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WO1995023164A1 - Sequence du recepteur de lymphocytes t specifiquement associee a une maladie immune - Google Patents

Sequence du recepteur de lymphocytes t specifiquement associee a une maladie immune Download PDF

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Publication number
WO1995023164A1
WO1995023164A1 PCT/EP1995/000670 EP9500670W WO9523164A1 WO 1995023164 A1 WO1995023164 A1 WO 1995023164A1 EP 9500670 W EP9500670 W EP 9500670W WO 9523164 A1 WO9523164 A1 WO 9523164A1
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Prior art keywords
seq
amino acid
polypeptide
cell receptor
antibody
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PCT/EP1995/000670
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English (en)
Inventor
Johan Mari Van Der Maaden
Antonius Wilhelmus Maria Rijnders
Johanna Paulina Maria Graus
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Akzo Nobel N.V.
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Priority to AU18120/95A priority Critical patent/AU1812095A/en
Publication of WO1995023164A1 publication Critical patent/WO1995023164A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to a polypeptide containing a variable region of a ⁇ chain of a T cell receptor associated with an immune disease, to a T cell receptor, immunogenic compounds and recombinant antibodies comprising this polypeptide, antibodies against this polypeptide, nucleic acids coding for the polypeptide, methods for detection of an immune disease, vaccines and pharmaceutical formulations for prevention and treatment of immune diseases.
  • T cells are often involved in immune diseases. In the case of autoimmune diseases these T cells are directed to autoantigens and they initiate an immune response to these antigens. However, in many autoimmune diseases the nature of the autoantigen is unknown.
  • autoreactive T cells An effective and specific therapy would be directed to the autoreactive T cells without affecting the normally active immune cells. But the autoreactive T cells are only discernible from other T cells by their ability to react with the autoantigen.
  • the antigen is unknown, however, it is virtually impossible to select the specific T cell from a pool of T lymphocytes.
  • the autoreactive T cell is characterized by it's receptors that mediate antigen recognition. These receptors are transmembrane proteins extending from the surface of the T cells. ⁇ he receptor recognizes a complex of an antigen and MHC (major histocompatibility complex) molecule on other cells. T cell receptors contain two subunits, designated TCR ⁇ and TCR ⁇ or TCR ⁇ and TCR ⁇ . These subunits, like iimmunoglobulin L and H chains, consist of variable and constant regions. Hypervariable seguences (CDRs), enabling the specificity of the T cell, are present in the so-called V-regions of TCR ⁇ and TCR ⁇ , forming the binding site for the epitope. The most variable CDR is CDR3.
  • CDRs Hypervariable seguences
  • the invention thus comprises a polypeptide comprising an amino acid seguence contained in the variable region of a ⁇ chain of a T cell receptor associated with an immune disease, characterized in that said amino acid seguence is the sequence according to SEQ ID NO:2 or a fragment thereof or a functional eguivalent thereof.
  • polypeptide refers to a molecular chain of amino acids, does not refer to a specific length of .;he product and if reguired can be modified in vivo or in vitro, for example by glycosylation, a idation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides and proteins are included within the definition of polypeptide.
  • the oligopeptides according to the invention have an amino acid sequence of at least 10 amino acids. More preferably the oligopeptides according to the invention have an amino acid seguence of 10-35, in particular 10-25 amino acids. Highly preferred are oligopeptides with an amino acid sequence of 10-15 amino acids.
  • fragment refers to any sequence of amino acids that is part of the amino acid sequence depicted in SEQ ID NO:2, which still displays the functional characteristics of the amino acid sequence of SEQ ID NO:2.
  • These functional characteristics of the sequence of SEQ ID NO:2 are the ability to react with the epitope to which the sequence of SEQ ID NO:2 is directed or the ability to raise antibodies in an immune reaction, which antibodies also will bind to the sequence of SEQ ID NO:2.
  • the amino acid sequence LFTGGS SEQ ID NO:12
  • Polypeptides comprising an amino acid seguence according to SEQ ID NO:12 are therefor within the scope of the invention.
  • polypeptide is in substantially pure form, which means that it is free of other biochemical moieties with which it is normally associated in nature.
  • substantially pure polypeptides for instance, can be synthesized, or produced recombinantly by means known to those skilled in the art.
  • whole T cell receptors can be treated enzymatically to produce polypeptides according to the invention.
  • amino acid sequence depicted in SEQ ID NO:2 is associated with rheumatoid arthritis (RA).
  • T cells associated with the disease are mainly located in the synovia of the joints which are affected.
  • the amino acid seguence depicted in SEQ ID NO:2 of the present invention enables the detection of RA-related T cells and of the autoantigen(s) with which these T cells are reactive.
  • Detection of the presence of the T cell receptor specific amino acid seguence depicted in SEQ ID NO:2 in patients or in a sample obtainable from patients enables an improved recognition of the autoimmune disease related T cell.
  • Such a detection can be performed by diagnostic methods using immunochemical reagents derived from the polypeptide of the invention.
  • the polypeptide of the invention not only enables the development of a diagnostic method indicating the presence of T cells having this receptor sequence, but the identified sequence can also serve as a target for specifically directed therapeutic compounds.
  • polypeptide of the invention can be used to identify the nature of the autoantigen responsible for the immune disease.
  • the present invention provides a method of immunotherapy for T cell receptor mediated pathologies, including autoimmune diseases, which avoids many of the problems associated with previously suggested methods of treatment.
  • the host's immune system is mobilized to suppress the autoaggressive T cells or, in case of passive immunization, antibodies directed to said T cells are administered.
  • the polypeptide according to the invention can be linked to an immunogenic carrier to further increase its immunogenicity , resulting in an immunogenic compound.
  • Suitable immunogenic carriers are, for instance, keyhole limpet haemocyanin (KLH), human or bovine serum albumine (HSA, BSA) or ovalbumine.
  • antibodies or fragments of antibodies which are specifically reactive with the amino acid sequence having SEQ ID NO:2 can be employed in immunotherapy for T cell receptor mediated pathologies in rheumatoid arthritis.
  • antibodies can be produced by performing an active immunization of a suitable mammal with the polypeptide according to the invention. This immunization with the polypeptide of the invention will give rise to the formation of antibodies directed to the variable region of this polypeptide.
  • a fragment of an antibody is defined as that part of an antibody which is able to react with and is specific for an antigen. These fragments can be obtained by enzymatic reactions from the original antibody but they also can be made by chemical synthesis or via recombinant DNA methods.
  • a functional derivative of an antibody is a compound which is also reactive with the variable region of the polypeptide according to the invention. Such compounds can be formed, for instance, by incorporating the amino acids constituting the complementary determining regions (CDR's) of an antibody in another amino acid seguence.
  • polyclonal antibodies When polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are known in the art (e.g. Mayer and Walter, eds, Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987). In short, a selected mammal, e.g. a rabbit is given (multiple) injections with one of the above-mentioned immunogenic compounds, e.g. corresponding to about 20 ⁇ g to about 80 ⁇ g of polypeptide per immunization. Immunization is carried out with an acceptable adjuvant, generally in equal volumes of immunogen and adjuvant.
  • Acceptable adjuvants include Freund's complete, Freund , s incomplete, alum-precipitate or water-in-oil emulsions, with a preference for Freund's complete adjuvant for the initial immunization. For booster immunization Freund's incomplete adjuvant is preferred.
  • the initial immunization consists of the administration of approximately 1 ml emulsion at multiple subcutaneous sites on the backs of the rabbits.
  • Booster immunizations utilizing an equal volume of immunogen are given at about one monthly intervals and are continued until adequate levels of antibodies are present in an individual rabbits serum. Blood is collected and serum isolated by methods known in the art.
  • Monospecific antibodies to each of the immunogens are affinity purified from polyspecific antisera by a modification of the method of Hall et al. (Nature 311, 379-387 1984), prepared by immunizing rabbits as described above with the purified polypeptides.
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen.
  • Homogeneous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, in this particular case binding to the amino acid sequence SEQ ID NO:2.
  • Monoclonal antibody reactive against the amino acid sequence depicted in SEQ ID NO:2 can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by technigues known in the art (Kohler and Milstein, Nature 256; 495-497, 1975). Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco's modified Eagle's medium (DMEM). Antibody producing hybridomas are cloned, preferably using the limiting dilution technique as described in Current Protocols in Immunology, Eds: J.A Coligan et al., Greene Publ. Ass. and Wriley-Interscience, 1992.
  • DMEM Dulbecco's modified Eagle's medium
  • Antibody producing cells are identified by screening with the appropriate immunogen.
  • Immunogen positive hybridoma cells are maintained by techniques known in the art.
  • Specific anti-monoclonal antibodies are produced by cultivating the hybrido as in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art.
  • Antibodies can be isolated from the culture of the immortalized lymphocytes.
  • Human antibodies can be produced by in vitro stimulation of isolated B-lymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a human being immunized with at least one polypeptide according to the invention.
  • Another aspect of the invention are molecules which can interact specifically with the epitope for which the T cell receptor of the invention is specific. These can be obtained by synthesizing recombinant antibodies through genetic engineering. In this case one of the complementary determining regions of such a recombinant antibody will be formed by the amino acid sequence according to SEQ ID NO:2. This can be accomplished by methods known in the art, preferably by CDR grafting (Jones et al., Nature 321, 522-525, 1986). In this way it is possible to prepare antibodies comprising the amino acid sequence having SEQ ID NO:2 according to the invention in one of their CDR's.
  • anti-idiotype antibodies which recognize the 'antigen' binding site of the antibody and therefore are an "internal image" of the 'antigen'.
  • anti-idiotypic antibodies against antibodies against the T cell receptor the 'antigen' is formed by the receptor.
  • anti-idiotypic antibodies against recombinant antibodies as discussed above the 'antigen' constitutes the antigen binding to these recombinant antibodies, i.e. the antigen for which the T cell receptor is specific.
  • anti-idiotype antibodies will be also very usef l for treatment and diagnostic purposes.
  • T cell receptors comprising the amino acid seguence of SEQ ID NO:2 are also within the scope of the invention.
  • sequence depicted in SEQ ID NO:2 or a fragment thereof or its functional derivative is included in a ⁇ chain of a T cell receptor.
  • This receptor can be used to search for the antigen(s) which specifically bind to this receptor. Methods to use this kind of receptors in such a way have been described by Hickling et al. (Eur. J. Immunol. .22, 1983-1987, 1992).
  • these receptors are particularly useful for the detection of the corresponding epitope of the receptor.
  • An epitope is defined as the specific surface of an antigen molecule, which is delineated by the area of interaction with a T cell receptor or antibody. Knowledge of this epitope, of the antigen on which it can be found and of the presence of the epitope in the various organs and tissues will give more insight in the onset, spread and development of the immune disease.
  • a T cell receptor comprising the amino acid seguence according to the invention will be reactive with the epitope-MHC complex. Localization of such a T cell receptor can be traced if said receptor has been labeled. Labeling can be done according to methods known in the art and labels that can be used are, for instance, enzymes, fluorescent compounds, dyes and radioactive compounds.
  • the antigen- T cell receptor complex can be isolated. It is possible then to characterize the antigen and to determine the epitope that binds to the polypeptide of the invention.
  • the T cell receptor can be used for diagnosis and therapy.
  • the T cell receptor is solubilized in aqueous solutions.
  • nucleic acid sequence coding for an amino acid seguence of the polypeptide of the invention is also covered by the invention.
  • the nucleic acid seguence coding for the polypeptide comprises the seguence shown in SEQ ID NO:l or a seguence which hybridizes to said seguence or its complementary strand under stringent conditions.
  • Said hybridizing sequences still encode for a polypeptide which is functional equivalent to the polypeptide of the invention.
  • "Stringent conditions" in hybridizing experiments are formed by a combination of a relatively high temperature and a relatively high concentration of salts in the solution. Next to the temperature and salt concentration the stringency is also determined by the number of nucleotides that are hybridized, by the A-T content and the number of mismatches of said hybridization reaction.
  • the degeneracy of the genetic code also means that the nucleic acid sequence of SEQ ID N0:1 codes for a certain amino acid sequence and the same amino acid sequence can be derived using a different nucleic acid seguence in which the codons for certain amino acids are different to those of the sequence of SEQ ID NO:2.
  • the various nucleic acid sequences coding for the amino acid sequence according to SEQ ID NO:2 also fall within the scope of the invention.
  • One of the aspects of the invention is to provide an immunochemical reagent which can be used in diagnostic test kits for detecting the presence of, or the predisposition for an immune disease.
  • Such an immunochemical reagent can comprise compounds such as a polypeptide according to the invention, a T cell receptor comprising the amino acid seguence according to SEQ ID NO:2 or an antibody comprising said amino acid sequence and antibodies reactive with said amino acid sequence having SEQ ID NO:2.
  • immunochemical reagent signifies that the compounds mentioned above are bonded to a suitable support or are provided with a labeling substance.
  • the supports which can be used are, for example, the inner wall of a microte ⁇ t well, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle.
  • Labeling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.
  • an immunochemical reagent according to the invention is used, which reagent is brought into contact with the test fluid, and the presence of immune complexes, formed between the immunochemical reagent and its counterpart in the test fluid, is detected and from this the presence of the immune disease or a predisposition for this disease can be derived.
  • the nature of the counterpart which is being detected in the test fluid varies. The following list shows which elements can be detected with the specified immunochemical reagents:
  • Immunochemical reagent comprising: counterpart:
  • polypeptide according to the invention epitope/antigen T cell receptor comprising amino acid sequence of SEQ ID NO:2: epitope/antigen antibody comprising amino acid sequence of SEQ ID NO:2: epitope/antigen antibody reactive with amino acid seguence of SEQ ID NO:2: T cell
  • the immunochemical reaction which must take place when using these detection methods is preferably a sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
  • test kit according to the invention must contain, as an essential constituent, an immunochemical reagent such as described above.
  • the test can consist of the - unlabelled - immunochemical reagent bonded to a solid support, for example the inner wall of a microtest well, it being possible to use a labeled immunochemical reagent for the detection.
  • the test kit can consist of the immunochemical reagent bonded to a solid support, a labeled antibody directed against this reagent then being used to compete with compounds in the test fluid.
  • an immunochemical reagent bonded to particles or sols must be brought into direct contact with the test fluid in which the counterpart to the immunochemical reagents which is to be detected is present.
  • a testkit can also comprise one or more nucleic acid seguences according to the invention.
  • the nucleic acid of the test kit will hybridize with complementary strands, if present, and the hybridized product can be detected by agents capable of discrimination of hybridized nucleic acids.
  • testkit comprising a nucleic acid
  • a testkit comprising a nucleic acid
  • amplification kit in which the hybridized strands are amplified in order to facilitate easy detection.
  • testkit in which one of the nucleic acid seguences which hybridizes with the DNA seguences of the invention is labeled so that it facilitates detection of the hybridized strands.
  • a diagnostic kit is the detection of circulating antibodies directed to vaccinated compounds to monitor a therapeutic effect of administration of vaccination with the polypeptide according to the invention.
  • Treatment of immune diseases can be established by the polypeptide of the invention, by nucleic acids according to the invention or by antibodies according to the invention. These compounds can be the basis of a pharmaceutical formulation.
  • vaccines comprising the polypeptide comprising the amino acid seguence of SEQ ID NO:2, the T cell receptor comprising the amino acid seguence of SEQ ID NO:2, an immunogenic compound comprising the amino acid sequence of SEQ ID NO:2 or an antibody specifically reactive with the amino acid sequence of SEQ ID NO:2.
  • polypeptide according to the invention can be formulated with pharmaceutical acceptable carriers for administration.
  • Pharmaceutical acceptable carriers include, for example, sterile salin, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, and water.
  • composition according to the invention may comprise one or more adjuvants.
  • Suitable adjuvants include, amongst others, aluminum hydroxide, aluminum phosphate, amphigen, tocophenols, monophosphenyl lipid A, muramyl dipeptide and saponins such as Quill a.
  • the amount of adjuvant depends on the nature of the adjuvant itself.
  • composition according to the invention may comprise one or more stabilizers such as , for example, carbohydrates, including sorbitol, mannitol , starch, sucrosedextrin, and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
  • stabilizers such as , for example, carbohydrates, including sorbitol, mannitol , starch, sucrosedextrin, and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
  • said polypeptide can be coupled to an immunogenic carrier.
  • immunogenic carriers include tetanus toxoid, Keyhole ly pet haemocyanin (KLH), Human and Bovine serum albumine (HSA, BSA) . This is in particular useful when oligopeptides comprising the amino acid seguence according to SEQ ID NO:2 are used.
  • Suitable administration routes are intramuscular, subcutaneous, intravenous or intraperitoneal injections, oral administration or nasal sprays.
  • the amount of active ingredient administered will depend on the route of administration, the time and frequency of administration, the age of the person treated as well as general health conditions and diet.
  • a dosage of 0.01 to 1000 ⁇ g, preferably 0.05 to 500 ⁇ g, more preferably 0.1 to 100 ⁇ g of polypeptide per kg of host can be used.
  • a pharmaceutical composition comprising a polypeptide according to the invention is administered to a patient suffering from an immune disease.
  • the administration will give rise to an immune response.
  • Antibodies or T cells will be stimulated which recognize this polypeptide as well as the corresponding amino acid seguence of the variable region on the ⁇ chain of the T cell receptor. These antibodies or T cells will specifically bind to this receptor thereby preventing that this T cell will initiate or maintain an immunological reaction. In this way only the specific T cell population carrying the amino acid seguence according to SEQ ID NO:2 of the invention will be influenced.
  • the antibodies reactive against the amino acid seguence of SEQ ID NO:2 or fragments or functional equivalents thereof are directly administered.
  • the antibodies specifically reactive with the amino acid sequence of SEQ ID NO:2 are very suitable for use in a vaccine in case of passive vaccination.
  • the antibodies can be formulated in essentially the same way as the polypeptides according to the invention.
  • antibodies have to be raised against the polypeptides according to the invention. This is achieved through an active immunization scheme of a suitable mammal as explained earlier. It will be clear to one skilled in the art that the polypeptide of the invention can be bound to a carrier molecule to enhance the immunogenicity.
  • passive immunization can be used for the treatment of immune disease.
  • complete antibodies will be produced by an immunized animal.
  • fragments of these antibodies, or functional derivatives of these antibodies or their fragments are useful.
  • a preferred embodiment is a conjugate of such an antibody, fragment, or functional derivative, with a cytotoxic substance.
  • the antibody is then used as targeting moiety which will bind to the 'antigen' for which it is specific.
  • the 'antigen' will be for instance the T cell receptor comprising the amino acid seguence depicted in SEQ ID NO:2 according to the invention.
  • the antibody will bind to it's 'antigen' and preferably be internalized.
  • the toxic compound is brought in or in the neighbourhood of the cell carrying the 'antigen'. In this way the toxin can act locally and thus specifically.
  • nucleic acid sequences of the invention for therapeutic purposes.
  • Anti-sense strands to the strand coding for the polypeptide of the invention will block translation of the DNA and thus prohibit the formation of the specific T cell receptor.
  • a conjugate of an anti-sense strand and a toxin can be administered. After hybridization to the target seguence the toxin can exert its toxic effects thereby destroying the DNA or other cell components.
  • these hybridization therapies only T cells expressing the specific target sequence are addressed because only in these T cells the specific genetic rearrangement takes place, which results in the expression of the T cell receptor comprising the amino acid seguence according to SEQ ID NO:2.
  • Such a therapy can prevent stimulation of pathological mechanisms without employing cell destruction mechanisms.
  • Conjugation of label and/or toxic compounds with a polypeptide, a nucleic acid, an antibody, fragment or analogue thereof or a receptor according to the invention can be performed by normal chemical methods or by recombinant DNA technigues.
  • Toxic compounds which can be coupled to the compounds according to the invention are for instance mitomycin, Pseudomonas exotoxin, diphteria toxin or derivatives, adria ycin, anthracycline derivatives, ricin, trichothecenes, calicheamycins, dynemycin, camptothecin, actinomycin D, ansamycins, amanitin, bleomycins, alpha emitting isotopes and all other compounds which are specifically cytotoxic.
  • the pharmaceutical compound is mixed with one or more pharmaceutical acceptable carriers, e.g. as described in the standard reference Chase et al., Remington's Pharmaceutical Sciences.
  • the compound can also be applied as an injection preparation in the form of a solution, suspension, emulsion, or as a spray, e.g. a nasal spray.
  • Synovia infiltrated with T cells were obtained from 11 patients suffering from rheumatoid arthritis (RA) . Patients fulfilled the ARA (American Rheumatology Association) criteria for definitive clinical RA and a clinical score of the joints from which the synovia were used was determined from X-ray photographs.
  • RA rheumatoid arthritis
  • Synovia were minced and incubated in complete medium (an equal mixture of Dulbecco's modiefied Eagle Medium (DMEM, Gibco 074-2100) and Nutrient mixture F12 (HAM's F12, Gibco 074-1100), supplemented with 2500 mg/1 sodium bicarbonate, 55 mg/1 sodium pyruvate, 2.3 mg/1 ⁇ -mercaptoethanol, 1.22 mg/1 ethanolamine, 260 mg/1 L- glutamine, 4.5-10 -4 mg/1 sodium selenite, 62.5 mg/1 sodium penicillin, 62.5 mg/1 streptomycin sulphate and 10% human pooled AB serum to which was added 200 U/ml collagenase and 0.1 mg/ml DNase I (obtained from Sigma) .
  • complete medium an equal mixture of Dulbecco's modiefied Eagle Medium (DMEM, Gibco 074-2100) and Nutrient mixture F12 (HAM's F12, Gibco 074-1100)
  • 2500 mg/1 sodium bicarbonate
  • the cells were filtered over a 200 ⁇ nylon filter and mononuclear cells were isolated by Ficoll Hypaque density centrifugation. The cells were incubated in a tissue culture flask in complete medium for 16 hours and non-adherent cells were collected and stimulated. Mononuclear cells from peripheral blood (PBL) for control experiments were isolated by Ficoll Hypague density centrifugation, collected and stimulated.
  • PBL peripheral blood
  • IL-2 Interleukine-2
  • 2-10 5 mononuclear cells per well were seeded in 96 wells roundbottom plates (obtained from Nunc) in 200 ⁇ l complete medium supplemented with 10 U/ml IL-2.
  • 2-10 4 T cells and 2*10 5 allogeneic irradiated (2500 rad) PBL were seeded in roundbottom microwells in 200 ⁇ l complete medium supplemented with 10 U/ml IL-2 and 1.25 ⁇ g/ml PHA (Phytohaemagglutinin, Wellcome) or 0.1 ⁇ g/ml anti-CD3 (OKT3, obtainable from ATCC).
  • PHA Physicalhaemagglutinin, Wellcome
  • anti-CD3 OKT3, obtainable from ATCC
  • T cell lines that could be used for V-gene analysis were derived from 9 patients. From
  • T cell lines derived from the synovia were characterized by FACS (Fluorescence Activated Cell Sorter) analysis for the expression of TCR-A ⁇ chains and gamma-_.
  • FACS Fluorescence Activated Cell Sorter
  • Table 2 Markers for B cells (CD19), NK cells (CD 16) and monocytes (CD 14) were absent in all lines, except SCRO.ll.CD3, which contained 36% CD 16 + cells.
  • the percentage of CD4 + and CD8 + , double positive and double negative T cells was determined. All lines expressed MHC class II antigens and were of the CD45RA- subtype. All lines with the exception of SCRO.04 and SCRO.08 consisted mainly of A ⁇ T cells. CD4/CD8 ratios varied and shifts in the ratio could not be consistently related with the different stimulation protocols.
  • RNAzol (Cinna/Bioteck) and 20 ⁇ l chloroform was added. The mixture was incubated on ice for 15 minutes and centrifuged. The RNA in the upper layer was precipitated with an equal volume isopropanol and the precipitate was washed once with 70% ethanol.
  • cDNA was synthesized using Avian Myeloblastoma Virus (AMV) reverse transcriptase and random hexanucleotides (Amersham cDNA synthesis system plus) according to the manufacturers instructions.
  • AMV Avian Myeloblastoma Virus
  • Table 3 a list is presented of the primers used for the amplification reactions of the VA and V ⁇ gene family from the cDNA preparations according to the method of Choi et al. (Y. Choi et al., Proc. Natl. Acad. Sci. USA 85, 8941- 8945, 1989; Rabat E.A. , u T.T. , Reid-Miller M, Perry H.M. and Gottesman K.S., in: Sequences of proteins of immunological intrest. 4th ed. Public Health Service, NIH, Washington DC, 1987) In each reaction a control amplification using TCR constant region primers was done.
  • Vfl3 GTCTCTAGAGAGAAGAAGGAGCGC 3.1-2
  • V ⁇ l6 AAAGAGTCTAAACAGGATGAGTCC 16.1
  • Vfil7 CAGATAGTAAATGACTTTCAG 17.1
  • E. coli DH5AF' cells obtained from Stratagene were transformed and minipreparations were made. Purified DNA was used for double strand DNA sequence reactions with T7 DNA polymerase (Pharmacia) using a C ⁇ specific primer (5'-TTCTGATGGCTCAAACAC-3' ) .
  • sequence -L-F-T-G-G-S-A-G-A-N- The results were confirmed in an independent experiment with clones derived from patient 14, where again the same sequence was found in 4 out of 5 clones.
  • the sequences flanking -L-F-T-G-G-S-A-G-A-N- are also found identical but not unique for the polypeptide of the invention because the leading V ⁇ sequence is specific for V ⁇ 14, which was meant to be amplified, and because the trailing sequence is coded on the J part of the receptor sequence, which is less variable than the D part, which codes for the polypeptide of the invention.
  • mice Five Balb/c mice were immunized with the N- acetylated peptide Ac-LFTGGSAGAN (SEQ ID NO:2) conjugated to the im unocarrier KLH (Keyhole Limpet hemocyanin) .
  • the peptide-KLH conjugate was dissolved in 40 mM phosphate, 0.9% NaCl, pH 7.4 to a concentration of 1 mg/ml.
  • An amount of peptide-KLH conjugate corresponding to 20 ⁇ g peptide was mixed with 20 ⁇ l complete (1st immunization) or incomplete (booster) Freunds adjuvant. In each case the total volume was brought to 100 ⁇ l by addition of PBS. and the preparations were injected intraperitoneal at 0 and 6 weeks.or at 0. 3, 6, 9 and 12 weeks.
  • mice were bled at 6 respectively 12 weeks and the titer of these antibodies against the N-acetyleted peptide was determined in an immunoassay. Plates were coated with the N-acetylated peptide Ac-LFTGGSAGAN coupled to BSA (Bovine Serum Albumine). The mice sera were diluted and 100 ⁇ l of the diluted sera was added for 1 hour. After several washes 100 ⁇ l of a sheep- anti-mouse-antibody labelled with HRP (Horse Raddish Peroxidase) diluted lOOOx was added for 1 hour, whereafter again several washes were performed. The binding of the HRP labelled antibody was detected by the enzymatic conversion of TMBS.
  • HRP Hexe Raddish Peroxidase
  • mice sera had an average titer of 1:3000, which is a very good titer.
  • a peptide comprising the amino acid sequence according to SEQ ID NO:2 is immunogenic and is able to raise antisera with very good titers.
  • From the afore-mentioned mice sera we were able to obtain at least five monoclonal antibodies which were highly reactive with the peptide comprising the amino acid sequence according to SEQ ID NO:2.
  • the monoclonal antibodies were obtained according to standard techniques well known in the art (Coligan et al. (eds), Current protocols in Immunology, 1992; Steenbakkers et al. , Mol. Biol. Rep. 11:125-134, 1994) .
  • the anti-peptide response was measured using plates coated with the N-acetylated peptided coupled to BSA. 100 ⁇ l of the monoclonal antibodies were added together with the free N-aceylated peptide in a concentration range of 0-0.155-0.313-0.626-1.25-2.5-5- 10 ⁇ g/well in duplo to the plate. After several wash steps sheep-anti-mouse-antibodies conjugated to HRP (lOOOx diluted) were added to each well, followed by incubation with the substrate TMBS at roomtemperature. 100 ⁇ l 4N H 2 S0 4 was added and the absorbance of the wells was determined at 450 nm. The free peptide was able to compete with the peptide coated to the plates and at concentrations varying from 200 ng/ml to 5 ⁇ g/ml of free peptide the monoclonal antibodies which were bound to the plate was reduced by 50%.
  • Fig. 1 Autoradiograph of amplification products of indicated patient derived T cell lines.
  • the synovium derived cell lines were stimulated by IL-2 (SCRO IL- 2), PHA + IL-2 (SCRO PHA) or anti-CD3 + IL-2 (SCRO aCD3) .
  • PBL peripheral blood lymphocytes. Indicated are the bands containing amplification products for C ⁇ , VA2, CA and V ⁇ l gene families.
  • Fig. 2a Expression of TCR VA genes as detected by amplification analysis. Indicated are strong, moderate and low expression observed from autoradiographs as shown in fig. 1 for each VA gene family (listed from VAl to vAl8) and for each T cell line. The synovium derived cell lines were stimulated by IL-2 (SCRO IL- 2), PHA + IL-2 (SCRO PHA) or anti-CD3 + IL-2 (SCRO aCD3). PBL IL-2 are peripheral blood lymphocytes stimulated by IL-2.
  • Fig. 2b Identical scheme for V ⁇ -gene families.
  • FIG. 3 Rearrangement of data in Figs. 2a and 2b to show differences in V gene expression.
  • TITLE OF INVENTION A T cell receptor sequence specificall associated with an immune disease.
  • ORGANISM Homo sapiens
  • G CELL TYPE: T-lymphocyte
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Homo sapiens
  • G CELL TYPE: T-lymphocyte
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ORGANISM Homo sapiens
  • G CELL TYPE: T lymphocyte
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Homo sapiens
  • G CELL TYPE: T-lymphocyte

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Abstract

L'invention se rapporte à un polypeptide comprenant une séquence d'acides aminés contenue dans une région variable d'une chaîne Vβ d'un récepteur de lymphocytes T associé à une maladie immune, et elle est caractérisée en ce que ladite séquence représente la séquence selon SEQ ID NO: 2 ou un fragment ou équivalent fonctionnel de celle-ci. L'invention se rapporte également à la molécule d'acide nucléique codant ce polypeptide, ainsi qu'aux anticorps faisant partie de la séquence d'acides aminés ou dirigés contre celle-ci. En outre, l'invention concerne des procédés de détection de la maladie immune ou de la prédisposition à celle-ci, des vaccins ainsi que des formulations pharmaceutiques destinés au traitement de cette maladie.
PCT/EP1995/000670 1994-02-23 1995-02-23 Sequence du recepteur de lymphocytes t specifiquement associee a une maladie immune WO1995023164A1 (fr)

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EP94200454.0 1994-02-23

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0816496A2 (fr) * 1996-06-24 1998-01-07 Roche Diagnostics GmbH Cellules T spécifiques pour le carcinome rénal
US20100311054A1 (en) * 2002-07-03 2010-12-09 Miller Jamie L Compositions and Methods for the Detection of Human T Cell Receptor Variable Family Gene Expression
US10377808B2 (en) 2013-01-29 2019-08-13 Max-Delbrück-Centrum Für Molekulare Medizin (Mdc) Berlin-Buch High avidity antigen recognizing constructs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001133A1 (fr) * 1989-07-19 1991-02-07 Arthur Allen Vandenbark Peptides recepteurs de cellules t constituant une therapie contre des maladies autoimmunes et malignes
WO1992012996A2 (fr) * 1991-01-22 1992-08-06 The Immune Response Corporation Vaccination et procedes de lutte contre des maladies causees par des reactions pathogenes de populations de lymphocytes t

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001133A1 (fr) * 1989-07-19 1991-02-07 Arthur Allen Vandenbark Peptides recepteurs de cellules t constituant une therapie contre des maladies autoimmunes et malignes
WO1992012996A2 (fr) * 1991-01-22 1992-08-06 The Immune Response Corporation Vaccination et procedes de lutte contre des maladies causees par des reactions pathogenes de populations de lymphocytes t

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0816496A2 (fr) * 1996-06-24 1998-01-07 Roche Diagnostics GmbH Cellules T spécifiques pour le carcinome rénal
EP0816496A3 (fr) * 1996-06-24 2002-01-09 Roche Diagnostics GmbH Cellules T spécifiques pour le carcinome rénal
US20100311054A1 (en) * 2002-07-03 2010-12-09 Miller Jamie L Compositions and Methods for the Detection of Human T Cell Receptor Variable Family Gene Expression
US10377808B2 (en) 2013-01-29 2019-08-13 Max-Delbrück-Centrum Für Molekulare Medizin (Mdc) Berlin-Buch High avidity antigen recognizing constructs
AU2018204891B2 (en) * 2013-01-29 2020-03-12 Max-Delbrück-Centrum Für Molekulare Medizin (Mdc) Berlin-Buch High avidity binding molecules recognizing mage-a1
US11447536B2 (en) 2013-01-29 2022-09-20 Max-Delbrück-Centrum Für Molekulare Medizin (Mdc) Berlin-Buch High avidity antigen recognizing constructs

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