JPH08500328A - Protective action of mutated superantigen - Google Patents

Abstract

  (57) [Summary] The present invention comprises a method for preventing or treating the toxic effects of a superantigen. The subject is treated with a molecule that interacts with the specific Vβ component of the T cell in a manner similar to the original superantigen. The molecules of the invention are mutated or modified superantigens that induce antibody production without inducing T cell proliferation.

Description

DETAILED DESCRIPTION OF THE INVENTION Protective action of mutated superantigen Field of the invention   The present invention relates to the prevention and treatment of antigen-mediated diseases and antigen-initiated diseases. Concrete In other words, it induces an antibody response to a superantigen that has no pathological effects Of a molecule that is a modified or mutated superantigen Administration provides protection against superantigen pathogens. Of the present invention The molecule may also be a T cell receptor in a manner that leads to modifications that make the T cell respond to antigen. May interact with the Vβ component. Background of the Invention   Vertebrate immune system protects vertebrates from infection by microorganisms and large parasites So evolved. This immune system responds to antigens in one of two ways: (1) proteins that circulate in the blood and specifically bind to the foreign antigen that induced them; B cell mediated humoral antibody response, including production of white antigen. By binding of the antibody to the antigen Blood protein system that helps phagocytes digest and destroy antigens (collectively supplemented) (Called the body); and (2) if the antigen is an infectious virus By killing the host cell, or other host cells (eg, phagocytic cells). ) To destroy the antigen, mainly with the foreign antigen on the surface of the host cell Cell-mediated immune responses mediated by T cells, including the production of specialized cells that respond Molecular Biology of the Cel 1) (1983), Alberts et al. (eds.), Chapter 17, 9 52).   Antibody production involves many pre-steps that lead to the stimulation of antibody-producing B cells. Is necessary. One of the key steps involved in antibody production is antigen recognition. This is the stage. Thymic (T) cell participation is required for antigen recognition.   T cells have an antigen-specific receptor, called a T cell antigen receptor (TCR), on their surface. Has a scepter. Before T cells can recognize a protein antigen, the antigen Must be presented on the surface of the presenting cell. The antigen is first macrophages or Must be processed by other antigen presenting cells. These cells are basically anti- Swallow the raw material, chop them into peptides, and the peptide is a major histocompatibility complex (MHC) Present on the cell surface together with molecules.   Major histocompatibility complex is formed by a complex of genes called major histocompatibility complex. Family of antigens to be loaded. MHC antigens are all higher vertebrate cells Is expressed on In humans these were first demonstrated on white blood cells , HLA antigens (human leukocyte-related antigens). The MHC molecule has two major Classes (Class I and Class II), each consisting of a set of cell surface glycoproteins . These two classes of MHC antigens stimulate different subpopulations of T cells. MHC Las II molecules are involved in many responses to pathogens. In contrast, MHC Ras I molecules are involved when the pathogen is a virus or a malignant cell. MHC When class I is involved, antibody stimulation does not occur and MHC class I treated antigen and T cells Vesicle interactions cause lysis of cells infected by the pathogen.   The processed antigenic peptide fits into the cleft of the MHC molecule. Antigen is presented And several T cells in the body that have receptors for that particular peptide Binds to the complex. Many T cells associate with autologous MHC glycoproteins on the same cell surface. Recognize antigens on the surface of cells only in the first case.   The ability of T cells to complex with the processed antigen and MHC complexes is It depends on the vesicle receptor (TCR). TCR consists of two protein chains (α and β) . Each chain has a constant domain and a variable domain. Two variable domains (α) or Is encoded in three (β) different gene fragments (variable (V), diversity ( D), bond (J)) (Siu et al. (1984) Cell 37: 393. Yanagi et al. (1985) Proc. Natl. Acad. S ci. ScL USA 82: 3430). In each T cell, V, D and Combination of J domains recognizes antigen in a manner characteristic of T cells only Specify the part. Marrack et al. (1988) Immunol. . Today 9: 308; Toyonaga et al. (1987) A. nn. Rev .. Immunol. 5: 585; Davis (19 85) Ann. Rev .. Immunol. 4: 529; Hendrick (H endrick) et al. (1982) Cell 30: 141; Babbit. b itt) et al. (1985) Nature 317: 359; Booth. (1987) Science 235: 1353; Townsend (Town) send) et al. (1986) Cell 44: 959; Bjor. (Kman) et al. (1987) Nature 329: 506). Typically α and β chains are involved in the recognition of ligands formed by processed antigens and MHC doing.   When T cells are stimulated by antigen, they divide and undergo various cell-mediated immune responses. Differentiate into activated effector cells involved in the response. At least 3 depending on T cells The following reactions are performed: (1) Cytotoxic T cells are exogenous or virus-infected vertebrae Specifically kills animal cells; helper T cells help B lymphocytes; and (3) Suppressor T cells suppress specific cell responses.   A new class of antigens, recently called “superantigens”, are specific Vβ components (such as Directly stimulates T cells by binding to the TCR β chain variable domain) (Kappler et al. (198) 7) Cell 49: 263; Kappler et al. (1987) C. ell 49: 273; MacDonald et al. (1988). Nature 332: 40; Pullen et al. (1988) Nat. ure 335: 796; Kappler et al. (1988) Nat. 332: 35; Abe et al. (1988) J. Am. Immunol.   140: 4132; White et al. (1989) Cell 56. : 27; Janeway et al. (1989) Immunol. Rev .. 107: 61; Berkov et al. (1980) J. Am. I mmunol. 139: 3189; Kappler et al. (1989) ) Science 244: 811). Unlike traditional peptide antigen recognition, Other components of the T cell receptor (ie, Dβ, Jβ, Vα, Jα) are superantibodies. It does not appear to play any role in original binding. Superantigens are generally T cells But only with specific Vβ components present on stimulated T cells Seems to interact. Because the relative number of Vβ genes is limited, T cells have specific Vβ components within individual cells, and therefore have specific superantigens. Can interact with most of the population of T cells. That is, it depends on the frequency of the Vβ population. Thus, 5-30% of all T cell populations are stimulated by superantigens, Similar responses to the original are usually much less than one thousandth. Super anti Progens interact with class II MHC molecules, but these are more in vivo than as peptides. It appears to act as a tact protein. That is, these are the usual peptide bonds. It does not appear to join the mating grooves. Instead, they are located on the outer wall of the coupling groove. It appears to interact with the acid residues. Known superantigens and their sequences and structures Are listed in Table I.   Two distinct classes of superantigens have been described. The first one was 20 years ago At this time, Festenstein is a certain M Significant responses were found in the mixed lymphocyte reaction between the same strains of HC. Irritating Minor lymphocyte stimulator (Ml) to distinguish antigens from MHC antigens Called antigen (Festenstein Transp lant Rev. 15:62). These superantigens are endogenous retro Encoded by the irs gene (Palmer (1991)   Curr. Bio. 1:74). These genes are present in mice This results in a marked loss of responding T cells, which is large in the T cell receptor population of the animal. A possible hole may be created (Pullen et al. (1988) supra). A second set of superantigens can produce various pathogenic effects after injection. Are provided by an ever-growing list of available bacterial or viral proteins. Racks and couplers (Marrack & Kappler) Science 248: 705).   Staphylococcus, a common human pathogen, cus aureus (S. aueus) is a SEA (staphylococcus) Several enterotoxins named from enterotoxin A) to SEE (Enterotoxin), which can cause human food poisoning and sometimes shock. Involved (Marrack & Kappler) ( 1990) supra; Bohach et al. (1990) Crit. Re v. Microbio. 117: 251). Some S.P. aureus only Strains are also thought to be involved in the majority of toxic shock syndromes in humans. Toxic shock syndrome toxin-1 (TSST-1) and dermabrasion syndrome (sc Related exfoliating toxins (e) associated with alded skin syndrome Produce xfolative toxins (ExTF). Another common Streptococcus spirogenes (Streptococcus), a pathogen of human skin and pharynx ococcus pyrogenes) (ie, group A Streptococcus) It also produces a toxin having superantigenicity (Abe et al. (199) 1) J. Immun. 146: 3747). These are Streptococcus Lithrogenic toxin (Streptococcal erythrogen) ic toxins) AC (SPEA-C).   S. The amino acid sequence of Aureus toxin shows some homology, It also shows significant differences (Bentley et al. (1988) J. Bac terol. 170: 34; Jones et al. (1986) J. . Bacteriol. 166: 29; Lee et al. (1988) J. Am. Bacteriol. 170: 2954; Bromster-Houtamaa (Blo Mster-Hautamaa) et al. (1986) J. Am. Biol. Chem. 261: 15783)). S. aureus has MHC class II molecule Antibodies that stimulate a potent T cell proliferative response in the presence of murine cells Carlson et al. (1988) J. Am. Immunol. 140 : 2848; White et al. (1989) Cell 56:27). . S. aureus protein selectively stabs rodent cells with specific Vβ components. Intense.   Toxin binding to class II MHC molecules is essential for T cell recognition, The process is more tolerant than found with normal antigens. Peptide antigens Although it is highly dependent on rele- sive (allelic) MHC residues, superantigens are widely used. Binds to MHC class II molecules of the releasable and isotypic type (Herman (Herman (mann) et al. (1989) Euro. J. Immunol. 19: 2171 Hermann et al. (1990) J .; Exp. Med. 172 : 709; Scholl et al. (1990), J. Am. Immunol. 144: 226; Molleck et al. Immu nol. 146: 463). T cells are rarely self MHC (allo MHC) (All-MHC)) does not recognize the peptide antigen bound to the molecule, but Cells are not only allele-type MHC but also different class II isotypes and xenogeneic (Xenogenic) may respond to toxin even bound to class II molecules it can. Such observations indicate that superantigens bind traditional peptide antigens. It is a relatively conserved part outside the hypervariable groove as an allele thought to be Suggests binding.   Superantigens can also be involved in autoimmune diseases, where components of the immune system Attack normal tissue. T cells that respond to themselves (potentially harmful self-reactivity The process of deletion of T cells) is called tolerance or negative selection (Kappl (Kappl (1987) Cell 49: 273; Kappler. (1988) Nature 332: 35; MacDonald. ld) et al. (1988) Nature 332: 40; Finke 1) et al. (1989) Cell 58: 1047). The immune system is usually self-reactive Cells are deleted, but some escape this monitoring mechanism at times. Human T thin The ability of the superantigen to stimulate 20% of the vesicle cluster is due to several circulating Has been suggested to lead to unwanted replication of T cells (Johnson hnson) et al. (1992) Scientific American 26 6:92). T cells with certain Vβ types are found in certain types of cells, such as arthritis and multiple sclerosis. Involvement in various autoimmune conditions has been suggested. These findings suggest that destructive cells Means activated by a superantigen that binds to the identified Vβ type ( Johnson, et al. (1992) supra).   Autoimmune diseases are the result of the immune system's inability to recognize itself. By the immune system Many diseases are caused by host cell attacks: multiple sclerosis and severe muscle Neurological disorders such as asthenia, joint disorders such as rheumatoid arthritis, systemic lupus Attack on nucleic acids as observed in matodes, other diseases involving various organs ( For example, psoriasis, juvenile diabetes, Sjogren's syndrome, and thyroid disease)). this These diseases have various symptoms, ranging from mild to life-threatening. An example For example, rheumatoid arthritis (RA) is a chronic, relapsing inflammatory disease, primarily involving joints. And affects 1-3% of the North American population, with a female to male ratio of 3: It is one. Severe RA patients have vasculitis, muscular atrophy, subcutaneous nodules, lymphoma, splenomegaly, And extra-articular symptoms such as leukopenia. About 15% of RA patients are fully lucky It is presumed to cause paralysis anesthesia.   T cells specific for autoantigens play a critical role in initiating autoimmune diseases Is suggested by some evidence. In the case of RA, MHC class II remains The link between the genes DR4 and DR1 Allele, and sometimes synovial fluid and affected Activating CD4 with minor clonality in nodal tissues⁺T cells (Stastony (Stas tny) et al. (1976) Engl. J. Med. 298: 869; Gibofus Gibofsky et al. (1978) Exp. Med. 148: 1 728; McMichael et al. (1977) Arth. Rh um. 20: 1037; Schiff et al. (1982) Ann. Rheum. Dis. 41: 403; Duguestoy et al. (1984) Hum. Immunol. 10: 165; Legrand (Leg) rand) et al. (1984) Am. J. Hum. Genet. 36: 690; Gregerse et al. (1987) Arth. Rheum. 3 2:15; Burmester et al. (1981) Arth. R heum. 24: 1370: Fox et al. (1982) J. Am. Im muno1. 128: 351; Hemler et al. (1986) J. . Clin. Invest. 78: 696; Stamencoic (Stamen) koic) et al. (1988) Proc. Natl. Acad. Sci. USA8 5: 1179) suggests that CD4⁺II with αβ TCR It suggests the involvement of restricted T cells. This view is based on the T cell portion in various ways. The finding that removal or inhibition can relieve the disease in some patients supports this. (Paulus et al. (1977) Art. Rheum.   20: 1249; Karsh et al. (1979) Arth. Rhe um. 22: 1055: Kotzin et al. (1989) N.A. Eng . J. Med. 305: 976; Herzog et al. (198) 7) Lancet ii: 1461; Yocum et al. (1989). Ann. Int. Med. 109: 863).   US patent application Ser. No. 07 / 732,114, which is specifically incorporated herein by reference. Is that specific Vβ components can be used for the diagnosis of autoimmune diseases, Physically, a high percentage of Vβ14 in synovial fluid to diagnose RA⁺Use of the presence of T cells Indicates that it can be used.   Many studies have focused on developing treatments for autoimmune diseases. example For example, European Patent Publication No. 340,109 (title: Anti-T Cell Receptor Determinants), and U.S. Patent No. 4, filed October 29, 1985. No. 550,086 (Reinherz et al. (Title: Human T cells Monoclonal antibodies that recognize T cells A method for detecting a specific sequence of a variable region gene and treating a disease with an antibody against the sequence Describes the law. U.S. Pat. No. 4,886,74, filed Dec. 12, 1989 No. 3 (Hood et al.) (Title: only one within the variable region of the T-cell receptor) Sequence-based diagnostic reagents and their use) are unique in the Vβ region associated with a particular disease. A method for diagnosing a disease based on the presence of a T cell having the sequence PCT special Patent Application Publication No. W090 / 06758 discloses a specific Vβ region (specification) associated with RA. Physically, Vβ3, Vβ9 and Vβ10) are detected, and Vβ3, Vβ9 and And treatment of RA with monoclonal antibodies recognizing Vβ10 and Vβ10 .Immunity   Animals that have not been exposed to the pathogen have specific protection against it. Absent. However, non-pathogenic agents that are similar in chemical structure to the pathogen but lack the ability to exhibit pathogenic effects Animals can be immunized against pathogens by injecting them . Animals produce antibodies specific to non-pathogenic forms of the pathogen, and these antibodies From pathogen-type pathogens. Summary of the Invention   The present invention includes a method for preventing the toxic effects of superantigens by treatment with molecules. In which the molecule elicits antibody production without T cell activation.   The present invention also relates to modified or mutated derivatives of superantigens. Molecules.   The present invention further provides the Vβ component alone or α chain of the T cell receptor (TCR). Antigen-induced, consisting of administering molecules that interact with both And methods of modifying a T cell response.   The molecules of the present invention may comprise at least one or more T cells that provide a particular Vβ component. It works by leading to the deletion or inactivation / desensitization of more subpopulations.   To prevent the toxic effects of superantigens, understand exactly how to achieve this effect. Need to be Prior to the present invention, how superantigens interact with T cells It is known whether it works, but the mechanism by which the target animal shows the disease and the disease progresses Whether or not was not fully understood. Various observations are made by several mechanisms. Suggest that some of them are responsible for toxicity.   A condition mediated or initiated by a superantigen is a mutant strain of the present invention. Is found to be prevented or treated by the administration of Was. By administering the mutant toxin of the present invention, antibody production against the mutant molecule May cause. Some of these antibodies are also found in normal non-mutated Reacts with Shin. Therefore, when an immunized individual faces a normal toxin, these Cross-reacts with normal toxin and inhibits its activity. BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic view of a ribbon having a three-dimensional structure of SEB. Region 1 (residues 9-23), Region 2 (residues 40-53) and region 3 (residues 60-61) are shaded Have been. The sites involved in MHC or TCR binding are indicated. MHC and And / or residues identified by mutational analysis important for TCR binding are indicated .   FIG. 2 a shows 2 μg of recombinant SEB purified from E. coli and S. aureus culture FIG. 4 shows SDS-PAGE analysis of wild-type SEB purified from the product. Molecular weight marker (KD): β-phosphorylase, 94; bovine albumin, 69; ovalbumin, 45; carboxylase, 30; soybean trypsin inhibitor, 21; lysozyme , 14. FIG. 2b is an SDS-PAGE analysis of SEB binding to DR on LG2 cells. Is shown. Molecular weight marker (kD): bovine albumin, 69; ovalbumin, 45 Chymotrypsinogen, 27; soybean trypsin inhibitor, 21; myoglo Bottle, 17; lysozyme, 14.   FIG. 3 shows the sequence of the SEB mutant. Dashed line is the unmutated part of SEB Indicates minutes. Position of the oligonucleotide used to generate the SEB mutant Are also shown.   FIG. 4 shows SEB and SEB mutation in lymphoblastoma TG2 cells having DR-1. Indicate variant binding.   FIG. 5 shows stimulation of T cell hybridomas by region 1 SEB mutant. Spirit Prepared SEB or region 1 mutant preparations are known to recognize SEB. Tested for the ability to stimulate a population of T-cell hybridomas with different Vβ components KS-20.15 (Vβ7), KS-6.1 (Vβ8.2), KS-47. 1 (Vβ8.3), K16-57 (Vβ8.1).   FIG. 6 shows stimulation of T cell hybridomas by the region 2 SEB mutant. Spirit The prepared SEB preparation was tested as in FIG.   FIG. 7 shows stimulation of T cell hybridomas by the region 3 SEB mutant. Spirit The prepared SEB preparation was tested as in FIG.   FIG. 8 shows the in vivo effects of SEB and its mutants. Group of 3 mice And then 0, 50 μg (left) or 100 μg (right) of recombinant SE B or mutant SEB BR-257 or SEB BR-358 A balanced salt solution (BSS).   FIG. 9 shows the protective effect of the mutant toxin against SEB challenge. mouse Received saline or BR-257 three months prior to wild-type SEB challenge. Was. Detailed description of the invention   The molecules of the invention prevent antigen-mediated and antigen-initiating diseases in different ways. Or to treat. The different ways in which the molecules of the invention may be useful include Modification of the T cell response that provides protection to the body and production of antibodies. Specifically, Ming is to prevent or treat superantigen-mediated diseases and superantigen-initiated diseases Give the law. The methods of the present invention are generally known in the art and described in the present invention. A mutated superantigen molecule is prepared by known methods and An antigen mutant capable of binding to the CR has been identified and has not been mutated. To be able to confer protection against exposure to new superantigens It comes from things.   The present invention is an implementation of the aforementioned method for achieving protection against known superantigens Describe the possibilities. Recombinant Staphylococcus enterotoxin B (SEB) Mutants were prepared and purified as described in Examples 1-4 below. HLA-D Different binding of mutant SEB molecules to R1 homozygous lymphoblastoid LG2 cells Examination of the stimulation of T cell hybridomas having a Vβ component SEB mutants capable of binding to R were selected. S not mutated Bind to LG2 cells to an extent indistinguishable from EB and stimulate T cell hybridomas 3 months before exposing SEB mutant BR-257 to SEB Showed complete protection against the toxic effects of SEB. Same for primates Was obtained.   The teachings of the present invention can protect animals against subsequent challenge with SEB. Although the production of a mutated SEB molecule has been described, The same can be applied to hyperantigens.   As a method for detecting and treating autoimmune diseases mediated by superantigens, Demonstration of the ability to utilize the presence of specific Vβ components in the diagnosis of autoimmune diseases Can be combined with Ming. The presence of a disease mediated by superantigens Pattern ”analysis (eg, whether there is a change in the Vβ component in the disease state). Can be. Changes in Vβ components (Vβ14 in rheumatoid arthritis synovial fluid)⁺T thin Vesicle increase) indicates the presence of a superantigen-mediated disease. Next suggested A method known in the art can be applied to identify a hyperantigen. Illness To determine whether superantigens may be involved in the initiation or progression of The fingerprint can be compared to that of a known superantigen. Virus infection or When bacterial infection is associated with the onset of the disease, You may search. If a superantigen is identified or isolated, exposure of the superantigen To produce mutant superantigen molecules that can provide protection against The method of the invention applies.   Various terms are used herein and it is useful to define them Will. These are shown below, which are used in the following examples. It should be noted.   As mentioned above, the major process in the immune response is that MHC molecules interact with antigens. To form a complex that is presented to T cells. Generally T cell response is very Of T cells that are specific for and respond to specific complexes of antigen and MHC molecules The population is very limited. This response is generally associated with most of the T cell receptors or Requires interaction of all components. However, in some cases, presentation The expressed antigen need only interact with the Vβ component of the receptor, and all other components Minutes are basically irrelevant. This is because the antigen reacts with more T cells than normal Means and can be.   The molecules of the present invention may be modified such that T cells respond to the superantigen, It can interact with the Vβ component of the T cell receptor. "Repair T cell responsiveness "Decorate" can occur when the subject's T cells are stimulated by the administered molecule or at the same time. Can change the way that responds to antigens administered before, after or after Means that For example, a subpopulation early in T cell development may Interact and be deleted. The molecules of the present invention thus provide a specific Vβ component. Deletion or inactivation of at least one or more subpopulations of the indicated T cells / (By leading to desensitization).   In certain embodiments of the invention, the molecule is a B cell response that normally occurs in the subject under consideration. Modifies the T cell response without changing the response. This type of substance is, for example, By conferring passive immunity on the subject or acting as a vaccine Is useful. When using superantigen derivatives, they have a limited T cell response. Response, and act as an antigen to elicit a B cell response. These derivatives no longer have superantigenicity. The superantigen derivative of the present invention Normal antibody production against the superantigen protein can be induced.   The antigens of the present invention can also be viewed as competitors of other antigens. Write here Molecule that produces an overall response to the antigen or superantigen otherwise. If they interact with the MHC components necessary to produce them, they prevent the extent of that response. Will stop or lower.   The molecules of the present invention can also be viewed as "enhancers" in some cases, Individual T cell responses are impaired or attenuated for several reasons You. The administration of the molecules encompassed by the present invention greatly expands the individual T cell population .   As used herein, the term “modifies T cell responsiveness” always refers to the second component (eg, For example, antigens), and is always identified as part of the T cell receptor To the response of T cells presenting the Vβ component of Is basically irrelevant.   The molecule of the present invention has an amino acid of at least sufficient size to bind to the MHC molecule. Contains an acid sequence. The rest of the molecule is from amino acids, or carbohydrate or lipid structures It may be.   The term “reduce responsiveness” refers to the part of a T cell that expresses a particular Vβ component. It also means deleting minutes.   As used herein, the term “superantigen derivative” means that at least its structure is MH The superantigen or part of the superantigen needed to bind to C or T cells Means a molecule containing an amino acid sequence substantially the same as the amino acid sequence presented I do.   A “modified” superantigen derivative (or fragment) is a “mutated” Different from per antigen (or fragment). The term "modified superantigen" Contains the same amino acid sequence as the superantigen, but It is defined as meaning a molecule that contains modifications not found in the original molecule itself. For example If the superantigen contains amino acids 1-250, a "modified" superantibody The original derivative has expanded amino acid sequence not found in the original superantigen molecule Contains the same sequence as amino acids 50-75, which is located in the portion. For example, additional modifiers For example, different glycosylation or lack of glycosylation, or usually nothing There is some glycosylation.   A “mutated” superantigen is the result of a mutation compared to its native form. In which the amino acid sequence is changed. For example, if the super antigen is amino When containing acids 1-250, the mutated superantigen will have the corresponding native configuration. Amino acids 50-68 and 72-7 which are the same as in the row but differ at amino acids 69-71 5 This difference is due to the “substitution” in which different amino acids are used, "Addition" longer than the original form by adding One of them.   "Vaccine" refers to the same type of vaccine generally administered to a subject. Refers to a formulation that provokes a response (eg, immunoprevention). Vaccine is adjuvant Or it may contain other substances.   A class of molecules known as superantigens are specific V-cells of the T cell receptor. It is known that it interacts with the beta component and increases the specific T cell subpopulation in large quantities. This interaction, assuming a previous interaction between the MHC molecule and the superantigen, is It is completely independent of the other areas of the cell receptor.   In connection with the interaction between MHC and peptides, MHC molecules can be of various "phenotypes". Different phenotypes are specific for various presented peptides and antigens. It should be noted that there is. For example, HLA-DR is related to SEB presentation. Is known. That is, different MHC phenotypes are valuable for different antigens However, correlation of HLA phenotype measurement with presentation of a particular antigen or antigen family Is well within the skill of the artisan.   That is, the present invention relates to an MHC molecule and at least one Vβ component of a T cell receptor. Includes modification of T cell responses through administration of a molecule that interacts with both . This interaction affects the T cell response in several ways. That may affect the response One of the most rudimentary methods is that a molecule interacts with an MHC molecule, To prevent the binding of Normal or superantigens are Because the complex that causes the answer cannot be formed with the MHC, If modified or does not induce T cell proliferation, there is a diminished or eliminated response. Will be.   Other methods of modifying the T cell response include "desensitizing", "inactivating" or It is "anergy" (immune weakness). This mechanism is based on MHC molecules, anti- Interaction with primordial and T cell receptors, following down-regulation (do wn regulation) or inactivation of T cells. This mechanism is mature Are more common in fetal deletions than in those who do. The latter phenomenon has three Various subpopulations of T cells are actually removed from an organism through the interaction of One.   Modification of a T cell response also includes stimulation of a T cell subpopulation. As described herein Information is available to those skilled in the art for substances that interact with MHC and for specific subpopulations of T cells. To allow the subpopulation of T cells to expand. This method is specific If the Vβ subpopulation is associated with a medical condition (eg, an autoimmune disease), Particularly preferred.   It should be understood that the immune response, when considered, includes both B and T cell responses. It is. One aspect of the invention is modifying a T cell response without modifying the B cell response. The use of decorating molecules. Such substances are particularly useful as vaccines, as described below. And useful.   The molecules of the invention are preferably (but not limited to) superantigen-derived Body. These derivatives may be modified or mutated as described above. These molecules or other molecules described herein may respond to T cell responses in the manner described. Is administered in an amount sufficient to modify The amount of substance used depends on the actual substance It depends on the response required and the subject being treated.   The molecule also acts as a vaccine. These vaccines have the usual molecular In the sense that they produce a B cell response without the total T cell response normally associated with the type, Provides protective immunity to elephants. Example 7 shows one of the effects of SEB. Again, those skilled in the art Depends on the variables within the regulatory range of the knowledge of the disease (conditions to be treated, Vβ molecules regulated, etc.) Thus, the substance selected as a vaccine is the responsibility of the person skilled in the art. This vaccine is Contains other substances commonly found in vaccine compositions (adjuvants, carriers, etc.) It may be.   The method of administration of the substances described herein (intravenous, intraperitoneal and intramuscular injection) As with all other standard methods of administering a therapeutic agent to a subject.   The present invention also discloses methods for making certain mutants useful in the aforementioned methods. (Eg, an isolated nucleic acid sequence that encodes a mutant; Cell lines transformed with the vectors and plasmids used, and isolated Mutant molecules (including mutant superantigens).   Other applications of the invention described herein will be apparent to those skilled in the art and will now be repeated. There will be no need to return.   The terms and expressions used are for description purposes only and are not limiting in any way. Features and equivalents that are shown and described when such terms and expressions are used Various modifications are possible without departing from the scope of the present invention. It should be understood that   Polymerase chain reaction (PCR) and standard molecular biological methods (see Example 1) Description) was used for the production and expression of recombinant SEB. Such a mutant is Prepared by one of the two methods described in Example 2. The first method is SE Random mutations were introduced over the entire length of the B gene. The second method is SEB Random mutations were introduced in a region defined by about 60-75 bases of the gene . For the first identification of potential mutant SEBs, a human DR-expressing cell line was Stimulation of rodent T cell hybridomas with different Vβ components in Lysates from the transformants were tested for the presence of a toxin. T cell hybrid The lysate, which was negative upon stimulation of the dormer, was a monoclonal antibody against SEB (mAbs). ) Was tested for the presence of SEB (Example 3). Produces non-functional SEB The transformants were sequenced and mutants were identified. Produce mutant SEB And transforming the mutant SEB as described in Example 4. Was. Mutation location and effects were analyzed. MHC for toxin recognition by T cells Since binding to class II molecules is essential, human MHC as described in Example 5 The ability of the mutant SEB to bind to the antigen HLA-DR1 was tested. Mutate Some hybridomas have specific Vβ components that are stimulated by non-toxins Mutant SEBs that selectively stimulate things (but not all) (eg, region 3 suddenly) Mutant). That is, T cells stimulated by wild-type superantigen Mutant superantigens of the invention for selectively stimulating only a small portion of a population Can be used.   Three regions at the N-terminus of SEB that affect MHC and / or TCR binding Was identified (Example 4 and FIG. 1). The mutation in region 1 (residues 9-23) is And both TCR binding. This result shows that 23N is particularly important. Suggested. S. Aureus enterotoxin may have maximum homology (Marrack & Kapple) (19 90), supra), which residues are all enterotoxins and toxic shock toxins Saved in Mutations in region 2 (residues 41-53) stimulate T cells Toxins have the ability to bind to MHC class II with similar effects Dramatically reduced. Approximately half of the mutations involved F44. Again, all This residue is conserved in enterotoxins, and this residue is the MHC of all toxins. Has shown to play a very important role in binding to. Interestingly area 1 Or any of the two mutations abolish binding of the toxin to MHC In all cases, the ability of the mutants to stimulate T cells Was recovered by adding Mutations in region 3 (60N, 61Y) Did not affect binding of the toxin to the MHC, but did not phase with the two Vβs (7 and 8.1). Influenced the interaction. This Vβ-specific action indicates that these amino acids Important for interaction with TCRVβ (probably not another Vβ component) Suggests.   The toxicity of the mutant SEB in animals was tested as described in Example 6. Ma Use balanced salt solution (BSS), recombinant SEB, or F44 (BR-35 8) or injected a region ISEB mutant at N23 (BR-257). I Mice receiving 50 μg of some mutant SEBs were compared to mice receiving BSS. Although indistinguishable, mice given recombinant SEB died within 5 days It was done.   The ability of the mutant SEB to confer immunoprotective capacity from SEB was tested in vivo ( Example 7). Three months before challenge with SEB, 100 μg of mutant SEB B Mice given R-257 were completely protected from the toxic effects of SEB, but BR Mice that did not receive -257 died 4-5 days after challenge with SEB. Similar results were obtained with primates. Sudden change to elicit a vomiting response in monkeys Different SEBBR-358 or BR-257 are ineffective or small even if effective (Example 7).   Example 8 describes the application of the above method to SEA toxins and the corresponding SEB mutant The production of a SEA mutant which behaves the same as described above is described. Embodiment 1 FIG.Preparation and expression of recombinant SEB Polymerase chain reaction (PCR)   PCR (Saiki et al. (1988) Science 239: 4 87) is compatible with ampritiq (AmPliTaq) recombinant Taq polymerase. Kin Elmer Cetus (Perkin Elmer Cetus) (Nor War Using a DNA thermal cycler from Norwalk, Connecticut went. 1 minute denaturation and annealing step, 1 minute for <500 bp synthesis Between 20 and 30 cycles for a 2 minute extension for> 500 bp synthesis I went. The template concentration was 1-10M and the oligonucleotide primer was -The concentration was 1 μM. Although the concentration of dNTPs is 200 μM, If so, the concentration of one dNTP was reduced to 20 mM.SEB creation body   The superantigen SEB gene was overexpressed as follows. Signal pepti Flanking the portion of the gene encoding mature SEB that does not contain In PCR using oligonucleotide primers, the line containing the SEB gene -Shaped plasmid (Ranelli et al. (1985) Proc. Nat) l. Acad. Sci. USA 82: 5850) as a template. . 5 'primer (SEQ ID NO: 1) It is.   This primer was used in plasmid pTZ18R (Pharmacia Fine Chemicals) Luz (Pharmaca Fine Chemicals), Piscataway ( Lac when cloned into Piscataway, NJ) It contains an EcoRI site that places the SEB gene in frame with the Z gene. this Oligonucleotide primers can also be used when the SEB gene has its own starting ATG LacZ gene fragment and SE so that it can be easily transferred to other primers having Contains an NcoI site that adds an ATG during the beginning of the B gene. This 3 'ply The mer contained a HindIII position after the stop code of the SEB gene (sequence Number 2):   This PCR fragment was digested with EcoRI and HindIII, and EcoRI / HindI was digested. Ligated to II digested pTZ18R. Escherichia coli XL1-Blue (Stratagene (S Tratagen), La Jolla, Calif.) The plasmid was transformed, a single transformant was collected, and the insert (pSEB2) was used. Was determined and the absence of mutation was confirmed.   Upon induction, the pSEB2 construct causes overproduction of almost cytoplasmic SEB. (About 10 μg / ml broth). However, it produces a LacZ / SEB fusion protein. Rather than this bacterium, aureus secreted SEB A protein having an abundance was produced (FIG. 1a). The LacZ portion of this fusion protein is majority Cleaved in vivo from the SEB of E. coli or introduced between LacZ and SEB ATG was more efficient than the LacZ translation initiation site. Example 2Production of SEB mutant   SEB mutants were produced by one of two methods. One person The method introduced random mutations over the entire length of the SEB gene. For this reason The SEB construct of Example 1 was prepared, but PCR was performed using Taq volimerase error. A 10-fold reduction in dATP or dTTP concentration was used to increase the rate ( Innis et al. (1988) Proc. Natl. Acad. Sci. ScL USA 85: 9436). This reduces the amount of product by a factor of 5-10. Two The products of the reaction were combined and cloned into pTZ18R as described in Example 1. Individual transformants were mutated as described for BR mutants in Example 3, Screened for EB. About 400 toxins produced Of the live transformants, 10 are functional mutants due to their ability to stimulate T cells. And identified. Low concentrations of dCTP and dGTP were treated similarly, but the product results The degree of the decrease was smaller in about 200 transformants. No mutant was detected.   Introduces random mutation to a region defined by about 60-75 bases of SEB gene A second PCR method was used to insert. 3 corrections with 1% each position The following oligonucleotide blocks, which are located as shown in FIG. Primers (A (SEQ ID NO: 3), B (SEQ ID NO: 4), and C (SEQ ID NO: 5)) Synthesized:   These mutated oligonucleotides are either vector (A and B) or internal SE The B (C) oligonucleotide was used as a pond primer and the SEB gene was used as a primer. It acted as a primer in a PCR reaction to form a template. Synthesized SEB fragment Each molecule has a few random bases in the region corresponding to the mutant primer. It was predicted to have the mutation. Mixed template during PCR reaction of mutant fragment Alone or with other fragments containing the 3 'part of the gene, And resynthesized the full-length SEB2 gene (Ho et al. (1989) Gene 77:51; Pullen et al. (1990) Cell. 61: 1365). Alternatively, digest with an appropriate restriction enzyme, bind to pSEB2, This was done by extracting the corresponding area from this.DNA sequencing   Plasmid insert was inserted into Sepuenase (USV). Chemical Company (U.S. Biochemical Corp.), Cleveland Sanger et al. (1977) (ProC. . Natl. Acad. Sci USA 74: 5463) Sequenced using modifications to the strand supercoil plasmid template (Weikert and Chamblis (1989) Editorial Members, U.S.A. S. Bioc chemical Corp. ), Cleveland, Ohio, pages 5-6). Example 3Screening of SEB mutant transformants Anti-SEB monoclonal antibody (mAbs)   B10. Immunized multiple times with SEB. From Q (βBR) by standard method Both produced 10 monoclonal antibodies specific for 5 SEB epitopes Was. One of these antibodies (B344.1) was used to quantify SEB and SEB mutants. And immunoaffinity purification. B344.1 is an IgG1 that binds to SEB. SEB with high affinity and bound to MHC class II molecules was detected and immunoprecipitated. And SEB bound to DR was selected because it did not interfere with T cell recognition (data Are not shown).Enzyme-linked immunosorbent assay for SEB   The amount of SEB in the preparation was measured by ELISA (enzyme-linked immunosorbent assay). Was. 6 μg / ml of natural SEB (Sigma Chemi) cal Co. ), St. Louis, MO) with a microtiter solution Rate wells were coated overnight. The wells are then incubated with 25% FCS Washed thoroughly. Varying concentrations of known and unknown SEB preparations with inhibitors To the wells and then a fixed amount of anti-SEB antibody (Toxin Technology (To xin Technology, Madison, Wisconsin (State) (polyclonal rabbit anti-SEB and BR, BB and BC The experimental monoclonal anti-SEB, B344) was added. After 1 o'clock the well was completely Washed, alkaline phosphatase conjugated to goat anti-rabbit IgG (Sigma Chem. (Sigma Chemical Co.) or p-nitrite as a substrate. Bound antibody was detected by standard methods using rophenyl phosphate. Computer analysis of the data indicates the OD of the reaction at 405 nm And the concentration of SEB in the unknown.Initial screening of potential mutant SEBs   For initial screening, potential mutant SEB genes Complete lysates were prepared as described from the individual transformants containing. Hi The DR-expressing cell line was used as a presenting cell, and a rod having an α / β receptor was used. Stimulating D-cell hybridomas with Vβ7 or Vβ8.3 to produce functional Toki Tested for the presence of syn. Affects the level or total length of SEB produced To eliminate any potential mutations Lysates deficient in stimulating were subsequently measured for the presence of SEB protein. Sudden We sequenced plasmids that produced proteins from them to find mutations Was. The sequence of the mutant is shown in FIG.   Three random mutants (BR) that induced errors with Taq polymerase In the region (1.2.4), all were collected at the amino-terminal 93 amino acids of the molecule (only And one case at the carboxyl end of the molecule, additional conservation in BR-374 Sex mutation). All of these except one as expected from their production method Are A to G or T to C mutations, but other variants of these sequences However, one silent mutation was seen. Was. With mutant oligonucleotide C or A (BC, BA mutant) Additional mutants were generated in region 1 or 2. Region 3 was originally an oligo A single mutant containing the last amino acid covered by nucleotide A (B A-62). This mutant is different from other BA mutants Had a phenotype. Added to this region along with mutant oligonucleotide B Mutants were produced (BB mutants). Disul conserved at position 93 Unpredictable and unpredictable interference with cysteine forming sulfide bonds Region 4 mutations were removed by further analysis. further Some mutants contain more than one region (Br-474, BA-72) Produce a highly degraded toxin (BR-267) or are already present Since the same mutant (BA-50) was used, further characterization was not performed. Example 4Preparation of recombinant SEB   Individual transformants taken from agar plates for initial screening Colonies were picked with 100 μl of 2XVT and carbenicillin lin) was transferred to a 96-well microtiter plate. BA is c The same plate was prepared except that it contained 1 mM IPTG as a well. Izu All were incubated overnight at 37 ° C. with stirring. For each well of the first plate 50 μl of glycerol was added, mixed and then stored at -70 ° C. SE To prepare the B-containing lysate, add 3 mg / ml to each well of the second plate. Lysozyme and 50 μl of HNM buffer containing 300 μg / ml DNAseI Buffer (10 mM Hepes, pH 7.0, 30 mM NaCl, 5 mM MagCl2) Nesium). Incubate the plate at 37 ° C. for 15 minutes, 3 times It was freeze-thawed and centrifuged into pellet fragments. Transfer the supernatant to a new plate Examines the presence of SEB by both ELISA and T cell hybridoma stimulation Was. In this way, preparations containing 0.3 μb / ml and 10 μb / ml of SEB are obtained. Produced.   96 wells stored at -70 C to produce purified mutant SEB From the microtiter plate. One containing IPTG Bacteria from the overnight culture (1 volume) were collected by centrifugation and 1-2 mg / ml 1:10 volume of HNM buffer containing team and 10 μg / ml DNAseI Resuspended in. The suspension is centrifuged at 15,000 g for 20 minutes to remove bacterial debris. Removed and the supernatant was collected and filtered (0.2μ). 2-3mg / ml monochrome 1:50 volume of Farose 4B beads bound to SEB (B344) with null antibody The filtrate was passed through a column containing Wash the beads thoroughly with PBS and remove toxin. Elution with 0.1 M glycine-HCl (ph 2.7) with 1 M sodium carbonate Neutralized. SEB was concentrated to 1 mg / ml, and Centricon 10 microconcentration was used. Totora (Centricon10 microconcentrators) ) (Amicon Corp., Denvers) , Mass.) Using BSS as the buffer. Bacterial culture in this way 3-10 mg of toxin per liter of product were obtained. SE thus produced B and its mutants were greater than 95% pure by SDS-PAGE. Territory The SEB mutants in Region 1 are shown in Table 2 and the Region 1 and Region 2 mutants Table 3 shows the region 3 mutants.   All of the mutants described have A to G or T to C nucleotide substitutions. Containing, as would be expected from the method used for their production.   When mutations are generated using oligonucleotides C or A, these The mutation was at amino acids 9-23 (region 1) or 41-53 (region 2) of the SEB sequence. ) (Table 3).   When oligonucleotide primer B was used, the mutants shown in Table 4 were produced. Was born.   In normal SEB, amino acid 93 is cysteine. Because of this, Mutations may interfere with disulfide bonds and are not considered further Was. That is, the mutants BR-30 and BR-311 were excluded.   Mutations that change in more than one region due to very degraded toxin Body (not more than one mutant) (ie, BR-474 and BA-72) It was removed as in BR-267. BA-50 is a known mutant, I did not consider it.SEB structural research   Probably the most studied staphylococcal enterotoxin, SE The three-dimensional structure of B was recently reported (Swaminathan et al.) (1992) Nature 359: 801). Fig. 1 shows a schematic diagram of SEB. . The SEB molecule contains two domains. First domain is residues 1-120 And the second consists of residues 127-239. As mentioned above, MHC class Three at the N-terminal portion of SEB that affect II binding and / or T cell activation (Kappler et al. (1992) J.E. xp. Med. 175: 387). The specific amino acids involved in each of these regions Acid has been measured. Some of the identified residues are associated with MHC class II binding and T cells Affect activation, others affect only T cell activation. Super antigen MH C class II binding is essential for T cells and thus affects MHC class II binding Since residues affect T cell activation, they do not provide T cell binding information. No. However, they provide information on MHC class II binding sites on superantigens. I can. On the other hand, it affects T cell activation but not MHC class II binding. The remaining residue may be in the T cell binding site on SEB.   Region 1, defined as 9-23 amino acid residues, contains TCR binding and MHC It is bifunctional because it affects both S-II binding. Mutations in this region The residues at positions 10, 14, 17, and 23 were included as single or two mutations. (Table 2). Asparagine at residue 23 (N23) has an α-helix (α2) Above, the side chains are facing the solvent. This is the most important residue and all Is conserved in staphylococcal enterotoxins and is most likely to activate TCR. is important. What interrupted MHC class II binding was a few five sudden changes at position 23 All, although variant, affected TCR activation. Of residues 14 and 17 Mutations affected both MHC class II binding and TCR activation. Residue S1 4 is a very short α-helix (α1), which has been exposed to solvent but F17 Located at the other end of the. S14, F17 and N23 on the surface of the toxin The location of (FIG. 1) makes the most important contacts with MHC class II molecules and / or Vβ. Is preferred. Residue F17 is inward, residues 174-179 and 203 -209 exists in a loop flanked by two separate loops. This is serine (F 17S) replaces MHC class II binding and cytokine production. It suggests that it may have introduced a structural change. S14 (on α1) and MHC Class II binding, and the association between N23 (on α2) and TCR activity, was Represents the structural basis upon which the role of potency is based. This area contains a small number of contiguous Another adjacent component of a second structure consisting of an amino acid but involved in a different function. Distributed over minutes. The close proximity of α1 and α2 indicates that the amino acid in region 1 is It is suggested that it is located in a trimolecular complex near the junction between Vβ and MHC class II. (Kappler et al. (1992) supra).   Region 2 is defined as residues 40-53 and contains all Staphylococcal entero Suggested to be important in mediating toxin binding to MHC class II in toxins ing. About half of the mutations in this region contain the conserved residue F44 (Table 3). Was. Other mutations contained residues 41, 45, 48, 52 and 53. That is, this region is probably specific for MHC class II binding. Residue 48- 52 is in the β chain (β2). Residue F44 is on the corner connecting β1 and β2 And the side chain is exposed to the solvent. This is an important hydrophobic bond with MHC class II It is conveniently located for making contact.   Region 3 is composed of two residues (60 and 61), one of which Differences affect TCR activation but not MHC class II binding. Remaining Groups 60 and 61 are exposed to the solvent, which is in the loop connecting β2 and β3 ( Table 4). Example 5Sudden binding of different SEBs to HLA-DR   Since binding to MHC class II is essential for toxin recognition by T cells, Mutations may be due to the ability of the toxin to bind to the DR molecule or the TCR-α / β May have affected his cognitive abilities. To distinguish between these two possibilities To help, the HLA-DR1 homozygous lymphoblastoma line LG2 was used (Gatch and Gatti and Leihold (1979) Tiss ue Antigens 13:35).¹²⁵50 μl of I-labeled LG2 cells Incubate at 37 ° C for 2 hours with or without g / ml recombinant SEB I did it. A cell-free lysate was prepared in 1% digitonin and 3 mg / ml B 344 anti-SEB monoclonal antibody-conjugated Sepharose beads and 4 Incubated for hours. Thoroughly wash beads and reduce labeled bound material SDS-PAGE below (Laemmlli (1970) Natu re 227: 680) and autoradiography. As a control, The DR monoclonal antibody L243 (Lampson and Levy) nd Levi) (1980) Immunol. 125: 293) Used (1/20 volume of lysate was used with anti-SEB beads).   SEB binds to the DR molecule on LG2 (FIG. 2b). Immunoaffinity purified ibis The same anti-SE used to prepare syn and purify SEB and its mutants Its binding to LG2 was determined by flow cytometry using monoclonal antibody B. The ability was evaluated. 3x10 in 100 μl tissue culture medium^FourOf LG2 cells Incubated overnight at 37 ° C. The cells are washed thoroughly and about 1 μg / ml anti-S Incubate with EB monoclonal antibody (B344.1) at 4 ° C for 30 minutes I did it. The cells were washed again and fluorescently labeled goat anti-mouse IgG1 (Fisher With Fisher Scientific Co. Both were incubated at 4 ° C. for 15 minutes. Wash cells again and fluoresce cell surface Of the toxin added to the second reagent alone. Corrected. The results (shown in Table 4) show the suddenness of each of Regions 1, 2 and 3 Shown for mutations.   Binding to LG2 by the four region 1 mutants resulted in unmutated SE B could not be distinguished from that of B. The other three mutants have a binding capacity of about 100 times lower. These results show that residues between residues 14 and 23 in region 1 Suggest that it is important for MHC binding. Five of the seven mutations 23N was involved. Only in one case (BR-29, 23N → S) does this sudden The mutation reduced MHC binding. These results indicate that residue 23N has NHC binding and Vβ Suggests that it is important for interaction. All mutations in region 2 are just Mutant in region 2 that binds LG2 shortly (about 1,000 times weaker than SEB) Defines a range of amino acids important for the binding of toxins to class II MHC (especially 44F). Indicates that it is regulated. Mutants in region 3 are basic for binding to LG2 This two amino acid region (60N, 61Y) is important for Vβ interaction Suggest that there is. Example 5Effects of mutations on T cells with different Vβ components   The SEB mutant was originally Vβ7⁺Or Vβ8.3⁺T cell hybridoma Identified to stimulate. Further elucidation of the effects of SEB mutants on T cell recognition For detailed evaluation, purified mutant toxin was treated with various doses of SEB. The four rodent Vβ components known to recognize (Vβ7, Vβ8.1-3 (White et al. (1991) supra; Callahan) (1989) supra: Herman et al. (1991) supra)) An additional T cell hybridoma with each was tested. Different concentrations of Toxin was added to 3 × 10 5 in 200 μl tissue culture medium.^FourDR⁺37 with cells Incubated overnight at ° C. 5 × 10 with essential Vβ specificity^FourT cells Hybridomas were added at 50 μl and the mixture was incubated overnight. coupler (Kappler) et al. Exp. Med. 153: 1198 Mosmann (1983) J. Mol. Immunol. Meth.   65:55, IL. Secreted T cell hybridoma response. -2 Measured. The results are shown in FIGS. 5-7.   Of the mutants in region 1 (FIG. 5), 5 with 23N (BR-257, B R-291, BC-6, BC-66, BC-88), these four are SEB Despite similarly binding to DR, only a small fraction of all hybridomas It was only stimulated. These results indicate that residue 23N is an important signal for Vβ interaction. The fifth mutant (BR-291) which is an amino acid but contains this amino acid is M This amino acid also has an effect on MHC binding because it only binds slightly to HC. Indicates what might be given. Mutants in the other two regions 1 are also It was only stimulated shortly. In the case of BR-75 this is mainly due to its weak DR binding May be due to the effect of the BR-210 mutation on T cells rather than binding to DR. It was several orders of magnitude larger for vesicle stimulation. Taken together, these results are: During T cell recognition of SEB bound to DR, the amino acids in region 1 are between Vβ and MHC. Located in the trimolecular complex of the junction, each residue interacts with each component. Evidence.   Mutations in other regions produced less complex phenotypes. Sudden change in area 2 All of the variants, despite the presence of the Vβ component in their receptors (FIG. 6) ), Did not stimulate all of the T cell hybridomas. There is a small difference However, in general, the effects of mutations on stimulation are the same as those seen with DR binding. Was. These results indicate that region 2 mutations primarily affect DR binding. Matches.   Mutants in region 3 of the two amino acids were the most different (FIG. 7). Adjacent to this area Random mutants in the range of 20 amino acids I was found. These mutants stimulate hybridomas with Vβ7 None, but hybridomas with Vβ8.2 or Vβ8.3 were stimulated. To confirm that this property is not specific for these hybridomas, The toxin was converted to four other T cell hybridomas (one Vβ7⁺Two Vβ8.1⁺ And one Vβ8.3⁺). The result can be distinguished from that of FIG. Failed (data not shown). Example 6Requirement of T cell interaction for in vivo action of SEB   How important is the superantigenicity of bacterial toxins to their in vivo toxicity The problem has not been solved. Previous experiments by the present inventors have shown that SEB Because sex is directly related to the frequency of T cells with an associated Vβ component, Of SEB in mice is related to its ability to stimulate T cells specifically for Vβ (Marrack et al. (1990) J. Exp. M ed. 171: 455). However, some S. aureus toxin Binds to class II on the sphere and stimulates the production of cytokines such as TNF and IL-1 Ability (Parsonnet (1988) Rev. Infe ct. Dis. 1: 263) suggests that direct monocyte stimulation Open the possibility that it may be enough to explain much of the pathogenicity of the disease.   To test this idea, it binds well to class II MHC but has extremely high concentrations. Region 1 mutant BR-257, which does not stimulate T cells other than Was injected into mice. Unmutated SEB and mutant BR-358 (this They bind slightly to class II MHC like all mutants in region 2 Only used as a control. LPS that may contaminate the preparation To minimize the effects, the strain lacking LPS responsiveness, C3H / HeJ mouse, Used. Rapid weight loss is most pronounced and rapid toxicity of SEB in mice (Marrack et al. (1990) supra), the injection was given on day 0. After that, the mice were weighed daily.   Groups of three mice were weighed and 0 μg, 50 μg or 100 μg of recombinant SEB, mutant SEBBR-257, or mutant SEBBR-35 8 to give a balanced salt solution (BSS). Body every day until the mouse dies The weight was measured. FIG. 8 shows the results. The results are based on the starting weight of surviving mice. Shown as the average rate of change.   Mice receiving 50 μg or 100 μg of recombinant SEB were rapidly treated in 3-4 days. All mice lost weight quickly and died within 5 days. Mutant BR-35 Mice given 8 were not affected and could not be distinguished from mice given BSS only Was. However, mice receiving 100 μg of BR-257 showed a slight weight loss And recovered later.   These results indicate that in mice most of the toxicity of SEB is due to its ability to stimulate T cells. Has been found to be dependent on force, including T cell-induced lymphokines themselves. Or those produced by other cells activated by T cells, Is very important for the mode of action of However, at high doses The slight effect of BR-257 is directly due to the bound SEB without T cell involvement. Figure 5 shows the potential contribution from stimulated cells with class II. Example 7Protective effect of SEB mutant   The protective effect of the SEB mutant was tested. In these experiments, wild-type S Before stimulating with EB, saline or 100 μg of BR-257 was added to mice. Was administered. On the day of administration (day “0”), 50 μg of SEB was administered intraperitoneally. Weight Change and survival were measured. The results are shown in FIG.   Mice given controls died 4-5 days after challenge with SEB, SEB Mice immunized with the mutant showed a protective effect. Example 8Production of SEB nad mutants and their protective effects in animals   A Staphylococcus enterotoxin A (SEA) mutant was constructed as described above. Produced according to the method. SEA amino acid sequence superimposed on SEA amino acid sequence The mutation at position 45 is similar to that found in the SEB mutant at position 45, It has been found that EA inhibits the ability to bind to MHC.   A similar test was performed in primates. Wild-type SEB, region ISEB mutant BR- 257 (mutated at F44) or BR-358 (mutated at N23) Mice were administered and the induction of an emetic response was evaluated. Both SEB mutants There was no effect in primates, or much less effective than wild-type SEB.   These results demonstrate that the mutant superantigen production methods described herein Is generally applicable to all superantigens and treats patients with superantigen disease It has been confirmed that it will provide a way to protect against the effects.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C12N 15/09 ZNA C12P 21/02 C 9282-4B (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG), AT, AU, BB, BG, BR, CA, CH, DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, US

Claims (1)

[Claims] (1) Use a molecule that induces antibody production without inducing T cell activation A method to prevent the toxic effects of superantigen by treatment. (2) The method according to claim 1, wherein the molecule is a mutated superantigen. Law. (3) The method according to claim 1, wherein the molecule is a modified superantigen. (4) A molecule consisting of a mutated superantigen. (5) A molecule consisting of a modified superantigen. (6) Inject molecules that interact with the specific Vβ component of the T cell receptor (TCR). A method of modifying a T cell response elicited by an antigen, the method comprising: (7) The method according to claim 6, wherein the molecule is a mutated superantigen. Law. (8) The method according to claim 6, wherein the molecule is a modified superantigen. (9) Use a molecule that induces antibody production without inducing T cell activation. To treat the toxic effects of superantigens by treatment.