JPH07501526A - MHC subunit conjugates useful in ameliorating adverse immune responses - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Closing of applications related to MMC subunit conjugates useful in ameliorating adverse immune responses reference This application is filed in U.S. Patent Serial No. 07/793, filed on November 19, 1991. ．． Part of No. 938 is pending. It includes the following co-pending and abandoned U.S. Patent application: (filed on April 23, 1991) Serial number 07/690/8 40: (filed on December 12, 1990) Serial number 07/635.8 40; (filed on June 23, 1988) Serial number 07/210.59 Related to 4. All of which are incorporated herein by reference in their entirety. nothing.

Background of the invention The invention relates to the treatment of diseases associated with adverse immune responses and to the treatment of such diseases. and materials and methods useful in diagnosis. In particular, it selectively binds to T cells. , and the major groups that complex with peptide fragments from antigens involved in these diseases. Relating to a complex comprising subunits from the tissue compatibility complex (MHC) glycoproteins . The complexes themselves are therapeutically useful or they are used for diagnostic purposes. to radioisotopes or other labels, or to specifically ablate target cells. It may further be conjugated to toxins or other substances.

MHC molecules are heterodimeric glycoproteins expressed on upper vertebrate cells. (heterodimeric glycoproteins), and Plays a role in the immune response. In humans, IHC molecules are It was first identified in white blood cells, so it is called HLA (human-leukocyte-associated). . In mice they are named H-2 antigens.

MHC molecules are divided into two groups, class I and class 11, which are structurally different from each other. Structurally and functionally different. Generally, the main function of MMC molecules is to binding to peptides and expressing them on the surface of cells . These peptides are peptide fragments that are as short as 8 to 20 amino acids. antigen-presenting cells that process the antigen into fragments that can be It is born from the presenting cell).

Class I MHC molecules are expressed on almost all nuclear cells and is recognized by cytotoxic T lymphocytes, which in turn antigen-containing cells (antig Destroy the en-bearing ce). Class [[MHC molecules are Cells involved in the initiation and maintenance of responses, e.g. T lymphocytes, B lymphocytes, macrophages Mainly expressed on phage, etc. Class If MHC molecules are helper T lymphocytes and the proliferation of helper T lymphocytes, and the specific antigen that appears induces an amplification of the immune response against the sex peptide. General discussion of the functions of MHC molecules For details, see Gray, Hlt, et al, 5 scientific. American November, 1989:56-64 (quote this incorporated herein by. )checking.

In addition to binding to antigenic peptides, MHC molecules It is also possible to bind to peptides that are conjugated to peptides (gous), or "self" peptides. Next When T lymphocytes respond to cells expressing "self" peptides, , autoimmune symptoms occur. Bus, S, et al, 5cien ce 242:1045-1047 (1988); Demotz, et. al., Nature 342:682-684 (1989).

(These publications are incorporated herein by reference). self over 30 A currently known immune disease is myasthenia gravis (myasthenia gravis). avis (MG)), multiple sclerosis (MG) (MS), systemic erythematosus ffi (MS), hemat.

5is (SLE)), rheumatoid arthritis itis (RS, insulin-dependent diabetes mellitus) ndent diabetes mellitus (IDDM)), etc. . A characteristic feature of these diseases is an attack by the victim's immune system on the tissues of the victim.

In individuals without disease, such attacks do not occur. Because the immunity A system is resistant to “self,” i.e., it cannot be treated as foreign. do not recognize “self” tissues; however, those suffering from autoimmune diseases In patients, such resistance does not occur and the tissue components are foreign. be recognized as such. For a general review of autoimmune diseases, see 5inha et al,, 5science 248:1380-1387 (this was published in 1990) , incorporated into this EA specification by reference. )checking.

The involvement of MHC class 11 proteins in autoimmune diseases has been demonstrated in numerous in vitro studies. and in animal models. For example, MHC class I [protein Antibodies against M1 itself or M1 (an agent that induces the expression of C class [1 gene)] Administration of antibodies against either prevents the development of autoimmune conditions. Helper T cells The role of monocloner for CD4, a characteristic helper T cell surface molecule, is also (Shizuru, J., A, et al., 5science (1988) 240:659-66 2). Additionally, in vitro assays may be used to determine whether anergy or reproductive nonresponsiveness ss) is an incompletely characterized costimulatory signal. ry signal) within lymphocytes by LlHC class II molecules. (Schwartz, Ce1 l(+989) +073-1081 and Quill et al, J, I mmunol, (1987) 138:3704-3712 (please refer to this This is incorporated herein by reference. )checking. ).

A number of other etiological responses involving unwanted immune responses are known. for example , many allergic diseases are associated with specific &IHC alleles or are autologous. It has been suggested that it has an autoimmune component. This invention is incorporated herein by reference. . )checking. Exemplary allergens include pigweed ( ragweed), cat allergens, and grass polle n).

Other deleterious T-cell-mediated responses occur in transplants from allogeneic hosts. intentionally introduced into the body as (grafts or transplants) It involves the destruction of foreign cells that have been introduced. “allograft rejection” This process, known as ``injection'', involves the interaction between host T cells and foreign MBC molecules. including interactions. Quite often, a wide range of MHC alleles is homologous. Involved in host response to explants.

Current treatments for autoimmune diseases and other immunopathologies are aimed at treating the symptoms. It is not about interfering in the etiology of the disease. Many unwanted side effects Broad spectrum chemotherapeutic agents with various uses are typically used. to helper T cells Compounds that can selectively suppress autoimmune responses are safer and more effective. provide more effective treatment. Additionally, such immunosuppressive compounds may be used to treat non-autoimmune diseases. diseases, such as graft versus host disease (graft versus host disease). ease (GVHD)) or in the treatment of various allergic responses.

For example, patients with chronic GVHD often experience symptoms and symptoms that mimic certain autoimmune diseases. and show signs of growth.

Inappropriate treatments currently available suppress MHC-restricted immune responses, but lead to unwanted side effects. identification of new agents that avoid overall nonspecific suppression of an individual's immune response, e.g. indicates an urgent need to do so.

Summary of the invention The present invention seeks to identify and suppress the above-mentioned aspects of the immune system that are responsible for unwanted immune responses. The present invention is directed to methods and compositions that can be used to. The set of the present invention The composition consists of a single-chain subunit of an MHC-encoded antigen-presenting glycoprotein and an antigenic peptide. A purified composition comprising an active portion of tide. These two components are covalently bonded. They can be bound by synthetic or non-covalent association. Third component, Sunawa Also, effector components can be included as well. The effector component is If the conjugate is used, for example, to diagnose autoimmune diseases, the labeled can be In addition, this effector component is Can be a toxin when used to selectively remove T cell populations Ru.

In another embodiment, a pharmaceutical composition in which a conjugate of the invention is the active ingredient. is directed towards. The composition may be used, for example, to treat certain autoimmune diseases associated with autoimmune diseases. down-regulate the parts of the immune system that are reactive with antigens ulate) or can be used for removal.

Brief description of the drawing Figure 1 shows the three-dimensional structure of human HLA-A2 antigen (class I).

Figure 2 shows a preferred second generation M)IC protein design.

Figure 3 shows the amino acid sequence for the alpha subunit of the acetylcholine receptor protein. and mRNA are shown.

Figure 4 shows the myelin basic protein protein. The amino acid sequence is shown.

Figure 5 shows the nucleotide sequence coating the [-A b-α chain.

Figure 6 shows the nucleotide sequence encoding the I-A''-β chain.

Figure 7 shows the list of DQ/DR haplotypes in humans. and their relationship with autoimmune diseases.

Figure 8 shows the preferred use of the conjugate of the invention for the diagnosis and/or treatment of autoimmune diseases. Indicates appropriate procedures.

Figure 9A shows the separation of α and β chains dissociated by heating for 5 minutes at 95°C. SDS polyacrylamide gel.

Figure 9B shows SOS polyacrylic acid showing separation of dissociated α and β chains due to low pH. It is mid-gel.

Figure 90 is a 5DS-PAGE analysis of single stranded preparations after reduction with 2-enzyme. .

Figure 10A depicts the results of a peptide binding assay using Sori-Gel TLC. Ru.

FIG. 10B depicts the results of a peptide binding assay using CAE.

Figure 11 shows the stability of single chain:peptide complexes of the invention.

Figure 12A shows binding of the α chain complex to cloned cells.

Figure 12B shows the preferential transfer of both heterodimeric and single-stranded complexes to T cells. Shows a bond.

Figure 12C shows that blocking experiments using anti-TCP monoclonal antibodies Figure 2 shows the ability of the conjugate of the invention to bind to TCP as evidenced.

FIG. 13A shows a microphysiometer induced by the interaction between the complex of the invention and T cells, as measured by The results show a sustained increase in the acidification of the culture medium.

Figure 13B shows that non-specific complexes do not induce a significant increase in acidification. are doing.

Figure 14 shows the significant induction of anergy in T cells by the complexes of the invention. It shows.

Figure 15 shows the induction of anergy as measured by [1-2 production] ing.

Figure 16 shows the attenuation of EAE in mice treated with the conjugate of the invention. ing.

Figure 17 shows that the dimeric Ml(C peptide complex exists as an aggregate). are doing. Because they pass through columns with void volume and therefore , 600. This is because it has a larger molecular weight than OOO.

Description of preferred embodiments The present invention comprises at least two components: an antigenic compound with associated effects on the immune system. of the λtHc-encoded glycoprotein associated with peptides or other antigenic sequences and antigenic representations. Contains the effective portion of the subunit. This peptide antigen and LIHC subunit The bond between the antigen binding site can be covalent or non-covalent. .

In other embodiments, the conjugate is an effector moiety that is typically a toxin or a label. It can also include. A portion of this effector may be an MHC subunit or an antigenic can be attached to any of the peptides. Each of the components of the system is Separately described: by which these complexes are then prepared, evaluated and used This section describes methods that can be used.

MHC-inducing components has been extensively studied. Generally they are on the surface of all cells class] glycoproteins: and are recognized primarily by cytotoxic T cells. It is found on the surface of some cells, including parasitic cells, e.g. macrophages; class 11, which relates to the presentation of antigens to helper T cells. It is. Many histocompatibility proteins have been isolated and characterized. M H. Nire is incorporated herein by reference. )checking.

Class I & IHC in humans are located on chromosome 6 and at three loci, H It has LA-1HLA-8 and HLA-C. The first two loci carry alloantigens. It has many alleles encoding it. Common to all antigenic specificities, 44 Kd heavyweight subunit and +2Kdβ2-microglobulin subunit? It has been found that it consists of For isolation of these detergent-soluble HLA antigens , Springer, T.A., et al.

Proc, Natl, Acad, Sci, USA (+976) 76 :2481-2485: Clementson.

K, J, et al, in″Membrane Proteins-Azz i, A,, ed: Bjorkman.

P, Ph, D, Thesis Harvard (1984) (these All incorporated herein by reference. ).

Further studies provide a detailed picture of the 3-D structure of HLA-A2, a class of human antigens. (Bjorkman, P. J., et al., Nature (1987) 329:506-518, which is incorporated herein by reference. It's crowded. ). In Figure 1, β2-microglobulin protein and the important α, upper segment You can see what's going on or what's going on. The C1 and α2 regions of the heavyweights are c ience (1987) 238:613-614. This is incorporated herein by reference. 252:7555-7567 (which is incorporated herein by reference). take in. ) homozygous human lymphoblastoid cell line J -Y can be purified after papain digestion of the plasma membrane. papain cleaves the 44Kd heavy chain close to the transmembrane region, creating C1, C2, C2 , and β2 microglobulin.

The three-dimensional structure of the antigen binding site in class II MHc antigen is Not known in great detail. However, class ■] glycoproteins are of class 1. It has a general domain structure similar to that of

The antigen-binding site of class 11 MHC is the N of two subunits (α and β chains). - Located on the terminal domain part. Separate antigen-binding sites are present in each subunit. located on the cut (luescher et at, J. Biol.

Chem, 265:11177-11184 (+990) (this is quoted from and is incorporated herein by reference. )checking. ). These two subunits are non- are held together by covalent forces, and each consists of two globular domains (that C1, α, and β1, β2), and they all exclude the α domain. It is stabilized by disulfide bonds.

MHC glycoproteins can be isolated from appropriate cells or produced recombinantly. can be done. A method for purifying murine [-A (class I) histocompatibility protein] , well known, and Turkewitz, A.P.

et at, Molecular Immunology (1983) 2 0:1139-1147 (incorporated herein by reference). are disclosed. By I-A and [-E subregions An isolated antigen encoded by two non-covalently linked pairs. Petide chain: It is shown that it consists of a 32-38 Kd α chain and a 26-29 Kd β chain. It was done. A third, constant, 31Kd peptide is noncooperative with these two peptides. Although associated by bonds, it is not pleomorphic and It does not appear to be a component of the antigen (Sekaly, R,P,, J, Exp, Med.

(1986) 164:1490-1504, which is incorporated herein by reference. Get into it. ).

(Described by Paul et at., supra, Chapters 16-18) Cloning of the MHC gene (as described below) Allows easy manipulation of the C subunit. In particular, seven alleles of the [-A region The α and β chains of the offspring mutants were cloned and sequenced (Este es, “T cell C1ones”, pp, 3-19).

As used herein, the term "isolated MHC subunit component" means HC glycoprotein subunit (i.e., Ml (α or β chain of CI or MHC [heavy chains of It refers to something that is not associated with the cell membrane of a cell. This term refers to full-length subunits. strands, as well as effective portions of MHC subunits. The effective part is the appropriate Contains antigen binding sites and sequences necessary for recognition by T cell receptors It is something. It typically contains at least about 60-80 of the full-length sequence. %, typically 90-95%. As detailed below, & +HC subunit components may be recombinantly produced or solubilized from appropriate cellular sources. I can do it. Although the IHc component is soluble in vivo ( That is, they retain sufficient salmonidity to function in vivo. Ru. ), they in certain embodiments associate with detergents to form micelles, or It associates with lipids to form liposomes.

The native form of the "mature" AIHC glycoprotein and subunit has one or more that may vary somewhat due to amino acid deletions, substitutions, and insertions or additions; is well known. Therefore, the &lHC subunit components are substantially naturally occurring. Although modified, they can still retain their respective activities.

Modified protein chains are well known to those skilled in the art and are described in detail below. can be easily designed and manufactured using a variety of recombinant DNA techniques. Wear. For example, a chain can change its primary structure by amino acid substitutions, additions, deletions, etc. It is possible to vary from the natural sequence in the bell.

These modifications can be used in numerous combinations to produce the final modified protein. Can create chains.

In general, modifications of genes encoding MHC subunit components can be Various well-known techniques, such as site-directed mutagenesis (87), is incorporated herein by reference. ). Those skilled in the art will appreciate that the effects of many mutations are You will understand that it is difficult to predict. I want to do it, I want to do it, I want to do it. by routine screening in a suitable assay for the desired characteristics. be evaluated. For example, the immunological characteristics of a subunit can be determined by competitive Can be detected by immunoassay. Ability of subunit to activate T cells The effect of modifications on force can be determined using standard inhydrocellular assays or as described below. Can be tested using the methods described in the Examples section . Other properties, such as redox or thermal stability, hydrophobicity, susceptibility to proteolysis, etc. Modifications for susceptibility or agglomeration tendency have been assayed according to standard techniques. Ru.

The present invention provides antigenic peptides and/or subunits for T cell receptors. questions, including increasing affinity, subunit stability, and facilitating purification and preparation. Amino acid sequence variants of MMC subunits prepared with various objectives of provide. This modified subunit modifies plasma half-life and therapeutic efficacy. improve and reduce the severity or occurrence of side effects during therapeutic use of the complexes of the invention. It is useful for making things easier. Amino acid sequence variants of subunits are usually A given variant that is not found in nature or in its natural alleles. This mutation The body typically has the same biological activity (e.g., antigenic peptides) as its natural homolog. connectivity). However, mutants that are unable to bind those ligands Variants and derivatives may nevertheless be used for (a) MHC hablotibe or Ml(C). (b) as a reagent in a diagnostic assay for antibodies that Anti-MMC antibodies from the antiserum or supernatant of hybridoma cultures when solubilized. and (C) at least one MMC epitope. as long as it remains active (as a competitive reagent for the native MHC/\protype). for MHC (labeled or unlabeled as a standard for MHC assays). useful as an immunogen for making antibodies or as a component of an immunoassay kit. .

Insertional mutants of the present invention are those in which one or more amino acid residues are present within a given MHC subunit. is introduced into the site and replaces a previously present residue. Ru. For example, insertional mutants are subunit mutants of a heterologous protein or polypeptide. Fusions can be at the mino or carboxyl terminus.

Immunological fusions can be made by in vitro cross-linking or by cross-linking with appropriate MHC subunits. using DNA encoding an immunogenic polypeptide linked to a target gene. Produced using recombinant cell culture.

These immunogenic fusions can, for example, Useful for making antibodies useful in diagnosis or purification of MHC by techniques .

Other fusions that may or may not be immunologically active include: Fusions of its subunits with heterologous signal sequences and enhancements with its subunits Polypeptides with a long plasma half-life (usually about 20 hours), e.g. including fusions with globulin chains or fragments thereof (co-pending application U.S. Serial No. 0 7/783.938, incorporated herein by reference. and. ).

Substitution variants are those in which at least l residues are removed and are different at that location. In this case, a residue is inserted. unnatural amino acids (i.e., naturally occurring proteins) (amino acids not normally found in humans) and isosteric Homologs (amino acids or others) are also suitable for use in the present invention.

Substantial changes in function or immunological identity may be due to changes in the structure of the polypeptide backbone (e.g. e.g. sheet or helical configuration), the charge or hydrophobicity of the molecule at the target site. Substituted residues that differ in their effect on maintaining properties, or side chain bulk. This is done by selecting . Generally creates larger changes in functionality Substitutions expected to occur include (a) hydrophilic residues, such as serine or thread; Onine is a hydrophobic residue such as leucine, isoleucine, phenylalanine, Substituted with valine or alanine: (b) cysteine or proline substituted with other substituted with (or by) any residue of (C) electropositive Residues with side chains such as lysine, arginine, or histidine are electrically negative. with a reactive residue such as glutamine or asparagine ( or (d) a residue with a bulky side chain, e.g. , phenylalanine has no side chain, such as glycine (or ri) that which is being replaced.

Subunit substitution variants are functionally homologous (at least about 70%) to other proteins. MHC subuni-nodomain, which has one or more domains (with homology to Mutants that have been substituted by a specific method.

including. A particularly preferred protein for this purpose is immunoglobulin superfluid. other members of the family.

Another class of mutants are deletion mutants. Is the deletion in the MHC subunit sequence? characterized by removing one or more amino acid residues from Typically, The transmembrane and cytoplasmic domains are deleted. cysteine or other Residues that are susceptible to chemical changes may also increase the oxidative stability of the MHC complex, for example. It can be desirable in that Potential proteolysis sites , for example, Arg. Deletion or substitution of Arg results in the deletion of one of the basic residues. , or by substituting glutamine or histidine residues. It will be done.

A preferred class of substitution or deletion mutants is the subunit transmembrane region. It consists of things that include areas. The transmembrane region of the MHC subunit node is , highly hydrophobic or oleophobic with appropriate size to span the lipid bilayer of cell membranes. It is a sexual domain. They bind Ml (C molecules) within the cell membrane. It is believed.

Typically, deletions or substitutions of hydroxylated residues in the transmembrane domain Inactivation of the transmembrane domain by oxidation can reduce the lipid content of the cell or membrane. recovery and distribution by reducing affinity and improving its aqueous solubility. This will make it easier to connect.

Alternatively, the transmembrane and cytoplasmic domains are potentially immunogenic agents. It can be deleted to avoid introducing pitopes. Inactivation of membrane binding function has a substantially hydrophilic hydrotherapy profile at the site. By deleting enough residues to create a or by substitution with a different residue that achieves the same result.

The fundamental advantage of transmembrane inactivated MHC is that it This means that it can be secreted into the nutrients.

This variant is soluble in body fluids, e.g. blood, and has no ability to enter cell membrane lipids. It has no appreciable affinity and therefore recombinant cell culture considerably simplifying its recovery from

Typically, the subunit variants of the invention will contain functional transmembrane molecules. and preferably have functional cytoplasmic sequences. Probably not. Such mutants have the presence of extracellular domains of MHC subunits. It will consist essentially of the effective part. In some situations, subunits may be lysed The transmembrane region (up to about IO amino acids) is contains an array from the area.

For example, a transmembrane domain can be combined with any amino acid sequence, e.g. 5-50 serine, threonine, lysine, arginine, glutamine, albalagi A random or predetermined sequence of hydrophilic residues, such as hydrophilic acid, which together are hydrophilic. may be substituted by those exhibiting a hydrotherapy profile of deleted Like (truncated) subunits, these mutants are Secreted into the nutrient base.

Glycosylation variants are within the scope of the invention. They are completely devoid of glycosylation (non-glycosylated). less glycosylated than the native form of the mutant and at least l Mutants with altered (deglycosylated) sites and altered glycosylation within them Including mutants that have been

Deglycosylate with native unmodified amino acid sequence d) and unglycosylated subunits. be caught.

For example, substitution or deletion mutations may result in subunit N- or membrane-bound glycodilation. used to remove the binding site, e.g. an asparagine residue is deleted, or substituted with other basic residues, such as lysine or histidine. or, Flanking residues that make up the glycosylation sites are substituted or deleted. however , its asparagine residue is glycosylated by removing its glycosylation recognition site. Unmodified to prevent lycodylation. In addition, the innate subunit Non-glycosylated subunits with amino acid sequences are found in recombinant prokaryotic cell cultures. Made. This is because prokaryotes cannot introduce glycosylation into polypeptides. This is because they cannot.

Glycosylation variants are conveniently produced by selecting appropriate host cells or in vitro. Made by Toro's method. For example, yeast is derived from mammalian systems. Introducing altered glycosylation.

Similarly, different species (e.g., hamsters, rats, insects, pigs, of bovine or ovine) or of tissue origin (e.g. lung, liver, lymph, mesenchyme) salivary mammalian cells (senchyma l) or epidermal (epidermal) However, for example, increased levels of mannose or increased proportions of mannose, fuco fucose, alkyl acids, and those typically found in mammalian glycoproteins. The ability to introduce glycosylation into mutants as characterized by other sugars This will be screened on a regular basis. In vitro production of subunits Processing involves enzymatic hydrolysis, such as neuraminidase (neuraminidase). This is done by minidase) digestion.

&IHC glycoproteins suitable for use in the present invention can be obtained by treatment with papain. ! Various treatments including solubilization by treatment with J KCI and by treatment with detergents It has been isolated from a variety of cells using various techniques.

In a preferred method, detergent extraction of class II proteins from lymphocytes followed by Use affinity purification. Next, the detergent, dialysis or selective binding beads, e.g. For example, it can be removed using BioBeads. This molecule is called MHC from any of the containing cells, e.g. from individuals suffering from targeted autoimmune diseases. It can be obtained by isolation from B lymphocytes. In particular, suitable MHC content The offspring were incubated with replication-defective Epstein-Ba cells using techniques known in the art. It can be isolated from B cells immortalized by transformation with rr virus. Wear.

Isolation of individual subunits from isolated MHC glycoproteins is known to those skilled in the art. Easily accomplished using standard techniques. For example, in the case of class [molecule] Then, the heavyweights were separated using 5DS-PAGE and the heavyweights were removed from the gel. electroeluting (e.g., Dornmaire et al., E. and Hunka et al. Piller, et al, Methods in Enzymol, 9 1:227-236 (1983) (incorporated herein by reference). . )).

Isolation of α and β subunits from MHCI+ molecules is also described below and 16( +989) (incorporated herein by reference). Isolate using 5DS-PAGE followed by electroelution.

Those skilled in the art will appreciate that there are many other standard methods for separating molecules, such as ion exchange chromatography. chromatography, size exclusion chromatography or affinity chromatography You will agree that the fee may be used.

Alternatively, the amino acid sequences of many class It proteins are known, and their remains are known. The gene has been cloned. Therefore, subunits can be produced using recombinant methods. can be done. These techniques allow for multiple modifications of the MHC subunits of the present invention. enable. For example, recombinant techniques have been used to create hydrophobic transmembrane domains. A method for carboxy-terminal truncation of deletions is provided. Also, this cal The boquin terminus can be used, for example, to introduce protein and/or lysine residues into the molecule. can be optionally chosen to facilitate binding of the toxin or label by Wear. Synthetic genes are typically produced by insertion into expression vectors and manipulation of the gene sequence. It will contain restriction sites to assist in the Encodes appropriate subunits The gene is then inserted into an expression vector and cultured in a suitable host, such as Escherichia coli (E, c oli), yeast, or other suitable cells, and the recombinant protein is expressed in obtain.

Availability of the gene makes it a preferred construct as it allows for quick manipulation of its sequence. The second generation of things consists of hybrid classes [and classes] as exemplified in Figure 2. Contains the features of II. Here, the class [[MHC α1 and β1 domains are Intermolecular dimers between these domains lead to edge-to-edge β-sheet contacts. combined through a flexible part that allows for There is. Next, β, the upper segment, was coexpressed to stabilize the complex. β, microglobulin-containing class] is fused to the α2 domain. class ]Although the transmembrane and intercellular domains of the gene may be included, If liposomes are not used to permeate the complex, such There can be no point in doing this. A simpler variant is a toxin-bound It contains only α1 and β1 domains with C-terminus for integration (Figure 2).

Construction of expression vectors and recombinants from appropriate NA sequences is well known in the art. It is carried out using the method specified. DNA and RNA isolation, amplification, and cloning For this purpose, standard techniques are used. In general, DNA rigger seven, DNA poly Enzyme reactions involving merase, restriction endonucleases, etc. or according to the manufacturer's instructions. will be carried out. These techniques and various other techniques are generally et al,, Alolecular Cloning-A 1aborat ory Manual, Co1d Spring Harbor Labor atory.

Co1d Spring Harbor, New York, 1989 ( This is incorporated herein by reference. ). other common The relevant literature is provided throughout this document. The steps therein are well known in this technical field. is believed to be well known and is provided for the convenience of the reader.

Expression can be in prokaryotic or eukaryotic systems.

Prokaryotes are often represented by various strains of E. coli.

However, other microbial strains, e.g. bacilli, can I can do it. In such prokaryotic systems, copies obtained from species compatible with the host A plasmid vector containing the site and control sequences is used. For example, Escherichia coli (E, coli) is typically I) BR322, i.e. Bolivar et al. According to Gene (1977) 2:95, Escherichia coli (E, coli) species A derivative of the plasmid, obtained from the above method, is used for transformation.

Commonly used are prokaryotic control sequences, herein referred to as transcriptional control sequences. A promoter for initiation, optionally with an operator, is attached to the liposome. Together with the binding site sequences, these are defined to contain β-lactamase (penicillinase) and lactose (lac) promoter system (Change) et al., Nature (+977) 198:1056) and Trippto Fan (trp) 0-E--ter system (Goeddel et al, Nucl eic Ac1ds Res, (1980) 8:4057) and lambda derivative P , promoter and N-gene lyfosome binding site (Shimatake et al. al., Nature (1981) 292, 128). Contains the promoter used.

Any available promoter system that is compatible with prokaryotes can be used. Wear. All documents cited herein either above or below are hereby incorporated by reference. , incorporated herein by reference.

Expression systems useful in eukaryotic hosts include proteins derived from appropriate eukaryotic genes. Consists of a motor. Classes of promoters useful in yeast include, for example: 3-phosphoglycerate kinase (Hitzeman, et al., J , Biol, Chem, (1980) 255:2073) Contains promoters for the synthesis of glycolytic enzymes, including promoters. other promoters is the enolase gene (Holland, M. J., e tal, J., Biol, Chem. (1981) 256:1385) and is the Leu2 gene obtained from YEp13 (Broach, J,, etc. Gene (+978) 8:121).

Suitable mammalian promoters include the early and late promoters from SV40 (Fi er, et al, Nature (1978) 273:113) or others viral promoters, e.g. polyoma, adenovirus Virus 1■, including those obtained from bovine papilloma virus or avian sarcoma virus. nothing.

Suitable viral and mammalian enhancers are cited above.

The expression system was modified using standard ligation and restriction techniques well understood in the art. operably linked to the MHC sequence using standard techniques using Constructed from control elements. Dissected plasmids, DNA sequences, or synthetic oligonucleotides Gates cleotides to be cleaved, tailored, and reformed into the desired form.

Site-specific DNA cleavage is generally understood in the field and includes specific a suitable restriction enzyme (or enzymes) under conditions specified by the manufacturer. Or by treatment with these commercially available restriction enzymes. For example, Ne w　England　Biolabs,　See Product Catalog About. Generally, approximately 1 μg of plasmid or DNA sequence is added to approximately 20 μm of buffer. cleaved by one unit of enzyme in a buffer solution. , typically an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. do. An incubation time of about 1 to 2 hours at about 37°C is performed. can.

However, variations can be tolerated. After each incubation, the protein The white was removed by extraction with phenol/chloroform and extracted with ether. Subsequent extraction can be performed and the nucleic acids are pre-precipitated with ethanol, followed by The water surface was measured by running it on a 5hephadex G-50 spin column. Collect from. If desired, size separation of the cleaved fragments can be performed using standard techniques. This can be done by polyacrylamide gel or agarose gel electrophoresis. Wear.

A general description of size separation can be found in &Ithods in Enzymology (1980) 65:499-560.

Restriction cleavage fragments were treated with 50mM Tris pH 7, 6, 50m&I NaCl, 20~20 in 6mM^IgCI, 6mM DTT and 5-10μM dNTPs. 4 days using an incubation time of approximately 15-25 minutes at 25°C. Treatment with large fragments of Klenow 1 (Klenow) It is possible to convert the terminal into a terminal. This Klenow fragment is 5゛attached. fill in the end, but despite the presence of 4 dNTPs, no protrusion Chews back the 3 single strands. desired allows selective repair of dNTPs within the limits dictated by the nature of their cohesive ends. This can be done by supplying only one of the dNTPs or selected dNTPs. I can do it. After treatment with Flenow, the mixture was treated with phenol/chloroform. extraction and ethanol precipitation followed by 5hephadex G-50 run on a column.

Synthetic oligonucleotides can be synthesized using commercially available automated oligonucleotide synthesis equipment. Synthesize using Single-stranded key prior to annealing or for labeling The enzyme treatment is carried out in excess of, for example, about 1 unit for 0.1 nmole of substrate. 50 ntl Tris pH 7.6 using polynucleotide kinase ,10m! J MgC12,5m1J dithiothreitol, l-2mM ATP , 1. ．． 7μmole” P-ATP (2,9mC1/mmole), 0. [mM spermidine (spermid 1ne), 0.1mM EDTA to do in the presence of.

Regenerate the following standard conditions and treatments: 20 ntl Tris-HCI p H7゜5.10mM hlgcl 2, l0mM DTT, 33μg/ ml BSA, 10mM-50mM NaCl, and 40μM at 0°C ATP, 0.01-0.02 (Weiss)-L knit T4 DNA rigger (for “sticky end” regeneration) or Intl at 14°C ATP, 0.3-0.6 (WeiSs)-L-: T4 DNA ligase (for “Plant End ° Liguenoyon”), either under 15-30 Perform in μl volume. Intermolecular "cohesive end" ligation is usually 33-1 Performed at a total DNA concentration of 00 μg/ml (5-100 nM total end concentration) .

Intermolecular plant end ligation (usually a 10- to 30-fold molar excess of ligation) (using a marker) is carried out at a total end concentration of 1μ81.

“In vector construction using vector fragments, vector fragments are commonly to remove the 5' phosphate and prevent re-gation of the vector. processed by bacterial alkaline phosphatase (BAP) to Ru. BAP digestion is performed at approximately 1 unit per μg of vector at 60″C for approximately 1 hour. In the presence of Na'' and Mg2- using BAP <150mM Tri s at pH 8.

To recover the nucleic acid fragments, the preparation was extracted with phenol/chloroform. , ethanol precipitated, and 5hephadex G-50 spin column. Desalt by application to lamb. Alternatively, you may want to re-gate the undesired fragments. can be prevented in double-digested vectors by additional restriction enzyme digestion. .

of the portion of the vector obtained from cDNA or genomic DNA where sequence modification is required. For this purpose, site-directed primer-directed mutagenesis can be used.

This is because the single-stranded phage D is mutated except for limited mismatching. This is done using primer synthetic oligonucleotides complementary to the NA and Representing natural variation. Simply put, synthetic oligonucleotides are used as primers. to synthesize a strand complementary to the phage, and the resulting double-stranded DNA A is transformed into a phage supporting host bacterium. transformed bacterium Cultures of phages were plated onto top agar and a single phage harboring culture was plated onto top agar. Allow plaque formation from cells.

Theoretically, 50% of new plaques carry the mutant form as a single strand. 150% will have the original sequence. obtained Plaques allow accurate match hybridization However, at that temperature, the mismatch caused by the original strand es) or at a temperature sufficient to prevent hybridization. hybridize with enzyme-treated synthetic primers. This probe and hybrid Plaques that develop are then picked, cultured, and the DNA recovered.

However, synthetic genes are conveniently used in the proteins of the invention. child The design of the gene replaces these encoding homologues with a portion of the coding sequence. Restriction sites may be included to allow easy manipulation of the gene for modification.

First transform with two ligation mixtures for plasmid construction. This can be ensured by Successfully transformed cells are treated with ampicillin, Tetracycline or other antibiotic resistance or as understood in the art The use of other markers depends on the mode of plasmid construction, such as selected. The plasmid from the transformed cells was then transformed into Clewe11. D, B, et al, Proc, Natl, Acad.

Sci, USA (1969) 62:1159, optionally chloride Mufenicol amplification (Clewe11. D, B, , J, Bacterio (1972) 110:667). The isolated DNA, analyzed by restriction enzyme treatment and/or Sanger, F., et al. , Proc, Natl, Acad, Sci, USA (1977) 74 :5463 dibuoxo method, Messing, et at, Nuc leic Aclds sequence.

The constructed vector is then transformed into a suitable host for protein production. . Depending on the host used, transformation can be carried out using standard techniques appropriate for such cells. It is done using Cohen, S.N., Proc. Calcium treatment used or Maniatis, et al., Molec ular Cloning: A Laboratory & Annual ( 1982) Co1d Spring Harbor Press, p, 2 The RbCl method described in 54 can be applied to prokaryotes containing substantial cell wall barriers or is used for other cells. For mammalian cells that do not have such a cell wall Graham and van der Eb, VirologY (1 978) 52:546 calcium phosphate precipitation method or electroboration is preferred. For transformation into yeast, use Van Soling transformed cells. , then cultured under conditions that favor the expression of MHC sequences, and the recombination The produced protein is recovered from the culture solution.

antigenic peptide Antigenic proteins or tissues for numerous deleterious immune responses are known. For example, fruit In experimentally induced autoimmune diseases, antigens associated with pathogenesis have not been characterized. In arthritis in rats and mice, innate type II collagen have been identified in collagen-induced arthritis, and adjuvant-related Microbial heat shock proteins have been identified in arthritis (Stuart et al. t, (1984).

It has been identified in experimental allergic thyroiditis (FAT) (^1ar on et al, (+988), J, EXP, Med, +52:1 115-1120): in experimental allergic myasthenia gravis (EA^IG) Acetylcholine receptor (A) Experimental allergy in mice and rats Myelitis (encephalitis) in the brain.

Miniphosphobasic protein (MBP) and protein in Mt. Myel 5 (EAj) proteolipid protein (PLP) (Ac (see Ha-Orbea et al., supra). Furthermore, for example, Antigens have been identified in humans Types of human rheumatoid arthritis ] I collagen (Holoshitz et al. (1986), Lan cet ratio 305-309): and acetyl choline ratio in myasthenia gravis. septa (Lindstrom et al. (1988), supra), these All are incorporated herein by reference.

antigen-presenting cells (APCs) The representation of antigens by MHC glycoproteins on the surface of It is believed to occur following hydrolysis to the tide unit. within antigenic protein The locations of these smaller segments can be determined empirically. child These segments are believed to be approximately 8-2o residues in length, and (& agretope (recognized by lHC molecules) and agretope (recognized by lHC molecules) and epitopes (recognized by T-cell receptors) . This epitope is a sequence of 5-6 amino acids that recognizes antigen-specific T cell receptors. Continuous or non-contiguous array. This allelicity is due to the association of the peptide with MHC glycoproteins. Continuous or non-contiguous sequences that are the cause of

An empirical method for determining the relevant 8-15 amino acid subunits is It has been described using the alpha subunit of the tyrcholine receptor. myasthenia gravis In MG (MG), the autoimmune response is directed against the region of this subunit. There is. an amino acid on the postsynaptic membrane at the nerve-muscle junction Loss of cetylcholine causes &IG symptoms.

^In IG, the α subunit of the acetylcholine receptor (AChl?) Autoantibodies to AChRs have been implicated in autoimmune responses directed against AChRs. ＾f Eighty-five percent of G patients have autoantibodies that react with the alpha subunit. child Among them, 60% have the main immunogen located between residues 6o and 80 It also contains an antibody that binds to the peptide segment of the α subunit, called ``fR''. (Tzartos and Lindstrom.

Proc, Natl, Acad, Sci, USAC1980) 77:7 55). Peptide segments recognized by autoreactive human T cells also Located on the α subunit (t(ohfield, et at, , Pra c, Natl, Acad, Sci, LISA (1987). These T Epitopes recognized by cells include residues 1-30, 125-147, 169 Lying in the darkness of -181, 257-271 and 351-368. Furthermore, human In AChR peptides 195-212 and 257-269, respectively , HLA-1) I'? 5 and NLA-1) R3, DQW2^fHc habrotibe has been partially characterized as an epitope in patients with myasthenia gravis ( Acha-Orbea (+989), see above. ).

Alleles that enable the representation of epitopes associated with the alpha subunit of this receptor Peptides that carry this property are easily determined.

For example, determination of suitable peptides in mouse models is performed as follows. Ru. The AChR of Torpedo californicus When immunized with human myasthenia gravis, the disease develops into a disease with many features of human myasthenia gravis. used as a stock or a model. MHC class 11 glycoprotein, lectin and This strain of mouse pancreas was isolated using a monoclonal antibody affinity support. isolated from cells. Purified^ (HC class 11 protein was purified by detergent dialysis) incorporated into lipid vesicles. The resulting vesicles are then stained with MH on each plane. To create a lipid bilayer containing C molecules, use a clean glass coverslip (cov Er　slim. )).

MHC class embedded within two adhesive planar lipid membranes [one cover containing one molecule] - Place the glass in each well of several 24-well culture plates. I put it in. A 20-residue synthetic peptide corresponding to the α subunit sequence overlaps, and one or more radiolabeled amino acid residues (prepared as described below). Each of the approximately 40 cells containing the group was placed in a well containing a cover glass and PBS. and incubate for several days. ^I) IC29711w1 protein anti The extent of peptide binding within the original binding site is determined by the coverage of the planar lipid membrane alone. - Due to the amount of radioactivity incorporated into the AIHC class 11 uniplanar lipid membrane on glass. ,Measure. The specific uptake of radioactivity is due to the binding of the bound peptide within the planar lipid membrane. Allelicity (MHC) of one of the several species of molecules of the AIHC class that exist class 11 peptide binding site).

In this way, a set of alleles for the α subunit of AChR can be generated from AfJ For mouse strains showing signs of MG during immunization with R or purified α subunit. Established.

Next, each of the alpha subunit synthetic peptide segments containing allelicity was , again, isolated λIHC clusters embedded within a planar lipid membrane on a coverslip. It is incorporated into the antigen-binding site of the S11 protein.

Place one coverslip in each well of a 24-well culture plate. and pancreatic cells from mice immunized against AChR (and their Adhesive & IHC class II proteins were isolated from this strain. ), the irradiated syngeneic (syngenic) to each well together with antigen-representing cells. . T cell activation, as measured by proliferation, is associated with MHC class II protein-binding. Synthetic peptides contain both alleles and epitopes for binding to T cells. It shows. In addition, T cell activation is influenced by lymphokines (IL-2, IL-3 ) is measured by measuring production (Quill et at, supra. checking ... ).

Rapid multiple peptide synthesis Dupon's equipment and technology for Synthesis (RA & IPS) α subunit of Torpedo catafornicus AChR To synthesize a large number of duplicated (10 residues overlap), two-leaf residue peptides from use it for. The sequence of this peptide is known and shown in Figure 3. There is. One or more radioactive amino acids are incorporated into each synthetic peptide. side chain The pentafluorophenyl active ester of FNIOC amino acid protected with Quasi-stepwise solid phase peptide synthesis followed by standard side chain deprotection and solid support To synthesize peptides while applying simultaneous desorption of peptides and amides from the body used for.

Alternatively, antigenic proteins, such as the 8-15 amino acid of acetylcholine receptor protein. Geysen, H.M.

et al, J., Immun, Meth, (1987) 102:27 4 (which is incorporated herein by reference). I'll come. This synthesized radiolabeled peptide is placed in a lipid membrane bilayer as described above. Ink them separately (on a plate) along with the purified & lHC protein formulated into test by nucleating.

In multiple sclerosis (MS), which results in the destruction of the miniphosphorus sheath within the central nervous system, Miniphosphorus basic protein (klBP), i.e., the main protein component of miniphosphorus, It is essentially an autoantigen. Also, directly related segments of MBP protein. cow ・Experimental allergic encephalitis (en) when immunized with miniphosphorus basic protein determined empirically using a mouse strain that develops . The sequence of ^IBP is shown in Figure 4.

Systemic lupus erythematosus (SLE) has a complex system+ogy or red blood It is caused by an autoimmune response to several tissues, including bulbar cells.

Peptides that are antigenic effectors of this disease, e.g. Found within proteins.

Rheumatoid arthritis (RA) is found in synovial fluid. It is a chronic inflammatory disease that results from an immune response to damaged proteins.

Insulin-dependent diabetes mellitus (IDDM) is responsible for the secretion of insulin. It results from an autoimmune attack on the beta cells within the islets of Langerhans. islet cell surface Circulation of antibodies to antigen and to intulin [known to precede DDM] It is being decisive in manifesting the immune response in IDDA [ The peptide is believed to be part of a beta cell membrane surface protein.

The related antigenic peptide subunits are relatively short, making the peptide It can be easily synthesized using standard automated methods for synthesis. on the other hand In some cases, they can be produced recombinantly using isolated or synthetic DNA sequences. can be However, this is the most It's not a valid approach.

Therefore, in summary, a set of labeled test peptides is prepared and What binds to UHC in planar lipid membranes containing MHC proteins contains isotopes. It has been shown that

The identified peptides are then synthesized by conventional solid-phase synthesis and treated with disease-inducing agents. The subset containing the epitope for the Luper T′m cell clone was identified as its candidate peptide. antigen-presenting cells (APCs) (or isolated^]HC complexes) and intact Incubate with #visceral or lymph node T cells immunized with long protein. Determined by. Successful candidates will be able to stimulate T cell proliferation in this system. Probably. A second smaller subset of two represents suitable peptide components.

effector component In one embodiment, the conjugate of the invention stimulates the immune response to the peptide of interest. Designed to destroy. In this case, the effector portion of the molecule is For example, toxins, chemotherapeutic agents, antibodies against cytotoxic T cell surface molecules, lipases, or “hard” radioisotopes, e.g., those that emit β-irradiation. It would be a loop. For example, a number of protein toxins are well known in the art; These are ricin, diphtheria, and vomit. gelonin, thyutomonas toxin, and abrin. include. Chemotherapeutic agents include, for example, doxorubicin, daunorubicin, methotrexate trexate), cytotoxins, and antisense RNA. use antibiotics You can also. Furthermore, radioisotopes such as yttrium-90 , phosphorus-32, lead-212, iodine-131, or balanrum-109. can be done. The emitted irradiation destroys potential T cells.

In some cases, the toxin or other effector component is delivered to a delivery system, e.g. , within a liposome or dextran carrier, in which case the active ingredient or a carrier can be bound within the complex.

The effector component can also be a labeled moiety.

The labeled complexes of the present invention can be used in various in vivo or in vitro applications. Can you use it? For either of these purposes, the complex can be directly labeled. can be identified. A wide variety of labels, such as radionucleotides (e.g. , gamma-irradiated inotobe, e.g. technetium-99 or indium- II+), fluorescent agent (e.g. fluorescein), enzyme, enzyme substrate, enzyme cofactor , enzyme inhibitors, chemiluminescent compounds, bioluminescent compounds, etc. . Those skilled in the art will know other suitable labels to bind to the complex, or use routine Experiments could be used to confirm this. Combining these signs is performed using standard techniques common to those skilled in the art.

In vitro uses include diagnostic applications, T cell typing, and isolation of specific cells. or labeling, etc. For example, the complex of the present invention may be used for MHC-T cell interaction. can be used to assay for potential inhibitors. possibility Certain inhibitors can be used in the complex of the invention in the microphysiometer device described above. can be used to assay the ability of conjugates to inhibit binding to T cells. Ru.

For in vivo diagnostic imaging, radioisotopes are typically , used according to well-known techniques. This radioisotope is Directly or indirectly using intermediate functional groups well known to those skilled in the art at the time of filing of the application. It can be bound to the protein by either. For example, chelating agents, For example, diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminete Triacetic acid (EDTA) and similar molecules are metal ion radioisotopes. It has been used to bind proteins.

In addition, this complex can be used in magnetic resonance imaging (magnetic resonance imaging). imaging (MR [)) or electron spin resonance (electron 5pi n resonance (ESR)) (both of which were filed at the time of filing this patent application). were well known to those skilled in the art. ) for in vivo diagnostic purposes, such as in They can also be labeled with paramagnetic isotopes. For example, these and related techniques have been used in the diagnosis of rheumatic diseases (Namey, in Textbook of Rheumatology, Kelley et at (eds,) 5unders, Ph1ladelphia , 1985. (which is incorporated herein by reference). ) . In general, use any of the conventional methods of visualization for diagnostic imaging. be able to. Gamma- and positron-irradiated radioisotopes are commonly used. , used for camera imaging, and paramagnetic isotopes for MRI. used for Therefore, the conjugates of the invention may be used to modify autoimmune responses in individuals. Can be used to monitor progress. Increase in number of exploratory T cells or specific therapeutic regimens aimed at ameliorating autoimmune diseases to measure reduction. It can decide whether the law is valid or not.

This effector component may be attached to an MHC subunit or if its properties are favorable. In some cases, it can be attached to a peptide moiety. For example, iodine-131 or Other radioisotope labels are often included within the determinant sequence of the peptide. can be included. The method for attaching this effector component to the complex is described below. Details are given below.

Formation of complex bringing together the elements of the complex by standard means known in the art; Can be done. Antigenic peptides can be prepared by, for example, MM by mixing the two components. It can non-covalently associate with the antigen binding site of the C subunit. Also, They can be identified using standard procedures, e.g. photo affinity discovery. (photo affinity labeling) (for example, Hall et al., Biochemistry 24:5702-57II (1 985) (incorporated herein by reference). ) to Therefore, they can be combined in a coexistence manner. This method allows antigens within peptides to Previously shown to be effective in covalently attaching to the antigen-binding pocket There is. For example, Leuscher eal, C Dan 1.59:247-255 (+989) (incorporated herein by reference).

Other methods of attachment will be apparent to those skilled in the art and include, for example, carboxylation on glycoproteins. trate group, e.g. the carbohydrate moiety of the α- and/or β-chains. can include binding through.

The protein effector component is purified by a standard dehydration reaction using carbodiimide. It can be attached to a C subunit or a peptide. Hetero bifunctional group Linker-1 such as 5PDP, glutaraldehyde, etc. can also be used.

The sequence for preparing the complex depends in each case on its components. example For example, in certain procedures, the peptide moiety and the MHC subunit components are By contacting the peptide with an MHC subunit component, e.g., by mixing. and associate non-covalently. Next, if desired, use this effector commercially. For example, 5PDP (Pierce Chemical s) to covalently bind to MHC. Or with an effector The MHC subunits are first bound using a dehydration reaction and the conjugate is , can be complexed with a peptide component.

If the effector is itself a protein, the complete complex can be constructed using recombinant methods. DNA can be made directly from suitable encoded DNA. example For example, AChR peptide +95-215 and RIG in humans and mice. What has been characterized as an epitope within the MG-associated & IHC antigen can bind to the antigen-binding site at the N-terminus of the polypeptide obtained from.

The amino acid sequence of the AChR peptide in the one-letter amino acid code is: DTPYL D [TYHF I9IQRIPLYFV. encodes a peptide The oligonucleotides are synthesized using known codons for amino acids, Preferably, such codons have a preferred use in the organism in which they are used. Used in oligonucleotide design. of various organisms and cell types Preferred codon usage is known in the art. For example, the expression If it is in Escherichia coli (E, coli), AChR195-2 A preferred oligonucleotide sequence encoding 15 is 5’ GACACCCCCG TACCTG GACATCACCTACCACT TCATCATG CAG CGT ATCCCG CTG TACTic C TG was able to be 3°. This array is then constructed using techniques known in the art. Sequences enfeeding subunits obtained from MHC antigens using techniques (with or without a sequence encoding a peptide loop region) ) can be incorporated. This uptake site is where the subunit is expressed and When folded, the AChR peptide antigen becomes an epitope for target T cells. This is something that could be used as a pool.

In one procedure, the AchR195-215 peptide is injected into the appropriate molecule. It is attached to the N-terminal end of the knit molecule. The recombinant complex is used in mice. For example, when the AChR peptide is [-A I′-α or I-A”- It can be incorporated into the sequence encoding any of the beta chains. these The sequences encoding the chains of are known and are shown in FIGS. (β chain). If the AChR peptide is incorporated into the β chain, e.g. For example, an oligonucleotide is inserted as a substituent for the leader peptide. can be done. Methods for replacing sequences within polynucleotides are well known in the art. Examples are given in the section on plasmid construction. It is listed.

A similar procedure can be used to encode peptides obtained from appropriate human HLA antigens. It can be used to incorporate AChR peptides into sequences. for example, In humans, habrotibe DR2W2 is associated with ^IG. Therefore, The AChR peptide encodes, for example, the β-chain of the DR2 allele. can be included in an array. Main serological specificity for DPI-9 The structural basis in the DR subregion is HLA-D from multiple DR hablotypes. The sequence encoding the R-β chain is also known. For example, Be1 Incorporated herein by et al., 81. )checking.

Evaluation of the complex The conjugates of the invention can be assayed using in vitro or in vivo systems. Ru. In one in vivo system, the conjugate is associated with the peptide of the conjugate. from patients who have been immunized with or exhibit immunity to the protein or antigen responsible for peripheral blood T cells. Successful complexes are combined with syngeneic T cells. selectively binds and prevents T cell proliferation even during stimulation with additional antigens. Probably. Alternatively, T cell activation can be triggered by 3H-thymidine incorporation or 3-( 4,5-dimethyl-thiazole-2-7')-2,5-diphenyltetrazoly [L-2 production as measured by the uptake of um bromide (&ITT) production or T cell proliferation.

A preferred method to assess the complex is to measure the rate of production of acidic metabolites within T cells. The use of a microphysiometer for this purpose. By using this device, the complex and T-cell receptor interactions can be detected faster and more easily. Ru. The physiological basis of this apparatus lies in the fundamental metabolites, lactate and and the acidity of carbon dioxide. The acidity of the culture medium containing a small sample of cells is determined by A light-addressable potentiometer sensor that can deliver can be measured by a patentiometric 5 sensor) . The rate of acidification is used as a measure of the metabolic rate of the cells to be assayed. .

Discussion of this technique for measuring changes in the metabolic rate of target cells Cite this incorporated herein by. )checking.

In in vivo systems, in the presence of APC, directed against isolated epitopes or completely T cells that proliferate in response to full-length antigen are cloned. This claw into non-immunized M tissue-compatible animals to reduce autoimmune disease. Inject. Symptoms associated with related complexes may improve or eliminate symptoms of the disease. It should be.

Any of the above types of complexes, i.e. those with effector components or You can use what you don't have. In one embodiment, the treatment comprises 2 It's double. containing an effective portion of an antigen that down-regulates the immune system of an individual. Processed by a complex of IHC subunits. Further down ~ Regulino John has discovered that MMC subunits contain antigens specific to the autoimmune disease being treated. This is achieved by treatment with an effective moiety and an effector component. Additionally, the complex A panel of can be used at the first stage of treatment. For example, one or more of the antigens if the peptide is suspected of being involved in an autoimmune response, and/or one or more antigens. treatment of the individual with several complexes if there is a suspicion of Can be done. This complex is combined with the appropriate &lHC subunit active portion and the antigen. Select from a panel containing valid parts. This complex optionally contains an effector It can further comprise components.

Selection of MHC complexes for treatment and/or diagnosis Diagnosis or treatment of individuals for diseases For selection of the MI (C subunit complex) of the present invention to be used in therapy , the type of LIHC antigen associated with the antigen or allergen representation is identified.

For example, specific autoimmune disorders are correlated with specific MHC types. to humans The list of DQ/DR halotypes and their relationship with autoimmune diseases is shown in Figure 7. Shown inside. Which of the alleles represents the MIC-encoded polypeptide? Methods for identifying whether a deviation is related to disease are known in the art. Ru.

Described in EP 286447, which is incorporated herein by reference. The method described above is preferred. In this method, several steps follow.

First, the relationship between MHC antigens and disease is determined based on genetic studies. this Methods for conducting such studies are known to those skilled in the art and are Information about all known related HLA diseases is available at Copenhagen's HLA and the Disease Registry. related to Saya loci encoding polypeptides that are likely to have the strongest association with disease. (See Figure 7).

Second, specific genes encoding diseases associated with LIHC antigens/polypeptides The target allele is identified. In allele identification, the susceptibility allele is Presumed to be dominant. Identification of alleles allows for strong association between specific subtypes and disease. This is done by measuring positive relationships. This can be done in a number of ways. All of these can be performed in a manner known to those skilled in the art. For example, Type classification is mixed lymphocyte response type (mixed lymphocyte response type). response (eight ILR) typing) and prime line Primed lymphocyte testing (PLT) ) is done by catequil. Both methods are Weir and Black kwell, eds, Handbook of Experimenta 1mmunology, (which is incorporated herein by reference) It is written in. It may also be specific to the MHC locus being examined. A probe is used to generate restriction fragments of DNA (restriction f). ragment length polymorphism) More often than not, this is done. For example, Nepom (1986), Anna ls N, Y, Acad, Sci, 475.1 (this is incorporated herein by reference) Incorporate it into the detailed document. ). Methods of making probes for MHC loci are known to those skilled in the art. It is. For example, Gregersen et al. (1986), Pr. oc, Natl, Acad, Sci, USA 79:5966; We issman et at, in Medicine in Transiti on: the Centennial of the University of 1llinois CoCo11e of Medicine (E, P , Cohen, ed., 1981) (all of which are incorporated herein by reference). Take it inside. )checking.

The most complete identification of subtypes conferring disease susceptibility is based on loci of genomic DNA or Performed by cDNA sequencing of mRNA encoded in-house. It will be done. The sequenced DNA shows that the supraaccessible region of the MHC-encoded polypeptide Contains the section being encoded.

Specific identification of desired DNA by probe for amplification of desired region Techniques for this are known in the art and include, for example, polymerase chain Including anti-RT (PCR) technology.

Once an allele conferring susceptibility to a specific autoimmune response is identified and the polypeptide encoded within that allele can also be identified . That is, the polypeptide sequence has D within the allele encoding it. can be derived from the NA sequence. used for diagnosis and/or treatment The IHC subunit complex of the present invention, which is associated with the symptoms of autoimmune diseases, Autoimmune antigens from the active part of the C subunit and associated with the same disease symptoms obtained from.

The next step is to identify a suitable antigen. Some related to autoimmune diseases autoantigens have been extensively studied. The identified autoantigens are associated with myasthenia gravis. acetylcholine receptor, miniphosphobasic protein in multiple sclerosis. original For primary biliary cirrhosis Mitochondrial dihydrolipoamide acetyltransferase, Type 11 collagen in equineoid arthritis, collagen in autoimmune thyroiditis roglobulin, S antigen in autoimmune uveitis, and Paraneoplastic pemphigus This includes desmoplakin f. Hatondo In autoimmune diseases, small autoantigenic peptide fragments of macromolecular self-antigens (epitope) is recognized by a defined subset of helper T cells. is shown.

Once macromolecular antigens are identified as targets for immunopathological responses, several distributed techniques used to identify and characterize the epitope. be done. These methods generally involve enzymatic digestion of whole self-antigens or produced by cloning and expressing a fragment of a gene encoding an antigen. Use autoantigenic fragments. The amino acid sequence of an autoantigenic peptide fragment was discovered. When a set of overlapping peptides is then synthesized. These methods use fragments or Synthetic peptides can stimulate disease-associated T cell clones or hybrids in a cognate antigen-representation system. Epitope sequences are identified by their ability to stimulate toma. For example, Livin gatone et al, Ann, Rev, Immun (all of these is incorporated herein by reference. )checking.

For example, more than 90% of patients with rheumatoid arthritis have DR4 (0w4), DR4 (D W14) or DPI habrotype (see Figure 7). human ・It is also known that type I+ collagen is the target antigen in rheumatoid arthritis. It is being Therefore, it should not be used for the treatment or diagnosis of patients with rheumatoid arthritis. The conjugates of the invention used enable antigenic representation for the induction of disease or DR4 (0w4), DRI and/or D disable antigen representation for suppression A polypeptide obtained from R4 (Dw14), which is type-11 collagen. will include those that form complexes with the effective portion of.

Suitable for use of the complex of the present invention for the diagnosis and/or treatment of autoimmune diseases The steps involved are shown in FIG. Simply put, if you have (or are suffering from) an autoimmune disease. identify individuals (prone to cancer) and identify autoimmune disorders. Identification is based on symptomatology and /Or an investigation of the family medical history. The MHC type of an individual is unknown in this field. serological determination by one or more of several methods known for by assay, and by DNA analysis (including RFLP and PCR techniques). Measured by cell typing. Individual T cells are transformed into autoreactive T cells. to determine the self-peptides recognized by the cells. , this is accomplished using the labeling complexes of the invention, as described above. multiple After binding or targeting of T cells is determined, individuals are treated with specific autoreactive T cells. The complex of the present invention and/or autoreactive T cells capable of suppressing cell replication. Dispose of it depending on what you want to kill. (as measured by residual autoreactive T cells) ) treatment using the labeling complex of the invention as described above. Monitor by research.

As used herein, the term "individual" refers to all mammals and all vertebrates with essentially equivalent MHC systems includes.

Model systems for in vivo testing include: for autoimmune diseases that can be used to evaluate the efficacy of the inventive complex. It is a model series.

systemic Lupus erythematosus s (SLE)) Autoimmune New Zealand black (NZB) Mice and phenotypically normal New Zealand White (NZW )? p of the U.S. strain, the hybrid is more resistant than that seen in the parent NZB strain. The disease develops into a fulminant, severe systemic autoimmune disease. these mice have several immunopathies, including antibodies to nuclear antigens and subsequent death. Disease development, female-dominated immune complexes with striking resemblance to SLH in humans Clarify those containing glomerulonephritis show. Night.

et al, J, EXp, Med, (+978) 147:1653 ．． (This is incorporated herein by reference.)

A strong association with the NIHC gene product has been reported in both humans and mice with this disease. It has been tell. HLA-DR2 and HLA-DR3 individuals are the ones that develop into SLE. are at higher risk than the general population (Reiku ertsen, et al., N.

Engl, J. Med (1970) 299:515), while NZB/W F+ mouse (H-2””), h-2 halotype obtained from NZW parent Genes that bind to nephritis contribute to the development of nephritis.

The effectiveness of the conjugates of the invention depends on survival rates and symptoms such as proteinuria and anti- The appearance of DNA antibodies can be measured by the progression of their development.

Proteinuria is treated with Uristix (Miles Laboratories, Inc. , Elkhart, IN). The approximate value is the following trace amount, lomg/dl; I+, 30mg/di: 2+ , 100mg/dl: 3+, 300mg/dl: and 4+, 1000m Give as g/dl. Development of high stage proteinuria in mice due to complex processing, it is significantly delayed.

The presence of anti-DNA specific antibodies in NZB/WF, li and 5tollar, J, Immunol, Methods ( 1986) 90:IO2 (which is incorporated herein by reference). using a modification of the enzyme-linked immunosorbent assay (ELISA), as described in It is measured by using

Myasthenia Gravis (MG) Myasthenia Gravis , one of several human autoimmune diseases associated with HLA-D. Safe nberg, et al, Ti5sue Antigens (1978 ) 12136: McDevitt, et at, Arth, Rhe um, (1977) 20:59 (incorporated herein by reference). nothing. ). In MG, for acetylcholine receptor (AcChoR) Antibodies induce neuromuscular effects by mediating loss of AcChoR in the postsynaptic membrane. Reduce transmission.

SJL/J female mice are a model system for human MG. these animal smells Experimental autoimmune myasthenia gravis (EAMG) has been linked to soluble A from other species. It is induced by immunizing mice with cChR proteins. against EAG sensitivity is partially MHC-related and mapped to a region within H-2. Ru. Christadoss, et al., J. Immu3 (this is referred to as Incorporated herein by use. ). Freund complete horse mackerel 15 µg of emulsified AcChR in a rejuvant on the back, hind legs, and base of the tail. Inject intradermally between 6 sites.

Animals are reimmunized 4 weeks later with this same regimen.

Evaluation can be performed by measuring anti-AcChoR antibodies, anti-AcCh.

R antibody levels were determined as described in Waldor, et al., supra. Sea urchin is measured by microtiter ELISA assay. Standard reagent volume is 50 μl per well. Reagents are usually placed in a well at 2 o'clock ISl! RTI: Incubate at room temperature. , dicarbonate buffer, pH 9.6. Add 5 μg of diluted AcCoR to each well. AcChoR and After incubation, the plates were treated with 0.05% Tween and 0.05% 4 times with a wash solution consisting of phosphate buffered saline containing NaN) rinse. Mouse serum was added to 0. OE PBS (pi(7,2), 1.5mM Mg Cl2.2. On+AI 2-mercaptoethanol, 0.05% Tween- diluted in 80.0.05% NaN3 (P-Tween buffer) and Incubate on plate. After washing the plate, place it in a P-Tween bag. A β-galactonderse-conjugated sheep-mouse antibody diluted in fur was added to the Add to each well. After the final wash, the enzyme substrate, p-nitrophenol Le-galactopyranosite was added to the plate and the degree of catalysis of the substrate was determined. . After 1 hour, the absorbance is measured at 405 nm.

Anti-AcCohR antibodies inhibit AcCohR-induced immunity when compared to non-immunized mice. It is predicted to be present in infected mice. Treatment with the complex expected to significantly reduce titers of anti-AcCohR antibodies in mice. .

The effect of treatment with the complex on clinical EA & IG can also be evaluated . Signs of myasthenia include a characteristic hunched posture with a drooping of the head and neck; This includes worsening arch shape, dislocated limbs, abnormal gait, and difficulty standing upright. nothing. Mild symptoms are present after standard stress tests and antibody titers Administration of the conjugate after a period after which the symptoms have fallen should improve.

Rheumatoid arthritis (RA) In humans, susceptibility to rheumatoid arthritis is related to HLA D/DR. are doing. The immune response in mice to innate type [1 collagen 1- Experiments for arthritis with multiple histological and etiological features similar to RA It has been used to establish a digital model. Collagen derivative association in mice Susceptibility to arthritis (CIA) is mapped to the H-21 region, particularly the [-A subregion. has been done. Mice from f(use, et-susceptible strain, DBA-1, Woo Ley and Luthra, J., [mmunol, (1985), 134.2366 (incorporated herein by reference).

), using the techniques described in By processing C[A].

In other models, adjuvant arthritis in rats is similar to human arthritis. is an experimental model for bacterial antigen-triggered autoimmunity. It is the prototype of arthritis. Ho1oschitz, etal, Prospec ts of [mmunology (CRCPressX1986): Pe arson Arthritis Rheum, (1964) 7:80 o This disease is caused by a clone of T cells that are reactive against Aji, /< Cyto (MT). as evidenced by its transmissibility by It is the result of a cell-mediated immune response.

Based on studies with identically cloned cells, the target autoantigen in the disease is cartilage ( This is because it is a part of the proteoglycan molecules of carti Iage).

Adjuvant disease in rats was described by Pearson, supra. sea urchin, i.e. at some storage site, preferably subcutaneously or at the base of the foot or tail. Freund’s adjuvant (dead Mycobacterium tuberculosis or its chemical derivative) given to (min., mineral oil, and emulsifier) in a single injection. This adjuvant is given in the absence of other antigens.

The effectiveness of the complex treatment on the manifestations of the above diseases will be monitored. These manifestations are related to histopathology and of synovial lining cells. Acute and subacute synovitis due to proliferation, predominantly associated Mononuclear infiltration of nodes and specific tissues, connectivetis invasion of bones and cartilage of joints by P. ue pannus) and especially adjacent to infected joints This includes periosteal (perio5teal) new bone formation. severe or chronic In cases of sexual intercourse, fibrotic or bone ankylosis may occur. Then, destructive changes occur. These histopathological signs are associated with Freund's expected to appear in control animals approximately 12 days after sensitization to the juvant. It will be done.

Insulin-dependent diabetes mellitus betes^l111itus([DDM)) rDDL+ selectively destroys insulin-secreting cells within the islets of Langerhans in the pancreas. observed as a result of. The involvement of the immune system in this disease is due to mononuclear cells. By morphological evidence of early infiltration of islets, by detection of anti-hypercellular antibodies, IDD&I Due to the high frequency of HLA-DR3 and -DR4 alleles in the population, and This is suggested by the clinical association between IDDAI and various autoimmune diseases. simultaneous An animal model for developmental IDDM and thyroiditis was developed in Be rats. It's been coming. As in humans, the rat disease is caused in part by the &lHC antigen. imposed by the encoding gene, characterized by islet invasion, and - Associated with the presence of islet antibodies. The l-E equivalent class II MHc antigen is This is because it is associated with the emergence of autoimmune diseases in cats. Biotard, et al, , Proc, Natl, Acad, Sci, USA ( 1985) 82:6627゜Insulitis (insulinitis) was found in morphological evaluation. ulitis) is characterized by the presence of mononuclear inflammatory cells within the islets. thyroid Thyroiditis, as a minimum standard, refers to the thyroid gland (thyroiditis). Focal-mediated lymphocytic infiltration (focal 1interst) within id grand) characterized by initial lymphocytic 1nfiltraje) be done. The most severe incident is a widespread, widespread lymphocytic infiltrate, i) destruction, fibrosis, and alteration of focal Hurthle cells. , indicates.

Treatment of 8B rats with the complex of the invention is associated with IDDAI and thyroiditis. It is expected to improve or prevent the manifestation of clinical and morphological signs.

Among other autochthonous models, the NOD mouse strain (H-2K　’D　　”) Immunization [DD^1 is a failed murine model. The strength of these animals is Regarding anti-hypercellular antibodies, severe intulinitis, and autoimmune destruction of β-cells. It is characterized by the evidence of Kanazawa.

et al, Diabetologia (1984) 27:113゜This disease The disease is passively metastasized by lymphocytes and treated with cyclosporine-A. (lkehara, et al, , Proc, Natl , Acad, Sci, USA (+985) 827743:^1ori, et al.), Diabetologia (1986) 29:244 , the number of untreated animals with deep glucose intolerance and ketosis and the onset of this disease Death develops within weeks (succumb) in 70-90% of females and males. 20-30% of animals develop diabetes within the first six months of life.

gJ Yoban research has identified at least two genetic loci that are responsible for disease susceptibility; One of them maps λIHC. both at the serological and molecular level. Characterization of NOD class II antigens can determine susceptibility to autoimmune diseases or IA This suggests that it is related to l. Acha-Orbea and McDev itt, Proc, Natl, Acad, Sci, USA (197 0) Treatment of flNOD mice with the 84:235° complex occurred before the onset of diabetes. It is expected to prolong the period of illness and/or improve or prevent the disease.

Experimental Allergic Brain Encephalomyelitis (EAE) Experimental allergic brain myelitis (EAE) contributes to the human disease of multiple sclerosis (MS) Causes autoimmune diseases of the central nervous system that are similar in many ways.

The disease can be caused in many species, including mice and rats. Wear.

The second disease is characterized by an acute onset of paralysis. . Perivascular infiltration by mononuclear cells in the CNS occurs in both mice and rats. It is observed at Methods of disease induction, as well as symptomatology, are described by Aranson (198 5) in The Autoimmune Disease (eds, R ose and N1ackay, Academic Press, Inc. ) pp, 399-427. and Acha-Orbea etal, (+9 89), Ann, Rev. 1mm, 7:377-405. There is.

One of the genes that mediates susceptibility is Ql, which is located within the MHC class 11 region. oore et al. (1990), J. Immunol, 124: 1815-1820). The best analyzed encephalitogenic (encephali togenic) protein is miniphosphobasic protein (&1BP), but in other encephalitis Inducible proteins have been found in the brain. Immunogenic epitopes are mapped (See Acha-Orbea et al., supra). PL mouse strain In (H-2''), two encephalitogenic proteins in MBP were characterized. MBP peptide p35-47 (8 IBP 35-47), and acetylation (8 IBP 1-9).

Book for ameliorating disease symptoms in individuals where EAE is induced The effectiveness of the inventive complex is measured by the survival rate and by the progression of the development of symptoms. can be

Formulation and administration When the transmembrane region of the MMC subunit is included, the composite of the present invention The body is conveniently administered after being incorporated into a lipid monolayer or bilayer. typical Although typically used for this purpose, any form of lipid membrane, e.g. Surface lipid membranes or cellular membranes of cells (eg, red blood cells) can be used.

The conjugate can also conveniently be incorporated into micelles. Example 2 below The data presented in the table are for Ml-IC-peptides comprising dimeric Ml(C molecules). This indicates that the complex exists primarily as an aggregate.

Liposor 14 is prepared according to standard methods, as described below. Can be done. However, if the transmembrane region is deleted, this The conjugate may be administered in a manner conveniently used for peptide-containing pharmaceuticals. I can do it.

Administration is systemic and preferably by intravenous injection, therefore , formulations are used that are compatible with the injection route of administration.

A preferred formulation is Remington's Pharmaceutical 5c iences, &1ack Publishing Company, Ph 1ladelphia, PA, 17th ed, (1985) (this , incorporated herein by reference. ) can be seen inside. Complex and medicament of the present invention A variety of pharmaceutical compositions can be prepared comprising a carrier effective as a drug.

This pharmaceutical composition is suitable in a variety of pharmaceutical delivery systems. medicine delivery For a brief review of current methods of - Langer, 5science 249:1527-1533 (1990) (which is incorporated herein by reference). Incorporate into. )checking.

In preparing the pharmaceutical compositions of the present invention, the complexes of the present invention are modified and their pharmaceutical compositions are Changing dynamics and biodistribution Numerous methods for modifying are known to those skilled in the art (e.g. Langer , see above. ). For example, suitable for increasing the serum half-life of the conjugate. The method involves treatment to remove carbohydrates involved in removing complexes from the bloodstream. including theory. Preferably, substantially all of the carbohydrate moieties are removed by this treatment. be done. Substantially all of the carbohydrate portion or at least about 7 of the carbohydrate portion When 5%, preferably about 90%, and most preferably about 99% are removed, removed. Soluble macromolecules, such as proteins, polysaccharides, or synthetic polymers, e.g. For example, binding to polyethylene glycol is also effective. Other methods include substances, e.g. For example, compounds made of proteins, lipids (e.g. liposomes), carbohydrates, or synthetic polymers. Involves protection of the complex within vesicles.

The liposomes of the invention are typically complexed or interacted with TwJ receptors. NIHc-peptide located on the surface of the liposome in such a manner that it can be utilized. including complexes. The transmembrane region is normally formed during membrane formation. The first is incorporated into the membrane. The liposome contains the desired drug (e.g., toxin or is used to target chemotherapeutic agents) to specific autoreactive T cells. I can do it. Alternatively, complexes embedded within liposomes can be activated within target cells. Can be used to induce indigestion.

Liposome charge is an important determinant in liposome clearance from the blood The negatively charged glue bonomes are more rapidly taken up by the reticuloendothelial threads (Ju liano, Biophys, Res, Commun, 63:651 ( (1975)) and therefore have a shorter half-life in the bloodstream. Delayed circulation Liposomes with a ring half-life are typically desirable for therapeutic and diagnostic applications. It is. For example, can it be maintained in the bloodstream from 8.12 or up to 24 hours? Particularly preferred are liposomes that can be used.

Typically, liposomes contain about 5-15 mole percent of negatively charged phospholipids. substances, such as phosphatidylglycerol, phosphatidylserine or hisphatidyl Prepared by tisyl inositol. Additionally, additional negatively charged phospholipids, e.g. , phosphatinolglycerol helps prevent spontaneous liposome aggregation and thus minimizing the risk of unwanted glueponome aggregate formation. Film-hardening agent (＾I embrane-rigidifying agents), e.g. The amount of allylganglioside (monosialylgangl　1oside) at a concentration of at least about 50 mole percent, and from 5 to 15 mole percent. Sphingomyelin or natural phospholipid is generally incorporated by reference in U.S. Pat. No. 4,837.028, herein incorporated by reference. Take it inside. ) increase of liposome preparations in the bloodstream as described in circulation.

Furthermore, liposome suspensions are free from free-radical and lipid-peroxidation during storage. Lipid-protecting agents that protect lipids against damage can be included. Lipophilic free Radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelates, such as ferrioxyanine (ferrioxia e) is preferred.

For example, 5zoka et at, Ann, Rev, Biophys, Bioeng, 9:467 (+980), U.S. Patent No. 4.235.871 , No. 4.501.728 and No. 4.837.028 (all of which are hereby incorporated by reference). Incorporated herein by use. ) using various methods as described in , can be utilized to prepare liposomes. One method is to produces multilamellar vesicles of is. In this method, the vesicle-forming lipid is dissolved in an organic solvent or solvent system and dried under vacuum or in an inert gas; Forms a thin lipid layer. Optionally, the film is coated in a suitable solvent, e.g. redissolved in tanol and then lyophilized for a more homogeneous lipid mixture. It forms a powder-like form that is more easily hydrated. This film is , coated with an aqueous solution of the targeted drug and targeting component, and typically , with stirring for a period of +5-60 minutes to hydrate. The resulting multi-lamme The size distribution of the LA vesicles can also be improved by hydrating the lipids under more vigorous agitation conditions. smaller sizes by the addition of solubilizing detergents, e.g. deoxycholate. Can be henovated.

The hydration medium transfers the targeted drug to the liposomes in the final liposome suspension. Contains at the concentration required in the internal volume. Typically, this doctor The drug solution is a complex between 10-]00 mg/m, l in buffered saline solution. Including the body.

After liposome preparation, the desired size range and relatively narrow distribution of liposomes can be determined. Liposomes can be sized to achieve the desired size. 1 One preferred size range is approximately 0°2-0.4 microns, which is , the liposome suspension is filtered through a conventional filter, typically a 0.22 micron filter. sterilization by filtration through a filter. This filter sterilization method when the liposomes are sized to about 0.2-0.4 microns. This can be done on a high throughput basis.

Several techniques may be utilized for sizing to the desired size.

One sizing method is described in U.S. Pat. No. 4,737,323 (which is incorporated by reference). Incorporated herein. ).

Sonication of liposome suspensions by either bath or probe sonication can be Progressive size down to small unilamellar vesicles, smaller than about 0.05 microns Create a decrease. Homogenization fragments large liposomes into smaller liposomes Another method relies on shear energy to In a typical homogenization procedure and multilamellar vesicles, typically between about 0.1 and 0.5 microns. Standard emulsion homogenization until the desired liposome size is observed. is recirculated through the In both methods, the particle size distribution is can be monitored by laser beam particle size separation.

Extrusion of liposomes through small-porous polycarbonate membranes or chiral ceramic membranes However, to reduce the liposome size to a relatively more narrow size distribution This is an effective method. Typically, the suspension is mixed until the desired liposome size distribution is achieved. The membrane is cycled through the membrane one or more times until it is formed. This liposome is a continuous liposomes are extruded through a small-pore membrane, resulting in a gradual decrease in liposome size. can be achieved.

Even under the most effective encapsulation methods, initially sized liposome suspensions The liquid may contain up to 50% or more of the complex in free (non-encapsulated) form. Ru.

Several methods have been utilized to remove untethered compounds from liposome suspensions. can be In one method, liposomes in suspension are subjected to high-speed centrifugation. The separation results in a pellet, and the supernatant contains free compounds and very small liposomes. leave a message. Another method is to concentrate the suspension by ultrafiltration and then replace it in a medium. resuspending the concentrated liposomes. Or gel filtration? It can be used to separate large liposome particles from dissolved molecules.

After the above treatments, the liposome suspension is transformed into the desired amount for use in intravenous administration. Bring it to concentration. This resuspends the liposomes in a suitable volume of injection medium. in which the liposomes are subjected to, for example, centrifugation or ultrafiltration. Concentrate the suspension where the filtration or drug removal step increases the total suspension volume. Therefore, it is concentrated. This suspension is then subjected to filtration as previously described. More sterilized. Liposomes comprising MHC-peptide complexes, e.g. , while delivering the drug in doses that vary according to the mode of administration. administered parenterally or topically while treating certain diseases, etc. be able to.

Micelles are a popular choice in the field for increasing the solubility of molecules with non-polar regions. commonly used. Therefore, one skilled in the art will appreciate that micelles are useful in the compositions of the present invention. You will admit that there is. Micelles comprising the complexes of the invention can be prepared by those skilled in the art. Prepared according to well known methods (e.g. Remington's Pha See Chapter 5, supra, Chap. 20. and. ). Micelles comprising the conjugates of the invention typically contain standard surfactant prepared using agents or detergents.

Micelles contain surfactants (hydrophobic moieties and one or more ionic or other strong moieties) in aqueous solution. (molecules containing hydrophilic groups). As the concentration of solid surfactant increases When the monolayer is adsorbed at the air/water or glass/water interface, further occupancy requires excessive compaction of the surfactant molecules already within the two monolayers. , it becomes more tightly packed. The amount of dissolved surfactant above that concentration A further increase in will increase the equivalent amount of new molecules that will assemble into micelles. bring about This process is called “critical micelle concentration” It starts at a characteristic concentration called "e concentration". Ru.

The morphology of micelles formed in dilute surfactant solutions is mostly spherical. interface The polar head groups of the activator molecules are arranged within an outer spherical shell, while their hydrocarbon The bare chains point toward the center and form the core of the micelle sphere. The hydrocarbon chain is The micelles are randomly coiled and intertwined, and the inside of the micelles is a non-polar, liquid-like substance. It has the characteristics of In micelles of polyoxyethylenated nonionic detergents, polio The xyethylene portion faces outward and is permeable to water. this The arrangement is energetically favorable. This is because the hydrophilic head group is in contact with water. and the hydrocarbon moiety is separated from the aqueous medium and its polar head This is because they are partially shielded from contact with water by the groups. placed inside the micelles. placed, the hydrocarbon tail of the surfactant molecule forms a weak van der Waalska They are interacting more with each other.

The size of micelles or their number of aggregates is largely influenced by geometrical factors. hydrocarbon water The radius of the elementary core can exceed the length of the extended hydrocarbon chain of the surfactant molecule. I can't. Therefore, an increase in chain length or an increase in homologous series increases the number of aggregated spherical micelles. increase. Surface activity whose hydrocarbon portion is one normal alkyl chain For agents, the maximum aggregate number structures with spherical morphology are C=, C+. , Ca2, CI4 and cps are approximately 27.39.54.72 and 92, respectively.

If the surfactant concentration increases by more than a few percent, and the electrolyte is added (for ionic surfactants) or the temperature was increased (for non-ionic surfactants). ), the size of the micelles increases. Under these conditions, the micelles remain As it grows, it becomes less spherical and becomes ellipsoidal, cylindrical, or finally lamellar. It will be in the shape of an a.

Common surfactants familiar to those skilled in the art are used in the micelles of the present invention. be able to. Suitable surfactants include sodium laurate, sodium oleate. um, sodium lauryl sulfate, octaoxyethylene glycol monodecyl Le Ether, Octoxynol 9 and PLLI RONICF-1270 (Wyandotte Chemicals Carp ,)including. Preferred surfactants are nonionic polyoxylic acids suitable for intravenous injection. Ethylene and polyquine propylene surfactants, such as PLURONICF- 127■, low octyl-β-glucopyranoside, etc. In addition, phosphorus quality, such as those described for use in the preparation of liposomes. It can also be used for cell formation.

The MHC subunits of the present invention have a lipophilic transmembrane region and a relatively hydrophilic The mixed micelles consist of the extracellular domain of a common surfactant. or formed in the presence of phospholipids and subunits. Mixed rice of the present invention may contain any combination of subunits, phospholipids and/or surfactants. can consist of Therefore, this micelle contains subunits and surfactants. Combinations of sex agents, phospholipids and surfactants or both subunits and phospholipids subunits in combination.

For pharmaceutical compositions comprising the conjugates of the invention, the dosage may be, e.g. the specific complex, the mode of administration, the particular disease being treated and its severity, the patient's overall It varies according to the individual's health and symptoms and the judgment of the prescribing physician. of rat specimen The dosage level for is generally between about IOμg and about 500μg. about A total dose of between 50 μg and about 300 μg is preferred. For example, in the course of a disease Treatment provided over three doses of 25 μg or 100 μg may be used. It is effective. The total dosage is between about 0.5 and about 25 mg/kg, preferably between about 3 and In the range between about 15 mg/kg.

Pharmaceutical compositions can be used parenterally, locally, for prophylactic and/or therapeutic treatment. , for oral or topical administration, e.g. by aerosol or transdermally. is intended. The pharmaceutical composition may be administered in various unit doses depending on the method of administration. It can be administered in a given form. For example, unit doses suitable for oral administration Forms include powders, tablets, pills, and capsules.

Preferably, the pharmaceutical composition is administered intravenously. Therefore, the present invention a solution of the complex dissolved or suspended in a carrier, preferably an aqueous carrier. A composition for intravenous administration is provided. Various aqueous carriers, e.g. Fur water, 0.4% physiological saline, etc. can be used. For example, phosphate Puffer saline (PBS) is particularly suitable for administering the soluble conjugates of the invention. Ru. These compositions are sterilized by conventional, well-known sterilization techniques.

or sterile filtered. The resulting aqueous solution was Prior to packaging or administering the lyophilized preparation for use as a sterile aqueous solution, It can be freeze-dried as well. This composition is pharmaceutically acceptable. auxiliary substances that are tolerated and are necessary to adapt to physiological conditions, e.g. , pH adjusting and buffering agents, osmotic pressure adjusting agents, hydrating agents, etc., e.g., sodium acetate. , sodium lactate, sodium chloride, potassium chloride, calcium chloride, lluvita monolaurate, triethanolamine oleate, etc. .

Concentrations of the conjugate may vary widely, i.e., less than about 0.05% by weight, usually about 1% by weight. or from at least about 1% by weight to as much as 10-30% by weight, Primarily, the liquid volume It can be selected depending on the amount, viscosity, etc. Preferred concentration for intravenous administration is about 0.02% to about 0.1% in PBS.

For solid compositions, conventional non-toxic solid carriers such as pharmaceutical grade of Mannitol, Lactose, Starch, Magnesium Stearate, Saccharin Na Thorium, talc, cellulose, glucose, nucrose, magnesium carbonate , etc. can be used. For oral administration, a pharmaceutically acceptable The non-toxic composition contained may contain any of the commonly used excipients, e.g. Incorporating as described, and generally 10-95% of the active ingredient. is formed by.

For aerosol administration, the complex is preferably combined with a surfactant and a propellant. Both are supplied in finely ground form.

The surfactant is, of course, non-toxic and preferably soluble in the propellant. Must. Representatives of such agents contain from 6 to 22 carbon atoms. Fatty acids, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid Phosphoric acid, linoleic acid, linuric acid, olesteric acid d) and oleic acid and an aliphatic polyhydro alcohol or its cyclic anhydride, e.g. For example, ethylene glycol, glycerol, erythritol, arabic Tol, mannitol, sorbitol, hexitol derived from sorbitol Esters or partial esters with water and polyoxyethyl esters of these esters lene and polyoxypropylene derivatives. mixed esters, e.g. mixed or Natural glycerides may also be used. The second surfactant is 0.1% of the composition. The weight can constitute 20% by weight, preferably 0.25-5% by weight. . The balance of the composition is usually a propellant. Liquefied propellants are is typically a gas and condenses under pressure. in a suitable liquefied propellant , lower alkanes containing up to 5 carbon atoms, such as butane and propane: Preference is given to fluorinated or fluorinated chlorinated alkanes. the above mixture , may be used. For aerosol production, containers with suitable valves , a suitable propellant containing a finely divided compound and a surfactant. death The components are thus maintained under elevated pressure until they are released by operation of the valve.

Compositions containing the complexes may be administered for therapeutic, prophylactic, or diagnostic uses. I can do it. In therapeutic use, the compositions may be used to treat symptoms of disease and its complications. in an amount sufficient to treat or at least partially halt, as noted above. , administered to patients already suffering from the disease. the appropriate amount to achieve this.” Amounts effective for this use are defined as "therapeutically effective doses." It will depend on the severity and overall condition of the patient. As noted earlier, This is typically between about 0.5 mg/kg and about 25 mg/kg, preferably will be between about 3 and about 15 mg/kg.

Administer to persons susceptible to or at risk of disease. Such amounts are considered “preventive” defined as the effective dose. In this use, the net amount is again Depends on patient's health condition and weight. This dosage is generally within the range mentioned above. It's within.

In diagnostic applications, compositions containing appropriate conjugates or cocktails thereof may be used to patients suspected of having an autoimmune disease to measure the presence of autoreactive T cells associated with the disease. Administer to patients who will be treated. Alternatively, the effectiveness of a particular treatment can be monitored. . An amount sufficient to accomplish this is defined as a ``diagnostically effective dose''. child The net amount will depend on the health condition of the patient, but it is generally from 0.01 to 1000 mg per dose, particularly from about 10 to about 100 mg per patient. In the range of 100mg.

Kits can also be provided for therapeutic or diagnostic uses. Did you do it? Therefore, the subject compositions of the present invention are usually provided in lyophilized form in a container. can do. A compound that can be conjugated or unconjugated with a label or toxin. The combination can be carried out using buffers such as Tris, phosphates, carbonates, etc., stabilizers, biocides, etc. agent, inert protein, such as serum albumin, and the use of one set. Included in the kit with instructions. Generally, these materials are composite present in less than about 5% by weight based on the amount and usually based again on its protein concentration. will be present in a total amount of at least about 0.001% by weight. often its life It may be necessary to include inert fillers or excipients to dilute the sexual ingredients; Therein, the excipient is present in a range from about 1 to about 99% by weight of the total composition. I can do something. When antibodies capable of binding to the complex are used in the assay , which will normally be present in separate vials. This antibody is typical typically conjugated to a label and formulated according to techniques well known in the art. It will be done.

The following examples illustrate the invention without limiting it.

Example example! This example is miniphosphobasic protein^! of the N-terminal peptide of BP(1-14)A', The binding of the purified Fuzumi+A” molecule to the α and β chains is shown. .

procedure: Purification of Fuzumi IA゜and]A" IAk and [A” are incorporated by reference in U.S. Pat. Incorporate it into the statement. ) Standard cyanogen bromide coupling described in By the method, monoclonal antibodies (specific for [A' and [A')], 10-2 ．． Prepared by cup-lengthening 16 with 5epharose 4B beads cultured CH27 cells and SJ cells, respectively. Purified from NP-40 extract of membranes prepared from L/J mouse spleen cells. a Affinity-purified IAk and IA" molecules were purified using 12% 1D SDS polyacrylic Characterized by amide gel electrophoresis.

Isolation and purification of α and β subunits of MHC molecules IAk was enriched with Am1con. The device was used to concentrate to Img/ml and for 12% separation (16 cm 18 cm) applied onto a slab gel unit.

Electrophoresis was performed at constant voltage (I00 volts) for 16 hours at room temperature, non-reducing. It was done under conditions. One lane was excised from the center of the gel and stained with silver. It turned dark. Using the stained gel lanes as a guide, identify the α and β chain balances. and the protein was isolated from Am1con in the presence of 0.1% SO5. Electrophoresis was performed at 400 mA for 4 hours in an Electroeluter apparatus. . The eluted strands were concentrated, dialyzed against PBS containing 0.1% OG, and native and characterized on a reduced SDS polyacrylamide gel.

Synthesis of peptides RIBP(1-14)A' with the sequence Ac-ASQARPSQRHGSKY rat miniphosphorus basic protein peptide homolog, sequence, YFKN[VTPRTP RIBP(89-101)Y” peptide representing PP and sequence, l5QAVHA Ovalbumin peptide with AHAE fNEAGRY, 0VA (323 -340) Y34' is replaced with side chain protected Fmoc amino acid and Applied Bi Standard solid-phase methods using the OSYSTEMS 431A automated peptide synthesizer It was synthesized from The deprotected crude peptide amide was purified by reverse phase HPLC. The homogeneity and identity of the prepared and purified peptides (identity) was confirmed by mass spectrometry assay.

Peptide binding assay Binding of peptides to α/β heterodimers or isolated chains of [A” was determined using thin layer chromatography. cellulose acetate electrophoresis (TLC) as previously described. CAE). Briefly, the synthetic MBP(1-14)A' and 0 VA(323-340)Y34' peptide was purified by Chlor Radiolabeled with [+25i] using the amine-T method. 200μg intact IAk at a concentration of /ml or each at a concentration of 100 μg/ml. Both chains were incubated with a 50-fold molar excess of radiolabeled peptide at 37°C for 48 hours. In a total volume of 100 μm.

Excess unconjugated peptide was transferred to PBS containing 0.1% OG detergent for 48 hours. It was removed by extensive dialysis at °C. Transfer 1 μl of the complex to a 5 cm silica plate. Applied in triplicate on gel TLC plates and 50% methanol and 5% acetic acid It was run in an ammonium solvent system. This plate is dried and radioactive Calculate the distribution, the percentage of IA' or chains occupied by the labeled peptide To do this, we estimated,R,0-0,2.

Peptide binding was also measured by CAE. Here, a 1 μm composite is Cellulose polyacetate paper strip (2.5 cm N15.2 cm) electrophoresis was performed using high-resolution Tris/Barbital (3 2.1 w/w%, 13.7 w/w% Barbital and 54.2 w/w% Nato barbiturate) for 10 min at 350° constant boiler in the presence of buffer pH 8.1. I went to Ruto. The strips are dried and coated with labeled peptide. To calculate the percentage of [Ak or chains occupied by in) were counted.

Chain: Preparation of peptide conjugates.

Two types of conjugates were prepared and purified. Peptide binding and T cell binding For assays of binding, unlabeled heterodimers or individual chains can be combined with radiolabeled peptides. The conjugate was prepared as previously described. Measurement of the rate of T cell extracellular acidification using unlabeled class I] or chains and unlabeled peptides under the same conditions, and the complex was mixed with ]mM sodium phosphate, penicillin and streptomycin. Dialyzed against dicarbonate-free, low-buffered RPMI 1640 media containing syn.

T cell binding assay.

Radiolabeled peptide and non-radioactive M! , Knowledge 1 Purification complex of a dimer or monomer The bodies were incubated with 2.6 × 105 resting T cells at 37 °C in a CO, incubator. 15 ml of polypropylene precoated with BSA g/ml in C ・Incubated in a tube. For antibody blocking experiments, cells were , 500 μg of affinity-purified hamster anti-magenta was added before addition of labeled complex. mouse α/β TCR monoclonal antibody (Pharmingen, San Di Ego, CA) was first incubated. TNP (Pharmin Gen, San Diego, CA) purified hamster [gG iso A type standard antibody was used as a negative control antibody. At the end of the incubation period Instead, 15ml of frozen PBS was added to each tube. calm the cells Yanakai re-F! ! turbid, centrifuged at 2.000 x g, and then The clear liquid was carefully removed. The washing procedure was repeated three times and the cell pellet was Counted in a gamma counter.

Measurement of T cell metabolic acidification rate Details of loading T cells into the microphysiometer for acidification rate measurements. as described in Owlcki et al., supra.

Briefly, cells were allowed to rest from antigen pulsing for IO days. , and in low serum (2,5%) medium to reduce their basic metabolic activity. Incubate overnight. The cells were harvested and placed in serum-free loading medium (10mM HEPES). Low buffering RPL containing buffer, pH 7.3! [1640) l　X　in Resuspend at 107 cells/ml. Microphysical measurement of extracellular acidification Perform in an Ometer and collect 1 minute potentiometer readings every 2.5 minutes. acid Sexualization rate data (volts 7 seconds) were mathematically targeted to 100% prior to cell stimulation. Standardized. This allows comparison of data from cells in separate chambers. make it possible.

result: Affinity-purified α and β chains of IA’ were purified on a non-reducing polyacrylamide gel. and an SDS polyacrylamide gel as shown in Figure 9. Characterized by electrophoresis. The dissociation of the α/β heterodimer into α and β chains is IA' was incubated at 95 °C for 5 minutes (as shown in panel A). or by exposure to pH 3.0 for 1 hour at 4°C (as in panel B). This can be achieved either by: Both conditions resulting in significant dissociation of the dimer into monomers. Ink at high temperatures vasion is said to further denature the MHC class I chain, so low pH treatment is recommended. was used as the method for monomer concentration. Purified by electroelution method The individual strands are free from any cross-contamination. In some cases, different bags Strand preparations from Ti contain 5-15% homodimer. As expected, homo The dimer site is mobile, close to the position of the α/β heterodimer. Moving. The high molecular weight band at approximately 60 kD in the native gel is either a chain The strand preparations shown in panel C were used to ensure that they were not a source of cross-contamination. Analysis was performed by 5OS-PAGE after reduction with 2-41H as shown. gel Silver staining showed no detectable cross-contaminating alpha or beta cytoplasms. . The relative amount of homodimer present in the chain preparation is calculated for both α and β chains. was quantified to scan the native gel lanes. As shown in panels D and E. The α and β polypeptide preparations used in this study were each 8° Contains 6% and 12.9% homodimers.

Binding of the peptide to the purified α and β chains of the &IBP(1-14)A′ peptide [At pH 8.0, where Ha is reported to bind to A' It was done at a later date. For both peptide assays, as shown in Figures 10A and 108. 30-34% of both α and β chains were occupied by this peptide. . The α/β heterodimer isolated under the same conditions was MBP(1-14)A′ It showed 25% occupancy by peptide.

The specificity of binding of the MBP(1-14)A' peptide to the individual chains of [Ak is together with the α and β chains of IA’, each of which binds only 2-3% of the peptide. [+251]-labeled ovalbumin peptide 0VA(323-340)Y ” was shown by incubating 0.

Stability of single chain:peptide complexes at 37 °C in PBS with 1% OG is represented in Figure It. β chain/MBP (1-1 4) Incubation of A' complex leads to 40% dissociation of L1.

In contrast, only 20% dissociation of the α-chain:&IBP(1-14)A' complex and [5% dissociation of the tBP(1-14)A' complex at 37 °C. Observed over a 24 hour period. IBP(1-14)A' to α and β:M The dissociation observed for BP(+-14)A' is clearly two-step (biph asic). In the initiation phase, β:&IBP(1-14)A' complex has a higher dissociation rate (k.

=6.2 x 10-@5ec-'). However, the second of dissociation For the phase, α/β^IBP(1-14)A' and α・^+BP(1-14 ) The slope of the A' complex is 5 x 10-' sec, respectively. -' and 7×10-'sec-', the values are almost the same. β:M The slope of the second phase of the BP(1-14)A' complex is 6.2 x 10-' 5e c-' had a value.

Kageichi This example shows that single-chain complexes of isolated α and β chains with antigenic peptides are This shows that it can be combined and stimulated.

Radiolabeled peptide and vermilion? ! Using a complex with Ji chain, chain/peptide complex and the direct binding of cloned T cells to the complex molecules that associate with the cells. The number of was calculated. T cell clone 4R3,9 and 9 IB within the context of IAk The one that recognizes P(+-14) to [+251]=IBP(1-14)A' Incubated with purified single chain complex containing peptide. T cells to detergent For cell binding and stimulation assays, detergent-free, low-resolution Dialyzed complexes against filtered RPMI 1640 medium or PBS buffer. ,used. The results presented in FIG. 12A show that IBP(1-14) A' Binding of the complex to 4R3,9 clone T cells or intact IAk:MBP(1- 14) Comparable to that of the A' complex. However, the β chain The ^IBP(+-14)A' complex has a lower binding affinity to 4R3,9T cells. demonstrated the integrity. The association of the lC class I+ peptide complex with T cells is 37 The number of complex molecules associated with T-cell carcinomas completed by 6 hours at °C. calculated, and IA″ 8tBP(1-14)A′ complex, α-chain BP(1 -14) Regarding A' complex and β chain, 8 IBP (1-14) A' complex, 2.55 x 10', 2゜I x 10', 1.0 x 1 respectively 0' was observed. The number of complex molecules bound to T cell cancer higher than the reported number of TCRs on the surface, 2.5-5. OX 10'~l x 10', which is due to the level of aggregation of the complex in the absence of detergent. I was able to do it. The specificity of the chain-peptide complex that binds to T cells is determined by the α and IA ' and MBP (90-103) complex treated with restriction enzymes. Confirmed by incubation with cells.

As shown in Figure 12B, IAk and λIBP(1-1 4) preferential binding to restriction enzyme-treated 4R3,9T cells for the peptide; Observations were made in comparison with HS-IT cells. In the positive control experiment, IA"・Yoke I Incubation of the BP(89-101)Y"" complex was carried out using the HS-1 clone. showed the expected strong binding to human T cells. Chain-peptide to 4R3,9T cells Binding of the complex was also observed in cells pretreated with α/β anti-TCR monoclonal antibodies. However, in an antibody blocking experiment, the antibody was unable to bind to the complex (Fig. 12C). confirmed. cells preincubated with isotype-matched antibodies under the same conditions. The cells showed no inhibition of binding.

Isolated chains, peptide complexes trigger T cell responses in vitro. To determine whether or not to use ligand-mediated cell triggering (tri using a microfinometer to measure the extracellular acidification rate of used.

Measurement of T cell metabolic acidification rate Details of loading T cells into a microphysiometer for acidification rate measurements. as described in Owlcki et al., supra.

Simply put, the cells are rested from the antigen pulse for 10 days and their salts are Culture overnight in low serum (2.5%) medium to reduce basal metabolic activity. cell were harvested and added to serum-incompatible loading medium (10mM HEPES buffer, pH 7). IX 10' cells/ml in low-buffered RP9111640) containing and resuspend. Extracellular acidification measurements were performed in a microphysiometer. Collect 1 minute potentiometer readings every 2.5 minutes. Acidification rate data (bottom) (7 seconds) was mathematically normalized to 100% prior to cell stimulation. This is different allows comparison of data from cells in different chambers.

A specific T cell response within minutes of the interaction of the complex with TCP occurs at resting 4R3,9 observed when T cells were exposed to single-chain, peptide conjugates. acidification rate A rapid, sustained increase in Characteristic interactions between the body and T cells are shown in Figure 13A for 4R3,9T cells. This was observed when exposed to the α chain~MBP(+-14)A' complex.

The complex of β-chain/MBP(+-14)A' peptide also appeared in 4R3,9 cells. induces an increased rate of acidification or is not as strong as, but slightly better than, the α-chain complex. He had the ability to The intact IA':MBP(1-14)A' complex has the highest level of T cell stimulation. This becomes clearer after about 2 hours of complex treatment. It became clear. [A” chain: The specificity of T cell activation of the peptide complex is shown in Figure 13. An equimolar mixture of α and β was used to treat T cells as shown in A and 13B. evidenced by the lack of increased acidification rate when used. for some experiments However, α and β chains without peptides increase the rate of acidification on medium alone. was not given at all. In the same set of experiments (Fig. 13B), several non-specific Heterogeneous^IHc complex, IA″2^+BP(89-1Of)YII, ]IAα chain: 9 IBP (89-101) Y'', and IA'β chain/MBP (89-101) Y''1 did not induce a significant increase in acidification rate.

Conclusion: In summary, the results presented here demonstrate that isolated chains of MHC class II molecules are and the isolated chains of MHC class 11 molecules and antigenic peptides. The complex with Tide is ^11 (C class [1 restriction enzyme-treated TCRs) on T cells. It has been proven that it can be recognized by In the case of T cell clone 4R3,9, This binding is similar to that induced by intact heterodimers in specific T cells. Lead to stimulation. This study demonstrated that intact α/β heterodimers on the cell surface represent antigens. This shows that it is not always necessary for

Example 3 This experiment demonstrated that the complex of the present invention was used in a proliferatively unresponsive state in a cloned T cell line. or has been shown to induce clonal anergy. To do this, The dimeric and non-dimeric forms of the individually isolated α and β chains, IA, were complex with BP90-101, and) presence of Is-1 cloned T cells. Incubated in water. The IA"・MBP90-101 complex is derived from murine T cells. specifically recognized by cell clone H3-1. unrelated used as control The complex consists of these two complexes that are not specifically recognized by one particular clone. It was the latter of the combinations.

Cloned T cell lines. The HS-I T cell line clone was transformed into IA'-expressing antigen-representing cells. Restriction enzyme treated with &IBP90-101 provided by 4R3,9 T-cell clones are provided by IA'-expressing antigen-representing cells^IBP rl- 14 and is treated with restriction enzymes. Both clones were exposed to antigen on a 10 day cycle. Subjected to pulse.

Induction of T cell unresponsiveness in inhydro. The remaining antigen-representing cells are transferred to T cell clones. (10 days after antigen pulse) was treated with 2 rounds of 19% metrizamito (met). rizamide) density gradient centrifugation, followed by RPA II 1640 medium. It was removed by washing twice. T cell clone (IX 10 @) the appropriate and unsuitable control complexes, as well as the complexes used in the preparation of the complexes. each for 24 h at 37°C in the presence of a 10-fold molar excess of the corresponding peptide. It was cultured. Additional control conditions were introduced when necessary. Hs-1a cells, 4 times Wash and incubate 8 x 10' T cells with 0.5, 10, or 1; t20 uM In the presence of MBP 9O-lot peptide, 5X10' freshly irradiated SJL/ In triplicate, by J nm1 FB cells and C 20 Ll/ml IL-strapping. , cultured. for the last 8 hours of a 72 hour incubation at 37°C. In between, 1 μCi of 3H-thymidine was added and the extent of proliferation was determined. It was measured by radioactivity.

Results and conclusions: Figure 14 shows the respective related complexes of N3-1 cells containing either dimer. , α-chain, or β-chain [A” component] Explaining. This antigenic proliferative responsiveness spans the range of observed regulatory responses; reduced by at least a factor of 2. Anel with α-chain complex and β-chain complex The G1 response is relatively similar and significantly different from that of the dimeric complex. It wasn't. [N15 is the L-2 responsiveness of these same cells after complex treatment] is depicted. Allergic cells have been reported to be sensitive to [L-2]. , but their proliferative response compared to non-anergized cells It is not reduced to consciousness. Dimeric forms of IA “complex and IA” α-chain complex exposed) (S-16BIIa showed a reduced response, whereas The complexes produced show a [shi-2 response] equivalent to that of the control.

This example demonstrates the ability of single-stranded complexes of the invention to induce anergy in vivo. It's proven. These experiments demonstrate prevention of εAE in SJL/J mice are doing. This experiment is generally performed by Sharma et al., Proc. Natf, Acad, Sci, USA 8811465-[469(1 991) (which is incorporated herein by reference). I went to sea urchin. Briefly, [α and β chains of Ao were prepared preparatively as described above]. It was purified from affinity purified IA' by electroelution method.

EAε is 1.2× as described in Sharma et al. Adoptive transfer of 107MBP (91-103)-reactive T cells transfer). This experiment was prepared as previously described. AIBP91-103 Forms a complex with peptide A” β-chain It was. On days 0.3 and 7, each mouse was treated with 7.5 μg of conjugate in 0.5 ml was received as described in .

The results are shown in FIG. 16 and Table 1.

Table 1 Acute EAE caused by injection of β-chain of [-As molecules bound to MBP p91-103 prevention of Number of paralysis, number of days of onset, average severity β・! 1lBP91-103 0/4 β9 IBPI-142/4 15 1.5 α8 IBP91-103 3/4 2 4 1.25β alone 2/4 18 1.5 Physiological saline 3/4 12 1.5 The results presented below demonstrate that the MHC-peptide complex comprising dimeric Ml (C molecules) This shows that the coalescence exists primarily as aggregates in the absence of detergent.

The IA'-P complex identified by 123I was described in U.S. Pat. , 297. Complex (1,5 mg/mI) was extensively dialyzed against phosphate-buffered saline (PBS) to remove detergent. and 6 ml b, v, 5 ephadex-G200 column (fraction size 5000-600.000). The results presented in Figure 17 show that the aggregation The complex passes through a column with a void volume of 600,000 This indicates that it has a larger molecular weight.

The dialyzed IA' complex (1.5 mg/ml) was also centrifuged and and the pellets were counted. To do this, add 200 μl of the complex (3 00 μg) in 5 ml PBS and +00.000 x g for 60 min. Centrifuged in a fixed angle rotating machine. The results are given in Table 2 below.

Table 2 - Detection experiment of complex aggregation by high speed rotation # Start cpm cpm in beer cpm in supernatant Agglomeration %1 514.576 317.717 205.797 60.682 519.340 321,304 209.108 60.57 The results of both chromatography and centrifugation experiments showed that AIHC-peptide This shows that the complexes are mostly present in aggregated or micelle form. this Their results showed that the single subunit complex of the present invention also did not aggregate in the absence of detergent. This strongly suggests that the molecule exists in a micellar or micellar form.

The present invention has been described in some detail in the above examples for purposes of clarity and understanding. However, certain changes and modifications may be made within the scope of the appended claims. That should be obvious.

FIG IA.

FIG, 2゜ FIG, 4° 1fu 0 koku αfu ζ < eMJ αfu C ro ゛ ko 3 bow 8 chance 5 6 wa sa 8 ηGo"arez w S"'; Φ　<<　--L)　<t, Dω　e)　al　　-　-　50　-　-　5　O F o)-CD <1- ← ←U)-CD)-(to OU Q U)-U(C) Hi ← 8 Effect White?　8　　Bi　　 ←　HCJ　(1-←　＜01- 8　Cao　　　8　冨　≦.　○ He <<< ← L) uuu (D　o　・　uo(g　已〕已 8 Tomi 8ah<< Φ　Q　　-0←　Φ　　-C)　＜ Meaning a = 1? Details HP IJ QOΦOΦ<) +5 Q (J j::::,,g,constant ■ Mi I eyes 1 Ql-(51-(hi 已　已　8 Ku 0) − δ constant 8 Ku 0 01-< <　(J　　)−(ku　(Q　(U 888 Blasphemy is <B> Number 88 Details 0l-CD<Tohi H(J() <5 CJ (J U g (′ U ″ is (. 0 8 effect ≦ 8 blasphemy (+uu Blind person 8 宕 b 193 Cao <HC,)Ql- １　■　−〇　■～　−　−〇 ? ”” (yN’l’ −～ Q To (Q (○ To Φ B(,)QU( oM pull. ← 0 1- (C) C) C KU　O←　L)、! -1-U CJ ω U d) − (J (ri L) < ((1 −″ 0 desecration b 3 B)-Q ? Ku . Ku Ku ←. Q Ku ro ← Ku Q Φ Q Ku 0←Φ0otpou (()-CD　O)-←　0 (1-C, +1-)-□L) 0 Φ Q C) 1-Φ O sh＜＜＜　Q　←　O ! ==1　舊　■3 Ku Ro Ku Q Hi Ku Ro Ku Q ″″　　　　　　to　. QΦ 1-U　C:)＜v　← Q UO) - (○ ku Φ Φ 00 0 (1: Base attack Toku 0 0 CJcpl-cpOU Lotto Do ← Ku Ku ○ Ku ← 已 0 0 S l-CD L) ← t-o　Q　<D　0　0 0 (shi 8 years old 3 8 = HO Ku　く　0　←　O已　　8　.

U Q U ← Bi.　u<　.

(L) <C) L) ((○　○　○　Hi　Oto Cr-00Φ B:ou+++ Ku a<a<LD CD ← Kuhi ← (<) t) tD < CD O Hi ← Ql-1-Ro CJ L:l) -(Jo Oku O H()-1-ri day. 0 0 0 0 Q o) - Q Φ Ku Φ ro ro ro ku) - 0ku 8　　　8　　00 Eye ■ l 1 Ku 8888 effect 8 fixed bi Ku　ku　○　ku　Φ　Q　Φ　− Do Tomi J Corpse 8 = 3 is H←u((C,)(Q o 0 ro oou ≦15 Blasphemy? Fixed wealth 8 8 blasphemy 000 Hee hee L) I-u < I-.

・ ・ ・ Ku ro ← Φ Uji Uji 8 Tomi 83 is Toki 〈　Φ　(CIO(J Q Hi 0 Ku Ro Q 0 0 =　=　　8　　　　　8　　8　　　　　　8　 OQΦΦU　U　C) ? Roto ← To ≦ 8 3 () CJ CD○O U (U is 8 fixed 8 yi 5 sudden Q (tD (Φ −■　−〇　−■ −? ■ Mouth 0) Feather δ -1-〜　-廿nω　-■　9=mortar mouth! Ritogi Sagi’j: :F! r2' 2 Gigi 3C・N～! 2B! ,. . εB -2 F/(38° FIG, 9A, FIG, 9B.

FIG, IOA, FIG 1011FI6.　1211゜ FIG. 13A.

FIG 1.5θ FIG, 15A FIG, l513゜ LJ/ml IL2 FIG 15C number of days FIG./6 F/, /7: Continuation of front page (51) Int, C1,6 identification code Internal office reference number C07K 14/74 8318-4HGOIN 33153 V 8310-2J331564 Z 9015-2J (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, SE), 0A (BF, BJ, CF, CG, CI, (Ji, GA, GN, ML, MR , SN, TD, TG), AT, AU, BB, BG, BR, CA, CH, C5゜DE, DK, ES, FI, GB, HU, JP, KP, KR, LK , LU, MG, MN, MW, NL, No, PL, RO, RU, S D.S.E. FI (72) Inventor Sharma, Tumesh D.

Studio Art, Os Altos, California 94022, United States Coat Vera (72) Inventor Matsu Cornell, Harden M.

United States, California 94305゜Each Tanford, El Esca -pad

Claims (40)

[Claims] 1. Contains an antigenic peptide and an isolated NHC subunit component with an antigen binding site. a substantially pure MHC-peptide complex consisting of associates with its antigen-binding site and causes its MHC-peptide complex to become a disease-associated T cell. An MHC-peptide complex that selectively binds to T cell receptors on cells. 2. Effector components that bind to antigenic peptides or NHC subunit components The composite of claim 1, further comprising. 3. 3. The conjugate of claim 2, wherein the effector component is a toxin. 4. 3. The conjugate of claim 2, wherein the effector component is a label. 5. Claim 1, wherein the peptide is non-covalently associated with the antigen binding site. complex. 6. Epitopes on Bebutide are recognized by T cells associated with autoimmune diseases , the composite according to claim 1. 7. The complex according to claim 1, wherein the MHHC subunit component is a class II alpha chain. body. 8. The complex according to claim 1, wherein the MHC subunit component is a class II β chain. . 9. a pharmaceutically acceptable carrier, and antigenic peptides and antigen binding sites; an isolated NHC-peptide complex comprising isolated MHC subunit components; the antigenic peptide associates with the antigen binding site and the MHC-peptide The complex binds to T-cell receptors on disease-associated T cells. , a pharmaceutical composition comprising. 10. Claim wherein the MHC-peptide complex further comprises an effector component. 9. The pharmaceutical composition according to 9. 11. 10. The complex according to claim 9, wherein the complex is incorporated into ribosomes or micelles. Pharmaceutical composition. 12. 10. The concentration of the complex is between about 0.02% and about 1% by weight. Pharmaceutical compositions as described. 13. Claims where the pharmaceutically acceptable carrier is phosphate buffered saline Pharmaceutical composition according to item 9. 14. A method of inducing anergy in a target T cell in a mammal, the method comprising: A complex capable of binding to a target T cell, the complex containing an antigenic peptide. and an isolated MHC subunit component having an antigen-binding site, and its antigenicity A therapeutically effective dose of the conjugate in which the peptide is associated with its antigen binding site , administering to the mammal. 15. 15. The method of claim 14, wherein the MHC component is a class II alpha chain. 16. 15. The method of claim 14, wherein the MHC component is a class II beta chain. 17. 15. The complex is incorporated into ribosomes or micelles. the method of. 18. 15. The method of claim 14, wherein the T cells are associated with an autoimmune disease. 19. A method for removing target T cells in mammals, the method comprising: A complex that can combine with an antigenic peptide and an antigen-binding site. an isolated MHC subunit component with a Therapeutically effective administration of complexes in which an antigenic peptide is associated with its antigen binding site A method comprising administering a dose to the mammal. 20. 20. The method of claim 19, wherein the MHC component is a class II alpha chain. 21. 20. The method of claim 19, wherein the MHC component is a class II beta chain. 22. 20. The method of claim 19, wherein the effector component is a toxin. 23. 23. The method of claim 22, wherein the toxin is associated with an antigenic peptide. 24. The toxins are adriamyin, mycophenolate ( mycophenolate) or 131. 25. 20. The complex is embedded in ribosomes or micelles. the method of. 26. 20. The method of claim 19, wherein the T cells are associated with an autoimmune disease. 27. A method of treating an autoimmune disease in a mammal, the method comprising: an isolated MHC subunit with an autoantigenic peptide and an antigen binding site. A purified MHC-peptide complex comprising A pharmaceutical composition comprising a complex in which a progenic peptide is associated with its antigen binding site. administering to the mammal a therapeutically effective dose of. 28. 28. The method of claim 27, wherein the MHC-peptide complex further comprises a toxin. 29. 28. The method of claim 27, wherein the pharmaceutical composition is administered intravenously. 30. The effective dose is about 3 mg MHC-peptide complex per kg body weight and 30. Between about 15 mg MHC-peptide complex per g. Method. 31. Autoimmune disease is rheumatoid arthritis, multiple sclerosis, or myasthenia gravis , the method of claim 27. 32. A method for diagnosing an autoimmune disease in a mammal, the method comprising: a single carrier containing a labeling moiety, an autoantigenic peptide, and an antigen-binding site. A purified MHC-bebutide complex comprising isolated MHC subunit components. contains a complex in which the autoantigenic peptide is associated with its antigen-binding site. administering a diagnostically effective dosage of a pharmaceutical composition comprising How to do it. 33. 33. The method of claim 32, wherein the labeling moiety is a radioisotope. 34. Antigenic bebutides and isolated MHC subunit components with antigen binding sites. A method for producing a complex comprising: MHC glycoprotein from a glycoprotein-producing cell. Isolate: isolate MHC subunit components from the MHC glycoprotein: isolate the MHC The subunit components are linked to the peptide in such a way that the peptide is bound to its antigen binding site. peptide, and the excess peptide not bound to its antigen binding site. a method comprising: removing the code; 35. 35. The step of removing excess peptide is performed by dialysis. the method of. 36. The step is to dialyze the complex in the presence of lipids to form ribosomes. 35. The method of claim 34, further comprising: 37. Claim 34, wherein the peptide is non-covalently bound to the antigen binding site. Method described. 38. A claim in which the MHC subunit component is a class II α chain or a class II β chain The method according to claim 34. 39. 35. The peptide of claim 34, wherein the peptide comprises amino acids 1-14 of MBP. Method. 40. Claim further comprising the step of attaching an effector component to the complex. 34.