KR19990022641A - Fused Soluble MHC Heterodimer: Peptide Complexes and Uses thereof - Google Patents

Abstract

Immune modulators such as soluble fused MHC heterodimer and soluble fused MHC heterodimer: peptide complex have been disclosed. In addition, associated methods and peptides have been disclosed. In a preferred aspect, these modulators and methods relate to autoimmunity.

Description

Fused Soluble MHC Heterodimer: Peptide Complexes and Uses thereof

There is currently great interest in the development of medicaments based on expressions that increase with respect to the structure and function of major histocompatibility complex (MHC) antigens. Such cell surface glycoproteins are said to play an important role in antigen presentation and inducing various T cell responses to antigens.

Unlike B cells, T cells do not directly recognize antigens. Instead, an accessory cell first processes the antigen and presents it in combination with the MHC molecule to elicit a T cell-mediated immunological response. The main function of MHC glycoproteins appears to be the binding and presentation of antigens treated in the form of short antigenic peptides.

In addition to binding to external or non-self antigenic peptides, MHC molecules can also bind to self peptides. Then, when T lymphocytes respond to cells providing magnetic or autoantigenic peptides, an autoimmune state is created. More than 30 autoimmune diseases are currently known, including myasthenia gravis (MG), multiple sclerosis (MS), systemic erythematosus (SLE), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM) have. The hallmark of this disease is the attack of the immune system against host tissues. In non-disease individuals, such an attack does not occur because the immune system recognizes this tissue as self. Autoimmunity occurs when a specific adaptive immune response occurs against autologous antigens.

Insulin-dependent diabetes mellitus (IDDM), known as type I diabetes, results from autoimmune destruction of insulin producing β-cells in the pancreas. Studies to identify autoantigens involved in β-cell destruction have found several candidates, including insulin (Palmer et al., Science 222: 1337-1339, 1983), less specific islet cell antigens (Bottazzo et al. , Lancet 11: 1279-1283, 1974), and 64 kDa antigens (Baekkeskov et al., Nature 298: 167-169 (1982); Baekkeskov et al., Nature 347: 151-156, 1990) that appear to be glutamic acid decarboxylase This includes. Antibodies to glutamic acid decarboxylase (herein referred to as GAD) have been found to exist 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 production of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter of the mammalian central nervous system. Little is known about the regulation of GAD activity or the expression of the GAD gene. Although widely distributed in the brain, GAD proteins are present in very small amounts and are very difficult to purify to homogeneity. GAD has a number of isoforms encoded by other genes. These isoforms of enzymes differ in molecular weight, motility, sequence (if known), and hydrophobic properties. For example, three different forms of GAD have been reported in the pig brain (Spink et al., J. Neurochem. 40: 1113-1119, 1983) and four forms have been published in the rat brain (Spink et al., Brain Res. 421: 235-244, 1987). Mouse brain GAD (Huang et al., Proc. Natl. Acad. Sci. USA 87: 8491-8495, 1990), and GAD clones isolated from cat brains (Kobayasi et al., J. Neurosci. 7: 2768-2772, 1987) It was also announced. At least two GAD isomers have also been published in the human brain (Chang and Gottlieb, J. Neurosci. 8: 2123-2130, 1988). Human pancreatic islet cell GAD has recently been characterized by molecular cloning (Lernmark et al., US patent application Ser. No. 07 / 702,162; PCT Publication WO 92/20811). This form of GAD is equivalent to a homogeneous form of the human brain, which is later revealed (Bu et al., Proc. Natl. Acad. Sci. USA 89: 2115-2119, 1992). The second GAD isoform found in the human brain does not exist in human islets (Karlsen et al., Diabetes 41: 1355-1359, 1992).

Inflammatory CD4 + (T _H 1) T cell responses to GAD are major autoantigen responsiveness, which has been suggested to coincide with the development of insulin salts in NOD mice and then to T-cell reactivity to other β-cell antigens. At the same time, early T cell responses to GAD have been reported to be limited to one region of the GAD polypeptide and to spread over time with additional GAD determinants (WO 95/07992; Kaufman et al., Nature 366: 69-71, 1993; and Tisch et al., Nature 366: 72-75, 1993).

Evidence suggests that in human and animal models, GAD is a major autoantigen involved in initiating β cell attack leading to diabetes. Three peptides derived from mouse and human GAD65, peptide # 17 SEQ ID NO: 246-266, peptide # 34 SEQ ID NO: 509-528, and peptide # 35 SEQ ID NO: 524-543, were induced by their ability to induce T cell responses in mice. It is associated as a candidate for antigen (Kaufman et al.).

Current treatment of autoimmune diseases and related conditions consists primarily in treating the manifestations of the disease but does not control the pathogenesis of the disease. Chemotherapeutic agents with a broad spectrum are typically used and these agents are usually associated with a number of undesirable side effects. Thus, there is a need for compounds that can block MHC binding to selectively inhibit autoimmune responses, thereby enabling safer and more effective treatment. Such selective immunosuppressive compounds are also required for the treatment of non-autoimmune diseases such as macrohost graft disease (GVHD) or various allergic reactions. For example, chronic GVHD patients often present conditions and signs similar to any autoimmune disease.

Inappropriate autoimmune disease treatments currently available block the MHC-restricted immune response, but present an urgent need to find new agents that can avoid undesirable side effects such as nonspecific inhibition of the individual's overall immune response. Preferred methods for treating autoimmune diseases and other MHC-mediated pathological conditions utilize soluble fused MHC heterodimer: peptide complexes to provide anergy or immune resistance to T cells in response to antigenic peptides. To achieve. The present invention satisfies such needs and provides related advantages.

The identification of synthetic antigenic peptides, and the fact that they selectively bind to and stimulate T cells associated with disease, may help to correlate specific peptides or peptide: MHC complexes in susceptibility to autoimmune diseases. will be. The present invention satisfies such needs and provides related advantages.

This application is directed to US patent application Ser. No. 08 / 480,002, filed Jun. 7, 1995, US patent application Ser. No. 08 / 483,241, filed June 7, 1995, and US, filed June 7, 1995. Claims the benefit of U.S. Provisional Application No. 60 / 005,964, filed on October 27, 1995, which is part of the pending application of patent application 08 / 482,133.

The present invention relates to immunomodulators such as soluble, fused MHC heterodimers and soluble, fused MHC heterodimer: peptide complexes. The invention also relates to related methods and peptides. These modulators and methods are involved in autoimmunity.

Summary of the Invention

A first aspect of the invention provides a kit comprising a first DNA fragment encoding at least a portion of a first domain of a selected MHC molecule; A second DNA fragment encoding at least a portion of a second domain of the selected MHC molecule; Encoding from about 5 to about 25 amino acids and encoding the first DNA such that the linkage of the first DNA fragment with the second DNA fragment by the first linker DNA fragment produces a fused first DNA-first linker-second DNA poly fragment A first linker DNA fragment that connects the fragment and the second DNA fragment to fit a frame; From about 5 to about 25 amino acids, such that the linkage of the fused first DNA-first linker-second DNA poly segment with the third DNA fragment by the second linker DNA fragment yields a soluble fused MHC heterodimer: peptide complex. A first DNA-first linker-second DNA polysegment that encodes an antigenic peptide capable of associating a second linker DNA segment encoding a peptide with a peptide binding groove of a selected MHC molecule and fused the third DNA segment A soluble fused MHC heterodimer: peptide complex is provided which consists of a third DNA segment linked in frame.

In one embodiment of the invention the selected MHC molecule is an MHC class II molecule.

In another embodiment, the first DNA fragment encodes a β1 domain.

In another embodiment, the second DNA fragment encodes an α1 domain or an α1α2 domain.

In other embodiments the selected MHC molecule is selected from the group consisting of IAg ⁷ , IA ^s , DR1β * 1501 and DRA * 0101.

In other embodiments, the selected MHC molecule is an MHC class I molecule.

In another embodiment, the first linker DNA fragment is GASAG (SEQ ID NO: 29) or GGGGSGGGGSGGGGS (SEQ ID NO: 36).

In another embodiment, the second linker DNA fragment is GGSGG (SEQ ID NO: 30) or GGGSGGS (SEQ ID NO: 31).

In another embodiment, the third DNA fragment encodes an antigenic peptide capable of stimulating an MHC-mediated immune response.

In another embodiment the peptide is a mammalian GAD 65 peptide, (SEQ ID NO: 59), (SEQ ID NO: 61), (SEQ ID NO: 40), (SEQ ID NO: 39), and a mammalian milein basic peptide (SEQ ID NO: 33).

The present invention further provides a soluble fused MHC heterodimer: peptide complex, wherein the MHC heterodimer: peptide complex further comprises a fourth DNA fragment encoding at least a portion of a third domain of the selected MHC molecule, and Encoding from about 5 to about 25 amino acids and generating second and fourth DNA to generate a fused third DNA-second linker-first DNA-first linker-second DNA-third linker-fourth DNA poly fragment A third linker DNA fragment that connects the fragments to fit the frame.

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 fragment is β2 chain.

In another embodiment, the third linker DNA fragment is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32).

The second aspect of the invention provides an isolated polynucleotide molecule encoding a soluble fused MHC heterodimer: peptide complex.

A third aspect of the invention provides a fusion protein expression vector capable of expressing a soluble fused MHC heterodimer: peptide complex, which comprises the following operably linked elements, a transcriptional promoter; A first DNA fragment encoding at least a portion of the first domain of the selected MHC molecule; A second DNA fragment encoding at least a portion of a second domain of the selected MHC molecule; Encoding from about 5 to about 25 amino acids and encoding the first and second amino acid fragments so that the linkage of the first DNA fragment and the second DNA fragment by the first linker DNA fragment yields a first DNA-first linker-second DNA poly fragment; A first linker DNA fragment connecting the second DNA fragment to fit the frame; A third DNA fragment encoding an antigenic peptide capable of associating with a peptide binding groove of a selected MHC molecule; From about 5 to about such that the linkage of the first DNA-first linker-second DNA poly fragment fused with the third DNA fragment by the second linker DNA fragment results in the expression of the soluble fused MHC heterodimer: peptide complex. A second linker DNA fragment encoding a 25 amino acid and linking in frame a first DNA-first linker-second DNA poly fragment fused with a third DNA fragment; And a transfer terminator.

In one embodiment of the invention there is provided an expression vector, wherein the MHC heterodimer: peptide complex further comprises a fourth DNA fragment encoding at least a portion of a third domain of the selected MHC molecule, and a fused third DNA-second A linker-first DNA-first linker-second DNA-third linker-fourth DNA encoding an about 5 to about 25 amino acid and linking the second and fourth DNA fragments in a frame to produce A third linker DNA fragment.

Another aspect of the invention is a soluble fused MHC heterodimer: peptide complex, wherein a soluble fused MHC heterodimer: peptide complex is cultured in a cell into which an expression vector has been introduced, whereby the cell is encoded by a DNA polyfragment. And recovering the soluble fused MHC heterodimer: peptide complex.

In another aspect of the present invention there is provided a pharmaceutical composition comprising a soluble fused MHC heterodimer: peptide complex in combination with a pharmaceutically acceptable excipient.

In another aspect of the invention there is provided an antibody that binds to the epitope of the soluble fused MHC heterodimer: peptide complex.

In another aspect of the present invention, there is provided a method of treating a patient to reduce an autoimmune response, comprising administering a therapeutically effective amount of the soluble fused MHC heterodimer: peptide complex of claim 1 It provides a method comprising inducing resistance.

In another aspect of the present invention, there is provided a method of making a responder cell clone that proliferates when bound with a selected antigenic peptide provided by a stimulator cell, the method comprising: Separating reactive non-adherent, CD56-, CD8- cells to form responder cells; Stimulating the responder cells with pulsed or primed stimulator cells; Restimulating the stimulated responder cells with pulse or prime stimulator cells; Consisting of isolating the responder cell clones.

In one embodiment the responder cells are isolated from prediabetes or new diabetic patients.

In a second embodiment the responder cell clone is a T cell clone.

In another aspect of the invention the selected antigenic peptide is a GAD peptide.

These and other aspects of the invention will become apparent upon reference to the following detailed description.

Detailed disclosure of the invention

Before describing the present invention, it will be helpful to understand it to provide definitions of some terms used herein.

Fused MHC heterodimer: peptide complex: As used herein it refers to a fusion protein, such as the fused MHC heterodimer: peptide complex of the invention. Such fusion proteins will be represented by colons (:). MHC-peptide complexes that are not fusion proteins are native MHC containing proteins or externally supplied MHC molecules and are denoted by dashes (-).

Domain of the selected MHC molecule: a portion of the MHC domain alone or in combination with other portions of the MHC domain that is sufficient to form a peptide binding site capable of providing an antigenic peptide for recognition by the T cell receptor. Such MHC domains comprise the extracellular portion of two polypeptide chains of class I or class II MHC. This includes all or either of the domains of the α chain (α1, α2, or α3) and β2-microglobulin subunits of class I MHC. For example, a class I MHC domain comprises any combination of three α chain domains and / or β2 domains, where the three α chain domains are independently of one another a combination of α1, α2, or α3, or tandem α1α2 , α2α3, α1α3. Also included are the α chains (α1, α2) and β chains (β1, β2) of class II MHC. It includes α1 or α2, or tandem α1 and α2 (α1α2), independently of the others. It also includes β1 or β2, or tandem β1 and β2 (β1β2), independently of the others.

Linker DNA Fragments: DNA fragments encoding about 5 to about 25 amino acids, forming a flexible link between two DNA fragments, circularly repeating glycine residues and interspersed with serine residues. This flexible link allows the two DNA fragments to form an appropriate arrangement, such as an MHC peptide binding groove, or allows the peptide to bind appropriately into such grooves.

Antigenic Peptides: Peptides comprising epitopes recognized by immune cells, in particular T cells, capable of stimulating MHC-mediated immune responses.

Major histocompatibility complex (MHC) is a very polymorphic class of proteins, divided into two classes, class I and class II, associated with the membrane and providing antigens to T lymphocytes (T cells). MHC class I and class II molecules are distinguished by the type of cell in which it is expressed and by the class of T cells that recognize it. Class I MHC molecules (eg, HLA-A, -B, and -C molecules in human systems) are expressed in almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs), which then become antigens Destroys the cells it contains. Class II MHC molecules (eg, HLA-DP, -DQ and -DR in humans) are mainly expressed on the surface of antigen-providing cells such as B lymphocytes, dendritic cells, macrophages and the like. Class II MHC is recognized by CD4 + T helper lymphocytes (T _H ). T _H cells induce proliferation of B and T lymphocytes, amplifying the immune response to the particular antigenic peptides presented (Takahashi, Microbiol. Immunol., 37: 1-9, 1993). Two distinct antigen processing pathways are associated with both MHC classes. For example, intracellular antigens synthesized inside cells, such as viral antigens or newly synthesized cellular proteins, are processed and presented by Class I MHC. Foreign antigens obtained by antigen-presenting cells (APCs) through endocytosis from the outside of the cells are processed and presented by class II MHC. After the antigenic material is processed by proteolysis by MHC-containing cells, the resulting antigenic peptide complexes with the antigen binding groove of the MHC molecule through various non-covalent bonds. MHC-peptide complexes on the cell surface are recognized by cytotoxicity or by specific T cell receptors on helper T cells.

Human MHC on chromosome 6 (also referred to as human leukocyte antigen (HLA)) has three loci, HLA-A, HLA-B and HLA-C, the first two of which encode many of the alloantigens It has a number of alleles. Adjacent regions known as HLA-D are subdivided into HLA-DR, HLA-DQ and HLA-DP. The HLA region is now known as the human MHC region and is equivalent to the H-2 region in mice. HLA-A, -B and -C are similar to mouse H-2K, -D and -L and are class I MHC molecules. HLA-DP, -DQ and -DR are similar to mouse I-A and I-E and are class II molecules. Two classes of MHC glycoproteins have been isolated and characterized (Fundamental Immunology, 2d Ed., WEPaul (ed.), Ravens Press, NY (1989); and Roitt et al., Immunology, 2d Ed., Gower Medical Publishing, London ( 1989), both of which are incorporated herein by reference).

Human MHC class I molecules consist of a polymorphic type I integral membrane glycoprotein heavy chain of about 46 kD, covalently bound to a 12 kD soluble subunit, β2-microglobulin. The heavy chain consists of two distinct extracellular domains, peptide binding regions formed by the α1 and α2 domains as membrane ends, and CD8-binding regions derived from the α3 domains as membrane bases. β2-microglobulin is a single dense immunoglobulin-like domain that lacks a membrane anchor and is associated with a class I heavy chain or free from plasma (Germain and Margulies, Annu. Rev. Immunol. 11: 403-50 , 1993).

Human MHC Class II is a heterodimeric integral membrane protein. Each dimer consists of one α and one β chain in a non-covalent state. The two chains are similar to each other, the α chain has a molecular weight of 32-34 kD and the β chain has a molecular weight of 29-32 kD. Both polypeptide chains contain N-linked oligosaccharide residues and have extracellular amino termini and intracellular carboxy termini.

The extracellular portions of the α and β chains that make up class II molecules are subdivided into two domains of about 90 amino acids each, which are called α1, α2, and β1, β2, respectively. α2 and β2 domains each contain a disulfide-linked loop. Peptide-binding regions of class II molecules are formed by the interaction of α1 and β1 domains. This interaction results in the formation of an open antigenic peptide-binding groove, which consists of two α helix and 8 strands of β-pleat sheet platform.

The α and β chains of class II molecules are encoded by other MHC genes and are polymorphic (see Addas et al., Cellular and Molecular Immunology, 2d Ed., WBSaunders Co., New York (1994), incorporated herein by reference). ). Preferred α chain in the present invention is DRA * 0101 and preferred β chain is DRβ1 * 1501.

The immunological properties of MHC histocompatibility proteins are mainly defined by antigenic peptides that bind to it. Antigenic peptides are those comprising amino acid sequences recognized by immune cells, such as T cells. Antigenic peptides against many autoimmune diseases are known. For example, in experimentally induced autoimmune diseases, the antigens involved in the onset have been characterized: in native arthritis in rats and mice, natural type II collagen has been identified in collagen-induced arthritis and heat shock of mycobacteria in supplemental arthritis. Proteins have been identified (Stuart et al., Ann. Rev. Immunol. 2: 199-218, 1984; van Eden et al., Nature 331: 171-173, 1988); Tyroglobulin has been identified in experimental allergic thyroiditis (EAT) in mice (Marion et al., J. Exp. Med. 152: 1115-1120, 1988); Acetylcholine receptor (AChR) has been identified in experimental allergy myasthenia gravis (EAMG) (Lindstrom et al., Adv. Immunol. 42: 233-284, 1988); Myelin basic protein (MBP) and proteolipid protein (PLP) have been identified in experimental allergic encephalomyelitis (EAE) in mice and rats (Acha-Orbea et al., Ann. Rev. Imm. 7: 377-405, 1989). Additionally, target antigens have also been identified in humans: type II collagen in human rheumatoid arthritis (Holoshitz et al., Lancet ii: 305-309, 1986) and acetylcholine receptors in myasthenia gravis (Lindstrom et al., Adv. Immunol. 42). : 233-284, 1988).

Soluble fused MHC heterodimer: peptide complexes of the invention can be used as antagonists to therapeutically block binding of certain T cells and antigen-presenting cells. In addition, these molecules can induce anergy or proliferative non-responsiveness in targeted T cells. Soluble fused MHC heterodimer: peptide molecules arising for the desired autoimmune disease are antigens associated with autoimmune disease that are suitably located in the binding groove of the MHC molecule, without the need for solubilization of the MHC or external supply of independently prepared peptides. Sex peptides.

Conventional methods for producing preferred MHC class II histocompatibility proteins provide materials comprising mixtures of antigenic peptides (Buus et al., Science 242: 1045-1047, 1988; Rudensky et al., Nature 353: 622-627, 1991), it can only be partially loaded with certain antigenic peptides (Watts and McConnel, Proc. Natl. Acad. Sci. USA 83: 9660-64, 1986; Ceppellini et al., Nature 339: 392-94, 1989). . Various methods have been developed for generating heterodimers that do not provide endogenous antigens that can be loaded with selected peptides (Stern and Wiley, Cell 68: 465-77, 1992; Ljunggren et al., Nature 346, 476-80, 1990; Schumacher et al., Cell 62: 563-67, 1990). WO 95/23814 and Kozono et al. Disclosed the production of soluble murine class II molecules, IE ^ah and IA ^d , where each of them has a peptide linked to the N terminus of the β chain by a linker. Ignatowicz et al. (J. Immunol. 154: 38-62, 1995) expressed membrane-bound IA ^d linked peptides. This method incorporates the use of membrane-bound heterodimers and soluble heterodimers.

The present invention provides the advantage of soluble fused MHC heterodimers composed of two or more MHC domains linked together via flexible linkages, wherein the antigen is capable of binding to peptide binding grooves provided by soluble fused MHC heterodimers. Sex peptides are constrained thereon (through additional flexible connections). Such complexes provide soluble MHC molecules and there is no need for complex heterodimeric cleavage or formation because the components of the heterodimer and the corresponding antigenic peptide are permanently linked in a single chain arrangement. This complex eliminates inefficient and nonspecific peptide loading. The preparation of the MHC: peptide complexes of the present invention by recombinant methods allows for specific and high yield of protein production, where the final product will only contain properly selected selected MHC: peptide complexes. Soluble heterodimer as used herein is one that does not include membrane-associated MHC. Soluble MHC heterodimers of the invention are not membrane-associated. Moreover, polypeptides contained within MHC heterodimers do not comprise amino acid sequences that can act as transmembrane domains or as cytoplasmic domains.

The present invention provides a soluble fused MHC heterodimer comprising an antigenic peptide covalently linked to the amino terminus of the α or β chain of MHC via peptide bonds, wherein the C terminus of the linked α or β chain is different from It can be linked to the N terminus of the β chain to produce two or three domain MHC molecules. The present invention also provides a linkage connecting additional domains to provide four domain MHC molecules. The α chain moiety may comprise: independently of one another, α1 or α2, or tandem α1 and α2 (α1α2), or may be linked together through an intermediate peptide linkage. The β chain moiety may also include the following: independent β 1 or β 2, β 1 β 2, tandem β 1 and β 2, or intermediate peptide linkages together. Combinations of [alpha] 1, [alpha] 2, [beta] 1 and [beta] 2 can also be generated via flexible linkers such as [beta] 1 [alpha] 1 or [beta] 1 [alpha] 1 [alpha] 2.

The soluble fused MHC heterodimer: peptide complex of the present invention comprises a first DNA fragment encoding at least a portion of a first domain of a selected MHC molecule; A second DNA fragment encoding at least a portion of a second domain of the selected MHC molecule; Encoding about 5 to about 25 amino acids and encoding the first DNA fragment and the second DNA fragment so that the linkage of the first DNA fragment and the second DNA fragment produces a fused first DNA-first linker-second DNA poly fragment A first linker DNA fragment to which a frame fits; A third DNA fragment encoding an antigenic peptide capable of associating with a peptide binding groove of a selected MHC molecule; From about 5 to about 25 amino acids, such that the linkage of the fused first DNA-first linker-second DNA poly segment with the third DNA fragment by the second linker DNA fragment yields a soluble fused MHC heterodimer: peptide complex. And a second linker DNA fragment linking the third DNA fragment to fit the frame to the fused first DNA-first linker-second DNA poly fragment. The invention also provides a fourth DNA fragment encoding at least a portion of a third domain of a selected MHC molecule, and a fused third DNA-first linker-first DNA-second linker-second DNA-third linker-agent A soluble fused MHC heterodimer: peptide complex comprising a third linker DNA fragment encoding about 5 to about 25 amino acids and linking the second and fourth DNA fragments in frame to generate a 4 DNA polysegment to provide.

The first, second, third and fourth DNA fragments of the selected MHC molecule may comprise a heavy chain of class I MHC or a portion of a β2-microglobulin subunit. This will include a portion of any combination of three extracellular domains (α1, α2, α3, α1α2, or α2α3) as well as the β2 domain. It also includes the α chain or β chain of class II MHC molecules. This would include portions of α1 or α2 or tandem α1 and α2 (α1α2) independently of the others. It will also independently include β1 or β2, tandem portions of β1 and β2 (β1β2). Soluble fused MHC heterodimer: peptide complexes of the invention can be represented by a combination of α1, α2, β1 and β2 generated via flexible linkers such as peptide-β1α1, peptide-β1α1α2 and the like.

The linkers of the present invention may have a length of about 5 to about 25 amino acids, depending on the molecular model of the MHC or MHC: peptide complex. Preferably, the flexible linker consists of repeating Gly residues separated by one or more Ser residues to enable any flexible movement. In the case of class II MHC complexes, this flexibility allows for good positioning of the α and β fragments so that a suitable binding groove is formed, which also allows for maximum positioning of the peptide within the groove. The linker position and length can be designed according to the crystal structure of MHC class II molecules (Brown et al., Nature 364: 33-39, 1993), where α1 and β1 are assembled to form peptide bond grooves. Linkers linking the segments of the α and β chains together are based on the geometries of the regions at the theoretical binding position and the distance between the C and N termini of the appropriate segment. Molecular modeling based on the X-ray crystal structure of class II MHC (Stern et al., Nature 368: 215-221, 1994) shows the length of the linker connecting the antigenic peptide, α chain fragment and β chain fragment.

Soluble fused heterodimeric MHC: peptide complexes of the present invention may include cDNA of any allele that increases or is prone to a certain autoimmune disease. Certain autoimmune diseases are associated with certain MHC types. Certain haplotypes have been associated with many autoimmune diseases. For example, HLA-DR2 + and HLA-DR3 + individuals have a higher risk of developing systemic lupus erythematosus (SLE) than the general population (Reinertsen et al., N. Engl. J. Med. 299: 515-18, 1970). Myasthenia gravis is associated with HLA-D (Safwenberg et al., Tissue Antigens 12: 136-42, 1978). Susceptibility to rheumatoid arthritis is associated with HLA-D / DR in humans. It is known in the art how to identify which alleles and then which MHC-encoded polypeptides are associated with which autoimmune disease. Exemplary alleles of IDDM include DR4, DQ8, DR3, DQ3.2.

The amino acid sequences of each of many Class I and II proteins are known and the gene or cDNA has been cloned. Thus, such nucleic acids can be used to express MHC polypeptides. If the desired MHC gene or cDNA is not available, cloning methods known to those skilled in the art can be used to isolate the gene. One such method that can be used is to purify a desired MHC polypeptide, obtain a partial amino acid sequence, synthesize a nucleotide probe based on that amino acid sequence, and use that probe to identify clones with the desired gene from cDNA or genomic libraries. will be.

The invention also provides a method of making a responder T cell clone that proliferates upon binding to a selected antigenic peptide provided by a stimulator cell. Such clones can be used to identify and map antigenic peptides associated with autoimmune diseases. This peptide can then be inserted into the soluble fused MHC heterodimer: peptide complex of the present invention. This method provides for isolation and enrichment of non-adherent, CD56-, CD8- T cells that are reactive with the selected antigenic peptide. These cells are referred to herein as responder cells. Suitable responder cells can be isolated, for example, from peripheral blood mononuclear cells (PBMNCs) obtained from patients before or after the onset of the autoimmune disease of interest. For example, PBMNC can be obtained from early and new diabetic patients. Such patients may be prescreened for specific HLA markers such as DR3-DR4 or DQ3.2 with the highest association with susceptibility to IDDM. A portion of the PBMNC obtained is stored for feeding to the stimulator cells. From the rest, the desired autoreactive responder cells are purified and separated by two platings to remove adherent cells from the population and then to remove monocytes and B cells with nylon fibers. Enrichment of non-adherent CD4 + T cells is completed by sequentially plating the cells on plates coated with anti-CD8 and anti-CD56 antibodies.

Stimulator cells are pulsed or primed with whole GAD or a suitable antigenic peptide. For example, stimulator cells from PBMNCs of IDDM patients can be stimulated with antigenic GAD peptides and then combined with PBMNCs or responder cells. After 7 to 14 days, responder cell (T cell) clones are generated via limiting dilution, which is tested for antigen reactivity.

Such responder cell (T cell) clones can then be used to map epitopes that bind to MHC and are recognized by specific T cells, for example. One such method utilizes duplicate peptide fragments of autoantigens produced by trypsin digestion, or more preferably, duplicate peptides are synthesized using known peptide synthesis techniques. The peptide fragments are then tested for their ability to stimulate responder T cell clones or cell lines (see, eg, Ota et al., Nature 346: 183-187, 1990).

Once such peptide fragments are identified, for example, synthetic antigenic peptides can be specifically designed to increase binding affinity for MHC and compete-remove any naturally processed peptides. Such synthetic peptides, when bound to the soluble fused MHC heterodimer: peptide complex, allow for manipulation of the immune system in vivo to render disease-associated activated T cells immune resistant or deficient in immune capacity. This improves autoimmune diseases.

Removing the functional role of each peptide and group of peptides in the interaction of MHC molecules with peptide ligands, and also in subsequent T-cell recognition and reactivity, is difficult due to the degeneracy of peptide binding to MHC. Changes in T cell recognition or changes in the ability of a modified peptide to associate with MHC can be used to establish that a particular amino acid or group of amino acids forms part of an MHC or T cell determinant. The interaction of the modified peptides can further be assessed through the development of direct peptide-MHC binding assays or by competition with the parent peptide for presentation to T cells. Changes to peptides not involved in MHC binding can also affect T cell recognition. For example, in peptides, the specific MHC contact point may be in the central core of only a few series or individual amino acids, but the amino acids involved in T cell recognition may comprise a completely different group of residues.

In a preferred method, residues that change T cell recognition are determined by substituting amino acids at each position in the peptide in question and by evaluating whether the change in such residues changes the peptide's ability to associate with MHC ( Allen et al., Nature 327: 713-15, 1987; Sette et al., Nature 328: 395-99, 1987; O'Sullivan et al., J. Immunol. 147: 2663-69, 1991; Evavold et al., J. Immunol. 148: 347-53, 1992; Jorgensen et al., Annu. Rev. Immunol. 10: 835-73, 1992; Hammer et al., Cell 74: 197-203, 1993; Evavold et al., Immunol. Today 14: 602-9, 1993; Hammer Et al., Proc. Natl. Acad. Sci. USA 91: 4456-60, 1994; Reich et al., J. Immunol. 154: 2279-88, 1994). One method involves generating a panel of modified peptides in which individual amino acid residues or groups of amino acid residues are replaced with conservative, semi-conservative or non-conservative residues. A preferred variant of this method is an alanine scan (Ala scan) in which a series of synthetic peptides have been synthesized in which each individual amino acid is replaced with L-alanine (L-Ala scan). Alanine is an amino acid which is found at every position (buried or exposed) in the secondary structure and does not cause space disturbances or adds additional hydrogen bonds or hydrophobic side chains. Alanine substitutions can be made independently or in groups depending on the information desired. If the information relates to specific residues involved in binding, then each residue of the peptide being studied can be converted to alanine and compared to an unsubstituted peptide with binding affinity. Further structural and morphological information about each residue and the peptide as a whole can be obtained, for example, by synthesizing a series of analogs in which each residue is substituted with D-amino acids such as D-alanine (D-Ala scan) (Galantino et al. , Smith, J. and River, J. (eds.), Peptides Chemistry and Biology (Proceedings of the Twelfth American Peptide Symposium), ESCOM, Leiden, 1992, pp. 404-05). Essential residues, and non-essential residues targeted for substitution, modification or deletion by other residues that may increase the desired properties can be identified (Cunningham and Wells, Science, 244: 1081-1085, 1989; Cunningham and Wells). Proc. Natl. Acad. Sci. USA, 88: 3407-3411, 1991; Ehrlich et al., J. Biol. Chem. 267: 11606-11, 1992; Zhang et al., Proc. Natl. Acad. Sci. USA 90: 4446-50, 1993; see also Molecular Design and Modeling: Concept and Applications Part A Proteins, Peptides, and Enzyme, Methods in Enzymology, Vol. 202, Langone (ed.), Academic Press, San Diego, CA, 1991) .

The cleaved peptide is modified or modified by synthesizing a peptide whose amino acid residues are cleaved from the N- or C-terminus to determine the shortest active peptide, or between the N- and C-terminus to determine the shortest active sequence. Can be generated from peptides. Such peptides can be specifically developed to stimulate a response when bound to specific MHCs to form peptide ligands for inducing anergi in suitable T cells in vivo or in vitro.

The physical and biological properties of the soluble fused MHC heterodimer: peptide complex can be assessed in various ways. Mass spectral analysis such as electrospray and matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI TOF) analysis are commonly used in the art to provide information such as molecular weight and to confirm disulfide bond formation. FAC analysis can be used to determine the proper folding of single chain complexes.

ELISA (enzyme-linked immunosorbent assay) can be used to measure the concentration of soluble fused MHC heterodimer: peptide complex and confirm correct folding. Whole cell; Solubilized MHC removed from the cell surface; Or this assay can be used with the free soluble fused MHC heterodimer: peptide complex of the present invention. In an exemplary ELISA, antibodies detecting the recombinant MHC haplotype are coated in wells of a microtiter plate. In a preferred embodiment, the antibody is L243, which is a monoclonal antibody that recognizes only correctly folded HLA-DR MHC dimers. Those skilled in the art will appreciate that other MHC class II-specific antibodies are known and available. Alternatively, if there are no suitable haplotypes of suitable antibodies, there are a number of easy techniques and methods in the art for generating antibodies (eg, Hurrell, JGR (ed.), Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boca Raton, FL, 1982). In addition, anti-MHC class II antibodies can be used as marker reagents to purify class II molecules through techniques such as affinity chromatography, or to detect the presence of class II molecules on cells or in solution. Such antibodies are also particularly useful for western analysis or immunoblotting of purified cell-secreting material. In this regard, polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use. Additionally, proteolytic and recombinant fragments and epitope binding domains can be used here. Also suitable are chimeric antibodies, humanized antibodies, conjugated antibodies, CDR-substituted antibodies, reshaped antibodies or other recombinant full or partial antibodies.

In ELISA format, bound MHC molecules can be detected using antibodies or other binding sites capable of binding the MHC molecules. This binding site or antibody can be labeled with a detectable label or can be detected using a detectably labeled secondary antibody or binding reagent. Detectable labels or labels are known in the art and include, for example, fluorescent, colorimetric and radiolabels.

Other assay methods may include specific T cell receptors for screening for corresponding MHC-peptide complexes, which may be done in vitro or in vivo. For example, in vitro anergy analysis determines whether non-reactivity is induced in the T cells tested. In summary, MHC molecules comprising antigenic peptides in peptide binding grooves can be mixed with the responder cells, wherein the responder cells preferably comprise peripheral blood mononuclear cells (PBMN) (B and T lymphocytes, monocytes and dendritic cells). Heterogeneous population), PBMNC lymphocytes, freshly isolated T lymphocytes, in vivo primed splenocytes, cultured T cells, or established T cell lines or clones. Particular preference is given to mammalian responder cells immunized with the antigenic peptide or having an apparent cellular immune response against the antigenic peptide.

Thereafter, such responder cells are combined with stimulator cells (antigen presenting cells; APCs) pulsed or primed with the same antigenic peptide. In a preferred embodiment, the stimulator cells are antigenic peptide-presenting cells such as PBMNC, PBMNC lacking lymphocytes, suitable antigenic peptide-presenting cell lines or clones (such as EBV-transformed B cells, etc.), EBV transformed autografts. And non-autologous PMNC, mouse L cells or exposed lymphocyte cells BLS-1 as genetically designed antigen presenting cells, in particular DRB1 * 0401, DRB1 * 0404 and DRB1 * 0301, etc. (Kovats et al., J. Exp. Med. 179). : 2017-22, 1994), or in vivo or in vitro primed or pulsed splenocytes. Particular preference is given to mammalian stimulator cells immunized with the antigenic peptide or having an apparent cellular immune response against the antigenic peptide. In some assay formats, it is desirable to inhibit the proliferation of stimulator cells prior to mixing with the responder cells. Such inhibition may be achieved, for example, by exposure to antimitotic agents such as mitomycin C or gamma irradiation. Suitable negative controls are included (absence; syngeneic APC; experimental peptide; APC + peptide; MHC: peptide complex; control peptide +/- APC). Furthermore, to demonstrate that non-responsiveness shows anergi, proliferation assays can be performed in +/- recombinant IL-2 duplex, since it has been demonstrated that IL-2 can recover immune deficient cells.

After approximately 72 hours of incubation, activation of responder cells is measured in response to stimulator cells. In a preferred embodiment, responder cell activation is determined by measuring proliferation using ³ H-thymidine acquisition (Crowley et al., J. Immunol. Meth. 133: 55-66, 1990). Alternatively, responder cell activation can be measured by production of cytokines such as IL-2 or by determining the presence of responder cell-specific, in particular T cell-specific activation markers. Cytokine production can be assayed by testing the ability of the stimulator + responder cell culture supernatant to stimulate cytokine-dependent cell growth. Responder cell- or T cell-specific activation markers can be detected using antibodies specific for such markers.

Preferably, the soluble fused MHC heterodimer: peptide complex induces non-reactive (eg anergi) in antigenic peptide-reactive responder cells. In addition to recognizing soluble fused MHC heterodimer: peptide complexes, responder cell activation requires the involvement of co-receptors on stimulator cells (APCs) stimulated with co-stimulatory molecules. By blocking or eliminating the stimulation of such co-receptors (e.g., exposing the responder cells to purified soluble fused MHC heterodimer: peptide complexes, blocking with anti-receptor or anti-ligand antibodies, or encoding such receptors) Knocking out a gene) can render the responder cells non-reactive to the antigen or to the soluble fused MHC heterodimer: peptide complex.

In a preferred embodiment, the responder cells are obtained from a source that exhibits an autoimmune disease or indication. Autoantigen-reactive T cell clones or cell lines are also preferred responder cells. In another preferred embodiment, the stimulator cells are obtained from a source that exhibits an autoimmune disease or indication. In addition, APC cell lines or clones that can be suitably treated and / or can provide autoantigens to the responder cells are preferred stimulator cells. In particularly preferred embodiments, the responder and stimulator cells are obtained from a source having diabetes or multiple sclerosis.

Here, Responder T cells can be selectively amplified and / or stimulated to generate a T cell population specific for the antigenic peptide. For example, antigenic peptide-reactive responder cells may be selected by flow cytometry, in particular by fluorescence activated cell sorting. This responder cell population can be maintained by repeated stimulation with APC providing the same antigenic peptide. Responder cell clones or cell lines can also be established from this responder cell population. Moreover, this responder cell population can be used to map epitopes of antigenic peptides and proteins from which they are derived.

Other methods of assessing the biological activity of soluble fused MHC heterodimer: peptide complexes are known in the art and have been developed using microphysiometers to measure the production of acidic metabolites in T cells following interaction with antigenic peptides. microphysiometer) and the like. Other assays include competitive assays comparing soluble fused MHC heterodimer: complex reactions to normal antigens. In addition, measurement of indicator production, such as cytokines or γ interferon, can provide an indication of complex responses.

Similar assays and methods can be developed and used for animal models of disease carried by the MHC: peptide complex. For example, a model system of insulin-dependent diabetes mellitus (IDDM), polynucleotides encoding I-Ag ⁷ MHC class II molecules in NOD mice, can be combined with autoantigenic peptides from GAD to study the induction of non-responsiveness in animal models. Can be.

Soluble fused MHC heterodimer: peptide complexes can be tested in vivo in many animal models of autoimmune disease. For example, NOD mice are spontaneous models of IDDM. Treatment with soluble fused MHC heterodimer: peptide complexes before or after the onset of disease is performed by assay of urine glucose levels in NOD mice as well as in vitro to assess reactivity to known autoantigens. T cell proliferation assays can be monitored (see, eg, Kaufman et al., Nature 366: 69-72, 1993). In addition, induced models of autoimmune diseases such as EAE can be treated with a suitable soluble fused heterodimer: peptide complex. Treatment in a retarded or inhibitory manner can be tracked by monitoring the clinical signs of EAE.

NOD mouse species (H-2g ⁷ ) is a murine model of autoimmune IDDM. In NOD mice the disease is characterized by anti-islet cell antibodies, severe insulin salts, and evidence of autoimmune destruction of beta-cells (see, eg, Kanazawa et al., Diabetologia 27: 113, 1984). This disease can be passively metastasized to lymphocytes and prevented by cyclosporin-A treatment (Ikehara et al., Proc. Natl. Acad. Sci. USA 82: 7743-47, 1985; Mori et al., Diabetologia 29: 244-47 , 1986). Untreated animals develop severe glucose intolerance and ketosis and die within weeks of the onset of the disease. The current colonies (# 11 NOD / CaJ) show a higher incidence of diabetes in males compared to other colonies, and 50-65% of males and 90-95% of females develop diabetes within the first 7 months after survival ( Pozzilli et al., Immunology Today 14: 193-96, 1993). Crossbreeding studies have defined at least two loci involved in disease susceptibility, one of which maps to MHC. Characterization of NOD class II antigens at the serological and molecular level suggests that susceptibility to autoimmune diseases is associated with I-Ag ⁷ (Acha-Orbea and McDevitt, Proc. Natl. Acad. Sci. USA 84: 2435 -39, 1987).

The development of diabetes can be studied in several ways, such as by manifesting naturally occurring diseases or in an adaptive metastasis model (Miller et al., J. Immunol. 140: 52-58, 1988). NOD mice naturally express autoimmune diabetes. In NOD / CaJ mice, diabetes in females is first observed at the age of three months. Young NOD / CaJ female mice can be treated with peptides, peptide: MHC complexes or control agents and then followed for 6 months to see if there is evidence of disease expression. NOD mice can be screened for diabetes by monitoring urine glucose levels, cutting the tails of animals exhibiting positive urine levels and further analyzing the blood for blood glucose levels with a glucometer. Mice with blood glucose levels of 250 mg / dl or higher are clearly classified as diabetic. This method involves treating autoreactive T cells.

IDDM can also be adaptively transferred by transplanting spleen cells from diabetic donors to non-diabetic recipients (Baron et al., J. Clin. Invest. 93: 1700-08, 1994). The method includes treating activated mature T cells in vivo. In summary, NOD / CaJ mice are examined (730 rad) and optionally divided into treatment groups. Preferably, about 1.5x10 ⁷ splenocytes are isolated from new diabetic mice and injected intravenously into recipient mice of 7-8 weeks of age with non-diabetic NOD, and amounts of 10, 5, or 1 μg / mouse after 6 hours. By intravenous injection of saline, peptide or MHC: peptide complex. Injections are repeated 4, 8 and 12 days after the first injection. The urine assay, and at the time of sacrifice, mice are tested for onset of diabetes by blood glucose. Treatment of these mice with the MHC: peptide complex is expected to prolong the period before onset of diabetes and / or to prevent or alleviate the disease. On the day when the first animal shows obvious signs of diabetes, mice in each treatment group are randomly selected and sacrificed, and the spleen and pancreas are removed for immunohistochemical analysis. The end of the study is the point at which all mice in the control group (saline) develop diabetes. Mice treated with saline usually develop diabetes within about 20 days.

Suitable expression systems for the production of suitable soluble MHC heterodimer: peptide complexes are available and known in the art. Various prokaryotes, fungi, and eukaryotic host cells are suitable for soluble fused MHC heterodimer: peptide complexes.

Prokaryotes useful as host cells in accordance with the present invention are most often various strains of Escherichia coli. However, other microbial strains, such as Bacillus, such as Bacillus subtilis, various Pseudomonas species, or other bacterial strains, may also be used.

According to the present invention, by operably linking a genetically designed nucleic acid coding sequence to a signal indicative of gene expression in a prokaryote, the soluble sequence from a nucleotide sequence designed by recombination encoding a soluble fused MHC heterodimer: peptide polypeptide The fused MHC heterodimer: peptide complex is expressed. A nucleic acid is one that is operatively linked when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is one that is operatively linked to a coding sequence when it affects the transcription of the sequence. In general, operably linked means that the nucleic acid sequences to be linked are contiguous, and that if they need to link two protein coding regions, they are contiguous and the reading frame is correct.

Genes encoding soluble fused MHC heterodimer: peptide complexes can be inserted into expression vectors, cloning vectors or vectors, which terms are used interchangeably herein and are usually plasmids or other that can be replicated in selected host cells. Represents a nucleic acid molecule. Expression vectors can be autonomously replicated, or can be replicated by insertion into the genome of a host cell by methods known in the art. Vectors that autonomously replicate will have an automatic replication sequence (ARS) or origin of replication that is functional in the selected host cell.

Plasmid vectors containing replication sites and control sequences extracted from species compatible with the selected host are used. For example, E. coli is typically transformed using a derivative of pBR322, which is a plasmid extracted from E. coli species by Bolivar et al., Gene 2: 95-113, 1977. Frequently, vectors are preferably available in one or more host cells, such as E. coli for cloning and construction, and Bacillus cells for expression.

Expression vectors typically include an expression cassette or transcription unit that contains all the elements necessary to express DNA encoding MHC molecules in a host cell. Typical expression cassettes include a DNA sequence encoding a soluble fused MHC heterodimer: peptide complex and a promoter operably linked to a ribosomal binding site. This promoter is preferably located away from the heterologous transcription start position approximately equal to the distance from the transcription start position in the natural setting. However, as is known in the art, some variation in this distance can be tolerated without loss of promoter function. In addition to the promoter sequence, the expression cassette may also include a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence, or may be obtained from another gene.

Commonly used prokaryotic regulatory sequences defined herein to include a promoter for transcription initiation, optionally in conjunction with a ribosomal binding site sequence, include betalactamase (penicillinase) and lactose (lac) promoter systems (Chang et al. , Nature 198: 1056, 1977) and tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8: 4057-74, 1980) and other commonly used promoters and lambda-extracted P _L promoters and N-gene ribosomes Binding sites (Shimataka et al., Nature 292: 128-32, 1981). Any available promoter system that functions in prokaryotes can be used.

Structural or regulatory promoters may be used in the present invention. Regulatory promoters are useful because host cells can be grown at high density before expression of the soluble fused MHC heterodimer: peptide complex is induced. High level expression of heterologous proteins slows cell growth in some situations. Particularly suitable regulatory promoters for use in E. coli include the bacteriophage lambda P _L promoter, the hybrid trp-lac promoter (Amann et al., Gene 25: 167-78, 1983), and the bacteriophage T7 promoter.

For expression of soluble fused MHC heterodimer: peptide complexes in prokaryotic cells other than E. coli, promoters that function in certain prokaryotic cell species are required. Such promoters may be obtained from genes cloned from the species, or heterologous promoters may be used. For example, the hybrid trp-lac promoter works on Bacillus as well as E. coli.

In addition, the expression of soluble fused MHC heterodimer: peptide complexes in prokaryotic cells requires a ribosomal binding site (RBS). For example, E. coli's RBS consists of a sequence of nucleotides 3-9 nucleotides in length located 3-11 nucleotides upstream of the start codon (Shine and Dalgarno, Nature, 254: 34-40, 1975; Steitz, In Biological regulation and development: Gene expression (ed.RF Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY.

Translation coupling can be used to increase expression. This method uses a short upstream open reading frame derived from a high expressing gene inherent in a translation system located downstream of the promoter, and a termination codon behind the ribosome binding site, followed by some amino acid codons. Immediately before the stop codon, there is a second ribosome binding site, followed by the stop codon followed by a start codon for initiation of translation. This system dissolves the secondary structure of RNA, which allows for efficient initiation of translation (see Squires et al., J. Biol. Chem. 263: 16297-16302, 1988).

Soluble fused MHC heterodimer: peptide complexes can be expressed intracellularly or secreted from cells. Intracellular expression usually shows high yields. However, some of the proteins may be in the form of insoluble inclusion bodies. Although some of the intracellular MHC polypeptides of the present invention may be active when obtained after cytolysis, the amount of soluble active MHC polypeptide can be increased by performing the refolding method (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor, NY, 1989; Marston et al., Bio / Technology 2: 800-804, 1985; Schoner et al., Bio / Technology 3: 151-54, 1985). Preferably, for purification and refolding, the cell pellet is lysed and refolded in urea-borate buffer followed by urea-borate-DTT buffer and silica gel based Vydac (Hewlett Packard, Wilmington, DE) or polymer based Poros-R2 Reversed phase HPLC purification is performed using PerSeptive Biosystems resin, where the bead size varies with the scale of the culture and is described in more detail below. Optionally, especially for large scale refolding, the sample is ultrafiltered into urea-borate buffer and then 0.2 μM to 1 mM, preferably 0.2 to 20 μM, of copper sulfate, followed by immediate folding. Refolding occurs over a range of 0.1 to 2.5 mg / ml protein.

One or more MHC: peptide complexes can be expressed in a single prokaryotic cell by placing multiple transcription cassettes in a single expression vector, or by using different selectable markers in each expression vector used in a cloning strategy.

The second method of expressing the MHC: peptide complex of the present invention is to allow the polypeptide to be secreted from the cell into the periphery or extracellular medium. The DNA sequence encoding the MHC polypeptide is linked to a cleavable signal peptide sequence. This signal sequence directs the migration of the MHC: peptide complex through the cell membrane. An example of a vector suitable for use in E. coli comprising a promoter-signal sequence unit is pTA1529, which has an E. coli phoA promoter and a signal sequence (eg, Sambrook et al., Oka et al., Proc. Natl. Acad. Sci USA 82: 7212-16, 1985; Talmadge et al., Proc. Natl. Acad. Sci. USA 77: 39892, 1980; Takahara et al., J. Biol. Chem. 260: 2670-74, 1985). Again, multiple polypeptides can be expressed in a single cell for periphery association.

MHC: peptide complexes of the invention can also be produced as fusion proteins. This method often shows high yield since normal prokaryotic regulatory sequences direct transcription and translation. In E. coli, lacZ fusion is used to express heterologous proteins. Suitable vectors such as the pUR, pEX, and pMR100 families are readily available (see, eg, Sambrook et al.). In some applications, it may be desirable to cleave non-MHC amino acids from the fusion protein after purification. This can be done by any of several methods known in the art, including cyanogen bromide, cleavage by proteases, or cleavage by factor X (eg, Sambrook et al., 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). Cleavage sites can be designed into the gene of the fusion protein at the desired cleavage point.

External genes such as soluble fused MHC heterodimer: peptide complexes can be expressed in E. coli as fusions with binding partners such as glutathione-S-transferase (GST), maltose binding protein, or thioredoxin. have. Such binding partners are translated in large quantities and can be used to overcome inefficient translation initiation of eukaryotic information in E. coli. Fusion with such a binding partner results in high level expression, and the binding partner is easily purified and then cleaved from the protein of interest. Such expression systems are available from myriad sources, such as Invitrogen Inc. (San Diego, Calif.) And Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

A method for obtaining recombinant proteins from E. coli that maintains N-terminal integrity has been described by Miller et al., Biotechnology 7: 698-704 (1989). The gene of interest in this system is generated as a C-terminal fusion with the first 76 residues of the yeast ubiquitin gene comprising a peptidase cleavage site. Cleavage at the junction of two sites results in a protein with intact N-terminal residues.

Vectors comprising nucleic acids encoding soluble fused MHC heterodimer: peptide complexes are transformed into prokaryotic host cells for expression. Transformation refers to inserting a vector containing a nucleic acid of interest directly into a host cell by well known methods. The particular method used to insert the genetic material into the host cell for expression of the soluble fused MHC heterodimer: peptide complex is not particularly important. Any of the well known methods of inserting foreign nucleotide sequences into host cells can be used. It is only necessary that the particular host cell used can express the gene.

Other transformation methods, depending on the type of prokaryotic host cell, include electroporation; transfection with calcium chloride, rubidium chloride calcium phosphate, or other materials; micropropellant shock; infection (if the vector is an infectious agent); And other methods. In general, see Sambrook et al., And Ausubel et al., (Eds.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. The cells into which the nucleic acids described above are introduced are intended to include the progeny of such cells. Also included in the present invention are transformed prokaryotic cells comprising the expression vector of the soluble fused MHC heterodimer: peptide complex.

Prokaryotic lines expressing large amounts of soluble fused MHC heterodimer: peptide complex polypeptides are generated using standard transfection or transformation methods, and then the polypeptides are purified using standard techniques. See, eg, Colley et al., J. Chem. 64: 17619-22, 1989; And Methods in Enzymology, Guide to Protein Purification, M. Deutscher, ed., Vol. See 182 (1990). Recombinant cells are grown and soluble fused MHC heterodimer: peptide complexes are expressed. The purification protocol will depend on whether the soluble fused MHC heterodimer: peptide complex is expressed intracellularly, in the periphery, or secreted from the cell. For intracellular expression, cells are obtained, lysed and recovered from cell lysates (Sambrook et al.). Peripheral MHC polypeptides are released from the periphery by standard techniques (Sambrook et al.). Once the MHC polypeptide is secreted from the cells, culture medium is obtained for purification of the secreted protein. Medium is typically purified by centrifugation or filtration to remove cells and cell fragments.

The MHC polypeptide is adsorbed onto any suitable resin (eg, CDP-Sepharose, Asialoprothrombin-Sepharose 4B, or Q Sepharose), or by ammonium sulfate fractionation, the use of polyethylene glycol precipitation, or by ultrafiltration. It can be concentrated by. Other methods known in the art may equally be suitable.

Further purification of the MHC polypeptide may be performed by standard techniques such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, reverse phase HPLC or other protein purification techniques used to obtain homogenates. The purified protein is then used to produce the pharmaceutical composition.

DNA constructs may also include DNA fragments necessary to direct secretion of the polypeptide or protein of interest. Such DNA fragments may comprise at least one secretory signal sequence. Secretory signal sequences, also called leader sequences, prepro sequences and / or pre sequences, are amino acid sequences that play a role in the secretion of a mature polypeptide or protein from a cell. Such sequences feature the core of hydrophobic amino acids and are typically (but not entirely) found at the amino terminus of newly synthesized proteins. Secretory signal sequences can be of the protein of interest, or can be extracted from other secreted proteins (eg, t-PA, a preferred mammalian secretory leader), or newly synthesized. Secretory signal sequences are linked to the correct reading frame to the DNA sequence encoding the protein of the invention. While any signal sequence may be located anywhere in the DNA sequence of interest, secretory signal sequences are often located 5 'to the DNA sequence encoding the polypeptide of interest (eg, Welch et al., US Pat. No. 5,037,743; Holland). Et al., US Pat. No. 5,143,830). Very often secretory peptides are cleaved from mature proteins during secretion. Such secretory peptides include processing sites that allow cleavage of secretory peptides from mature proteins as they pass through the secretory pathway. Examples of such processing sites are those recognized by the Saccharomyces cerevisiae KEX2 gene as dibasic cleavage sites or Lys-Arg processing sites. Processing sites can be encoded in secretory peptides or added to the peptides, eg, by mutagenesis in vitro.

Secretion signals include α factor signal sequences (prepro sequences; Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjan et al., US Pat. No. 4,546,082; Brake, EP 116,201), PHO5 signal sequences (Beck et al., WO 86 / 00637), BAR1 secretory signal sequence (Mackay et al., US Pat. No. 4,613,572; Mackay, WO 87/002670), SUC2 signal sequence (Carlsen et al., Molecular and Cellular Biology 3: 439-447, 1983), a-1- Antitrypsin signal sequence (Kurachi et al., Proc. Natl. Acad. Sci. USA 78: 6826-6830, 1981), a-2 plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102: 1033- 1042, 1987) and tissue plasminogen activator signal sequences (Pennica et al., Nature 301: 214-221, 1983). In addition, secretory signal sequences are established by, for example, von Heinje (European Journal of Biochemistry 133: 17-21, 1983; Journal of Molecular Biology 184: 99-105, 1985; Nucleic Acids Research 14: 4683-4690, 1986). Can be synthesized according to the scheme. Another signal sequence is the synthetic signal LaC212 spx (1-47)-ERLE described in WO 90/10075.

Secretory signal sequences can be used alone or in combination. For example, the first secretory signal sequence can be used in combination with a sequence encoding the third domain of the barrier (as described in US Pat. No. 5,037,243, incorporated herein by reference). The third domain of the barrier can be located in the appropriate reading frame on the 3 'side of the DNA fragment of interest or on the 5' side of the DNA fragment, and in the appropriate reading frame with the DNA fragment and secretory signal sequence of interest.

Selection of suitable promoters, terminators and secretion signals of all expression systems is within the skill of one of ordinary skill in the art. Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (Gene Expression Technology, Methods in Enzymology, Vol. 185, Goeddel (ed.), Academic Press, San Diego, CA, 1990 and Guide to Yeast See Genetics and Molecular Biology, Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San Diego, CA, 1991; all of which are incorporated herein by reference). Proteins of the invention can also be expressed in fibrous fungi, such as the fungus Aspergillus strain (McKnight et al., US Pat. No. 4,935,349, which is incorporated herein by reference). Expression of cloned genes in cultured mammalian cells and in E. coli is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; incorporated herein by reference). It has been described in detail. As will be apparent to those skilled in the art, regulatory sequences, vectors and methods well established in the literature can be used to express the proteins of the invention in other host cells such as algae, insects and plant cells.

Yeast vectors suitable for use in the present invention in yeast include YRp7 (Struhl et al., Proc. Natl. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), POT vectors (Kawasaki et al., US Pat. No. 4,931,373, which is hereby incorporated by reference), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978) and derivatives thereof. Preferred promoters for use in yeast include promoters of yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or promoter of alcohol dehydrogenase gene (Young et al., Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., (Eds.), P.355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). Other promoters TPI1 promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4 ^c promoter (Russell, etc., Nature 304: 652-654, 1983; Irani and Kilgore, U.S. Patent Application No. 07 / No. 784 653, and CA 1,304,020 EP 284 044, all of which are incorporated herein by reference. Expression units may also include transcriptional terminators such as TPI1 terminators (Alber and Kawasaki).

Yeast cells, especially cells of Saccharomyces species, are suitable hosts for producing compounds of the invention. Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins therefrom are described, for example, in Kawasaki, US Pat. No. 4,599,311; Kawasaki et al., US Pat. No. 4,931,373; Brake, US Pat. No. 4,870,008; Welch et al., US Pat. No. 5,037,743; And Murray et al., US Pat. No. 4,845,075, all of which are incorporated herein by reference. Transformed cells are selected by a selectable marker, often a phenotype that is determined by drug resistance or the ability to grow in the absence of certain nutrients (eg, leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (US Pat. No. 4,931,373), which allows for the selection of cells transformed by growth in glucose-containing medium. Preferred secretory signal sequences for use in yeast are those of the S. cerevisiae MFα1 gene (Brake; Kurjan et al., US Pat. No. 4,546,082). Promoters and terminators suitable for use in yeast include glycosylase genes (see, eg, Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat.No. 4,615,974; and Bitter, U.S. Pat.No.4,977,092) and alcohol dehydrogenase genes. It includes. See also US Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, incorporated herein by reference. Transformation systems for other hosts are known in the art including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragillis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa. For example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986; Cregg, US Pat. No. 4,882,279; And Stroman et al., US Pat. No. 4,879,231.

Other fungal cells are also suitable as host cells. For example, Aspergillus cells can be used according to the method of US Pat. No. 4,935,349, McKnight et al., Incorporated herein by reference. Methods for transforming Acremonium chrysogenum have been disclosed by US Pat. No. 5,162,228, Sumino et al., Incorporated herein by reference. Methods for transforming Neurospora are disclosed by Lambowitz, US Pat. No. 4,486,533, incorporated herein by reference.

The host cell containing the DNA construct of the present invention is then cultured to produce a heterologous protein. Cells are cultured according to standard methods in culture medium containing the nutrients necessary for the growth of certain host cells. Various suitable media are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins, inorganic salts and growth factors. Growth media will generally be selected for cells containing the DNA construct, for example by drug resistance or lack of essential nutrients supplemented with selectable markers on the DNA construct or co-transfected with the DNA construct. .

Yeast cells are, for example, preferably cultured in a chemically defined medium consisting of non-amino acid nitrogen sources, inorganic salts, vitamins and essential amino acid supplements. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 6.5. The method of maintaining a stable pH preferably includes buffering and constant pH control through the addition of sodium hydroxide. Preferred buffers include succinic acid and bis-tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells with defects in the genes required for asparagine-linked glycosylation are preferably grown in medium containing an osmotic stabilizer. Preferred osmotic stabilizers are sorbitol supplemented into the medium at a concentration between 0.1M and 1.5M, preferably at a concentration of 0.5M or 1.0M. Culture Mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium suitable for the particular host cell used is within the skill of the art.

Methods of inserting exogenous DNA into mammalian host cells include calcium phosphate-mediated 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, 1982) and DEAE-dextran mediated transfection (Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), all of which are incorporated by reference. Boehringer Mannheim transfection-reagent (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethyl ammoniummethylsulfate; Boehringer Mannheim, Indianapolis, IN) or Lipofectin Reagent (N- [1- (2,3-Dioleyloxy) propyl] -N, N, N-trimethylammonium chloride and dioleyl phosphatidylethanolamine; GIBCO-BRL, Gaithersburg, MD) Cationic lipid transfection using commercially available reagents can be used. The preferred mammalian expression plasmid is Zem229R (this was deposited on September 28, 1993 as an E.coli HB101 transformant in the Rockville Parklon Drive 12301 American Type Culture Collection, Maryland, USA under the name of the Budapest Treaty and Accession No. 69447 was issued. Was granted). The production of recombinant proteins in cultured mammalian cells is described, for example, in Levinson et al., US Pat. No. 4,713,339; Hagen et al., US Pat. No. 4,784,950; Palmiter et al., US Pat. No. 4,579,821; Ringold, US Pat. No. 4,656,134, all of which is incorporated by reference. Preferred cultured mammalian cells include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), DG44, and 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) cell lines. Additional suitable cell lines are known in the art and are available from public depositories such as the Rockville American Type Culture Collection, Maryland, USA. In general, strong transcriptional promoters, such as the promoter of SV-40 or cytomegalovirus, are preferred. See, eg, US Pat. No. 4,956,288. Other suitable promoters include those of the metallothionein gene (US Pat. Nos. 4,579,821 and 4,601,978, incorporated herein by reference) and adenovirus major late promoters.

Drug resistance is generally used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are often referred to as transformants. Cells that are cultured in the presence of a selective reagent and capable of delivering the gene of interest to its offspring are shown as stable transformants. Preferred selectable markers are genes that encode resistance to the antibiotic neomycin. The selection is carried out in the presence of neomycin-type drugs such as G-418 and the like. Selection systems can also be used to increase the expression level of the gene of interest, which is the process represented as amplification. Amplification is performed by culturing the transformant in the presence of low levels of selective reagents and increasing the amount of selective reagents, thereby selecting for cells that produce high levels of the product of the introduced gene. Preferred amplifiable and selectable markers are dihydrofolate reductases that confer resistance to methotrexate. Other drug resistance genes can also be used (eg, hygromycin resistance, multi-reagent resistance, puromycin acetyltransferase).

Soluble fused MHC: peptide complexes of the present invention are first isolated from a cell, followed by conventional purification methods, such as Coy et al. (Peptides Structure and Function, Pierce Chemical Company, Rockford, IL, pp369-72, 1983). By ion-exchange and partition chromatography described in, for example, reverse phase chromatography described in Andreu and Merrifield (Eur. J. Biochem. 164: 585-90, 1987), or Kofod et al. (Int. J. Peptide and Protein Res. 32: 436-40, 1988). Further purification may be by other conventional purification means such as liquid chromatography, gradient centrifugation, gel electrophoresis. Protein purification methods are known in the art (see generally, Scopes, R., Protein Purification, Springer-Verlag, NY, 1982, incorporated herein by reference) and may be applied to the purification of recombinant polypeptides described herein. Soluble fused MHC heterodimer: peptide complexes of at least about 50% purity are preferred, with at least about 70-80% purity being more preferred, with purity of about 95-99% or more being particularly preferred for pharmaceutical use. . Once partially or homogeneously purified as desired, the soluble fused MHC heterodimer: peptide complex can then be used for diagnostic or therapeutic purposes, as described in detail below.

Soluble fused MHC heterodimer: peptide complexes of the invention can be used in a method of reducing-modulating a portion of the immune system that is responsive to autoimmune disease. Soluble fused MHC heterodimer: peptide complexes of the present invention are intended for use as an immunotherapeutic agent for inducing immune resistance or non-responsiveness (anergy) in patients who are likely or will be immune to a specific autoantigen. I find it useful. By methods known in the art, patients who have or are likely to exhibit certain autologous diseases are identified and the MHC type is determined. T cells of a patient can be examined in vitro to determine autoantigenic peptides recognized by the patient's autoreactive T cells using the complexes and methods described herein. The patient can then be treated with the complex of the present invention. Such methods will generally comprise administering the soluble fused MHC heterodimer: peptide complex in an amount sufficient to prolong the period prior to the onset of autoimmune disease and / or alleviate or prevent the disease. Thus, soluble fused MHC heterodimer: peptide complexes of the present invention are believed to be useful for therapeutic and diagnostic applications associated with autoimmune diseases.

The methods of treatment of the present invention may include, for example, oral tolerance (Weiner et al., Nature 376: 177-80, 1995), or intravenous resistance. Conditions of induction of resistance depend on a variety of factors, but resistance can be induced in mammals. The exact dosage and frequency of administration will vary in order to induce immunological resistance in adults at risk of or suffering from autoantigen-related diseases such as IDDM. For example, in adults, about 20-80 μg / kg may be administered by various routes, ie, parenterally, orally, by aerosol, by transdermal injection, or the like. In newborns, resistance can be induced by parenteral injection or, more commonly, by oral administration in a suitable formulation. The exact dosage and route and frequency of administration will vary.

Soluble fused MHC heterodimer: peptide complexes will typically be more resistant when administered in soluble form than in aggregate or particle form. The persistence of the soluble fused MHC heterodimer: peptide complex of the present invention is generally required to maintain resistance in adults and, therefore, may require administration of more common complexes, or in forms that extend the half-life of the complex. Can be. For example, Sun et al., Proc. Natl. Acad. Sci. See USA 91: 10795-99, 1994.

In another aspect of the invention, for prophylactic and / or therapeutic treatment, according to conventional methods, for topical administration such as a pharmaceutically acceptable carrier or parenteral, topical, oral, or aerosol or transdermal administration, etc. Pharmaceutical compositions are provided that comprise soluble fused MHC heterodimer: peptide complexes of the invention contained in excipients. This composition is typically in a form suitable for systemic injection or infusion and may be formulated intact with sterile water or isotonic saline or glucose solution. The formulation may further comprise one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in the form of, for example, liquids, powders, emulsions, suppositories, liposomes, transdermal patches, and tablets. Those skilled in the art will formulate the compounds of the present invention in a suitable manner and in accordance with established practices, as described in Remington's Pharmaceutical Science, Gennaro (ed.), Mack Publishing Co., Easton, PA 1990, which is incorporated herein by reference. can do.

Pharmaceutical compositions of the invention are administered at one to weekly intervals. An effective amount of such pharmaceutical composition is an amount that clinically significantly reduces harmful T cell-mediated immune responses to autoantigens, such as those associated with IDDM, or provides other pharmacologically beneficial effects. Such amount will depend in part on the particular condition being treated, the age, weight and general state of health of the patient, and other factors apparent to those skilled in the art. Preferably, the amount of soluble fused MHC heterodimer: peptide complex administered will be in the range of 20-80 μg / kg. Compounds with significantly increased half-life can be administered at lower doses or at less frequent frequencies.

In addition, kits for therapeutic or diagnostic use may be supplied. Thus, the compositions of the present invention may be provided in lyophilized form, usually in a container. Soluble fused MHC heterodimer: peptide complexes are included in the kit with instructions when used and optionally with buffers, stabilizers, biocides, and inactive proteins. Generally, this selective material will be present in up to about 5% by weight, based on the amount of soluble fused MHC heterodimer: peptide complex, usually the total amount based on the concentration of soluble fused MHC heterodimer: peptide complex It will be present at least about 0.001% by weight. If an excipient is present at about 1-99% by weight of the total composition, it may be desirable to include an inert extender or excipient to dilute the active ingredient.

In one aspect of the invention, soluble fused MHC heterodimer: peptide complexes are used to prepare antibodies for diagnostic or therapeutic use. The term antibody as used herein includes antigen-binding fragments such as polyclonal antibodies, monoclonal antibodies, F (ab ') ₂ and Fab fragments, as well as recombinantly produced binding partners. This binding partner inserts a variant or CDR region from a gene encoding a specific binding antibody. The affinity of the monoclonal antibody or binding partner can be readily determined by one skilled in the art (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).

Methods of making polyclonal and monoclonal antibodies are well described in the literature (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, JGR, Ed., Monoclonal Hybridoma Antibodies Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which is incorporated herein by reference). As will be apparent to those skilled in the art, polyclonal antibodies can be produced from various warm blooded animals, such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice, or rats. Immunogenicity of soluble fused MHC heterodimer: peptide complexes may be increased through the use of adjuvants, such as Freund's complete or incomplete adjuvants. Various assays known to those skilled in the art can be used to detect antibodies that specifically bind to soluble fused MHC heterodimer: peptide complexes. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include simultaneous immunoelectrophoresis, radio-immunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assay, dot blot assay, inhibition or competition assay, and sandwich assay.

Other techniques of monoclonal antibody preparation can be used to prepare and express recombinant monoclonal antibodies. In summary, we isolated mRNA from B cell populations and used it to construct heavy and light chain immunoglobulins in suitable vectors such as λIMMUNOZAP (H) and λIMMUNOZAP (L) vectors that can be obtained from Stratogene Cloning Systems (La Jolla, Calif.). Create a cDNA expression library. The vectors are then screened or coexpressed individually to form Fab fragments or antibodies (Huse et al., Science 246: 1275-81, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32 , 1989). Subsequently, positive plaques are converted to non-soluble plasmids, which allows for high level expression of monoclonal antibody fragments in E. coli.

Antibodies of the invention can be generated by immunizing animals selected from various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice, and rats with recombinant soluble fused MHC heterodimer: peptide complexes. The serum of such animals is a source of polyclonal antibodies. In addition, antibody producing cells obtained from immunized animals are immortalized and screened. Since the development of human monoclonal antibodies against human antigens, such as soluble fused MHC heterodimer: peptide complexes, may differ from conventional immortalization techniques, it may be desirable to make non-human antibodies first. Using recombinant DNA technology, the antigen binding region of a non-human antibody is transferred to the corresponding site of the human antibody coding region, resulting in substantially human antibody molecules. Such methods are generally known in the art and described, for example, in US Pat. Nos. 4,816,397 and EP Publications 173,494 and 239,400, both incorporated herein by reference.

In another aspect of the invention, soluble fused MHC heterodimer: peptide complexes can be used to clone T cells having a specific receptor for the soluble fused MHC heterodimer: peptide complex. Generally, soluble fused MHC heterodimer: peptide complex-specific T cells are isolated and cloned using techniques available to those of ordinary skill in the art, using T cells and their membrane preparations, soluble fused on T cells. Animals can be immunized to produce antibodies of the MHC heterodimer: peptide complex receptor. The antibody may be polyclonal or monoclonal. If polyclonal, the antibody may be of a murine, hare mammal, horse, sheep, or various other mammals. Monoclonal antibodies will typically be murines produced according to known techniques, or combinations thereof, such as in human, or chimeric or humanized antibodies as described above. The anti-soluble fused MHC heterodimer: peptide complex receptor antibody thus obtained can then be administered to the patient to reduce or eliminate the T cell subpopulations representing the receptor. Such populations of T cells recognize cells involved in, or are involved in, autoimmune diseases associated with autoantigenic peptides or have already undergone autoantigenic peptides and are involved in immunological destruction of these cells.

Coupling of solid supports with antibodies and their use in protein purification is well described in the literature (see, e.g., Methods in Molecular Biology, Vol. 1, Walker (Ed.), Human Press, New Jersey, 1984, which herein Incorporated by reference). Antibodies of the invention can be used as marker reagents to detect the presence of MHC heterodimer: peptide complexes on cells or in solution. Such antibodies are also particularly useful for western analysis or immunoblotting of purified cell-secreting material. Polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use in this regard. In addition, proteolytic and recombinant fragments and epitope binding domains can be used herein. Also suitable are chimeric, humanized antibodies, conjugated antibodies, CDR-substituted antibodies, reshaped or other recombinant whole or partial antibodies.

The following examples are provided by way of example and not by way of limitation.

Example 1

Preparation of DNA Sequences Encoding Human Soluble Fused MHC Heterodimer: Peptide Complexes

Plasmid pLJ13 contains an MHC class II β chain (DR1β * 1501) signal sequence; Myelin basic protein coding sequence (bp 283 to 345 encoding amino acid DENPVVHFFKNIVTPRTPPPS 82 to 102) (SEQ ID NO: 33); A DNA sequence encoding a flexible linker represented by the amino acid sequence (GGGSGGS SEQ ID NO: 31); Class II MHC DR1β * 1501 (SEQ ID NO: 50) β1 region coding sequence; A DNA sequence encoding a flexible linker represented by the amino acid sequence (GASAG SEQ ID NO: 29); Class II MHC DRA * 0101 (SEQ ID NO: 51) α1 region coding sequence. This plasmid directs the secretion of a soluble fused MHC heterodimer, represented by β1-α1, wherein the myelin basic protein peptide involved in multiple sclerosis is attached to the N terminus of β1 (Kamholz et al., Proc. Natl. Acad. Sci. USA 83: 4962-66, 1986), designed to form soluble fused MHC heterodimer: peptide complexes.

To prepare pLJ13 (SEQ ID NO: 49), PCR was used to introduce a DNA sequence encoding MPB at the junction of the signal sequence with the β1β2 sequence of the β chain of DR1β * 1501. The MBP-containing β1 region was then linked to the α1 region via a linker sequence introduced by PCR.

As a first step, full length α chain, cDNA encoding DRA * 0101, and cDNA encoding full length β chain were inserted into the expression vector pZCEP. DNA encoding this molecule can be isolated using standard cloning methods, such as Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982); Sambrook et al., (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989); Or Mullis et al., US Pat. No. 4,683,195, all of which are incorporated herein by reference.

pZCEP (Jelineck et al., Science, 259: 1615-16, 1993) was digested with Hind III and Eco RI and a 0.85 kb Hind III-Eco RI fragment containing cDNA encoding the b chain of DR1β * 1501 was inserted. The resulting plasmid is denoted pSL1.

PZCEP was digested with Bam HI and Xba I, ~ 0.7 kb SacI-SSP I fragments containing cDNA encoding the chain of DRA * 0101 were isolated by agarose gel electrophoresis, Bam HI-SacI and SSP I-XbaI It was inserted with a polylinker sequence (SEQ ID NO) comprising the ends. The resulting plasmid is shown as pSL2.

The cloning site of the linker sequence uses PCR to generate a signal sequence of class II b DR1B * 1501, which connects the sequence encoding the first 5 amino acids (DPVVH) of MBP (82-104) to the 3 'end of the signal sequence. Amplified ˜100 bp Hind III / Cla I fragments were generated. DNA sequences encoding amino acid VH were selected to generate unique ApaLI sites.

A second ClaI / XbaI fragment of 750 bp was generated using PCR, a class II β chain DR1β * 1501, which linked the DNA sequence encoding the last 2 amino acids (GS) of the linker to the 5 ′ end of the β1 sequence. Sequences encoding the transmembrane domain of and the β1β2 region. DNA sequences encoding amino acid GS were selected to generate unique Bam HI positions.

This fragment was digested with Hind III / Cla I and Cla I / XbaI, separated by agarose gel electrophoresis and inserted into Hind III / XbaI-digested pCZEP. The resulting shuttle plasmid was digested with ApaLI and BamHI, and the oligonucleotides encoding the remainder of the MBP sequence (represented as amino acid sequence FFKNIVTPRTPPPS) and the initiation site of the flexible linker GGGSG were inserted. The resulting construct contained an MBP sequence linked to the β1β2 sequence of DR1β * 1501 via an intermediate linker.

In addition, the construct comprising the signal sequence of DR1β * 1501 is linked to the N terminus of an MBP peptide (DENPVVHFFKNIVTPRTPPPS sequence identification number 33) linked to the N terminus of the β1 domain of DR1β * 1501 via a flexible linker (GGGSGGS SEQ ID NO: 31) It became. Six overlapping oligonucleotides were prepared by recombining the signal sequence, MBP peptide flexible linker and connecting to the N terminus of the β1 domain through a unique Bam HI position. Oligos were kinase treated before ligation. For each oligo, 50 pmol of oligo (ZC7639 (SEQ ID NO: 2), ZC7665 (SEQ ID NO: 6), ZC7663 (SEQ ID NO: 4), ZC7640 (SEQ ID NO: 3), ZC7666 (SEQ ID NO: 7), and A 50 ml reaction was prepared comprising ZC7664 (SEQ ID NO: 5), 22.4 ml TE, 5 ml TMD, 5 ml ATP and 5 ml kinase The reaction was incubated at 37 ° C. for 1 hour and then incubated at 65 ° C. for 10 minutes. Kinase treated oligos were stored until needed at −20 ° C. Then 0.5 mg Eco RI-BamHI linearized pSL1, 20 pmol of each kinase treated oligonucleotide (ZC7639 (SEQ ID NO: 2), ZC7665 (SEQ ID NO: 6) ), ZC7663 (SEQ ID NO: 4), ZC7640 (SEQ ID NO: 3), ZC7666 (SEQ ID NO: 7), and ZC7664 (SEQ ID NO: 5), 1 ml TE, 1 ml TMD, 1 ml ATP, and 0.5 ml ligase A 10 ml ligation reaction was prepared, which was incubated at 37 ° C. for 1 hour. 1 μl of the stain was electroporated into cells that meet DH10B requirements (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions, plated in LB plates containing 50 mg / ml ampicillin and incubated overnight. Was identified by restriction and sequencing and denoted as pSL21.

To prepare pLJ13, a ~ 0.48kb encoding DNA sequence from the signal sequence to the b1 region of pSL21 to which the DNA encoding the sequence of the second flexible linker (as amino acid sequence GASAG (SEQ ID NO: 29)) is linked PCR fragments were generated.

1 mg full length linearized DR1β * 1501 signal / MBP / linker / β chain (pSL21), 200 pmol ZC7511 (SEQ ID NO: 1), 200 pmol ZC8194 (SEQ ID NO: 8), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 1 wax 100 ml PCR reactions were prepared containing beads (AmpliWax-, Perkin-Elmer Cetus, Norwalk, CT). After an initial cycle of 95 ° C. for 5 minutes, 5 U Taq polymerase was added and the reaction was amplified for 30 minutes of 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 72 ° C. DR1β * consisting of a 29 amino acid DR1β * 1501 β chain signal sequence, a 21 amino acid MBP peptide sequence, a 6 amino acid flexible linker (GGGSGGS SEQ ID NO: 31), a 83 amino acid β1 domain, a 5 amino acid flexible linker (GASAG SEQ ID NO: 29) 1501 signal sequence / MBP peptide / linker / β1 / linker fragment was obtained. An estimated size of 374 bp band was separated by low melting agarose gel electrophoresis.

Generating a second ˜0.261 kb PCR fragment encoding the α1 portion of DRA * 0101 with DNA encoding the second flexible linker added at the 5 ′ end and DNA sequence encoding the stop codon at the 3 ′ end It was.

1 mg full length linearized DRA * 0101 (pSL2), 200 pmol ZC8196 (SEQ ID NO: 9), 200 pmol ZC8354 (SEQ ID NO: 14), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 1 wax bead (AmpliWax-, Perkin-Elmer) 100 ml PCR reactions were prepared including Cetus, Norwalk, CT). After an initial cycle of 95 ° C. for 5 minutes, 5 U Taq polymerase was added and the reaction was amplified for 30 cycles of 1 minute at 94 ° C., 2 minutes at 55 ° C., and 3 minutes at 72 ° C. The linker / DRA * 0101 a1 domain was obtained by containing a 5 amino acid flexible linker (GASAG SEQ ID NO: 29) linked to the N terminus of the 81 amino acid DRA * 0101 a1 domain and adding a stop codon and an XbaI restriction site to the C terminus. Bands of expected size 261 bp were separated by low melting agarose gel electrophoresis.

These two PCR fragments were used to generate the final Hind III / Xba I PCR product, which encodes the signal sequence of DR1β * 1501 linked to MPB peptide and linker peptide DNA, followed by β1, and β1 is a flexible peptide (GASAG). Through the DNA encoding SEQ ID NO: 29) to the 5 'end of α1.

1 mg 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), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5 U Taq polymer 100 ml PCR reactions containing the enzyme were prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C., 1 minute at 50 ° C., and 1 minute at 72 ° C. The 5-amino acid 3 'linker (GASAG SEQ ID NO: 29) of the signal sequence / MBP / linker / β1 / linker fragment is the same 5-amino acid linker of the linker / α1 fragment that links β1 and α1 domains in frame through the 5-amino acid linker. Overlapped with The resulting 730 bp MBP-β1α1 PCR product was located at the 5 'HindIII position, followed by the DR1β * 1501 β chain signal sequence, 21 amino acid MBP peptide DENPVVHFFKNIVTPRTPPPS (SEQ ID NO: 33), DRA * 0101, a 5 amino acid linker at the N terminus of the α1 domain. A 8 amino acid flexible linker (GGGSGGSG) linked to the N terminus of the DR1β * 1501 β1 domain linked by GASAG SEQ ID NO: 29) and ends with an XbaI restriction position. MBP β1α1 fragments are inserted into HindIII / XbaI pZCEP. Recombinant clones were identified by restriction and sequencing and labeled as pLJ13 (human MBP-β1α1).

Example 2

Synthesis of NOD Mouse α and β MHC cDNA

Maniatis et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982 and Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987, all incorporated herein by reference. Total RNA was isolated from splenocytes of NOD MOUSE NAME using homogenization in guanidinium thiocyanate and CsCl centrifugation by the method of O. oligo d (T) cellulose chromatography (Mini-Oligo (dT). ) Poly (A) + RNA was isolated using Cellulose Spin Column Kit (5 Prime-3 Prime), Boulder, CO).

The first strand cDNA was synthesized using the Superscript-RNase H reverse transcriptase kit according to the manufacturer's instructions. 1 μl of solution containing 1 mg total NOD RNA was mixed with 1 ml oligo dT solution and 13 ml diethylpyrocarbonate-treated water. The mixture was heated at 70 ° C. for 10 minutes and cooled on ice.

The first strand cDNA synthesis was performed in a RNA-primer mixture with 4 ml Superscript-buffer, 4 ml 0.1 M dithiothreitol, 2 ml deoxynucleotide triphosphate solution containing 10 mM dATP, dGTP, dTTP, dCTP, respectively, and 2 ml of 200 U / It was initiated by the addition of ml Superscript-reverse transcriptase. The reaction was incubated at room temperature for 10 minutes, then at 42 ° C. for 50 minutes, then at 70 ° C. for 15 minutes and then cooled on ice. The reaction was terminated by addition of 1 ml RNase H, incubated at 37 ° C. for 20 minutes and cooled on ice.

Then two 100 ml PCR reaction mixtures were prepared. One reaction was performed using the primers ZC8198 (SEQ ID NO: 10, antisense α chain primer, XbaI position) and ZC8199 (SEQ ID NO: 11, sense α chain primer, EcoRI position) to form α chains of class II MHC NOD (IAg ⁷ ). Amplified or β chains of class II MHC NOD (IAg ⁷ ) using primers ZC8206 (SEQ ID NO: 12, antisense β chain primer, XbaI position) and ZC8207 (SEQ ID NO: 13, sense β chain primer, EcoRI position) Was amplified. In both cases, addition of XbaI at the 5 'end of the fragment, EcoRI and 3' end, allowed for cloning into the expression vector. Each reaction mixture contained 10 ml of the first strand template, 8 ml 10 × synthetic buffer, 100 pmol sense primer, 100 pmol antisense primer, 65 ml dH ₂ O and 1 wax beads (AmpliWax-, Perkin-Elmer Cetus, Norwalk, CT). After an initial cycle of 5 minutes at 95 ° C, 1U Taq polymerase was added and the reaction was amplified for 30 minutes of 1 minute at 94 ° C, 2 minutes at 55 ° C, and 3 minutes at 72 ° C. The resulting a and b chain fragments were digested with EcoRI-XbaI, treated with RNAse, then separated by low melting agarose gel electrophoresis, and ligated to EcoRI-XbaI linearized pZCEP (Jelineck et al., Science 259: 1615-16, 1993). The full length β chain pZCEP was expressed as pLJ12, and the full length α chain pZCEP was expressed as pLJ11.

Example 3

Preparation of Mouse Soluble Single Chain MHC Molecules Containing Antigenic Peptides Linked Through Flexible Linkers

I. Peptide-β1α1

To prepare a molecule comprising an antigenic peptide linked via a flexible linker to the N terminus of a single chain MHC molecule containing a b1 domain linked to a1 domain via a second flexible linker, a four step preparation was performed.

A. GAD-β1α1 IAg ⁷

1) The β1 domain (SEQ ID NO: 43) of the IAg ⁷ NOD mouse β chain was isolated from the β2 domain and fused to linker fragments at the 5 ′ and 3 ′ ends using PCR.

100 ml full length, EcoRI / XbaI linearized, IAg ⁷ b chain, 200 pmol ZC9478 (SEQ ID NO: 16), 200 pmol ZC9480 (SEQ ID NO: 18), 100 ml with 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase PCR reactions were prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C, and 1 minute at 72 ° C. Β1 / linker consisting of a 91 amino acid b1 domain, an 8 amino acid portion of the flexible linker (GGSGGGGS SEQ ID NO: 34) fused to the 5 'end, a 5 amino acid flexible linker (GGSGG SEQ ID NO: 30) fused to the 3' end A fragment was obtained. Bands of expected size 330 bp were separated by low melting agarose gel electrophoresis.

2) PCR was used to add GAD 65 peptide (SRLSKVAPVIKARMMEYGTT (SEQ ID NO: 59)) and additional linker fragments to the b1 / linker fragment from 1. Additionally, the only BamHI site, and the second ribosome binding site followed by The last 16 nucleotides of the phi 10 coupler with the stop codon (RBS SEQ ID NO: 48) was added to the 5 ′ end of the GAD peptide to facilitate cloning and expression.

1 ml of the above eluted b1 / linker fragment, 200 pmol ZC9473 (SEQ ID NO: 15), 200 pmol ZC9479 (SEQ ID NO: 17), 200 pmol ZC9480 (SEQ ID NO: 18), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymer 100 ml PCR reactions containing the enzyme were prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C, and 1 minute at 72 ° C. These fragments are all designed to contain overlapping 5 'and / or 3' fragments, both of which can be annealed to their complementary strands and serve as primers for the reaction. The final 15 3 'nucleotide of ZC9499 (SEQ ID NO: 23) is a seamless linkage of the GAD peptide to the β1 domain in frame with the 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36), thereby providing a β1 / linker fragment ( overlapping with the first 15 nucleotides of ggaggctcaggagga) (SEQ ID NO: 35). ZC9479 (SEQ ID NO: 17) served as the 5 'primer by adding the BamHI position followed by RBS (SEQ ID NO: 48) at the 5' end of the GAD peptide sequence. A 15 nucleotide gap (gaggatgattaaatg) between the 3 'end of ZC9479 (SEQ ID NO: 17) and the first 15 nucleotides of ZC9473 (SEQ ID NO: 15) added sites to align with the peptide. The resulting 450 bp GAD / β1 fragment was isolated by low melting agarose gel electrophoresis.

3) The α1 domain (SEQ ID NO: 44) of IAg ⁷ was isolated from the α2 domain and fused to the linker fragment at the 5 ′ end and to the serine residue at the 3 ′ end, followed by the SpeI and EcoRI positions using PCR.

100 ng full length, EcoRI / XbaI linearized, including I-Ag ⁷ α chain, 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9493 (SEQ ID NO: 20), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase 100 ml PCR reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. An a1 / linker fragment consisting of a 87 amino acid a1 domain, a 5 amino acid flexible linker (GGSGG) (SEQ ID NO: 30), a serine residue fused to the 3 'end, a SpeI and EcoRI position was obtained . Bands of expected size 300 bp were separated by low melting agarose gel electrophoresis.

4) To complete the construct, 2 ml of GAD / β1 fragments from 2), 2 ml of α1 / linker fragments from 3), 200 pmol ZC9479 (SEQ ID NO: 17), 200 pmol ZC9493 (SEQ ID NO: 20), 10 ml 10X polymerase Final 100 ml PCR reactions were prepared comprising buffer, 10 ml dNTPs and 5U Taq polymerase. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. The 5 amino acid 3 'linker (GGSGG SEQ ID NO: 30) of the GAD / β1 fragment overlaps with the 5 amino acid linker of the α1 / linker fragment, which links β1 and α1 domains in frame through the 5 amino acid linker. The resulting GAD-β1α1 PCR product was located at the 5 ′ BamHI position, followed by RBS (SEQ ID NO: 48), 20 amino acid GAD65 peptide (SRLSKVAPVIKARMMEYGTT (SEQ ID NO), 5 amino acid linker (GGSGG sequence identification) at the N terminus of the IAg ⁷ α1 domain A 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36) linked to the N terminus of the IAg ⁷ β1 domain linked by No. 30) and ends with the SpeI and EcoRI restriction sites GAD-β1α1 fragments to BamHI and EcoRI Restriction digested and separated by low melting agarose gel electrophoresis The restriction digested fragments were then subcloned into BamHI-EcoRI linearized expression vector p27313 (WO 95/11702) Accurate recombinant clones were subjected to restriction and sequencing. This was confirmed by pLJ18 (GAD-β1α1 IAg ⁷ SEQ ID NO: 42).

B) MBP-β1α1 IA ^s

The β1 domain (SEQ ID NO: 46) of IA ^s was isolated from the β2 domain and fused to linker fragments at the 5 ′ and 3 ′ ends using PCR.

1) 100 ng full length, EcoRI / XbaI linearized, IAg ^s β chain (p40553), 200 pmol ZC9478 (SEQ ID NO: 16), 200 pmol ZC9497 (SEQ ID NO: 22), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymer 100 ml PCR reactions containing the enzyme were prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. IA ^s β1 consisting of an 91 amino acid β1 domain, an 8 amino acid portion of the flexible linker (GGSGGGGS SEQ ID NO: 34) fused to the 5 'end, and a 5 amino acid flexible linker (GGSGG SEQ ID NO: 30) fused to the 3' end Linker fragments were obtained. Bands of expected size 330 bp were separated by low melting agarose gel electrophoresis.

2) PCR was used to add the myelin basic protein (MBP) peptide (FFKNIVTPRTPPP (SEQ ID NO: 37) and the remainder of the 5 'linker to the IA ^s β1 / linker fragments from above, in addition, the unique BamHI position, and Ribosome binding sites Then a stop codon (RBS SEQ ID NO: 48) was added to the 5 'end of the MBP peptide to facilitate cloning and expression.

1 ml of eluted IA ^s β1 / linker fragment from 1), 200 pmol ZC9499 (SEQ ID NO: 23), 200 pmol ZC9479 (SEQ ID NO: 17), 200 pmol ZC9497 (SEQ ID NO: 22), 10 ml 10X polymerase buffer, 10 ml dNTPs And 100 ml PCR reaction comprising 5U Taq polymerase. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C, and 1 minute at 72 ° C. These fragments are all designed to contain overlapping 5 'and / or 3' fragments, both of which can be annealed to their complementary strands and serve as primers for the reaction. The final 15 3 'nucleotide (ggaggctcaggagga) (SEQ ID NO: 35) of ZC9499 (SEQ ID NO: 23) seamlessly connects the IA ^s β1 domain and MBP peptide via a 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36). Thus, the first 15 nucleotides of the IA ^s b1 / linker fragment are duplicated. ZC9479 (SEQ ID NO: 17) overlaps the first 32 nucleotides of ZC9499 (SEQ ID NO: 23), generates a BamHI restriction site, and adds RBS (SEQ ID NO: 48) and a termination codon to frame the MBP peptide , 5 'primer. The resulting 400 bp MBP / IA ^s β1 fragment was isolated by low melting agarose gel electrophoresis.

3) The α1 domain (SEQ ID NO: 47) of the IA ^s was isolated from the α2 domain and fused to the linker fragment at the 5 ′ end and to the serine residue at the 3 ′ end, followed by the SpeI and EcoRI positions using PCR.

100 ml PCR comprising 100 ng full length, linearized IA ^s α chain (p28520), 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9496 (SEQ ID NO: 21), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase The reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. 87 amino acid IA ^s α1 domain, a 5 amino acid flexible linker (GGSGG) (SEQ ID NO: 30) fused to the 5 'end, a serine residue fused to the 3' end, an IA ^s α1 / linker consisting of the SpeI and EcoRI positions A fragment was obtained. Bands of expected size 300 bp were separated by low melting agarose gel electrophoresis.

4) To complete the construct, 2 ml of MBP / IA ^s β1 fragment from 2), 2 ml of IA ^s α1 / linker fragment from 3), 200 pmol ZC9479 (SEQ ID NO: 17), 200 pmol ZC9496 (SEQ ID NO: 21), Final 100 ml PCR reactions were prepared comprising 10 ml 10 × polymerase buffer, 10 ml dNTPs and 5U Taq polymerase. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 53 ° C, and 1 minute at 72 ° C. MBP / IA ^s β1 5 amino acid 3 'linker fragment (GGSGG SEQ ID No. 30) 5-amino acid linker of the IA ^s α1 / linker fragment connecting the IA ^s β1 and IA ^s α1 domain so that the frame is fit through a 5 amino acid linker Overlapped with The resulting 673bp MBP-β1α1 IA ^s PCR product was located at the 5 'BamHI position, then RBS (SEQ ID NO: 48), 13 amino acid MBP peptide (FFKNIVTPRTPPP (SEQ ID NO: 37), 5 amino acid linker at the N terminus of the IA ^s α1 domain) A 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36) linked to the N terminus of the IA ^s β1 domain linked by (GGSGG SEQ ID NO: 30) and ends with SpeI and EcoRI restriction sites. Restriction digestion with BamHI and EcoRI and separation by low melting agarose gel electrophoresis The restriction digested fragments were then subcloned into BamHI-EcoRI linearized expression vector p27313 (WO 95/11702). Confirmed by sequencing, it was designated pLJ19 (MBP β1α1 IA ^s SEQ ID NO 45).

II. Peptide-β1α1α2β2

An antigenic peptide linked via a flexible linker to the N terminus of a single chain MHC molecule containing a β1 domain, linked to the N terminus of the β2 domain via a second flexible linker, and linked to the N terminus of the α1α2 domain via a flexible linker In order to manufacture a molecule containing, four steps of preparation were performed.

A. GAD-β1α1α2β2 IAg ⁷

1) The α1α2 domain of IAg ⁷ was fused to the 5 amino acid linker at the 5 ′ end and to the 15 amino acid linker at the 3 ′ end using PCR.

100 ml full length, linearized IAg ⁷ α chain (pLJ11), 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9722 (SEQ ID NO: 27), 100 ml with 5 ml 10X polymerase buffer, 5 ml dNTPs, and 2.5 U Taq polymerase PCR reactions were prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. IAg consisting IAg ⁷ α1α2 domain, the 5 'fused to a terminus, 5 amino acid flexible linker (GGSGG SEQ ID No. 30), 3', the 15 amino acid flexible linker (GGGGSGGGGSGGGGS SEQ ID NO. 36) fused to the terminal ^7, the linker / α1α2 / linker fragments were obtained. Bands of expected size were separated by low melting agarose gel electrophoresis.

2) Isolating the β2 domain of I-Ag ⁷ from the β1 domain and using PCR, a 15 amino acid linker was fused at the 5 ′ end of the β2 domain and at the 3 ′ end with a stop codon followed by an EcoRI restriction site.

100 ng full length linearized I-Ag ⁷ β chain (pLJ12), 200 pmol ZC9721 (SEQ ID NO: 26), 200 pmol ZC9521 (SEQ ID NO: 24), 5 ml 10X polymerase buffer, 5 ml dNTPs, and 2.5 U Taq polymerase 100 ml PCR reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. I-Ag ⁷ consisting of a β2 domain (SEQ ID NO: 58), a 15 amino acid flexible linker (GGGGSGGGGSGGGGS) fused to the 5 'end (SEQ ID NO: 36), a termination codon fused to the 3' end, and an EcoRI restriction site Linker / β2 fragments were obtained. Bands of expected size were separated by low melting agarose gel electrophoresis.

3) the α1α2 domain (SEQ ID No. 57) of the ^I-7 Ag using fusion PCR was a β2 domain of the ^I-7 Ag. The 15 amino acid linker sequence at the 3 'end of the α1α2 fragment was completely overlapped with the same 15 amino acid sequence at the 5' end of the β2 fragment by linking domains to fit the frame, via a flexible linker.

5 ml I-Ag ⁷ linker / α1α2 / linker fragment from 2), 5 ml I-Ag ⁷ linker / β2 fragment from 3), 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9721 (SEQ ID NO: 26), 10 ml 10X 100 ml PCR reactions were prepared comprising polymerase buffer, 10 ml dNTPs and 5 U Taq polymerase. The reaction was carried out for 30 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C, and 2 minutes at 72 ° C. I-Ag ⁷ α1α2 domain, a 5 amino acid flexible linker (GGSGG) fused to the 5 'end (SEQ ID NO: 30), 15 amino acid flexible fused to the 3' end, linked to the 5 'end of the β2 domain An I-Ag ⁷ linker / α1α2 / linker / b2 fragment consisting of a linker (GGGGSGGGGSGGGGS SEQ ID NO: 36) was obtained. Bands of expected size were separated by low melting agarose gel electrophoresis.

4) To complete the construct, 5 ml of GAD-β1α1 fragment from A-4, 3 ml of I-Ag ⁷ linker / α1α2 / linker / β2 fragment, 200 pmol ZC9521 (SEQ ID NO: 24), 200 pmol ZC9479 ( SEQ ID NO: 17), a final 100 ml PCR reaction was prepared comprising 10 ml 10 × polymerase buffer, 10 ml dNTPs and 5U Taq polymerase. The reaction was carried out for 30 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C, and 2 minutes at 72 ° C. The entire linker / a1 portion of the GAD / β1α1 and linker / α1α2 / linker / β2 fragments are framed via I-Ag ⁷ β1 and I-Ag ⁷ α1α2 through a 5-amino acid flexible linker (GGSGG SEQ ID NO: 30) Linking / linker / β2 domains overlap. The resulting GAD-β1α1α2β2 I-Ag ⁷ PCR product was located at the 5 'BamHI position, followed by RBS (SEQ ID NO: 48), 20 amino acid GAD peptide (SRLSKVAPVIKARMMEYGTT (SEQ ID NO: 59), 5 amino acid linker at the N terminus of the α1α2 domain). A 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36) linked to the N terminus of the IAg ⁷ β1 domain linked by GGSGG SEQ ID NO: 30) and ends with a β2 domain and an EcoRI restriction site GAD-β1α1α2β2 fragment Restriction digestion with BamHI and EcoRI and separation by low melting agarose gel electrophoresis The restriction digested fragments were then subcloned into BamHI-EcoRI linearized expression vector p27313 (WO 95/11702). Confirmed by sequencing, this was designated pLJ23 (GAD-β1α1α2β2 IAg ⁷ SEQ ID NO: 56).

B. MBP-β1α1α2β2 IA ^s

1) The α1α2 domain of IA ^s was fused to a 5 amino acid linker at the 5 ′ end and to a 15 amino acid linker at the 3 ′ end using PCR.

100 ml PCR with 100 ng full length, linearized IA ^s α chain (p28520), 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9722 (SEQ ID NO: 27), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase The reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. 196 amino acid IA ^s α1α2 domain, 5 IA ^s consisting of 'fused to a terminus, 5 amino acid flexible linker (GGSGG SEQ ID No. 30), 3', the 15 amino acid flexible linker (GGGGSGGGGSGGGGS SEQ ID NO. 36) fused to the terminal Linker / α1α2 / linker fragments were obtained. The expected size, band of 650 bp, was separated by low melting agarose gel electrophoresis.

2) The β2 domain of IA ^s was isolated from the b1 domain and, using PCR, a 15 amino acid linker was fused at the 5 'end and at the 3' end with the stop codon and then the EcoRI restriction site.

100 ml PCR comprising 100 ng full-length linearized IA ^s β chain (p40553), 200 pmol ZC9721 (SEQ ID NO: 26), 200 pmol ZC9521 (SEQ ID NO: 24), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase The reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. IA ^s consisting of a 105 amino acid β2 domain (SEQ ID NO: 55), a 15-amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36), a termination codon fused at the 3 'end, and an EcoRI restriction site Linker / β2 fragments were obtained. The expected size, band of 374 bp, was separated by low melting agarose gel electrophoresis.

3) to the domain of α1α2 IA ^s using a PCR was fused to a β2 domain of the IA ^s. The 15 amino acid linker sequence at the 3 'end of the α1α2 fragment was completely overlapped with the same 15 amino acid sequence at the 5' end of the β2 fragment by linking domains to fit the frame, via a flexible linker.

5 ml IA ^s linker / α1α2 / linker fragment from 2), 5 ml IA ^s linker / β2 fragment from 3), 200 pmol ZC9481 (SEQ ID NO: 19), 200 pmol ZC9721 (SEQ ID NO: 26), 10 ml 10X polymerase buffer, 100 ml PCR reactions were prepared comprising 10 ml dNTPs and 5 U Taq polymerase. The reaction was carried out for 30 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. Fused to the 5 'end of the 5 amino acid flexible linker (GGSGG) (SEQ ID NO: 30), 106 amino acid β2 domain, fused at the 3' end, fused to the 5 'end of the IA ^s α1α2 domain of 196 amino acids An IA ^s linker / α1α2 / linker / β2 fragment consisting of a 15 amino acid flexible linker (GGGGSGGGGSGGGGS SEQ ID NO: 36) was obtained. The expected size, 977 bp band, was separated by low melting agarose gel electrophoresis.

4) To complete the construct, 2 ml of MBP-β1α1 fragment from B-4, 3 ml of IA ^s linker / α1α2 / linker / β2 fragment, 200 pmol ZC9479 (SEQ ID NO: 17), 200 pmol ZC9521 (SEQ ID NO: 17) No. 24), a final 100 ml PCR reaction was prepared comprising 10 ml 10 × polymerase buffer, 10 ml dNTPs and 5 U Taq polymerase. The reaction was carried out for 30 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. The entire linker / a1 portion and the linker / α1α2 / linker / β2 fragment of MBP / β1α1 are IA ^s β1 and IA ^s α1α2 / linker / β2 so that they are framed through a 5-amino acid flexible linker (GGSGG SEQ ID NO: 30). Linking domains was duplicated. The resulting 1360 bp MBP-β1α1α2β2 IA ^s PCR product was located at the 5 'BamHI position, then RBS (SEQ ID NO: 48), 13 amino acid MBP peptide (FFKNIVTPRTPPP SEQ ID NO: 37), at the N terminus of the full length IA ^s α domain. 15 amino acid flexible linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 36) linked to the N terminus of the IA ^s β1 domain linked by amino acid flexible linker (GGSGG SEQ ID NO: 30) and ends with the β2 domain and EcoRI restriction site. MBP β1α1α2β2 fragments were restriction digested with BamHI and EcoRI and separated by low melting agarose gel electrophoresis. Restricted digested fragments were then subcloned into BamHI-EcoRI linearized expression vector p27313 (WO 95/11702). Recombinant clones were identified by restriction and sequencing and designated as pLJ20 (MBP β1α1α2β2 IA ^s SEQ ID NO 54).

III. MBP-α1α2

To generate a molecule comprising an antigenic peptide linked via a flexible linker to the N terminus of a single chain MHC molecule consisting of an alpha 1 alpha 2 domain, a two step process was performed.

1) The α1α2 domain of IA ^s (SEQ ID NO: 53) was fused to 25 amino acid linkers at the 5 ′ end and to the stop codon and the SpeI and EcoRI restriction sites at the 3 ′ end using PCR.

100 ng full length, including EcoRI-XbaI linearized IA ^s α chain (p28520), 200 pmol ZC9720 (SEQ ID NO: 25), 200 pmol ZC9723 (SEQ ID NO: 28), 10 ml 10X polymerase buffer, 10 ml dNTPs, and 5U Taq polymerase 100 ml PCR reaction was prepared. The reaction was carried out for 35 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. 196 amino acid IA ^s α1α2 domain, 25 amino acid flexible linker (GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 32) fused to the 5 ′ end, IA ^s linker / α1α2 comprising a stop codon fused to the 3 ′ end and a SpeI and EcoRI restriction site A fragment was obtained. The expected size, band of 672 bp, was separated by low melting agarose gel electrophoresis.

2) 5 ml linker / α1α2 IA ^s from 1), 200 pmol ZC9723 (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 and 5U 100 ml PCR reactions were prepared comprising Taq polymerase. The reaction was carried out for 30 cycles of 1 minute at 94 ° C, 1 minute at 54 ° C, and 2 minutes at 72 ° C. IA ^s MBP / consisting of a 196 amino acid IA ^s α1α2 domain, a 25 amino acid flexible linker (GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 32), a termination codon fused to the 3 ′ end, and a SpeI and EcoRI restriction site Linker / α1α2 fragments were obtained.

There was a 12 amino acid duplication (GGGGSGGGGSGG SEQ ID NO: 38) between the 5 'end of the 25 amino acid linker of the linker / α1α2 fragment and the 3' end of ZC9499 (SEQ ID NO: 23). ZC9499 (SEQ ID NO: 23) added the BamHI restriction site, RBS (SEQ ID NO: 48), and MBP peptide (FFKNIVTPRTPPP SEQ ID NO: 37) at the 5 'end of the 25 amino acid flexible linker. ZC9479 (SEQ ID NO: 17) overlapped the first 32 nucleotides of ZC9499 (SEQ ID NO: 23) and served as a 5 'primer. The resulting 743 bp MBP-α1α2 IA ^s PCR product was a 25 amino acid chain linked to the N 'end of the 5' BamHI position, then RBS (SEQ ID NO: 48), 13 amino acid MBP peptide (FFKNIVTPRTPPP SEQ ID NO: 37), IA ^s α1α2 domain Sex linker (GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 32) and ends with SpeI and EcoRI restriction sites. MBP-α1α2 fragments were restriction digested with BamHI and EcoRI and separated by low melting agarose gel electrophoresis. Restricted digested fragments were then subcloned into BamHI-EcoRI linearized expression vector p27313 (WO 95/11702). Recombinant clones were identified by restriction and sequencing and designated as pLJ21 (MBP-α1α2 IA ^s SEQ ID NO 52).

Example 4

Transfection and Induction of Soluble Fused MHC Heterodimer: Peptide Complexes in E.coli

Transfection

E. coli K-12 strain W3110 was obtained from ATCC and has 1 copy of the phage lambda-DE3 (which is a T7 RNA polymerase gene) using Novagen (Madison, WI) DE3 solubilization kit according to the manufacturer's instructions. )). Plasmids pLJ18 (GAD β1α1 IAg ⁷ ), pLJ23 (GAD β1α1α2β2 IAg ⁷ ), pLJ19 (MBP β1α1 IA ^s ), and pLJ20 (MBP β1α1α2β2 IA ^s ) were described by Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 1982) was used to transform into host strain W3110 / DE3 using Ca ⁺⁺ transformation.

Judo

All four plasmid transformants were derived as described below. pLJ18 will be used as a prototype example. Single colonies containing pLJ18 (GAD β1α1 IAg ⁷ ) were used to inoculate 5-6 ml LB containing 50 mg / ml carbenicillin (Sigma), usually until the OD ₆₀₀ of the culture was between 0.45 and 0.60. The culture was spun at 37 ° C. for 3 hours. Glycerol stock was made from a portion of each culture and 1 ml of culture was centrifuged at 5,000 × g for 5 minutes at 4 ° C. To initiate induction, isopropyl-bbD-thio-galactopyranoside (IPTG) was added at a final concentration of 1 mM and the culture was spun at 37 ° C. Aliquots were taken from each culture at 0, 1, 2, and 3 hours and overnight time points and OD ₆₀₀ was measured. Aliquots were obtained by centrifugation at 5,000 × g for 5 minutes at 4 ° C. The pellet was resuspended in an appropriate volume of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to afford 0.02 OD ₆₀₀ / ml. Aliquots of each time point were then stored at −20 ° C. until needed.

50 μl of each time point aliquot was electrophoresed on 4-20% Tris-glycine SDS polyacrylamide gel in denatured (reduced) sample buffer followed by Coomassie blue staining. One band was present at about 33 kD.

For Western blot analysis, 1/60 dilutions of each time point aliquot were electrophoresed on 4-20% Tris-glycine SDS polyacrylamide gel in denaturing (reduced) sample buffer. The protein was transferred to nitrocellulose by electroblotting. The blot was reacted with mouse anti-IAg ⁷ MHC antiserum followed by rabbit anti-mouse antibody / horseradish peroxidase conjugate (BioSource International, Camarillo, Calif.) And ECL ^™ detection reagent (Amersham Corp.) to visualize the protein. . The blot was then exposed to the radiographic film. One band was present at about 33 kD.

Example 5

Purification from Inclusion Body and Refolding of GAD-β1-α1

Two liter cultures of GAD-β1-α1 were grown at 37 ° C. until reaching 0.77 OD ₆₀₀ . The initial culture volume was increased for large scale production of the protein. Cultures were grown for 3 hours and 15 minutes after induction until reaching 0.97 OD ₆₀₀ . The whole cell pellet was stored in 20 ml TE (50 mM Tris-HCl, pH 8.0, 2 mM EDTA) at -20 ° C until needed.

The pellet is resuspended in 0.1% Triton X-100, 100 mg / ml lysozyme and 1/10 initial culture volume TE, incubated at 30 ° C. for 20 minutes, then cooled on ice and then slowly mixed between pulses Sonication was performed with three 20 second pulses at power setting 5 (Branson 450).

The pellet lysates were then centrifuged in an SS34 rotor at 12,000 xg for 10 minutes at 4 ° C. The pellet was washed with 1/10 initial culture volume of 1% NP-40 in TEN (50 mM Tris-HCl pH8.0, 2 mM EDTA, 100 mM NaCl) and centrifuged in an SS34 rotor at 12,000 × g for 10 minutes at 4 ° C. The pellet was then washed with TEN of 1/10 initial culture volume without detergent. The pellet was centrifuged as before and the supernatant was removed. The pellet was resuspended in extraction buffer (8M urea, 25 mM borate pH8.5, 10 mM DDT) at a concentration of approximately 200 mg / ml and incubated at 37 ° C. for about 2 hours. Additional 38 ml urea / borate / DTT buffer was added to the supernatant, and the whole sample was 3.5L 4M urea, 50 mM borate pH8. For 48-72 hours at 4 ° C. or until recrystallized (this is confirmed by analytical HPLC). It was dialyzed against 1 and then dialyzed against 3.5L 50mM borate pH8.1 at 4 ° C. This material was subjected to preparative reverse phase chromatography using a Vydac C-18 column (Hewlett Packard, Wilmington, DE) or Poros-R2 (PerSeptive Biosystems) and warmed to 40 ° C. The column was eluted with (A) 98% water / 0.1% TFA, and (B) 100% CH ₃ CN / 0.09% TFA for 28 minutes at a flow rate of 1 ml / min to give the final purified product.

Four desalted and purified samples of GAD-β1-α1 were independently injected into a triple quadrupole electrospray mass spectrometer to determine the mass of intact recombinant protein. The average mass obtained from these 4 measurements was 24434.67 +/- 2.72 Da. The mass obtained is in good agreement with the mass expected in the cDNA-translated sequence, 24432.89 Da. The percent error of the measurement is 0.007% and this is typical of the error associated with this type of mass spectrometry.

Additionally, peptide mapping of the proteins was performed by proteolytically desalted and purified samples of GAD-β1-α1 with trypsin. The resulting digest was analyzed using a MALDI-TOF mass spectrometer. This analysis confirms the presence of disulfide bridges between Cys50 and Cys112 as expected for properly folded molecules. N-terminal sequence analysis also confirms the expected sequence and removal of Met.

Example 6

Isolation and Proliferation Protocol of GAD Reactive Human T Cell Clones and Cell Lines

I. Isolation of Responder Cell Populations

Peripheral blood mononuclear cells (PBMNC) from prediabetes or newly developing diabetic patients with a source of autoreactive T cells were isolated by density centrifugation on ficoll-hypaque. Cells were washed several times and from normal non-transfused male donors tested positive in 15% PHS medium (RPMI-1640, 15% heat inactivated normal male human serum (mixed lymphocyte cultures using established techniques). ), 2 mM L-glutamine, 5 × 10 ⁻⁵ M beta-mercaptoethanol). Portions of PBMNCs were stored for use as antigen pulse antigen presenting cell APCs (under stimuli, see below) and portions were frozen for subsequent stimulation. The rest was plated on tissue culture plates and incubated for 1 hour at 37 ° C. to remove adherent cells. Non-adherent cells were removed from the plate with medium, added to a new plate, and incubated overnight at 37 ° C., 5% CO ₂ to remove any remaining adherent population.

A non-adhesive cell population was obtained and concentrated for T cells by passing the cells on nylon fibers, which removes remaining monosite and B cells. Non-adherent cells were enriched for T cells and natural killer cells by removing CD56 + and CD8 + cells. This was done by obtaining non-adhesive cells (lacking CD56 + and CD8 +) by sequentially culturing the cells in anti-CD8 antibody coated plates and anti-CD56 antibody coated plates.

II. Preparation of Stimulator Cell Population: 0 days

PBMNC was incubated in 0.5 ml volume of 15% PHS medium overnight at 37 ° C., 5% CO ₂ with 1:20 GAD65 (approximately 50 mg / ml). This can also be done using frozen cells, which were thawed, washed twice and incubated with GAD65 for 5-7 hours. Cells were irradiated at 3000 rads, washed twice and counted.

III. Stimulation of T Cells

1-2 x 10 ⁶ CD4 + enriched T cells or nylon fiber enriched T cells or PBL's, 15% PHS medium, a pulsed or pulsed with total GAD 1-2 x 10 without antigen in 1.5ml ⁶ josa Mixed with stimulator. After 6 days, 100 μl of cells were transferred from all conditions of stimulation to two separate wells of 96 well plates. 1 μCi of 3H-thymidine was added to each well and obtained for 5 hours to determine the proliferative response of each responder cell population to the GAD pulsed stimulator compared to the pulsed stimulator without antigen. By day 7 the cells were frozen or obtained. The obtained cells were washed twice and re-stimulated with 1-2 × 10 ⁶ stimulators prepared as described in II, using fresh or frozen autografted or non-autografted HLA-matched PBMNCs.

Around 8 and 11 days, 10 U / ml human recombinant IL-2 (Research and Development Systems, Minneapolis, Minn.) Was added to the culture. Cultures were increased as needed with the medium, with the cells divided by 1: 2 or 1: 3 to maintain <8 x 10 ⁵ cells / ml. Cells split too quickly and extra IL-2 was added if exogenous IL-2 was needed. By day 14, the cells were restimulated as above to maintain T cell lines and make frozen stocks. T cell clones and cell lines can be generated by limiting dilution to antigen stimulation as described above, or the cells can be tested for peptide and MHC responses as described below.

IV. Cloning of T Cells

Around 14 days T cells were obtained, washed, resuspended in 15% PHS medium with 10 U / ml IL-2, and 1 × 10 ⁴ stimulators (above) in 15 ml total volume of Terrasaki plates (Research and Development Systems). Prepared). Cloning may also be initiated around 7 days.

Cells were examined for growth and transferred to wells in 200 ml 15% PHS medium containing 1 × 10 ⁵ stimulators with a cell volume of about 1/2 of the well volume of a 96 well round bottom plate. Additional aliquots of IL-2 were added to the cultures after 24 hours so that the final concentration of 15% PHS medium was 10 U / ml.

As the cells grew in the wells, around 4 and 5 days, the antigen reactivity was tested and 1: 1 divided into additional wells containing 10 U / ml 15% PHS medium as the cells fused.

Cell stocks were frozen from 96 well cultures or expanded to 24 wells, 1.5 ml cultures using 1.5 × 10 ⁶ stimulators and one or several T cells in the wells.

V. Testing Reactivity to GAD

T-cell clones were left (2 days, at least 7 days after antigen stimulation without IL-2), washed, counted and resuspended in 15% PHS medium. They were plated at a concentration of 25,000 cells / well in 100 ml 15% PHS medium. Autografts or HLA-class II-matched PBMNCs were incubated with GAD (about 50 mg / ml) for at least 5 hours and loaded with GAD. Cells were washed and irradiated at 3000 rads. The cells were washed and resuspended in 15% PHS medium and added to T cells at a concentration of 1 × 10 ⁶ cells / well in 100 ml 15% PHS medium. Cells were incubated for 48 hours, then pulsed with 1 mCi 3H-thymidine and harvested. Positive response is thought to be stimulation index> 3 (mean index cpm / average cpm of the sample stimulated with antigen or without control antigen or the average cpm of the sample of cells stimulated with the control antigen). Some controls include T cells alone, stimulators alone, purified negative antigens, GAD purified from baculovirus, PHA, IL-2.

Other methods known in the art for testing clones and cell lines include the following methods: dose response to antigen; Response to this antigen or negative antigen control; Determination of HLA-class II restriction by adding blocking anti-HLA class II antibodies to the plate; And using the peptide to load the stimulator to determine peptide specificity, as described above, except that the peptide is tested by dose titration and the peptide is left behind in the assay. Dose reactions in combination with peptide specificity tests can also be done.

Antigen presenting cells used to determine HLA-limiting include autograft and non-autologous PMNBCs, which may have matches and mismatches at the HLA locus, and genetically engineered antigen presenting cells include BLS-1 and Mouse L cells or other APCs expressing only one HLA class II molecule.

VI. Reactivity Tests for Synthetic GAD Peptides

Four separate T cell lines extracted from one HLA-DRB1 * 0404 patient (ThHo) were used to map a duplicate set of 74 synthetic GAD peptides, 20-mers, including the full length of GAD 65 (SEQ ID NO: 59). . Antigen incubating cells, BLS-DRB1 * 0404 and / or BLS-DRB1 * 0401 (Kovats et al., J. Exp. Med. 179: 2017-22, 1994) by incubating with peptides (about 50 mg / ml) for at least 5 hours. Loaded with peptide. The reactivity of the T cells was determined as above. One peptide, hGAD 33 (PGGAISNMYAMMIARFKMFP SEQ ID NO: 40), stimulated 3 or 4 cell lines with BLS-B1 * 0404. The COOH terminal cleavage of the peptide of 20 amino acid to 11 amino acid fragment (PGGAISNMYAM SEQ ID NO: 39) stimulated only one T cell line when provided by BLS-B1 * 0404 or BLS-DRB1 * 0401. The 10 amino acid fragment (PGGAISNMYA SEQ ID NO: 41) stimulated the same T cell line only when provided by BLS-B1 * 0404. In addition to identifying GAD epitopes that stimulate T cells extracted from diabetic donors, the method quickly identifies peptide and HLA restriction of T cell lines and clones.

Example 7

Synthesis of GAD Peptides

Peptides amidated at the C terminus were synthesized by solid phase peptide synthesis (SPPS) using Fmoc chemistry. Chemicals used in the synthesis were obtained from Nova Biochem (La Jolla, Calif.). This peptide was assembled on a Rink amide MBHA resin (0.25 mM size) starting from the C terminal end using a 432A Applied Biosystems, Inc. (Foster City, CA) automated peptide synthesizer and solid phase technique. This synthesis required double coupling to ensure completion of the coupling reaction and used HBtu-HOBT coupling chemistry. The bold residues required at least double coupling (SRLSKVAPVIKARMMEYGTT-NH ₂ (SEQ ID NO: 59).) Each cycle included Fmoc deprotection of amines from dendritic amino acid residues, and coupling of incoming Fmoc-amino acids. After successful assembly of the resin, the resin was washed with dichloromethane and dried under vacuum for two hours The peptide resin as a scavenger contained 1 ml of 4-methoxybenzenethiol and 0.7 g of 4-methylmercaptophenol. Resuspend in 10 ml trifluoroacetic acid (TFA) This suspension was slowly mixed at room temperature for 2 hours, then filtered through a PTFE filter and the filtrate was mixed with 1 liter organic solvent (pentane: acetone = 4: 1). The white precipitate was allowed to settle for 1-2 hours at room temperature, after which the crude precipitated peptide was separated by centrifugation. The crude peptide was washed three times with an organic solvent mixture and vacuum dried overnight.

Reversed phase HPLC of crude peptide showed one major peak and smaller impurities, which may be the deletion peptide. The main peak was separated by preparative reverse phase HPLC using a solvent gradient consisting of Start Buffer A (0.1% TFA) and Stop Buffer B (70% acetonitrile in 0.1% TFA). Fractions were obtained (10-15 ml) and lyophilized to remove all solvents. Fractions were analyzed by reverse phase HPLC and pure fractions were further characterized by mass spectrometry.

Peptides having a carboxyl group of the last amino acid at the C-terminus were prepared using solid phase Fmoc chemistry. Peptides were assembled on the Wang resin starting from the C-terminal end using a 431A Applied Biosystems automated peptide synthesizer. Wang resin (Fmoc-Thr (tBu) -Wang) to which the first amino acid was attached was loaded into the synthesizer and coupling was done from the next amino acid at the C-terminus. In order to ensure the completion of the coupling reaction, double coupling was performed on the amino acids indicated above. HBtu-HOBt coupling chemistry was used for this purpose. Each cycle involved Fmoc deprotection of amines from dendritic amino acid residues and coupling of incoming Fmoc-amino acids. After successful assembly of the peptide, the resin was washed with dichloromethane and dried for 2 hours. Cleavage and purification of peptides are as described above.

Relative affinity of all synthetic peptides for MHC was tested using DELFIA assays and response to IL-2 production by C-terminal amidated GAD65-restricted T cell hybridomas, as described in later examples. CTLL cell proliferation was used to measure T-cell binding by peptide: MHC complex.

Example 8

Synthesis of Ala Scan Peptide

A series of 20 C-terminal amidated GAD65 peptides comprising amino acids 524 to 543 were synthesized by substituting a single alanine instead of each non-alanine residue and naturally substituted tyrosine in place of the residue where alanine is present. Peptides were synthesized by solid phase peptide synthesis (SPPS) technique using ABIMED-Gilson AMS 422 multiple peptide synthesizer (Middleton, WI). The synthesizer consisted of an X-Y-Z mobile Gilson auto-sampler, a 48 column reactor module, amino acid and activating reagent reservoir. Reagents and solvents were added to each column sequentially by micro-injector, but washing of the resin in all reaction columns was performed simultaneously.

Peptides were simultaneously assembled and synthesized on AMS-422 in 0.025 mM size using Rink amide MBHA with a substitution level of 0.55 mM per gram. 20 columns were set in the synthesizer with 0.025 mM of the resin activated in each column. The first step involved the removal of Fmoc using 20% piperidine in dimethylformamide (DMF). This step was carried out simultaneously on the resin phase in each reaction column. Sequential mixing protocols were introduced to maximize deprotection (Thong Luu, Pham Son and Shrikant Deshpande, Automated Multiple Peptide Synthesis: Improvements in Obtaining Quality Peptides, Int. J. Peptides Proteins, 1995, in press). Double deprotection was also used to obtain complete deprotection of the Fmoc group. The resin washing step was done simultaneously using DMF.

The first amino acid coupling involves the activation of a specific amino acid in a reaction column labeled by an autoinjector with pyBOP / HOBt / N-methyl morpholine in DMF (ratio of active site of resin to activated amino acid = 1: 6). By introducing. The resin was mixed by slowly bubbling nitrogen for 20 seconds in the reaction column. Dichloromethane (DCM) was added to the reaction mixture so that the ratio of DMF: DCM was 3: 1. The resins were mixed before different amino acid coupling was initiated in different reaction columns. The most hydrophobic amino acids were first coupled to these amino acids so that the coupling time was maximum. After the first amino acid was coupled, all reaction columns were washed simultaneously with DMF. Double coupling techniques are often used to complete amino acid coupling to the resin. After the double coupling was completed, the resin was washed with DMF and activated the next cycle of Fmoc deprotection and amino acid coupling.

After the final Fmoc deprotection, the peptide resin was washed with DCM and the reaction column was dried by applying a vacuum to the synthesizer. The column was removed from the synthesizer and a stopper at one end was closed using a syringe stopper (# 3980025, Gilson). 1.5 ml of TFA containing 0.07 g of 4- (methylmercapto) phenol and 0.1 ml of 4-methoxybenzenethiol were added to each column and mixed for 2 hours at room temperature. After the cleavage was completed, the stopper at one end of the reaction column was removed, the reaction mixture was filtered and the filtrate was collected in 100 ml of pentane: acetone (4: 1). Peptides were precipitated for 2 hours at room temperature and then separated by centrifugation. The pellet was washed three times with pentane: acetone and twice with pentane. The crude peptide was vacuum dried for 2 hours and then subjected to analytical reverse phase HPLC and mass spectrometry. Peptides that did not precipitate from the pentane: acetone solution within 2 hours were cooled to −20 ° C., then separated and washed as above.

Example 9

Synthesis of cleaved C-terminal amidated GAD65 peptide

A series of C-terminal amidated GAD65 (SEQ ID NO: 59) peptides were synthesized in which one or more N- or C-terminal amino acids were systematically cleaved.

Peptides were synthesized by solid phase peptide synthesis using the ABIMED-Gilson AMS 422 multiple peptide synthesizer, as described in Example 8.

Example 10

Cleaved C-terminal Amidated GAD65 Core Peptides

Testing of the cleaved C-terminal amidated GAD65 peptide of Example 9 showed that the C-terminal truncated peptide (including amino acids 528-543) and the N-terminal truncated peptide (including amino acids 524-539) were I-. It has been shown that it can still bind Ag and peptides comprising amino acids 528-539 can also stimulate C-terminal amidated GAD65 peptide restricted T cell hybridomas. Based on this information, a second series of cleaved peptides was synthesized based on this core sequence (Table 4), which was analyzed for MHC affinity and involvement of C-terminal amidated GAD65 restricted T cell hybridomas. Can be.

Peptides were synthesized by solid phase peptide synthesis in a 433 A Applied Biosystems automated peptide synthesizer. Peptides were assembled from the carboxy terminus to 0.05 mM size on Rink Amide MBHA resin (0.55 mM per gram of substitution level). The HOBt / HBTU coupling technique was used for the acylation of amines on the dendrite, and piperidine was used for the deprotection of Fmoc-protected a-amines of amino acids on the dendrite. N-methylpyrrolidinone (NMP) was used as a solvent for the coupling / deprotection reaction, and dichloromethane (DCM) was used for the final washing of the peptide resin. Deprotection was monitored by measuring the conductivity of the released Fmoc. If deprotection is difficult, the coupling is also difficult, and thus double coupling and / or acetylation after coupling have been introduced into the synthesis.

After assembly of the peptide chains on the resin, the peptide resin was vacuum dried for 2 hours and subjected to a deprotection protocol. The resin was resuspended in 2 ml of trifluoroacetic acid (TFA) containing 0.14 g of 4-methylmercaptophenol and 0.2 ml of 4-methoxybenzenethiol. The suspension was mixed for 2 hours and filtered through 200 ml of organic solvent (pentane: acetone 4: 1). Peptide suspensions were incubated overnight at -20 ° C. The suspension was left and a peptide film on the inner surface of the vial was observed. The clear solvent was decanted off and the film was slowly washed with 50 ml of pentane: acetone mixture. After a total of three washes, a 50 ml wash of pentane was repeated twice. The film was dissolved in 10 ml of 70% aqueous acetonitrile with 0.1% TFA and the solution was diluted to 30 ml with distilled water. The peptide solution was lyophilized and the resulting white powder was characterized by reverse phase HPLC and mass spectrometry. This product was used for peptide binding and T cell activation without further purification.

Example 11

Generation of C-Terminal Amidated GAD65 (aa524-543) Restricted Hybridoma T Cell Lines

NOD mouse hybridoma cell lines expressing T cell receptors specific for the C-terminal amidated GAD65 peptide were generated. The method of obtaining this hybridoma derives from the generation of mouse T cell hybridomas of Green Protocol, Current Protocols in Immunology, Wie. In summary, three 9-week-old female NOD mice were injected into the plantar feet with 50 μg C-terminal amidated GAD65 peptide in 100 ml CFA (complete Freund's adjuvant), resulting in proliferation of T cells limited to this peptide. After 8 days, mice were sacrificed by cervical dislocation and the spleen and lymph nodes (Sul, subcutaneous groin) were removed. In the Falcon 3002 Petri dishes, the lymph nodes were thinned between the two glass slides in suspension. The spleens were ground in cell suspension in a separate dish and then centrifuged at 12,000 RPM for 5 minutes at room temperature. The supernatant was removed and the plenosite was removed from the erythrocytes by dissolution: the splenocytes were resuspended in 0.9 ml sterile water for about 5-10 seconds, after which 0.1 ml 10X PBS was added quickly and then approximately 4 ml of Bruff medium (Click Medium EHAA; Irvine Scientific, Santa Ana, Calif.), 200 ml penicillin / streptomycin (BioWhittaker, Walkersville, MD), 200 ml L-glutamine (L-Glut, BioWhittaker), 15 g sodium bicarbonate (Sigma, St. Louis) , MO), 43 ml β-mercaptoethanol (Sigma), 11.6 ml gentamycin sulfate solution (Irvine Scientific), 10 liter sterile water including 10% fetal bovine serum (FBS, Hyclone, Logan, UT) was added. The cells were resuspended using a 5 ml pipette and the lipid material was filtered off. Cells were counted, adjusted to a concentration of 2 × 10 ⁶ cells / ml, and then stimulated in vitro with C-terminal amidated GAD65 peptide at a concentration of 10 mg / ml. Once the cells began to die (approximately 3-5 days), lymphocytes and splenocytes were harvested from the culture. Dead cells were removed by centrifugation through Ficoll-Hippark. Cells were adjusted to a density of 5 × 10 ⁶ to 2 × 10 ⁷ and covered with Ficoll-Hippa at a ratio of 5 ml to 5 ml. Cells were then centrifuged at 2000 RPM for 20 minutes at 4 ° C., washed twice with Bruff medium and finally washed with Bruff medium containing 0% FBS. BW5147 cells, lymphoma cell line (ATCC, Tumor Immunology Bank 48), were harvested and washed in wash medium. BW5147 cells were mixed in 1: 1 ratio with splenocytes and lymphocytes in Bruff medium containing 20% FBS. The cell mixture was centrifuged at 2000 RPM for 5 minutes at room temperature. The supernatant was aspirated off and 1 ml medium preheated to 37 ° C was added. 50% polyethylene glycol (PEG) solution (Sigma) was added dropwise to the cell pellet for 1 minute to facilitate cell fusion. After every dropwise addition, the pellet was stirred gently and then for a further 1 minute. 2 ml of preheated wash medium was added dropwise to the PEG / cell mixture for 2 minutes with a 2 ml pipette and gently stirred after each drop. The mixture was then centrifuged at 2000 RPM for 5 minutes and the supernatant was discarded. Thymus of unprimed NOD mice were removed and ground to Bruff medium containing 20% FBS. Thymic cells were counted and adjusted to a concentration of 5 × 10 ⁶ cells / ml. The number of thymus cells added was calculated so that the splenocytes were 100 ml / well, the number of 0.1-1 × 10 ⁵ cells / well. This number of thymic cells in Bruff medium with 20% FBS was strongly released onto the cell pellet. The cell mixture was then plated in 100 ml / well 96 well plates, leaving most of the wells empty to ensure sterilization. Plates were incubated at 37 ° C., 7.5% CO ₂ . The following day, 100 ml 2 × HAT (Sigma) in Bruff medium containing 20% FBS was added to each well and the plate was transferred to the incubator. The following day, cells were observed for the death of the fusion of both lymphocytes. Only fusions between lymphomas and lymphocytes should survive. At day 6 100 ml 2 × HAT (Sigma) in Bruff medium with 10% FBS was added to each well. The next day the cells were examined for expansion. Cells that appeared to have expanded were transferred to 24 well plates in 1 ml 1 × HAT (Sigma) in Bruff medium containing 20% FBS. Double sets were made and examined daily. Growing cells were transferred to T-25 flasks. This T cell hybridoma was gradually transferred to Bruff medium containing 20% FBS and 0% HAT and held for some time until screened for specificity for C-terminal amidated GAD65 peptide.

Example 12

Screening of C-terminal Amidated GAD65 Restricted T Cell Hybridoma Cell Lines

To determine the specificity of T cell hybridomas, antigen-presenting cells (APCs) were prepared by grinding and lysing NOD mouse spleen as in Example 11. Splenocytes were made up in 3 ml with Bruff medium containing 10% FBS. To prevent DNA synthesis, mitomycin C (Sigma) was added at 0.3 ml per 3 ml of cell suspension. APC was incubated for 30 minutes in a 37 ℃ water bath and washed three times in Bruff medium containing 10% FBS, each time centrifuged for 5 minutes at 1200 RPM. After the final wash, APCs were adjusted to a concentration of 2 × 10 ⁶ cells / ml in Bruff medium with 10% FBS. C-terminal amidated GAD65 peptides were titrated from 333 μg / ml to 0.15 μg / ml in round bottom 96 well plates. 50 μl (1 × 10 ⁵ ) APC was added to the peptide. Hybridomas were counted and adjusted to a concentration of ¹ × 10 ⁶ cells / ml in Bruff medium containing 10% FBS, and 100 μl (1 × 10 ⁵ ) cells were added to each well. Hybridomas were also tested for the following: peptides other than I-Ag ⁷ MHC + C-terminal amidated GAD65; MHC + C-terminal amidation GAD65 other than I-Ag ⁷ ; I-Ag ⁷ MHC alone; And C-terminal amidated GAD65 alone. Plates were incubated overnight at 37 ° C., 5% CO ₂ . The next day, 150 μl of spent medium was removed from each well, transferred to a flat bottomed 96 well plate and frozen to kill live cells. Only media consumed from wells in which T cells were activated will contain IL-2. IL-2 dependent CTLL cells (ATCC TIB-214) for survival were centrifuged, washed three times with Bruff medium containing 10% FBS and concentration of 5 × 10 ³ cells in 50 μl in a flat bottom 96 well plate. Plated. Supernatants collected from APC / hybridoma were thawed and 50 μl of supernatant was added to similar wells including CTLL cells. Two rows were plated as controls of CTLL cells. Dual control wells included medium and cells alone or cells, medium and titrated IL-2. Plates were incubated overnight at 37 ° C., 5% CO ₂ . The following day, cells were pulsed with 1 μCi / well of ³ H-thymidine. The plate was incubated overnight to insert ³ H-thymidine into the cells. The following day, cells were harvested with Skatron Basic 96 Cell Harvester (Carlsbad, CA) according to the manufacturer's instructions. The filter mat was dried overnight and then placed in a sample bag. Approximately 10 ml Beta Scint scintillation solution (Wallac, Turku, Finland) was added and the bag sealed. Insertion of ³ H-thymidine into DNA was measured by Wallac 1205 Betaplate Beta Counter (Turku, Finland). Insertion of ³ H-thymidine by CTLL cells showed IL-2 in the spent medium, and the native hybridomas in the medium were directed to the I-Ag ⁷ MHC + C terminal amidated GAD65 peptide of NOD-extracted APC. Is activated. Thus, wells containing CTLL cells with a high proliferative response correspond to hybridomas specific for the peptide: MHC complex. Initial fusion produced hybridoma MBD.1, which showed a strong proliferative response with ³ H-thymidine injected> 5000 cpm. This indicates that it is specific for the C terminal amidated GAD65 peptide + I-Ag ⁷ . It also showed less response> 2000 CMP to the same GAD65 peptide lacking C terminal amidation. However, no response was seen with any of the other MHC / peptide combinations. All other cells showed a stimulus response of <500 cpm. The second fusion produced several additional hybridomas showing specificity for the C-terminal amidated GAD65 peptide + I-Ag ⁷ MHC, which were MBD2.3, MBD2.7, MBD2.8, MBD2.11 and MBD2 .14.

Example 13

Identification of amino acid residues required for binding of peptides with C-terminal amidated GAD65 peptide + NOD MHC class II, I-Ag ⁷ restricted T cell hybridomas

C-terminal amidated GAD65 + I-Ag as described above⁷Specific hybridomas ( MBD.1, MBD2.3, MBD2.7, MBD2.8, MBD2.11 and MBD2.14) were subjected to I-Ag using the method described in Example 12.⁷Screen for specificity for Ala scan peptide or truncated peptide. In summary, Ala scan peptides or truncated peptides were tested at a series of concentrations between 333 and 0.15 μg / ml. Proliferation of CTLL cells indicates that certain alanine substitutions (or cleavage of specific amino acids) do not affect the binding of the specific hybridoma's T cell receptor and MHC-peptide complexes. Lack of proliferation indicates that the substituted (or truncated) residue is involved in binding of the complex by the T cell receptor. Proliferation was severely affected by single substitution of alanine at amino acid positions 524, 526, 527, 528, 529, 531, 532, or 533, or tyrosine substitution at positions 530 or 535 as compared to unsubstituted control peptides. . With cleaved peptides comprising amino acids 527-539, activation of T cell hybridomas with at least one T cell hybridoma that recognizes peptides containing amino acids 529-539 has been shown, which indicates that these residues have been tested for T Important for binding to cellular hybridomas.

Example 14

Peptide Binding with NOD MHC Class II I-Ag ⁷

The relative affinity of a given peptide (Ala scan or truncated) for MHC is Jensen et al., J. Immunol. Meth. Measured by Europium-Streptavidin dissociation enhanced Lanthanon Element Fluoroimmunoassay (DELFIA) developed by 163: 209, 1993. This assay can utilize whole cells or solubilized MHC molecules. Each peptide was analyzed in triplicate. For example, for Ala scan peptides, NOD spleen cells were fixed with 1% paraformaldehyde for 10 minutes at room temperature or 30 minutes on ice, followed by RPMI 1640, 1% PSN (GIBCO-BRL, Gaithersburg, MD), 200 mM L Wash once with glutamine (Hazelton Biologics, Lenexa, KS) and 10% heat inactivated fetal bovine serum (FCS) and wash twice with DPBS (Dulbecco's PBS, BioWhittaker, Walkersville, MD). 1 × 10 in 0.15M NaCl with 1:50 dilution of protease inhibitor stock solutions D, E, and F (Tables 5a and 5b), 0.01% sodium azide, and 1M citrate / PO ₄ , pH5.5 ^{Resuspend the} cells at ⁷ cells / ml.

Protease Inhibitor Stock Solution Stock D 50X 150 mg Phenanthroline 108 mg PMSF (phenylmethylsulfonyl fluoride)

Stock D 50X 1.8 mg Pepstatin 30 mg TPCK (N-Tosyl-L-phenylalanine Chloromethyl Ketone) 120 mg Benzamidine 150 mg Iodoacetamide 126 mg NEM 3 ml Soluble in methanol Stock E 50X 1 mg Roypeptin 15 mg TLCK (N-α-p-tosyl-L-lysine chloromethyl ketone) Dissolve in 3 ml H ₂ O with 15 μl of 1M citrate / PO ₄ , pH5.5 Stock F 50X 8.76 mg EDTA Dissolve in 15 μl 1M Tris, 3ml H ₂ O with pH8.0.

100 μl of cell-protease inhibitor mixture was added to a 96-well round bottom plate (Costar, Pleasanton, Calif.). Immobilized NOD cells were incubated at 37 ° C. with biotinylated and C terminal amidated GAD65 peptides at concentrations of 10,000 nM and unlabeled Ala scan peptides at concentrations of 100,000, 1,000 and 10 nM. Known allele-specific peptide (SEQ ID NO: 61), mouse serum albumin (MSA), with high affinity for I-Ag ⁷ was used as a positive control, binding to IA ^d but binding to I-Ag ⁷ Ea not used was used as negative control (Reich et al., J. Immunol. 154: 2279-88, 1994). After incubation, the plates were vortexed and centrifuged in a Beckman GA-6R centrifuge (Beckman, Fullerton, CA) for 10 minutes at 1500 rpm. Supernatant was removed and cells were treated with 60 μl / well NP-40 lysis buffer (0.5% NP40, 0.15M NaCl, 50 mM Tris, pH 8.0, 0.01% sodium azide, protease inhibitor stock D, E, F (Table 1:50 dilution of 3)). Cells were incubated on ice for 30 minutes with mixing every 15 minutes. Thereafter, the resultant was centrifuged at 1500 rpm for 10 minutes to obtain a clear melt.

Assay plates were prepared by coating 96-well flat bottom plates (Costar) with 100 μl / well anti-I-Ag ⁷ antibody (10.2.16, 50 μg / ml, TSD Bioservices, Germantown, NY) in DPBS. The plates were incubated at 4 ° C. for 12-18 hours. Unbound antibody was removed and plates were plated in 200 μl / well MTB (1% BSA, 5% powdered scheme milk, TTBS (0.1% Tween 20, 0.5M Tris, 1.5M NaCl, pH 7.5) for 30 minutes at room temperature). 0.01% sodium azide). 50 μl of MTBN (1% BSA, 5% powdered scheme milk, 0.01% sodium azide, NP40 in TTBS) was added to the wells and 50 μl of the above clear lysate. Plates were incubated at 4 ° C. for 2 hours and washed 7 times with TTBS. Europium-labeled streptavidin (Wallac # 1244-360) diluted 1: 1000 with DELFIA assay buffer (Table 6a and Table 6b) was added to the plate at 100 μl / well.

DELFIA Assay Buffer Buffer stock 0.1 M Tris 0.15 M NaCl 0.05% sodium azide 0.01% Tween-20 pH 7.75

10 mM DTPA Stock 20 mM Na ₂ CO ₃ DTPA (diethylenetriaminepentaacetic acid, Sigma, St. Louis, MO) DELFIA Assay Buffer 200 μl 10 mM DTPA stock 100 ml buffer stock 0.5 g BSA (bovine serum albumin)

The plate was incubated at 4 ° C. for 1 hour and then washed 7 times with TTBS. 100 μl of Reinforcement Solution A (Table 7) was added to each well, taking care not to foam the reagents, and the plate was vibrated for 3 minutes at room temperature. Plates were measured with a time-lapse fluorometer (Wallac 1234 DELFIA Research Fluorometer).

Reinforcing Solutions A and B Solution A 2 mM sodium acetate, pH 3.1 0.05% Triton X-100 60 μM BTA (benzoyl trifluoroacetone, Sigma # B5875) 8.5 μM yttrium oxide (Sigma # Y3375) ddH ₂ O, stored in dark container at 4 ° C. Solution B 250 mM Tris-HCl, pH 7.0 250 Phen (1,10-phenanthroline, Sigma # P1294) ddH ₂ O, stored in dark container at 4 ° C

Single substitution of alanine at amino acid positions 524, 526, 527, 528, 529, 531, 532, or 533, or substitution of tyrosine at amino acid positions 530 or 535 is unsubstituted for the NOD MHC (I-Ag ⁷ ) binding site It resulted in a peptide that could no longer compete with the biotinylated C terminal amidated GAD65 peptide. Substitution of alanine instead of arginine at position 536 prevented activation in 4 of 6 T cell hybridomas. Substitution of alanine for methionine at position 537 prevented activation in 5 of 6 hybridomas. The substitution of alanine for methionine at position 538 prevented the activation of one of the T cell hybridomas. As determined by peptide cleavage, the GAD65 epitope binding to I-Ag ⁷ includes amino acids 527-539. This is associated with hybridoma data, indicating that amino acids 527-539 are involved in binding to the NOD MHC class II molecule, I-Ag ⁷ . Suitable GAD peptides will be amino acids 525-540 (SEQ ID NO: 60).

Example 15

In vitro induction of anergy to peptide-MHC complex

This assay examines whether specific peptide-MHC complexes induce anergy in C-terminal amidated GAD65 restricted T cell clones or in vivo primed lymphocytes.

Flat bottom 96 well plates (Costar) were coated with 100 μl / well (5 μg antibody / well) anti-class II antibody (10.2.16, 50 μg / ml, TSD Bioservices, Germantown, NY) in DPBS and 4 ° C. Incubated for 12-18 hours. Unbound antibody was removed and plates were blocked with 5% BSA (bovine serum albumin, Sigma), incubated for 30 minutes at room temperature and washed 5-7 times in Bruff medium with 10% FBS. Peptide-MHC complexes, preferably I-Ag ⁷ , or Ala scan or truncated GAD peptides complexed with C-terminal amidated GAD65, were added at 2 and 10 μg / ml. Controls include peptide-MHC complexes such as I-Ag ⁷ -MSA-OH; Peptides alone or MHCs alone, each of which may be added at the same concentration as the peptide-MHC complex. Plates were then incubated at 4 ° C. for 8-10 hours. C-terminal amidated GAD65-limited T cell clones were counted and diluted with Bruff medium containing 10% FBS to plate 6 × 10 ⁵ cells per well in 200 μl medium. Plates were incubated 12-18 hours at 37 ° C.

In vivo primed lymphocytes can also be used in place of T cell clones. In summary, as described in Example 11, NOD mice were primed with 30-50 μg peptide / 150 μl complete Freund's adjuvant in the plantar. After 8 days, mice were sacrificed and the spleen, slaw and superficial inguinal lymph nodes were removed. The tissue was ground, prepared, mitomycin C treated, and then ready for analysis as in Example 11.

The next day, the plates were washed to remove unbound complexes. Cells were pipetted from plate to separate labeled Eppendorf tubes, centrifuged at 1200 RPM for 5 minutes and washed three times with Bruff medium containing 10% FBS. The cells were counted and each tube was divided into two tubes, one tube containing one third of the total cell number and the other tube containing two thirds. Cells were centrifuged again and tubes containing 1/3 of the cells diluted to 200 μl with Bruff medium containing 10% FBS and 10 U / ml IL-2. The other tube was diluted to 400 μl with Bruff medium containing 10% FBS without IL-2.

The second 96-well plate contains 0.1 μg / ml anti-CD3 (CD3-e cytochrome antibody, Pharmingen) such that for each sample analyzed, at least two wells contain α-CD3 and at least four wells contain peptides. , San Diego, CA), or by adding peptides such as C-terminal amidated GAD65 in amounts of 10 μl / well in 0.6 μg / μl stock. Antigen presenting cells (APC) were prepared as described in Example 12 and diluted to 5 × 10 ⁶ cells / ml with Bruff medium containing 10% FBS and 100 μl was added only to wells containing peptides. Without IL-2, 100 μl of previously prepared T cell clones or in vivo primed lymphocytes were added to wells containing α-CD3 and to half of the wells containing peptide and APC. T cell clones or lymphocytes treated with IL-2 were added only to the remaining wells, including peptides and APCs, and the final form consisted of three treatments: α-CD3; Peptide + APC with IL-2; And for each of peptide + APC without IL-2, was in the form of a dual well comprising peptide-MHC complex or control peptide-MHC. T cell / lymphocyte concentration should be at least 5 × 10 ⁴ cells / well, preferably about 2.3 × 10 ⁵ to about 5.3 × 10 ⁵ . Plates were incubated at 37 ° C. for 3 days.

Cells were then pulsed with ¹ μCi / well of ³ H-thymidine. The plates were incubated for 5 hours to insert ³ H-thymidine into the cell DNA. Cells were then harvested from Skatron Basic 96 Cell Harverster according to the manufacturer's instructions. The filter mat was dried overnight and then placed in a sample bag. Approximately 10 ml of Beta Scint scintillation solution (Wallac, Turku, Finland) was added and the bag sealed. Insertion of ³ H-thymidine into DNA was measured by Wallac 1205 Betaplate Beta Counter (Turku, Finland). Insertion of ³ H-thymidine by T cells indicates that T cells have recovered from anergy by the addition of IL-2. If T cells become anergic and add APC and peptide (but not IL-2), it will not respond to APC and peptide and no insertion of ³ H-thymidine will occur. Using α-CD3 as a control it was shown that the cells were still alive and normally responding to other stimulators.

Example 16

Selective delivery

IDDM can be selectively delivered by injecting spleen cells from a diabetic donor into a non-diabetic recipient. Female NOD / CaJ mice were screened for diabetes by monitoring urine glucose levels. Animals with positive urine values of at least 250 mg / dl glucose were analyzed for blood glucose levels by tail cutting. In addition, if blood glucose was 250 mg / dl or more, mice were classified as obvious diabetes.

Newly diabetic NOD mice were examined (730 rad) and randomly divided into four treatment groups and splenocytes were isolated as described above. Non-diabetic and 7-8 week old NOD recipient mice were divided into four groups. The first group received 1 × 10 ⁷ splenocytes by intravenous injection. Six hours after injection, a second intravenous injection of saline, 10 μg / mouse C-terminal amidated GAD65 peptide, or 10, 5, or 1 μg / mouse C-terminal amidated GAD65 peptide-MHC complex was received. The second group received 2 × 10 ⁷ splenocytes, then saline, 10 μg / mouse C-terminal amidated GAD65 peptide-MHC complex, or 5 μg / mouse MSA-MHC complex. The third group received injections of 1 × 10 ⁷ splenocytes and saline, 10 μg / mouse C-terminal amidated GAD65 or 200 μg / mouse 10.2.16, anti-class II antibody. The fourth group was receiving injections of 1x10 ⁷ leno spool site, then brine, 20μg / mouse C- terminal amidated peptide GAD65, or 1, 5, or 10μg / mouse C- terminal amidated peptide GAD65 -MHC complex. Group 4 mice received only two treatments with peptides or peptide-MHC complexes, one at day 0 and the second at day 4. The other group received further treatment around 8 and 12 days. Mice were tested for onset of diabetes by urinalysis. As determined by urine and blood glucose levels, on the day when the first animal shows clear signs of diabetes, mice from each treatment group are randomly selected and urine and blood glucose levels are determined for all selected mice and then the animals And spleen and pancreas were removed for immunohistochemical analysis. Saline-treated mice developed diabetes within about 12-20 days. The first group of mice that received four treatments of the 10 μg peptide-MHC complex did not show significant expression of the disease until day 30 and did not express the disease until day 75. Receipt of 5 μg peptide-MHC complex stabilized in 40% diseased mice by day 30, gradually increased disease expression by day 80, and showed 100% disease in mice by day 80. The fourth group of mice, which received only two treatments of the peptide-MHC complex, showed some delay in disease initiation. That is, less than 50% of mice receiving 10 μg peptide-MHC showed disease by 30 days. Blocking with anti-MHC antibodies in group 3 delayed the onset of the disease but provided less protection. That is, at least 75% of mice receiving 10 μg peptide alone expressed disease by day 30. While the C terminal amidated GAD65 (SEQ ID NO: 59) peptide alone promoted the onset of diabetes in this selective delivery model, the peptide-MHC complex prevented the onset of diabetes.

From the foregoing description, while specific embodiments of the invention have been described herein for purposes of illustration, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Sequence list

(1) General Information:

(i) Applicant: Zymogenetics Incorporated

United States 98102 Washington Seattle Eastlake

Avenue East 1201

Anergen Incorporated

United States of America 94063 California Redwood City

Penobscote Drive 301

(ii) Title of the Invention: Immune Modulators and Methods Associated With Them

(iii) number of sequences: 61

(iv) communication address:

(A) Recipient: Zymogenetics Incorporated

(B) Street: Eastlake Avenue East 1201

(C) City: Seattle

(D) State: Washington

(E) Country: United States

(F) ZIP Code: 98102

(v) computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.25

(vi) the present application data:

(A) Application number:

(B) filing date:

(C) Classification:

(vii) Priority application data:

(A) Application number: US 08 / 480,002

(B) Application date: June 7, 1995

(vii) Priority application data:

(A) Application number: US 08 / 483,241

(B) Application date: June 7, 1995

(vii) Priority application data:

(A) Application number: US 08 / 482,133

(B) Application date: June 7, 1995

(vii) Priority application data:

(A) Application number: US 60 / 005,964

(B) Application date: October 27, 1995

(viii) Agent Information:

(A) Name: Gary E. Parker

(B) Registration Number: 31-648

(ix) Telecommunication Information:

(A) Phone: 206-442-6673

(B) Fax: 206-442-6678

(2) Information about SEQ ID NO: 1

(i) Sequence Features:

(A) Length: 33 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 1:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 58 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 2:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 58 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 60 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 4:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 60 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 5:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 59 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 6:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 59 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 7:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 27 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 8:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 25 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 9:

(2) Information about SEQ ID NO: 10:

(i) Sequence Features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 10:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 11:

(2) Information about SEQ ID NO: 12:

(i) Sequence Features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 12

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 13

(2) Information about SEQ ID NO: 14:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 14

(2) Information about SEQ ID NO: 15:

(i) Sequence Features:

(A) Length: 111 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 15

(2) Information about SEQ ID NO: 16:

(i) Sequence Features:

(A) Length: 39 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 16:

(2) Information about SEQ ID NO: 17:

(i) Sequence Features:

(A) Length: 32 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 17

(2) Information about SEQ ID NO: 18:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 18:

(2) Information about SEQ ID NO: 19:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO 19:

(2) Information about SEQ ID NO: 20:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 20

(2) Information about SEQ ID NO: 21:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 21

(2) Information about SEQ ID NO: 22:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 22

(2) Information about SEQ ID NO: 23:

(i) Sequence Features:

(A) Length: 107 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 23

(2) Information about SEQ ID NO: 24:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 24

(2) Information about SEQ ID NO: 25:

(i) Sequence Features:

(A) Length: 72 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 25

(2) Information about SEQ ID NO: 26:

(i) Sequence Features:

(A) Length: 63 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 26

(2) Information about SEQ ID NO: 27:

(i) Sequence Features:

(A) Length: 63 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 27

(2) Information about SEQ ID NO: 28:

(i) Sequence Features:

(A) Length: 33 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 28

(2) Information about SEQ ID NO: 29:

(i) Sequence Features:

(A) Length: 5 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 29

(2) Information about SEQ ID NO: 30:

(i) Sequence Features:

(A) Length: 5 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 30

(2) Information about SEQ ID NO: 31:

(i) Sequence Features:

(A) Length: 7 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 31

(2) Information about SEQ ID NO: 32:

(i) Sequence Features:

(A) Length: 25 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 32

(2) Information about SEQ ID NO: 33:

(i) Sequence Features:

(A) Length: 21 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 33

(2) Information about SEQ ID NO: 34:

(i) Sequence Features:

(A) Length: 8 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 34

(2) Information about SEQ ID NO: 35:

(i) Sequence Features:

(A) Length: 15 base pairs

(B) Type: nucleic acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 35

(2) Information about SEQ ID NO: 36:

(i) Sequence Features:

(A) Length: 15 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 36

(2) Information about SEQ ID NO: 37:

(i) Sequence Features:

(A) Length: 13 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 37

(2) Information about SEQ ID NO: 38:

(i) Sequence Features:

(A) Length: 12 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 38

(2) Information about SEQ ID NO: 39:

(i) Sequence Features:

(A) Length: 11 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 39

(2) Information about SEQ ID NO: 40:

(i) Sequence Features:

(A) Length: 20 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 40

(2) Information about SEQ ID NO: 41:

(i) Sequence Features:

(A) Length: 10 amino acids

(B) Type: amino acid

(C) strands: single strand

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 41

(2) Information about SEQ ID NO: 42:

(i) Sequence Features:

(A) Length: 654 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..654

(xi) Sequence content: SEQ ID NO: 42

(2) Information about SEQ ID NO: 43:

(i) Sequence Features:

(A) Length: 273 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..273

(xi) Sequence content: SEQ ID NO: 43

(2) Information about SEQ ID NO: 44:

(i) Sequence Features:

(A) Length: 261 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..261

(xi) Sequence content: SEQ ID NO: 44:

(2) Information about SEQ ID NO: 45:

(i) Sequence Features:

(A) Length: 633 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..633

(xi) Sequence content: SEQ ID NO: 45

(2) Information about SEQ ID NO: 46:

(i) Sequence features:

(A) Length: 273 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..273

(xi) Sequence content: SEQ ID NO 46:

(2) Information about SEQ ID NO: 47:

(i) Sequence features:

(A) Length: 261 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..261

(xi) Sequence content: SEQ ID NO: 47

(2) Information about SEQ ID NO: 48:

(i) Sequence features:

(A) Length: 16 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(xi) Sequence content: SEQ ID NO: 48

(2) Information about SEQ ID NO: 49:

(i) Sequence features:

(A) Length: 621 base pairs

(B) Type: nucleic acid

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..621

(xi) Sequence content: SEQ ID NO: 49

(2) Information about SEQ ID NO: 50:

(i) Sequence features:

(A) Length: 279 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..279

(xi) Sequence content: SEQ ID NO: 50:

(2) Information about SEQ ID NO: 51:

(i) Sequence features:

(A) Length: 243 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..243

(xi) Sequence content: SEQ ID NO: 51

(2) Information about SEQ ID NO: 52:

(i) Sequence features:

(A) Length: 702 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..702

(xi) Sequence content: SEQ ID NO 52:

(2) Information about SEQ ID NO: 53:

(i) Sequence features:

(A) Length: 588 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..588

(xi) Sequence content: SEQ ID NO: 53

(2) Information about SEQ ID NO: 54:

(i) Sequence features:

(A) Length: 1323 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..1323

(xi) Sequence content: Sequence ID: 54

(2) Information about SEQ ID NO: 55:

(i) Sequence features:

(A) Length: 318 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..318

(xi) Sequence content: SEQ ID NO: 55

(2) Information about SEQ ID NO 56:

(i) Sequence features:

(A) Length: 1341 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..1341

(xi) Sequence content: SEQ ID NO 56:

(2) Information about SEQ ID NO: 57:

(i) Sequence features:

(A) Length: 588 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..588

(xi) Sequence content: SEQ ID NO 57:

(2) Information about SEQ ID NO 58:

(i) Sequence features:

(A) Length: 312 base pairs

(B) Type: Hexane

(C) Number of strands: double strand

(D) form: linear

(ii) Molecular Type: cDNA

(ix) Features:

(A) Name / Key: CDS

(B) Location: 1..312

(xi) Sequence content: SEQ ID NO 58:

(2) Information about SEQ ID NO: 59:

(i) Sequence features:

(A) Length: 20 amino acid

(B) Type: amino acid

(C) Number of strands: single

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO: 59

(2) Information about SEQ ID NO: 60:

(i) Sequence features:

(A) Length: 16 amino acids

(B) Type: amino acid

(C) Number of strands: single

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO 60:

(2) Information about SEQ ID NO: 61:

(i) Sequence features:

(A) Length: 15 amino acids

(B) Type: amino acid

(C) Number of strands: single

(D) form: linear

(ii) Molecular Type: Peptide

(xi) Sequence content: SEQ ID NO 61:

Claims (26)

A first DNA fragment encoding at least a portion of a first domain of a selected MHC molecule; A second DNA fragment encoding at least a portion of a second domain of the selected MHC molecule; Encoding from about 5 to about 25 amino acids and binding to the first and second DNA fragments by a first linker DNA fragment to produce a fused first DNA-first linker-second DNA poly fragment; A first linker DNA fragment connecting the second DNA fragment to fit the frame; A third DNA fragment encoding an antigenic peptide capable of associating with a peptide binding groove of a selected MHC molecule; From about 5 to about 25 amino acids to link the first DNA-first linker-second DNA poly fragment and the third DNA fragment fused by the second linker DNA fragment to produce a soluble fused MHC heterodimer: peptide complex A soluble fused MHC heterodimer: peptide complex consisting of a second linker DNA fragment encoding a and a third DNA fragment in frame with the fused first DNA-first linker-second DNA poly fragment. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the selected MHC molecule is an MHC class II molecule. The soluble fused MHC heterodimer: peptide complex of claim 2 wherein the first DNA fragment encodes a β1 domain. The soluble fused MHC heterodimer: peptide complex of claim 2 wherein the second DNA fragment encodes an α1 domain or an α1α2 domain. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the selected MHC molecule is selected from the group consisting of I-Ag ⁷ , IA ^s , DR1β * 1501 and DRA * 0101. The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the selected MHC molecule is an MHC class I molecule. The soluble fused MHC heterodimeric: peptide complex of claim 1, wherein the first linker DNA fragment is GASAG (SEQ ID NO: 29) or GGGGSGGGGSGGGGS (SEQ ID NO: 36). The soluble fused MHC heterodimer: peptide complex according to claim 1, wherein the second linker DNA fragment is GGSGG (SEQ ID NO: 30) or GGGSGGS (SEQ ID NO: 31). The soluble fused MHC heterodimer: peptide complex of claim 1, wherein the third DNA fragment encodes an antigenic peptide capable of stimulating an MHC-mediated immune response. The peptide consists 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 of claim 9, wherein the antigenic peptide is selected from. The fourth DNA fragment of claim 1, wherein said MHC heterodimer: peptide complex further encodes at least a portion of a third domain of the selected MHC molecule, and a fused third DNA-second linker-first DNA- A third linker DNA fragment encoding about 5 to about 25 amino acids and linking the second and fourth DNA fragments in frame to produce a first linker-second DNA-third linker-fourth DNA poly fragment; Soluble fused MHC heterodimer: peptide complex, characterized in that it comprises a. 12. The soluble fused MHC heterodimer: peptide complex of claim 11 wherein the selected MHC molecule is an MHC class I molecule. 12. The soluble fused MHC heterodimer: peptide complex of claim 11 wherein the selected MHC molecule is an MHC class II molecule. 12. The soluble fused MHC heterodimer: peptide complex of claim 11 wherein the fourth DNA fragment is a β2 chain. 12. The soluble fused MHC heterodimer: peptide complex of claim 11, wherein the third linker DNA fragment is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32). An isolated polynucleotide molecule encoding the soluble fused MHC heterodimer: peptide complex of claim 1. A fusion protein expression vector capable of expressing a soluble fused MHC heterodimer: peptide complex of claim 1 comprising the following operably linked elements: Transcriptional promoters; A first DNA fragment encoding at least a portion of a first domain of a selected MHC molecule; A second DNA fragment encoding at least a portion of a second domain of the selected MHC molecule; Encoding from about 5 to about 25 amino acids and binding to the first and second DNA fragments by a first linker DNA fragment to produce a fused first DNA-first linker-second DNA poly fragment; A first linker DNA fragment connecting the second DNA fragment to fit the frame; A third DNA fragment encoding an antigenic peptide capable of associating with a peptide binding groove of a selected MHC molecule; From about 5 to about 25 amino acids to link the first DNA-first linker-second DNA poly fragment and the third DNA fragment fused by the second linker DNA fragment to produce a soluble fused MHC heterodimer: peptide complex A second linker DNA fragment that encodes a linker and connects the third DNA fragment to frame with the fused first DNA-first linker-second DNA poly fragment; And Warrior Terminator. The fourth DNA fragment of claim 17, wherein said MHC heterodimer: peptide complex further encodes at least a portion of a third domain of the selected MHC molecule, and a fused third DNA-second linker-first DNA- A third linker DNA fragment encoding about 5 to about 25 amino acids and linking the second and fourth DNA fragments in frame to produce a first linker-second DNA-third linker-fourth DNA poly fragment; Expression vector comprising a. A cell into which the expression vector according to claim 17 is introduced, wherein the cells expressing the soluble fused MHC heterodimer: peptide complex encoded by the DNA polyfraction are collected, and the soluble fused MHC heterodimer: peptide complex is collected. Soluble fused MHC heterodimer: peptide complex produced by. A pharmaceutical composition comprising the soluble fused MHC heterodimer: peptide complex of claim 1 in combination with a pharmaceutically acceptable excipient. An antibody that binds to the epitope of the soluble fused MHC heterodimer: peptide complex of claim 1. A method of treating a patient to reduce an autoimmune response, the method comprising inducing immunological inhibition in the patient by administering a therapeutically effective amount of the soluble fused MHC heterodimer: peptide complex of claim 1. Way. A method of making a responder cell clone that proliferates when bound with a selected antigenic peptide provided by a stimulator cell, wherein the non-adhesive, CD56-, CD8- cells that are reactive with the selected antigenic peptide are isolated to separate the responder cells. Forming a; Stimulating the responder cells with pulsed or primed stimulator cells; Restimulating the stimulated responder cells with pulsed or primed stimulator cells; And Separating the responder cell clones. The method of claim 23, wherein the responder cells are isolated from prediabetes or newly disclosed diabetic patients. The method of claim 23, wherein the responder cell clone is a T cell clone. The method of claim 23, wherein the selected antigenic peptide is a GAD peptide.