JPH11507238A - Fused soluble MHC heterodimer: peptide conjugates and uses thereof - Google Patents

Abstract

  (57) [Summary] Immunization modulators are disclosed, such as soluble fused MHC heterodimers and soluble fused MHC heterodimers: peptide complexes. Related methods and peptides are also disclosed. In a preferred aspect, these modulators and methods are associated with autoimmunity.

Description

DETAILED DESCRIPTION OF THE INVENTION     Fused soluble MHC heterodimer: peptide conjugates and uses thereofRelated cases   No. 08 / 480,002 (filed on Jun. 7, 1995) and U.S. Pat. No. 83,241 (filed on June 7, 1995) and US patent application Ser. No. 08 / 482,133 (June 1995) U.S. Prov., Which is a continuation-in-part application of U.S. Prov. isional Aplication) No. 60 / 005,964 (filed on October 27, 1995) Insist on enjoying the benefits.Background of the Invention   Based on increasing understanding of the structure and function of major histocompatibility complex (MHC) antigens At present, there is a great interest in developing pharmaceuticals based on this. These cells Surface glycoproteins are involved in antigen presentation and in various T cell responses to antigens It is known to play an important role in inducing.   T cells, unlike B cells, do not recognize antigens directly. Instead, accessories Cells first process the antigen and present it for the MHC molecule Elicit a T cell-mediated immune response. The primary function of MHC glycoproteins is Binds to the labeled antigen and presents the antigen in the form of a short antigenic peptide. And seems to be.   In addition to binding to foreign or “non-self” antigenic peptides, MHC molecules also Can bind to "self" peptides. T lymphocytes are then presenting themselves In response to cells or autoantigenic peptides, it becomes autoimmune. More than 30 Autoimmune diseases are now known, including myasthenia gravis (MG), multiple sclerosis (M S), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), insulin dependence Including diabetes mellitus (IDDM). The features of these diseases are associated with host tissue. Attack by the immune system. Such attacks do not occur in non-diseased individuals. No. Because the immune system recognizes these tissues as "self." Autoimmunity Occur when a specific adaptive immune response is mounted against self-tissue antigens. You.   Insulin-dependent diabetes mellitus (IDDM) (also known as type I diabetes) , Resulting from autoimmune destruction of the insulin-producing beta cells of the pancreas. responsible for beta cell destruction Studies aimed at identifying self-antigens have identified several candidates. It Insulin (Palmer et al.,Science 222: 1337-1339,1983) Is an uncharacterized islet cell antigen (Bottazzo et al.,Lancet ii: 1279-1283, 1974) , And a 64kDa antibody that has been shown to be glutamate decarboxylase Hara (Baekkeskov et al.,Nature 298: 167-169 (1982); Baekkeskov et al.Nature 347: 151 -156, 1990). Glutamate decarboxylase (hereinafter referred to herein as Antibodies (known as "GAD") are present in patients prior to clinical signs of IDDM (Baekkeskov et al.,J . Clin. Invest. 79: 926-934, 1987).   GAD catalyzes the rate-limiting step in the synthesis of γ-aminobutyric acid (GABA). γ-amino Butyric acid (GABA) is the major inhibitory neurotransmitter of the mammalian central nervous system. GAD Little is known about regulation of activity or GAD gene expression Not in. Very low GAD protein, despite its wide distribution in the brain And is very difficult to purify uniformly. GAD depends on different genes. It has a number of isoforms encoded by These many forms of this enzyme are It differs in quantity, kinetic properties, sequence (if known), and hydrophobic properties. An example For example, three different forms of GAD in pig brain (Spink et al.,J . Neurochem. 40: 111 3-1119, 1983), and four forms in rat brain (Spink et al.,Brain Res. 42 1 : 235-244, 1987). Mouse brain GAD (Huang et al.,Proc . Natl. Acad. Sci. USA  87: 8491-8495,1990) and GAD clones isolated from cat brain (Kobayashi et al.J . Neurosci. 7: 2768-2772, 1987). GAD At least two isomers have been reported in the human brain (Chang and Gott lieb,J . Neurosci. 8: 2123-2130, 1988). Human pancreatic islet cell GAD has recently been (Lernmark et al., US Patent Application No. 07 / 702,162; P CT application publication WO92 / 20811). This form of GAD is one of the subsequently identified human brains. Identical to the isoform (Bu et al.,Proc . Natl. Acad. Sci. USA 89: 2115-2119, 1992) . The second GAD isoform identified in human brain is absent in human islet cells (Ka rlsen et al.Diabetes 41: 1355-1359, 1992).   Inflammatory CD4⁺(T_H1) The response of T cells to GAD is a major autoantigen reactivity and NO D coincides with the onset of insulitis in mice and subsequently responds to other β-cell antigens It has been suggested that some T cell reactivity occurs. At the same time, the first T for GAD Cellular response is restricted to one region of the GAD polypeptide and becomes more pronounced over time. Have been reported to propagate to GAD determinants (WO95 / 07992; Kaufman et al.,Nature 366 : 69-71, 1993; and Tisch et al.Nature 366: 72-75, 1993).   GAD initiates beta-cell attack leading to diabetes in both human and animal models Evidence suggests that it is a major autoantigen responsible for Mouse and human 3 peptides from GAD65, peptide No. 17 sequence 246-266, peptide No. 34 sequence 509-528 and peptide number 35 sequences 524-543 elicit a T cell response in mice Has been suggested as a candidate for self-antigen by its ability to direct (Kaufman et al., Ibid.) .   Current treatments for autoimmune diseases and related conditions mainly treat symptoms And does not intervene in the etiology of the disease. Broad spectrum chemotherapeutic agents Although used ostensibly, this drug often leads to many undesirable side effects ing. Therefore, blocking the MHC binding selectively suppresses the autoimmune response. Compounds that can be controlled and thereby provide safer and more effective treatments It is. In addition, such selective immunosuppressive compounds are useful for graft-versus-host disease (GVHD) or Is required in the treatment of non-autoimmune diseases such as various allergic responses . For example, chronic GVHD patients often have conditions and symptoms similar to certain autoimmune diseases. Present.   Improper autoimmune disease treatments currently available block MHC restricted immune response To avoid unwanted side effects such as non-specific suppression of the individual's overall immune response. Illustrate the need to identify new drugs immediately. Self-mediated by MHC A desirable approach to the treatment of autoimmune diseases and other pathological conditions is that soluble Fusion MHC heterodimer: T responding to antigenic peptide using peptide complex Achieving tolerance or anergy to cells. The present invention Meet the needs and provide related benefits.   The identification of synthetic antigenic peptides, and these peptides are associated with disease and Demonstrating that they selectively bind to MHC molecules that stimulate T cells Linking peptides or peptide: MHC complexes to susceptibility to autoimmune diseases Help you. The present invention fulfills these needs and provides related advantages. Offer.Summary of the Invention   In a first aspect, the present invention provides a soluble fusion MHC heterodimer comprising: : Providing a peptide complex: at least the first domain of the selected MHC molecule First DNA segment that also encodes a portion of the second domain of the selected MHC molecule A second DNA segment encoding at least a portion of: about 5 to about 25 amino acids The first that encodes and connects the first and second DNA segments in-frame One linker DNA segment; wherein the first linker DNA segment The ligation of one DNA segment to the second DNA segment is the fusion of the first DNA-first Linker-becomes the second DNA polysegment; peptide linkage of the selected MHC molecule A third DNA segment encoding an antigenic peptide capable of binding to a mating groove; Encodes 5 to about 25 amino acids, and encodes a third DNA segment in-frame Fused first DNA-first linker-second DNA polysegment A linker DNA segment; wherein the third linker DNA segment DNA segment fused first DNA-first linker-second DNA poly segment Ligation into a soluble fused MHC heterodimer: peptide complex.   In one embodiment, the selected MHC molecule is an MHC class II molecule.   In another embodiment, the first DNA segment encodes a β1 domain .   In yet another embodiment, the second DNA segment comprises an α1 domain or α1 domain. Encodes the 1α2 domain.   In another embodiment, the selected MHC molecule is IA^g7, IA^s, DR1β * 1501, and And DRA * 0101.   In a further embodiment, the selected MHC molecule is an MHC class I molecule.   In yet another embodiment, the first linker DNA segment is GASAG (SEQ ID NO: No. 29) or GGGGSGGGGSGGGGS (SEQ ID NO: 36).   In yet another embodiment, the second linker DNA segment is GGSGG (SEQ ID NO: No. 30) or GGGSGGS (SEQ ID NO: 31).   In a further embodiment, the third DNA segment stimulates an MHC-mediated immune response Encodes a possible antigenic peptide.   In another embodiment, the peptide is a mammalian GAD65 peptide (SEQ ID NO: 59), ( SEQ ID NO: 61), (SEQ ID NO: 40), (SEQ ID NO: 39), and mammalian myelin basic Selected from the group consisting of peptides (SEQ ID NO: 33).   The present invention further provides a soluble fusion MHC heterodimer: peptide complex. . Here, the MHC heterodimer: peptide complex is the third of the selected MHC molecules. A fourth DNA segment encoding at least a portion of the domain, and from about 5 Encodes about 25 amino acids, and inflates the second and fourth DNA segments A third linker DNA segment further connected by a Second linker-first DNA-first linker-second DNA-third linker-fourth D Becomes NA poly segment.   In one embodiment, the selected MHC molecule is an MHC class I molecule.   In a second embodiment, the selected MHC molecule is an MHC class II molecule.   In another embodiment, the fourth DNA segment is a β2 strand.   In yet another embodiment, the third linker DNA segment is GGGGSGGGGSGGG GSGGGGSGGGGS (SEQ ID NO: 32).   In a second aspect, the invention relates to a soluble fused MHC heterodimer: pep Provided is an isolated polynucleotide molecule that encodes a tide complex.   In a third aspect, the present invention provides a fusible material comprising the following operably connected elements: Fused MHC heterodimer: fusion capable of expressing a peptide complex Provide a protein expression vector: transcription promoter; selected MHC molecule A first DNA segment encoding at least a portion of a first domain; selected DNA segment encoding at least a part of the second domain of the MHC molecule Encoding about 5 to about 25 amino acids, and in-frame first and second DNs; A first linker DNA segment connecting the A segments; wherein the first linker -Ligation of a first DNA segment to a second DNA segment by a DNA segment Becomes the fused first DNA-first linker-second DNA polysegment; Encoding an antigenic peptide capable of binding to the peptide-binding groove of the selected MHC molecule A third DNA segment that encodes about 5 to about 25 amino acids, and First DNA-first linker-second DNA poly- A second linker DNA segment connecting the segments; where the second linker D First DNA-first linker fused to third DNA segment by NA segment -Ligation to the second DNA polysegment is a soluble fused MHC heterodimer : Expressing a peptide complex; and a transcription terminator.   In one embodiment, the present invention provides that the MHC heterodimer: peptide complex is A fourth DNA sequence encoding at least a portion of the third domain of the selected MHC molecule. And about 5 to about 25 amino acids, and the second and fourth DN Also includes a third linker DNA segment that connects the A segment in-frame. Only the fused third DNA-second linker-first DNA-first linker-second DNA- A third linker-an expression vector that becomes a fourth DNA polysegment is provided.   In another aspect, the present invention provides a method of culturing cells into which an expression vector has been introduced, Causes the cells to undergo soluble fused M encoded by the DNA polysegment. HC heterodimer: expressing a peptide complex; and soluble fused MHC Rhodimer: A soluble fusion produced by recovering the peptide complex. MHC heterodimer: peptide complex is provided.   In yet another aspect, the invention relates to a soluble fused MHC heterodimer: Pharmaceutical compositions comprising a peptide conjugate together with a pharmaceutically acceptable vehicle .   In another aspect, the invention relates to soluble fused MHC heterodimers: pepti Antibodies that bind to an epitope of the host complex.   In yet another aspect, the invention relates to treating a patient to reduce an autoimmune response. A method of placing a soluble, fused MHC promoter according to claim 1 in a therapeutically effective amount. Rhodimer: Peptide conjugates induce immune tolerance in patients Providing a method comprising directing.   In yet another aspect, the present invention relates to a method for selecting a selected antigen presented by a stimulator cell. Methods for preparing responsive cell clones that proliferate when combined with a native peptide A non-adherent CD56-, CD8- cell reactive with the selected antigenic peptide Isolating and thereby forming a responder cell; Stimulating with cells or primed stimulator cells; pulsing the stimulated responder cells Restimulating with primed or primed stimulator cells; and response Isolating a cell clone.   In one embodiment, the responder cells are prediabetic. Or from patients who have newly developed diabetes.   In a second embodiment, the responder cell clone is a T cell clone.   In another aspect, the selected antigenic peptide is a GAD peptide.   These and other aspects of the invention will be apparent from and elucidated with reference to the following detailed description. become.Detailed description of the invention   Before describing the present invention, some of the uses used herein Providing definitions for words may help in understanding.   Fusion MHC heterodimer: Peptide complex (fusion MHC (major histocompatibility factor) hetero Dimer: peptide complex)   In this specification, this term is used to refer to MHC (major histocompatibility factor) in the present invention. Multimer: Refers to a fusion protein, such as a peptide conjugate. Such a fusion tamper The quality is described with a colon (:). MHC-peptide complex is a fusion protein Instead of quality, MHC in its original form or bound to exogenous peptide Is described with a dash (-).   The domain of the selected MHC molecule: A pair with itself or another MHC domain By combination, the antigenic peptide is presented in a form that the T cell receptor can recognize. Refers to the portion of the MHC that is sufficient to create a peptide binding site for This They protrude extracellularly in both class 1 and class 2 MHC. Contained in the two existing polypeptide chains. In other words, a chain (a1, a2, a3) Part or whole, and class 1 MHC b_Two-Includes microglublin subunit No. For example, in a class 1 MHC domain, the a chain has three a Various forms of German (a1, a2, a3) or tandem forms (a1a2, a2a3, a1a3) And / or comprises the b2 domain. In addition, the a chains (a1, a2) and And b chains (b1, b2). a1 and a2 each alone or in tandem form (a1a2) included. Each of b1 and b2 alone or in tandem (b1b2) is also included.   Linker DNA segment: In the DNA region encoding 5 to 25 amino acids The typical case is that consecutive glycine residues are composed of several serine residues. Is divided and the area at both ends is a link for flexible movement I have. This flexible link is located at both ends, for example, the MHC Or the peptide is suitable for the antigen recognition groove. It allows you to get stuck.Antigenic peptide : Contains an amino acid sequence recognized by immune cells, especially T cells It is a peptide that can elicit an immune response by MHC.   Major histocompatibility factors (MHCs) are a class of proteins with many polymorphisms and are class 1 And T2 lymphocytes (T cells) with membrane-bound proteins Has the function of presenting. Class 1 and class 2 MHC molecules are Are distinguished by the type of cells present and the type of T cells that recognize them. Kula MHC molecules (for example, HLA-A, HLA-B and HLA-C molecules in humans) are almost all Expressed in cells with all nuclei and recognized by cytotoxic T-type lymphocytes (CTL) Finally, cells having the antigen are destroyed by CTL. Class 2 MHC molecules (eg For example, in the case of humans, HLA-DP, HLA-DQ, and HLA-DR molecules) It is mainly expressed on the surface of antigen-presenting cells such as process cells and macrophages. K Lass 2 MHC is CD4⁺Helper T lymphocytes (T_H). T_Hcell Promotes the proliferation of both B cells and T cells, thereby providing a specific anti- Has the effect of amplifying the immune response to the original peptide (Takahashi, Microbiol. un ol., 37: 1-9, 1993). The process of cleaving two different antigens is the Related to Russ. Intracellularly synthesized antigens, such as viral proteins And newly synthesized intracellular proteins are converted to class 1 MHC after being cleaved. Presented. Extracellular antigen presenting cells (APC) by endocytosis Is presented by class 2 MHC after undergoing cleavage. Antigen peptides produced as a result of proteolysis of antigenic substances in cells with MHC Ptides enter the groove for binding to antigens in the MHC molecule and form various non-covalent bonds. A complex is formed by combination. The cell surface MHC-peptide complex is a cytotoxic T Recognized by specific T cell receptors on cells or helper T cells.   Three human MHCs (or human leukocyte-associated antigens (HLA)) on chromosome 6 Loci exist. They are HLA-A, HLA-B and HLA-C, of which HLA-A and HLA-B has many alleles that encode allogeneic (heterologous) antigens. HLA-D The adjacent region known as HLA-DR, HLA-DQ, HLA-DP is further redifferentiated. HLA area Is now known to be the human MHC region, and H-2 It is equivalent to an area. HLA-A, HLA-B and HLA-C are the same as mouse H-2K, H-2D and H-2L. Similar and class 1 MHC molecules. HLA-DP, HLA-DQ, and HLA-DR are It is a class 2 MHC molecule, similar to IA and IE of class I. Class 1 with sugar chains , Class 2 MHC proteins have been isolated and characterized (Fundamental Immunology, 2d Ed., W.E. Paul (ed), Ravens Press, N.Y. (1989); and Roitt et al., Immunology, 2d ed., Gower Medical Publishing, London (1989), Et al. Are both incorporated herein by reference).   The composition of human class 1 MHC molecules is a polymorphic glycoprotein with a molecular weight of about 46 kDa. Type 1 membrane-bound heavy chain molecules_TwoMolecular weight 1 called -microgloblin It is in the form of non-covalently bound soluble 2 kDa subunits. Heavy chain The region consists of two distinct extracellular regions: a1, away from the membrane, Peptide-binding region formed by a2 domain and a3 domain adjacent to membrane Is a CD8 binding region consisting of b_Two-microgloblin is a single with no anchor to the membrane It is a compact, immunoglobulin-like domain that combines the heavy chain region of class 1 with Bound or present alone in plasma (Germain and Margulies , Annu. Rev. Immunol. 11: 403-450, 1993).   Human class 2 MHC is a protein embedded in a heterodimeric membrane. individual Is a non-covalent bond of one a chain and one b chain. Two of them Are similar to each other, the a-chain has a molecular weight of 32-34 kDa, and the b-chain has a molecular weight of 29-32 kDa. Both proteins have several N-linked sugar chains attached, The N-terminus faces out of the cell and the C-terminus faces in the cell.   The extracellular regions of the a and b chains that make up the class 2 MHC molecule also contain approximately 9 Redifferentiated into two domains of 0 amino acids, which are a1, a2 and b1, respectively. It is called b2. The a2 domain and b2 are each formed by a disulfide bond. It has a loop structure. The peptide binding region of the class 2 molecule consists of the a1 domain and b1 It is shaped by domain interactions. This interaction opens one side. An antigen peptide binding groove consisting of two a-helices and eight b-sheets is created.   The a and b chains of class 2 molecules are encoded by different MHC genes, respectively. And show gene polymorphism (Addas et al., Cellular and Molecular Immunology, 2d Ed., W.B. Saunders Co., New York (1994). Incorporated as a reference). In the present invention, the a chain has DRA * 0101 and the b chain has DRb1 * 15. 01 was used.   The immunological properties of MHC that are involved in histocompatibility Is determined. Antigen peptides are defined by immune cells, such as T cells. Is a peptide containing an amino acid sequence that is recognized. For many autoimmune diseases The presence of the corresponding antigenic peptide is previously known. For example, experimentally Antigens involved in the pathogenesis of autoimmune diseases : In the case of arthritis in rats and mice, natural type 2 collagen is collagen Is an antigen of arthritis caused by Heat shock protein is an arthritis antigen in adjuvants (Stuart et al., Ann. Rev. Immunol. 2: 199-218, 1984). Also, thyroglobulin (thyrog loblin) is an experimentally induced mouse allergic thyroiditis (EAT) (Maris on et al. Exp. Med. 152: 1115-1120, 1988), acetylcholine receptor (A (ChR) is an experimentally induced allergic myasthenia gravis (EAMG) (Lindstrom et al., Adv. Immunol. 42: 233-284, 1988), basic myelin protein (MBP) and Roteolipid protein (PLP) is an experimentally induced antigen in mice and rats. For allergic encephalomyelitis (EAE) (Acha-Orbea et al., Ann. Rev. Imm. 7: 377-405). , 1989), and each is known to be an antigen. In humans, Several target antigens are known. Type 2 collagen is used for human chronic joint (Holoshitz et al., Lancet ii: 305-309, 1996) Receptors are myasthenia gravis (Lindstrom et al., Adv. Immunol. 42: 233-284). , 1988), and each is known to be an antigen.   The soluble fusion MHC heterodimer: peptide conjugates of the present invention may be useful in therapeutic applications. Can be used as an antagonist to inhibit the binding of specific T cells to antigen-presenting cells Noh. In addition, this molecule provides anergy to target T cells, a specific antigen Causes reduced immunity to Prepared for the desired autoimmune disease Soluble fused MHC heterodimer: peptide molecules have a corresponding antigenic peptide Of the MHC solubilized or separately prepared. There is no need to add peptides from outside.   Methods previously used to regulate the desired class 2 MHC proteins Used a mixture of antigenic peptides as a raw material (Buus et a., Science 2 42: 1045-1047, 1988; and Rudensky et al., Nature 353: 622-627, 1991), purpose Can form a complex with only a small portion of the antigenic peptide (Watts and McConnel, P. roc. Natl. Acad. Sci. USA 83: 9660-64, 1986; and Ceppellini et al., Natur. e 339: 392-94, 1989). Incorporates target peptides that do not present endogenous antigens Various methods have been developed to prepare heterodimers that can be used (Stern and  Wiley, Cell 68: 465-77,1992; Ljunggren et al., Nature 346: 476-80, 1990; and Schumacher et al., Cell 62: 563-67, 1990). International Publication No.WO95 / 23814 and And Kozono et al., A class 2 molecule of the mouse, I-E^dkAnd I-A^d, Each pep Adjusting the soluble form by linking the tide to the N-terminus of the b-chain with a linker States the law. Ignatowicz et al. (J. Immunol. 154: 38-62, 1995) describe membrane-bound I -A^dIs expressed in a form in which a peptide is bound. These methods are membrane-bound and And a soluble heterodimer in combination.   A soluble bound MHC heterodimer according to the invention comprises two or more MHC domains Are connected by a flexible linker and Another flexible linker connects the antigen peptide to the peptide binding domain. They are connected so that they can be combined, and have some advantages. like this The complex is soluble and the heterodimer and antigenic peptide are single-stranded They are always linked in the form of a lipopeptide, so they can separate complex heterodimers. There is no need to stick. In this complex, inefficient and non-specific antigen peptide There is no binding. MHC: peptide complexes prepared by recombinant DNA methods are specific And produce high yields of protein, and the final product is Contains only the folded MHC: peptide complex. As used here Soluble heterodimers do not contain membrane-bound MHC. The soluble MHC hete of the present invention Rhodimers are not membrane-bound. In addition, polypep contained in MHC heterodimer Tides contain amino acid sequences that can function as transmembrane and cytoplasmic domains. Not in.   The soluble MHC heterodimer described in the present invention is the N-terminal of the MHC a-chain or b-chain. An antigenic peptide is covalently fused to the end by a proteinaceous amide bond, The end is followed by the N-terminus of the other a or b chain, resulting in two or three domains. Forming MHC molecules with ins. The present invention also includes yet another domain. Link to form an MHC molecule with four domains Existing. The a-chain region contains only a1 or a2, or a1 and a2 in tandem Linked or with a peptide linker incorporated between them. Or one of them. The b chain region contains only b1 or b2, or b1 and b2 Are connected in tandem, or a peptide linker is Any of the indented forms. Also, the combination of a1, a2, b1, b2 (For example, b1: a1 or b1: a1: a2) Can be   Encodes a soluble fusion MHC heterodimer: peptide complex as described in the present invention The following DNA contains the following regions: The first DNA region is the first of the MHC molecule of interest. Contains at least a domain portion; the second DNA region is the second of the MHC molecule of interest. The first linker DNA region comprises 5 to 25 The length encoding an amino acid, and the first and second DNA regions are connected in frame. Connect, by this connection, the first DNA region, the first linker region, the second The third DNA region binds to the MHC molecule of interest And a second linker DNA region. The region is 5 to 25 amino acids long and covers the third DNA region. To the first DNA region, the first linker region, and the second DNA region This connection encodes a soluble fusion MHC heterodimer: peptide complex DNA fragment is obtained. The present invention also relates to another type of soluble fusion MHC heterozygote. Monomer: also gives a peptide complex, ie it is the third of the MHC molecule of interest A third linker D having a fourth DNA region containing at least a domain portion; The NA region is 5 to 25 amino acids long and is the second and fourth DNA Connect the regions in-frame. With this connection, the third DNA region, the first Linker region, first DNA region, second linker region, second DNA A region, a third linker region, and a fourth DNA region are connected in this order.   The first to fourth DNA regions of the target MHC molecule are class 1 MHC b_Two-microglub Contains the lin subunit or part of the heavy chain. In this case, along with the b2 domain, 3 There may be a combination of two extracellular domains (a1, a2, a3, a1a2, a2a3). Ma A chain and b chain of class 2 MHC. Include only a1 or a2, or type a1 and a2 It also includes the case where it is connected to Ndem. Include only b1 or b2, or b1 and b2 It also includes the case where the tandem is attached. Soluble fusion MHC heterodimer: Peptide complexes were formed using a1, a2, b1, and b2 domains using a flexible linker. It can be represented by a combination, for example, peptide-b1-a1, peptide-b1-a1-a 2, peptide-b1-a1-a2-b2 and so on.   The linker region is 5 to 25 amino acids in length, and its length is determined by MHC or MHC: It depends on the molecular model of the peptide complex. What we want is a flexible linker -Indicates that consecutive glycine residues are interrupted by several serine residues, That is, random and flexible movements can be performed. Class 2 In the case of the MHC complex of It contributes to the proper shaping of the bond-like groove, and the peptide It is possible to fit to the maximum. The insertion location and length of the linker are a1 Crystal structure of a class 2 MHC molecule in which p1 and b1 form a peptide binding domain (Brown et a l., Nature 364: 33-39, 1993). Each part of a chain and b chain Is the structure of the virtual binding region and the C-terminal and N-terminal of each part. Is determined based on the distance to X-ray crystal structure of class 2 MHC molecule (Stern et al., (Nature 368: 215-221, 1994) , A chain, and the length of the binding part to the b chain are determined.   The soluble fusion MHC heterodimer: peptide complex of the present invention Can use any allelic cDNA that predisposes to autoimmune disease it can. Certain autoimmune diseases are associated with certain MHC types. Specific haplo Types are associated with many autoimmune diseases. For example, HLA-DR2⁺And HLA-DR3⁺Also Are more likely to develop systemic lupus erythematosus (SLE) (Reinertsen et al., N . Eng. J. Med. 299: 515-18, 1970). Myasthenia gravis is associated with HLA-D (Safwenberg et al., Tissue Antigens 12: 136-42, 1978). Human joint liu Machi is associated with HLA-D / DR. Which allele, or what type of MHC tan Methods for determining whether park is involved in autoimmune diseases are known in the art. Have been. Typical alleles in IDDM include DR4, DQ8, DR3, DQ3.2 I have.   The amino acid sequences of many class 1 and class 2 proteins are known, and Genes and cDNAs have also been cloned. Therefore, these nucleic acid fragments are It can be used for protein expression. If the target MHC gene or cDNA has not been isolated In other cases, those genes can be cloned using techniques possessed by those skilled in the art. And it is possible. One method is to purify the desired MHC, Determine the sequence, synthesize a nucleic acid probe corresponding to the amino acid sequence, and use it To select a clone containing the target gene from a cDNA library or genomic library. The method of choice is known to those skilled in the art.   The present invention is present and augmented with specific antigenic peptides presented by stimulator cells. Also provided are methods of modulating T cells that cause (react) proliferation. like this Clones are used to identify and map antigenic peptides involved in autoimmune diseases. Can be used. The peptide so identified is then the soluble fusion MH according to the invention. C heterodimer: can be introduced into a peptide complex. This method is specific to Non-adhesive CD56 reactive to antigenic peptides^-And CD8^-And enrichment of T cells Can be used. These cells are referred to herein as responder cells. Call. Appropriate responder cells, for example, before or at the onset of Can be isolated from peripheral mononuclear leukocytes (PBMNC) of later patients. And For example, peripheral mononuclear leukocytes can be isolated from patients before or shortly after the onset of diabetes . Such patients may be associated with specific HLA markers, e.g. Screening can be performed in advance using DR34-DR4 or DQ3.2. Gathered A part of the obtained PBMNC is kept as a responder cell. Using the rest, Purify and isolate self-reactive responder cells by plating twice You. The first is to remove the adherent cells from the population and then to remove mononuclear leukocytes and B cells. Remove with lone wool. Non-adhesive CD4⁺T cell enrichment is anti-CD8 and Complete by plating sequentially on plates coated with anti-CD56 antibody Complete.   The stimulator cells are stimulated with the full length of GAD or an appropriate antigen peptide. An example For example, stimulated cells collected from PBMNC of an IDDM patient are punctured by an antigenic GAD peptide. 7-14 days when given intense and then with PBMNC or responder cells After that, responding cells (T cell) clones are generated by limiting dilution, and antigen reactivity is tested. Strike.   Such a responder cell (T cell) clone is, for example, a specific T cell Can be used to identify the epitope portion that can be recognized by the like that One method is cleavage with trypsin or, more preferably, peptide synthesis. An overlapping peptide fragment of the autoantigen produced by the synthesis is used. this Such peptide fragments can stimulate responsive T cell clones and lineage cells Used in the test of lucidity (see Ota et al., Nature 346: 183-187, 1990, etc.) .   If such a peptide fragment is obtained, the binding strength to MHC will be increased, and the natural type Design and synthesize antigenic peptides that act in opposition to peptides You. Such synthetic peptides are soluble in fusion MHC heterodimers: peptide complexes Manipulates the immune system in vivo, that is, activation associated with disease Induces tolerance or anergy in committed T cells, resulting in autoimmunity May improve the disease.   Individual peptides in association with MHC molecules and in T cell recognition and reactivity A detailed examination of what functions are being performed This is a very difficult attempt due to the denaturation in the binding to the tide. T cell recognition Changes, or changes in the ability to partially bind the peptide to MHC, How specific amino acids and groups of amino acids are involved in determining MHC and T cell recognition Can be determined. The interaction with the partially altered peptide is Competition experiment using the presentation of the original peptide to T cells as an index and the direct peptide and MHC Further evaluation can be performed by performing a binding assay. Involved in binding to MHC Peptide changes that do not affect T cell recognition. For example, pepti In the case of a peptide, the point of contact with a particular MHC is a series of several in the middle of the peptide, or Or may occur only with individual amino acids, but not in T cell recognition. Amino acids may be completely different residues.   The preferred method is to replace each amino acid in the peptide with another amino acid. By evaluating the effect of the change on the binding of the peptide to MHC, Residues that alter T cell recognition are determined (Allen et al., Nature 327: 713-15). Sette et al., Nature 328: 395-99, 1987; O'Sullivan et al., J. Am. Imm unol. 148: 347-53, 1992; Jorgensen et al., Annu. Rev. Immunol. 10: 835-73 Hammer et al., Cell 74: 197-203, 1993; Evavold et al., Immunol. T oday 14: 602-9, 1993; Hammer et al., Proc. Natl. Acad. Sci. USA 91: 4456- 60, 1994; and Reich et al. Immunol. 154: 2279-88, 1994). One way and For example, an individual or group of amino acids can be replaced by similar amino acids, How to create a group of amino acids, a group of peptides with completely different amino acids Is the law. One type of this method, Alan scan, is a synthetic pen. It is a method to replace each amino acid in the peptide one by one with L-alanine (L -Ala scan). Alanine is selected because it is all parts of the protein Distribution (surface and interior in protein) and steric hindrance in two-dimensional structure No harm and no extra hydrogen or hydrophobic bonds It is. Alanine substitutions can be applied to individual amino acids depending on the information sought. Or for consecutive amino acids. The required information is If it is a conferring residue, the individual residue of the peptide is changed to alanine to increase the binding strength. Compare with the original peptide. For individual amino acid residues of the peptide, It is also possible to obtain further structural information of the whole peptide, for example individual peptides There is a method of synthesizing by substituting the amino acid residue with a D amino acid, D alanine, or the like (D -Ala scan) (Galantino et al., In Smith, J. and Eivier, J. (eds.), Peptide s Chemistry and Biology (Preceedings of the Twelfth American Peptide Symp osium), ESCOM, Leiden, 1992, pp. 405-05). This identifies essential residues You can modify, remove, or replace non-essential residues to The quality of the objective can be improved (Cunningham and Wells, Science, 244: 10 81-1085, 1989; Cunningham and Wells, Proc. Natl. Acad. Sci. USA 88: 3407- 3411, 1991; Ehrlich et al., J. Amer. Biol. chem. 267: 11606-11, 1992; Zhang et. al., Proc. Natl. Acad. Sci. USA 90: 4446-50, 1993; see also "Molecular De sign and Modeling: Concepts and Applications Part A Proteins, Peptides, and Enzymes, "Methods in Enzymology, Vol. 202, Langone (ed.), Academic Pr ess, San Diego, CA, 1991).   Peptide sequence in which the amino acid has been replaced Then, by synthesizing the amino acid sequence from the N-terminus or C-terminus, By synthesizing the shortest region of the active region, and also cutting the one between the N-terminal and C-terminal, The shortest amino acid sequence that serves as the active region can be determined. like this By binding the peptide to a specific MHC, in vivo or in vivo against appropriate T cells  Used as peptide ligand to induce anergy reaction in vitro Can be developed for the purpose of   Soluble fusion MHC heterodimers: physical and biochemical properties of peptide complexes Sex can be assessed in a number of ways. electrospray and Mtatrix-Assisted Laser Desorpt Mass for ion / ionization time of flight mass spectrometry (MALDI TOF) analysis, etc. Analytical analysis is routinely used in the art, and may include protein molecular weight and Used to confirm sulfide bond formation. Single chain complex is appropriate for FACs analysis Used to determine if it is folded.   ELISA (Enzyme-linked Immunosorbent Assay) Dimer: Determination of peptide complex concentration and whether it is properly folded You can check. In this assay, whole cells were excised and solubilized from the cell surface. Either MHC or the fusion MHC heterodimer of the present invention: peptide complex Can also be used. Haplotype with recombinant MHC in typical ELISA Is coated on the bottom of the microtiter plate. concrete In the example, a model that recognizes only the properly folded HLA-DR MHC dimer L243. Noclonal antibodies are used. Those skilled in the art will appreciate that other class 2 MHC specific antibodies I know it is available. If an antibody against a specific haplotype MHC Many routine techniques for preparing antibodies, even if they are not present And methods exist (eg, Hurrel, J.G.R. (ed)., Monoclonal Hybr idoma Antibodies: Techniques and Applications, CRC Press Inc., Boca Rato n, FL, 1982). If an antibody against class 2 MHC is used, affinity chromatography Chromatography methods and markers for the presence of class 2 molecules in cells and solutions -By using it as a reagent, it is possible to purify class 2 molecules. like that Antibodies are secreted from cells, especially in immunoblot analysis such as Western analysis. It is useful when analyzing what has been done. Polyclonal antibody or affinity protein Manufactured polyclonal, monoclonal or single-chain antibodies are also used for this purpose it can. Furthermore, fragments degraded by protease or prepared by recombinant methods Alternatively, an epitope binding domain or the like can also be used herein. Chimera anti Antibodies, veneered antibodies, CDR exchange (-rep) laced) antibodies, reshaped antibodies, and all or part of other recombinant antibodies. Applicable.   For use in ELISA, the bound MHC molecule can bind to the antibody or MHC. Can be detected using other molecules that can These molecules and antibodies are detectable Detectable by secondary antibody or molecule labeled with a detectable or detectable Is done. Detectable probes known in the art include fluorescence, absorbance, and radioactivity. And so on.   The corresponding MHC-peptide complex using specific T cell receptor Alternatively, there are other methods for screening in vitro. For example, in  In vitro anergy assay shows non-reactivity to T cells under test It can be determined whether the user has been induced. Briefly, an antigen-binding groove also contains an antigen peptide. One MHC molecule is mixed with the responder cells. At this time, responding cells include peripheral mononuclear Cells (PBMN) (mixture of B-type lymphocytes, T-type lymphocytes, mononuclear cells, dendritic cells, etc.) Cell group), PBMNC lymphocytes, freshly prepared T-type lymphocytes, in In vivo stimulated spleen cells, cultured T cells, established T cell lineages, A clone is desired. Immunize the target antigen, or Responsive cells from individuals with a pronounced cellular immune response are particularly desirable.   These responder cells then stimulated by the same antigenic peptide It is mixed with cells (antigen presenting cells; APCs). Specifically, punctures that are antigen-presenting cells Intense cells include PBMNCs, PBMNCs from which lymphocytes have been removed, and antigen peptides. Appropriate cell line or clone (EBV-transformed B cells, etc.) sformed autologous or non-autologous PMNCs, genetically engineered Original presentation cells such as mouse L cells and BLS-1 which is a bare lymphocyte cell, Among them, DRB1 * 0401, DRB1 * 0404, DRB1 * 0301 (Kovats et al., J. Exp. Med. 179: 2017-22, 1994) or spleen cells stimulated in vivo or in vitro No. Immunize the antigen of interest or have significant cellularity already to the antigen of interest Stimulatory cells from individuals with an immune response are particularly desirable. Some assays and First, it is desirable that the growth of stimulator cells be suppressed first before mixing with responder cells. It is. This can be done by gamma irradiation or cell fractions such as mitomycin C. An agent that inhibits cleavage is used. With the appropriate negative controls (No addition, syngeneic APC, experimental peptide, APC + peptide, MHC: pep tide complex, control peptide +/- APC). In addition, non-reactivity is anergy This growth experiment was performed in the presence and absence of recombinant IL-2 to confirm that Simultaneously, because in the presence of IL-2 cells do not become anergic Is known.   After about 72 hours, the activation of responder cells by stimulator cells is measured. Specific implementation In a similar manner, activation of responder cells is dependent on cell proliferation using 3H-labeled thymidine. (Crowley et al., J. Immunol. Meth. 133: 55-66, 19). 90). Alternatively, production of cytokines such as IL-2, or response cells By confirming the presence of an activation marker specific for T cells, in particular, Activation can be measured. Cytokine production depends on stimulator cells and responder cells. Mixed culture supernatants can cause cytokine-dependent cell growth Can be assayed by measuring Responsive or T cell specific activity The sex marker is detected using an antibody specific to it.   Preferably, the soluble fusion MHC heterodimer: peptide complex is an antigenic peptide Causes non-responsiveness (such as anergy) to responsive cells. Soluble Fusion MHC heterodimer: In addition to the recognition of peptide complexes, it also activates responder cells Receives the stimulus provided by co-stimulatory molecules Involvement of the receptor on stimulator cells (APC) is required. These types of receptors Block or eliminate stimulation (e.g., by purifying responder cells). Exposure to soluble fusion MHC heterodimers: peptide complexes or such Blocking with antibodies to the Responding cells can disrupt antigen or soluble fusion MH by disrupting the gene encoding Causes non-reactivity to the C-heterodimer: peptide complex.   In a preferred embodiment, the responder cells cause an autoimmune disease / syndrome. Prepare from what is rubbed. Or T cells that are reactive to autoantigens It is desirable to prepare from loans and affiliates. In another embodiment, the stimulator cells are Prepared from those causing autoimmune disease / syndrome. Or APC Properly cleave self-antigens in cell lines and clones and / or Use what can be shown. Particularly preferred embodiments include responder cells And the stimulator cells are prepared from those from diabetes or multiple sclerosis.   At this point, the responding T cells are specifically expanded and / or stimulated, resulting in A T cell group specific to the antigenic peptide has been created. For example, flow cytometry By means of a tree or, in particular, a fluorescence activated cell sorter (FACS), the antigenic peptide Reactive responder cells can be selected. This group of responding cells contains the same antigen It can be maintained by repeated stimulation with APCs presenting tides. Or Alternatively, responder cell clones and lineages can be created from this group of responder cells. further The position of the epitope of the antigenic peptide or protein given using this responder cell group. Position can be determined.   Measuring the biological activity of soluble fusion MHC heterodimers: peptide complexes Other methods are known in the art and can be used herein. For example, acidic metabolic products synthesized by T cells after the interaction of antigen cells with T cells For example, there is a method using a microphysiometer for measuring an object. Another assay There is a competitive assay method. This is a soluble fusion MHC heterodimer: pepti The response to the complex is compared with that of a normal antigen. In addition, Measuring production of ins and gamma interferons is also an indicator of complex response Becomes   MHC: Model animal of disease caused by peptide complex is created And they can be used in similar assays and methods. For example, Class 2 MHC molecule of NOD mouse, a model of insulin-dependent diabetes mellitus (IDDM) A certain I-A^g7When the nucleic acid sequence encoding is used together with the autoimmune antigen GAD, It can be used to study the induction of non-reactivity in product models.   Soluble fused MHC heterodimer: peptide complex is a model for many autoimmune diseases Can be used in vivo on animals. For example, NOD mice are a spontaneous model of IDDM. It is. Pre-morbidity due to soluble fusion MHC heterodimer: peptide complex After the onset of the disease, administration of the glucose concentration in the urine of mice and Monitor reactivity to self-antigen by in vitro T cell proliferation assay (Eg, Kaufman et al., Nature 366: 69-72, 1993). Induction such as EAE Soluble fusion MHC heterodimer: peptide complex also responds to selected autoimmune conditions Coalescence administration can be performed. Effective for preventing EAE or suppressing disease progression The symptoms of EAE can be observed and monitored after administration to produce   NOD mouse (H-2g⁷) Is a model of IDDM in rodents. NOD mouse Conditions include the presence of antibodies against islet cells of Langerhans, severe insulitis And autoimmune destruction of b cells (Kanazawa et al., Diabetol). ogia 27: 113, 1984). The disease is transmitted passively by lymphocytes and It can be prevented by treatment with sporin-A (Ikehara et al., Proc. Natl. A cad. Sci. USA 82: 7743-47, 1985; Mori et al., Diabetologia 29: 244-47, 198. 6). Untreated mice have a lack of glucose tolerance and ketosis. Wake up and die within weeks of illness. The colony (# 11NOD / CaJ) used this time is Causes a higher incidence of diabetes in males than in other colonies, 50-65% in males and females Develops within 7 weeks after birth in 90-95% of individuals (Pozzilli et al., Immunology T oday 14: 193-96, 1993). Breeding studies have identified at least two loci It is associated with onset, one of which is the MHC region. NOD mouse class 2 From the results of serological and molecular analysis of the antigen, I-A in this autoimmune disease^g7Noseki (Acha-Orbea and McDevitt, Proc. Natl. Acad. Sci. USA 8 4: 2435-39, 1987).   The progression of diabetes can be studied in several ways, including spontaneous These include disease progression and adaptive transfer models (Miller et al., J. . Immunol. 140: 52-58, 1988). NOD mice spontaneously cause IDDM. NOD / Ca In J mice, female diabetes is observed from about three months after birth. Young female NOD / CaJ mice with peptide, peptide-MHC complex, and control You can give and see the progress of the condition for 6 months. NOD mouse urine Observe if the patient has diabetes by measuring the glucose concentration. When the glucose level reaches the level of diabetes, the tail of the mouse is cut off to determine the amount of glucose in the blood. Measure further with a lucometer. If the blood glucose level is more than 250mg / dl If so, it is classified as overt diabetes. This method involves the administration of self-reactive natural T cells. Need.   IDDM does not show diabetes by transplanting spleen cells from a diabetic donor It is also caused in individuals (Baron et al., J. Clin. Invest. 93: 1700-08, 1994). The method involves the administration of mature T cells that have been activated in vivo. Simple Briefly, NOD / CaJ mice were exposed to radiation (730 rad) and treated with several therapeutic groups. Arbitrarily distribute to loops. Approximately 1.5x10 from mice just after diabetes⁷Individual spleen Cells were removed and injected intravenously into NOD mice that did not develop diabetes 7-8 weeks old 6 hours later, saline, peptide, complex of MHC and peptide was added to each animal for 1 hour. 0, 5, and 1 microgram are injected intravenously, respectively. 4 days, 8 days, 12 days after treatment The same treatment is performed after a day. Examining the onset of diabetes by examining mouse urine Finally, the glucose concentration in the blood is examined. MHC and peptide in these mice Administration of the complex can delay the onset of diabetes. (Or) can be expected to improve the disease. Diabetes is apparent in the first animal When there was a tendency, one animal was randomly selected and killed from each treatment group, The spleen and pancreas are removed for weaving chemistry analysis. This survey was conducted by the control group Terminate when all mice in the (saline) develop diabetes. Administered saline Mice almost develop diabetes within 20 days.   Expression systems suitable for producing soluble fusion MHC heterodimers: peptide complexes Are known in the art. Hosts of various prokaryotes, yeasts, molds, and eukaryotes Is suitable for the production of soluble fusion MHC heterodimer: peptide complexes.   Prokaryotes are useful as host cells, and in the present invention, Escherichia co The various strains of li are most frequently used. But other microorganisms, such as Bacillus s Various strains of bacilli such as ubtilis and Pseudomonas, and other bacterial strains Can be used.   In the present invention, the expression of the soluble fusion MHC heterodimer: peptide complex is Recombinant soluble fusion MHC heterodimer: peptide complex The encoding nucleic acid sequence should include a signal sequence that causes gene expression in prokaryotic cells. This is accomplished by operably connecting the encoding nucleic acid sequences. One nucleic acid sequence is another When placed in a functional relationship with the array of (Operably linked). For example, promoters and enhancers When connected to affect the transcription of amino acid encoding regions, they Connected as practicable. Generally, nucleic acid sequences are connected so that they are adjacent. When connecting two protein coding regions, When a connection is made, it is referred to as being operably connected.   The gene encoding the soluble fusion MHC heterodimer: peptide complex was The terms “current vector”, “cloning vector”, and “vector” It is used confusedly in the textbook, but is generally a plasmid or Refers to a nucleic acid sequence capable of replication in a host cell. Expression vectors are autonomous Replicated or integrated into the host cell's genome using methods known in the art. Duplicate by being done. An autonomously replicating vector contains a replica that works in the target host Has an origin or autonomously replicating sequence (ARS).   Vectors with replication and control sequences from species compatible with the host cell of interest Is used. For example, E. coli is typically Boliver et al. (Gene 2: 95-113, 1977). ). Transformation with a vector derived from pBR322 isolated from a strain of E. coli Is done. Often, using vectors that can be used in more than one host cell Perform cloning and plasmid construction in desired E. coli, for example, Bacillus For example, when expression is performed.   A typical expression vector has a portion for transcription or a portion required for gene expression. Contains all essential elements and can be used for MHC expression in host cells. Typical A typical expression cassette is a fusion MHC heterodimer that is operable with a promoter: The ribosome binding region is connected to the gene part encoding the peptide complex . So that the distance between the transcription start site and the gene in the original promoter is the same Determine the location of the introduced promoter. But well known in the art Use some flexibility in the distance without causing a decrease in promoter function. Can be scraped. In addition to the promoter sequence, the expression cassette is located below the structural gene. The stream contains a transcription termination region for efficient transcription termination. The transcription termination region It is derived from the same gene as the promoter or from a different gene.   The commonly used prokaryotic control regions set forth herein are: A promoter region for transcription initiation, and optionally an operator region, followed by Betalactamase (penicillase) containing a ribosome binding sequence and commonly used inase) promoter and lactose (lac) promoter (Change et al., Nature 198: 1) 056, 1977), tryptophan (trp) promoter (Goeddel et al., Nucleic Acids Re s. 8: 4057-74, 1980), P from lambda phage_LPromoter and N gene ribosomes And the chromosome binding region (Shimatake et al., Nature 292: 128-32, 1991). Prokaryotic cells Any promoter available within can be used.   In the present invention, both constitutive and regulated promoters are used. It is. In regulated promoters, soluble fusion MHC heterodimers: pepti The advantage is that host cells can be cultured at high concentrations before inducing complex expression. is there. In some situations, high expression of a heterologous protein slows cell growth. E. col Some promoters particularly suitable for use in i include the lambda bacteriophage P_L Promoter and trp-lac hybrid promoter (Amann et al., Gene 25: 167- 78 1983), and the bacteriophage T7 promoter is included.   Soluble fusion MHC heterodimers in prokaryotes other than Escherichia coli: Expression requires a promoter that functions in that particular prokaryotic species. That's it Such promoters are derived from genes already cloned in that species. Alternatively, a promoter of a heterologous organism is used. For example, trp-lac hybrid The promoter is E. It works not only in E. coli but also in Bacillus.   The ribosome binding region (RBS) is a soluble fusion MHC heterodimer in prokaryotes: pep Necessary for expressing the tide complex. For example, E. E. coli RBS is 3-9 salt Group length 3 to 11 bases upstream from the start codon (Shine and D algarno, Nature, 254: 34-40, 1975; Steitz, In Biological regulation and d evelopment: Gene Expression (ed. R.F. Goldberger), Vol. 1, p.349, 1979, P lenum Publishing, NY).   Expression can be increased by coupling in translation. This strate Gee is a short open library upstream of a gene that is highly expressed in the natural translation system. Use a loading frame. This short open reading frame is professional Motor, after ribosome binding sequence, stopped after a few amino acid codons The codon enters. Immediately before the stop codon is the second ribosome binding sequence The top codon is followed by the next translation start codon. In this system, the two-dimensional structure of RNA is almost After that, efficient translation starts. Squires et al., J.S. Biol. Chem. Two 63: 16297-16302, 1988.   Soluble fusion MHC heterodimer: peptide complex is intracellular or from cell It can be expressed by secretion. Expression in cells often produces high yield results. However Some of them may be insoluble inclusion bodies. The present invention Some of the intracellularly expressed MHC proteins are activated by cell lysis Although it can be recovered as a sex form, MH of the active form can be recovered by combining the refolding process. C protein can be increased (Sambrook et al., Molecular Cloning: A Laboratory Mannual Second Edition, Cold Spring Harbor, NY, 1989 .; Marsto n et al., Bio / Technology 2: 800-804,1985; Schoner et al., Bio / Technology. 3: 151-54, 1985). For purification and refolding, ideally The urea fraction is dissolved and the urea / boric acid / DTT solution is used, followed by the urea / boric acid solution. After refolding, silica gel-based Vydac (Hewlett Packard, Wilming ton, DE) or polymer-based Poros-R2 resin (PerSpective Biosystems) Purification is performed by reversed phase HPLC. The size of these beads depends on the culture scale. And will be described in detail later. Especially on a large scale In the case of refolding, the sample is optionally treated with urea / boric acid. Ultrafiltration of the solution, followed by 0.2 micromolar to 1 millimolar, ideally 0 Add 2 to 20 micromoles of copper sulfate and then immediately Let it go. Refolding occurs at protein concentrations from 0.1 to 2.5 mg / ml .   By integrating multiple transcription cassettes into one expression vector, or The different cloning strategies used for each expression vector By using a selectable marker, one or more MHC-peptide complexes can be combined into one It can be expressed in prokaryotic cells.   The second method for expressing the complex of MHC and peptide in the present invention, Periplasm, or secretion of proteins into extracellular cultures is there. A cleavable signal peptide sequence is connected to the DNA sequence encoding MHC. The signal sequence acts to move the complex of MHC and peptide across the cell membrane Have E. pTA1529 is an example of a suitable vector used in E. coli . This vector is E. coli. E. coli phoA promoter and signal sequence (Samb rook et al., supra; Oka et al., Proc Natl Acad. Sci. USA 82: 7212-16,198 Five; Talmadge et al., Proc Natl Acad. Sci. USA 77: 39892, 1980; Takahara et al ., J. Biol. Chem. 260: 2670-74, 1985). Again, meeting in periplasm For this purpose, a plurality of proteins can be expressed in one cell.   The complex of MHC and peptide in the present invention is also expressed in the form of a fusion protein be able to. This method often results in high yields, because ordinary prokaryotes This is because the control sequence of the cell performs transcription and translation. E. In E. coli, lacZ and Is used for exogenous protein expression. pUR, pEX, pMR100 series (Sambrook et al., supra) are readily available for this purpose. Special For certain applications, cleavage of amino acids other than MHC after purification of the fusion protein Things are desired. This is achieved by several methods known in the art it can. This includes cleavage by cyanogen bromide, protease, Factor X, etc. (Sambrook et al., Supra; Goeddel et al., Proc Natl Acad. Sci. USA 76 : 106-10,1979; Nagai et al., Nature 309: 810-12,1984; Sung et al., Proc. Natl Acad. Sci. USA 83: 561-65, 1986). The cut is made by gene recombination technology It can be introduced to the desired location in the fusion protein.   Like the soluble fusion MHC heterodimer: peptide complex gene in the present invention A foreign gene is E. coli. Expression in Escherichia coli fused to other binding partner proteins Can be done. Glutathione-S-transf This includes erase (GST), maltose binding protein, thioredoxin, etc. like this Binding partners have high translational efficiency, and therefore require genetic information from eukaryotic cells. E. Resolves low translation initiation efficiency when translating in E. coli. Such a knot Fusion with a partner protein results in increased expression and -Toner proteins and other such proteins are easily purified and released from the target protein. Is possible. Such expression systems are available from many sources, for example, Invitrogen Inc. (San Diego, CA) and Pharmacia LKB Biotechnology Inc. (Pisca taway, NJ).   (Biotechnology 7: 698-704, 1989). N-terminal using E. coli Methods for preparing intact recombinant proteins are described. This Target system is yeast ubiquitin with a protease cleavage site It is made by fusing to the C-terminus of the first 76 amino acid residues of the gene. This By cutting the connecting part of the two proteins, the original N-terminal protein Is produced.   A vector containing a soluble fusion MHC heterodimer: peptide complex gene has been developed. Introduced into host cells for realization. "Transformation" is A vector containing a nucleic acid sequence is directly transferred to a host cell by a well-known method. To enter. For soluble fusion MHC heterodimer: peptide complex expression In some cases, a vector or the like is introduced into a host cell by a specific method. Is not particularly important. Use any of the well-known methods for introducing foreign nucleic acids be able to. The only important thing is that the specific host cell used expresses the gene Is it possible?   Transformation methods vary depending on the prokaryotic host used, but electroporation. Method, calcium chloride, rubidium chloride and calcium phosphate or Transfection method using other substances, induction by microprojector Entry, infection (if the vector is infectious), or other methods is there. Common cases include Sambrook et al., Supra and Ausubel et al., (E ds.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N See Y, 1987. In reference to which nucleic acid shown above was introduced into the cell The progeny of such cells are also included. Soluble fusion MHC heterodimer: peptide complex A transformed prokaryotic cell having a vector having a combined gene is also included in the present invention.   By common transfection or transformation, soluble fusion MHC After preparing prokaryotic cells that express large amounts of dimeric: peptide complex proteins The expressed protein is purified by a general method. For example, Colley et al. , J. et al. Chem. 64: 17619-22, 1989; and Methods in Enzymology, "Guide to Prote in Purification ", M. Deutscher, ed., Vol. 182 (1990). To express a soluble fusion MHC heterodimer: peptide complex. Expression The method of protein purification is based on the fact that the soluble fusion MHC heterodimer: Secreted out of cells when expressed in the periplasm It depends on the case. For intracellular expression, collect cells and lyse Sa The protein is recovered from the cell lysate (Sambrook et al., Supra). Perip MHC proteins in lasm are released from the periplasm by common means ( Sambrook et al., Supra). If the MHC protein is secreted extracellularly, The culture is collected for protein purification. Typically, the medium is centrifuged. Remove cells and cell debris by separation or filtration.   MHC protein is prepared using a suitable resin (CDP-Sepharose, Asialoprothrombin-Sepharose 4B , Q-Sepharose, etc.), ammonium sulfate fractionation and polyethylene glycol precipitation Or using ultrafiltration. Others known in the art The method is used as well.   Further purification of the MHC protein is performed according to a general method, for example, affinity purification. Tea chromatography, ion exchange chromatography, size chromatography Use laffy, reverse-phase HPLC, and other methods used to obtain homogeneous proteins I have. The purified protein is subsequently used in a pharmaceutical composition.   The DNA to be constructed also contains a DNA region necessary for secreting the target protein. Such a DNA region contains at least one secretory signal sequence. Secretory signa Sequence, also called leader sequence, prepro sequence, or pre sequence, It has the function of secreting out of the cell. Such sequences are characterized by a hydrophobic core region. Marked, typically (but not all) novelly synthesized Located at the N-terminus of the protein. Is the secretory signal sequence for the protein of interest? And other secreted proteins (for example, t-PA is often used as the secretory leader sequence in mammalian cells). Used) or synthesized in a novel way. The secretory signal sequence is used in the present invention. The target protein DNA and the frame are connected together. The secretory signal sequence is It is usually connected to the 5 'end of the DNA sequence encoding the protein of interest. Some kind The signal sequence may be ligated at an appropriate non-5 'end (Welch et al. al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). secretion During the process, the secretory signal peptide is often cleaved from the mature protein. This. Such secretory signal peptides include cleavage from the mature protein during the secretion process. There is a cutting site to be released. As an example of such a cutting site There is a cleavage sequence in which two basic amino acids are arranged, for example, Saccharomyces cere vi There is a lysine-arginine cleavage sequence recognized by the KEX2 gene product of siae . Cleavage sequences can be integrated into secretion signals or introduced in vitro It can be added to the peptide by a method or the like.   The secretion signal includes an a-factor secretion signal sequence (prepro sequence: Kuja n and Herskowitz, Cell 30: 933-943, 1982; Kurjan et al., U.S. Patent No. 4,546, 082; Brake, EP 116,201), PHO5 signal sequence (Beck et al., International Publication No. WO86 / 00637), a BARI secretory signal sequence (MacKay et al., U.S. Patent No. 4,613,572; Mac Kay, WO 87/002670), SUC2 signal sequence (Carlsen et al., Molecul) ar and Cellular Biology 3: 439-447, 1983), a-1-antitrypsin signal sequence (K urachi et al., Proc. Natl. Acad. Sci. USA 78: 6826-6830, 1981), a-2 plasm in inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102: 1033-1042 , 1987), tissue plasminogen activator signal sequence (Pennica et al., Natur e 301: 214-221, 1983). Alternatively, a secretory signal sequence, such as von Rules established by Heinje (European Journal of Biochemistry 133: 17-21 , 1983; Journal of Molecular Biology 184: 99-105, 1985; Nucleic Acids re. search 14: 4683-4690, 1986). With another synthetic signal LaC212 spx (1-47) -ERLE is described in WO 90/10075. .   Secretory signal sequences may be used alone or in combination. For example, The first secretory signal sequence is used in conjunction with the sequence encoding the third domain of the barrier. (US Pat. No. 5,037,243, herein incorporated by reference in its entirety). ). The third domain of the barrier is in frame with the 3 'end of the DNA sequence of interest. Bound or in-frame with the secretion signal and the 5 'end of the DNA sequence of interest Are combined.   Suitable promoters, terminators and secretion signals in all expression systems For the selection, a general technique in the field may be used. Saccharomyces cere Expression of cloned genes in visiae is a well-known area ("Gene Expression Technology," Methods in Enzymology, Vol. 185, Go eddel (ed.), Academic Press, San Diego, CA, 1990 and "Guide to Yeast Gene tics and Molecular Biology, "Methods in Enzymology, Guthrie and Fink (e ds.), Academic Press, San Diego, CA, 1991, incorporated herein by reference. ). The protein of the present invention is also used in filamentous fungi such as Aspergillus. (McKight et al., US Pat. No. 4,935,349 and References used here). The cloned gene can be used in cultured mammalian cells or E. coli. c oli and the like are described in detail by Sambrook et al. (Molecular C loning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 19 89, incorporated herein by reference). Obviously in that area, In the present invention, the protein is used to separate other host cells, such as birds, insects, and plants. Expression using regulatory cells, vectors and methods developed in the field using cells It is also possible to make it.   A suitable yeast vector for use in the present invention is YRp7 (Strul et al., Proc. N atl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121). -133, 1979), a POT vector (Kawasaki et al., U.S. Pat. PJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1). 978), and those derived therefrom. Glycolysis of yeast as the promoter used System promoter (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; A lber and Kawasaki, J.S. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, United States Patent No. 4,599,311) or alcohol dehydrogenase promoter (Young et al., in Genetic Engineering of Microorganism for Chemicals, Hollaender et al. , (Eds.), P.355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101: 192 -201, 1983). including. Other promoters include the TPI1 promoter (Kawasaki U.S. Pat. No. 4,599,311, 1986) and ADH2-4^CPromoter (Russell et al., Natu re 304: 652-654, 1983; Irani and Kilgore, U.S. Patent Application Serial No. 0 No. 7 / 784,653, CA 1,304,020 and EP 284 044, incorporated herein by reference. ). The expression unit contains a transcription termination sequence such as the TPI1 terminator (Albe r and Kawasaki, ibid).   Yeast cells, especially Saccharomyces, are suitable hosts for the production of the substances according to the invention. It can be called a cell. Transform yeast cells with foreign DNA to produce recombinant protein This method is disclosed in, for example, the following literature. Kawasaki, US Patent No. 4,59 9,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,00 No. 8, Welch et al., US Pat. No. 5,037,743; Murray et al., US Pat. Four No. 845,075, which is incorporated herein by reference. Transformed cells are selection markers Phenotype determined by the individual, generally drug resistance or specific auxotrophy (Such as leucine). Vectors used in yeast The POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. In this system, the transformed cells are selected on a medium containing glucose. It is possible. Secretory signal sequences used in yeast include S. cerevisiae. cer There is a secretory signal sequence of Mfa1 of Evisiae (Brake, ibid .; Kurjan et al., USA Patent No. 4,546,082). Suitable promoters for use in expression in yeast , As a terminator, a glycolytic enzyme gene promoter (for example, Kawasaki U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,645,974; Bitter No. 4,977,092, which is incorporated herein by reference), and alcohol de hydrogenase gene promoter. Also U.S. Pat.Nos. 4,990,446 and 5,063,154 See also, Nos. 5,139,936, 4,661,454, and references used therein Please. Hansenula polymorpha, Schizosaccharomyces ponbe, Kluyveromyces lac tis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia me against other species of yeast such as thanolica, Pichia guillermondii, Candida maltosa Transformation systems are also known in the art. For example, Gleeson et al. Gen. Mi crobiol. 132: 3459-65, 1986; Cregg, U.S. Patent No. 4,882,279, Stroman et al. See U.S. Patent No. 4,879,231.   Other fungal cells are also suitable as host cells. For example, Aspergillus is M cKnight et al. method (US Pat. No. 4,935,349, incorporated herein by reference). Can be used by Transformation method to Acremonium chrysogenum is Sumi published by no et al. (US Pat. No. 5,162,228, incorporated herein by reference). Have been. Transformation method into Neurospora is described in Lambowitz (U.S. Pat. No. 4,486,533, Which are incorporated herein by reference).   Host cells containing the constructed DNA are cultured to produce foreign protein. You. Cells are grown using a medium containing the nutrients necessary for the growth of their host cells. Cultured by the method. A variety of suitable media are known in the art, Are generally carbon and nitrogen sources, necessary amino acids, vitamins, minerals, Contains children. By culturing in these media, the DNA Cells grow selectively, i.e. by drug selection or on the introduced DNA or Is a nutrient source required for host cells by a selectable marker on the simultaneously introduced DNA A method such as complementing the deficiency of is used.   For example, yeast cells contain nitrogen sources (other than amino acids), inorganic salts, vitamins, Cultured in a chemically prepared medium consisting of amino acids, etc. PH of the medium is from 2 During 8, it is ideally kept at 6.5. How to control the pH of the medium Methods include using a buffer solution and always controlling the pH, ideally Add sodium hydroxide. Ideal buffers include succinic acid and Bis- Tris (Sigma Chemical Co., St. Louis, MO) and the like. Asparagine binding Yeast cells deficient in genes required for the formation of glycans are osmotic stabilizing media Incubate with Ideal osmotic pressure stabilizers have a concentration of 0.1M to 1.5M (preferably Sorbitol (0.5M or 1.0M conditions) is used. Cultured mammalian cells are generally Incubate with commercially available culture media with or without serum. Specific host details Selection of a culture medium suitable for the vesicles is performed by ordinary selection in the art.   As a method for introducing foreign DNA into mammalian cells, calcium phosphate Transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson , Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-45, 198). 2), transfection with DEAE-dextran (Ausubel et al., (Eds), C urrent Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 19 87, incorporated herein by reference). Tran with cationic lipid The sfection method is also included. In this case, Boehringer Mannheim TRANSFECTION-R EAGENT (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl ammoniummethylsulfa te; Boehringer Mannheim, Indianapolis, IN) or LIPOFECTIN reagent (N- [1- (2,3- dioleoyloxy) propyl] -N, N, N-trimethyl ammonium chloride and dioleoyl phosp hatidylethanolamine; GIBCO-BRL, Gaithersburg, MD) May be used in a manner specified by the manufacturer. A commonly used mammalian cell expression vector is Zem229R (Ameri during the Budapest Treaty) Deposited with can Type Culture Collection, 12301 parklawn Drive, Rockville, MD And on September 23, 1993, E. Accession Number as a transformant of E. coli HB101  69447). Production of recombinant protein in cultured mammalian cells For example, see Levinson et al., US Pat. No. 4,784,950; Palmiter et al. al., US Patent No. 4,579,821; Ringold et al., US Patent No. 4,656,134, the present invention. Incorporated as a reference in the detailed text, published in Commonly used mammalian cells COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1650) RL 1632), BHK 570 (ATCC No. CRL 10314), DG44 and 293 (ATCC No. CRL 1573; Grah am et al., J. Am. Gen. Virol. 36: 59-72, 1977). Other suitable Cell lines are known in the art and are available from the American Type Culture Collection, Ro Available from public depositories such as ckville and Maryland. Generally, SV-40 or Strong promoters such as cytomegalovirus are used. US Patent See No. 4,956,288. Other suitable promoters include metallothionein Genes (U.S. Pat.Nos. 4,579,821 and 4,601,978, incorporated herein by reference) And the adenovirus major late promoter.   Drug selection was used to select cultured mammalian cells into which the foreign gene had been incorporated. Will be Such cells are commonly referred to as "transfectants". be called. Cultivated under the selection drug, inherit the target gene to daughter cells properly Cells that can do so are called "stable transfectants". Often used The selectable marker is a resistance gene to the antibiotic neomycin. Choice is G-41 It is performed in the presence of a neomycin-type drug such as 8. Such cell selection method Is also used to increase the expression level of the target gene. This is "Amplify A process called “Amplification”. Transfection is performed using a culture solution containing a low concentration of the selective drug. Culture and then increase the drug concentration to select cells that produce more of the desired gene product How to Selectable marker for commonly used amplification Cars include dihydrofolate reductase, which provides resistance to methotrexate. You. Other drug resistance genes (hygromycin resistance, multidrug resistance, puromy cin acetyltransferase) also exists.   The soluble fusion MHC heterodimer: peptide complex in the present invention is as follows: It is purified in the process. First, proteins are extracted from the cells, Size chromatography (Coy et al., Peptide Structure and Function, Pie rce Chemical Company, Rockford, IL, pp 369-72, 1983), reversed-phase chromatography Raffy (Andreu and Merrifield, Eur. J. Biochem. 164: 585-90, 1987, etc.), HPLC (Kofod et al., Int. J. Peptide and Protein Res. 32: 436-40, 1988, etc.) For example, a method commonly used for protein purification is used. The next step purification method Liquid chromatography, density gradient centrifugation, gel electrophoresis and others. Method is used. Protein purification methods are known in the art (general In the story, Scopes, R., Protein Purification, Springer-Verlag, NY, 1982, book , Incorporated herein by reference), the recombinant protein in this case. It is also applied during purification. Soluble fusion MHC heterodimer: peptide complex refinement Production rate should be at least 50%, especially 70-80%, especially when used for medication purposes. Ideally, 95-99% or more is desired. Depending on the purpose, soluble fused MHC he After the terodimer: peptide complex has been partially or completely purified, As mentioned, it is used for diagnosis or treatment of disease.   The soluble fusion MHC heterodimer: peptide conjugates of the present invention can be used in autoimmune diseases. Is used to suppress some of the immune system that has been activated. The soluble melt of the present invention Synthetic MHC heterodimer: peptide conjugates elicit an immune response to specific self-antigens Immune tolerance or non-responsiveness to prone or already-causing patients It is expected to be an immunotherapeutic agent that induces sex (anergy). A certain self Patients who have or are susceptible to an immune disease can be identified by methods in the art. , MHC types have been identified. The patient's T cells are shown here with this complex. Self-reactivity recognized by patient's self-reactive T cells in vitro using Tested to determine the original peptide. The patient then uses the conjugate of the invention. Can be treated. Such methods are commonly used until the onset of an autoimmune disease. Sufficient to extend the duration of the disease and / or to improve or prevent the disease What Amount of soluble fusion MHC heterodimer: peptide conjugate may also be included U.   Oral tolerance (Weiner et al., Nature 376: 177-80, 199) 5) and venous tolerance are also included. Tolerance is a phenomenon induced in mammals, However, the conditions under which tolerance occurs depend on many factors. Self-antigens such as IDDM Immunize adults who are susceptible to or have already developed the disease involved. The exact amount and number of doses of the drug to induce volume also vary. For example About 20-80 mg / kg for adults, parenterally or orally, by aerosol or skin It is administered by various means such as internal injection. For neonates, intradermal injection Alternatively, oral administration can induce tolerance according to an appropriate prescription. Correction of drug administration The exact amount and method of administration also vary.   Typically, the soluble fusion MHC heterodimer: peptide complex is in a soluble state When administered in the form of aggregated or particulate, It is more tolerant. Soluble fusion MHC heterodimer in the present invention The persistent presence of the body: peptide complex can maintain tolerance in adults Frequent administration of the conjugate or half-life of the conjugate Administration in a prolonged form is required. For example, Sun et al., Proc. Natl. Acad. Sci. USA 91: 10795-99, 1994.   In another aspect, the present invention provides a soluble fusion MHC heterodimer according to the present invention: Contains peptide conjugates and carriers or excipients that are recognized as drugs Contains, for subcutaneous, topical, oral administration such as aerosol or skin penetration Thus, it provides a composition of drugs used for the prevention and / or treatment of the disease. medicine Composition is typically in a form suitable for injection or infusion into tissue. And is therefore formulated with sterile water, isotonic solution, or glucose solution You. When formulating, diluents, extenders, emulsifiers, preservatives, buffers, And liquids, powders, emulsions, suppositories, liposomes, subcutaneous patches, tablets, etc. Given in the form. Those skilled in the art will be able to use appropriate methods and accepted practices (Remington 's Pharmaceutical Sciences, Gennaro (ed.), Mack Publishing Co., Easton, P A 1990, incorporated herein by reference) to formulate the compounds of the present invention. .   The pharmaceutical compounds of the present invention are administered at intervals of from one day to one week. Like that The effective amount of a pharmaceutical compound is the harm caused by T cells to self antigens When a significant immune response, such as that occurring in IDDM, is clinically significantly reduced, Or the dose at which other pharmacologically valuable effects appear. Like that Effective doses may depend, in part, on the particular context of treatment, age, weight, It depends on the state of health, other factors which are obvious to the person skilled in the art. Preferably soluble Sex MHC heterodimer: dose of peptide conjugate is within 20-80 mg / kg . Administer compounds with significantly longer half-lives in smaller doses or less frequently .   Kits can also be supplied for therapeutic and diagnostic purposes. With the main composition in the present invention The product is generally supplied in a lyophilized state in a container. The kit contains soluble fusion Synthetic MHC heterodimer: with peptide conjugate, with instructions for use and optional It also includes buffers, stabilizers, biocides, and inactive proteins. Typically, These appendages comprise approximately 5 to 5 soluble fusion MHC heterodimer: peptide complexes. At or below the weight of the normally soluble fusion MHC heterodimer: peptide complex And a weight of 0.001% or less. Inert bulking agents and excipients to dilute the active ingredient It is desirable to include an agent (present from 1% to 99% of the total amount).   In one aspect of the present invention, a soluble fusion MHC heterodimer: peptide complex May be used in the preparation of diagnostic or therapeutic antibodies. This specification As used in the text, "antibody" includes polyclonal antibody, monoclonal antibody Antibody, F (ab ')_TwoAntigen-binding fragments such as and Fab fragments, and recombinantly produced Binding partners are also included. Some of these binding partners are It incorporates the variable and CDR regions of a matching antibody. Monoclonal antibodies and binding proteins -Toner affinity is measured by methods common in the art (Scatc hard, Ann. NY Acad. Sci. 51: 660-72, 1949).   Methods for preparing monoclonal and polyclonal antibodies are described in detail in the literature (Eg, Sambrook et al., Molecular Cloning: A Laboratory M anual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. et al. G.R ., Ed., Monoclonal Hybridoma Antibodies: Technique and Applications, CRC  Press, Inc., Boca Raton, FL, 1982, incorporated herein by reference). A variety of homeothermic animals, such as cattle, can be , Goat, sheep, chicken, rabbit, mouse, rat, etc. Antibodies are prepared. Immunogenicity of soluble fusion MHC heterodimers: peptide complexes Is an adjuvant, such as Freund's complete or incomplete adjuvant Rise by the use of. Various assays can be performed by one skilled in the art. Detection of antibodies that bind to the soluble fusion MHC heterodimer: peptide complex is performed. Exemplary assays are detailed in the following book (Antibodies: A Labor atory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press , 1988). Representative examples of such assays include co-immunoelectrophoresis, radio- Munoassay, immunoprecipitation with radioactivity, using immunosorbent linked to enzyme Assays, dot blot assays, inhibition or competition assays, sandwich And other assays.   Other techniques may be used to construct and express recombinant monoclonal antibodies. It is. Briefly, mRNA was isolated from a population of B cells, and lIMMUNOZAP (H) Or a suitable vehicle such as lIMMUNOZAP (L) (Stratagene Cloning System, La Jolla, CA). A cDNA expression library for immunoglobulin heavy and light chains. You. These vectors can then be screened individually or with Fab fragments or anti- Co-expressed to form the body (Huse et al., Science 246: 1275-81, 1989;  Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989). Positive DNA obtained from Lark was from E. coli. Generating high levels of monoclonal antibody fragments in E. coli Introduce into a plasmid that does not have cell lysis that can be expressed.   Antibodies in the present invention may be of various types of thermostatic animals such as horses, cows, goats, sheep , Dog, chicken, rabbit, mouse, rat, etc. MHC heterodimer: produced by immunization with a peptide complex. Another Immortalization of antibody-producing cells prepared from immunized animals Performing Human monoclonal antibodies to human antigens, such as fusion MHC Making terror dimers: peptide conjugates is difficult with ordinary immortalization techniques So it's better to make a non-human antibody first. Recombinant DNA technology Using a technique, the antigen-binding region of a non-human antibody to the coding region of a human antibody In To create a substantially human antibody molecule. Such a method is Well known in the art, for example, US Patent No. 4,816,397 and EP publications 1 73,494 and 239,400, which are incorporated herein by reference.   In another aspect of the invention, the soluble fusion MHC heterodimer: peptide complex is It is used to clone T cells that have a specific receptor for it. So Once a skilled person has access to the soluble fusion MHC, Telomere: T cells once peptide-complex-specific T cells have been isolated and cloned Fusion membrane MHC heterodimer on T cells by immunizing animals with their membrane fractions: Pepti Antibody to the complex receptor is prepared. Antibodies are polyclonal or monoclonal Ronal is also possible. For polyclonal antibodies, rodents, lagomorphs, It can be made in horses, sheep, or various other species of mammals. Monochrome In the case of antibodies, in cases typically made using previously known techniques , Originally from a rodent, or human by the method shown above, Create a combination of these in the form of chimeric or human-adapted antibodies . The thus obtained anti-soluble fusion MHC heterodimer: peptide complex Scepter antibodies also reduce the population of T cells presenting such receptors. Or administered to patients for the purpose of removal. This group of T cells is involved in autoimmune antigens In people who are or are already susceptible to diseases such as autoimmune diseases Recognizes cells with self-antigen peptides and is involved in their immune cell destruction .   Methods of linking antibodies to a solid support and using them for protein purification are well known. (Eg, Methods in Molecular Biology, Vol1, Walker (ed.), Hum ana Press, New Jersey, 1984, incorporated herein by reference). In the antibody of the present invention, the MHC heterodimer: peptide complex is It can be used as a marker reagent for detecting presence in a liquid. like that Antibodies can also be used for Western analysis and immunoblotting, especially for purified secreted proteins. Can be used for Polyclonal antibody, affinity purification Polyclonal, monoclonal, and single-chain antibodies have also been developed for this purpose. Suitable for use. It can be further degraded by proteases or prepared by recombinant methods. Prepared fragments or epitope binding domains can also be used. Chimeric antibody, human Antibodies, veneered antibodies, CDR-replaced antibodies Applicable to all or part of the body, reshaped antibodies and other recombinant antibodies It is.   The following examples are provided to illustrate this patent and are not intended to be limiting. It's not because of restrictions.                                  Example                                 Example 1 Construction of DNA sequence encoding human soluble fusion MHC heterodimer: peptide complex   Plasmid pLJ13 contains the MHC Class II β chain (DR1β * 1501) signal sequence, A sequence encoding a basic protein (bp 283 to 345, amino acids 82 to 102 D ENPVVHFFKNIVTPRTPPPS) (SEQ.ID.NO.33), amino acid sequence (GGG SGGS SEQ. ID. NO. DNA sequence encoding the flexible linker represented by 31) Sequence, coding sequence for class II MHC DR1β * 1501 (SEQ. ID. NO. 50) β1 site , A flexible linker represented by an amino acid sequence (GASAG SEQ. ID. NO. 29) DNA sequence encoding the class II MHC DRA * 0101 (SEQ.ID.NO.51) α1 site Has an array to code. This plasmid is a soluble plasmid represented by β1-α1. Created to secrete fusion MHC heterodimers and associated with multiple sclerosis (Kamholz et al., Proc. Natl. Acad. Sci. USA 83: 4962-66). , 1986) Myelin basic protein is attached to the N-terminus of β1. in this way As a result, a soluble fusion MHC heterodimer: peptide complex is formed.   PCR was used to construct pLJ13 (SEQ. ID. NO. 49), and the signal sequence and D A DNA sequence encoding MBP was introduced between the β1β2 sequences of the β chain of R1β * 1501. Thereafter, the β1 site and the α1 site including MBP are inserted through the linker sequence introduced by PCR. Connected.   As a first step, the cDNA encoding the full length of the α chain, DRA * 0101, The cDNA to be loaded was inserted into the expression vector pZCEP. DNA encoding these molecules Is Maniatis et al. Chopsticks (Molecular Cloning: A Laboratory Manual, Cold Spring Harbo r, NY, 1982) and those with chopsticks from Sambrook et al. (Molecular Cloning: A Laboratory) Manual, Second Edition, Cold Spring Harbor, NY, 1989), and this specification Mullis et al. Chopsticks incorporated by reference in U.S. Patent No. 4,683,195, It can be isolated by standard cloning methods.   pZCEP (Jelineck et al., Science, 259: 1615-16, 1993) was processed with HindIII and EcoRI. 0.85 kb HindIII-EcoRI with cDNA encoding the β1 chain of DR1β * 1501 The fragment was inserted. The resulting plasmid was named pSL1.   pZCEP was treated with Bam HI and XbaI. CD encoding α-chain of DRA * 0101 A 0.7 kb SacI-SSPI fragment containing NA was separated and isolated by agarose gel electrophoresis. And a polylinker having a BamHI-SacI end and an SSP I-XbaI end (SEQ. ID. NO.). -Inserted with the sequence. The resulting plasmid was named pSL2.   The cloning site of the linker sequence was determined by PCR using Class II b DR1b * 1501. A signal sequence, at the 3 'end of which the first 5 of MBP (82-104) Amplify an approximately 100 kb Hind III / Cla I fragment to which a sequence encoding amino acids is linked Was created. Single ApaLI cleavage using DNA sequence encoding amino acids VH The site was introduced.   Another approximately 750 bp ClaI / XbaI fragment was generated using PCR. This cut The fragment encodes the β1β2 site of the Class II β chain DR1β * 1501 and the transmembrane domain. Two amino acids (GS) at the 5 'end of the β1 sequence DNA sequences to be linked. The DNA sequence encoding the amino acid GS was selected This was to create a single BamHI cleavage site.   These fragments were treated with HindIII / ClaI and ClaI / XbaI and then agarose gel. Separated by electrophoresis, inserted into HindIII / XbaI treated pCZEP Was. The shuttle plasmid thus produced was treated with ApaLI and BamHI, and the MBP sequence Oligos encoding the remainder of the amino acid sequence FFKNIVTPRTPPPS The nucleotide and the first part of the flexible linker, GGGSG, were inserted. Like this The MBP sequence linked to the β1β2 sequence of DR1β * 1501 via a linker including. The plasmid thus produced was named pSL21.   Separately, DR1β is added via a flexible linker (GGGSGGS SEQ. ID. NO. 31). * MBP peptide linked to the N-terminus of the 1501β1 domain (DENPVVHFFKNIVTPRTPPPS SE Q. ID. NO. 33) The DR1β * 1501 signal sequence is linked to the N-terminus Was done. Reconstruct the signal sequence, MBP peptide flexible linker, and Six overlapping sites, such as binding to the N-terminus of the β1 domain through the Bam HI cleavage site Ligonucleotides were prepared. Oligos are kinased before ligation It was. 50 pmol of each oligo (ZC7639 (SEQ. ID. NO. 2), ZC7665) (SEQ. ID. NO. 6), ZC7663 (SEQ. ID. NO. 4), ZC7640 (SEQ. ID. NO. 3), Z C7666 (SEQ.ID.NO.7) and ZC7664 (SEQ.ID.NO.5), 22.4 ml TE, 5 ml  A reaction solution containing TMD, 5 ml ATP and 5 ml kinase was prepared. Reaction at 37 ° C for 1 And then incubated at 65 ° C. for 10 minutes. Kinase treated oligos Stored at -20 ° C until needed. Next, 0.5 linearized with Eco RI-Bam HI. mg pSL1, each with a 20 pmol oligonucleotide kinased (ZC7639 (SEQ . ID. NO. 2), ZC7665 (SEQ. ID. NO. 6), ZC7663 (SEQ. ID. NO. 4), ZC764 0 (SEQ.ID.NO.3), ZC7666 (SEQ.ID.NO.7) and ZC7664 (SEQ.ID.NO.5) ), 10 ml ligation containing 1 ml TE, 1 ml TMD, 1 ml ATP and 0.5 ml ligase A reaction solution was prepared. The reaction was performed by incubating at 37 ° C. for 1 hour. Baa This ligation reaction is performed by electroporation according to the instructions of the car. DH10B competent cells (GIBCO BRL, Gaithersbu rg, MD). Then, place on 50mg / ml ampicillin-containing LB plate Spray and incubate overnight. Properly recombined clones undergo restriction digestion Analysis and sequence analysis identified it and was named pSL21.   To construct pLJ13, include from the signal sequence to the end of the b1 site of pSL21. The second flexible linker (amino acid sequence GASAG (SEQ.ID.NO.29)) A DNA fragment encoding the DNA sequence encoding the DNA fragment was prepared.   DR1β * 1501 signal / MBP / linker / β chain (pSL21) with full length linearized mg, 200 pmol ZC7511 (SEQ. ID. NO. 1), 200 pmol ZC8194 (SEQ. ID. NO. 8) , 10 ml 10 × polymerase buffer, 10 ml dNTPs and wax beads (AmpliWax- , Perkin-Elmer Cetus, Norwalk, CT). Was. After the first cycle at 95 ° C for 5 minutes, add 5U Tag Polymerase and heat at 94 ° C for 1 minute. The amplification reaction was performed for 30 cycles at 55 ° C. for 1 minute and at 72 ° C. for 1 minute. this As a result, a 29 amino acid DR1β * 1501β chain signal sequence, a 21 amino acid MBP peptide sequence, 6 amino acid flexible linker (GGGSGGS SEQ. ID. NO. 31), 83 amino acid β 1 domain, including 5 amino acid flexible linker (GASAG SEQ. ID. NO. 29) DR1β * 1501 signal sequence / MBP peptide / linker / β1 / linker cleavage A piece was obtained. Low melting point agarose gel electrophoresis shows expected size, That is, a fragment of a 374 bp band was separated and isolated.   Another one encodes the α1 site of DRA * 0101, and another 5 ' The DNA encoding the flexible linker is a D encoding a stop codon at the 3 'end An approximately 0.261 kb PCR fragment to which each of the NA sequences was bound was generated.   1 mg of DRA * 0101 (pSL2) having a full length linearized, 200 pmol of ZC8196 (SEQ. ID.N. O. 9), 200 pmol of ZC8354 (SEQ. ID. NO. 14), 10 ml of 10 × polymerase buffer Fur, 10 ml dNTPs and one wax bead (AmpliWax-, Perkin-Elmer C etus, Norwalk, CT) was prepared. First 95 ° C for 5 minutes After 5 cycles, 5U Tag polymerase is added, 1 minute at 94 ° C, 2 minutes at 55 ° C, The amplification reaction was performed at 72 ° C. for 3 minutes for 30 cycles. 81 amino acid DRA * 0101 Includes a 5-amino acid flexible linker linked to the N-terminus of the α1 domain Linker / DRA * 0101 a termination codon at the C-terminus of the a1 domain and XbaI restriction enzyme cleavage site The one with the added rank was obtained. Predicted by low melting point agarose gel electrophoresis The fragment of the band of 261 bp was isolated.   DR1β * 1501 signal sequence, MPB peptide and linker The peptide DNA, followed by β1, and the DNA encoding the flexible peptide Final HindIII / XbaI PCR product encoding α1 linked to β1 at the 5 ′ end These two PCR fragments were used to generate (GASAG SEQ. ID. NO. 29) 1 ml signal sequence / MBP / linker / β1 / linker fragment, 1 ml linker / a1 fragment, 200 pmol ZC7511 (SEQ. ID. NO. 1), 200 pmol ZC8196 (SEQ. ID. NO. 9), 10ml of 10x polymerase buffer, 10ml dNTPs and 5U Tag polymerase A 100 ml PCR reaction solution containing was prepared. The reaction is performed at 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 1 minute. Performed in 35 cycles. Signal sequence / MBP / linker / β1 / linker fragment The 5 amino acid 3 'linker is over the same 5 amino acid linker in the linker / α1 fragment -Inflation of β1 domain and α1 domain via 5 amino acid linker Connected with the The 730 bp MBP-β1α1 PCR product thus obtained is 5 ′ HindI Following the II cleavage site, DR1β * 1501 β chain signal sequence, 21 amino acid MBP peptide DEN PVVHFFKNIVTPRTPPPS (SEQ.ID.NO.33), 8 amino acid flexible linker (GGGSGGSG). This linker is linked to the N-terminus of the DR1β * 1501 β1 domain Further, this domain is connected via a 5-amino acid linker (GASAG SEQ. ID. NO. 29). ) DRA * 0101, linked to the N-terminus of the α1 domain. And finally, XbaI restriction enzyme The elementary cleavage site comes. The MBP β1α1 fragment was introduced into HindIII / XbaIpZCEP. set Recombinant clones were identified by restriction digest analysis and sequence analysis, and pLJ1 3 (human MBP-β1α1).                                 Example 2                    NOD Mouse Synthesis of α and β MHC cDNA Total RNA was isolated from NOD MOUSE NAME spleen cells according to the method of Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982  And Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wi Guinezini, ley and Sons, Inc., NY, 1987, incorporated herein by reference. Homogenize in thiothioacetate and use CsCl centrifugation. Poly (A) + RNA Is oligo d (T) cellulose chromatography (Mini-Oligo (dT) Cellulose S It was separated using a Pin Column Kit (5 Prime-3 Prime), Boulder, CO).   Superscript-Rnase H according to manufacturer's instructions^- Reverse Transcriptase Kit (GIBCO BRL), the first single-stranded cDNA was synthesized. Contains 1mg total NOD RNA One microliter of the solution was treated with 1 ml of oligo dT solution in diethyl pyrocarbonate. The mixture was mixed with 13 ml of water and heated at 70 ° C. for 10 minutes, and then cooled on ice.   First single-stranded cDNA was synthesized using 4 ml of Superscript-buffer, 0.1 M dithiotrate Deoxynucle containing 10 mM each of dATP, dGTP, dTTP, and dCTP 2 ml of reotide triphosphatase solution and 200 U / ml Superscript-revers The procedure was started by adding 2 ml of etranscriptase to the RNA primer mixture. Reaction time is 10 minutes After incubation at room temperature, incubate at 42 ° C for 50 minutes and at 70 ° C for 15 minutes, Then, it was cooled on ice. The reaction was incubated at 37 ° C for 20 minutes and then ice-cold RN. Termination was performed by adding ase H.   Next, two 100 ml PCR reaction mixtures were prepared. One reaction is primer ZC81 98 (SEQ ID NO: 10, antisense α chain primer, XbaI cleavage site) and ZC8199 ( Class II MHC using SEQ ID NO: 11, sense α chain primer, EcoRI cleavage site) NOD (IAg⁷), And the other is primer ZC8206 (SEQ. ID. NO. 12). Antisense β chain primer, XbaI cleavage site) and ZC8207 (SEQ. Antisense β chain primer, EcoRI cleavage site) Amplification was performed. In both cases, cloning into expression vectors is possible. A single restriction enzyme cleavage site, ie, an EcoRI 3 'end at the 5' end of the fragment. An XbaI cleavage site was introduced at the end. Each reaction mixture has a first strand And 10 x synthesis buffer, 8 ml of sense primer, 100 pmol of sense primer, Antisense primer 100pmol, d.H_TwoO65ml, 1 wax bead (AmpliWax- , Perkin-Elmer Cetus, Norwalk, CT). First 95 ° C for 5 minutes Following the cycle, 1U Tag polymerase is added, 1 minute at 94 ° C, 2 minutes at 55 ° C, 72 ° C The cycle of 3 minutes was repeated 30 times to perform the amplification reaction. The α chain thus created The fragment and the β-chain fragment were digested with EcoRI-XbaI, treated with RNase, PZCEP (Jelis) separated by Rose gel electrophoresis and linearized with EcoRI-XbaI neck et al., Science, 259: 1615-16, 1993). Full-length β-chain pZCEP is pL The full-length α-chain pZCEP, designated J12, was designated pLJ11.                                 Example 3          Contains antigenic peptides linked by flexible linkers                      Construction of mouse soluble single-chain MHC molecule IPeptide-β1α1   Has a b1 domain linked to the a1 domain via another flexible linker An antigen peptide linked to the N-terminus of a single-chain MHC molecule via a flexible linker Four steps were taken to construct the containing molecule.   A. GAD-β1α1 IA^g7   1) IA^g7NOD Is the β1 domain (SEQ ID NO.43) of the mouse β chain a β2 domain? The 5 ′ and 3 ′ ends were fused to the linker fragment using PCR.   Full length IAg linearized with EcoRI / XbaI⁷  b chain 100ng, 200pmol ZC9478 (S EQ. ID. NO. 16), 200 pmol of ZC9480 (SEQ. ID. NO. 18), 10 ml of 10 × Tag poly 100 ml of merase buffer, 10 ml of dNTPs and 5U Tag polymerase A PCR reaction solution was prepared. Reaction cycle at 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute Was cycled 35 times. 8 amino acids linked to the 91 amino acid b1 domain and the 5 'end Amino acid flexible linker site (GGSGGGGS SEQ, ID No. 34), and 3 'end Including a 5 amino acid flexible linker (GGSGG SEQ. ID. NO. 30) linked to A 1 / linker fragment was obtained. Low melting point agarose gel electrophoresis , A 330 bp band was isolated.   2) GAD 65 peptide (SRLSKVAPVIKARMMEYGTT (SEQ. ID. NO. 59) and another peptide A lexible linker was ligated to the b1 / linker fragment obtained in 1 using PCR. Ku In addition, a single BamHI cleavage site to facilitate cloning and expression, and And a second ribosome binding site before the stop codon (RBS SEQ. ID. NO. 48) The last 16 nucleotides of the phi10 coupler are linked to the 5 'end of the GAD peptide. Was.   1 ml of the eluted β1 / linker fragment obtained above, 200 pmol of ZC9473 (SEQ. ID. NO. 1) 5), 200 pmol of ZC9479 (SEQ. ID. NO. 17), 200 pmol of ZC9480 (SEQ. ID. NO. 18) , 10 ml of 10X polymerase buffer, 10 ml of dNTPs, and 5U Tag polymer A 100 ml PCR reaction solution was prepared using the enzyme. The reaction is performed at 94 ° C for 1 minute and at 50 ° C for 1 minute. And 35 cycles of 1 minute at 72 ° C. All fragments are 5 'and / or 3' parts Was annealed to the complementary strand as it was made to have overlapping parts Could work as a primer for the reaction. 3 'of ZC9499 (SEQ.ID.NO.23) The terminal 15 nucleotides are β1 / linker fragment (ggaggctcaggagga) (SEQ. ID. NO. 17) overlaps the first 15 nucleotides of To the β1 domain through the Xibull Linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36) Connected seamlessly. ZC9479 (SEQ.ID.NO.17) acts as a 5 'primer The BamHI cleavage site following RBS (SEQ. ID. NO. 48) was added to the 5 'end of the GAD peptide sequence. It was connected to the end. The 3 'end of ZC9479 (SEQ. ID. NO. 17) and ZC9473 (SEQ. ID. 5 nucleotides overlap between 5) and site in-frame with the peptide Was introduced. The resulting 450 bp GAD / β1 fragment was electrophoresed on a low melting point agarose gel. Separated by motion.   3) IAg⁷Α1 domain (SEQ.ID.NO.44) was separated from the α2 domain and Using a linker fragment at the 5 'end and Sele at the 3' end and the serine residue before EcoRI. Fused to the group.   I-A whose entire length has been linearized with EcoRI / XbaI^g7100 ng of α1 chain and 200 pmol of ZC9481 ( SEQ. ID. NO. 19), 200pmol of ZC9493 (SEQ.ID.NO.20), 10ml of 10x polymer 100ml PCR reaction containing 10ml dNTPs and 5U Tag polymerase A liquid was prepared. The reaction was performed at 94 ° C for 1 minute, 53 ° C for 1 minute, and 72 ° C for 1 minute for 35 cycles. 87 amino acid a1 domain at 5 'end and 5 amino acid flexible linker (GGSGG) (SEQ.IN.NO.30), a serine residue at the 3 'end and SpeI and EcoRI cleavage sites An a1 / linker fragment fused to the position was obtained. For low melting point agarose gel electrophoresis Thus, a band of the expected size, ie, 300 bp, was separated.   4) 2 ml of GAD / β1 fragment obtained in 2) to complete the target 3) 2 ml of α1 / linker fragment, 200 pmol ZC9479 (SEQ. ID. NO. 17), 200 pmol ZC9493 (SEQ.ID.NO.20), 10ml 10x polymerase buffer, 10ml dNTP A PCR reaction containing s and 5U polymerase was prepared. Reaction is 94 ° C for 1 minute, 53 ° C 35 cycles were performed at 72 ° C. for 1 minute for 1 minute. 5 amino acid 3 'linker of GAD / β1 fragment -(GGSGG SEQ. ID. NO. 30) is a 5-amino acid linker of α1 / linker fragment Burlap and link β1 domain and α1 domain via 5 amino acid linker did. The GAD-β1α1 PCR product thus generated has 5 ′ BamHI and RBS (SEQ. ID. NO. 48), a 20 amino acid GAD65 peptide (SRLSKVAPVIKARMMEYGT T (SEQ. ID. NO.), 15 amino acid flexible linker (GGGGSGGGGSGGGGS (SEQ. I. D. NO. 36), which is linked to the Iag by a 5 amino acid linker (GGSGG SEQ. IS. NO. 36).⁷ Is linked to the N-terminus of the β1 domain of -(GGSGG SEQ. IS. NO. 30) by Iag⁷At the N-terminus of the α1 domain. So Finally, the restriction sites for SpeI and EcoRI are included. The GAD-β1α1 fragment Restriction enzyme treatment with BamHI and EcoRI was performed by low melting point agarose gel electrophoresis. Isolated. The restriction digested fragment was then linearized with BamHI-EcoRI. It was subcloned into the current vector p27313 (WO95 / 11702). Correctly recombined Clones were identified by restriction digest analysis and sequence analysis, and pLJ18 ( GAD-β1α1 Iag⁷SEQ.ID. NO.42).   B) MBP-β1α1 IA⁸   IA⁸Β1 domain (SEQ. ID. NO. 46) is separated from the β2 domain and used for PCR. However, both the 5 'end and the 3' end were fused to a linker fragment.   1) IA whose entire length is linearized with EcoRI / XbaI⁸  β chain (p40553) 100ng, 200pmol ZC9478 (SEQ. ID. NO. 16), 200 pmol ZC9497 (SEQ. ID. NO. 22), 10 ml 10 × polymer 100 ml of PCR with enzyme buffer, 10 ml dNTPs and 5U Tag polymerase A reaction solution was prepared. The reaction is performed at 35 cycles of 94 ° C for 1 minute, 53 ° C for 1 minute and 72 ° C for 1 minute. Was. An 8 amino acid flexible linker (GGSGGGGS SEQ. ID. NO. 34), a 5 amino acid flexible linker (GGSGG SEQ. ID. NO. 30) at the 3 'end IA containing a bound 91 amino acid β1 domain⁸  The β1 / linker fragment was obtained. The expected 330 bp band was separated and isolated by low melting gel electrophoresis.   2) Myelin basic protein (MBP) peptide (FEKNIVTPR TPPP SEQ. ID. NO. 37) and the remainder of the 5 'linker were analyzed by PCR using IAs β1 / linker frag Added to ment. In addition, a unique Bam HI cleavage site and a termination codon Bosome binding site (RBS SEQ. ID. NO. 48) is also rapidly cloned and expressed Added to the 5 'end of the MBP peptide in order to   100 ml of PCR reaction was performed with 1 ml of β1 / linker fragment eluted in 1), 200 pmol ZC949 9 (SEQ. ID. NO. 23), 200 pmol ZC9479 (SEQ. ID. NO. 17), 200 pmol ZC9497 ( SEQ. ID. NO.22), 10ml 10X polymerase buffer, 10ml dNTPs, 5 U Taq polymer Prepared using ase. The reaction is repeated at 94 ° C for 1 minute, 50 ° C for 1 minute and 72 ° C for 1 minute 35 times. I did it. These fragments overlap both on the 5 'and 3' side, or on one side It is designed so that complementary strands of each other can anneal, and plays the role of primer in the reaction. Have a split. The last 15 nucleotides (ggaggctcaggag) of ZC9499 (SEQ ID NO: 23) ga SEQ. ID. NO. 35) is a gap between the first 15 nucleotides of IAs β1 / linker fragment No overlap, flexible linker of 15 amino acids (GGGGSGGGGSGGGGS SEQ. ID NO. 36 ) Binds the MBP peptide to the IAs β1 domain. ZC9479 (SEQ.ID.NO.17) ) Serves as the 5 'primer and acts as the first 32 nucleotides of ZC9499 (SEQ. ID. NO. 23). Otides completely overlapped and terminated with the Bam HI cleavage site and also RBS (SEQ. ID. NO. 48). A stop codon has been introduced in frame with the MBP peptide. 400 bp MB obtained P / IAs β1 fragment was separated and isolated by low-dissolution gel electrophoresis.   3) The α1 domain of IAs (SEQ. ID. NO. 47) is isolated from the α2 domain, By PCR, the linker fragment was placed at the 5 'end and SpeI and Eco RI cleavage sites at the 3' end, Further serine residues were fused.   A 100 ml PCR reaction was performed with 100 ng of full-length linear I-As α chain (p28520), 200 pmol ZC9481 (SEQ. ID. NO. 19), 200 pmol ZC9496 (SEQ. ID. NO. 21), 10 ml 10X polymeras It was prepared using e buffer, 10 ml dNTPs, and 5 U Taq polymerase. The reaction is at 94 ° C One minute, 53 ° C. for 1 minute, and 72 ° C. for 1 minute were repeated 35 times. 87-residue IAs α1 domain A 5 amino acid flexible linker (GGSGG SEQ. ID NO. 30), 3 ' IAs α1 / linker fragment fused with a serine residue, SpeI, and EcoRI cleavage site at the end  Was acquired. Expected 300 bp band isolated by low-dissolution gel electrophoresis Was done.   4) To complete the target, the final 100 ml of the PCR reaction was performed using 2) 2 ml of MBP 2ml IAs α1 / linker fragment of / IAs β1 fragment, 3), 200pmol ZC9479 (S EQ. ID. NO. 17), 200pmol ZC9496 (SEQ.ID.NO.21), 10ml 10X polymerase b buffer, 10 ml dNTPs, 5 U Taq polymerase. The reaction is performed at 94 ° C Min, 53 ° C for 1 minute and 72 ° C for 1 minute were repeated 35 times. 5 amino acids of MBP / IAs β1 fragment The 3'linker (GGSGG SEQ. ID. No. 30) of the noic acid is 5 members of the IAs α1 / linker fragment. IAs β1 overlaps with the same linker part of amino acid, and through this 5-amino acid linker The domain and the IAs α1 domain were linked in frame. Obtained679 The base-paired MBP-β1α1 IAs PCR product has a BamHI cleavage site at the 5 ′ end, followed by RBS (SEQ . ID. NO. 48), a 13 amino acid MBP peptide (FEKNIVTPRTPPP SEQ. ID. NO. 37), A 15 amino acid flexible linker (GGGGSGGGGS) linked to the N-terminus of the IAs β1 domain GGGGS SEQ. ID NO. 36), and the IAs β1 domain is a 5-amino acid linker (GGSG G SEQ. ID NO. 30) and binds to the N-terminus of IAs α1 domain via Spe I and Ec o The RI cleavage site is included last. This MBP-β1α1 IAs fragment contains Bam HI and E After restriction enzyme treatment with coRI, it was separated and isolated by low-dissolution gel electrophoresis. next This fragment, which had been digested with restriction enzymes, was digested with Bam HI and Eco RI to obtain a linear expression vector. And subcloned into the vector p27313 (WO95 / 11702). Obtained recombinant clone Was confirmed by restriction enzyme treatment and sequence analysis, and pLJ19 (MBP β1α1 IAs EQ . ID. NO. 45). II.Peptide-β1α1α2β2   The β1 domain is linked via a flexible linker at the N-terminus of the α1α2 domain. The flexible linker of the eye also binds to the N-terminus of the β2 domain. Antigen bound to the N-terminus of a single-chain MHC molecule composed of a flexible linker via a flexible linker A four-step construction approach was taken to create molecules containing tide. A. GAD-β1α1α2β2 IA^g7   1) I-A^g7Α1α2 domain is a 5 amino acid linker at the 5 ′ end using PCR and 3 ′ end A 15 amino acid linker was fused at the end.   A 100 ml PCR reaction requires 100 ng of full-length linear I-A^g7 α chain (pLJ11), 200pmol ZC948 1 (SEQ. ID. NO. 19), 200 pmol ZC9722 (SEQ. ID. NO. 27), 5 ml 10X polymeras It was prepared using e buffer, 5 ml dNTPs, and 2.5 U Taq polymerase. The reaction is 94 C. for 1 minute, 54.degree. C. for 1 minute and 72.degree. C. for 2 minutes were repeated 35 times. I-A^g7 α1α2 domain 5 ′ flexible linker (GGSGG SEQ. ID NO. 30) at the 5 ′ end, 3 ′ end Flexible amino acid linker (GGGGSGGGGSGGGGS SEQ. ID NO. 36) of 15 amino acids fused to the end I-A^g7 linker / α1α2 / linker fragment was obtained. Expected size Band was isolated by low dissolution gel electrophoresis.   2) I-A^g7Β2 domain was isolated from the β1 domain and A 15 amino acid linker is fused at the 5 'end of the An Eco RI restriction enzyme site was introduced.   A 100 ml PCR reaction requires 100 ng of full-length linear I-A^g7 β chain (pLJ12), 200pmol ZC972 1 (SEQ. ID. NO. 26), 200 pmol ZC9521 (SEQ. ID. NO. 24), 5 ml 10X polymeras It was prepared using e buffer, 5 ml dNTPs, and 2.5 U Taq polymerase. The reaction is 94 C. for 1 minute, 54.degree. C. for 1 minute and 72.degree. C. for 2 minutes were repeated 35 times. β2 domain (SEQ. ID. NO .58) at the 5 'end of a 15 amino acid flexible linker (GGGGSGGGGSGGGGS SEQ. ID NO. . 36) I-A with a stop codon and Eco RI restriction enzyme site introduced at the 3 'end^g7 link The er / β2 fragment was obtained. The expected size band is a low melting gel Separated and isolated by movement.   3) I-A^g7The α1α2 domain (SEQ. ID. NO. 57) of^g7Β2 de It was fused to the main. The 15 amino acid linker sequence at the 3 'end of the α1α2 fragment is It completely overlaps the same 15 amino acid sequence at the 5 'end of the β2 fragment, The domains were joined in-frame via r.   100 ml of the PCR reaction is performed in the 2) I-A^g7 linker / α1α2 / linker fragment 5ml, 3) I-A^g7 linker / β2 fragment 5ml, 200pmol ZC9481 (SEQ.ID.NO.19), 200p mol ZC9721 (SEQ. ID. NO. 26), 10 ml 10X polymerase buffer, 10 ml dNTPs, 5 U  It was prepared using Taq polymerase. The reaction is performed at 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 2 minutes. Repeated 30 times.   The I-Ag7 α1α2 domain has a 5 amino acid flexible linker (GGSGG SEQ . ID NO. 30) 15 amino acid flexible linker (GGGGSGGGGSGGGGS SE at the 3 'end Q. ID NO. 36) fused to I-A bound to the 5 'end of the β2 domain^g7 linke r / α1α2 / linker / β2 fragment was obtained. Expected size band is low Isolated by melting gel electrophoresis.   4) In order to complete the target, the last 100 ml of the PCR was performed using the 5m l 5ml I-A of GAD-β1α1 fragment, 3)^g7 linker / α1α2 / linker / β2 fragmen t, 200 pmol ZC9521 (SEQ. ID. NO. 24), 200 pmol ZC9479 (SEQ. ID. NO. 17), Prepare using 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5 U Taq polymerase. Was. The reaction was repeated at 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 2 minutes 30 times. GAD- β1α1 and linker / α1α2 / linker / β2 completely overlap the linker / α1 part IA via a 5-amino acid flexible linker (GGSGG SEQ. ID NO. 30)^g7 β1 and I- A^g7 α1α2 / linker / β2 bound in frame. The resulting GAD-β1 α1α2β2 I-A^g7 The PCR product had a BamHI cleavage site at the 5 'end followed by RBS (SEQ. ID. NO. . 48), a GAD peptide of 20 amino acids (SRLSKVAPVIKARMMEYGTT SEQ. ID. NO. 59), I -A^g7A 15 amino acid flexible linker (GGGGSGGGGSG) linked to the N-terminus of the β1 domain GGGS SEQ. ID NO. 36) and I-A^g7β1 domain is a 5-amino acid flexible link er (GGSGG SEQ. ID NO. 30), binds to the N-terminus of the α1α2 domain, The main contains the Eco RI cleavage site last. This GAD-β1α1α2β2 fragment Is digested with Bam HI and Eco RI, separated by low-resolution gel electrophoresis, Released. This fragment, which had been digested with restriction enzymes, was then digested with Bam HI and Eco RI. It was subcloned into the linear expression vector p27313 (W095 / 11702). Obtained Recombinant clones were confirmed by restriction enzyme treatment and sequence analysis, and pLJ23 (GA D-β1α1α2β2 I-A^g7 SEQ. ID. NO. 56). B. MBP-β1α1α2β2 IAs   1) The α1α2 domain of I-As is a 5 amino acid linker at the 5 ′ end using PCR, and the 3 ′ end Was fused with a 15 amino acid linker.   A 100 ml PCR reaction was performed with 100 ng of full-length linear I-Asα chain (p28520), 200 pmol ZC9481 (SEQ. ID. NO. 19), 200 pmol ZC9722 (SEQ. ID. NO. 27), 10 ml 10X polymeras e buffer, 10 ml dNTPs, and 5 U Taq polymerase. The reaction is at 94 ° C One minute, 54 ° C. for 1 minute and 72 ° C. for 2 minutes were repeated 35 times. 196 amino acid I-As α1 A 5 amino acid flexible linker (GGSGG SEQ. ID NO. 30) is located at the 5 'end of the α2 domain. ), A flexible linker (GGGGSGGGGSGGGGS SEQ. ID NO. An I-As linker / α1α2 / linker fragment fused with 6) was obtained. Expected The 650 bp band was separated and isolated by low dissolution gel electrophoresis.   2) The β2 domain of I-As is isolated from the β1 domain, and the β2 domain is determined using PCR. Is fused with a 15 amino acid linker at the 5 'end and a stop codon at the 3' end followed by Ec o RI restriction enzyme sites have been introduced.   100 ml of the PCR reaction solution is 100 ng of full-length linear I-As β chain (p40553), 200 pmol ZC9 721 (SEQ. ID. NO. 26), 200 pmol ZC9521 (SEQ. ID. NO. 24), 10 ml 10X polymer ra It was prepared using se buffer, 10 ml dNTPs, and 5 U Taq polymerase. The reaction is 94 C. for 1 minute, 54.degree. C. for 1 minute and 72.degree. C. for 2 minutes were repeated 35 times. 105 amino acid β2 domain 55 amino acid flexlble linker (GGGGSGGGG) at the 5 'end of SGGGGS SEQ. ID NO. 36) A stop codon and an Eco RI restriction enzyme site are introduced at the 3 'end. The inserted I-As linker / β2 fragment was obtained. Band of expected size Was isolated by low melting gel electrophoresis. Expected low band of 374 bp Isolated by gel electrophoresis.   3) The α1α2 domain of I-As was fused to the β2 domain of I-As using PCR. The 15 amino acid linker sequence at the 3 'end of the α1α2 fragment is the 5' end of the β2 fragment. It completely overlaps with the same 15 amino acid sequence at the end, Cats were combined in-frame.   100 ml of the PCR reaction was performed using the 2) I-As linker / α1α2 / linker fragment 5 ml, 3) I-As linker / β2 fragment 5 ml, 200 pmol ZC9481 (SEQ. ID. NO. 19), 200 pmol  ZC9721 (SEQ. ID. NO. 26), 10 ml 10X polymerase buffer, 10 ml dNTPs, 5 U Ta Prepared using q polymerase. The reaction was performed at 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 2 minutes. Repeated times.   The 196 amino acid I-As α1α2 domain is a 5 amino acid flexible linke at the 5 'end. r (GGGSGG SEQ. ID NO. 30) 15 amino acid flexible linker (GGGGSG) at the 3 'end GGGSGGGGS SEQ. ID NO. 36) is fused to the 5 'of the 106 amino acid β2 domain. An I-As linker / α1α2 / linker / β2 fragment bound to the end was obtained. Expectation The 977 bp band isolated was isolated by low melting gel electrophoresis.   4) In order to complete the target, the final 100 ml PCR reaction was performed using the 2m l 2 ml of MBP-β1α1 fragment, 3) I-As linker / α1α2 / linker / β2 fragmen t, 200 pmol ZC9521 (SEQ. ID. NO. 24), 200 pmol ZC9479 (SEQ. ID. NO. 17), 1 Prepare using 0 ml 10X polymerase buffer, 10 ml dNTPs, and 5 U Taq polymerase. Was. The reaction was repeated 30 times at 94 ° C. for 1 minute, 54 ° C. for 1 minute, and 72 ° C. for 2 minutes. MBP- β1α1 and linker / α1α2 / linker / β2 completely overlap the linker / α1 part I-As β1 and I via a 5-amino acid flexible linker (GGSGG SEQ. ID NO. 30) -As α1α2 / linker / β2 domains bound in frame. The resulting 1 The 360 base pair MBP-β1α1α2β2 I-As PCR product has a BamHI cleavage site at the 5 ′ end followed by a No. 48, a 13 amino acid MBP peptide (FFKNIVTPRTPPP SEQ. ID . NO. 37), a 15 amino acid flexible linker attached to the N-terminus of the I-Asβ1 domain (GGGGSGGGGSGGGGS SEQ. ID NO. 36), and further I-A^g7The β1 domain is 5 amino acids With the flexible linker (GGSGG SEQ. ID NO. 30) through the N-terminal of the α1α2 domain Binding, the β2 domain has an Eco RI cleavage site at the end. This MBP-β1α1α2β2 The fragments are digested with Bam HI and Eco RI, and subjected to low melting gel electrophoresis. Separated and isolated. Next, this fragment treated with restriction enzymes was used for Bam HI and Eco. RI-treated and subcloned into a linear expression vector p27313 (WO95 / 11702) Was. The obtained recombinant clone was confirmed by restriction enzyme treatment and sequence analysis. , PLJ20 (MBP-β1α1α2β2 I-As SEQ. ID. NO. 54). III.MBP-α1α2   Through a flexible linker at the N-terminus of a single-chain MHC molecule composed of α1α2 domain A two-step construction approach was taken to create the linked antigenic peptide molecules.   1) The α1α2 domain of I-As (SEQ.ID.NO.53) has 25 amino acids at the 5 ′ end using PCR. A stop codon and Spe I and Eco RI cleavage sites were introduced at the 3 'end of the amino acid linker. Was.   A 100 ml PCR reaction is performed with 100 ng of full-length linear Eco RI-Xba I I-As α chain (p28520), 200 pmol ZC9720 (SEQ. ID. NO. 25), 200 pmol ZC9723 (SEQ. ID. NO. 28), 10 ml  Prepared using 10X polymerase buffer, 10ml dNTPs, 5U Taq polymerase . The reaction was repeated at 94 ° C. for 1 minute, 54 ° C. for 1 minute and 72 ° C. for 2 minutes 35 times. 196 nets 25 amino acid flexible linker (GGGGSGGG) at the 5 'end of the I-As α1α2 domain GSGGGGSGGGGSGGGGS SEQ. ID NO. 32) is fused with a stop codon and Spe I at the 3 'end. And I-As linker / α1α2 fragment into which Eco RI cleavage site was introduced . The expected 672 bp band was separated and isolated by low-dissolution gel electrophoresis. .   2) 100 ml of PCR reaction is performed using 5 ml of linker / α1α2 I-As fragment of 1), 200 pmol ZC 9723 (SEQ. ID. NO. 28), 400 pmol ZC9499 (SEQ. ID. NO. 23), 200 pmol ZC9479 (SEQ.ID.NO.17), 10 ml 10X polymerase buffer, 10 ml dNTPs, 5 U Taq pol Prepared using ymerase. The reaction was repeated 30 times at 94 ° C for 1 minute, 54 ° C for 1 minute, and 72 ° C for 2 minutes. I returned. The 196 amino acid I-As α1α2 domain has a 25 amino acid flex at the 5 'end. ible linker (GGGGSGGGGSGGGGSGGGGSGGGGS SEQ. ID NO. 32) fused to the 3 'end I-As MBP / linker / α with a termination codon and Spe I and Eco RI cleavage sites at the ends 1α2 fragment was obtained.   The 5 'end of the 25 amino acid linker of the linker / α1α2 fragment and ZC9499 (SEQ. ID . NO.23), there was a 12 amino acid overlap between the 3 'ends. ZC9499 (SEQ. ID. NO.23) has a BamHI cleavage site at the 5 'end of a 25 amino acid flexible linker, RBS (SEQ. . ID. NO. 48) and the MBP peptide (FEKNIVTPRTPPP SEQ. ID. NO. 37) Was. ZC9479 (SEQ ID NO.17) is the first 32 nucleotides of ZC9499 (SEQ ID NO.23). Overlaps with otide and serves as a 5 'primer. The resulting 743 base pair MBP- The α1α2 IAs PCR product has a BamHI cleavage site at the 5 ′ end, followed by RBS (SEQ. ID. NO. 48). ), 13 amino acid MBP peptide (FEKNIVTPRTPPP SEQ. ID No. 37), IAs α1α2 25 amino acid flexible linker (GGGGSGGGGSGGGGSGGG) attached to the N-terminus of the domain GSGGGGS SEQ. ID NO. 32) Furthermore, the IAs α1α2 domain is composed of Spe I and Eco RI The cleavage site is included last. This MBP-α1α2 IAs fragment contains Bam HI and Eco R It was treated with restriction enzyme I and separated and isolated by low-dissolution gel electrophoresis. Next, This fragment, which had been digested with restriction enzymes, was treated with Bam HI and Eco RI and had a linear expression vector. And subcloned into the vector p27313 (WO95 / 11702). Obtained recombinant clone Was confirmed by restriction enzyme treatment and sequence analysis, and pLJ21 (MBP-α1α2 IAs SEQ . ID. NO. 52).                                 Example 4         E.coli Of a soluble fusion MHC heterodimer: peptide complex in Escherichia coli                      Transfection and induction Transfection   E.coli K-12 W3110 strain was obtained from ATCC and was obtained from Novagen (Magison, WI) DE3 lysogenizat. Lysogen of phage lambda-DE3 (T7 RNA polym with a copy of the erase gene). Plasmid pLJ18 (GAD β1α1 IAg7), pLJ23 (GAD-β1α1α2β2 I-A^g7), PLJ19 (MBP β1α1 IAs), pLJ20 (MBP-β1 α1α2β2 I-As) for Ca ++ transformation (Maniatis et al. Molecular Coloning; A L aboratory Manual, Cold Spring Harbor, NY, 1982) to isolate host strain W3110 / DE3. Transformation.Guidance   All four plasmid transfectants were derived as follows. Model pLJ18 Here is a simple example. A single colony having pLJ18 (GAD β1α1 IAg7) was subjected to 50 mg / ml calben Inoculated into 5-6 ml of LB containing icillin (Sigma), and the culture solution was cultivated for about 3 hours with an OD600 of 0.45 to Shaking culture was performed at 37 ° C. until the temperature reached 0.60. Glycerol-preserved strains are And 1 ml of the culture was centrifuged at 5000 xg for 5 minutes at 4 ° C. Start guidance Isporopyl-beta-D-thio-galactopyranoside (IPTG) to a final concentration of 1 mM To 37 ° C. with shaking culture. 0, 1, 2, 3 hours from each culture Thereafter, and after one night, a certain amount of the culture solution was taken out, and OD600 was measured. These are 4 The cells were collected by centrifugation at 5000 xg for 5 minutes. Pellet to 0.02 OD600 / ml Resuspend in E (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and store at -20 ° C until needed. Was.   50 μl of each of these samples is denatured (reduced) in sample buffer. After mixing with 4-20% Tris-glycine SDS polyacrylamide gel, electrophoresis, C Staining with oomassie Blue was performed. There was a band at about 33 kDa.   For Western blot analysis, 1/60 diluted sample is used for denaturing (reducing) sample After dissolving in buffer, mix with 4-20% Tris-glycine SDS polyacrylamide gel, Electrophoresis was performed. Protein is nitrocellulose by electroblotting Transferred to membrane. Mouse anti-I-A^g7 MHC antiserum, rabbit anti-mouse antibody / horseradi Peroxidase conjugate (BioSource International, Camarillo, CA) In addition, react the ECLTM detection reagent (Amarsham Corp.) with the blot. Thus, the proteins could be visualized. The blot was then autoradiographed. The film was exposed. There was a band at about 33 kDa.                                 Example 5               Purification from inclusion bodies and refolding of GAD-β1α1   21 cultures of GAD-β1α12 were cultured with shaking at 37 ° C. until the OD600 reached 0.77. Scale up from initial culture volume to large-scale protein production it can. Induction was initiated by adding IPTG to a final concentration of 1 mM. Invitation OD600 reached 0.97 3 hours and 15 minutes after the introduction. The pellet is 20 ml of TE (10 mM Tris-HC l, 1 mM EDTA, pH 8.0) and stored at -20 ° C until needed.   The pellet is resuspended in 1/10 the volume of TE in the initial medium and lysozyme at a concentration of 100 mg / ml. Then, add Triton X-100 at a concentration of 0.1%, incubate at 30 ° C for 20 minutes, and cool on ice. Was. Adjust the output to 5 (Branson 450) and mix gently for 20 seconds. The sonication was performed three times.   The lysate of the pellet was centrifuged at 12000 × g and 4 ° C. for 10 minutes using an SS34 rotor. The pellet was prepared by adding 1% NP-40 to TEN (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 100 mM NaCl). The obtained solution was washed by adding 1/10 volume of the initial medium, and 12000 × g using an SS34 rotor, Centrifuged at 4 ° C for 10 minutes. In addition, the pellet contains 1/10 the volume of detergent TEN-free TEN. This was centrifuged as before and the supernatant was discarded. pellet Is about 200 mg / ml in extraction buffer (8 M urea, 25 mM borate pH 8.5, 10 mM DDT). The cells were resuspended at a concentration and incubated at 37 ° C. for about 2 hours. In addition, 38 ml of urine in the supernatant Add Boron / Borate / DDT Buffer and add 3.5L 4M Urea, 50mM Borate pH 8.1 solution was reoxidized at 4 ° C for 48-72 hours or analyzed by HPLC. Dialysis was performed until observed. Then, dialyzed at 4 ° C against 3.5 L of 50 mM borate pH8.1 solution did. The sample was a Vydac C-18 column (Hewlett Packard, Wilmington , DE) or preparative reversed-phase chromatography using Poros-R2 (PerSeptive Biosystems) It was subjected to graphy. Columns are (A) 98% water / 0.1% TFA and (B) 100% CH_ThreeCN /0.09% TFA, eluted at a flow rate of 1 ml / min for more than 28 minutes .   Four desalted and purified samples of GAD-β1-α1 contain intact recombinant protein Each to a quadrupole electrospray mass spectrometer to determine the molecular weight of Was. The average molecular weight obtained from these four measurements was 24434.67 +/- 2.72 Da. Profit The calculated molecular weight is the molecular weight predicted from the cDNA translated sequence, 24432.89 Da. And a very good match. The percent error of measurement is 0.007%, this kind of mass Typical values for analytical methods.   In addition, a desalted and purified sample of GAD-β1-α1 To generate a tide map, it was subjected to proteolysis using tryptone. The obtained digest was analyzed using a MALDI-TOF mass spectrometer. As a result of analysis, Disulfide between Cys50 and Cys112 as predicted from the folded molecule The presence of crosslinks was confirmed. The sequence was predicted as a result of N-terminal sequencing. It was clear that Met was removed.                                 Example 6 GAD Protocol for isolation and expansion of reactive human T cell clones and lines I. Isolation of immune response cells   Pre-diabetic or diabetic patients have sources of autoreactive T cells Perhaps, peripheral blood mononuclear cells (PBMNCs) were obtained from these patients in ficol-hypaq Isolated by density centrifugation in ue. Cells are washed several times and 15% PHS medium (RPMI-1640, serum inactivated with 15% heat (from normal male donor without transfusion) Was positive in mixed lymphocyte culture medium), 2mM L-glutamine, 5 × 10^-FiveM be ta-mercaptoethanol). Some of the PBMNC have pulsed labeled antigens Stored for use as one antigen-presenting cell (APC), some continue to stimulate Frozen for. The rest is cultured in a tissue culture plate to remove adherent cells For 1 hour at 37 ° C. Plate non-adherent cells with medium Remove and transfer to a new plate and remove 5 remaining adherent cells. % CO_TwoIncubated overnight at 37 ° C in the presence.   Non-adherent cell populations were collected and cells were passed through nylon wool to enrich for T cells. This operation removes the remaining monocytes and B cells. Cells that did not adhere were CD56 + and T cells and natural killer cells were enriched by removing CD8 + cells. This procedure consists of a plate coated with anti-CD8 antibody and a plate coated with anti-CD56 antibody. Incubate the cells on a plate to remove non-adherent cells (except for CD56 + and CD8 +). Performed by collecting.II . Preparation of stimulated cell group: Day 0   PBMNC in 0.5 ml of 15% PHS medium in 5% CO_Two1:20 GAD65 (about 50mg / ml) in the presence In addition, it was incubated overnight at 37 ° C. This cell thaws the frozen cells It can also be obtained by washing twice and incubating for 5-7 hours in the presence of GAD65. This Cells were irradiated at 3000 rad, washed twice and counted.III . T cell stimulation   1-2 × 10⁶Enriched CD4 + T cells, T cells enriched in nylon wool, PBL Any of 1-2 x 10⁶Mix with irradiated stimulator cells, 1.5 ml of 15% The cells were pulsed in PHS medium in the absence of antigen and in the state containing the entire GAD. 6 days later For all aggressive conditions, duplicate 100 μl cells per well of a 96-well plate Moved to Add 1 microCurie of 3H-thymidine to each well and allow 5 hours Allow the cells to recover and pulse for all conditions without antigen The proliferative response of the stimulated cells pulsed with the whole GAD compared to the cells was observed. On the seventh day Cells were cryopreserved or harvested. Wash the collected cells twice, and then proceed as described in Chapter II. 1-2 × 10 prepared by the method⁶Stimulated again with single stimulator cells.   10 U / ml recombinant human IL-2 (Research and Development Systems, Minneapolis , MN) were added to the day 8 and day 11 cultures. Divide the cells into 1: 2 or 1: 3 <8 × 10^FiveDiluted with medium to give cells / ml. Cell division is very fast, outside If native IL-2 was required, additional IL-2 was added. On day 14 cells are The T cell line was maintained by restimulation with the method and a frozen stock was prepared. T cell clones and strains Was prepared using the limiting dilution method described above, and the reaction test for peptides and MHC described later was performed. went.IV . T cell cloning   On day 14, T cells are harvested, washed, and resuspended in 15% PHS with 10 U / ml IL-2. 1 × 10^FourThe total volume of each sample was adjusted to 15 ml and terasaki plate (Research and Development Syste ms). On day 7, cloning could begin.   After observing the growth of the cells, 1/2 the volume of the wells of a 96-well round bottom flask with 1 × Ten^FiveThe cells were transferred to wells containing 200 ml of 15% PHS medium containing individual stimulator cells. 24 hours later Then, further add IL-2 to the culture solution so that the final concentration of IL-2 is 10 U / ml in a 15% PHS medium. Was added.   Reactivity of cells with antigen on day 4 or 5 as cells grow in wells Was measured. Cells are fused at a ratio of 1: 2 to 15% PH with 10 U / ml IL-2 for cell fusion. Transferred to other wells containing in S medium.   T cells from one or more of the wells described above, or 1.5 × 10⁶Using stimulator cells, Created by freezing stored cells from 96-well culture, or 24 wells, The cells were transferred to a 1.5 ml medium.V. Testing for reactivity to GAD   T cell clones are rested (without IL-2 for 2 days, at least after stimulation with antigen) The culture was suspended for 7 days), washed, counted, and resuspended in 15% PHS medium. Cells The culture was adjusted to 25,000 cells / well in 100 ml of 15% PHS medium. Same PBMNC of organism or belonging to HLA class II for 5 hours with GAD (about 50mg / ml) Stimulation was given by the above incubation. Wash the cells 3000 rad Was irradiated. Resuspend again in 15% PHS medium and in 100 ml of 15% PHS medium 1 × 10⁶Added to cells / well concentration of T cells. Incubate for 48 hours, 1m The 3H-thymidine of Ci was pulsed and collected. A positive response is a stimulation index> 3 (Stimulation index SI = average cpm of sample stimulated by antigen) (Average cpm of cells stimulated with / without antigen or control). T as control Cells only, stimulator cells only, purified negative antigen, GAD purified from baculovirus, The test was performed under the conditions of PHA and IL-2.   Other methods for testing clones and strains are known in the art. The method is anti Dose response to the antigen, immune response to antigens and antigen-negative controls, anti-HLA Identification of HLA class II molecular restriction by adding And so on. Peptides are measured by titration and remain in the assay Can be performed by the method described above. Dose response combined with peptide specificity test It may be done.   The antigen-presenting cells used to determine HLA-restriction Self-, non-self, non-self PBMNC or BLS-1 or mouse L cells or other APCs Includes those genetically designed to express only HLA class II molecules.VI . Testing for reactivity to synthetic GAD   Using four T cell lines from one HLA-DRB1 + 0404 patient (ThHo), GAD65 (SEQ . ID. NO.59) over the entire length of the 20mer A map of 74 synthetic peptides was created. Antigen presenting cells BLS-DRB1 + 0404 and And / or BLS-DRB1 + 0401 (Kovats et al., J. Exp. Med. 179: 2017-22, 1994) ) Was stimulated by incubating with the peptide for at least 5 hours. T cells Was measured by the method described above. One of the peptides, hGAD33 (PGGAISNMYAMMIARFK MFP SEQ. ID. NO.40) is presented on BLS-B1 + 0404 and stimulates 3 or 4 T cell lines Was. The COOH terminus of this peptide, from residues 20 to 11 (PGGAISNMYAM SEQ. ID.N. O.39), and show it to BLS-B1 + 0404 or BLS-DRB1 + 0401, Only one T cell line was stimulated. Fragment of 10 amino acids (PGGAISNMYA SEQ. ID . NO.41) stimulated its T cell line only when presented on BLS-B1 + 0404. this Using methodology to identify GAD antigenic determinants that stimulate T cell lines from pre-diabetic patients Method to quickly identify HLA restriction in T cell lines and clones. Noh.                                 Example 7                            GAD Peptide synthesis   C-terminal amidated peptide is used for solid phase peptide synthesis utilizing the properties of Fmoc group (SPPS). The reagents used for the synthesis were from Nova Biochem (La Jolla , CA). Peptides were purchased from 432A Applied Biosystems, Inc. (Foster Cit y, CA) using a Rink amide MBHA resin (0. twenty five It was synthesized from the C-terminal side on a millimolar scale). In this synthesis, the coupling reaction Double coupling is necessary for complete operation, and the coupling properties of HBtu-HOBt It's being used. Unmodified residues require at least double coupling (SRL SKVAPVIKARMMEYGTT-NH2 (SEQ ID NO: 59). Each cycle contains amino acid residues on the resin Includes deprotection of Fmoc amino acids from the group and couples with the next Fmoc amino acid. After the peptide is automatically synthesized, the resin is washed with dichloromethane and vacuumed for 2 hours. Dried. Peptide resin is 1 ml of 4-methoxybenzenethiol as a scavenger 10 ml of trifluoroacetic acid (TFA), containing 0.7 g of 4-methylmercaptophenol Resuspended in. The suspension was gently stirred at room temperature for 2 hours and filtered through a PTFE filter. The filtrate to a lidded tube containing 1 L of mixed organic solvent (pentane: acetone = 4: 1). Added to the lath bottle. If left at room temperature for 1-2 hours, a white precipitate will precipitate, Coarse peptides can be separated by decantation after centrifugation. Wear. The crude peptide was washed three times with a mixed organic solvent and dried in vacuo overnight.   Reversed phase HPLC of the crude peptide shows a major peak and a small impurity peak. Are believed to be due to incomplete peptides. Main peak is reverse phase HPLC for preparation Using start buffer A (0.1% TFA) and end buffer B (70% acetonitrile)  in 0.1% TFA). Fraction (10-15ml) Was recovered and lyophilized to remove all solvent. About fractions Analyzed by reversed phase HPLC and the purified fractions were analyzed by mass spectrometry. Was.   Peptides with a carboxyl group at the C-terminus are for the solid-phase method using Fmoc amino acids Was prepared. Peptides were analyzed using an automated peptide synthesizer from 431A Applied Biosystems. Was synthesized from the C-terminal side on Wang resin. Wang resin with the first amino acid attached When (Fmoc-Thr (tBu) -Wang) is applied to the synthesizer, the amino acid next to the C-terminal is coupled. Cause a reaction. With such amino acids, the coupling reaction is completely performed as described above. Requires a double coupling. Therefore, the nature of coupling of HBtu-HOBt Is used. In each cycle, Fmoc amino acids from amino acid residues on the resin Includes deprotection and couples with the next Fmoc amino acid. Automatic peptide synthesis After that, the resin was washed with dichloromethane and dried under vacuum for 2 hours. Cut peptide Cutting and washing are as described above.   The relative affinity of synthetic peptides for MHC molecules is determined by the DELFIE method Measured by interlocking action. In other words, C-terminal amidated GAD65-restricted T cell hybrids Quantification of MHC complex using proliferation of CTLL cells associated with IL-2 production by lidoma It is possible.                                 Example 8                        Ala Synthesis of scan peptide   The 20 C-termini corresponding to the amino acid sequence from 524 to 543 of GAD65 are amidated. The synthesized peptide was synthesized. These peptides add one non-alanine residue at a time. It is a peptide in which alanine is substituted for tyrosine in the case of an alanine residue. this The peptide was synthesized using the solid-phase method ABIMED-Gilson AMS 422 multiple peptide synthesizer (M iddleton, WI). This synthesizer is Gilson auto-sampl that moves in X-Y-Z directions er, consisting of a 48 column reactor module and a storage for amino acids and activating reagents. Reagents and solvents are continuously added to each column by microinjector During that time, washing of the resin in all columns is performed simultaneously.   Peptides are collected on a 0.025 millimolar scale Rink amide MBHA resin of the synthesizer And synthesized with a substitution rate of 0.55 millimoles per gram. 0.025 millimol 20 columns filled with the activated resin of e were attached to the synthesizer. In the first stage Fmoc removal was performed using 20% piperidine in dimethyl formamide (DMF). This operation was performed simultaneously on the resin in each reaction column. Deprotection with maximum efficiency Protocols to perform (Thong Luu, Pham Son and Shrikant Deshpande, Automated Multiple Peptide Synthesis: Improvements in Obteining Quality P eptides, Int. J. Peptides Proteins, 1995, in press) did. The double deprotection method was also used to completely remove the Fmoc group. Tree using DMF The operation of washing the fat was performed simultaneously in each column.   Coupling of the first amino acid is performed by adding the amino acid to the reaction column with an auto injector. Performed. While slowly bubbling nitrogen through the reaction column, Was stirred for 20 seconds. Add dichloromethane (DCM) to the reaction mixture and add DMF: DCM The ratio was adjusted to be 3: 1. Coupling of amino acids in the next column Before that, the resin was again stirred. Strongly hydrophobic amino acids maximize coupling time Coupling first to do. After the removal of the first Fmoc group, all reaction columns are Washed simultaneously with DMF. Double coupl to fully couple amino acids to resin The ing method was always used. After completion of the double coupling, the resin is washed with DMF and the Fmoc group Desorption and amino acid coupling were started.   After the final removal of the Fmoc group, the peptide-bound resin is washed with DCM and the reaction column Was evacuated to dry the resin. Remove the column from the synthesizer and place it on one side of the column The lid was closed with a syringe cap (# 3980025, Gilson). 0.07 g of 4- (methy lmercapto) Phenol and 1.5 ml of TFA containing 0.1 ml of 4-methoxybenzenethiol The mixture was stirred at room temperature for 2 hours. After the cleavage of the peptide is complete, Take the cap, filter the reaction mixture and filter the filtrate with 100 ml of pentane: acetone (3: 1) Added to the solution. Leave the peptide for 2 hours at room temperature to precipitate the peptide. The precipitate was separated by filtration. The precipitate is washed three times with pentane: acetone solution and then with acetone Washed twice. The crude peptide is dried in a vacuum for 2 hours and analyzed by reversed-phase HPLC or quality. It was subjected to a mass spectrometer. pentane: A pen that did not precipitate within 2 hours with an acetone solution The peptide was cooled overnight at −20 ° C., and separated and washed by the method described above.                                 Example 9                 Synthesis of truncated C-terminal amidated GAD65 peptide   A series of C-terminal amidated GAD65 (S EQ. ID. NO.59) The peptide was synthesized (Table 3).   The peptide is ABIMED-Gilson AMS 422 multip as described in Example 8. The peptide was synthesized by a solid phase peptide synthesis method using a le peptide synthesizer.                               Example 10                 Truncated C-terminal amidated GAD65 core peptide   When examining the truncated C-terminal amidated GAD65 Core Peptide, The truncated peptide (containing 528-543 amino acids) and the N-terminal truncated peptide (524-53 amino acids) 9 amino acids) and I-A^g7Was found to bind. And 528 ~ 539 Peptides containing amino acids are also restricted to the C-terminal amidated GAD65 core peptide. Stimulated T cell hybridomas. Based on this information, Synthesis of a series of truncated peptides based on the core sequence (Table 4) And the involvement of T-cell hybridoma restricted to C-terminal amidated GAD65. Can be analyzed.   Peptides are synthesized in solid phase using Applied Biosystems' automatic peptide synthesizer, 433A It was synthesized by the method. Peptide is 0.05 mm from carboxy terminus with rink amide MBHA resin Synthesized on a molar scale (substitution level 0.55 mmol / g). Amine on the tree Use the HOBt / HBTU coupling strategy to silylize the amino acid F on the resin. Piperidine was used for deprotection of the moc-protected amine. N-methylpyrrolidinone ( NMP) was used as the solvent for the coupling / deprotection reactions. Dichloromethane (DCM) Used for final washing of peptide resin. Deprotection increases conductivity when Fmoc is released Monitored by measuring. If deprotection is difficult, coupling Also difficult and hence double coupling and / or post-coupling Cylation was performed during the synthesis.   After synthesizing the peptide chain on the resin, the peptide resin is dried under vacuum for 2 hours. And subjected to the deprotection protocol. 0.14 g of 5-methylmercaptopheno The suspension was suspended in trifluoroacetic acid (TFA) containing l and 0.2 ml of methoxybenzenethiol. The suspension is mixed for 2 hours and then mixed with 200 ml of organic solvent (pentane: acetone 4: 1) Added through filter. The peptide suspension was kept at -20 degrees overnight. Purification When the suspension was allowed to stand, a peptide film was observed on the inner surface of the glass bottle. Decant off clear solvent and carefully with 50ml pentane: acetone mixture Was washed. Washing was performed three times in total, and then twice with 50 ml of pentane. Dissolve the film in 10 ml of 70% aqueous acetonitrile containing 0.1% TFA, and add distilled water. To 30 ml. The peptide suspension was lyophilized and the resulting white powder was reversed phase. Analyzed by HPLC and mass-spectrometry. This product does not undergo further purification And used for peptide binding and T cell activity measurements.                                Example 11             C- Restricted to terminal amidated GAD65 (amino acids 524-543)                       Generation of hybridoma T cell line   NOD mouse expressing T-cell receptor specific for C-terminal amidated GAD5 peptide A hybridoma cell line was prepared. The procedure for obtaining these hybridomas is described in this book. Greene, Wiley Interscience Current Protocol, referenced in the specification. Using "Production of Mouse T Cell Hybridomas" in s in Immunology . Briefly, three 9-week-old female NOD mice were given 100 ml CFA (Complete Fre und's Adjuvant) and 50 μg of C-terminal amidated GAD65peptide Injection caused a sharp increase in T cells restricted to this peptide. mouse Was killed 8 days later by amputation of the neck, spleen and lymph nodes (popliteal, superficialin guinal). Lymph nodes are broken apart between two glass plates, Suspended in on4002 Petri dish. After rubbing the spleen into a cell suspension in another dish, Centrifuged at room temperature, 12,000 RPM for 5 minutes. Remove the supernatant and leave with the spleen cells. Red blood cells were removed by hemolysis: splenocytes were resuspended in 0.9 ml of sterile water, -10 seconds later, quickly add 0.1ml 10X PBS, and then add about 4ml of Bruff's medium (Click ' s medium EHAA; Irvine Scientific, Santa Ana, CA) or 200 ml of penicillin / stre ptomycin (BioWhittaker), 15g of sodium bicarbonate (Sigma, St. Louis, MO), 43m l β-mercaptoethanol (Sigma), 11.6 ml gentamycin sulfate solution (Irvine Scientific), 10% fatal bovine serum (FBS, Hyclone, Logan, UT) Bacterial water was added. The cells were resuspended with a 5 ml pipette and the lipid was filtered off and discarded. Fine 2 × 10 vesicle concentration⁶ cells / ml, and in vitro C-terminal am Stimulation was performed with 10 mg / ml of the idated GAD65 peptide. If cells blast (about 3-5 days ), Lymphocytes and splenocytes were harvested from culture. Dead cells are centrifuged with Ficoll-Hypaque To remove. 5 x 10 cells⁶~ 2 × 10⁷Become a Fico The ll-Hypaque was overlaid so as to have a ratio of 5 ml-5 ml. The cells are then 2,000 RPM at 4 ° C , Centrifuge for 20 minutes, wash twice with Bruff's medium, and finally with Bruff's medium without FBS And washed. BW5147, a lymphoma cell (ATCC, Tumor Immunology Bank 48) Was cultured and washed with a washing medium. Transfer cells BW5147 to Bruff's medium containing 20% FBS. Cells and lymphocytes were mixed at a ratio of 1: 1. Centrifuge the cell mixture at 2,000 RPM for 5 minutes at room temperature. I thought. The supernatant was aspirated and 1 ml of medium previously warmed to 37 ° C. was added. 50% p olyethylene glycol (PEG) solution (Sigma) was added dropwise to the cell pellet for 1 minute. To promote cell fusion. Gently agitate the pellet with each addition and for an additional minute Stirred. 2 ml of pre-warmed medium is added dropwise to the PEG / cell mixture solution. The mixture was added dropwise with a pipette for 2 minutes, and each mixture was gently stirred. 5 at 2000 RPM Centrifuged for minutes and discarded the supernatant. Obtained from unprimed NOD rats The thymus was crushed and removed with a Bruch's medium containing 20% FBS. Count thymocytes at a concentration of 5 × 10⁶ cells / ml. The number of thymocytes to be added is 100 ml / cm 0.1 to 1 × 10 per well^FiveIt was calculated and calculated to be cells / well. B with 20% FBS Thymocytes in ruff's medium were forcibly added onto the cell pellet. Cell mix solution The solution was dispensed into 96 well plates so as to be 100 ml / well. No end of well Bacteria are not dispensed to keep the bacteria. Incubate the plate at 37 ° C, 7.5% CO2 Nourished. The next day, 2xHAT (Sigma) in Bruf's medium containing 20% FBS in each well 100 ml was added and the plate was returned to the culture tank. At a later date, the cells are fused by two thymocytes Observed as death. Only the fusion between lymphoma and lymphocytes grows Should do it. Six days later, 100 ml of 2 × HAT (Sigma) in Bruff's medium containing 10% FBS was added. Added to each well. At a later date, it was determined whether the cells had increased. Increase These cells were observed as 1x HAT in Bruff's medium containing 20% FBS. Transferred to 1 ml of 24 well plates. Two sets were made and checked every day. Grow up Some were transferred to T-25 flasks. These T-cell hybridomas gradually become 20% FB Transfer to Bruff's medium containing S and 0% HAT and have specificity for C-terminal amidated GAD65. It was kept until screening.                               Example 12 C- Screening of T cell hybridoma cell lines restricted to terminal amidated GAD65   Antigen-presenting cells (APCs) were used to determine the specificity of T cell hybridomas. The mouse spleen was ground and dissolved by the method of Example 11 to prepare. Splenocytes contain 10% FBS Was added to 3 ml of Bruff's medium. Mitomycin C (Sigma) 0.3m per 3ml of cell suspension l added to inhibit DNA synthesis. Incubate APCs in a 37 ° C water bath for 30 minutes and add 10% FBS Was washed three times with Bruff's medium containing After washing, Bruch's medium containing 10% FBS for APC's 2 × 10⁶Cells / ml were added. C-terminal amidated GAD65 pep Tide was titrated from 333 μg / ml to 0.15 μg / ml in a round bottom 96 well plate. 50 μl (1 × 10^Five) APCs were added to the peptide. Including the number of hybridomas, including 10% FBS 1 × 10 in Bruff's medium⁶cells / ml, 100μl (1 × 10^Five) Cells Added. Hybridomas were also tested as follows. I-A^g7 MHC + C-terminal a Peptides other than midated GAD65, I-A^g7Other than MHC + C-terminal amidated G65, I -A^g7 MHC only, C-terminal amidated GAD65 only. Plate at 37 ° C under 5% CO2 Cultured overnight. On the next day, remove 150 μl of medium from each well, flat bottom 96 wells Transferred to plate and frozen to kill all cells. T cells were activated Only medium from the wells contains IL-2. CTLL cells that depend on IL-2 for their survival (ATCC TIB-214) was centrifuged, and washed three times with Bruff's medium containing 10% FBS. 5 × 10 per^ThreeCells were placed in a flat bottom 96 well plate. APC / Hybrid Thaw the supernatant collected from the dome and add 50 μl to a similar well containing CTLL cells. I got it. Two rows were cultured as a control for CTLL cells. Two control wells: medium and cells Only or contains cells, medium and titrated IL-2. Plate at 37 ° C, Cultured overnight at 5% CO2. At a later date, cells were stimulated with 4H-thymidine 1 μCi / Well. Step The rate was cultured overnight to allow 3H-thymidine to be incorporated into the cells.   Cultivate cells with Skatron Basic 96 cell incubator (Carlsbad, CA) according to manufacturing policy did. The filter mat was dried overnight and placed in a sample bag. About 10 Add Beta Sciont scintillation fluid (Wallac, Turku, Finland) Closed. Incorporation of 3H-thymidine into DNA is performed by Wallac 1205 Betaplate Beta Coun ter (Turku, Finland). The uptake of 3H-thymidine by T cells That the medium was present, and that the medium was C-terminal amidated GAD65 peptide + NOD-derived A PCs I-A^g7 Indicates that it was activated by MHC. Therefore showed a high proliferative response Wells containing CTLL cells correspond to hybridomas specific for MHC complex peptides I do. Hybrids showing high proliferative response due to> 5000 cpm incorporation of 3H-thymidine , The initial fusion that occurred in MBD.1 was C-terminal amidated GAD65 peptide + I-A^{g 7} Is specific to GAD65 peptide without C-terminal amidation Low response but> 2000 CMP, but other MHC / peptide pairs There was no response in the combination. The stimulus response in other cells was <500 cpm. After the second fusion, C-terminal amidated GAD65 peptide + I-A^g7 Special for MHC Hybrids named MBD2.3, MBD2.7, MBD2.8, MBD2.11, MBD2.14 with opposite sex Ma got more.                                Example 13       C- T cells restricted to terminal amidated GAD65 + NOD MHC class II, IA ^g7             Identification of binding site of peptide binding to hybridoma   C-terminal amidated GAD65 + I-A as mentioned above^g7Specific hybrid cells (MB D.1, MBD2.3, MBD2.7, MBD2.8, MBD2.11, MBD2.14) are as described in Embodiment 12. I-A by way^g7 + Ala Scanning or branched peptide specificity Cleaned. Briefly, alanine scanning peptides or branched peptides , 333 to 0.15 μg / ml. Proliferation of CTLL cells Is Alanine substitution (or specific amino acid cleavage) No effect on binding between T cell receptor and MHC-peptide complex . Lack of proliferation means that replacement (or truncation) residues are bound by the T cell receptor to form a complex pair. Has been shown to be relevant. Control peptide without substitution In other words, one amino acid residue of alanine at positions 524,526,528,529,531,532,533 Proliferation was severe due to group substitution or substitution of tyrosine at positions 530 and 535. Affected. Activation of T-cell hybridomas requires cleavage of amino acids from 527 to 539 amino acids. Found in truncated peptides. At least 529-539 T-cell hybridomas Recognizes a peptide with an amino acid, which is the T This indicates that it is essential for cell hybridoma binding.                                Example 14                 NOD MHC Peptides that bind to class II IA ^g7   The relative affinity of the peptide (alanine scanning or cleavage) for MHC is sen et al. Immunol. Meth. 163: 209, 1993, developed by Europiumstreptav idin dissociation enhanced lanthanide fluoreoimmunoassay (DELFIA) Specified. The assay used whole cells or solubilized MHC molecules. peptide Was measured three times each. For example, in case of alanine scanning peptide, NOD spleen Cells are fixed with 1% paraformaldehyde for 10 minutes at room temperature and 30 minutes on ice. , RPMI 1640, 1% PSN (BIBCO-BRL, Gaithersburg, MD), 200 mM L-glutamine (Haze lton Biologics, Lenexa, KS), wash once with 10% heat-inactivated fetal calf serum (FCS) And washed twice with CPBS (Bulbecco's PBS, BioWhittaker, Walkersville, MD) . Cells are diluted 1:50 protease inhibitor stock solutions D, E, F and 1M citrate / PO4, pH 1 × 10 in 0.15M NaCl containing 5.5⁷ redissolved in cells / ml   100 μl of the cell protease inhibitor mixture was added to a round bottom plate (Costar, Pleasanto) n, CA). C-terminal am with biotin-labeled immobilized NOD cells 10,000 g of idated GAD65 peptide and 100,000 unlabeled alanine scanning peptide, 1 The cells were cultured at 37 ° C. for 12-20 hours together with 10,000 and 10 nM. I-A^g7Allele features with high specificity Positive for Mouse serum albumin (MSA) known as isomeric peptide (SEQ.ID.NO.61) As a control, binds to I-Ad but I-A^g7Ealpha that does not bind to (Reich et al., J. Immunol. 154: 2279-88, 1994). Further Continue feeding, vortex the plate, and use a Beckman GA-6R centrifuge at 1500 rpm for 10 minutes Centrifuged (Beckman, Fullerton, CA). Remove supernatant and wash cells with NP-40 lysis buffer (0.5% NP40, 0.15 M NaCl, 50 mM Tris, pH 8.0, 0.01% sodium azide, 1:50 Diluted proteinase inhibitor stocks D, E, F (Table 3)) were added at 60 μl / well. And dissolved. Incubate the cells on ice for 30 minutes with mixing every 15 minutes, 1500 Centrifugation at rpm for 10 minutes gave a clear lysate.   Assay plate: 96-well flat bottom plate (Coaster) 100 μl / well in DPBS Anti-I-A^g7Antibody (10.2.16, 50 μg / ml, TSD Bioservices, Germantown, NY) To prepare. Plates were incubated at 4 ° C. for 12-18 hours. Remove unbound antibody 200μl / well MTB (1% BSA, 5% powdered skin milk, 0.01% sodium azide Mixed with TTBS (0.1% Tween20, 0.5M Tris, 1.5M NaCl, pH7.5) for 30 minutes Blocked at room temperature and washed 7 times with TTBS. 50 μl MTBN (1% BSA, 5% Milk, 0.01% sodium azide, NP40 dissolved in TTBS) per well In addition, 50 μl of clear lysate was added from above. Plates are incubated at 4 ° C for 2 hours, Washed 7 times with TBS. Europium-labeled streptavidin (Wallac # 1244-360) and DELF 100 μl / well of 1: 1000 dilution of IA measurement buffer (Table 6) added.   Plates were incubated at 4 ° C. for 1 hour and washed 7 times with TTBS. Avoid whipping the reagent Carefully add 100 μl of solution A (Table 7) to each well and allow the plate to come to room temperature. For 3 minutes. Add 20 micol / well of Enhancement Solution B (Table 7), Leave at room temperature for 30 minutes. Place the plate on a time-delay fluorometer (Wallac 12 34 DELFIA Research Fluorometer).   Amino acids at positions 524, 526, 527, 528, 529, 531, 532, 533 are converted to alanine Biotin labeling when substitution or substitution of amino acid at position 530,535 with tyrosine NOD MHC of C-terminal amidated GAD65 peptide (I-A^g7In competition with binding sites I can no longer do it. When the arginine at position 536 is replaced with alanine, four of the six T Inhibited the activation of vesicular hybridomas. Replace methionine at position 538 with alanine This inhibited the activation of five of the six hybridomas. Methionine at position 538 Substitution with alanine inhibited activation of one T cell hybridoma. peptide The antigenic determinant of GAD65 that binds to IAG7 determined by cleavage has amino acids 527-539. Including. 527-539 amino acids are NOD MHC class II molecule, I-A^g7Included in the site that binds to It also correlates with hybridoma data suggesting a rare occurrence. Suitable GAD pep The tide will be from aa525 to aa540.                                Example 15             Induction of peptide-MHC complex versus anergy in vitro   This assay uses a C-ter where a specific peptide-MHC complex is restricted to T cell clones. minal amidated GAD65 or anel in lymphocytes primed in vivo To see if it induces energy.   Flat bottom 96-well plates (Costar) were prepared using antibody class II (10.2.16, 50 μg / ml) in DPBS. , TSD Bioservices, Germantown, NY) 100 μl / well (5 μg per well) And incubated at 4 ° C. for 12-18 hours. Remove unbound antibody and plate Was protected with 5% BSA (bovine serum albumin, Sigma) and incubated at room temperature for 30 minutes. The cells were washed once with a bruff's medium containing 10% FBS. Peptide-MHC complex, preferably C-t erminal amidated GAD65 and I-A^g7Complex or Ala scan or cut GAD pep Tide was added at 2 and 10 μg / l. Control is I-A^g7Peptide MHC complex such as -MSA-OH, medium Only, peptide only, or MHC only equal to the concentration of peptide-MHC complex. Was added. Thereafter, the plate was incubated at 4 ° C. for 8-10 hours. C-terminal amid ated GAD65-restricted T cell clones were counted and diluted in Bruff's medium containing 10% FBS. I shouted. 6 × 10^FiveOf cells came out in one well with 200 μl of medium.   Lymphocytes primed in vivo were also used instead of T cell clones . Briefly, NOD mice were applied to the footpad in the manner described in Example 11 by 30-50 μl. g peptide / 150 μl primed with complete Freund's adjuvant. 8 days later The mice were sacrificed and the spleen, popliteal and inguinal nodes were removed. The organization was changed to Example 11 Pulverized, prepared, treated with Mitomycin C, and ready for measurement as described in Prepared as follows.   At a later date, wash the plate to remove unbound complexes and remove cells from the plate. Pipette and transfer to another, labeled Eppendorf tube at 1200 RPM For 5 minutes, and washed three times with a Bruff's medium containing 10% FBS. Count the number of cells, Divide each tube into two tubes, one of which contains 1/3 cells The other tube contained the remaining 2/3 of the cells. Centrifuge cells again The tube containing 1/3 of the cells to 10% FBS and 10 U / ml IL-2 in Bruff's medium. And diluted to a total volume of 200 μl. The other tube contains 10% FBS But diluted to 400 μl with Bruff's medium without IL-2.   The second 96-well plate contains 0.6 μg / μl C-terminal amidated GAD65. 10 μl / well or 0.1 μg / ml of anti-CD3 (CD3-e cytochrome antibody, Pharmingen, San Diego, CA). At least 2 Wells contain α-CD3 and at least 4 wells contain peptide, each containing The sample was measured. Antigen presenting cells (APCs) were prepared as described in Example 12 and 5 × 10 in Breff's medium containing% FBS⁶Wells containing 100 μl peptide diluted to cells / ml Only added. 100 μl of pre-prepared T cell clone or first in vivo Time-stimulated IL-2 free lymphocytes from α-CD3 containing wells and peptides and AP Added to half of the wells containing Cs. Those cells treated with T cells or IL-2 Companion spheres were added only to leave wells containing peptide and APCs. The final arrangement is There are two wells containing peptide-MHC complex or control peptide-MHC. And APCs without peptide-IL2 and APCs without peptide-IL2, respectively. Did some kind of processing. T cell / lymphocyte concentration at least 5 × 10^FourBecome cells / well Should preferably be about 2.3 × 10^FiveFrom 5.3 × 10^FiveIt is. Plate at 37 ° C Cultured for 3 days.   Cells were stimulated with 3H-thymidine 1 μCi / well. Incubate the plate for 5 hours , 3H-thymidine was incorporated into cellular DNA.   If the addition of APCs and peptides (but not IL2), T cells become anergic APCs and peptide reactions do not occur, and 3H-thymidine incorporation does not occur . Α-CD3 as a control, whether the cells are alive and only by other stimuli Used to indicate if it is reacting.                                Example 16                                Adoptive transfer   IDDM injects spleen cells from diabetic donor to non-diabetic recipient Can be adopted by adoption. Urinary glucose levels in female NOD / CaJ mice They screened for diabetes by monitoring. At least glucose in it Analyzed blood glucose using tail clipping for those showing a positive urine level of 250 mg / dl Those with a blood glucose level exceeding 250 mg / dl were classified as overt diabetes.   Newly irradiated diabetic NOD mice (730 rad) and randomly divided into 4 treatment groups Splenocytes were isolated as described above. Non-diabetic 7-8 week NOD recipient The mice were divided into four groups. Group 1 is 1 × 10⁷Splenocytes from the vein It is a thing. After 6 hours from the injection, 10 μg / mouse C-terminal amidate d GAD65 or 10,5,1 μg / mouse C-terminal amidated GAD65 peptide-MHC Complex salts were each injected intravenously. Group 2 is 2 × 10⁷Inject splenocytes, 10  μg / mouse C-terminal amidated GAD65 peptide-MHC complex, or 5 μg / mou se MSA-MHC complex salts were injected respectively. Group 3 is 1 × 10⁷Give the spleen cells 10 μg / mouse C-terminal amidated gAD65 or 200 μg / mouse 10.2.16, a Each of the nti-class II antibodies was injected. Group 4 is 1 × 10⁷Give the spleen cells 20μg / mouse C-terminal amidated GAD65 peptide, or 1,5,10 μg / mouse  Each C-terminal amidated GAD65 peptide-MHC complex was injected. group The mice of No. 4 were treated only with peptide or peptide-MHC complex for the first time. Was received on day 0 and a second time on day 4. Other groups will be processed on the 8th and 12th days Was made. Mice were tested for the development of diabetes by urine analysis. Urine and blood glue On the day when overt diabetes was first detected by analysis based on course concentration, Select mice at random from the group and select urine and blood glucose levels All mice were examined and then sacrificed for spleen analysis for immunohistochemical analysis. And took out the pancreas. The saline-treated mice developed diabetes in about 12-20 days. Ten Group 1 mice that received 4 treatments with μg peptide-MHC complex were prominent by day 30 No onset was found, even after 75 days. If you are 100% sick in mice The mice injected with 5 μg peptide-MHC complex were 40% And the onset of illness gradually increased until day 80. Of peptide-MHC complex Group 4 mice receiving only two treatments had a somewhat late onset of disease. Was. That is, 50% or less of mice injected with 10 μg of peptide-MHC were I became ill. Group 3 mice blocked with anti-MHC antibody have late onset As noted, their protection was insignificant. That is, 10μg peptide More than 75% of the injected mice developed disease by day 30. This adoptive transfer model According to the C-terminal amidated GAD65 peptide (SEQ.ID.NO.59) alone, The onset was accelerated, while the peptide-MHC complex suppressed it.   As noted above, specific embodiments of the invention are described herein for illustrative purposes only. However, various changes may deviate from the intended purpose and scope of the invention. Will be done. Therefore, the invention is limited without additional claims. Absent.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/74 C07K 14/74 19/00 19/00 C12N 5/10 C12N 5/00 B // (C12N 5/10 C12R 1 (31) Priority claim number 08 / 483,241 (32) Priority date June 7, 1995 (33) Priority claim country United States (US) (31) Priority claim number 60 / 005,964 ( 32) Priority Date October 27, 1995 (33) Priority Country United States (US) (81) Designated State EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS) , MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventor Cainsbogel, Wayne Washington, USA 98115, Seattle, 24T Avenue N. E. 6014 (72) Inventor Litchi, Eva Pier United States of America 94024, Los Altos, Paine Drive 1255 (72) Inventor Gross, Jane A. United States of America Washington 98125, Seattle, NH. E. 110 T. H. Street 4224 (72) Inventor Despande, Shrinkant United States of America 94555, Fremont, Lang Avenue 34619 (72) Inventor Sheppard, Paul O. Washington 98053, Redmond, N. USA. E. 2nd Street 20717

Claims (1)

[Claims] 1. A soluble fused MHC heterodimer: peptide complex comprising: a first DNA segment encoding at least a portion of a first domain of a selected MHC molecule; A second DNA segment encoding at least a portion thereof; a first linker DNA segment encoding about 5 to about 25 amino acids and connecting the first and second DNA segments in-frame; The linking of the first DNA segment to the second DNA segment by the first linker DNA segment becomes a fused first DNA-first linker-second DNA polysegment; A third DNA segment encoding an antigenic peptide capable of binding to the peptide-binding groove of the MHC molecule; encoding about 5 to about 25 amino acids, and in-frame with the third DNA A second linker DNA segment connecting the first DNA-first linker-second DNA polysegment fused to the third DNA segment; The linkage to the fused first-first linker-second DNA polysegment results in a soluble fused MHC heterodimer: peptide complex. 2. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the selected MHC molecule is an MHC class II molecule. 3. 3. The soluble fused MHC heterodimer: peptide complex of claim 2, wherein said first DNA segment encodes a β1 domain. 4. 3. The soluble fused MHC heterodimer: peptide complex of claim 2, wherein the second DNA segment encodes an α1 domain or an α1α2 domain. 5. The selected MHC molecules, IA ^g7, IA ^5, is selected from the group consisting of DR1β * 1501, and DRA * 0101, fused MHC heterodimers of soluble claim 1: peptide complex. 6. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the selected MHC molecule is an MHC class I molecule. 7. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the first linker DNA segment is GASAG (SEQ ID NO: 29) or GGGGSGGGGS GGGGS (SEQ ID NO: 36). 8. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein said second linker DNA segment is GGSGG (SEQ ID NO: 30) or GGGSGGS (SEQ ID NO: 31). 9. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein said third DNA segment encodes an antigenic peptide capable of stimulating an MHC-mediated immune response. 10. The peptide is selected from the group consisting of a mammalian GAD65 peptide (SEQ ID NO: 59), (SEQ ID NO: 61), (SEQ ID NO: 40), (SEQ ID NO: 39), and a mammalian myelin basic peptide (SEQ ID NO: 33). The antigenic peptide according to claim 9, wherein 11. The MHC heterodimer: peptide complex encodes a fourth DNA segment encoding at least a portion of a third domain of the selected MHC molecule, and about 5 to about 25 amino acids; A fused third DNA-second linker-first DNA-first linker-second DNA-second further comprising a third linker DNA segment connecting the fourth DNA segment in-frame. 2. The soluble fused MHC heterodimer: peptide complex of claim 1, which becomes a three linker-fourth DNA polysegment. 12. 12. The soluble fused MHC heterodimer: peptide complex of claim 11, wherein said selected MHC molecule is an MHC class I molecule. 13. 12. The soluble fused MHC heterodimer: peptide complex of claim 11, wherein the selected MHC molecule is an MHC class II molecule. 14． 12. The soluble fused MHC heterodimer: peptide complex of claim 11, wherein said fourth DNA segment is a β2 chain. 15. 12. The soluble fused MHC heterodimer: peptide conjugate of claim 11, wherein the third linker DNA segment is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32). 16. An isolated polynucleotide molecule encoding a soluble fused MHC heterodimer: peptide complex according to claim 1. 17． The soluble fused MHC heterodimer of claim 1, comprising the following operably linked elements: a fusion protein expression vector capable of expressing a peptide complex: a transcription promoter; a selected MHC molecule. A first DNA segment encoding at least a portion of a first domain; a second DNA segment encoding at least a portion of a second domain of a selected MHC molecule; encoding about 5 to about 25 amino acids; And a first linker DNA segment connecting said first and said second DNA segments in-frame; wherein said first DNA segment is linked to said second DNA segment by said first linker DNA segment. Becomes a fused first DNA-first linker-second DNA polysegment; capable of binding to the peptide binding groove of the selected MHC molecule A third DNA segment encoding an antigenic peptide; a first DNA-first linker-second DNA polysegment encoding from about 5 to about 25 amino acids and fusing the third DNA segment in frame. A connecting second linker DNA segment, wherein the linkage of the third DNA segment to the fused first DNA-first linker-second DNA polysegment by the second linker DNA segment is soluble Expresses a fused MHC heterodimer: peptide complex; and a transcription terminator. 18. The MHC heterodimer: peptide complex encodes a fourth DNA segment encoding at least a portion of a third domain of the selected MHC molecule, and about 5 to about 25 amino acids; Further comprising a third linker DNA segment connecting the fourth DNA segment in-frame, the fused third DNA-second linker-first DNA-first linker-second DNA-second 18. The expression vector of claim 17, wherein the expression vector is a three linker-fourth DNA polysegment. 19. Culturing cells into which the expression vector of claim 17 has been introduced, whereby said cells express a soluble fused MHC heterodimer: peptide complex encoded by said DNA polysegment; Soluble fused MHC heterodimer: peptide complex produced by recovering the soluble fused MHC heterodimer: peptide complex. 20. A pharmaceutical composition comprising the soluble fused MHC heterodimer: peptide conjugate of claim 1 together with a pharmaceutically acceptable vehicle. 21. An antibody that binds to an epitope of the soluble fused MHC heterodimer: peptide complex of claim 1. 22. A method of treating a patient to reduce an autoimmune response, comprising administering a therapeutically effective amount of the soluble fused MHC heterodimer: peptide conjugate of claim 1 to the patient, thereby tolerizing immune tolerance in the patient. Inducing. 23. A method for preparing a responder cell clone that proliferates in combination with a selected antigenic peptide presented by a stimulator cell, comprising: a non-adherent CD56-, CD8 reactive with the selected antigenic peptide Isolating cells, thereby forming responsive cells; stimulating the responsive cells with pulsed or primed stimulator cells; pulsing the stimulated responsive cells with pulsed stimulator cells or primed stimulator cells Stimulating again with the stimulated cells; and isolating a responder cell clone. 24. 24. The method of claim 23, wherein the responder cells are isolated from a patient who is pre-diabetic or has newly developed diabetes. 25. 24. The method of claim 23, wherein said responder cell clone is a T cell clone. 26. 24. The method according to claim 23, wherein said selected antigenic peptide is a GAD peptide.