 429-435 for a review) during which peptides very antigenic short sizes (about 7-13 amino acids) are produced by degradation of a more complex polypeptide against which the final immune reaction will be directed. These cottrts peptides are then associated with CMHI or CMHII molecules to form a protein complex which is transported to the cell surface so to present said peptides to circulating cytotoxic T lymphocytes or to lymphocytes T
circulating helper, respectively. It should also be noted that the specificity of CMH I or CMH II molecules vis-à-vis the antigenic peptides varies in function of CMH I or CMH II molecules (example for CMHI: HLA-A, HLA-B, ...) and the allele (example for MHC I: HLA-A2, HLA-A3, HLA-A11) considered. Within 2o of the same animal species, from one individual to another, there is a large variability of genes coding for the molecules of the MHC system (on this subject, see in particular George et al., 1995, Immunology Today 16: 209-212).
According to a particular embodiment, the cells, 'such as the cells dendritics, macrophages, astrocytes, CD4 + T lymphocytes, CD8 + T lymphocytes are modified so as to express on their surface antibodies specific for the target peptide. The peptide is neutralized by antibody expressed on the surface of cells. These cells are preferably immune, of patient preference, preferably cytotoxic, modified to express all or part of an antibody specific for the target polypeptide.
Isolation of mononuclear cells from peripheral blood In 1968 Boyum described a rapid technique which allows centrifugation of blood on density gradient, to separate cells mononuclear (lymphocytes and monocytes) with a good yield (theoretical yield 50%, it is-i.e. 106 cells / ml of blood). 50 ml of peripheral blood collected sterile in heparinized tubes are centrifuged for 20 minutes at 150g at 20 C. The cells recovered are diluted in two volumes of initial peripheral blood of sterile PBS. 10 ml of this suspension are deposited on 3m1 of a Ficoll-Hypaque solution (medium of separation of lymphocytes, Flow). After centrifugation for 20 minutes at 400g and 20 C without deceleration braking, the mononuclear cells sediment at the PBS-Ficoll interface, in a dense, opalescent layer, while almost all of red blood cells and polymorphonuclear cells sediment at the bottom of the tube. The cells 10 mononuclear cells are recovered and washed in sterile PBS.
Internalization of antigens by cells presenting the antigen:
Preliminary treatment of antigen presenting cells: cells presenting the antigen are washed beforehand with a PBS-BSA buffer at 0.5% (w / v) then listed then they are preincubated in the presence of different 0.5% reduction inhibitors in PBS-BSA 0.5% containing 10 M to 10 mM
final DTNB (5,5'-dithio-bis-2-nitrobenzoic acid) or NEM (N-ethylmaleimide). The subsequent steps of fixing antigens to the surface cellular or internalization of antigens are also carried out in the presence of different inhibitor concentrations.
Protocol for internalization of antigens by cells presenting the antigen:
8.10 'cells are internalized in the presence of a saturated amount of 125 Iodine radiolabelled proteins (1 g) in microwells in 70 l.
After one incubation time at 4 ° C. with agitation, the antigens are fixed to the surface of cells. The cell suspension is washed twice with PBS-BSA and the caps cells are taken up in 70 l of buffer and incubated. at 37 C for different periods up to 2 hours. Cells and supernatants are separated by centrifugation at 800g for 5 minutes 4 C. For longer incubation periods, the stage pre-fixation of antigens on the cell surface is deleted. The cells are diluted in RPMI-10% FCS medium in the presence of 20 mM Hepes, at 10'iml cells. Cells are incubated in the presence of excess antigen while different periods at 37 C (1 gg of molecules / 5.10 'cells monocytes / macrophages or / 108 B-EBV cells).
All therapeutic agents defined within the framework of this invention are used to prevent and / or treat a degenerative disease and or neurological and / or autoimmune, such as multiple sclerosis, alone or in combination combination. They can also be used to assess their effectiveness in vitro or in vivo.
Administration of therapeutic agents in humans:
The biological material according to the invention can be administered in vivo 10 in particular in injectable form. We can also consider an injection by way epidermal, intravenous, intra-arterial, intramuscular, intracerebral by syringe or any other equivalent means. According to another embodiment by administration oral or any other means perfectly known to those skilled in the art and applicable to the present invention. Administration can take place as a single dose or repeated, one or more several times after a certain interval. Route of administration and the dosage the most suitable ones vary according to different parameters such as by example the individual or the disease to be treated, the stage and / or the evolution of the illness, or nucleic acid and / or protein and / or peptide and / or molecule and / or cell to transfer or target organ / tissue.
For the implementation of the treatment of the mammal mentioned in the present invention, it is possible to have compositions pharmaceutical containing biological material as previously described, advantageously associated with a pharmaceutically acceptable vehicle for administration to man or animal. The use of such supports is described in the literature (see for example Remington's Pharmaceutical Sciences 16 '''ed. 1980, Mack Publishing Co). This pharmaceutically acceptable vehicle is preferably isotonic, hypotonic or has low hypertonicity and has ionic strength relatively low, such as a sucrose solution, for example. Furthermore, said composition can contain solvents, aqueous or partially aqueous vehicles such as some water sterile, free of pyrogenic agents and dispersing media for example. The pH of these pharmaceutical compositions is suitably adjusted and buffered according to conventional techniques.

Figures FIG. 1 represents the amino acid sequence of the GM2AP protein, and the localization of the peptides, which is underlined, and which are used for the antibody production GM2AP anti-peptides.
FIG. 2 represents the amino acid sequence of the protein MRP14, and the localization of the peptides, which is underlined, and which are used for the antibody production anti-MRP peptides 14.
FIG. 3 represents the amino acid sequence of the protein Saposine B, and the localization of the peptides, which is underlined, and which are used for the production of 10 Saposin B anti-peptide antibodies FIG. 4 represents the assay of the protein MRP8 (ng / ml - on the ordinate) in the urine of patients with multiple sclerosis (MS), in the urine of patients with other neurological diseases (AMN) and in the urine of controls considered healthy (TS). not means the number of urines tested by category.
FIG. 5 represents the assay of the protein MRP 14 (ng / ml - in ordered) in the urine of patients with multiple sclerosis (MS), patient urine other neurological diseases (AMN) and in control urine considered healthy (TS). n means the number of urines tested by category.
FIG. 6 represents the assay of the protein MRP8 / 14 (ng / ml - on the ordinate) 20 in the urine of patients with multiple sclerosis (MS) patient urine other neurological diseases (AMN) and in control urine considered healthy (TS). n means the number of urines tested by category.
FIG. 7 represents the average concentrations of the MRP8 proteins, MRP14, MRP8 / 14 (ng / ml - on the ordinate) in the urine of patients with sclerosis in 25 plaques (MS), in the urine of patients with other diseases neurological (AMN) and in the urine of controls considered healthy (TS). n means the number urine tested by category..
FIG. 8 represents the assay of the GM2AP protein (ng / ml - in prescribed) in the urine of patients with multiple sclerosis (MS), in the urine 30 from patients with other neurological diseases (AMN) and in witness urine considered healthy (TS). n means the number of urines tested by category.

FIG. 9 represents the assay of the protein Saposine B (g / ml - in ordered) in the urine of patients with multiple sclerosis (MS), patient urine other neurological diseases (AMN) and in control urine considered healthy (TS). n means the number of urines tested by category.
FIG. 10 represents the co-detection of the Saposin B proteins (g / ml - in order) and GM2AP (ng / ml - abscissa) in urine samples from MS patients, from supposed healthy witnesses and patients with other neurological diseases and the correlation observed between the levels of the two proteins.
FIG. 11 represents: FIG. 11A, the assay of the GM2AP protein in ng / ml in the urine of a progressive remitting MS patient (curve claire) and the gliotoxicity as a percentage of dead cells estimated by the MTT test (dark curve);
FIG. 11B the assay of the protein Saposine B in g / ml in the urine of a MS patient in progressive remitting form (clear curve) and gliotoxicity in percentage cells dead estimated by the MTT test (dark curve).
FIG. 12 represents the product of the concentrations of the GM2AP proteins and saposin B in ngx g / m12 in the urine of a relapsing-remitting MS patient progressive (clear curve) and gliotoxicity as a percentage of dead cells estimated by the MTT test (dark curve).
FIG. 13: FIG. 13A, the assay of the GM2AP protein in ng / ml in the urine from a progressive MS patient (clear curve) and the gliotoxicity in percentage dead cells estimated by the MTT test (dark curve); figure 13B on dosage of Saposin B protein in g / ml in the urine of a fit MS patient progressive (curve clear) and gliotoxicity as a percentage of dead cells estimated by the MTT test (curve dark).
FIG. 14 represents the product of the concentrations of the GM2AP proteins and saposin B in ngx g / m12 in the urine of a progressive MS patient (clear curve) and gliotoxicity as a percentage of dead cells estimated by the MTT test (dark curve).

Figure 15 shows the correlation between GM2AP concentrations in ng / ml (abscissa) and gliotoxicity in percentage of dead cells estimated by the MTT test (ordered) determined in the urine of MS patients and controls.
Figure 16 shows the correlation between Saposin concentrations B in g / ml (abscissa) and gliotoxicity in percentage of dead cells estimated by the MTT test (ordered) determined in the urine of MS patients and witnesses.
Figure 17 shows the correlation between the product of the concentrations GM2AP and Saposin B in ngx g / ml '(abscissa) and gliotoxicity in percentage of dead cells estimated by the MTT test (ordered) determined in urine from to MS and control patients.
Figure 18 shows the correlation between GM2AP concentrations (ng / ml - on the y-axis), the concentrations of Saposin B (g / ml -orderly right) and gliotoxicity as a percentage of dead cells estimated by the MTT test (abscissa). Two estimated correlation lines are shown on the graph. The bold lines relate to saposin B concentrations; the lines in light black relate to GM2AP concentrations.

Examples:

Example I: Collection and pool of urine.
Urine samples of different volumes were taken from healthy individuals (negative MS) with no neurological disease a priori or self-immune. The toxic activity of each sample with respect to cells astrocytic murines was tested in vitro using the MTT test. In total a pool of 20 liters urine was constituted (negative SEP pool). At the same time, samples urine from different volumes were taken from individuals with sclerosis in plates (MS positive) at different stages of the disease. The ticking activity of each sample taken from murine astrocytic cells has been tested in vitro using the MTT test. A total of 80 liters of urine was pooled (SEP pool positive).

Example 2: Purification of urinary proteins.

SEP positive and SEP negative urine pools, collected and tested according to exeinple 1, have been purified to obtain a protein concentration high and eliminate high molecular weight proteins as much as possible.
Precipitation: precipitation with ammonium sulphate (Prolabo - ref. 21 333 365) were performed on the SEP positive and SEP negative urine pools.
The 60% percentage of. saturated ammonium sulphate for 40% urine, i.e. 390 grams of ammonium sulfate per liter of urine was used. Each pool is distributed in 1.8 liter fractions in 2 liter bottles to improve precipitation. The precipitation was carried out for 2 x 8 hours, at room temperature, under agitation sweet. After centrifuging the urine pools at 3000 rpm for 10 min., At a temperature of 10 C, the pellet obtained is taken up in a 20 mM Tris buffer containing CaCl, 1 mM and 0.25 M urea. The mixture was then centrifuged at 3 000 rpm for 10 min. The supernatant contains the concentrated proteins. He is either used immediately for the next step, or frozen if the next step cannot to be performed continuously.
Ion exchange chromatography: the protein-containing solution has then passed over a DEAE fast Flow gel (marketed by PHARMACIA).
This step is carried out at low pressure on a PHARMACIA column filled of gel. The pads are brought to the column by a peristaltic pump which allows a regular flow. The column equilibration buffer is the Tris 20 buffer mM, pH
7. The fraction corresponding to the precipitation supernatant and containing a number of too high salt is dialyzed against this buffer before depositing on the column. A
elution by a salt gradient makes it possible to recover the proteins. The elution gradient East carried out in steps of NaCI 100, 200, 300, 500 mM in the buffer balancing the column. The elution fractions are tested by the MTT test and will not be preserved as the positive fractions, that is the fraction eluted at 200 Mm NaCl. These fractions may be processed immediately or stored in a freeze-dried state.
Purification: A size exclusion chromatography based on the difference in size and shape of the proteins to be eluted was used. The fraction corresponding to the 200 mM NaCl elution is deposited on the column. During elution, low molecular weight proteins are retained and therefore eluted more late than large molecules. The purifications have been carried out on HPLC

with a TosoHaas TSK Prep G 3000 SW column, with a diameter of 21.5 mm and of a length of 300 mm, the molecular weight exclusion limit is 500,000 daltons. The elution buffer used contains 100 mM phosphate, sulfate of 100 mM sodium, pH 6.8. Separation of the protein mixture has been carried out in 60 min. Only the fraction corresponding to a mass of 15-20,000 daltons has been preserved.
This fraction is dialyzed in a 20 mM Tris buffer containing CaCl, 0.2 mM, pH
7.2, then lyophilized.
At each stage, only the fractions exhibiting toxic activity have been selected for the next step. A control of toxic activity 1o of proteins was carried out at each step, using the MTT test. Alone fractions with significant toxic activity were selected for the additional purification described in Example 3.

EXAMPLE 3 Additional Purification of the Urinary Proteins by reverse phase chromatography.
Urine pools from MS patients (MS pool positive) and non-MS patients (negative MS pool), obtained after purification according to example 2, have was taken up in distilled water, then diluted with a 0.2% TFA / 10% solution acetonitrile to obtain a final concentration of approximately 130 to 140 g / ml.
Separation by reverse phase C8 HPLC was carried out on a column Brownlee Aquapore (trade name) marketed by Perkin Elmer (column characteristics: 300 angstroms / 7 m / (100x4.6) mm). Of them columns separate were used for the positive and negative pools, respectively.
The injections were made by multi-injections of 250 l. Proteins have been elected with a linear gradient of 5% to 15% of buffer B in 5 min., then from 15% to 100% of buffer B in 95 min., at a flow rate of 0.5 ml / min. Separation buffers A and B
used respectively are the 0.1% TFA buffer (Pierce n 28904) / Mi11iQ water and the 0.09% TFA / 80% acetonitrile buffer (Baker). Detection was performed by measured absorbance W at 205 and 280 nm. The fractions have been collected in 1.5 ml and 0.5-1 ml fractions in the area of interest. The fractions have been frozen after collection in dry ice.

The collected fractions were then dried under vacuum and taken up in 100 l 0.1% TFA / 30% acetonitrile. 20 1 of the fractions were transferred to eppendorfs from 500 l, dried and washed twice with 100 l of MilliQ water, then dried again.
The toxic activity of the proteins contained in each fraction collected after 5 elution was determined using the MTT test. Only fraction 21 presenting an activity significant toxic was retained. The number of this fraction corresponds to the order of elution according to the elution conditions set out in this example.

Example 4 Analysis of Proteins Obtained by Separation on HPLC on Gel 10 SDS-TRICINE.
The collection pool for fraction 21 obtained by HPLC, as described in Example 3, and originating from 20 injections of the positive SEP pool, was deposited on an SDS gel-TRICIN 16% precast from 10 wells and 1 mm thick (sold by the society Novex). The conditions of use of the gel correspond to those recommended speak 15 supplier. The sample is taken up in 75 g of sample buffer 1 once concentrated (SDS-TRICINE N LC 1676, 1 ml twice concentrated + 50 1 of (3-mercaptoethanol (Pierce) diluted 1/2 in water) and 251i1 of the sample are placed on the gel in thrice. The pool of collection of fraction 21 from 6 injections of the negative SEP pool was deposited on the gel under the same conditions as those described for the positive SEP pool. The migration on 20 two gels were made in parallel in the same migration tank (XCELL II NOVEX
(trade name)) at a constant voltage of 125 mV for 2 hours. Tank is placed in a container with ice. The gels were stained directly after migration by staining with zinc / imidazole (staining kit 161-0440 sold by the society BIORAD) to obtain a reversible negative coloring. The strips of proteins are 25 translucent on opaque background.

Example 5: Trypsin digestion of the gel bands.
All the protein bands visualized in the deposits of fraction 21 have were cut and subjected to proteolysis with trypsin.

The gel strips are cut with a scalpel into 1 mm slices and transferred to eppendorf tubes. The eppendorfs are subject to a peak of centrifugation to drop the gel pieces and after centrifugation 100 gl of washing buffer (100 Mm NHIC03 / 50% CH3CN) are added to the gel pieces.
After 30 min. stirring at room temperature, the supernatant is removed by 20 l fractions and the washing step is repeated twice. The eppendorfs are dried for 5 min. in speed vac. 20 g of trypsin (Modified sequenal grade PROMEGA V5111) are taken up in 200 g of digestion buffer (5 mM TRIS, pH
8) and are dissolved for 30 min. at room temperature, with stirring intermittent and 20 to 30 l of resuspended trypsin are added to the gel pieces. The eppendorfs are centrifuged and stored in a hot room at 28 C overnight. After digestion gel strips can be used immediately for measures of mass or frozen for later use.

Example 6: Chemical digestion at CNBR of the gel bands.
In the event of a protein resistant to enzymatic cleavages, in particular to the action of trypsin as described in Example 5, the bands between 16kD and 20kD were processed with CNBR. The gel bands, already used for the digests with trypsin, are dried 5 to 10 min. in speed vac.
A solution of CNBR (FLUKA) at 200 mg / ml was prepared in 70% acid formic (BAKER). 20 l of this solution was used to rehydrate the pieces of gel. The reaction was carried out for 20 h at room temperature and to darkness. The peptides are extracted 3 times 30 min. with 100 gl of 0.1%
VAT / 60%
Acetonitrile. The extraction solutions are combined and concentrated to 20 l.
These samples are diluted 5 times in 0.1% TFA / water. The conditions of separation are those described for the peptides of digestion with trypsin.

Example 7: Analysis by MALDI-TOF spectrometry.
l of extraction buffer (2% TFA / 50% acetronitrile) are added 30 to samples. The eppendorfs to be analyzed are subjected to a 5 min cenirifugation, then a 5 min sonication. and finally to a centrifugation of 1 min.

On a stainless steel disc, 14 deposits of 0.5 l of matrix (acid a-cyano-4-hydroxy-trans-cinnamic to saturation in acetone) are produced. A fine microcrystalline layer "
uniform is obtained. 0.5 l of a 2% TFA / water solution are deposited on this sub-layer on the 14 deposits, then 0.5 l of sample to be analyzed are added.
In this drop thus formed, 0.5 l of an a-cyano-4- acid acid saturation solution hydroxy-trans-cinnamic in 50% acetonitrile / water are added. After drying at ambient temperature for 30 min., the crystal deposits are washed with 2 l of water which are at once evacuated by a breath of air. All spectra are obtained on a mass spectrometer BRUKER BIFLEX (trademark) equipped with a reflectron. The measures (90 to 120 shots laser on the entire deposit) are accumulated to obtain a spectrum of mass which is the more representative of all the peptides present in the sandwich sample matrix.
For each deposit, a calibration with the peptides of the autolysis of the trypsin was made so to be able to use a measurement accuracy of less than 100 ppm.
Databases searches were performed in MS-FIT
PROTEINPROSPECTOR. The common parameters used in this research are (1) database: NCBInr, (2) a tolerance of 100-50 ppm, (3) cysteines are not modified, (4) methionines can be oxidized, (5) weight range molecular: 1000-100,000 Da, (6) up to 3 switching sites can be ignored.

Example 8: N-terminal sequencing of digestion peptides.
(i) Extraction and separation by HPLC of the digestion peptides.
After mass measurements on the entire digestion, the rest of the peptides is extracted in 3 times 30 min. in a sonication bath with 0.1% TFA / 60%
acetonitrile.
The extraction solutions are combined and dried to 20 l under vacuum.
After dilution in 80 l of buffer A (0.1% TFA / water), the extractions of the bands gel, digested with trypsin, are injected into a C18 / MZ- column Vydac / (125x1.6) mm / 5 m. The elution of the peptides is done at a rate of 150 l / min. and in a gradient ranging from 5% buffer B (0.09% TFA / 80% acetronitrile) to 40% of buffer B in 40 min., then from 40% buffer B to 100% buffer B in 10 min.
The detection is made by measuring the UV absorbance at 205 nm. Collecting peaks is carried out in 500 l eppendorf tubes. Fractions are kept on the ice and for the 18-20 kD band of the positive SEP 21 pool analyzed by spectrometry of MALDI-TOF mass.
(ü) N-terminal sequencing.
Fractions corresponding to only one mass peak were analyzed by Edman degradation on a sequencer (Model 477A PERKIN ELMER / Applied Biosystems). The sequencing conditions are those described by the constructor. A
micro cartridge was used for depositing samples and PTH-AminoAcid to are identified with an online HPLC system (Model 120A PERKIN
ELMER / Applied Biosystems).
The deposit of the fraction to be sequenced was done in several deposits of 15 gl with intermediate drying. The tube containing the peptide is washed with 15 l 85% formic acid (BAKER). The amino acid sequences correspond always at the measured masses. Peptides, whose masses do not correspond not at the main protein identified, were sequenced in priority. Of this way it was possible to identify up to three proteins in a gel strip.

Example 9: Results and discussion.
After reverse HPLC of the negative SEP control pool and of the positive SEP pool, the toxic activity of each elution fraction was determined using the MTT test.
Only fraction 21 of the positive SEP pool exhibits toxic activity in vitro. The fraction 21 of the negative SEP control pool shows no toxic activity.
The activity toxic of fraction 21 of the positive SEP pool was confirmed in vitro by FACS, like described in patent application WO 98/11439 on astrocytic cells murines.
The protein content of fraction 21 of the negative SEP control pool and of the positive MS pool was observed after separation on SDS-TRICINE 16% gel and staining of the zinc / imidazole gel. Molecular weight proteins apparent were found in both fractions. By cons five bands different and from low apparent molecular weights are only visible in fraction 21 from the pool MS positive (bands 8, 14, 18 and 20 kD). Each band corresponds at least a protein and variants of said proteins which have molecular weight apparently close that of the native protein. These variant sequences present a percentage of homology or identity with the native sequences of at least 70%, preferably at least minus 80% and advantageously at least 98%.
The proteins of interest of fraction 21 of the positive SEP pool were then analyzed by mass spectrometry and / or sequencing and search for homology in the databases. The results show the presence of five bands of migrating protein between 22 and 5 kD in fraction 21 of the positive SEP pool and variants of said proteins.
These proteins are the C-terminal fragment of Perlecan, which begins with acid amino 3464 and ends at amino acid 3707 of the protein sequence complete, identified in the sequence identifier SEQ ID N 2, the precursor of the protein plasma binding to retinol whose sequence is given in SEQ ID N 4, the protein activator of GM2 identified in SEQ ID N 8, calgranulin B identified in SEQ ID N 17 and the saposin B
represented in SEQ ID N 24. As described above, homologs or variants said proteins have also been identified by sequencing. These sequences homologous proteins or variants are the product of mutations in genes encoding said proteins. AT
As an example, SEQ ID N 9 has 99% homology or identity with of the GM2 activator protein and the fragment of SEQ ID N 9 which begins to acid amino 34 and ends at amino acid 202 has 98.88% homology or identity with the corresponding fragment of the native protein identified in SEQ ID N 8.
Example 10: Demonstration of proteins in a urine sample.
Urine samples from an MS negative individual and a patient Positive MS have been collected. These urine samples were purified according to the protocol described previously.
The final elution fractions 21 were analyzed separately by mass spectrometry. The mass profile of each fraction corresponding to each urine sample was compared the mass profile obtained for the proteins identified in the examples previous. The results show that for the urine sample from the MS patient positive the masses correspond to the molecules (i) C-terminal fragment of Perlecan, (ii) activator protein of GM2, (iii) calgranulin B and (iv) saposin B
previously identified.
However, none of these masses has been identified in the mass profile obtained after analysis of the urine sample from the individual MS negative. The process described is usable as a diagnostic test.

Example 11: Western Blot test.
Western blots were carried out on different fractions of raw urine or purified as described in Example 2. Urine samples from healthy individuals and patients with multiple sclerosis are being tested in parallel. The samples are 10 deposited on an electrophoresis gel to separate the different protein based of their molecular mass under the action of an electric field. Westerners Blot are made after transfer of the proteins from the gel to a membrane. To reveal the transferred proteins, the membrane is saturated in saturation buffer, then incubated with an antibody directly labeled with alkaline phosphatase. The antibody used is an anti-calgranulin 15 (mouse monoclonal antibody, clone CF 145 subtype IgG 2b marketed by the company Valbiotech: reference MAS 696p lot PC96G696). The substrate of the enzyme is dichloride 3.3 '- (1,1'-biphenyl) 4,4'diazonium and sodium 2-naphthalenyl phosphate (marketed under the name (3 Naphtyl acid phosphate Sigma ref. N7375 and ô dianisine Tetrazotized D3502) is added for the revelation of the bands and the visualization of the proteins related to 20 antibody. A molecule with an apparent molecular mass of approximately 14,000 Da is revealed in purified urine from MS patients, with a signal relatively intense. This protein corresponds to calgranulin B (apparent molecular mass: 14 kD).
On the other hand, no signal is observed from the urine of healthy individuals. This observation confines the presence of this protein specifically in the urine of patients with of SEP and the setting 25 implementing a detection method using an antibody recognizing the protein.

Example 12: Production of monoclonal antibodies.
The production of monoclonal antibodies by ascites requires compatibility of the H-2 system between the hybridoma and the producing mouse. 20 female mice Balb / c, aged 6 30 weeks, undergo an injection of 0.5ml of Pristane (2-6 10-14 tetramethylpentadecane acid) in their peritoneal cavity, for the ascites production (Porter et al., 1972). One week to 10 days later, 5.106 to 10.106 hybridomas diluted in 0.5ml sterile buffer NaCI 0.145M, NaZHPO410 mM, KC1 2.7 mM, KH2PO4 1.5 mM at pH
7.4 are injected intraperitoneally. Ascites appears one by two weeks later. The ascites liquids present in the peritoneal cavity are then collected with a syringe after incision of the peritoneum. The collected liquid is centrifuged at 3000g for 15 minutes at room temperature, filtered through gauze to remove fat, then buffered adding 1/20 ' of its volume of tris-HCI 1M at pH 8Ø This method makes it possible to obtain quantities antibodies 10 times higher than those obtained by hybridoma culture.
The immunoglobulins present in the ascites fluid are released by salts (sulfate ammonium or sodium sulfate). The ascites liquid is precipitated by the sulfate 40% ammonium. After 20 minutes in the cold the solution is centrifuged 15 8000g minutes to 4 C. The precipitate is washed and resuspended cold in a sulfate solution 40% ammonium then centrifuged again. The new precipitate enriched in IgG is put back in solution in PBS buffer and dialyzed overnight against 25 mM Tris-HC1 buffer, 150 mM NaC1 pH
7.4.
In parallel, an agarose-Protein A (or protein G) column (sold form lyophilized, Pierce) is washed extensively with 25 mM Tris-HCl buffer, NaCI 150mM
pH7.4. The solution enriched in IgG is deposited on the column then the column is washed. IgG
retained by the column are eluted at acid pH (glycine 200 mM pH 2.8). The eluted fractions are neutralized with a volume of Tris-Base 1 M pH 10.5. The content in immunoglobulins of each fraction collected is quantified by reading absorbance at 280 nm (e 1%, lcm = 14.0 Prahl and Porter 1968). The rich fractions are pooled. The degree of purification of IgGs pooled is analyzed by acrylamide gel electrophoresis in the presence of SDS.
IgGs purified are dialyzed overnight against the 25 mM Tris-HCl buffer, 150 mM NaCl pH7.4, sterile filtered, aliquoted and stored at -20 C. Their concentration final is determined by reading the absorbance at 280 nm or by micro-BCA assay. Peptides immunogens referenced SEQ ID N 58, SEQ ID N 54, SEQ ID N 55, SEQ ID N 56, SEQ ID N
57, SEQ
ID N 58, SEQ ID N 59 and SEQ ID N 65 were used for production antibody monoclonal, according to the protocol described above. But, it is within reach of the man of profession to define other protocols for the production of antibodies monoclonal, by example from the techniques described by Kôhler and Milstein and by Galfre G. et al.
previously cited or techniques derived from them.
Production of recombinant proteins and polyclonal antibodies and monoclonals.
Recombinant proteins Recombinant proteins GM2AP (SEQ ID NO: 73) and Saposin B
(SEQ ID NO: 74) used to carry out the standard range of this study were produced in a prokaryotic system and purified from the clones of these two protein obtained in our laboratory using the methods and protocols well known to one skilled in the art.
Anti-GM2AP or anti-Saposin B antibodies Anti-GM2AP or anti-Saposin B antibodies used to make the study were either produced in our laboratory or donated generously.
Anti-Saposin B and anti-GM2AP polyclonal antibodies (Li et al, Glycoconjugate, 1984) were used for the study (see the examples below): they are denounced SAP84 and GM2AP84.
Anti-GM2AP or anti-Saposin B polyclonal antibodies have been produced and purified in the laboratory using protocols and methods well known to those skilled in the art: 50 g of GM2 protein or prokaryotic Saposin B
purchased were injected into rabbits on days OJ, D28 and D56; of them booster shots were performed once a month for two consecutive months. Both antibody anti-GM2AP polyclonal antibodies and two anti-Saposin B polyclonal antibodies have summer obtained and their specificity with respect to the recombinant protein was verified by Western blot and by Elisa.

Polyclonal antibodies against GM2AP or Saposin B peptides have been produced and purified in the laboratory using protocols and methods well known to those skilled in the art; 75 g of defined GM2AP or Saposin B peptides, products and coupled with KLH in our laboratory were injected daily OJ, J28 and J56; several boosts were made once a month for 5 months consecutive with injection of 75 g each time. Four anti-polyclonal antibodies peptides GM2AP, four polyclonal anti-Saposin B peptides and four antibody polyclonal rabbits anti-peptides MRP 14 were thus obtained and their specificity with respect to the recombinant protein was checked by Western blot and by Elisa. The peptide sequence GM2AP, Saposin B and MRP14 chosen are described in Figures 1 to 3.
It was obtained:
- an anti-mixture antibody of two peptides of 13 and 15 amino acids of GM2AP: 189-190; an anti-peptide antibody of 18 amino acids from GM2AP: 191-192 (cf.
Figure 1), - an anti-mixture antibody of two peptides of 13 and 19 amino acids of MRP14: 193; an anti-peptide antibody of 17 amino acids of MRP14: 195-196 (cf. Figure 2), - an anti-mixture antibody of three peptides of 12, 15 and 15 amino acids of Saposin B: 74-75; another anti-mixing antibody of 3 peptides of 12, 15 and 15 acids Saposin B amines: 72-73 (see Figure 3).
Native anti-fraction monoclonal antibodies were produced and purified in the laboratory using well-known protocols and methods art. The native fraction corresponds to the cytotoxic elution fraction obtained at from the pool of 80 liters of urine from MS patients and after purification. It's the last elution fraction which contains the three proteins GM2AP, Saposine B, MRP14. 30 g of this fraction of purification were injected into three mice on days OJ, D14, D28 and the sample has been taken at D38. After screening and cell fusion, protocols known to man art for establishing hybridomas and monoclonal antibodies, hybridomas have been re-injected at the mouse and the ascites fluid were recovered 10 days later. Antibodies have been purified on Sepharose-Protein A column and the specificity with respect to the fraction used for the immunization was checked by Western blot and by Elisa. So four antibody monoclonals were obtained: 19C1A7, 3D3F9, 18C8C5 and 7D12A8.
EXAMPLE 13 Determination of the MRP14 Proteins in the Urine by Technique ELISA.
The proteins MRP14, MRP8 and the heterocomplex MRP8 / 14 were assayed in human urine using (i) either an Elisa assay technique according to the known method of the art and using anti-MRP antibodies described in previous examples; (ii) either the 'MRP Enzyme Immunoassay' kit marketed by BMA
Biomedicals AG, Augst, Switzerland, using the antibodies in the kit, the protocol being carried out according to the kit instructions.
Detection of MRP14 and MRP8 / 14 in urine.
The assays were carried out from 17 urines of individuals from the population active (TS), from 27 urines from multiple sclerosis (MS) patients and 7 urine patients with other neurological diseases (AMN).
- Figure 4 illustrates the levels of MRP8 assayed in this urine: while the MRP8 concentration is almost zero in AMN urine, there is no really from difference in distribution between TS and MS urine. Note however that the differences observed are almost negligible because the concentrations measured are extremely weak.
- Figure 5 illustrates the levels of MRP14 measured in the same urine: then that he there are not really any differences in the distribution of concentrations between the TS urine and AMN, the concentrations are higher in some MS urine.
- Figure 6 illustrates the MRP8 / 14 heterodimer rates measured in the same urine: while there is not really a difference between the concentrations TS urine and AMN, we observe higher concentrations in some MS urine, corresponding perhaps to a subpopulation of MS patients characterized by an activity of the disease.
MRRP8 / 14 measured in the urine is a marker for MS disease activity (characterized by an inflammation peak).
- Figure 7 summary confirms that there is no difference significant of concentration of MRP8 and MRP14 between TS, AMN and MS urine, then that a weak difference in MRP8 / 14 concentration is observed between these urines, this concentration being higher on average in MS urine and being a marker for the activity of the illness (peak inflammation).

Example 14: ELISA Protocols Used for the Assay of the GM2AP and Proteins Saposin B.
The GM2AP or Saposin B proteins were measured in human urine in using anti-GM2AP or anti-Saposin B polyclonal antibodies in following the Elisa protocol described by Gardas et al. (Glycoconjugate Journal 1, 37-42, 1984). The main steps are briefly described below:
At each step, the wells of a 96-well microplate are filled with 200 l of the designated solution. The wells are first covered with a solution from GM2AP
5 (recombinant prokaryotic protein) diluted to 50 ng / ml in buffer carbonate-bicarbonate, pH 9.6. After incubation overnight at 4 ° C., the solution is eliminated and the wells are washed four times with PBS pH 7.4 buffer containing 0.05% Tween-20 (PBS-Tween). The microplates thus coated are stored at 4 C for approximately 2 weeks.
Urine samples at three different dilutions (20x, 40x and 80x or others 10 appropriate dilutions) are incubated with an appropriate dilution of the polyclonal antibody of anti-GM2AP or anti-Saposin B rabbit overnight at 4 C. A series of standard dilutions a recombinant protein ranging from 2.0 to 62.5 ng / ml is used for realize the range standard and are treated the same. All dilutions are made in PBS-Tween buffer containing 1 mg / ml ovalbumin. So 0.2 ml of each incubated solution is added in 15 of the wells covered in duplicate and the plates are left for 2 hours at temperature ambient. The wells are then washed four times in PBS-Tween and filled still with a solution of goat anti rabbit IgG antibodies coupled to peroxidase and diluted around 1200 time. After a 2 hour incubation at room temperature, the wells are washed four times in PBS-Tween and again filled with the staining reagent. The reagent of coloring 20 consists of 100 mg of 2,2'-azino-di- (3-ethylbenzothiazoline) acid sulfonic and 10 1 of 30%
of hydrogen peroxide for one hour at room temperature and the degree coloring of each microwell is estimated by reading the absorbance at 405 nm.
A standard curve is constructed by abscissa the concentration of GM2AP of the standard range or of Saposin B with a logarithmic scale and in ordered on 25 percent absorbance with a linear scale. The percentage absorbance of the sample is the absorbance ratio between the urine sample and the control which contains only the antiserum, without the soluble antigen.
A GM2AP recombinant protein solution produced in a prokaryotic system, and concentration 3 mg / ml is diluted in 50 mM carbonate buffer, pH 9.6 and 50 1 are 30 added to each well of a 96-well microplate, i.e. 50 1 per well of a 0.5 g / ml solution. The plates thus prepared are incubated overnight at room temperature, The anti-GM2AP polyclonal antibody produced in the laboratory (rabbit 79) was purified and diluted in 0.05% PBS-Tween buffer in the presence of serum horse 10%. This solution is diluted to 1/8000 '"u The solution is used to realize a standard range with 8 points of range covering concentrations from 0 to 500 ng / ml. Pre-incubation is carried out overnight at room temperature between 100 l of antibody and 100 l of urine sample to be assayed or protein solution GM2AP or Saposin B recombinant used for the standard range. After washing the microplate in PBS-Tween, 50 l of the incubation mixture are added per well, then lo incubated for two hours at room temperature. The microplate is new washed in PBS-Tween, then 50 l of rabbit anti-IgG antibody coupled to the peroxidase and diluted 1/5000 are added to each microwell of the plate and incubated while two hours at room temperature. After further washes of the microplate, 100 l of OPD are added to each well and incubated for 20 minutes at ambient temperature. The coloring of - each well, proportional to the concentration of GM2AP or Saposin B recognized by the specific antibody used, is estimated by reading absorbance.

A GM2AP or Saposin B recombinant protein solution produced in prokaryotic system, and concentration 3 mg / ml is diluted in buffer carbonate 50 mM, pH 9.6 and 501il are added to each well of a 96 microplate well either 50 1 per well of a 1.5 g / ml solution. The plates thus prepared are incubated overnight at room temperature. GM2AP anti-peptide polyclonal antibodies purified laboratory products (rabbit 190 and rabbit 191) are used alone or in mixture diluted to 1/1000 for each in 0.05% PBS-Tween buffer in the presence of horse serum 10%. The standard range is carried out using protein GM2AP or Saposin B prokaryotic recombinant diluted to cover the range concentration 0 to 1500 ng / ml with 8 points. 100 l of antibody (one antibodies or two together) are pre-incubated in the presence of 100 l of urine sample to test or GM2AP or recombinant Saposin B solution, overnight at temperature ambient. After washing the microplate in PBS-Tween, 50 l of the mixture incubation is added per well and then incubated for two hours at temperature ambient. The microplate is again washed in PBS-Tween, then 50 l anti-IgG antibodies of rabbit coupled with peroxidase, diluted to 1/5000, are added to each microwells of the plate and incubated for two hours at room temperature. After washing the microplate, 100 1 of OPD are added to each well and incubated for 20 minutes to ambient temperature. The coloring of each well, proportional to the concentration of GM2AP or Saposin B recognized by the specific antibody used, is estimated by reading absorbance.

Example 15: Determination of GM2AP proteins in the urine.
The GM2AP protein was measured in the urine of 22 patients with multiple sclerosis (MS), 5 patients with other neurological diseases (AMN) and 9 individuals, controls considered healthy (TS), chosen from the active population and collected during a medical visit, following the Elisa protocol described below, using anti-GM2AP polyclonal antibodies. The SEP patietits selected for this study are all-round patients, i.e. with different stages and profiles of the diseases, and different treatments, etc.
The results of the assay are reported in Figure 8. While only AMN urine and 2/9 so-called 'TS' urine have a GM2AP concentration greater than 200 ng / ml, 10/22 (i.e. 45%) have a concentration greater than 200 ng / ml.
These results indicate that if the GM2AP protein is present in very low concentration ( <400 ng / ml) in the urine of individuals in the population active she is present in higher concentration in the urine of MS patients.
However 12 MS urine also have low levels of GM2AP. Of these 12 patients, 10 are Processing.
High urinary concentrations of GM2AP appear to be a pathology MS, and more precisely a marker of a stage or a form of the disease, of the activity of the disease, and is certainly influenced by any treatment in progress. Note that two individuals in the workforce have high concentrations of GM2AP (these two cases have were voluntarily included in the study because they both had a gliotoxic activity in their urine unlike other individuals in this same category).
It is impossible to know if they are healthy individuals, or suffering from a pathology, or people with multiple sclerosis because samples from so-called TS individuals were taken anonymously, without knowledge of the clinical record.
Higher urinary concentrations of GM2AP are detected in urine from MS patients; a high GM2AP concentration can then be a marker of the MS pathology, and more specifically a form of the disease, a stage of disease, of period of activity, and can be influenced by any treatment in progress. These concentrations urinary tract high in GM2AP can also have a predictive value of a start worsening of the disease, or mild MS at the start of its development, etc.
The absolute values of GM2AP concentrations detected in the urine are dependent on the affinity and specificity of the antibody used, but in general, the trend between the three groups of individuals is preserved whatever the antibody used.

Example 16: Determination of Saposin B proteins in the urine.
The protein Saposine B was detected in the same urine samples as those used for the study of GM2AP detection. The dosages were carried out in parallel with those of GM2AP, in the same study, following the Elisa protocol described below, using polyclonal anti-Saposin B antibodies.
The results of the Saposine B assay are shown in Figure 9. 0/5 urine AMN and 2/9 TS urine have a Saposin B concentration greater than 2 g / ml, then that 6/22 (or 27%) have a concentration greater than 2 g / ml.
These results indicate that the protein Saposine B is present in each urine (so-called healthy population or so-called sick population) has concentrations no negligible, i.e. <2 g / ml. These assay results are compatible with those described in the bibliography. However even if Saposin B is present in each urine, it seems to be present in higher concentration in some urine SEP. This increased concentration of saposin B in Sep urine is can-be masked by the basal concentration of this protein in the ordinary state.
So the high urinary concentrations of Saposin B appear to be a marker of MS pathology, and more precisely a marker of a stage or a form of the disease, disease activity, and is certainly influenced by everything ongoing treatment.
Saposin B measured alone seems to be a slightly less marker discriminant of a form or activity of the disease than GM2AP. We also note a two times Labor force individuals have high concentrations of Saposin B
and that these are the same two individuals who also had a high concentration of GM2AP in their urine.
In conclusion, higher urinary concentrations of Saposin B are detected in the urine of MS patients; a high concentration of Saposin B can be then a marker of MS pathology, and more precisely of a form of the disease, of stage of the disease, a period of activity, and can be influenced by any treatment in Classes. These high urinary GM2AP concentrations may also have a value predictive of an onset of worsening of the disease, or mild MS in early evolution, etc. But generally speaking, the high concentrations of Saposin B alone seem to be less discriminating markers than high concentrations of GM2AP
alone.
Absolute values of Saposin B concentrations detected in urine are dependent on affinity. and the specificity of the antibody used, but in a way general, the trend between the three groups of individuals is maintained whatever the antibody used.

Example 17: Co-assay of the GM2AP and Saposin B proteins in the urine.
Figure 10 shows the concentrations of GM2AP assayed in the samples of urine described in Figure 5 versus the concentration of Saposin B
dosed in these same samples and described in Figure 6. In this graph are reported the samples SEP (dark diamonds) and the OND and 'Healthy' samples (white diamonds).
In this graph, it is clear that:
- the higher the GM2AP concentration in the urine, the more Saposin B concentration is high. (We have shown that it is not a case general with other proteins and that that doesn't translate a disturbance renal, with the creatinine assay in parallel for each of the samples tested.);

- high concentrations of GM2AP and Saposin B are characteristic MS samples (with the exception of two urines from the workforce, above) .These joint high concentrations of GM2AP and Saposin B are markers of MS pathology, more precisely of a window of the 5 disease (quadran on the right and at the top of the graph).
In conclusion, this analysis confirms that urinary concentrations of GM2AP (> 400 ng / ml) and Saposin B (> 2 g / ml) are co-detected in the urine of MS patients and may represent markers of MS pathology, more precisely of a form of the disease, a stage of the disease, a period lo of activity, and can be influenced by any treatment in progress: It is advantageous to measure the two proteins in parallel in each sample, and consider the two concentrations.
Assay of GM2AP and Saposin B in the urine of two patients kinetic.
15 Patient MS # 1 - Progressive Remittent Form.
Urine from this patient was collected during the course of his sickness. The patient was hospitalized at JO for an attack. He suffered on D1 a flash of corticosteroids then was followed in time from a clinical point of view (the flash brought clinical improvement). Figure 11 shows the GM2AP dosage profile and some 20 Saposin B in this urine during evolution, and Figure 12 shows the profile of produces two concentrations of GM2AP and Saposin B, reflecting a co-detection high concentrations. High GM2AP and Saposin B concentrations at moment of relapse and hospitalization, gradually decrease in time after the corticosteroid flash up to 90 days.
25 Patient MS # 2 - Progressive Form.
Urine from this patient was collected during the course of his sickness. The patient was hospitalized at JO for an attack. He suffered at JI
a flash of Endoxan then was followed in time from a clinical point of view (the flash provided clinical improvement and at D60, signs of worsening of the disease summer 30 observed). Figure 13 shows the dosage profile of GM2AP and Saposin B in this urine during evolution, and Figure 14 shows the profile of the product of both GM2AP and Saposin B concentrations, reflecting co-detection of concentrations high. Elevated GM2AP and Saposin B concentrations at the time of thrust and hospitalization; gradually decrease over time after the flash Endoxan (or also called cyclophosphamide) up to 23 days and appear to increase for become high at D60, thus showing a perfect correlation with the evolution of the signs clinics.
These results confirm that:
- high concentrations of GM2AP and Sapsoine B in the urine are markers of MS pathologies, and in particular the co-detection of strong concentrations of two proteins together (translated by the product of the two concentrations);
- high concentrations of GM2AP and Saposin B in the urine are disease activity markers (here during the outbreak) or are influenced markers by immunosuppressive treatments such as corticosteroids or Endoxan which lower them concentrations.
This example illustrates that these markers can be used between other:
- to carry out therapeutic monitoring of a patient and assess the benefit therapeutic treatment for a given patient; or - predict a worsening of the disease, predict a peak in activity. etc ...
- decide on an early therapeutic (re) take on clinical signs EXAMPLE 18 Correlation Between the Detection of the Proteins MRP14, GM2AP and Saposin B in the urine and gliotoxicity measured in this urine.
In order to verify a correlation between the presence of these proteins alone or in combination in the urine and gliotoxicity of the urine, were dosed in parallel them protein concentrations of interest and there gliotoxicity of a sampling patient urine with multiple sclerosis (MS), patients with other diseases neurological (AMN) and individuals from the workforce known as TS. From patients. SEP, we note patients with different forms and stages of the disease, under treatment or not, at different disease activities.
The proteins MRP, GM2AP and Saposine B were measured in urine following the Elisa protocols described above. The dosages analyzed in this examples are those described in the previous examples. Each urine sample analyzed in Elisa was analyzed by the MTT test to measure the gliotoxicity of each sample. Gliotoxicity is expressed as a percentage of dead cells (estimated by colorimetry using tetrazolium salts) from a cell line murine astrocytic (CLTT1.1) after 48 hours of incubation in the presence of centrifuged urine.
FIG. 15 represents the concentration of GM2AP as a function of the gliotoxicity of urine determined by MTT test.
22 MS urine (gray diamonds), 5 AMN urine (black diamonds) and 9 so-called urines TS (black diamonds) have been plotted on the graph. It's the same urine who have been studied in Examples 15 and 16. It is observed that all of the control urines (AMN and TS) have low GM2AP levels ( <400 ng / ml) and low gliotoxicity ( <15%), except of a control TS urine (already commented on in Example 15) for which observe a strong GM2AP concentration and gliotoxicity.
MS urine is divided into three sub-populations:
- urine with low GM2AP concentration ( <400 ng / ml) and low gliotoxicity ( <15%), - urine with low GM2AP concentration ( <400 ng / ml) and gliotoxicity (> 15%), or essentially 3 urines, - urine with a high concentration of GM2AP (> 400 ng / ml) and high gliotoxicity (> 15%).
These three sub-populations perhaps reflect MS sub-populations, that is say different forms or stages of the disease, different activities of the disease, different therapeutic benefits, ...
However it can be noted that all urine presenting a strong concentration in GM2AP also exhibit high gliotoxicity.
In conclusion: there is a correlation between urinary concentration high in GM2AP and gliotoxicity (all urine with high concentration in GM2AP are gliotoxic (10/10), and all urine with low concentration in GM2AP are not gliotoxic ( <15%), with the exception of 3 urines / 1 2 MS).
This translates the implication of. GM2AP protein in the mechanism of gliotoxicity, only or in combination, in its natural or modified form, but recognizable by an antibody anti-GM2AP. In addition, the co-detection of a high concentration of GM2AP in urine and high gliotoxicity correlates with a subpopulation of patients with of SEP.
FIG. 16 represents the concentration of Saposin B as a function of the gliotoxicity of urine determined by MTT test.
22 MS urine (gray diamonds), 5 AMN urine (black diamonds) and 9 so-called urines TS (light gray diamonds) have been plotted on the graph. They are the same urine that has been studied in Examples 15 and 16. We observe. that the more urine is rich in Saposin B, the more gliotoxic they are. There is a fairly clear correlation between concentration of Saposin B
and gliotoxicity of urine.

In conclusion: there is a correlation between high urinary concentration in Saposin B and gliotoxicity. This reflects the involvement of the protein Saposine B in the gliotoxicity mechanism, alone or in combination, in its natural form or modified but recognizable by the anti-saposin B antibody used for the assay.

FIG. 17 represents the product of the concentrations of GM2AP and Saposin B
as a function of the urine toxicity determined by MTT test.
22 SEP urines (gray diamonds), 5 AMN urines (black diamonds) and 9 urines say TS (light gray diamonds) examples 15 and 16 have been reported in Figure 17. The gliotoxicity of this urine is analyzed according to the product of GM2AP concentrations and Saposin B, i.e. based on the co-detection of the two proteins in the urine. We very clearly observes a correlation between the product of the two GM2AP concentrations and Saposin B and gliotoxicity, much more important than considering it only one protein.
It is observed that 5/5 of the AMN urines have a product of GM2AP concentration and Saposin B
low and low gliotoxicity; 8/9 TS urine has a product of GM2AP concentration and Weak SaposineB and / or low gliotoxicity. However, there are basically three MS urine subpopulations:
- urine with low GM2AP.Saposin B concentration and low gliotoxicity ( <15%), - urine with a high concentration of GM2AP. Saposin B and high gliotoxicity (> 15%).
These two subpopulations perhaps reflect MS subpopulations, that is to say different forms or stages of malaria, different activities of the illness, various therapeutic benefits, .... However, it is very important to note that all urine with a high concentration of GM2AP and Saposin B, i.e. jointly having a high concentration of GM2AP and Saposin B, also exhibit high gliotoxicity. The two subpopulations of MS patients are all the more marked and clear when we jointly consider the three 1o markers: gliotoxicity, high GM2AP concentration and concentration high in Saposin B. This is confirmed in Figure 18.
In conclusion: there is a correlation between urinary concentration high of GM2AP and Saposin B and Gliotoxicity. All urine with a strong GM2AP and Saposin B levels are gliotoxic, and all urine with a low concentration of GM2AP and Saposin B are not gliotoxic ( <15%), at the exception of 2 urines / 22 SEP. This reflects the involvement of the two proteins and Saposin jointly or in combination in the mechanism of gliotoxicity, under their natural or modified form but recognizable by the anti-GM2AP and anti-saposin B used for the assay. Plus the co-detection of a strong urinary GM2AP and Saposin B concentration and high gliotoxicity correlates with a subpopulation of MS patients (stage, form, activity, treatment of disease?), compared to another subpopulation. These three markers considered jointly allow to discriminate between two subpopulations of MS patients.
Evolution of gliotoxicity and GM2AP and Saposin concentrations B depending on the course of the disease of two patients after and during treatment.
The correlation between gliotoxicity, high concentration of GM2AP AND
Saposin in urine and MS pathology has also been confirmed in measuring these three parameters in the urine of two patients during the course of their nialadie.
Patient # 1: MS remittent-progressive form, hospitalized at OJ for a 10 thrust and received a corticosteroid flash at JI. After the flash, he shown a clinical improvement until D90 - (see Figures 11,12), Patient # 2: MS progressive form, hospitalized at OJ for a flare-up and having received a flash from Endoxan (also called cyclophosphamide) on D1. At D60, there presents new clinical signs of worsening of his disease - (cf.
figures 13.14). .
5 For both patients, it was shown:
- a correlation between urinary gliotoxicity and the clinical course of disease (when clinical signs are severe, gliotoxicity is high ; when the clinical signs decrease following treatment, gliotoxicity decreases and bECOMES
stationary; when signs of worsening appear after treatment, the 10 gliotoxicity seems to increase again), - a correlation between the gliotoxicity rate in the urine of patients and GM2AP and Saposin B concentrations, and - a correlation between the high concentrations of GM2AP and Saposine B and the clinical course of the disease.
15 In conclusion: the dosage of GM2AP AND Saposin B proteins in urine is a good discriminatory marker of a subpopulation of MS
(Stadium, form, activity, treatment of the disease). GM2AP and / or Saposin proteins B are involved in the mechanism of gliotoxicity, alone or in combination, under their natural form or in a form recognizable by polyclonal antibodies used 20 for their dosage. As GM2AP and Saposin proteins are co-detected in strong concentration in. gliotoxic urine it's possible that these two protein act in combination to induce gliotoxicity.

EXAMPLE 19 Immunohistochemical Analysis of Protein Expression 25 GM2A, SAPB, MRP14 and MRP8 in a cropping system producing gliotoxin in vitro (monocyte cultures), as well as in normal brain tissue and pathological MS and witnesses.
Protocol: Monocyte cultures from a patient with MS and a healthy control were carried out in parallel, according to the present protocol described briefly.
30 From the peripheral blood of these two volunteers taken from ACD, the PBMC
(Peripheral Blood Mononuclear Cel1s) are isolated on Ficoll using the technical known to those skilled in the art. Recovered cells (at the level of the ring) are washed twice in RPMI environment. The cells are then listed on Kovas-slide and are sown in a 25 cmZ primary bottle or on a Labteck slide (8 cups) (in permanox) in RPMI medium supplemented with 15% human AB serum at OJ. The cells are grown on dimpled Labtek type blades to provide direct support for analysis of monocytes which adhere to the support and subsequently differentiate into macrophages. For the slides, 2,106 cells are thus seeded at a rate of 0.25 106 cells / well. For the vials, 4,106 cells are seeded at the rate of 0.25 106 cells / well.
At D1, the cells in suspension are recovered and the Labteck wells or flasks are washed twice in RPMI (previously heated to 37 C) before adding RPMI medium supplemented with 5%
human AB serum. AJ 1, J3, J6, J9, J 12 or 14, J 15 the culture medium is changed; the supernatants are removed and cells fixed on slides using the known techniques of one skilled in the art. At each change of environment, at least two slides were fixed in paraformaldehyde and stored for immunohistochemical analysis.
Composition of the medium: RPMI (500 ml) with 15 ml of 200 mM glutamate, 5 ml 100 M sodium pyruvate, 5 ml non-essential amino acids (100x), antibiotics penicillin and streptomycin 100,000 U / l and anti-interferon antibodies humans at 100 U / l.
Results: Four cultures of monocytes in vitro were thus studied in kinetics: two cultures of monocytes from the blood of control individuals and two cultures of monocytes from MS patients. At different times of cultivation (OJ, JI, J3, J6, J9, J12), the Corresponding supernatants were also recovered. Once the kinetics completed, the slides corresponding to the different culture days were incubated in presence of antibodies polyclonal anti-GM2A, SAP-B, MRP-8 and MRP14. The gliotoxicity of each floated thus recovered was estimated by MTT test. The GM2AP protein concentration, MRP14 and Saposin B was also determined in each supernatant by protocol Elisa like described in Examples 13 and 14.
The results of immunofluorescence on fixed cells are summarized below ;
we can note :
- an absence of expression of MRP8 at all stages of the 2 cultures - a clear expression of MRP-14 in the period between D9 and D15, found in both cultures, although stronger in the MS culture.
This expression seems to correlate a step of macrophagic differentiation.
- very low expression (low intensity and low number of cells) is observed at the start of culture in the control culture and corresponds presumably physiological presence of GM2A in lysosomes macrophagic.
- In MS culture, a much clearer expression of GM2A (more high intensity and larger number of cells) is observed, with a marking relatively homogeneous cytoplasmic t0 between D3 and D6, disappears on D9 and is at new noted on J 14-J 15 with intense marking and located at the periphery cytoplasmic, drawing the internal contour of the plasma membrane. These observations do not are not found in all of the control slides.
Analysis with the anti-SAP-B antibody did not yield a interpretable immunohistochemical labeling.
In the cultures of MS monocytes already carried out, 3/3 presented a gliotoxicity peak on D9 and 2/3 a lower peak on D6. No peak being detected in cultures of 2/2 non-MS control monocytes analyzed in parallel. Likewise, the determination of the proteins MRP 14, GM2AP and Saposin B in the supernatant of cultures during kinetics showed that the proteins SapB and GM2AP
are detected by Elisa in the supernatants of MS monocytes and not in those of control monocytes, on days D6 and especially D9 of the culture; proteins don't are not detected beyond these kinetics. Note that the antibodies used to the dosage can recognize the physiological forms of proteins, but also of complexed and / or modified forms.
We therefore observe that the period J6-J9 during which we observe a most important gliotoxicity in the supernatant, is covered by the period J3-J15 during which we observe a less differentiated production of the control negative of GM2A in cells with quantitative and qualitative fluctuations in his = 0 cellular expression (amount of expression and cellular localization).

Example 20: Immunohistology technique on brain sections in paraffin.
Histological sections prepared in paraffin are dewaxed in xylene and alcohol before undergoing a pretreatment which aims to unmask antigens; this pretreatment can correspond to (i) twice 5 minutes in the microwave (750W) in presence of a sodium citrate buffer, citric acid, (ii) treatment with acid by incubation for 15 minutes in a 1% periodic acid solution or by 5 minute incubation in 99% formic acid solution. The endogenous peroxidases are then blocked by incubating the slides for 30 minutes in 1% hydrogen peroxide then extensive washing in water during 15 minutes. The background noise is blocked by incubating the slides for 30 minutes in presence of PBS
Triton 0.03%, 10% donkey serum (for polyclonal antibodies) or 10% donkey serum goat (for monoclonal antibodies). Labeling with the primary antibody is made in applying 100 to 200 l of primary antibody solution per slide (0.5 to 5 g / mi depending on the title) in 0.03% Triton PBS and then incubating for 2 hours at room temperature. The blades are then rinsed 3 times in PBS-Triton for 10 minutes. Antibody labeling secondary is performed using biotinylated antibodies capable of binding specifically to primary antibodies, for example anti-rabbit IgG. or anti-mouse IgG
diluted in PBS-Triton 0.03%. the slides are washed and incubated in a solution for 2 hours (2 l streptavidin-biotin-peroxides complex, 1600 l PBS-Triton 0.03%). The blades are from washed again before being revealed protected from light in buffer A
then rinsed out water before microscopic observation. Buffer A for 5 slides: 25 ml Tris0.05M
pH 7.6, 2.5 ml Inzidazole 1M, 15 ml sterile water, 2 ml DAB 5 mg / ml, 5 ml Ammonium nickel 10%, 30 l HZ02 1%.
The same antibodies were used for an immunohistochemical study, according to the technique described briefly below, on paraffin slides obtained by cut to microtome of brains collected post-mortem from MS and controls deceased from pathologies non-neurological.
The results of the analysis are summarized below:
There is no labeling of non-MS and MS brains in this substance gray and normal white matter (not damaged) with the different anti antibodies MRP8, MRP14, GM2A. Non-specific reactivity did not allow to interpret the results with the anti-saposin B antibody in this application immunohistochemical.
On the other hand, we note, in the areas of MS brain plates - anti-MRP14 reactivity in macrophagic cells and microglial, having a relatively homogeneous distribution over the whole extent of demyelination zones (plaques), - a lower (less frequent) anti-MRP8 reactivity linked essentially to perivascular lymphoid infiltrates - clear anti-GM2A reactivity in macrophages and microgliocytes to areas of plates, with a particular density in the areas constituting the wall glial in peripheral edge of plate. A marking of some astrocytes has also found in demyelination areas.
These various observations show that there is hyperexpression particular of the proteins MRP-14 and GM2A in the cultures of monocytes of Sep producing gliotoxic activity in their supernatant, as well as in zones defining demyelination plaques in MS brains. They testify therefore of the reality of the coincidence between their abnormal co-expression, the production of gliotoxic activity and demyelination lesions.
In addition, their abnormal production in the context of MS, in macrophage cells in the blood as well as in the brain, indicates that it is founded, to carry out their dosage in biological fluids to correlate their amount with the lesional and inflammatory activity of MS.

Example 21 Measurement of the Activity of T Cells by Proliferation of T cells (Sredni et al., 1981).
T cells are washed twice in culture medium to remove any trace of IL2 present in the initial culture medium. Lymphocytes B (EBV-LCL) or monocytes / macrophages taken as cells presenting the antigen, are irradiated at 10,000 rads, washed twice with culture medium (RPMI). 2.10 ' T cells (2: W cells / ml) and 2.10 'autologous irradiated B cells (2.10' cells / ml) are incubated together in the presence of a concentration range growing from the antigen in a final volume of 200 l in microwells. After 48 hours of culture at 37 ° C., 1 Ci of 3H-thymidine in 50 l of RPMI medium is added in each well. T cells, the only ones to divide, incorporate thymidine tritiated in DNA. After 18 hours of culture, the cells of each microwell are harvested on glass wool pellets by aspiration. After osmotic lysis of cells, the radioactivity incorporated into DNA is absorbed on the pellets (cell Harvester 530, Inotech). Each dried tablet is placed in a plastic tube which contains 2 ml of scintillating; the radioactivity b adsorbed on each of the pellets is quantified in a liquid scintillation beta meter (LKB Rackbeta 1217). The results are expressed lo as arithmetic mean of cpm / culture ('counts per minute').

Example 22: Protocol for detecting the association between the peptides and histocompatibility molecules (APC approach transformed with a peptide is setting at CMH 1).
1) Material The sources of histocompatibility molecules are currently two main types: mutant cells and histocompatibility molecules purified.
The mutant cell used is the human T2 cell which is a variant of the TI line produced by fusion of CEM T lymphoma and B lymphoma 721.174 (Salter and Cresswell Embo J 1986, 5 .: 943-949). This cell which is devoid of peptide transporters contains heavy chains of class molecules I free from peptides which will be able to accept exogenous peptides.
Class I histocompatibility molecules purified by affinity chromatography from human B cell lines transformed by EBV can also be used. In this case the endogenous peptides have to be eliminated by treatment with urea. 5 M and sodium hydroxide 12.5 mM (pH
11.7) for 1 hour at 4 C, followed by their elimination by a desalting column (PDLO, Pharmacia). The histocompatibility molecules are immediately put back into presence of the peptides to be tested in a PBS buffer with 0.05% Tween 20, 2 mM
3o EDTA, 0.1% NP40 and 6mM CHAPS, in the presence of 2 giml B2m to facilitate the reassociation (Gnjatic et al., Eur J Immünol 1995 25: 1638-1642).

The peptides tested generally have 8 to 10 residues, sometimes 11 or 12. They have been synthesized by Néosystems (Strasbourg), or by Chiron mimotopes (Victoria, Australia). They are used at concentrations varying from 100 M to 0.1 nM.
2) Assembly protocol (Connan et al., Eur J Immunol 1994, 24:
777; Couillin et al. Eur J Immunol 1995, 25: 728-732).
Aliquots of 8.105 cells in a volume of 64 l, distributed in Eppendorf microfuge tubes, are placed in the presence of a lysis buffer containing mM PBS, pH 7.5 1% NP40, protease inhibitors (1 mM PMSF, 100. M
iodoacetamide, 2 g / ml aprotinin, 10 M leupeptin, 10 M pepstatin and 10 g / ml to trypsin inhibitor). Lysis takes place in the presence of the peptides to be tested for 30 minutes or 1 hour at 37 C. After removal of the non-solubilized material by a centrifugation at 15,000 rpm at 4 ° C., the supernatant and added with 140 l of PBS containing 0.05% Tween 20, 3 mM sodium azide, 1 mM PMSF and 10 mg / ml bovine albumin. Each sample is incubated for 20 hours at 4 VS
t5 in 2 wells of a microtiter plate of the Nunc, Maxisorb type, beforehand coated with a monoclonal antibody (10 g / ml in PBS) which recognizes the molecules histocompatibility with a conforming conformation (s) for the presentation peptides and similar to that (s) present on the surface of cells.
The plaque coated with antibodies is previously saturated with bovine albumin at 10 mg / ml 20 in PBS-Tween before placing the sample. The second antibody that allows the detection of the assembly of histocompatibility molecules is directed against the B2m.
It is coupled either to biotin (NHS-LC biotin, Pierce) or to phosphatase alkaline (P-552, Sigma) and is incubated at 2 g / ml for one hour at 37 C. In the case of the job biotin, 45 minute incubation at 20-25 C with streptavidin coupled 25 with alkaline phosphatase (E-2636, Sigma) is carried out. The activity of the phosphatase alkaline is measured using as substrate 4-methyl-umbelliferyl-phosphate (M-8883, Sigma) at 100 M in 50 mM diethanolamine, pH 9.5 with MgC12 1 mM. The reading is made at 340/460 nm using a cytofluorimeter.
3) Stability of HLA / peptide complexes:
30 The stability of the aforementioned complexes has been studied because it conditions the good presentation of the antigen and induction of the T response. To this end, We used either purified HLA or the T2 cell lysate. With purified HLA, we have eliminated the endogenous peptides (as described in 2) then it was placed in the presence of the peptide to test in Eppendorf tube at 37 C, for varying times from a few minutes to several days. The next incubation phase on a 96-well plate (as described in 2) with anti-antibody HLA is done for one hour at 37 C. The revelation is carried out so classic. With T2 cell lysate, all incubations are also done at 37 C, after adding all protease inhibitors.

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Claims (35)

1. ~ Use of at least one polypeptide comprising at least one fragment a protein to obtain a diagnostic, prognostic composition, prophylactic or therapy intended to detect, prevent or treat a pathological condition associated with sclerosis in plates, said protein being chosen from proteins whose sequence peptide to the native state corresponds to SEQ ID N ° 17, SEQ ID N ° 18, SEQ ID
N ° 19, SEQ ID N ° 20, SEQ
ID N ° 21, SEQ ID N ° 22, SEQ ID N ° 23, and the sequences peptides that present at at least 70% identity, preferably at least 80% identity and advantageously at least 98% identity with any of the SEQ ID peptide sequences N ° 17 to SEQ ID N °
23, and the peptide sequences or the fragments of said sequences belonging to the same family than calgranulin B. 2. ~ Use according to claim 1, of at least two polypeptides in combination as defined in claim 1. 3. ~ Use according to claim 1, characterized in that said protein is chosen from proteins whose peptide sequence in the native state corresponds to SEQ ID
N ° 17 and the peptide sequences which present at least 70%
identity, preferably less 80% identity and advantageously at least 98% identity with the sequence peptide SEQ ID No. 17. 1 4. ~ Use according to any one of claims 1 to 3, characterized in this that the peptide sequence of said polypeptide comprises SEQ ID No. 17. 5. ~ Use according to any one of claims 1 to 3, characterized in this that the peptide sequence of said polypeptide consists of SEQ ID No. 17. 6. ~ Use of at least one nucleotide fragment, to obtain a diagnostic, prognostic, prophylactic or therapeutic composition intended to detect, predict, prevent or treat a medical condition associated with sclerosis in plates, according to which said nucleotide fragment is chosen from fragments which code for at least a fragment of a protein as defined in claim 1. 7. ~ Use according to claim 6, characterized in that said fragment nucleotide code for said protein. 8. ~ Use of at least one nucleotide fragment to obtain a diagnostic, prognostic, prophylactic or therapeutic composition intended to detect, predict, prevent or treat a medical condition associated with sclerosis in plates, according to which said fragment is a fragment of a nucleic sequence chosen from Moon any of the sequences SEQ ID N o 42, SEQ ID N o 43, SEQ ID N o 44, SEQ ID N
o 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ
ID No. 51, SEQ ID N o 52 and their complementary sequences. 9. Use of a specific ligand for a polypeptide or a fragment nucleotide according to any one of the preceding claims for get a diagnostic, prognostic, prophylactic or therapeutic composition intended to detect, predict, prevent or treat a medical condition associated with sclerosis in plates. 10. Method for detecting at least one protein associated with sclerosis in plates, in a biological sample, characterized in that one puts in contact the biological sample with at least one specific ligand of at least one polypeptide such as defined in claim 1, then detecting the formation of a complex between said polypeptide and said ligand. 11. Method according to claim 10, characterized in that said ligand is a monoclonal antibody, polyclonal antibody, receptor, substrate of activity enzymatic or an enzyme of which said polypeptide is a cofactor. 12. Method according to claim 10, characterized in that the sample biological consists of cells or supernatants of said cells of a patient likely to be affected by multiple sclerosis. 13. The method of claim 10 or 12, wherein the sample biological consists of monocyte or macrophage cells or supernatants of these cells. 14. Method according to any one of claims 10, 12 or 13, in which the biological sample consists of cultured cells or supernatants of these cells in culture, after a period of between 6 and 12 days of culture, preferably after 9 days. 15. Method according to any one of claims 10, 12 or 13, in which the biological sample consists of cells, ex vivo, preferably monocytes or macrophages. 16. Method for detecting at least one ligand associated with multiple sclerosis, in a biological sample, characterized in that one puts in contact the sample biological with at least one polypeptide as defined in claim 1, then we detect the forming a complex between said polypeptide and said ligand. 17. The method of claim 16, characterized in that the ligand is a antibody, receptor, enzyme activity substrate or an enzyme whose said polypeptide is a cofactor. 18. Method according to any one of claims 10 to 17, characterized in this that the sequence of said polypeptide comprises a chosen peptide sequence among one any of SEQ ID Nos. 17 to 23. 19. Method according to any one of claims 10 to 17, characterized in this that the sequence of said polypeptide consists of a chosen peptide sequence among one any of SEQ ID Nos. 17 to 23. 20. Method according to any one of claims 10 to 19, characterized in this that the biological sample is urine, cerebrospinal fluid or serum. 21. Method for detecting, in a sample of a biological fluid, the less a polypeptide as defined in any one of claims 1 to 5, according to which, optionally after purification of said sample, it is analyzed by mass spectrometry the mass profile obtained from said sample and compared to a profile of mass of reference. 22. Use of at least one polypeptide as defined in any one of claims 1 to 5, to obtain a diagnostic, prognostic composition, prophylactic or therapeutic intended to detect, prevent or treat a pathological condition associated with the multiple sclerosis, and preferably at least one polypeptide as defined to the claim 4. 23. Use according to claim 22, in which the sequences peptide include the peptide sequences or fragments of said sequences belonging to the same family of proteins as calgranulin B. 24. Use according to claim 22 or 23, which is associated with use detection of gliotoxic activity. 25. Method for detecting, in a sample, a concentration value from at at least one polypeptide according to any one of claims 1 to 5, said concentration being associated with a pathological condition, characterized in that said dose polypeptide, the assay to obtain a concentration value which is compared to a threshold value representative of multiple sclerosis. 26. Use of at least one polypeptide as defined in claim 1 for test the effectiveness of a therapeutic agent. 27. Use of at least one polypeptide comprising at least one fragment of a protein for the preparation of a pharmaceutical composition intended to treatment multiple sclerosis, said protein being chosen from proteins whose sequence peptide in the native state corresponds to SEQ ID No. 17, SEQ ID No. 18, SEQ ID No.
o 19, SEQ ID
No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, peptide sequences who have at least 70% identity, preferably at least 80% identity, and advantageously at least 98% identity with any of the sequences peptide SEQ ID N o 17 to 23, and the peptide sequences or the fragments of said sequences belonging to the same family of proteins as calgranulin. 28. Use according to claim 27, characterized in that the polypeptide a the sequence SEQ ID NO: 17. 29. Use of at least one nucleotide fragment, to test the effectiveness of a therapeutic agent for a pathological condition associated with sclerosis in plates, according to which said nucleotide fragment is chosen from the fragments which code for at least a fragment of a protein, said protein being chosen from proteins whose sequence peptide in the native state corresponds to SEQ ID No. 17, SEQ ID No. 18, SEQ ID No.
o 19, SEQ ID
No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, peptide sequences who have at least 70% identity, preferably at least 80% and advantageously at minus 98% identity with any of the peptide sequences SEQ ID N
o 17 to 23, and the complementary fragments of said fragments and the fragments which encode for the peptide sequences or the fragments of said sequences belonging to the same family of proteins than calgranulin B. 30. Use to test the efficacy of a therapeutic agent for a condition pathology associated with multiple sclerosis, recombinant proteins and / or coded by all or part of the nucleotide fragments defined in claim 29. 31. Use of at least one nucleotide fragment for the preparation of a pharmaceutical composition intended for the treatment of multiple sclerosis, that said nucleotide fragment is as defined in claim 29. 32. Use for the preparation of a pharmaceutical composition intended for the treatment of multiple sclerosis, recombinant proteins and / or coded by all or part of the nucleotide fragments defined in claim 29. 33. Use according to any one of claims 29 to 32, characterized in what said nucleotide fragment codes for said protein. 34. Use according to any one of claims 29 to 31, characterized in that said fragment is a fragment of a nucleic sequence chosen from Moon any of SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ
ID No. 46 and SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49 and SEQ ID No. 50, SEQ ID No.
51, SEQ ID
No. 52, and their complementary sequences. 35. Use according to claim 34, characterized in that the sequence nucleic acid is SEQ ID No. 42.