Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Monoclonal antibodies recognizing a peptide associated with a major histocompatibility antigen Monoclonal antibodies recognizing a peptide associated with a major histocompatibility antigen.
• the invention also relates to the applications of these antibodies to diagnosis and treatment and, where appropriate, to the prevention of certain pathologies.
• MHC molecules Major Histocompatibility Complex
• the complex formed by the MHC molecule and the antigen is recognized respectively by cytotoxic T cells or by helper T cells capable of activating antibody synthesis by B cells.
• the inventors are interested in the role that could be restricted monoclonal antibodies for the diagnosis but also the treatment of different pathologies.
• the subject of the invention is therefore restricted monoclonal antibodies recognizing antigens associated with MHC molecules. Also within the scope of the invention are strains of hybridomas producing restricted monoclonal antibodies.
• the invention also relates to compositions for the diagnosis of the presence of an infection or of a cellular disturbance capable of generating a tumor state or an autoimmune disease.
• the invention also relates to compositions for the treatment of infections or cellular disturbances as described above.
• the antigens referred to in the preceding paragraphs and which constitute one of the two entities of the complex recognized by the restricted antibodies according to the invention, are either peptides resulting from the degradation of proteins originating from an infectious agent or pathogen either peptides from cellular dysfunction (deregulation) or even self-degrading proteins.
• These peptides can for example be obtained by enzymatic cleavage for example by the enzymes of the lysosome or of the Golgi apparatus.
• these peptides are advantageously obtained by chemical synthesis according to conventional methods of synthesis of peptides.
• the monoclonal antibodies of the invention are characterized by their ability to specifically recognize a complex formed by a peptide characteristic of an antigen of a pathogenic agent or a peptide characteristic of cell deregulation and by a molecule of the Major Histocompatibility Complex (MHC) having the capacity to recognize and fix this peptide, said antibodies being restricted monoclonal antibodies, since they do not recognize said peptide in association with a MHC molecule of haplotype which does not fix the peptide (not specific for the peptide).
• MHC Major Histocompatibility Complex
• a test can be carried out as described by BOUILLOT et al, (Nature, 1989, vol. 339, p.473-475).
• a peptide whose specificity is sought with respect to the MHC molecule is incubated with a known MHC molecule, on cells labeled with a radioactive isotope such as chromium 51. After this incubation, the suspension is brought into the presence of cytotoxic lymphocytes (CTL) specific for the peptide.
• CTL cytotoxic lymphocytes
• the antibodies defined above are designated by the term restricted monoclonal antibodies.
• the restricted monoclonal antibodies are further characterized in that they are devoid or practically devoid of reaction with respect to the peptide against which they are directed, present in the isolated state and / or in that they recognize weakly or not at all, the MHC molecule alone.
• An antibody practically devoid of the capacity to recognize a peptide alone is according to the invention, an antibody which exhibits with this peptide a reaction less than twice the signal corresponding to the background noise.
• An antibody weakly recognizing a MHC molecule alone recognizes this MHC molecule according to a reaction which is of the order of 1/10 to 1/5 of the reaction existing under the same conditions with the specific peptide-MHC complex.
• an ELISA type immunoenzymatic reaction as described below by way of example, can be carried out.
• the immunoenzymatic reaction is carried out first using the reagent based on conjugated anti-mouse Ig antibody coupled either to urease or to alkaline phosphatase, on a culture supernatant of hybridomas. The reaction is then confirmed by the anti-mouse Ig conjugated antibody coupled for example to alkaline phosphatase.
• the cells serving as targets are, for example, spleen cells previously incubated with the peptide against which the antibody is to be formed (5 to 50 ⁇ g / ml for 10 7 cells at 37 ⁇ C for one to two hours).
• the cells are fixed on polyvinyl plates according to the technique described by Ternynck and Avrameas (Ternynck and Avrameas: Immuno-techniques enzy atiques - Les circumstances INSERM, 1987) using poly-L-lysine.
• optical density O.D
• O.D optical density
• the restricted monoclonal antibodies of the invention have the advantage of having a high selectivity which makes them particularly advantageous in their application to diagnosis, treatment and, where appropriate, vaccination.
• the peptides recognized by the antibodies of the invention can be characteristic of pathogenic agents such as viruses, bacteria, parasites, for example plasmodium or fungi, in particular pathogenic yeasts. It may also be peptides representing minor antigens of histo compatibility, peptides characteristic of cellular disorders of the tumor or cancer type or even of the autoimmune type.
• the restricted monoclonal antibodies are characterized in that the recognized peptides have from 7 to 25, preferably about ten amino acids.
• the presence of a peptide having an amino acid length close to that given in the above may be sufficient for the binding reaction with MHC molecules and for that of recognition by the antibodies of the invention.
• antigens constituted by a peptide associated with a solubilized and / or solubiated form of the MHC molecule which is specific to it for a given species.
• this is prepared by solubilization with an ionic detergent such as triton X100 or NP40 or with a nonionic detergent such as octyl -3D-glycopyranoside, the solubilization being followed affinity chromatography.
• an ionic detergent such as triton X100 or NP40
• nonionic detergent such as octyl -3D-glycopyranoside
• This soluble molecule can be prepared by a modified cellular host by insertion of the coding gene for the MHC molecule which it is desired to express, this gene being devoid of the sequence coding for the transmembrane part of the molecule. Given the deletion of this sequence, the heavy chain and the light chain of the MHC molecule are linked with a GGGS-type polylinker. The gene is incorporated into the host under conditions such that its regulatory elements are recognized by the cellular host.
• a suitable cellular host is for example an insect cell.
• Particular restricted monoclonal antibodies are characterized in that the recognized peptide is specific for a human HIV retrovirus and in particular in that it is peptides of the gag protein for example the peptide gag5 (GHQAAMEMLKE) or the peptide gag p25 synthesized by Neosystem (Strasbourg, reference SP89-158) (sequence 263-277), or peptides of the internal protein pl4 (nucleoprotein) in particular the peptide K16F described by Claverie JM et al in Eurley Imm 1988 vol 18 p 1547-1553 .
• the recognized peptide is specific for a human HIV retrovirus and in particular in that it is peptides of the gag protein for example the peptide gag5 (GHQAAMEMLKE) or the peptide gag p25 synthesized by Neosystem (Strasbourg, reference SP89-158) (sequence 263-277), or peptides of the internal protein pl4 (nucleoprotein) in particular the peptide K16F
• Antibodies according to the invention can also be characterized in that the human MHC molecule recognized is of various haplotypes (for example HLA-A2, HLA-A3, HLA-B7, HLA-B12, HLA-B27, HLA-B37, HLA-C 3) of class I as of class II (for example HLA-DR2 , HLA-DR3, HLA-DR4, HLA-DR5).
• haplotypes for example HLA-A2, HLA-A3, HLA-B7, HLA-B12, HLA-B27, HLA-B37, HLA-C 3) of class I as of class II (for example HLA-DR2 , HLA-DR3, HLA-DR4, HLA-DR5).
• a particular restricted monoclonal antibody according to the invention is the antibody which recognizes a peptide of the protein p25 (gag) of HIV, in particular the peptide gag5, in association with a MHC molecule of haplotype HLA-B 7 or HLA-A11 .
• the invention also relates to a process for the preparation of restricted monoclonal antibodies of the invention.
• a first process suitable for the production of these restricted monoclonal antibodies is characterized in that:
• - spleen cells from an animal previously immunized are fused with isogenic spleen cells coated with a determined peptide, with myeloma cells in the presence of a fusion promoter, for example polyethylene glycol, the spleen cells being in excess compared to myeloma cells, for example in a proportion of 10 to 1 approximately,
• a fusion promoter for example polyethylene glycol
• a screening is carried out in order to select those of the hybridomas capable of producing restricted monoclonal antibodies, for example by the ELISA technique, carried out with a reagent based on conjugated anti-mouse Ig antibody coupled to an enzyme, for example urease , or alkaline phosphatase or peroxidase,
• an enzyme for example urease , or alkaline phosphatase or peroxidase
• Another technique such as the cytotoxicity technique can be used for screening.
• hybridomas producing the above restricted monoclonal antibodies.
• These hybridomas are the products of fusion of myeloma cells and spleen cells of an animal previously immunized with the peptide-antigen, under conditions such that the antigen against which the antibodies are formed, consists of a complex comprising the above peptide associated with a MHC molecule.
• the hybridomas result from the fusion of the spleen cells described above with a myeloma of the HGPRT * type, for example: X63-Ag8, NS-1 or SP2 / 0.
• Hybridomas are produced according to the protocol of Kohler and Milstein (Nature, 1974, 256: 495-497).
• the lymphocyte cells or not, normal or tumor bringing the MHC molecule are preferably of haplotype H-2 d , H-2 b , H-2, H-2 C 'or H-2 S in mice, but it may also be cells expressing molecules of human MHC (or of another species) in transgenic (expressing the same human MHC) or non-transgenic mice.
• the invention also relates to lymphocyte cells or not, human or animal, for example of mice, coated with a peptide or a polypeptide preferably immunogenic, against which one wants to prepare restricted monoclonal antibodies.
• a second method for producing the monoclonal antibody according to one restricted "invention is to achieve the fusion of the immortalized B blood cells with Epstein Barr virus and human B lymphocytes placed beforehand in contact with the peptides against which it is desired to form restricted monoclonal antibodies.
• the B cells previously brought into contact with the peptides against which it is sought to form monoclonal antibodies can be taken from the peripheral blood of a donor previously immunized with the peptide, when it is not toxic to this donor.
• These B lymphocytes can also be obtained by in vitro culture in contact with the peptides, the recovery of the B cells covered with peptides being preceded by one or more stimulation cycles.
• restricted antibodies or analogous molecules such as the Fab part of these antibodies, or T cell receptor analogs capable of recognizing the MHC-peptide complex can be obtained in E. coli or other microorganisms , according to the techniques of Huse et al (Science, (1989), 246, 1275), ard et al (Nature, (1989), 341, 544) Bird et al (Science, (1988), 242, 423); or other authors.
• the genes amplified by the PCR technique coding for restricted monoclonal antibodies or the genes coding for receptors for specific murine or human T cells or other species, are introduced using suitable vectors. E.coli, in conditions allowing their expression. Then, a screening is carried out in order to determine which of the expressed molecules which have the specificity required for the recognition of the peptide-molecule complex of the MHC. The screening can be carried out according to the method described above.
• the above technique can also be implemented by introducing gene sequences coding for restricted antibodies or specific T cell receptors, or even mutants of these sequences.
• the genes or fragments of genes coding for the restricted monoclonal antibodies are obtained by extraction of DNA from immune cells, the preparation of which is described above.
• the invention therefore relates to antibodies or analogues produced in E. coli or in other microorganisms, to restricted monoclonal antibodies to humans or to other species (for example obtained in rats or hamsters by techniques analogous to those described above or obtained according to the methods of Borrebaeck et al, PNAS (1988), vol. 85, p.3995-3999).
• the antibodies of the invention are of great interest for different applications. They can be used, for example, for applications in the diagnosis of infections or for the diagnosis of cellular disorders such as they manifest in certain cancers or autoimmune diseases.
• the invention relates in this regard to a composition for diagnosing the presence of peptides associated with MHC molecules in a biological sample tested, characterized in that it comprises restricted monoclonal antibodies as described above.
• This diagnostic composition can be implemented either on a biological sample or in a living organism.
• the restricted monoclonal antibody is labeled with a radioactive substance or with a substance which can be made visible by fluorochrome or LASER techniques.
• the invention also relates to a kit for the in vitro diagnosis on a biological sample of an infection by a pathogenic organism or for the diagnosis of cancer, an autoimmune disease or another cellular disorder, characterized in that it comprises: restricted monoclonal antibodies according to the invention, having the specificity sought taking into account the diagnosis to be made,
• the biological sample used is advantageously a sample of serum or any other biological fluid.
• test for the in vitro detection of an infection or a cellular disturbance comprising the following steps:
• antibodies of the invention can be used to detect a virus such as the HIV AIDS virus, or even to detect tumors or autoimmune disorders.
• the restricted monoclonal antibodies can also be made cytotoxic.
• the antibodies of the invention can then enter as an active ingredient in a pharmaceutical composition for the treatment of the above infections or disorders, for example by competition, in particular in the case where they are not cytotoxic. In this case, they are mixed with an acceptable pharmaceutical vehicle.
• Substances capable of making cytotoxic antibodies are toxins, antibiotics, radioactive isotopes.
• Such antibodies can be used in immunotherapy.
• the invention also relates to a pharmaceutical composition, characterized in that it comprises, as active principle, restricted monoclonal antibodies in accordance with the invention.
• a pharmaceutical composition characterized in that it comprises, as active principle, restricted monoclonal antibodies in accordance with the invention.
• Such antibodies formulated in a pharmaceutical composition could be used in particular as competitive agents to block the action of specific T cells, particularly in autoimmune pathologies.
• the restricted monoclonal antibodies as a vaccine, to stimulate the production of anti-idiotypic antibodies, in particular in the case of autoimmune diseases.
• Vaccinating compositions according to the invention therefore comprise, as active principle, the restricted mAbs described above.
• the anti-idiotypic antibodies produced by the injection of the restricted mAbs recognize and eliminate the self-reactive T cells.
• BALB / c line mice (H-2 d ) are immunized by ip intraperitoneal injection of isogenic spleen cells covered with peptide. It is in this case the peptide of the nucleoprotein of the influenza virus called NP147-158R " .
• the spleen cells are incubated beforehand with the peptide at a rate of 10 7 cells per 100 ⁇ g of peptide in a volume of 1 ml of medium. culture (DMEM + 2% fetal calf serum) The incubation is carried out in an oven with CO 2 (5%) for 1 hour.
• the mice receive 3 intraperitoneal injections at the rate of one injection per week.
• the 4th injection is done by two routes: 0.5 ml of the suspension is injected intraperitoneally, 0.5 ml intravenously.
• the spleen cells are obtained from the spleen after dissociation in DMEM medium + 5% fetal calf serum.
• the red blood cells are lysed using 0.83% ammonium chloride (NH 4 C1) and the lymphocytes are then washed three times with DMEM + 5% fetal calf serum.
• the fusion partner consists of 3 HGPRT " myelomas, namely X63-Ag8, NS-1 and SP2 / 0.
• the lymphocytes are fused with either myeloma X63, NS-1 or SP2 / 0 separately.
• PEG 1500 polyethylene glycol
• the protocol is based on that described by Kôhler and Milstein (Nature, 1974, 256: 495-497) below: the lymphocytes and myeloma cells (X63, NS-1 and SP2 / 0) are put together in the proportion indicated above.
• the three preparations (lymphocytes plus X63 or more NS-1 or more SP2 / 0) are centrifuged for 10 minutes at 250g and at 4 * C. The supernatants are removed, however, leaving a film of liquid above the cell pellet.
• the tubes are placed at 37 ⁇ C in a water bath. 1 ml of a solution of polyethylene glycol 1500 (50% solution of PEG 1500 in RPMI at pH 7) is added dropwise to the cell pellet. The operation, which should last 1 minute, is followed by the addition of 20 ml of DMEM plus 10% fetal calf serum. The whole is left for another three minutes in the water bath at 37 ⁇ C, then the tubes are centrifuged for 15 minutes at 250 g and at + 4 ° C.
• the supernatants are removed and the cells are washed once with 20 ml of DMEM + 10% fetal calf serum.
• the cells are then returned to a DMEM solution plus 10% fetal calf serum to which the HAT medium is added (HAT 100X mother solution: Hypoxanthine at 1.36 mg / ml, Aminopterin at 0.018 mg / ml and Thymidine at 0.76 mg / ml).
• HAT 100X mother solution Hypoxanthine at 1.36 mg / ml, Aminopterin at 0.018 mg / ml and Thymidine at 0.76 mg / ml.
• the cell suspensions are distributed in the wells of plastic culture plates (NUNCLON, 96 wells) at a rate of 200 ⁇ l per well and containing 10,000 cells.
• the supernatant from each well is removed and subjected to screening by the immunoenzymatic reaction "ELISA". It is carried out firstly using the reagent based on conjugated anti-mouse Ig antibody coupled to urease and then confirmed by the conjugated anti-mouse Ig antibody coupled to alkaline phosphatase.
• the cells serving as targets are spleen cells previously incubated with the peptide NP 147-158R " (5
• the wells having given an optical density (OD) value greater than at least twice that of the negative control reaction of the target cells with the conjugated antibody without culture supernatant are selected.
• OD optical density
• Supernatants from the same wells are also tested on cells not covered with peptide and the same haplot-ype to ensure that the reaction observed is not due to "autoantibodies”.
• the test on the peptide in question, fixed alone (without cells) directly on the plate thus confirms that the selected supernatants react only with the cells covered with peptide.
• Three hybridomas were obtained having the characteristics of the so-called restricted type antibodies, in the sense that they react only with spleen cells of a precise haplotype (H-2 d ) covered with peptide (NP 147-158R " ).
• three clones are named N6.6, S.2.2 and X5.3 of the isotype IgG 2b , K, IgG 3 , K and IgG 2b , K respectively.
• the purpose of the experiment is to test the capacity of a restricted anti-peptide antibody, to inhibit the in vivo growth of tumor cells which have been brought into contact with the specific peptide.
• DBA / 2 mice (H-2 d ) are divided into 4 groups each comprising four mice. They received, by subcutaneous injection, a suspension of cells of the mastocytoma P815 (these are transplantable tumor cells which, once injected into syngeneic hosts (DBA / 2 mice) cause the development of a lethal tumor).
• mice in the second group received P815 cells mixed with restricted antibody at 5 / .g / 10 6 cell (this is the antibody X5.3 under X.5.3.7.T clone ). These mice serve as controls having received cells and antibodies alone.
• mice of the 3rd group received P815 cells incubated for 1 hour at 37 ⁇ C with the peptide NPR " of the nucleoprotein of the influenza virus at a rate of 10 ⁇ g / 10 6 cells. It is the control group which received the cells and the peptide alone.
• mice of the 4th group received P815 cells incubated with the peptide and then placed in the presence of the restricted antibody X5.3.7.T under the same conditions and proportions as the control groups 2 and 3.
• the same number of P815 cells (10 5 cells / mouse) is injected into each mouse which is monitored individually for the development of a subcutaneous tumor. The results are expressed on the one hand in average diameter of the tumors for each group, in percentage of mice having a visible tumor and finally in percentage of lethal tumors.
• the number of lethal tumors for each group at the end of the experiment is as follows:
• a hybridoma (9.3.2) was obtained, which recognizes in ELISA test and in complementary dependent cytotoxicity test, the peptide 120K in the presence of lymphocytes expressing H- 2 K 6 .
• the controls constituted either by cells expressing H-2 K 6 in the absence of the peptide or preparations where the peptide (I20K) is incubated with cells carrying a different haplotype (H-2 Y?) Are not recognized.
• the technique was modified by adding a secondary antibody, in this case, the rabbit anti-mouse immunoglobulin antibody. Then the Rabbit supplement is added.
• the secondary antibody which has recognized the X5.3 antibody activates the complement which allows lysis.
• the restricted antibody called X5.3, subclone X5.5.7 / T lyses 30% of specific targets (P815-H-2 d cells + peptide) and 0% when the cells P815 are not brought into prior contact with the peptide.
• Target cells (cells + peptide) or controls (cells without peptide) are labeled with radioactive iodine 125 by the method using the enzyme lactoperoxidase (Marchalonis, JJ, Biochem. J., 1969, 113: 291). After labeling, the cells are lysed with a detergent, Triton X-100 in the presence of enzyme inhibitors. The lysate is incubated with an unbound antibody (control antibody) in the presence of the sepharose-protein A beads. After centrifugation, the lysate is incubated with the specific antibody (restricted antibody). The complex is adsorbed on Sepharose-protein A beads, washed, recovered and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).
• SDS-PAGE polyacrylamide gel electrophoresis
• a soluble H-2 molecule (devoid of the transmembrane part) obtained by genetic engineering was produced and emptied of its endogenous peptide. It is used to house the peptide of the nucleoprotein of the influenza virus inside. This molecule serves as a target for the antibody restricted in the ELISA or radioimmunoassay (RIA) technique using protein A labeled with iodine 125 for example.
• RIA radioimmunoassay

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