WO1991012332A1 - Monoclonal antibodies for recognizing a peptide linked to a major histocompatibility antigen - Google Patents

Monoclonal antibodies for recognizing a peptide linked to a major histocompatibility antigen Download PDF

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Publication number
WO1991012332A1
WO1991012332A1 PCT/FR1991/000121 FR9100121W WO9112332A1 WO 1991012332 A1 WO1991012332 A1 WO 1991012332A1 FR 9100121 W FR9100121 W FR 9100121W WO 9112332 A1 WO9112332 A1 WO 9112332A1
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Prior art keywords
peptide
monoclonal antibodies
restricted
cells
restricted monoclonal
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PCT/FR1991/000121
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French (fr)
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Guy Huynh Thien Duc
Pierre Rucay
Philippe Kourilsky
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Institut National De La Sante Et De La Recherche Medicale
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Publication of WO1991012332A1 publication Critical patent/WO1991012332A1/en

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    • 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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Abstract

Restricted monoclonal antibodies characterized by their ability to recognize specifically a complex consisting of a peptide which is characteristic of a pathogenic agent antigen or a cell derangement, and a Major Histocompatibility Complex (MHC) molecule having the ability to recognize and fix this peptide, said antibodies being restricted monoclonal antibodies in that they are unable to recognize said peptide combined with a non peptide specific haplotype MHC molecule. These antibodies can be used to diagnose conditions caused by infectious agents or cell derangements to be found for example in tumors or autoimmune diseases.

Description

Anticorps monoclonaux reconnaissant un peptide associé à un antigène majeur d'histocompatibilite.Monoclonal antibodies recognizing a peptide associated with a major histocompatibility antigen.
L'invention concerne des anticorps monoclonaux restreints reconnaissant un peptide associé à un antigène majeur d'histocompatibilite. L'invention concerne aussi les applications de ces anticorps au diagnostic et au traitement et le cas échéant à la prévention de certaines pathologies.Disclosed are restricted 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.
On connait l'intérêt des molécules du Complexe Majeur d'Histocompatibilité (molécules du CMH) en tant que structures me branaires de reconnaissance permettant au système- immunitaire de reconnaître le soi du non-soi. Une fonction majeure des molécules de CMH est de présenter des antigènes aux lymphocytes T.We know the interest of the molecules of the Major Histocompatibility Complex (MHC molecules) as me bran structures of recognition allowing the immune system to recognize the self from the non-self. A major function of MHC molecules is to present antigens to T lymphocytes.
Selon l'appartenance de la molécule du CMH à la classe I ou à la classe II, le complexe formé par la molécule du CMH et l'antigène est reconnu respectivement par dès cellules T cytotoxiques ou par des cellules T auxiliaires capables d'activer la synthèse de l'anticorps par les lymphocytes B.Depending on whether the MHC molecule belongs to class I or to class II, 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.
Des travaux ont déjà permis de mettre en évidence l'existence d'anticorps reconnaissant un antigène associé à une molécule de CMH. Par exemple. Van Leuveen et al (J\ Exp. Med. (1979) 150, 1075-1083) ont décrit l'existence, chez une malade atteinte d'anémie, d'un sérum polyclonal contenant des anticorps capables de lyser spécifiquement des lymphocytes portant 1•antigène H-Y associé à une molécule de CMH du type HLA-A2.Studies have already made it possible to demonstrate the existence of antibodies recognizing an antigen associated with a MHC molecule. For example. Van Leuveen et al (J \ Exp. Med. (1979) 150, 1075-1083) described the existence, in a patient suffering from anemia, of a polyclonal serum containing antibodies capable of specifically lysing lymphocytes carrying 1 • HY antigen associated with a MHC molecule of the HLA-A2 type.
Cependant les résultats connus jusqu'à présent montrent qu'il n'a pas été possible de produire ni d'identifier et de * caractériser des anticorps monoclonaux restreints, c'est-à-dire spécifiques du complexe formé par un antigène déterminé et une molécule de CMH spécifique de cet antigène, également déterminée.However, the results known so far show that it has not been possible to produce, identify and * characterize restricted monoclonal antibodies, that is to say specific for the complex formed by a determined antigen and a MHC molecule specific for this antigen, also determined.
A cet égard, plusieurs équipes ayant tenté de mettre au point des modèles expérimentaux en vue de produire des anticorps monoclonaux restreints, ont publié des résultats négatifs.In this regard, several teams having tried to develop experimental models with a view to producing restricted monoclonal antibodies, have published negative results.
Par exemple, l'équipe de Tamminen et al utilisant comme antigène du virus grippal Influenza type A, souche H3N2 (X-31) (Tamminen et al., Eur. J. Immunol. (1987) JL7, 999-1006) et également le groupe de Rubin, Malissen, Jorgensen et Zeuthen utilisant comme antigène l'insuline (Rubin et al., Res. Immunol. (1989) 140, ...) ont publié des résultats négatifs, c'est-à-dire qu'ils n'ont pas réussi à produire des anticorps restreints. L'équipe de Klinman a décrit des résultats positifs ( ylie et al., J. Exp. Med. (1982) 155, 403-414 ; Frescher and Klinman, J. Exp. Med. (1986) 164, 196-210) . Cependant les travaux de Klinman et al. montrent que les anticorps monoclonaux produits reconnaissent des cellules transformées par un virus oncogène SV40, sans que l'antigène reconnu, ni la molécule du CMH impliquée, soient identifiées.For example, the team at Tamminen et al using the influenza A type H3N2 (X-31) influenza virus antigen (Tamminen et al., Eur. J. Immunol. (1987) JL7, 999-1006) and also the group of Rubin, Malissen, Jorgensen and Zeuthen using insulin as antigen (Rubin et al., Res. Immunol. (1989) 140, ...) published negative results, that is to say that they failed to produce restricted antibodies. Klinman's team described positive results (ylie et al., J. Exp. Med. (1982) 155, 403-414; Frescher and Klinman, J. Exp. Med. (1986) 164, 196-210) . However, the work of Klinman et al. show that the monoclonal antibodies produced recognize cells transformed by an oncogenic virus SV40, without the recognized antigen, nor the MHC molecule involved, being identified.
Les difficultés rencontrées pour la préparation des anticorps monoclonaux restreints selon l'invention peuvent être attribuées à différents facteurs et en particulier à la mise au point d'une technique particulière de préparation des anticorps et également à des techniques très sensibles pour le criblage.The difficulties encountered in the preparation of the restricted monoclonal antibodies according to the invention can be attributed to various factors and in particular to the development of a particular technique for preparing the antibodies and also to very sensitive techniques for screening.
Les inventeurs se sont intéressés au rôle que pourraient présenter des anticorps monoclonaux restreints pour le diagnostic mais aussi le traitement de différentes pathologies.The inventors are interested in the role that could be restricted monoclonal antibodies for the diagnosis but also the treatment of different pathologies.
Ils ont été capables de remédier aux difficultés rencontrées jusqu'à présent et ont obtenu des anticorps monoclonaux restreints caractéristiques d'un complexe constitué par un antigène en association avec une molécule du CMH reconnaissant spécifiquement cet antigène.They have been able to remedy the difficulties encountered so far and have obtained restricted monoclonal antibodies characteristic of a complex constituted by an antigen in association with a MHC molecule specifically recognizing this antigen.
L'invention a donc pour objet des anticorps monoclonaux restreints reconnaissant des antigènes associés à des molécules du CMH. Entrent également dans le cadre de l'invention, des souches d'hybridomes productrices des anticorps monoclonaux restreints.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.
L'invention concerne aussi des compositions pour le diagnostic de la présence d'une infection ou d'un dérèglement cellulaire- susceptible de générer un état tumoral ou une maladie autoimmune.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.
L'invention se rapporte aussi à des compositions pour le traitement d'infections ou de dérèglements cellulaires tels que décrits ci-dessus.The invention also relates to compositions for the treatment of infections or cellular disturbances as described above.
Elle vise de plus un procédé pour la production des anticorps monoclonaux restreints de l'invention.It further relates to a process for the production of the restricted monoclonal antibodies of the invention.
Les antigènes dont il est question dans les paragraphes précédents et qui constituent l'une des deux entités du complexe reconnu par les anticorps restreints selon l'invention, sont soit des peptides tels qu'issus de la dégradation de protéines provenant d'un agent infectieux ou pathogène soit des peptides issus d'un dysfonctionnement (dérégulation) cellulaire ou encore des protéines dégradées du soi. Ces peptides peuvent par exemple être obtenus par coupure enzymatique par exemple par les enzymes du lysosome ou de 1'appareil de Golgi.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.
La formation du complexe nécessite également que le peptide et la molécule du CMH soient capables de se lier entre elles. »The formation of the complex also requires that the peptide and the MHC molecule are capable of binding together. "
Pour la préparation d'anticorps monoclonaux restreints, ces peptides sont avantageusement obtenus par voie de synthèse chimique selon les méthodes classiques de synthèse des peptides.For the preparation of restricted monoclonal antibodies, these peptides are advantageously obtained by chemical synthesis according to conventional methods of synthesis of peptides.
Ils peuvent aussi être préparés à partir de protéines naturelles par exemple des protéines issues _They can also be prepared from natural proteins, for example proteins from _
4 d'agents pathogènes ou des protéines mises en jeu lors de dérèglements cellulaires de type tumoral ou de type auto-i mun, notamment des protéines mutées.4 pathogens or proteins involved in cellular disorders of the tumor type or of the auto-mun type, in particular mutated proteins.
Les anticorps monoclonaux de l'invention sont caractérisés par leur capacité à reconnaître spécifiquement un complexe formé par un peptide caractéristique d'un antigène d'un agent pathogène ou un peptide caractéristique d'une dérégulation cellulaire et par une molécule du Complexe Majeur d'Histocompatibilité (CMH) ayant la capacité de reconnaître et de fixer ce peptide, lesdits anticorps étant des anticorps monoclonaux restreints, dès lors qu'ils ne reconnaissent pas ledit peptide en association avec une molécule de CMH d'haplotype qui ne fixe pas le peptide (non spécifique du peptide) .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).
Pour déterminer la spécificité du couple peptide-molécule du CMH de classe I, on peut effectuer un test tel que décrit par BOUILLOT et al, (Nature, 1989, vol. 339, p.473-475) . Conformément à ce test, un peptide dont on recherche la spécificité vis-à-vis de la molécule de CMH est incubé avec une molécule de CMH connue, sur des cellules marquées avec un isotope radioactif tel que le chrome 51. Après cette incubation, la suspension est mise en présence de lymphocytes cytotoxiques (CTL) spécifiques du peptide. Lorsque les cellules CTL lysent les cellules mises en présence du peptide et de la molécule du CMH, on peut en déduire que la molécule du CMH a une affinité spécifique pour le peptide.To determine the specificity of the MHC class I peptide-molecule pair, a test can be carried out as described by BOUILLOT et al, (Nature, 1989, vol. 339, p.473-475). In accordance with this test, 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. When the CTL cells lyse the cells brought into contact with the peptide and the MHC molecule, it can be deduced therefrom that the MHC molecule has a specific affinity for the peptide.
De la même manière, il existe divers tests qui permettent de repérer l'association fonctionnelle et spécifique d'un peptide à un antigène du CMH de classe II. On peut par exemple, mettre en oeuvre le test de Sette et al (P.N.A.S. (1989), Vol. 86, p3296-3300) .Similarly, there are various tests that identify the functional and specific association of a peptide with a MHC class II antigen. One can for example use the test of Sette et al (P.N.A.S. (1989), Vol. 86, p3296-3300).
Les anticorps ci-dessus définis sont désignés par le terme anticorps monoclonaux restreints. Selon un mode de réalisation particulier de l'invention, les anticorps monoclonaux restreints sont encore caractérisés en ce qu'ils sont dépourvus ou pratiquement dépourvus de réaction à l'égard du peptide contre lequel ils sont dirigés, présent à l'état isolé et/ou en ce qu'ils reconnaissent faiblement ou pas du tout, la molécule du CMH seule.The antibodies defined above are designated by the term restricted monoclonal antibodies. According to a particular embodiment of the invention, 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.
Un anticorps pratiquement dépourvu de la capacité à reconnaître un peptide seul est selon l'invention, un anticorps qui présente avec ce peptide une réaction inférieure à deux fois le signal correspondant au bruit de fond.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.
Un anticorps reconnaissant faiblement une molécule de CMH seule reconnaît cette molécule de CMH suivant une réaction qui est de l'ordre de 1/10 à 1/5 de la réaction existant dans les mêmes conditions avec le complexe peptide-CMH spécifique.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.
Pour déterminer si un anticorps monoclonal obtenu répond aux définitions de l'invention, une réaction immunoenzymatique de type ELISA, telle que décrite ci-après à titre d'exemple, peut être réalisée.To determine whether a monoclonal antibody obtained meets the definitions of the invention, an ELISA type immunoenzymatic reaction, as described below by way of example, can be carried out.
La réaction immunoenzymatique est effectuée d'abord au moyen du réactif à base d'anticorps conjugué anti-Ig de souris couplé soit à l'uréase, soit à la phosphatase alcaline, sur un surnageant de culture d'hybridomes. La réaction est ensuite confirmée par l'anticorps conjugué anti-Ig de souris couplé par exemple à la phosphatase alcaline. Les cellules servant de cibles sont par exemple des cellules spléniques préalablement incubées avec le peptide contre lequel on veut former les anticorps (5 à 50μg/ml pour 107 cellules à 37βC pendant une à deux heures) . Les cellules sont fixées sur plaques de polyvinyle selon la technique décrite par Ternynck et Avrameas (Ternynck et Avrameas: Techniques immuno- enzy atiques - Les éditions INSERM, 1987) au moyen du poly-L-lysine.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 μg / ml for 10 7 cells at 37 β 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 éditions INSERM, 1987) using poly-L-lysine.
Des témoins utilisés pour vérifier l'appartenance des anticorps monoclonaux restreints à 1'invention et qui par conséquent ne doivent pas donner une réaction positive avec les anticorps monoclonaux retreints de 1'invention sont constitués d'une part par 1•un ou l'autre des constituants pris séparément, à savoir le peptide contre lequel on souhaite former des anticorps monoclonaux restreints ou les cellules portant la molécule du CMH correspondante seule sans ledit peptide, et d'autre part par 1*un ou 1'autre de ces constituants en association avec un partenaire non relié c'est-à-dire non spécifique (le peptide différent de celui contre lequel on veut obtenir des anticorps monoclonaux restreints associé à la molécule de CMH étudiée ou le peptide contre lequel on souhaite produire des anticorps monoclonaux restreints, associé à une molécule du CMH non spécifique) .Witnesses used to verify the membership of the monoclonal antibodies restricted to the invention and which therefore should not give a positive reaction with the monoclonal antibodies restricted from the invention consist, on the one hand, of one or the other. constituents taken separately, namely the peptide against which it is desired to form restricted monoclonal antibodies or the cells carrying the corresponding MHC molecule alone without said peptide, and on the other hand by one or the other of these constituents in combination with an unrelated partner, that is to say nonspecific (the peptide different from the one against which we want to obtain restricted monoclonal antibodies associated with the MHC molecule studied or the peptide against which we want to produce restricted monoclonal antibodies, associated to a non-specific MHC molecule).
La réaction positive ci-dessus est marquée par une densité optique (D.O) supérieure à au moins deux fois celle observée pour les puits qui contiennent tous les composants de la réaction excepté les anticorps restreints.The above positive reaction is marked by an optical density (O.D) greater than at least twice that observed for the wells which contain all of the components of the reaction except the restricted antibodies.
Les anticorps monoclonaux restreints de l'invention, ont l'avantage de présenter une sélectivité importante ce qui les rend particulièrement intéressants dans leur application au diagnostic, au traitement et le cas échéant à la vaccination.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.
Les peptides reconnus par les anticorps de 1•invention peuvent être caractéristiques d'agents pathogènes tels que des virus, des bactéries, des parasites, par exemple le plasmodium ou des champignons notamment des levures pathogènes. Il peut s'agir également des peptides représentant des antigènes mineurs d'histo compatibilité, de peptides caractéristiques de dérèglements cellulaires de type tumoral ou cancéreux ou encore de type auto-immum.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.
Selon un mode de réalisation préféré de l'invention, les anticorps monoclonaux restreints sont caractérisés en ce que les peptides reconnus présentent de 7 à 25, de préférence une dizaine d'acides aminés.According to a preferred embodiment of the invention, the restricted monoclonal antibodies are characterized in that the recognized peptides have from 7 to 25, preferably about ten amino acids.
Pour la présentation de certains antigènes par les molécules du CMH, la présence d'un peptide ayant une longueur en acides aminés voisine de ce qui est donné dans ce qui précède peut suffire à la réaction de fixation avec les molécules de CMH et à celle de reconnaissance par les anticorps de l'invention.For the presentation of certain antigens by MHC molecules, 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.
Pour la préparation et la détermination des anticorps monoclonaux restreints selon 1'invention on peut également avoir recours à des antigènes constitués par un peptide associé à une forme solubilisée et/ou solubie de la molécule de CMH qui lui est spécifique pour une espèce donnée. Dans le cas de l'utilisation de la forme solubilisée, celle ci est préparée par solubilisation avec un détergent ionique tel que le triton X100 ou le NP40 ou avec un détergent non ionique tel que l'octyl -3D-glycopyranoside, la solubilisation étant suivie d'une chromatographie d'affinité. Pour préparer la molécule sous forme solubilisée on se référera à la technique décrite par STALLCUP K et al (J. of I munology, 1981, vol. 127, page 923) .For the preparation and determination of the restricted monoclonal antibodies according to the invention, it is also possible to use 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. In the case of the use of the solubilized form, 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. To prepare the molecule in solubilized form, reference will be made to the technique described by STALLCUP K et al (J. of I munology, 1981, vol. 127, page 923).
Dans le cas de l'utilisation de la molécule soluble de CMH celle ci est obtenue dépourvue de sa partie traπsanembranaire par génie génétique.In the case of the use of the soluble MHC molecule, this is obtained devoid of its traπsanembrane component by genetic engineering.
Cette molécule soluble peut être préparée par un hôte cellulaire modifié par insertion du gène codant pour la molécule de CMH que l'on souhaite exprimer, ce gène étant dépourvu de la séquence codant pour la partie transmembranaire de la molécule. Compte tenu de la suppression de cette séquence, on lie la chaîne lourde et la chaîne légère de la molécule de CMH avec un polylinker de type GGGS. Le gène est incorporé dans l'hôte dans des conditions telles que ses éléments de régulation sont reconnus par l'hôte cellulaire. Un hôte cellulaire approprié est par exemple une cellule d'insecte.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.
Des anticorps monoclonaux restreints particuliers sont caractérisés en ce que le peptide reconnu est spécifique d'un rétrovirus humain HIV et notamment en ce qu'il s'agit des peptides de la protéine gag par exemple le peptide gag5 (GHQAAMEMLKE) ou le peptide gag p25 synthétisé par Neosystem (Strasbourg, référence SP89-158) (séquence 263-277) , ou des peptides de la protéine interne pl4 (nucléoprotéine) notamment le peptide K16F décrit par Claverie JM et al dans Eur jour Imm 1988 vol 18 p 1547-1553.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 Eur jour Imm 1988 vol 18 p 1547-1553 .
D'autres anticorps monoclonaux restreints selon l'invention sont dirigés contre des peptides tels que les suivants :Other restricted monoclonal antibodies according to the invention are directed against peptides such as the following:
.le peptide de 1'autoantigène de la protéine basique de myéline MPB 1 à 11 décrit par raith.D et al dans (Cell 1989 vol. 59 p 247-255),the peptide of the autoantigen of the basic protein of myelin MPB 1 to 11 described by raith.D et al in (Cell 1989 vol. 59 p 247-255),
.le peptide de la sous-unité α du récepteur de l'acétylcholine décrit par Mozes et al (EMBO journal 1989 vol 8 p 4049-4052),the peptide of the α subunit of the acetylcholine receptor described by Mozes et al (EMBO journal 1989 vol 8 p 4049-4052),
•le peptide de la chaîne α de l'insuline décrit par Gradehandt et al, (Immunological Reviews, (1988) vol. 106, p59 à 75) ,The insulin α chain peptide described by Gradehandt et al, (Immunological Reviews, (1988) vol. 106, p59 to 75),
.le peptide de l'enveloppe du virus de l'hépatite B, Chisari et al (Cell 1989 vol 59 p 1145-1156) .the hepatitis B virus envelope peptide, Chisari et al (Cell 1989 vol 59 p 1145-1156).
Des anticorps selon l'invention peuvent aussi être caractérisés en ce que la molécule du CMH humain reconnue est de divers haplotypes (par exemple HLA-A2, HLA-A3, HLA-B7, HLA-B12, HLA-B27, HLA-B37, HLA-C 3) de classe I comme de classe II (par exemple HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5) .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).
Un anticorps monoclonal restreint particulier selon l'invention est l'anticorps qui reconnaît un peptide de la protéine p25 (gag) de HIV, notamment le peptide gag5, en association avec une molécule de CMH d'haplotype HLA-B 7 ou HLA-A11.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 .
L'invention concerne aussi un procédé pour la préparation d'anticorps monoclonaux restreints de l'invention. Un premie procédé adapté à la production de ces anticorps monoclonaux restreints est caractérisé en ce que: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:
- on fusionne des cellules spléniques d'un animal préalablement immunisé avec des cellules spléniques isogèniques recouvertes d'un peptide déterminé, avec des cellules de myélόme en présence d'un promoteur de fusion, par exemple le polyéthylène glycol, les cellules spléniques étant en excès par rapport aux cellules myélomateuses, par exemple dans une proportion de 10 pour 1 environ,- 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,
- on réalise un criblage afin de sélectionner ceux des hybridomes capables de produire des anticorps monoclonaux restreints, par exemple par la technique ELISA, effectuée avec un réactif à base d'anticorps conjugué anti-Ig de souris couplé à une enzyme par exemple l'uréase, ou la phosphatase alcaline ou la peroxydase,- 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,
- on récupère et on clone les hybridomes ainsi sélectionnés.- the hybridomas thus selected are recovered and cloned.
Une autre technique telle que la technique par la cytotoxicité peut être utilisée pour le criblage.Another technique such as the cytotoxicity technique can be used for screening.
Entrent également dans le cadre de 1'invention les hybridomes producteurs des anticorps monoclonaux restreints ci-dessus. Ces hybridomes sont les produits de fusion de cellules de myélomes et de cellules spléniques d'un animal préalablement immunisé avec le peptide-antigène, dans des conditions telles que l'antigène contre lequel sont formés les anticorps, est constitué par un complexe comportant le peptide ci-dessus associé à une molécule de CMH.Also within the scope of the invention are the 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.
De préférence les hybridomes résultent de la fusion des cellules spléniques ci-dessus décrites avec un myélome de type HGPRT* par exemple: X63-Ag8, NS-1 ou SP2/0.Preferably, 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.
La réalisation des hybridomes est faite d'après le protocole de Kohler et Milstein (Nature, 1974, 256:495- 497) .Hybridomas are produced according to the protocol of Kohler and Milstein (Nature, 1974, 256: 495-497).
Les cellules lymphocytaires ou non, normales ou tumorales apportant la molécule de CMH sont de préférence d'haplotype H-2d, H-2b, H-2 , H-2C' ou H-2S chez la souris, mais il pourra s'agir également de cellules exprimant des molécules de CMH humaines (ou d'une autre espèce) chez des souris transgéniques (exprimant le même CMH humain) ou non transgéniques.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.
L'invention se rapporte aussi à des cellules lymphocytaires ou non, humaines ou animales, par exemple de souris, recouvertes d'un peptide ou d'un polypeptide de préférence immunogène, contre lequel on veut préparer des anticorps monoclonaux restreints.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.
Un second procédé pour la production des anticorps monoclonaux restreints selon 1»invention consiste à réaliser la fusion entre des cellules B du sang immortalisées avec le virus d'Epstein Barr et des lymphocytes B humains préalablement mis en contact avec des peptides contre lesquels on cherche à former des anticorps monoclonaux restreints.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.
Les cellules B préalablement mises au contact des peptides contre lesquels on cherche à former des anticorps monoclonaux, peuvent être prélevées dans le sang périphérique d'un donneur préalablement immunisé avec le peptide, lorsqu'il n'est pas toxique pour ce donneur. Ces lymphocytes B peuvent aussi être obtenus par culture in vitro au contact des peptides, la récupération des cellules B recouvertes de peptides étant précédée d'un ou plusieurs cycles de stimulation.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.
Enfin, les anticorps restreints ou des molécules analogues telles que la partie Fab de ces anticorps, ou des analogues des récepteurs des cellules T ayant la capacité de reconnaître le complexe CMH-peptide peuvent être obtenus dans E.coli ou d'autres micro - organismes, selon les techniques de Huse et al (Science, (1989) , 246, 1275) , ard et al (Nature, (1989), 341, 544) Bird et al (Science, (1988), 242, 423); ou d'autres auteurs.Finally, 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.
Conformément à ces techniques, on introduit à l'aide de vecteurs appropriés, les gènes amplifiés par la technique PCR, codant pour les anticorps monoclonaux restreints ou les gènes codant pour les récepteurs de cellules T spécifiques murins ou humains ou d'autres espèces, dans E.coli, dans des conditions permettant leur expression. Puis, on réalise un criblage afin de déterminer celles des molécules exprimées qui présentent la spécificité requise pour la reconnaissance du complexe peptide-molécule du CMH. Le criblage peut être effectué selon la méthode décrite précédemment.In accordance with these techniques, 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.
La technique ci-dessus peut également être mise en oeuvre en introduisant des séquences de gènes codant pour les anticorps restreints ou les récepteurs de cellules T spécifiques, ou encore des mutants de ces séquences.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.
Les gènes ou fragments de gènes codant pour les anticorps monoclonaux restreints sont obtenus par extraction de l'ADN des cellules immunes, dont la préparation est décrite ci-dessus. L'invention se rapporte donc à des anticorps ou à des analogues, produits dans E.coli ou dans d'autres micro-organismes, à des anticorps monoclonaux restreints humains ou d'autres espèces (par exemple obtenus chez le rat ou le hamster par des techniques analogues à celles décrites précédemment ou obtenus selon les méthodes de Borrebaeck et al, P.N.A.S. (1988), vol. 85, p.3995-3999) .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).
Les anticorps de l'invention présentent un grand intérêt pour différentes applications. Ils peuvent être utilisés par exemple pour des applications au diagnostic d'infections ou pour le diagnostic de dérèglements cellulaires tels qu'ils se manifestent dans certains cancers ou maladies autoimmunes.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.
L'invention se rapporte à cet égard à une composition pour le diagnostic de la présence de peptides associés à des molécules de CMH dans un échantillon biologique testé, caractérisée en ce qu'elle comprend des anticorps monoclonaux restreints tels que décrits précédemment.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.
Cette composition de diagnostic peut être mise en oeuvre soit sur un échantillon biologique, soit dans un organisme vivant.This diagnostic composition can be implemented either on a biological sample or in a living organism.
Selon cette dernière application, on aura recours aux techniques de diagnostic pratiquées en imagerie médicale.According to this latter application, diagnostic techniques practiced in medical imaging will be used.
Dans cette hypothèse, l'anticorps monoclonal restreint est marqué par une substance radioactive ou par une substance pouvant être rendue visible par les techniques de fluorochrome ou de LASER.In this hypothesis, the restricted monoclonal antibody is labeled with a radioactive substance or with a substance which can be made visible by fluorochrome or LASER techniques.
Pour le diagnostic d'une pathologie déterminée, dans laquelle interviennent des antigènes tels que décrits précédemment, il est nécessaire de prendre en considération l'haplotype des molécules du CMH présentes chez le patient chez lequel est réalisée la détection. L'invention vise aussi un kit pour le diagnostic in vitro sur un échantillon biologique d'une infection par un organisme pathogène ou pour le diagnostic d'un cancer, d'une maladie autoimmune ou d'un autre dérèglement cellulaire, caractérisé en ce qu'il comprend: des anticorps monoclonaux restreints selon l'invention, ayant la spécificité recherchée compte tenu du diagnostic à ïeffectuer,For the diagnosis of a specific pathology, in which antigens as described above intervene, it is necessary to take into consideration the haplotype of the MHC molecules present in the patient in whom the detection is carried out. 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,
- des moyens pour révéler la présence d'un conjugué de type complexe (antigène-molécule ou peptide-molécule du CMH) lié avec l'anticorps.- Means for revealing the presence of a complex type conjugate (antigen-molecule or peptide-molecule of the MHC) linked with the antibody.
L'échantillon biologique utilisé est avantageusement un échantillon de sérum ou tout autre fluide biologique.The biological sample used is advantageously a sample of serum or any other biological fluid.
Entre également dans le cadre de l'invention un test pour la détection in vitro d'une infection ou d'un dérèglement cellulaire, comprenant les étapes suivantes:Also included within the scope of the invention is a test for the in vitro detection of an infection or a cellular disturbance, comprising the following steps:
- mise en coptact de l'échantillon biologique avec des anticorps selon l'invention,- placing the biological sample in coptact with antibodies according to the invention,
- détection de la présence d'un conjugué du type anticorps-complexe (antigène-molécule ou peptide- molécule du CMH) .- detection of the presence of an antibody-complex type conjugate (antigen-molecule or peptide-molecule of the MHC).
L'application au diagnostic, des anticorps de l'invention peut être utilisée pour détecter un virus tel que le virus HIV du SIDA, ou encore pour détecter des tumeurs ou des dérèglements autoimmuns.Application to the diagnosis, 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.
Selon un mode de réalisation de l'invention on peut aussi rendre les anticorps monoclonaux restreints cytotoxiques.According to one embodiment of the invention, the restricted monoclonal antibodies can also be made cytotoxic.
Les anticorps dé 1•invention peuvent alors entrer à titre de principe actif dans une composition pharmaceutique pour le traitement des infections ou des dérèglements ci-dessus par exemple par compétition notamment dans le cas où ils ne sont pas cytotoxiques. Dans ce cas, ils sont mélangés avec un véhicule pharmaceutique acceptable.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.
Des substances capables de rendre les anticorps cytotoxiques sont des toxines, des antibiotiques, des isotopes radioactifs.Substances capable of making cytotoxic antibodies are toxins, antibiotics, radioactive isotopes.
De tels anticorps peuvent être utilisés en immunothérapie.Such antibodies can be used in immunotherapy.
L'invention vise aussi une composition pharmaceutique, caractérisée en ce qu'elle comprend, à titre de principe actif, des anticorps monoclonaux restreints conformes à l'invention. De tels anticorps formulés dans une composition pharmaceutique pourraient être utilisés notamment comme agents compétiteurs pour bloquer l'action de cellules T spécifiques, particulièrement dans des pathologies autoimmunes.The invention also relates to 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.
Entre également dans le cadre de 1*invention l'application des anticorps monoclonaux restreints à titre de vaccin, pour stimuler la production d'anticorps anti-idiotypiques notamment dans le cas des maladies autoimmunes.Also within the scope of the invention is the application of the restricted monoclonal antibodies as a vaccine, to stimulate the production of anti-idiotypic antibodies, in particular in the case of autoimmune diseases.
Des compositions vaccinantes selon 1*invention comprennent donc à titre de principe actif, des AcM restreints ci-dessus décrits. Les anticorps anti- idiotypiques produits par l'injection des AcM restreints reconnaissent et éliminent les cellules T autoréactives.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.
D'autres caractéristiques et avantages de l'invention apparaissent dans les exemples qui suivent. EXEMPLESOther characteristics and advantages of the invention appear in the examples which follow. EXAMPLES
ETABLISSEMENT DES HYBRIDOMES SECRETANT DES ANTICORPS DE TYPE RESTREINT.ESTABLISHMENT OF HYBRIDOMAS SECRETING ANTIBODIES OF THE RESTRICTED TYPE.
ImmunisationImmunization
Des souris de lignée BALB/c (H-2d) sont immunisées par injection intra-péritonéale i.p. de cellules spléniques isogéniques couvertes de peptide. Il s'agit en l'occurrence du peptide de la nucléoprotéine du virus grippal dénommé NP147-158R". Les cellules spléniques sont incubées au préalable avec le peptide à raison de 107 cellules pour lOOμg de peptide dans un volume de 1ml de milieu de culture (DMEM + 2% de sérum de veau foetal). L'incubation se fait dans une étuve à C02 (5%) pendant 1 heure. Les souris reçoivent 3 injections intra-péritonéales à raison d'une injection par semaine. Un intervalle de 3 semaines sépare la 3ème de la 4ème injection qui précède le sacrifice et le prélèvement de la rate. Trois jours séparent la 4ème injection du prélèvement de la rate. La 4ème injection se fait par deux voies: 0,5ml de la suspension est injectée par voie intra- péritonéale, 0,5ml par voie intra-veineuse. Fusion cellulaireBALB / 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 μg 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. interval of 3 weeks separates the 3rd from the 4th injection which precedes the sacrifice and the removal of the spleen. Three days separate the 4th injection from the removal of the spleen. The 4th injection is done by two routes: 0.5 ml of the suspension is injected intraperitoneally, 0.5 ml intravenously. Cell fusion
Les cellules spléniques sont obtenues à partir de la rate après dissociation en milieu DMEM + 5% de sérum de veau foetal. Les globules rouges sont lysés au moyen du chlorure d'ammonium (NH4C1) à 0,83% et les lymphocytes sont ensuite lavés trois fois en DMEM + 5% de sérum de veau foetal.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.
Le partenaire de fusion est constitué par 3 myélomes HGPRT", à savoir X63-Ag8, NS-1 et SP2/0. Autrement dit, les lymphocytes sont fusionnés soit avec le myélome X63, NS-1 ou SP2/0 séparément. La fusion se fait au moyen du polyéthylène glycol (PEG 1500) de la firme MERCK, à raison de 10 lymphocytes pour une cellule de myélome. Le protocole est basé sur celui décrit par Kôhler et Milstein (Nature, 1974, 256:495-497) rappelé ci-après: les lymphocytes et les cellules de myélome (X63, NS-1 et SP2/0) sont mis ensemble dans la proportion indiquée en haut. Les trois préparations (lymphocytes plus X63 ou plus NS-1 ou plus SP2/0) sont centrifugées pendant 10 minutes à 250g et à 4*C. Les surnageants sont enlevés en laissant cependant un film de liquide au-dessus du culot cellulaire. Les tubes sont mis à 37βC dans un bain marie. On ajoute goutte à goutte 1ml d'une solution de polyéthylène glycol 1500 (solution de 50% de PEG 1500 dans RPMI à pH 7) sur le culot cellulaire. L'opération devant durer 1 minute, est suivie par l'addition de 20ml de DMEM plus 10% de sérum de veau foetal. Le tout est laissé encore trois minutes dans le bain marie à 37βC puis les tubes sont centrifugés pendant 15 minutes à 250g et à +4'C. Les surnageants sont enlevés et les cellules sont lavées une fois avec 20ml de DMEM +10% de sérum de veau foetal. Les cellules sont remises ensuite dans une solution de DMEM plus 10% de sérum de veau foetal auquel est ajouté le milieu HAT (solution mère HAT 100X: Hypoxanthine à l,36mg/ml, Aminoptérine à 0,018mg/ml et Thymidine à 0,76mg/ml). Les suspensions cellulaires sont distribuées dans les puits de plaques de culture en plastique (NUNCLON, 96 puits) à raison de 200 μl par puits et contenant 10.000 cellules. On ajoute ensuite 50.000 cellules spléniques normales, ou de préférence de cellules spléniques normales irradiées à 300 rad, dans chaque puits à titre de "cellules de remplissage". Les cellules sont incubées ainsi pendant 7 à 10 jours dans une étuve à C0 (5%) et à 37βC. CRIBLAGEThe fusion partner consists of 3 HGPRT " myelomas, namely X63-Ag8, NS-1 and SP2 / 0. In other words, the lymphocytes are fused with either myeloma X63, NS-1 or SP2 / 0 separately. is carried out using polyethylene glycol (PEG 1500) from the firm MERCK, at the rate of 10 lymphocytes for a myeloma cell. 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 β 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 β 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). The cell suspensions are distributed in the wells of plastic culture plates (NUNCLON, 96 wells) at a rate of 200 μl per well and containing 10,000 cells. 50,000 normal spleen cells, or preferably normal spleen cells irradiated at 300 rad, are then added to each well as "filling cells". The cells are thus incubated for 7 to 10 days in an oven at C0 (5%) and at 37 β C. SCREENING
Au bout de 7 à 10 jours, le surnageant de chaque puits est prélevé et soumis au criblage par la réaction immunoenzymatique "ELISA". Elle est effectuée d'abord au moyen du réactif à base d'anticorps conjugué anti-Ig de souris couplé à l'uréase puis confirmée ensuite par l'anticorps conjugué anti-Ig de souris couplé à la phosphatase alcaline. Les cellules servant de cibles sont des cellules spléniques préalablement incubées avec le peptide NP 147-158R" (5
Figure imgf000019_0001
After 7 to 10 days, 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
Figure imgf000019_0001
17 à 50μg/ml pour 107 cellules) à 37*C pendant une à deux heures. Elles sont fixées sur plaques de polyvinyle (Dynatech) selon la technique décrite par Ternynck et Avrameas (Ternynck et Avrameas Techniques immuno- enzymatiques- Les éditions INSERM, 1987) au moyen du poly-L-lysine.17 to 50μg / ml for 10 7 cells) at 37 ° C for one to two hours. They are fixed on polyvinyl plates (Dynatech) according to the technique described by Ternynck and Avrameas (Ternynck and Avrameas Immuno-enzymatic techniques - INSERM editions, 1987) using poly-L-lysine.
Les puits ayant donné une valeur en densité optique (D.O) supérieure au moins de deux fois celle du témoin négatif (réaction des cellules cibles avec l'anticorps conjugué sans surnageant de culture) sont sélectionnés. Les surnageants des mêmes puits sont testés aussi sur les cellules non couvertes de peptide et de même haplot-ype pour s'assurer que la réaction observée n'est pas due aux "autoanticorps". De même le test sur le peptide en question, fixé seul (sans cellules) directement sur la plaque, confirme ainsi que les surnageants sélectionnés réagissent seulement avec les cellules couvertes de peptide.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. 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". Similarly, 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.
La sélection étant effectuée, les puits sont soumis à des clonages et sous-clônages pendant 3 à 4 fois. A chaque clonage et sous-clônage, les surnageants sont examinés de nouveau sur les mêmes critères décrits en haut. En même temps, la réaction immunoenzymatique est appliquée pour déterminer l'isotype de l'hybridome. A ce stade est effectuée parallèlement la réaction en vue de déterminer la restriction, autrement dit, la réaction devant être observée seulement avec 1'„haplotype recherché, en l'occurrence H-2d. EN CONCLUSION:Once the selection has been made, the wells are subjected to cloning and subcloning for 3 to 4 times. With each cloning and subcloning, the supernatants are examined again on the same criteria described above. At the same time, the enzyme immunoassay is applied to determine the isotype of the hybridoma. At this stage the reaction is carried out in parallel to determine the restriction, in other words, the reaction to be observed only with the desired haplotype, in this case H-2 d . IN CONCLUSION:
Trois hybridomes ont été obtenus présentant les caractéristiques des anticorps de type dit restreint, en ce sens qu'ils réagissent seulement avec des cellules spléniques d'un haplotype précis (H-2d) couvertes de peptide (NP 147-158R") . Ces trois clones sont dénommés N6.6, S.2.2 et X5.3 d'isotype IgG2b,K, IgG3,K et IgG2b,K respectivement.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.
EFFETS IN VIVO D'UN ANTICORPS RESTREINT SPECIFIQUE DE PEPTIDE SUR LA CROISSANCE TUMORALE.IN VIVO EFFECTS OF A RESTRICTED SPECIFIC ANTIBODY OF PEPTIDE ON TUMOR GROWTH.
L'expérience a pour but de tester la capacité d'un anticorps restreint anti-peptide, d'inhiber la croissance in vivo des cellules tumorales ayant été mises en contact avec le peptide spécifique.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.
PROTOCOLE EXPERIMENTALEXPERIMENTAL PROTOCOL
Des souris DBA/2 (H-2d) sont réparties en 4 groupes comportant chacun quatre souris. Elles ont reçu, par injection sous-cutanée, une suspension de cellules du mastocytome P815 (il s'agit de cellules tumorales transplantables qui, une fois injectées aux hôtes syngéniques (souris DBA/2) provoquent le développement d'une tumeur létale) .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).
Dans le 1er groupe, des cellules p815 seules sont injectées aux souris DBA/2, ce groupe sert de témoin de croissance des cellules P815 sur l'hôte syngénique.In the 1st group, p815 cells alone are injected into DBA / 2 mice, this group serves as a control for the growth of P815 cells on the syngeneic host.
Les souris du second groupe ont reçu des cellules P815 en mélange avec l'anticorps restreint à raison de 5 /.g/106 cellules (il s'agit de l'anticorps X5.3, sous clone X.5.3.7.T). Ces souris servent de témoins ayant reçu des cellules et des anticorps seuls.The 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.
Les souris du 3ème groupe ont reçu des cellules P815 incubées pendant 1 heure à 37βC avec le peptide NPR" de la nucléoprotéine du virus grippal à raison de 10 μg/106 cellules. C'est le groupe témoin ayant reçu les cellules et le peptide seul.The mice of the 3rd group received P815 cells incubated for 1 hour at 37 β C with the peptide NPR " of the nucleoprotein of the influenza virus at a rate of 10 μg / 10 6 cells. It is the control group which received the cells and the peptide alone.
Les souris du 4ème groupe ont reçu des cellules P815 incubées avec le peptide et mises ensuite en présence de l'anticorps restreint X5.3.7.T dans les mêmes conditions et proportions que les groupes témoins 2 et 3. Un même nomb*e de cellules P815 (105 cellules/souris) est injecté à chaque souris qui est suivie individuellement quant au développement d'une tumeur sous-cutanée. Les résultats sont exprimés d'une part en diamètre moyen des tumeurs pour chaque groupe, en pourcentage de souris présentant une tumeur visible et enfin en pourcentage de tumeurs létales.The 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.
RESULTATSRESULTS
1) Evolution des tumeurs sur l'hôte svnqénique injecté avec les cellules P8151) Evolution of tumors on the neurogenic host injected with P815 cells
Le développement de la tumeur sur les souris DBA/2 injectées avec différentes préparations est représenté par la courbe des diamètres moyens des tumeurs de chaque groupe en fonction du temps (figureThe development of the tumor in DBA / 2 mice injected with different preparations is represented by the curve of the mean diameters of the tumors of each group as a function of time (FIG.
1).1).
En particulier on peut noter, au 24ème jour après l'injection des cellules :In particular, it can be noted, on the 24th day after the injection of the cells:
- la présence de 4 tumeurs sur 4 souris dans chacun des trois groupes témoins (cellules P815 seules, cellules P815 + anticorps seuls, cellules P815 + peptide seul), avec un diamètre moyen de 7,5; 10,25 et 7,75 mm respectivement.- the presence of 4 tumors on 4 mice in each of the three control groups (P815 cells alone, P815 cells + antibodies only, P815 cells + peptide alone), with an average diameter of 7.5; 10.25 and 7.75 mm respectively.
- la présence de deux tumeurs sur 4 souris dans le groupe expérimental (cellules P815 + peptide + anticorps) avec un diaatiètre moyen de 4,5 mm.- the presence of two tumors on 4 mice in the experimental group (P815 cells + peptide + antibody) with an average diameter of 4.5 mm.
2) Développement des tumeurs létales2) Development of lethal tumors
Le nombre de tumeurs létales pour chaque groupe en fin d'expérience est le suivant :The number of lethal tumors for each group at the end of the experiment is as follows:
Groupe 1 (cellules P815 seules) : 3/4Group 1 (P815 cells only): 3/4
Groupe 2 (cellules P815 + anticorps seuls) : 4/4Group 2 (P815 cells + antibodies only): 4/4
Groupe 3 (cellules P815 + peptide seul) : 4/4Group 3 (P815 cells + peptide alone): 4/4
Groupe 4 (cellules P815 + peptide + anticorps) :Group 4 (P815 cells + peptide + antibody):
2/4.2/4.
Pour l'ensemble des témoins (groupe 1 + 2 + 3) on observe : 11 tumeurs létales sur 12, soit un pourcentage de 92% comparé à 2 tumeurs sur 4 ou 50% pour le groupe expérimental.For all of the controls (group 1 + 2 + 3), we observe: 11 of 12 lethal tumors, i.e. one percentage of 92% compared to 2 tumors out of 4 or 50% for the experimental group.
HYBRIDOMES SECRETANT DES ANTICORPS RESTREINTS ANTI- PEPTIDE DE L'ALBUMINE DE L'OEUF DE POULE 0VA,ς.,^ OU 12OK ASSOCIE A H-2 Kb.HYBRIDOMAS SECRETING ANTIBODIES ANTI-PEPTIDE OF CHICKEN EGG ALBUMIN 0VA, ς ., ^ OR 12OK ASSOCIATED WITH H-2 Kb.
Ce peptide OVA253.273 ou plus simplement 12OK de 20 acides aminés dont la composition est la suivante IINFEKLTEWTSSNVMEERK est reconnu en association avec la molécule CMH de classe I (H-2 Kb) par les lymphocytes T cytotoxiques spécifiques (Carbone et Bevan, J. Exp. Med., 1989, 169:603; J. Exp. Med., 1990, 171:377) .This OVA 253 peptide. 273 or more simply 12OK of 20 amino acids the composition of which is as follows IINFEKLTEWTSSNVMEERK is recognized in association with the MHC class I molecule (H-2 Kb) by specific cytotoxic T lymphocytes (Carbone and Bevan, J. Exp. Med. , 1989, 169: 603; J. Exp. Med., 1990, 171: 377).
En utilisant le même procédé que celui décrit pour le peptide du virus grippal NPR* un hybridome (9.3.2) a été obtenu, qui reconnaît en test ELISA et en test de cytotoxicité complément dépendante, le peptide 120K en présence de lymphocytes exprimant H-2 K6. Les témoins constitués soit par des cellules exprimant H-2 K6 en l'absence du peptide ou des préparations où le peptide (I20K) est incubé avec des cellules portant un haplotype différent (H-2 Y?) ne sont pas reconnus.Using the same method as that described for the peptide of the influenza virus NPR * 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.
Les études relatives à ces anticorps monoclonaux restreints anti-peptide OVA253_273 reprennent les mêmes schémas expérimentaux que ceux appliqués dans l'étude des anticorps restreints anti-peptide NPR". De plus il est particulièrement intéressant dans le cas de la molécule K6 de pouvoir étudier la reconnaissance du peptide en association avec les molécules Y? (K6"1) mutantes provenant des souris de la série C57B1/6.The studies relating to these restricted monoclonal antibodies anti-peptide OVA 253 _ 273 follow the same experimental schemes as those applied in the study of the restricted anti-peptide antibodies NPR " . In addition it is particularly interesting in the case of the molecule K 6 to be able to study the recognition of the peptide in association with the mutant Y? (K 6 " 1 ) molecules originating from mice of the C57B1 / 6 series.
TECHNIQUES UTILISEES DANS L'ETUDE DES ANTICORPS RESTREINTS, AUTRES QUE CELLES DECRITES PRECEDEMMENT 1) Cytotoxicité complément dépendante utilisant des cibles marquées au 51Cr .TECHNIQUES USED IN THE STUDY OF RESTRICTED ANTIBODIES OTHER THAN THOSE PREVIOUSLY DESCRIBED 1) Complementary cytotoxicity using targets labeled with 51 Cr .
21 La technique dérivant de celle utilisée par Cerottini et al dans l'étude de la cytolyse à médiation cellulaire ou CMC a été appliquée (Cerottini et al, 1974, J. Exp. Med., 140 : 703). Brièvement les cellules cibles (cellules du CMH d'haplotype déterminé + peptide) sont marquées par le chrome (Cr) radioactif 51 sous forme de chromate de sodium (NA25iCrθ4) . On incube les cellules cibles (ou les cellules témoins sans peptide) en présence du monoclonal spécifique. Le pourcentage de lyse spécifique est déduit en mesurant le chrome radioactif libéré dans le surnageant. Dans le cas des expériences avec l'anticorps monoclonal restreint X5.3 la technique a été modifiée en ajoutant un anticorps secondaire, en l'occurence, l'anticorps de Lapin anti-Immunoglobuline de Souris. Ensuite est ajouté le complément de Lapin. L'anticorps secondaire ayant reconnu 1'anticorps X5.3, active le complément gui permet la lyse. A titre d'exemple, dans notre cas, l'anticorps restreint dénommé X5.3, sous-clone X5.5.7/T lyse 30% de cibles spécifiques (cellules P815-H-2d + peptide) et 0% quand les cellules P815 ne sont pas mises au contact préalable avec le peptide.21 The technique derived from that used by Cerottini et al in the study of cell-mediated cytolysis or CMC has been applied (Cerottini et al, 1974, J. Exp. Med., 140: 703). Briefly, the target cells (MHC cells of determined haplotype + peptide) are marked by radioactive chromium (Cr) 51 in the form of sodium chromate (NA 2 5iCrθ 4 ). The target cells (or control cells without peptide) are incubated in the presence of the specific monoclonal. The percentage of specific lysis is deduced by measuring the radioactive chromium released in the supernatant. In the case of the experiments with the restricted monoclonal antibody X5.3, 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. For example, in our case, 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.
2) Immunoprécipitation après marquage de surface par l'iode radioactif 1252) Immunoprecipitation after surface labeling with radioactive iodine 125
Des cellules cibles (cellules + peptide) ou témoins, (cellules sans peptide) sont marquée à l'iode radioactif 125 par la méthode utilisant l'enzyme lactoperoxydase (Marchalonis, J.J., Biochem. J. , 1969, 113 : 291) . Après marquage, les cellules sont lysées par un détergent, le Triton X-100 en présence d'inhibiteurs d'enzyme. Le lysat est incubé avec un anticorps non-relié (anticorps témoin) en présence des billes de sépharose-protéine A. Après centrifugation, le lysat est incubé avec l'anticorps spécifique (anticorps restreint) . Le complexe est adsorbé sur billes de sépharose-protéine A, lavé, récupéré et analysé en électrophorèse sur gel de polyacrylamide (SDS-PAGE) .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).
Les résultats ont montré que l'anticorps restreint donne une bande de précipitation, après autoradiographie, quand les cellules portant l'haplotype correspondant sont incubées avec le peptide. (En l'absence du peptide les résultats, pour cette expérience, sont extrêmement faibles mais ne semblent pas absolument négatifs) .The results showed that the restricted antibody gives a precipitation band, after autoradiography, when the cells carrying the corresponding haplotype are incubated with the peptide. (In the absence of the peptide, the results for this experiment are extremely weak but do not seem absolutely negative).
3) Utilisation de la molécule H-2 soluble obtenu par génie génétique et vidée de son contenu peptidique3) Use of the soluble H-2 molecule obtained by genetic engineering and emptied of its peptide content
Une molécule H-2 soluble (dépourvue de la partie transmembranaire) obtenue par génie génétique a été produite et vidée de son peptide endogène. Elle est utilisée pour loger à l'intérieur le peptide de la nucléoprotéine du virus grippal. Cette molécule sert de cible pour l'anticorps restreint dans la technique ELISA ou radioimmuno-essai (RIA) utilisant la protéine A marquée à l'iode 125 par exemple. 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.

Claims

2323
REVENDICATIONS 1/ Anticorps monoclonaux restreints caractérisés par leur capacité à reconnaître spécifiquement un complexe formé par un peptidé caractéristique d'un antigène d'un agent pathogène ou un peptide caractéristique d'une dérégulation cellulaire et par une molécule du Complexe Majeur d'Histo-compatibilité (CMH) ayant la capacité de reconnaître et de fixer ce peptide, lesdits anticorps étant des anticorps monoclonaux restreints, dès lors qu'ils ne reconnaissent pas ledit peptide en association avec une molécule de CMH d'haplotype non spécifique du peptide. CLAIMS 1 / Restricted monoclonal antibodies 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 cellular deregulation and by a molecule of the Major Histo-Compatibility 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 not specific for the peptide.
2/ Anticorps monoclonaux restreints selon la revendication 1, caractérisés» en ce qu'ils sont dépourvus ou pratiquement dépourvus de réaction à l'égard du peptide seul et/ou en ce qu'ils reconnaissent faiblement ou pas du tout la molécule du CMH seule.2 / Restricted monoclonal antibodies according to claim 1, characterized "in that they are devoid or practically devoid of reaction with respect to the peptide alone and / or in that they weakly or not at all recognize the MHC molecule alone .
3/ Anticorps monoclonaux restreints selon la revendication 1 ou 1^ revendication 2, caractérisés en ce que les peptides reconnus sont caractéristiques d'agents pathogènes ≈des virus, des bactéries, des parasites par exemple le plasmodîum, des champignons, notamment des levures pathogènes, ou des peptides représentant des antigènes mineurs d'histo- co patibilité, des antigènes spécifiques de tumeurs ou des antigènes spécifiques de dérèglements autoimmuns. 3 / Restricted monoclonal antibodies according to claim 1 or 1 ^ claim 2, characterized in that the recognized peptides are characteristic of pathogenic agents ≈viruses, bacteria, parasites for example the plasmodium, fungi, in particular pathogenic yeasts, or peptides representing minor antigens of histopatibility, specific antigens of tumors or specific antigens of autoimmune disorders.
4/ Anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 3, caractérisés en ce que les peptides reconnus présentent de 7 à 25, de préférence une dizaine d'acides aminés. 4 / Restricted monoclonal antibodies according to any one of claims 1 to 3, characterized in that the recognized peptides have from 7 to 25, preferably about ten amino acids.
5/ Anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 4, caractérisés en ce que le peptide reconnu est spécifique d'un rétrovirus humain HIV et notamment en ce qu'il s'agit de peptides de la protéine gag, de la nucléoprotéine ou du peptide K16F.5 / Restricted monoclonal antibodies according to any one of claims 1 to 4, 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, of the nucleoprotein or of the peptide K16F.
6/ Anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 5, caractérisés en ce que la molécule du CMH reconnue est de différents haplotypes de classe I comme de classe II. 6 / Restricted monoclonal antibodies according to any one of claims 1 to 5, characterized in that the recognized MHC molecule is of different haplotypes of class I as of class II.
7/ Procédé pour la préparation d'anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 6, caractérisé en ce que:7 / A method for the preparation of restricted monoclonal antibodies according to any one of claims 1 to 6, characterized in that:
- on fusionne des cellules spléniques d'un animal préalablement immunisé avec des cellules spléniques isogèniques recouvertes d'un peptide déterminé, avec des cellules de myélome en présence d'un promoteur de fusion, par exemple le polyéthylène glycol, les cellules spléniques étant en excès par rapport aux cellules myélomateuses, par exemple dans une proportion de 10 pour 1 environ,- spleen cells from an animal previously immunized are fused with isogenic spleen cells covered 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,
- on réalise un criblage afin de sélectionner ceux des hybridomes capables de produire des anticorps monoclonaux restreints, par exemple par la technique ELISA effectuée avec un réactif à base d'anticorps conjugué anti-Ig de souris couplé à une enzyme par exemple l'uréase ou la phosphatase alcaline ou la peroxydase,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,
- on récupère et on clone les hybridomes ainsi sélectionnés.- the hybridomas thus selected are recovered and cloned.
8/ Hybridomes tels qu'obtenus par fusion de cellules de myélomes et de cellules spléniques d'un animal préalablement immunisé avec le peptide contre lequel on souhaite produire des anticorps monoclonaux restreints, dans des conditions telles que l'antigène contre lequel sont formés les anticorps, est constitué par un complexe comportant le peptide ci-dessus associé à une molécule de CMH qui le reconnaît spécifiquement. 8 / Hybridomas as obtained by fusion of myeloma cells and spleen cells of an animal previously immunized with the peptide against which it is desired to produce restricted monoclonal antibodies, under conditions such as the antigen against which the antibodies are formed , consists of a complex comprising the above peptide associated with a MHC molecule which specifically recognizes it.
9/ Composition pour le diagnostic de la présence de peptides associés à des molécules de CMH, dans un échantillon biologique ou dans un organisme vivant, caractérisée en ce qu'elle comprend des anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 6.9 / Composition for the diagnosis of the presence of peptides associated with MHC molecules, in a biological sample or in a living organism, characterized in that it comprises restricted monoclonal antibodies according to any one of claims 1 to 6.
10/ Composition pour le diagnostic selon la revendication 9, caractérisée en ce qu'elle permet de détecter une infection par un agent pathogène, notamment un virus.10 / A composition for diagnosis according to claim 9, characterized in that it makes it possible to detect an infection by a pathogenic agent, in particular a virus.
11/ Composition pour le diagnostic selon la revendication 9, caractérisée en ce qu'elle permet la détection de tumeurs.11 / A composition for diagnosis according to claim 9, characterized in that it allows the detection of tumors.
12/ Composition pour le diagnostic selon la revendication 9, caractérisée en ce qu'elle permet la détection de dérèglements autoimmuns. 12 / Composition for diagnosis according to claim 9, characterized in that it allows the detection of autoimmune disorders.
13/ Anticorps monoclonaux selon l'une quelconque des revendications 1 à 6, caractérisés en ce qu'ils sont couplés à des substances capables de les rendre cytotoxiques par exemple à une toxine, un antibiotique ou un isotope radioactif.13 / Monoclonal antibodies according to any one of claims 1 to 6, characterized in that they are coupled to substances capable of making them cytotoxic, for example to a toxin, an antibiotic or a radioactive isotope.
14/ Composition pharmaceutique, caractérisée en ce qu'elle comprend à titre de principe actif, des anticorps monoclonaux restreints selon la revendication 13.14 / Pharmaceutical composition, characterized in that it comprises, as active principle, restricted monoclonal antibodies according to claim 13.
15/ Kit pour le diagnostic in vitro sur un échantillon biologique d'une infection par un organisme pathogène ou pour le diagnostic d'un cancer, d'une maladie autoimmune ou d'un autre dérèglement cellulaire, caractérisé en ce qu'il comprend: - des anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 6, ayant la spécificité recherchée compte tenu du diagnostic à effectuer, - des moyens pour révéler la présence d'un conjugué de type complexe (antigène-molécule du CMH) lié avec 1'anticorps.15 / 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 any one of claims 1 to 6, having the specificity sought taking into account the diagnosis to be made, - Means for revealing the presence of a complex type conjugate (antigen-MHC molecule) linked with the antibody.
16/ Composition pharmaceutique comprenant à titre de principe actif, des anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 6, pour la modulation de l'action de cellules T, notamment dans les pathologies autoimmunes.16 / A pharmaceutical composition comprising, as active principle, restricted monoclonal antibodies according to any one of claims 1 to 6, for modulating the action of T cells, in particular in autoimmune pathologies.
17. Composition vaccinante caractérisée en ce qu'elle comprend à titre de principe actif, des anticorps monoclonaux restreints selon l'une quelconque des revendications 1 à 6. 17. Vaccinating composition characterized in that it comprises, as active principle, restricted monoclonal antibodies according to any one of claims 1 to 6.
PCT/FR1991/000121 1990-02-14 1991-02-14 Monoclonal antibodies for recognizing a peptide linked to a major histocompatibility antigen WO1991012332A1 (en)

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WO1996020215A2 (en) * 1994-12-23 1996-07-04 Laboratoires Om S.A. Use of mhc-ii binding and/or mhc-ii mimicking molecules for the prevention and/or treatment of inflammatory diseases
WO1997002342A1 (en) * 1995-06-30 1997-01-23 Københavns Universitet Recombinant antibodies from a phage display library, directed against a peptide-mhc complex
WO1999058693A1 (en) * 1998-05-14 1999-11-18 Centre National De La Recherche Scientifique - Cnrs Complex formed by a peptide and a major histocompatibility complex at the surface of phages
US6153408A (en) * 1991-11-15 2000-11-28 Institut Pasteur And Institut National De La Sante Et De La Recherche Medicale Altered major histocompatibility complex (MHC) determinant and methods of using the determinant
EP1474120A2 (en) * 2002-02-13 2004-11-10 Technion Research And Development Foundation, Ltd. Antibody having a t-cell receptor-like specificity, yet higher affinity, and the use of same in the detection and treatment of cancer, viral infection and autoimmune disease
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US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
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US6153408A (en) * 1991-11-15 2000-11-28 Institut Pasteur And Institut National De La Sante Et De La Recherche Medicale Altered major histocompatibility complex (MHC) determinant and methods of using the determinant
WO1996020215A2 (en) * 1994-12-23 1996-07-04 Laboratoires Om S.A. Use of mhc-ii binding and/or mhc-ii mimicking molecules for the prevention and/or treatment of inflammatory diseases
WO1996020215A3 (en) * 1994-12-23 1996-10-10 Om Lab Sa Use of mhc-ii binding and/or mhc-ii mimicking molecules for the prevention and/or treatment of inflammatory diseases
WO1997002342A1 (en) * 1995-06-30 1997-01-23 Københavns Universitet Recombinant antibodies from a phage display library, directed against a peptide-mhc complex
WO1999058693A1 (en) * 1998-05-14 1999-11-18 Centre National De La Recherche Scientifique - Cnrs Complex formed by a peptide and a major histocompatibility complex at the surface of phages
FR2778669A1 (en) * 1998-05-14 1999-11-19 Centre Nat Rech Scient Genetically modified microorganism, especially phage, useful for screening for antigenic peptides recognized by T cells
US9095533B2 (en) 2000-03-27 2015-08-04 Technion Research & Development Foundation Limited Antigen-presenting complex-binding compositions and uses thereof
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EP1474120A2 (en) * 2002-02-13 2004-11-10 Technion Research And Development Foundation, Ltd. Antibody having a t-cell receptor-like specificity, yet higher affinity, and the use of same in the detection and treatment of cancer, viral infection and autoimmune disease
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