JPH11510375A - Recombinant antibody from phage display library directed to peptide-MHC complex - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for producing an antibody or an antibody fragment that specifically recognizes a peptide-MHC complex. It also relates to the antibodies and antibody fragments of the present invention conjugated to a drug or superantigen. Furthermore, the present invention also relates to a pharmaceutical composition comprising the antibody or antibody fragment according to the present invention for preventing or treating infectious diseases and autoimmune diseases and cancer, and a composition for diagnosing the disease. .

Description

DETAILED DESCRIPTION OF THE INVENTION                       Directs peptide-MHC complex             Recombinant antibody from phage display library Field of the invention   The specific recognition of the peptide-MHC complex by the T cell receptor is responsible for the specific immune response. Important reaction. Thus, antibodies to the peptide-MHC complex May be a useful tool for studying T cell function and T cell recognition Could lead to new approaches in diagnosis and immunotherapy. However, Antibodies or antibody fragments with T cell specificity by upcoming hybridoma technology Has proven difficult to produce. The invention is antigen-specific and Recombinant antibodies having specificity limited by MHC possessed by T cells and cleavage of antibodies About a piece.Background of the Invention   T cells and B cells are two fundamentally different modes of recognition in the specific immune system To represent. T cells reside on the surface of antigen-presenting cells during the alternation selection process Presented with a major histocompatibility complex (MHC) Gain the ability to recognize the original peptide. In contrast, B cells are restricted to their own MHC B cell receptor (antibody) is soluble or membrane bound Recognize the three-dimensional structure of the target structure, regardless of its form.   The apparent difference in the mode of capacity acquisition of B cells and T cells indicates that T cells possess M Antibodies with HC-specific specificity are rare and rarely occur in conventional B-cell Theory of why it was difficult to produce such antibodies using bridoma technology It will be clear. Report of producing antibody restricted to own MHC by conventional means Are only slightly sporadic (Wylie et al., 1982; van Leeuwen et al., 1979) Froscher et al., 1986), other reporters report that their attempt failed. (Tamminen et al., 1987; Rubin et al., 1989). More recently, a few anti-peptide-MH The C antibody has been reported (Murphy et al., 1989; Duc et al., 1993; Aharoni et al., 1991). The molecular basis of the difficulties so far is the recently elucidated peptide-MHC class I complex. It appears to be shown in the structure (Fremont et al., 1992; Madden et al., 1993; Young Et al., 1994 ). The peptide is deeply embedded inside the MHC, and the extension of the peptide residue It is presented as a mosaic mixed with its own MHC residues (see above). Surprisingly, peptides bound to MHC class I are also 100-300Å^TwoBelow but outside Facing the table and can therefore be used for direct recognition (Fremont et al., 199 2; Young et al., 1994). Autologous MHC is a peptide-MHC complex presented to T cells It is thought to occupy most of the body surface (Fremont et al., 1992; Davis et al., 1988). . An antibody that recognizes a protein molecule is about 800Å of its ligand^Two(Davis Et al., 1990), peptide-MHC complex also recognizes antibodies by its own MHC Things need to dominate. In addition, antibodies are said to be restricted to their own MHC The specificity of such antibodies is rather eliminated or suppressed. Being silenced (Nemazee et al., 1989) is specific for the peptide-MHC complex This explains why it is difficult to prepare antibodies. T cells are dominated by their own MHC Faced with the same problem of recognizing ligands, empowering T cells For the whole body organs are provided. The positive and negative selection process The majority of mature thymocytes are removed and non-self-reactive, but by their own MHC Only a small fraction of those that enter the circulation as restricted mature T cells (v o Boehmer et al., 1989).   When faced with the problem of isolating rare antibody specificities, phage display technology High selection of surgery (McCafferty et al., 1990; Clackson et al., 1991; Barbas III et al. 1991; Winter and Milstein, review in 1991, Winter et al., 1994) are particularly useful. It is for. This technology is a library that has the desired specificity abundantly through immunization procedures. Leeches, or have a very large variety and size, Any of the libraries considered to have antibody specificity for the epitope (Griffiths et al., 1994). Whether the chosen immunization protocol worked Germline with an affinity in the μM range in the initial immune response Antibodies from variable genes account for the majority, followed by increased somatic hypermutation It is important to note that results in an affinity in the nM range (Fo ote and Milstein, 1991). Similarly, isolated from immunization-based libraries The affinity of selected phage display antibody fragments generally indicates that the unimmunized Ibra Higher than the affinity of those isolated from B. Lee (Marks et al., 1991; Bye et al., 1992; Ho ogenboom and Winter, 1992). WO 95/15982, Phage Dis Using prey technology, immunologically recessive epitopes )).   In WO 91/12332 (Kourilsky et al.), A virulence antigen or Contains peptides characteristic of cytotoxicity and major histocompatibility complex (MHC) molecules A limited monoclonal antibody characterized by the ability to specifically recognize the complex. Thus, the above-mentioned peptide was bound to the non-peptide-specific haplotype MHC molecule. Antibodies that are limited by their inability to recognize You.   In DE 42 24 542 A1 (Hammerling), (a) a single histocompatibility class I molecule (B) the gene encoding the major histocompatibility molecule to express the gene. Mouse genome, (c) binding of major histocompatibility class I molecules to peptides, (d) Immunization of transformed mice with the combined molecule, (e) immunized mice Isolation of the splenocytes and (f) a major set of peptides bound by conventional means Includes the production of monoclonal antibodies against the tissue-matched gene complex class I molecules, Monoc directed to major histocompatibility complex class I molecules with peptide antigen binding. Methods by which lonal antibodies can be produced are outlined.   WO 91/12332 and DE 42 24 542 A1 are both recombinant means. Therefore, it is considered that restricted antibodies can be produced, but the disclosure content Is by inference and vague, and suggests that one of skill in the art would produce such antibodies. It does not make it possible.Summary of the Invention   The present invention provides certain pre-determined by the use of phage display technology. For producing antibodies and antibody fragments that recognize the modified peptide-MHC complex Provide the law. The speed and feasibility of this technology is due to MHC-limited T cell recognition Is useful for the study of cancer and leads to new approaches in diagnosis and immunotherapy. Production of antibodies and antibody fragments against various specific peptide-MHC complexes Be pragmatic. In addition, the present invention relates to antigen-specific and MHC-restricted recombinants. And the antibody itself.Detailed description of the invention   One important aspect of the present invention takes advantage of the high selectivity of phage display technology. Using a recombinant that recognizes a predetermined peptide-MHC complex This is a method for producing an antibody or an antibody fragment.   In its broadest aspect, the invention specifically recognizes peptide-MHC complexes A method for producing an antibody or antibody fragment, (a) a file in which at least one nucleic acid fragment encoding the antibody or antibody fragment is expressed Provision of a library, (b) producing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex, At least one fragment encoded by at least one nucleic acid fragment expressed by Selection of images, and (c) at least one nucleic acid encoding said antibody or antibody fragment of said phage Introduction of the fragment into cells, and the above peptide-M as an expression product by the cells Including producing the above antibody or antibody fragment that specifically recognizes the HC complex About the method.   The term "nucleic acid" occurs as either DNA or RNA and is single- or double-stranded. A high molecular weight polynucleotide which may be any of Nucleic acid fragments Is a "teraphage" library, or a method described in detail below. Can be obtained from any of the antibody libraries from animals immunized with Wear.   A teraphage library contains a large number of human antibody gene fragments, May have a gene fragment encoding the desired antibody or antibody fragment Means a library that is considered expensive. Such a very large library Lee's examples are described, for example, in Waterhouse, P.G. et al., 1993, Griffith. (Griffiths, A.) et al., 1994. This method is applied to the patient's body. Has one or more antibodies specifically reactive with the MHC-peptide complex present If treatment is planned for an individual patient, Directs a preselected MHC-peptide complex thought to be unique to the individual This is very useful because a specific antibody can be produced.   Methods for producing antibodies or antibody fragments that specifically recognize peptide-MHC complexes The currently preferred embodiment includes the following steps: (a) isolating RNA from an animal immunized with the peptide-MHC complex, (b) converting the RNA isolated in step (a) into a nucleic acid encoding an antibody or an antibody fragment; Antibody or antibody cleavage by using as a starting material in the amplified reaction Amplifying at least one nucleic acid fragment encoding the fragment and expressing said nucleic acid fragment Creating a phage library to perform, (c) specifically recognizing the peptide-MHC complex, at least one of the amplified At least one antibody producing antibody or antibody fragment encoded by the nucleic acid fragment Selecting phage; and (d) at least one of said phage encoding said antibody or antibody fragment The introduction of the nucleic acid fragment into cells, and the expression of Producing the above antibody or antibody fragment that specifically recognizes the peptide-MHC complex Floor.   This approach is described in detail in the examples for Fab fragments. In this application PSAN13.4.1 described in detail, but the number of genes is about 10⁷Relatively small in size with the seed But from a highly selective library with an affinity in the nM range. The fact is that the mice used for immunization produce high affinity antibodies. Ha_255-262-K^kThis strongly indicates that it actually reacted with the complex. Therefore, The immunization protocol chosen may be an important factor in the production of Fab 14.3.1 Very high.   One skilled in the art will recognize techniques similar to those outlined in the Examples, or Waterhouse ( Waterhouse, G.P.) et al. It is clear that the method can be used for other antibody fragments or whole antibodies. It is white. Use of phage display technology is preferred, but single-chain Fv antibody fragment display Alternative methods such as plasmid (Cull et al., 1992) or cells (Francisco et al., 1) 993) to prepare a recombinant peptide-MHC specific antibody by using It is thought that we can do it.   As used herein and in the claims, an antibody has a binding specificity for a particular antigen. Is a glycoprotein. Antibodies are generally heterotetramers with a molecular weight of about 150,000 daltons A glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains . Each light chain is linked to one heavy chain by a single covalent disulfide bond However, the number of disulfide bonds between heavy chains depends on the immunoglobulin isotype. Variously different. In addition, each heavy and light chain is the same regularly arranged It also has intra-chain disulfide bridges. Each heavy chain has at one end a variable region (V_H) Followed by a long constant region. Each light chain can be at one end Variable area (V_L) With a constant region at the other end. The constant region of this light chain Is aligned with the first constant region of the heavy chain, and the variable region of the light chain is It is aligned with the area. Certain amino acid residues remain between the light and heavy chain variable regions. The groups are believed to form a link.   The term "variable" refers to the fact that the sequence of certain portions of the variable Means the binding and specificity of each particular antibody to its particular antigen. Used as Variability is the complementarity present in both the light and heavy chain variable regions. It is concentrated in three areas called the decision regions (CDRs) or hypervariable regions. The relatively highly conserved portion of the variable region is called the framework (FR). So In each chain, the individual CDRs are located close to each other via the FR region, Contributes to the formation of the antigen-binding site of the antibody together with the CDRs of other chains (see Kabat et al., 1987). See). The constant region is not involved directly in binding an antibody to an antigen, but is antibody dependent. Shows a variety of effector functions, including the involvement of antibodies in cytotoxicity.   When antibodies are subjected to papain digestion, Fab Fabs each have a single antigen-binding site. Two identical antigen-binding fragments, called fragments, and easily as residues. The "Fc" fragment, named for its ability to crystallize, is obtained. Also, Pepsi When treated, has two antigen-binding sites and retains the ability to crosslink with antigen F (ab ')_TwoA fragment is obtained. "Fv" represents a complete antigen recognition and binding site Includes the smallest antibody fragment. This region contains one heavy and one light chain variable region. It consists of dimers tightly associated by non-covalent bonds. Three of each variable region CDRs interact with V_H-V_LWhat defines the antigen binding site on the surface of the dimer Exactly according to this configuration. By assembling the above six CDRs, Binding specificity Is given to the antibody. However, a single variable region (or three CDRs specific for an antigen) Only half of the Fv) or scFv also has lower affinity than the entire binding site However, it has the ability to recognize and bind to antigens. Fab fragment is also light chain It contains the constant region and the first constant region of the heavy chain (CH1). Fab 'fragment is a heavy chain CH1 domain At the carboxy terminus of the antibody, one or more cysteines from the antibody hinge region This differs from the Fab fragment in that a few residues including a residue are added. F (ab ')_TwoAnti Body fragments are initially paired with Fab 'fragments with a cysteine in the hinge region in the middle. It happened.   The “light chain” of antibodies from all vertebrate species is based on the constant region amino acid sequence. Based on two distinctly different types called kappa (κ) and lambda (λ) Can be applied to any one. Antibodies have amino acids in the constant region of their heavy chains. Depending on the acid sequence, it can be assigned to any of the different classes, including IgA, I There are five major classes: gD, IgE, IgG and IgM. Some of these are IgG Subclasses such as -1, IgG-2, IgG-3 and IgG-4, IgA-1 and IgA-2 (Ip).   The term “antibodies and antibody fragments” is the most broad term used herein and in the claims. Single monoclonal antibodies, and their use as peptide-MHC Fragments of such antibodies (eg, Fab, F ab ', F (ab')_Two, Fv and scFv). Therefore, the present invention relates to an antibody Fragment is Fab, Fab ', F (ab')_Two, Fv, scFv and other antigen binding sequences of antibodies A method according to the invention selected from the group consisting of: Also, IgM or IgG complex Complexes of the antibody or antibody fragment of the present invention, such as, are also included in the scope of the present invention.   As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody. Populations, i.e., naturally occurring variants may be present in small amounts Except that the individual antibodies that make up the population were obtained from the same population. Antibody. Monoclonal antibodies are highly specific, having a single antigenic site Be oriented. In addition, each monoclonal antibody binds a single determinant on the antigen Be oriented.   The monoclonal antibodies herein are, in particular, one of heavy and / or light chains. The antibody is from a specific species, or the class or subclass of the specific antibody Identical or homologous to the corresponding sequence in an antibody belonging to the rest of the chain Is an antibody from another species, or the class or subclass of another antibody. An antibody belonging to the class of Las, or such an antibody that exhibits a desired biological activity. "Chimeric" antibodies that are identical or homologous to the corresponding sequence in a fragment of the antibody including.   A “humanized” form of a non-human (eg, mouse) antibody is one that is derived from a non-human antibody. Chimeric antibodies containing small sequences or fragments thereof (Fv, Fab, Fab ', F (ab')_TwoOr sc Other antigen-binding sequences of antibodies, such as Fv). Mostly humanized antibodies Indicates that the residues in the complementarity determining region (CDR) of the recipient have the desired specificity, affinity and potency. Residues from CDRs of non-human species such as mice, rats or rabbits with (Recipient antibody). In some cases, The Fv framework residues of the human antibody are replaced by corresponding non-human residues. In addition, humanized antibodies can include recipient antibodies and introduced CDRs or frameworks. May contain residues not found in any of the amino acid sequences. Such modifications can Added to further improve or optimize the performance of Generally, humanized An antibody binds to all or substantially all of its CDR regions with the CDR regions of a non-human antibody. In response, all or substantially all of its FR regions are consensus Substantially at least one, and typically two, variable regions, such as the FR regions of a row. Including all. Optimally, a humanized antibody has at least one of the constant regions (Fc) of the antibody. Includes two parts. For more details, see Winter and Mill Recognized by Milstein, 1991, or mouse monoclonal antibody L243 A humanized antibody having specificity for a recognized epitope and a method for producing the same. See WO 94/29451, cited above.   Antibodies or antibody fragments that specifically recognize peptide-MHC complexes If produced by one of ordinary skill in the art, for example, the In addition to the original binding sequence and the FR region, e.g., the consensus sequence of a human antibody, Such as a "humanized" antibody, optionally comprising at least a portion of the constant region of the antibody. It is envisioned that suitable analogs of the antibodies or antibody fragments described can be prepared. Changes are possible, as evident from the above overview of "humanized" antibody production technology. There are many different types of antibodies, and those skilled in the art can make appropriate selections depending on the purpose of a particular antibody. It seems that you can do it.   Human major histocompatibility complex (MHC) is also named human leukocyte antigen (HLA) system First using maternal antiserum to identify paternal transplant antigens expressed in offspring The features were examined. HLA typing initially involves organ and tissue transplantation, Developed to facilitate planting. Alleles include HLA class I alleles And HLA class II alleles. Most frequently expressed The HLA gene is one of the most polymorphic loci in the genome, It is concentrated at the peptide binding site.   Of the 15 class I genes detected, the so-called classic HLA class I inheritance Only three HLA-A, HLA-B and HLA-C loci make up the nucleus of the offspring. that's all Are highly polymorphic, expressed in virtually all nucleated cells, and have at least HLA-A And HLA-B are known to limit T cell responses to intracellular antigens. Have been. Currently, a total of 125 alleles for HLA-A, -B and -C genes Has been publicized as an HLA-related factor by the WHO Nomenclature Committee. Allowed in the ceremony.   β_TwoTo associate with microglobulin and be transported to the cell surface for expression there HLA class I molecules require the presence of a peptide that binds to that specific HLA allele. I need it. This peptide is provided by a process called antigen processing. It is. All peptides examined so far are single amino acids or closely related `` Anchor sites '' where only a few amino acids with different side chains are detected Including at least two. One such anchor is always at the C-terminal site. Natural ring At the border, the length of this peptide is generally 8-10 residues (Madden, D.R., 1995). .   HLA class II molecules are structurally strongly related to class I molecules. In this connection Contains the domain organization and probably also the antigen binding site. K Ras II molecules are specific cell types that regulate immune responses, especially B cells and activated T cells. Mainly involved in the interaction between dendritic cells and myeloid monocytic lineage cells (macrophages) Involved in. Class II proteins consist of three isotypes, called DR, DQ, and DP. Step It is divided into. There is a strong linkage disequilibrium between DR and DQ, with HLA-B gene This is often observed in some individuals of any ethnic origin. Specific DR alleles in the entire set of DR / DQ / B alleles Not a few. The term "DR haplotype" refers to this typical class II (and Sometimes used to represent a set of class I) alleles. With class II molecules Early data giving insight into the properties of peptides found in the coupled state? Compared to, for example, peptides of 13-17 amino acids that bind to class I molecules, It is suggested that such peptides tend to be longer and unrestricted (Madd en, D.R., 1995).   As used herein and in the claims, the term "peptide" has a reduced length. Short peptides, both 2 and up to 10 amino acid residues, and In addition to containing both gopeptides (11-100 amino acid residues), proteins (at least A functional entity comprising one peptide, oligopeptide or polypeptide Undergo glycosylation or lipidation, or chemical modification to include prosthetic groups. Digits may be used). The definition of peptide is found in the body of animals, including humans Transforms any type of host as well as native forms of peptides / proteins A recombinant protein or peptide contained in any type of expression vector that results in And chemically synthesized peptides.   The term "MHC molecule" is as described above. `` Peptide-MHC complex '' The term has the ability to bind to a particular MHC molecule, thereby Means a peptide capable of forming a de-MHC complex. From the above, the problem Specific peptides and MHC molecules used for the production or selection of antibodies By making appropriate changes with respect to the method of the invention, a number of different It is clear that antibodies can be produced.   The term "specifically recognizes the peptide-MHC complex" refers to the equilibrium dissociation constant K_DIs 10^-7 ~Ten^-TenIn the range of M, the antibody has the ability to bind to the peptide-MHC complex. means. Equilibrium dissociation constant of antibody or fragment thereof produced by the method of the present invention K_DProvides a kinetic binding analysis of pSAN13.4.1, for example, by using surface plasmon resonance. Selected by one skilled in the art, such as the method outlined in Example 3 described in detail. To It can be determined by any suitable method.   An important step in the method of the invention is to specifically recognize the peptide-MHC complex The purpose is to select phage expressing the antibody or antibody fragment. Current favor In a preferred embodiment, this selection comprises cells expressing the peptide-MHC complex. And / or phage Incubate library with beads conjugated with peptide-MHC complex It is implemented by doing. In a more general sense, this choice depends on the antibody or Shares a peptide-MHC complex with a library of phage expressing antibody fragments By panning on an alternating matrix that is listed as a factor, Implemented. In a preferred embodiment, the alternation matrix comprises peptide-M Cells expressing HC complex and beads to which peptide-MHC complex is bound is there. Alternatively, use two different tubes, such as immunotubes (NUNC) or latex beads. Plastic surface coated with the selected MHC / peptide complex It may be used as a trick. In addition, each time a panning is performed, The combined phage is eluted and amplified in cells such as Escherichia coli. F binding to cells expressing MHC molecules in complex with the peptide It is preferred to analyze by ACS.   One important reason this approach can be successful is the extremely large antibody library Antibody that allows efficient and rapid screening of proteins It is believed that the ability to select phage display technology for production exists. further Purified MHC molecules enriched in one particular peptide are immunized and selected Used in order. When using the procedures described in the examples herein, About 80% of the MHC molecules bound to the desired peptide, ie, per mole of MHC molecules It has been calculated that 0.8 mole of the specific peptide binds. This high content is exempt It is also considered an important factor for the success of epidemic and subsequent panning, , The content of MHC molecules is, for example, about 90%, about 95%, or even about 98% or about 99%. I.e., further increase to substantially 100% specific peptide-MHC conjugate It is considered convenient. Immunization step by using teraphage technology When avoiding levels, purified MHC molecules enriched in one particular peptide can be used. To use Is considered to be much more important in the success of the selection procedure. But desired The content of the peptide is as low as about 20%, 30%, 50%, 66% or 75%, for example. It is unlikely that the selected MHC molecules will yield beneficial results.   The method of the present invention further comprises an antibody or a protein that specifically recognizes the desired peptide-MHC complex. Alternatively, to produce the antibody or antibody fragment, the nucleic acid encoding the antibody or antibody fragment Introducing into cells. These cells are microbial cells, including bacteria such as E. coli. Cells derived from multicellular organisms such as organisms, yeast, protozoa, and fungi, insect cells, It is selected from the group consisting of plant cells, mammalian cells or cell lines. Or organization Transgenic activity producing antibody fragments under the control of a specific promoter The DNA encoding the ScFv fragment of the antibody is introduced into embryonic stem cells to produce (Logan, 1993).   Regardless of the type of organism used, the nucleic acid encoding the antibody or antibody fragment The DNA fragment containing the DNA can be either directly or by using a suitable vector. Introduced into the body or cells. Subsequently, cells producing the desired antibody or antibody fragment are produced. Cells are selected based on the level of productivity under conditions appropriate for the vector and cell. Select. The selected cells are further expanded to produce the desired antibody or antibody It is an extremely important and continuous source of fragments.   If appropriate, clone the DNA fragment encoding the antibody or antibody fragment. And its nucleotide sequence can be determined. Then, if necessary One skilled in the art would then be aware of, for example, site-directed mutagenesis or random Have the nucleotide sequence initially determined using conventional techniques such as mutagenesis Antibody with the same specificity as the antibody but with thermodynamic properties (k_a, K_d, K_DDifferent), Nucleic acids encoding novel antibodies or fragments thereof can be prepared. like this The selection of a new antibody is essentially the same as outlined for the original antibody. Alternative procedures must be used. In the same manner, if the selection procedure is modified accordingly Genetically engineered antibodies with binding specificity for another peptide-MHC complex Can also be prepared.   In a preferred embodiment, the present invention relates to a nucleic acid encoding the above antibody fragment, In accordance with the provisions of the Plague Treaty, on June 28, 1995, Les (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (DSM) with plasmid pSAN13.4.1 deposited under accession number DSM10070. How to be loaded.   Thus, one important aspect of the present invention is that of the host organism or cell line. A restricted antibody of the invention contained within an expression vector capable of replicating internally Or a DNA fragment encoding an antibody fragment or an analog or subsequence thereof. Related to This vector is particularly useful for plasmids, phages, cosmids, mini-stains. It can be a chromosome, or a virus. In one interesting aspect of the invention, this Vectors are vectors that, when introduced into a host cell, integrate into the host cell genome. It may be a tar.   In another aspect, the present invention relates to a peptide prepared by the method of the present invention. An antibody or an antibody fragment that specifically recognizes a C-MHC complex. In particular, the present invention A non-glycosylated antibody or antibody that specifically recognizes the peptide-MHC complex Includes antibody fragments. Such an antibody or antibody fragment comprises the antibody or antibody It may be produced when the plasmid encoding the fragment is expressed in E. coli. .   Included within the scope of the invention are antibiotics, cytotoxic agents and antineoplastic tumors. The present invention is combined with a pharmaceutical preparation such as a pharmaceutical preparation selected from the group consisting of preparations. There are antibodies or antibody fragments according to the invention. Alternatively, the antibody or antibody fragment can be Bacterial superantigen staphylococcal enterotoxin with the ability to activate spheres A (SEA) (Dohlstein M. et al., 1994) and the like. Anti Confers superantigenicity on cells recognized by the body, thereby providing Induce T cells to suppress tumor growth or autoimmune disease as described It can also be emitted. Another approach is to use antibodies or antibody fragments to activate complement. Conjugated with a carbohydrate polymer having the ability to To desired cells expressing a peptide-MHC complex recognized by body fragments Targeting.   For some purposes, antibodies specifically recognize a particular peptide-MHC complex One binding site and, for example, an epitope on a cytotoxic T cell or a pharmaceutical Containing another site that specifically recognizes another antigenic epitope, such as a drug Machine A potent antibody would be advantageous.   Bifunctional antibodies are two monoclonal cell lines that produce two related antibodies. Can be produced by chemical bonding of two antibody fragments, or two antibody fragments. it can.   The term "binding site" refers to the antigen recognition structure present in the variable region of the antibody molecule It is interpreted as follows. The use of bifunctional antibodies allows the detection of peptide-MHC complexes in a sample. A pharmaceutical preparation, a biologically active molecule or another antigen, A special technique for targeting to the site where the reagent has the greatest effect Order is possible.   In one advantageous embodiment, the other antigen to which the bifunctional antibody reacts is Effector cells such as cytotoxic T cell differentiation antigens (Staertz et al., 1985; van Ravenswaay-Claasen et al., 1993; see Fanger et al., 1989). Hybrid antibody Pharmaceutical preparations that react with are preferably cytotoxic preparations, antibiotics or antineoplastic Selected from the group consisting of tumor preparations (see Collar and Kaplan, 1984).   Clinically, the use of the antibodies and antibody fragments of the present invention manipulates the T cell response. You can devise new ways to make it. A virus located inside the cell, Bacteria or parasites are generally presented by MHC class I molecules and are more susceptible than antibodies. Recognized by cytotoxic T cells. Present in the form of a complex with MHC class I molecules Antibodies specific for the peptide to be conjugated to the toxin and lost by T cells Used to mimic the response of T cells to remove lost virus-infected cells, etc. It is thought that it is possible. One example of a disease for which such an approach would be useful is HI V. Such antibodies may direct complement lysis to T cell ligands Could suggest another novel approach to immunotherapy.   The idiotypic (antigen-binding) structure of an antibody is antigenic, and Specific antibodies directed to a biotype structure can be generated. Idio tie Antibodies raised against the loop are called anti-idiotypic antibodies. Such anti The body may mimic the structure of the original antigen, and therefore May have the same function as the original. Such antibodies have functions, utilities and properties Has the ability to replace the original antibody in part or in its entirety. Therefore The scope of the present invention also includes anti-idiotype antibodies directed to the antibody fragment of the present invention. included.   Antibodies are antibodies directed to the site of the antibody that reacts with the epitope of the antigen And anti-anti-idiotype antibodies directed against anti-idiotype antibodies. Anti-idiotype antibodies can be prepared by methods well known to those skilled in the art. In addition, anti-idiotype antibodies directed against the above-mentioned anti-idiotype antibodies were also developed. It is included in the range of Ming.   Accordingly, the present invention is directed to a method selected from the group consisting of viruses, bacteria and parasites. Antibodies or antibody fragments according to the present invention for eradication of pathogens present in cells And a pharmaceutically acceptable excipient. The above pharmaceutical group The product is a monoclonal antibody or a monoclonal antibody that specifically recognizes the peptide-MHC complex. Recognize a mixture of noclonal antibodies or different peptide-MHC complexes A mixture of antibodies may be included.   Individuals with a particular HLA genotype may be susceptible to a very wide variety of diseases Has been found to selectively increase. Today, the association with HLA genotypes More than 30 diseases have been identified, and their location in the HLA region is determined by mapping. More than 35-50 types of expressed genes have been identified. HLA class I and class II molecules Binds to antigenic peptides and presents them to CD8 + and CD4 + T cells, respectively It works by: Because of this, certain antigen peptides that do not bind other alleles By binding to the tide, the HLA allele can be It is very likely that the disease has caused susceptibility. For more information on this, see Fu See gger et al., 1995.   Alternatively, anti-peptide-MHC antibodies may lead to inappropriate immunity T leading to autoimmunity Cellular responses may be inhibited (Akahori et al., 1991). MHC has many self-exempt An obvious risk factor in epidemic disease is disease induction restricted to self MHC. Indicates that a single or multiple onset self antigens are present. If that is the case, Antibody fragments directed to a ligand such as It is thought to be a potent inhibitor of response.   Block antibody production requires disease-induced T cell epitopes and restricted MHC Knowledge of such issues, but such requirements will be met in the future (Tisch et al., 1993; Kaufman et al., 1993; Michaeleksson et al., 1994; Wurcherpfennig et al. And Strominger, 1995).   Thus, in a further aspect, the invention relates to an antibody or an antibody according to the invention. Block inappropriate T cell reactions, including fragments and pharmaceutically acceptable excipients To a pharmaceutical composition. In particular, the present invention has a high risk for the development of malignant tumors. Prophylaxis after being established as a member of the risk group, and treatment or recurrence of malignant tumors Antibodies or antibody fragments according to the present invention and pharmaceutically acceptable A pharmaceutical composition comprising the excipient to be added. This pharmaceutical composition is well known to those skilled in the art. It can be formulated by a method.   Another aspect of the invention is a method for treating or preventing recurrence of an autoimmune disease. A pharmaceutical comprising an antibody or antibody fragment according to the invention and a pharmaceutically acceptable excipient Composition.   In a further aspect, the present invention relates to HLA class I-related diseases (ankylosing myelitis, Ster disease, psoriatic spondylitis, idiopathic hemochromatosis, psoriasis vulgaris and Beche Disease and HLA class II-related diseases (rheumatoid arthritis, oligoarticular juvenile joints) Rheumatism, systemic lupus erythematosus, Sjogren's disease, IDDM, Addison's disease, gray Bouzu's disease, Hashimoto's disease, pediatric steatosis, primary biliary cirrhosis, pemphigus vulgaris, acquired Epidermolysis bullosa, Hodgkin's disease, squamous cell carcinoma of the cervix, multiple sclerosis, optic neuritis, null Colepsy, myasthenia gravis, Goodpasture syndrome and alopecia areata) The pharmaceutical composition according to the present invention for preventing or treating a disease selected from the group consisting of Regarding the use of In addition, the present invention provides for wiping by directing complement lysis. Eradicate intracellular pathogens selected from the group consisting of The use of a pharmaceutical composition according to the present invention for   Antibodies directed against specific peptide-MHC conjugates can It can also be used to detect the presence of such a peptide-MHC complex. Pepti The prerequisites for creating a de-MHC complex are that the cells express the MHC in question, And it has the ability to produce the peptide in question. The former is a cell type And whether the cells are being stimulated by various cytokines. one Generally, all somatic cells express MHC class I molecules, but do not express MHC class II. Is a more restricted group of immune cells (dendritic cells, B cells, etc.). Irritation Examples of MHC expression in cells induced by γ-interferon on macrophages MHC class II expression. In the latter, cells use It depends on the amount of protein stored and the cellular machinery for antigen degradation. Generally, antigen processing results in mutated self-proteins and infectious agents. Short peptide fragments from all intracellular proteins, including incoming protein antigens . Thus, MHC molecules are derived from all self and foreign protein antigens Binds to the peptide and presents it.   The normal function of T cells is to attack foreign compounds and ignore self compounds To examine the self-identity of peptide-MHC complexes presented by cells in the body And An antibody capable of recognizing the self-identity of a peptide-MHC complex is It is thought to have the same self-non-self discrimination ability. Such antibodies May have several advantages over T cells, such as: Easier to prepare and label, easier to transport and store, more sensitive Various non-physiological factors that are considered to have high affinity to enable powerful analysis Inspection in the target buffer is possible. Using antibodies specific for peptide-MHC More mutated self-proteins (such as mutated oncogenes) or exogenous infectious agents (Such as a virus) to determine the presence or absence of protein inside or outside the cell Can be detected irrespective of the reason. In particular, antibodies are usually Intracellular antigens that may be spared from detection "" Antibodies.   Thus, peptide-MHC-specific antibodies are mentioned by way of example and not limitation. Then, a tumor cell having a mutated oncogene or an infected cell having a virus Since it can be identified, it has the potential as a diagnostic agent. Its detection is of interest Fluorescence-activated cell selection for detection, quantification, characterization and purification of target cells It can be performed by another device (FACS, Becton Dickinson's FACS Star, etc.) it can. Peptides are also used for large-scale cell sorting and purification of cells of interest. De-MHC specific antibodies can also be attached to magnetic particles. High with high throughput Feeling In order to perform detection, a peptide-MHC specific antibody is labeled (for example, biotin). Using an antigen directly bound to a microtiter plate, Or a solid phase enzyme via another antibody (the latter is known as the sandwich method) A prime immunoassay (ELISA) may be performed.   Of cells from blood or tissue obtained by biopsy (live or fixed) Inspection is also possible. Recently demonstrated that MHC can detach from cells and enter the bloodstream (See Puppo et al., 1995). Soluble MHC at concentrations up to 0.5-25 μg / ml It is reported to be present. This concentration is locally elevated around the site of inflammation I do. Experiments show that such peptide-MHC complexes retain the W6 / 32 epitope (W6 / 32 is an anti-MHC class I monoclonal antibody. TCC HB95). This epitope is β_TwoM for both microglobulin and peptide Depends on binding properly to HC class I. Therefore, such soluble M HC molecules are representative of the peptide-MHC complex, and circulating soluble peptide-M The HC complex carries an overall imprint of intracellular protein metabolism in the body It is possible to conclude. Such specificity simply means taking a blood sample, Evaluation by performing an ELISA using a peptide-MHC specific antibody for detection Can be valued. This is a method that enables the evaluation of intracellular metabolism using blood samples. As such, it is considered to have great diagnostic implications.   Thus, recombinant antibodies that are peptide specific and restricted by MHC, Its solubility, high affinity and stability make it a T cell or recombinant soluble T cell receptor Under conditions not using (eg, immunoprecipitation, immunohistochemistry, etc.) Scientific because it is very suitable for detecting the presence of T cell epitopes Is also considered clinically useful. Therefore, during the antigen presentation process It is also possible to directly address questions about the mechanism and site where events occur. It also allows for the visualization and quantification of T cell epitope expression on antigen-presenting cells. It is thought that. Clinically, antibodies with peptide / MHC specificity are Mutations or intracellular infections usually retained for recognition by antibodies can also be used for antibodies Is considered to be useful in diagnosis. Accordingly To detect specific T cell epitopes by using antibodies (or antibody fragments). Diagnostic The kit allows the use of an antibody having the specificity described in the present invention. Dependent.   The present invention provides the antibody or antibody fragment according to the present invention, an anti-idiotype antibody or Diagnostics for detecting the presence of peptide-MHC complexes containing anti-anti-idiotype antibodies Disclosed compositions are provided. In particular, the invention consists of viruses, bacteria and parasites Diagnostic composition for detecting a pathogen in a cell selected from the group, and malignancy It relates to a diagnostic composition for the detection of a tumor. Various recognized by T cells Human tumor antigens are known, which are useful for the use of the antibodies or antibody fragments of the present invention. Would be a useful target for specific immunotherapy.   The invention also provides a diagnostic composition for detecting the presence of an autoimmune disease, and And individuals were assigned to high-risk groups for developing malignancies or autoimmune diseases The invention also relates to a diagnostic composition for:Description of the drawings Figure 1. Phage selection by biopanning   A library of phage expressing Fab fragments of immunoglobulin_{255-2 62} Panning on alternating matrices with -MHC complex as the only common factor Was. The first and third panning are Ha_255-262RMA-S-K combined with^kReal against cells And the second and fourth pannings were_255-262-K^kCoating with composite This was performed on beads (Interfacial Dynamics, Oregon). Each pa Each time the phage is eluted, the bound phage is eluted and amplified in E. coli.^k Of FAC binding to RMA-S cells bound to peptides by gene transfer Analyzed by S. The peptide is Ha_255-262(Unfilled bar) or NP_50-57(Filled bar). Black arrows indicate the level of the background signal. average Fluorescence intensity = MFI. Figure 2. Purification and binding properties of Fab13.4.1 (A) Purified and reduced Fab 13.4.1 was purified after polyacrylamide-SDS gel electrophoresis. Analyzed by detecting using silver staining method. (B) Purified Fab 13.4.1, Ha_255-262/ K^k(○), NP_50-57/ K^k(●), K^k(△) Or Ha_255-262(▲) for binding to latex beads coated Seki FACS analysis. Purified H-2K bound to endogenous peptide^bInspection of molecules However, no binding to Fab 13.4.1 was observed (results not shown). Suffered The coated latex beads were incubated with various concentrations of Fab 13.4.1. , FACS using FITC-labeled rabbit anti-mouse IgG (DAKO, Denmark) as detection antibody Was analyzed by (C) Competition for binding. Ha_255-262/ K^kLatex beads coated with 3nM Fab 13.4.1, and various concentrations of soluble Ha_255-262/ K^kComplex (○), NP₅₀-₅₇ / K^kComplex (●), K^k(△) or Ha_255-262Latte coated with (▲) After incubation with the beads, FACS analysis was performed in the same manner as in FIG. 2B. . FIG. Results of BIAcore (BIAcore) measurement   Ha_255-262-K^kAnd NP_50-57-K^kComplex, immobilized (A) Fab 13.4.1 or (B) H10 0-27R55 (anti-K^kBIAcore sensorgr (BIAcore sensorgr) am). The conjugate was diluted to a concentration of 500 nM and placed on the immobilized antibody for 3 minutes. (C) Ha_255-262 -K^kKinetic binding curve for binding to Fab 13.4.1. Various concentrations The complex was applied to the association phase for 3 minutes and the dissociation phase for 6 minutes. Actual concentration is a sensor Displayed in the program. All binding curves are displayed as resonance units (RU) over time. Indicated. FIG. Specific suppression of peptide-specific and MHC-restricted T cell responses RMA-S-K^kCells are sub-optimally dosed with Ha_255-262Or NP_50-57At 26 ℃ with one Incubated overnight. RMA-S-K with attached peptide^kCells in different doses In the presence of Fab 13.4.1, Ha_255-262With specific T cell hybridoma Co-cultured. As a control, NP_50-57The maximum dose of specific T-cell hybridoma (390 nM) in the presence (black bar) or absence (white bar) of Fab13.4.1 And co-cultured (insert). The sub-optimal dose used was adjusted to HK8.3-6F8 (●). Ha for presentation_255-262Then 6μM, Ha for presentation to HK8.3-5H3 (●)_255-262 0.3 μM, NP for presentation to HK9.5-24_50-57Then 0.3μM, presentation to HK9.5-162 NP for_50-57Then, it was 3 μM. FIG. Panning results for epitope-specific selection   Purified K^kAnd Ha_255-262(Figure 5A) or NP_50-57(Fig. 5B) , Coated on plastic surfaces as described in Example 1a. This figure is continuous As a result of the panning performed in^k/ Ha_255-262 Fab phage with specificity for the complex (K^k-NP_50-57For complex (Unlike that).   FIG. 5A shows (K^k/ Ha_255-262Individual after 3 pannings (for libraries) 18 of the 20 clones selected for the antigen (K^k/ Ha_255-26220) Shows that this binding was peptide-specific in 16 of the 16 clones. (sand That is, two of the twenty clones have K in a non-peptide specific manner.^k/ Ha_255-262When Joined)   FIG. 5B shows (K^k/ NP_50-57Individually after 3 pannings (for libraries) Of the 20 selected clones, seven were antigen (K_k/ NP_50-57) And these 20 species Indicates that only one of the clones had peptide-specific binding.Example Method: Purification of MHC:   K^kThe production of AKR-derived resources was performed as previously described (Olsen et al., 1994). RDM-4, which is an emphysema cell, was used. About 1 × 10^TenCells in lysis buffer (1% in PBS)  Nonidet P-40, 25 mM iodoacetamide, 1 mM PMSF (SIGMA, Catalog No. P-76 26), resuspend in 100 ml containing 5 mM sodium orthovanadate) and 10 minutes at room temperature For a while. Lysate is cleaned by centrifugation and stored at -80 ° C And thawed by incubating overnight at + 4 ° C before use. To the melt Is a filter by Millipore filter (pore size 8 ~ 0.45μ) -Perform several treatments to^kMonoclonal antibody 11.4.1 (TIB95, American Type C affinity collection (obtained from ATCC) By K^kThe molecule was purified. Affinity columns from manufacturers (Pharmacia) Activated cyanogen bromide Sephadex matrix according to 11.4.1 It was prepared by covalently binding the proteins. After passing the lysate through the column several times , (I) Column containing 0.1% SDS, 0.5% Nonidet P-40 and 0.02% sodium azide 20 times of Column containing volume of PBS, (ii) 0.05% Nonidet P-40 and 0.1% sodium azide 20 times the volume of PBS, and (iii) 20 times the volume of the column containing 0.1% sodium azide. Washed thoroughly with a volume of PBS. Combined K^kMolecule is 5 times the volume of the column (50 ml) Elution buffer (0.05M diethanolamine, 0.15M NaCl, 0.1% sodium azide, Eluted with 0.1% sodium deoxycholate, pH 11) and eluted binder The material is immediately neutralized with 2.5 ml of 2M Tris-HCl, pH 6.3, 0.1% sodium azide solution did. The eluate was collected using a Collodion bag (Satorius, catalog number 13200E). Concentrated by empty dialysis. When the volume of the eluate reaches about 0.5 ml, 1% octyl Add 10 ml of PBS containing glycosides (Sigma) and 0.1% sodium azide, and add Vacuum dialysis was continued until the final volume was 1 ml. K^kProtein concentration of SDS-PAGE and BCA Assessed by assay.   Human β2-microglobulin is obtained from the urine of uremic patients and should be homogenous. Purified by gel filtration and isoelectric focusing (Stryhn et al., 1994). Peptide synthesis:   Influenza virus-derived nucleoprotein peptide (NP_50-57, 1 character code: SD YEGRLI) and the hemagglutinin peptide (Ha255-262 = FESTGNLI) Synthesized manually with a RaMPS synthesizer (Dupon) using the C protection strategy. Preparation of peptide-MHC class I complex:   Affinity-purified MHC class I molecules dissolved in 15 μM detergent were 4 μM NP_50-57Or Ha_255-2625μM human β2-microglobulin with peptide And incubated at 18 ° C for 30-48 hours in the presence of This reaction mixture 1 mM PMSF (Sigma, P-7) in PBS (0.14 M NaCl / 0.01 M sodium phosphate buffer). 626), 8 mM ethylenediaminetetraacetic acid (EDTA) (BDH, 10093), 1.2 mM 1.10 phena Centroline (Sigma, P-9375), 69 μM Pepstatin A (Sigma, P-4625), 128 μM  Na-p-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma, T-7254), 135 μM  Na-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (Sigma, T-4376 ) And 1 mM N-ethylmaleimide (NEM) (Sigma, E-3876). Reaction mixture The final concentration of the surfactant in NP-40 is 0.05%, and almost complete peptide exchange PH was adjusted to 5.5 by adding citrate buffer to this PBS so that Unbound The type of peptide was isolated as previously described (Olsen et al., 1994) by Sephadex. Sephadex G25 span column chromatography. Immobilization of purified MHC-peptide conjugate on latex beads:   The peptide-MHC complex was prepared according to the method described above. Low activator concentration (in PBS Column chromatography with 0.05% Nonidet P-40) did. In a low concentration activator buffer, conjugate was⁷Pieces of sulfurized latex beads (5μ diameter, Interfacial Dynamics Corporation, batch number 436) and mix lightly Incubate overnight at + 4 ° C with shaking. The remaining part of the beads is 1% bovine serum Blocked with albumin PBS solution. After washing the beads once, FACS buffer (1% BSA, PBS, 0.1% sodium azide) Resuspend in 1 ml, panning or FAC Used for any of the S analyses. The bead specimen was stable at + 4 ° C for about one week. Production of MHC-peptide complex on cells:   RMA-S.K^kA cell line (donated by W. Ortiz-Navarette) converts the peptide Peptide transport from the endoplasmic reticulum to the endoplasmic reticulum, where the peptide-MHC complex is formed Impaired processing of MHC class I molecules due to body deletion. High concentration By growing the cells in a medium containing the peptide of^kAlmost all It can be expressed on the surface. Since this effect is a result of the peptide, Part K^kThe molecule is thought to form a complex with the attached peptide on the cell surface You. Therefore, RMA-S.K with peptide^kCells will have the desired peptide on their surface. Expresses only the tide-MHC complex. 10 cells in 5 ml of growth medium containing 0.1 mM peptide⁷ Conjugates on the cell surface by adding Is formed. Cells are harvested by centrifugation, washed once and then in 1 ml of growth medium. Resuspend in Immunization:   Inbred BALB / k mice were given one human unit of BCG (M. tuberculosis bovis, The  Immunization was performed by intraperitoneal administration of the State Serum Institute). Ha_255-262When K formed a complex of^k0.5 mg of 0.1 μM phosphate buffer containing 0.05% Nonidet P-40 1 mg of PPD (supplied by The State Serum Institute) in pH 7.5 Mix to bring the total volume to 0.5 ml. Glutaraldehyde solution of the same volume Liquid (0 .2% glutaraldehyde, 0.1 mM NaHPO_Four, 0.05% Nonidet P-40, pH7.5) And the mixture was placed at 4 ° C. for 2 days with reciprocal shaking. Add PBS 12.3 to this mixture ml, 6 mg / ml Al (OH)_Three Add 6.7 ml, mix well and use for immunization Was. Three weeks after BCG priming, mice were immunized twice with 0.5 ml of the antigen mixture. gave. Immunizations were performed by subcutaneous injection at two week intervals. Second immunization Ten days after the transformation, spleens were collected. Preparation of a phage library expressing an immunoglobulin fusion protein:   Received two immunizations according to the method of Chirgwin et al., 1979 Total RNA was isolated from spleens collected from mice. Super Script Plus (Sup er script Plus) (Gibco BRL) Reverse transcriptase and incubate the mixture for 1 hour at 42 ° C. Oligo-dT was added to the first strand cDNA in the reaction to_{n = 18}Was added. Transcription yeast After heat inactivation of the DNA template, the cDNA template was subjected to ethanol precipitation, and VH, Fd and And used for the primary PCR amplification of the light chain gene. Details of the primers used below Are described in Prum et al., 1993. The PCR reaction was dNTP (0.2 mM), Includes reaction buffer, primers and cDNA supplied by manufacturer (Cetus) Performed in 100 μl of solution. For VH reaction, use MVH1-25 and MJH1-4 primers. Each included 5pmole. MVH1-25, and MCHγ1, γ2A and γ for Fd reactions 6.7 pmole of each of the 2B primers was included. MVK1-25 and MCK Included Limer 20pmole. Mineral oil was placed on top of the reaction mixture and kept at 94 ° C for 2 minutes . Next, Supertaq (HT Biotechnology) or Ampritak ( Amplitaq) (Cetus) 1.5 U was added, and the mixture was subjected to a reaction cycle (94 ° C for 1 minute, 55 ° C For 30 minutes at 72 ° C. for 2 minutes) and then incubated at 72 ° C. for 10 minutes. Gene The primary PCR amplification product can be used in combination with the Clean (GeneClean) (Bio101, Inc) procedure. Was purified by agarose gel electrophoresis. Assembly of VH gene and CH1-LINK-D: 100 μl of the reaction solution was prepared using the above buffer and dNTP, 5 ng of purified Fd gene fragment, 20 ng of purified CH1-LINK-D, FabTAG. BA CK 1-2 (25 pmole each), and FabLINK.FORW. 50pmole included. Fd heredity Assembling of LINK-D with the daughter: 100 μl of the reaction solution was mixed with the above buffer and dNTP and purified 5 ng of Fd gene fragment prepared, 2 ng of purified LINK-D, FabTAG.BACK 1-2 (each Re2 5pmole), and FabLINK.FORW. 50pmole included. Assembly of L-chain gene and LINK-D Chemistry: 100 μl of the reaction solution contains the above buffer and dNTP, and purified light chain gene 5 ng, 2 ng of purified LINK-D, and 5 FabLINK.BACK and FabTAG.FOR. 0pmole included. The heating cycle program starts as described above and 25 cycles ( (2 minutes at 94 ° C, 1 minute at 55 ° C, 2 minutes at 72 ° C) to gel-assemble the aggregated product. Made. Final assembly: buffer and dNTPs described above, and purified VH-CH1-LINK-D fragment 5 100 μl of the reaction solution containing 5 ng or 5 ng of the purified Fd-LINK-D fragment was added to the purified light chain-LI. It was mixed with 5ng of NK-D fragment and 20pmole of ASSEMBLY-1 primer. Heating cycle pro Grams are started as described above and 25 cycles (94 ° C for 1.5 minutes, 69 ° C for 1 minute, (72 ° C. for 2 minutes) to gel purify the assembled product.   DNA fragments from the final assembly reaction were incubated for 2 hours at 37 ° C with 10 U NotI / μg DNA. Digested. After phenol extraction and ethanol precipitation, place the DNA in SfiI buffer. Dissolve and incubate under oil layer at 50 ° C for 2 hours using 10 U of enzyme per 1 μg of DNA. I did it. Purify DNA using the Geneclean procedure and Ligation to purified NotI and SfiI cut pFAB5c. 20 μl of the ligation reaction was digested. 0.5 μg of inserted vector and 0.5 μg of DNA for insertion,_Four-DNA ligase (Ame (rsham) 1.5 U and incubated overnight at 15 ° C. Then, consolidation mixing The product was purified by phenol extraction and ethanol precipitation, and reconstituted in 20 μl of water. Suspended. Bio-Rad set at 25μF, 2.5kV and 200 ohms Electroporation using an E. coli pulser set (E. coli pulser set) Was transfected into E. coli TOP 10F 'cells (British Biotechnology). Pal Immediately after the addition of 1 ml of freshly prepared SOC medium (Sambrook et al., 1989), Cells were shaken at 37 ° C. for 1 hour. To get an estimate of the size of the entire library, This was serially diluted and plated on LB-ampicillin plate medium. VH and light chain inheritance Child library is 1.6 × 10⁷Live Fd and L chain genes, including species clones Rally is 6 × 10⁶Species clones were included. 1 ml each of the transformation mixture, Ampicillin 100 μg / ml, tetracycline 8 μg / ml, and 1% glucose Transferred to 40 ml of 2 × TY medium containing OD₆₀₀Cultures under shaking until 37 reached 1-2 Incubated at ° C. An aliquot of the library was stored at -80 ° C as a glycerin-added sample. Superinfection against library: 200 ml 2 × TY, 100 μg / ml ampicillin and Transformed cells were placed in a 2 liter flask containing 8 μg / ml tetracycline.⁹Plant an individual And shaken at 37 ° C. at 250 rpm. OD₆₀₀When the value reaches 0.5, the helper fur Di-R408 (Stratagene) is added in multiples of 50 and left at 37 ° C for 20 minutes to infect Proceeded. Subsequently, cells were treated with isopropyl-β-D-thiogalactopyranoside (IPTG ) Is added to a final concentration of 0.1 mM and is shaken (250 rpm) at room temperature. Incubation was continued overnight. Beret cells and plum (Φrum) et al. , 1993 and Engberg et al., 1995, by PEG precipitation. The phage supernatant was concentrated. Selection of antigen conjugate by panning: Panning on cells: K^kRM transfected A-S cells (RMA-S.K^k) Ha_255-262Was prepared. 80 μl of cells 20μl (about 10¹¹cfu) and mix at room temperature with gentle shaking. Incubated for hours. The cells are washed three times with growth medium and trypsin (Worthingt) on) Lysis of bound phage by treatment with 1 mg / ml for 1 hour at 37 ° C. Let out. Remove cells by centrifugation and remove OD₆₀₀Finger in the range of 0.8 to 1.0 400 μl of exponentially growing E. coli TOP10F 'cells (British Biotechnology) The supernatant was transferred into the containing vial. The following co-infections were performed as described in the previous paragraph did. Panning on beads: purified Ha_255-262-K^kLatex coated composite Beads 1 × 10⁶Phage library (20 μl) in PBS buffer containing 1% BSA About 10¹¹cfu) and incubated at + 4 ° C. for 3 hours with slight permeation. Centrifugation Beads were precipitated by treatment and washed three times. After the last wash, remove the beads Resuspend in 100 μl of lysine elution buffer (0.1 M glycine hydrochloride, 0.1% BSA, pH 2.2) The bound phages were eluted by incubation and left at room temperature for 10 minutes. Centrifugation Then, the beads were removed, and 8 μl of 2M Tris-base was added to neutralize the supernatant. This mix Transfer the mixture to a vial containing 400 μl of exponentially growing cells and Superinfection was performed according to the method. FACS analysis:   FACS analysis showed Fab-phage and RMA-S.K pulsed with peptide.^kFine Evaluation of binding to vesicles, or Fab fragments and various peptides immobilized on beads- It was performed for the purpose of detecting the binding to the MHC complex. FACS analysis using cells: RMA-S.K pulsed with peptide^kCell 10^FivePieces With FACS buffer (1% bovine serum albumin (BSA), PBS, 0.1% sodium azide) About 4 × 10 in 100 μl⁹Mix with cfu Fab phage or Fab13.4.1 and place on ice After 2 hours of incubation, the plate was washed three times with FACS buffer. Detection antibody is FITC labeled Goat anti-phage or rabbit anti-M13-phage serum treated with DAKO, Denko mark) and used diluted 1: 100 in FACS buffer. Samples are placed on ice for 30 minutes. Cubbed. Unbound antibody is removed by washing and the remaining cells are Resuspended in 200 μl of FACS buffer containing mualdehyde and analyzed by FACS. FACS analysis using beads: Various concentrations of purified Fab13.4.1 fragment were Beads coated with de-MHC complex 10^FiveAnd mixed. Incubate for 2 hours on ice After beating, the beads were washed three times, followed by a 1:50 dilution of FI in FACS buffer. Incubate with TC-labeled rabbit anti-mouse IgG serum (DAKO, Denmark) for 30 minutes did. The sample was further processed in the same manner as above.   FACS data is available from FacScan (Becton Dickinson Immunocyto measurement). Three parameters were obtained for each analysis: (i) forward scattered light, (ii) side scattered light, and (iii) fluorescein fluorescence emission (515 545 nm). The forward scatter threshold should be set to exclude cell or bead debris. Set. Ten^FourCollect data on individual beads or cells and use Lysis II (Lys is II) (registered trademark) software (Becton Dickinson Immunocytometry) And analyzed. Production and purification of soluble Fab fragments from selected clones:   To prepare the Fab fragment of pSAN13.4.1, the Fab-encoding cassette was His at the position of the gene III element of the C terminal (Engberg et al., 1995)₆expression vector to add -taq Transferred to the reactor. Using this new construct, prepare a Fab fragment as follows Performed: 2 × TY 500 ml, ampicillin 100 μg / ml and tetracycline 8 μg / ml Inoculate 10 ml of the transformed cells into two flasks each having a capacity of 21 and containing 2 ml at 37 ° C. 50rpm Shaking was applied. OD₆₀₀When is 0.5, IPTG was adjusted to a final concentration of 1 mM. Addition and incubation under shaking (250 rpm) continued for 4 hours at room temperature. Ice cold TES buffer (200 mM Tris-HCl, 5 mM EDTA (ethylenediaminetetraacetic acid Um dihydrate), 500 mM sucrose, pH 8.0) by resuspending the cells in 10 ml Periplasmic proteins were isolated. After 5 minutes, add 15 ml of 1: 4 TES buffer and mix Objects were incubated on ice for 30 minutes. Remove cell debris by centrifugation, MES buffer (10 mM 2- [N-morpholino] ethanesulfonic acid (SIGMA, catalog number M-8 2-50), 20mM NaH_TwoPO_FourThe periplasmic fraction was dialyzed against 4 Was. Periplasmic fraction was transferred to ABx matrix (JT Baker Research Products) The material passed through and retained was graded with 0.5 M NaH at pH 7.0._TwoPO_FourElute using Was. Fab fragments were further purified by immobilized metal ion chromatography (IMAC). Made. The Fab positive fraction of the ABx purified product was buffered with IMAC buffer using 5 times concentrated buffer stock solution. Adapted to solution A (PBS, 0.5M NaCl), 5 volumes of 0.1M CuSO_FourIn advance And then subjected to column equilibration with buffer A and chelating Superose HR 10 / Loaded on two columns (Pharmacia Biosystems). The bound protein is imidazole Dissolve using a gradient (0-0.5M) with buffer (IMAC buffer A, 0.5M imidazole). Issued. The amount of purified Fab fragments was assessed by SDS-PAGE and BCA assay. Was. All purifications were performed using the High Load® system (Pharmacia).  Biosystems). Generation of murine T cell hybridoma restricted to class I and cytotoxic T cells Cell analysis:   Mouse T cell hybridomas restricted by class I, HK9.5-24 and And HK9.5-162 (NP_50-57Specific and K^k(Stryhn et al., 1994) ) And HK8.3-5H3 and HK8.3-6F8 (Ha_255-262Specific and K^kRestrictions (Stryhn et al., 1994)) and its specificity have been described previously.   K^kRMA-S.K, an RMA-S cell line transfected with^k(Stryhn et al., 199 4) The Ha_255-262Or NP_50-57With 26 ° C overnight. Inn After cubation, wash the cells thoroughly and culture with various concentrations of Fab13.4.1. Resuspended in the ground. T cell hybridoma cells 10^FiveRMA-S with additional processing. K^kFine Cell 10^FiveIncubate for 24 hours at 37 ° C in a total volume of 250 μl with The supernatant was tested for release of IL-2 according to Kappler et al., 1981. BIAcore analysis:   The formed complex (ie, K^kTo determine the concentration of Traces of known specific activity¹²⁵I-labeled NP_50-57And add G-25 span column chromatography Complex formation was measured by chromatography (2). 20μ of purified Fab13.4.1 g / ml in 10 mM acetate buffer and from the manufacturer (Pharmacia, Sweden). As recommended, dextra of the BIAcore chip is performed by standard amine coupling chemistry. Attached to the surface. The amount of Fab fragments bound to the chip is slightly less than 1000 RU Value was maintained. Purified and quantified peptide / MHC complex Into the chamber, where the limitations of mass transport could interfere with the calculation of kinetic indicators. The flow rate was set at 10 μl / min to avoid All measurements are 0.1% NP-40 Performed at 22 ° C in PBS containing PBS. Stimulation of T cell hybridomas, T cell receptor specificity   Ha_255-262/ K^kTo evaluate the fine specificity of specific T cell hybridomas, An in vitro stimulation assay was used (21). RMA-K for antigen presenting cells^k(K^kThe gene Introduced thymoma). 1 well per 96-well microtiter plate 1 × 10^FiveRMA-K^kCells, Ha_255-262Specific and K^kHybrids restricted by Doma HK8.3-5H3 or HK8.3-6F8 1 × 10^FivePlanted with individual. Various Mana concentration of Ha_255-262Peptide or similar substitute was added and 10% FCS was added RPMI₁₆₄₀Inside, 5% CO_TwoIncubate the culture at 37 ° C for 24 hours in a humidified atmosphere containing Was added. The supernatant was collected and first described by Kappler et al. (26). According to the method described, use 4,000 HT-2, an IL-2-dependent cell line, per well. To evaluate the content of IL-2. Required to obtain 100 units / ml of IL-2 The concentration of the required peptide was determined. Specificity of recombinant pSAN13.4.1 antibody   Specific Ha_255-262-K^kAfter preparing the complex and purifying it, the Maxisorp 96-well flat-bottom microtaper at a concentration of 10 pg (10 nM) Coat was applied to the interplate (Nunc, Denmark). Then, add 2% skim milk Including After blocking the plate with PBS buffer, It was exposed to pSAN13.4.1, which was expressed as a fusion protein located. Wash plate After washing, expose to HRP conjugated with anti-phage antibodies (Pharmacia, Uppsala) and wash After purification, imaging and interpretation at 490 nm was performed with an ELISA reader. pSAN13 In order to assess the fine specificity of .4.1, graded concentrations of analog-K^kComplex (K^kof Prepared using 450 μM of the analog to obtain saturation) with the pSAN13.4.1 phage Was added beforehand as a competitor. Required to get 50% inhibition Competitor complex concentration (IC₅₀)It was determined. I c₅₀Are lower, pSAN13.4.1 Analog of -K^kThe binding to the complex is high. Example 1 Generation and selection of peptide-specific, MHC-restricted antibodies   K, a purified mouse MHC class I^kAnd K^kInfluence restricted by Hemagglutinin (Ha_255-262) And the complex Prepared as described in the method section. Tuberculin pep for immunization Peptide-derived purified protein (PPD) binds to the purified complex and is used as a B cell immunogen. K^kIs primarily tolerated, but highly reactive to PPD as a T-cell immunogen. First stimulus by Bacille-Calmette-Guerin (BCG) H-2 received^k(BALB / k) was used for immunization of mice.   Facilitate antibody responses directed to epitopes restricted by their own MHC For their syngeneic nature and, due to their high efficiency, according to Lachmann et al., 1978. The BCG-PPD immunization method described first was selected. Total m of the spleen 10 days after immunization RNA was isolated and cDNA was made by reverse transcription. Compatible with immunoglobulin Fab fragments Using a specific set of degenerate primers to PCR amplify the cDNA fragment This fragment was cloned into the pFab5c vector (Φrum et al., 1993) and Expression of Cteriophage as a fusion with minor virus coat protein pIII Was. First library is 2 × 10⁷Species of phage that are subjected to a panning procedure. After binding, the bound phages were eluted and regrown in E. coli. Effect of selection procedure To increase the rate, the phage^k-Transfected RMA-S cells (RMA-S.K^k) To H a_255-262Panned and refined homogenized Ha_{255- 262} -K^kPanning and alternation on latex beads coated with composite gave. The purpose of the panning strategy is to swap matrices every other panning And a common selection epitope, in this case Ha_255-262-K^kIs to maintain You. Phage regrown in each selection round were 2x10^Tencfu / ml Adjust, Ha_255-262Or NP_50-57RMA-S.K stimulated by pulse^kConnection with cells Compatibility tested by FACS analysis using FITC-labeled anti-phage antibody as detection antibody (NP used for comparison_50-57Peptide is also K^kLimited by). Shown in Figure 1 As mentioned above, after performing panning alternately 2 × 2 times, Ha_255-262-K^kRecognize It was observed that the content of Fab phage gradually increased. Perform 1 to 3 pannings NP while giving_50-57RMA-S.K stimulated by pulse^kCells (fill The increase in binding to is relatively small, probably due to a common cellular epitope. It is thought that this reflects the increase in Fab phage having low binding affinity. This population is efficiently removed as a result of the fourth and final panning of the beads. It is. Example 1a   The description about the antibody Fab fragment (pSAN13.4.1) described in detail above Using a technique similar to that of pSAN13.4.1, it has the same specificity but with lower affinity Other antibodies (eg, pSAN KHO_Four) Has been isolated. Furthermore, NP_50-57With peptides Complexed MHC-K^kFab fragment that specifically recognizes a molecule (eg, pSAN KN36) Pieces have also been isolated. The latter antibody, K^k-NP_50-57Starting with the composite Fab live generated by the above procedure using mice immunized with Isolated from Rally. For the latter two antibodies, an alternate panning procedure The technique used in-between is slightly different from that described above: the selected MHC / Peak Express the same complex as the plastic surface coated with the peptide complex Instead of performing the experiment while rotating between cells, a Fab phage library (K^k / Ha_255-262Made from mice immunized with)^k-NP_50-57Complex K in the presence of a competing amount of latex beads coated with^k/ Ha_255-262 Panning was performed on the plastic surface covered by the composite. Similarly, K^k -NP_50-57Fab-phage library from mice immunized with , K^k/ Ha_255-257In the presence of latex beads coated by the conjugate, K^k-NP_50-57 Panning on the plastic coated by Alternate panning strategy It is believed that some variations on it can be used successfully. The important thing is that the book The applicability and generality of our immunization and alternation panning procedures is already It is shown. Example 2 Specificity of clone pSAN13.4.1   Individual phages were isolated from the phage population obtained by final panning and Rescreening for specificity was performed using an A-based assay. Consideration Of the 50 clones we did, seven were Ha_255-262-K^kReacted specifically with the complex (de Data not shown). The 15 clones are Ha_255-262-K^kAnd NP_50-57-K^kComplex Can bind to both and recognize epitopes shared by the two complexes Sex was considered the highest. The remaining 28 clones are Ha_255-262-K^kAnd NP_{50-5 7} -K^kDid not bind to any of the complexes. The CDR3 heavy chains and DNA sequences corresponding to the light and light chain regions were determined to be identical (data Data are not shown), all of which are single productive antibody light / heavy chain combinations. It was suggested that it came from the event. Soluble from one clone, pSAN13.4.1 Production and purification of soluble Fab molecules (Figure 2A), and various combinations of peptides and MHC Specificity was determined by FACS analysis using latex particles coated by lamination. And analyzed. The specificity of the Fab molecule (Fab13.4.1) depends on its reactivity_255-262And K^k Only for the complex formed between_{50 -57} -K^kThe complex did not recognize the MHC-restricted T cell specificity It had obvious features (FIG. 2B). Furthermore, in connection with Fab 13.4.1, Soluble Ha_255-262-K^kThe complex was coated on beads with Ha_255-262-K^kAbility to compete with Ha that is isolated_255-262Peptide and isolated K^k, And NP_50-57 -K^kThis interaction is saturated, as demonstrated by the inability of the complex to And specific (FIG. 2C). Fab13.4.1 and Ha_255-262-K^kInteraction with 10-100 nM soluble Ha to inhibit_255-262-K^kA complex is needed and this Therefore, Ha_255-262-K^kAffinity KD of Fab13.4.1 for the complex is between 10-100 nM It was shown that there is. Example 2a MHC class I K^kBy Ha_255-262Epitope recognition   K^kTo assess the specificity of the influenza virus hemagglutinin Ha_{twenty five 5-262} K derived from (FESTGNLI)^kT cell epitome restricted by A complete set of peptide analogs with individual amino acid substitutions in the Was. For all positions within this epitope, each of the parent molecule's amino acids They were replaced one by one with another natural amino acid (excluding cysteine). these Analog K^kWas tested in a biochemical inhibition assay (25) -Log (IC₅₀(Analog)) (data not shown). Surprised What should be done is that most replacements are more or less K^kAllowed by. In particular, third and fourth And substitution at position 6^kDid not significantly change the binding to Et al. K for mutations at these sites^kWas considered neutral. 144 seeds Only 14 species (or 10%) have less than 100% less binding Met. All such harmful substitutions are concentrated in positions 2 and 8, Preference was given to amino acids E and I, respectively. Data show 10 minutes of binding at 8th place 9 substitutions dropped to less than 1 in the second place, 6 substitutions? The authors suggest that the eighth is the most decisive of the two. 1st, 5th The effects of the substitutions on positions 7 and 7 are less clear, with F being preferred in position 1. In the fifth position, charged amino acids and Y have a disadvantage, and in the seventh position F and And L were considered preferential. Example 2b By T cells, K^kHa restricted by_255-262Epitope recognition   To examine the fine specificity of the T cell response restricted by MHC class I, a_255-262Specific and K^kHK8, two types of T cell hybridomas restricted by In bioassays using 3-6F8 and HK8.3-5H3, each single amino acid The stimulatory ability of the substituted analog was evaluated. This assay uses K^kConnectivity with Note that it is dependent on both T cell recognition. Each analog Perform a dose-response analysis on the Used to determine analog concentration. The most critical peptide residue is in the first position F (with any hybridoma) and T at the 4th position (with any hybridoma) With dorma ) And N in 6th place (HK8.3-6F8 only). In the above position, T Cell specificity was very strict, allowing only the same amino acids as the parent molecule . In other words, the stimulatory activity was completely abolished in any substitution (T at position 4 was replaced with S). Except for a 10-fold reduction with conservative substitutions). 3rd place (any ha 6th (HK8.3-5H3 only) and 7th (both hybridomas) (With dorma), the specificity was rather high but less strict. Was. At these sites, only a few substitutions, mostly conservative or semi-conservative Was accepted. Fifth place (both hybridomas) slightly irregular Highly specificity was observed. The least definitive peptide residue is K^kTo Is the anchor site, followed by I at position 8 (any hybridoma ) And E in the second place (with any hybridoma). This Multiple substitutions are more or less tolerated at these sites, leading to loss of T cell stimulatory activity. Many such substitutions are^kThis was thought to be due to the loss of connectivity. Example 2c K with pSAN13.4.1 antibody^k-Ha_255-262Epitope recognition   pSAN13.4.1 is associated with peptide-specific and MHC-restricted T cell specificity Ha_255-262And K^kRecognize a complex comprising This point further In order to investigate, the immobilized Ha of pSAN13.4.1_255-262-K^kTo inhibit binding to the complex -K soluble analog^kIn biochemical assays using conjugates, The ability of each of the substituted amino acids to bind to pSAN13.4.1 was evaluated. Analog -K^kThe complex is K^kFocus on pSAN13.4.1 specificity To squeeze, it was made under peptide saturation conditions. No. 4 is the most critical residue , Which could only be replaced with a conservative substitution, S. No. 2 The critical residues in the eye are L at position 7 and S at position 3, mostly conservative or semi-conservative. Some substitutions that were viable were tolerated (data not shown). More deterministic than this The residue that was low is F at position 1, which is mostly conserved or semi-conserved10 The loss of binding did not reach one-tenth in species substitutions. In contrast, Less determinant residues are G at position 5 and E at position 2, which are 13-14 species Substitution with amino acids was possible. The residue with the least decisiveness was ranked 8th I at This was substitutable for all other amino acids (except R).   Using the same peptide-MHC complex,_255-262Specific and K^kLimited by Unique T cells against two different T cells and the same peptide-MHC complex The fine specificity of the body, pSAN, was similarly evaluated. As a result, the characteristics of T cells It became clear that there was a remarkable similarity between the isomerism and the specificity of the pSAN antibody, Most of the peptide residues that can be recognized by cells are also recognized by this antibody It was thought. Example 3 Kinetic binding analysis on pSAN13.4.1   Surface plasmon resonance (ie, detection of changes in refractive index on a surface; BIAcor e, Pharmacia, Sweden) using Fab 13.4.1 to confirm the specificity and interaction Kinetic index was determined. Purified Fab13.4.1 on BIAcore sensor chip Ha fixed and pre-formed_255-262-K^kOr NP_50-57-K^kCreate complex Was used. Purified Fab13.4.1 was adjusted to 20 μg / ml with 10 mM acetate buffer at pH 4.5. Dilute in solution and follow standard amine coupling chemistry according to the manufacturer (Pharmacia, Sweden). The reaction bound to the dextran surface of the BIAcore chip. Preformed Keep K^kA complex of and was prepared in the same manner as described above. Formation Complex (ie, K^kComplex to determine the concentration of the active binding site) Traces of iodinated NPs of known specific activity during the formation of_50-57Was added . After separation of the unbound peptide on a G-25 span column (Stryhn et al., 1994) Bound and free¹²⁵The amount of I was measured by gamma counting and the complex formed The concentration was calculated. The amount of Fab fragments bound to the chip is slightly less than 1000 RU And set the flow rate to 10 μl / min, thereby limiting the To avoid interference with the calculation of the kinetic index. All measurements are 0.1% NP-40 And 22 ° C. in phosphate buffered saline.   As shown in FIG. 3A, according to the sensorgram, Fab 13.4.1_255-262-K^kWhat is Binds but NP_50-57-K^kDoes not bind. As a control, K^kSpecific H100-27R55, a monoclonal antibody provided by Dr. G. Hammerling ) Is immobilized on a BIAcore sensor chip and the same peptide-K^kActivated specimen . Figure 3B, H100-27R55 is any peptide-K^kBinds to the complex as well And an equivalent amount of functional K for the chip^kThat the protein was available Is supported. The slope of the first bonded phase is Ha_255-262-K^kDepends on the concentration of FIG. 3C), which allows us to determine the association rate constant K_aIs 1 × 10^FourM^-1s^-1Is calculated as We were able to.   The sensorgram in FIG. 3C revealed two experimental problems: (i) blank note Signal increased by about 19 RU and decreased almost linearly at the end of the meeting period. (Ii) The baseline decreased about IRU / min during the course of the experiment. This indicates a gradual loss of the immobilized ligand. These experimental deviations Are linearly interpolated to correct the association phase and the total sensorgram, respectively. Used for These corrections and the following calculations are made in Gentofte, Denmark. Of the Hagedorn Research Laboratories (Gentofte) This was done using a mathematical model developed by Ron Shymko. Dissociation and 21 data from each sensorgram for mathematical analysis of both sides of the meeting Points were used. First, a two-sided exponential model fitting is performed for the dissociation phase. went. Calculated k_dThe value is 7.3 × 10^-Fours^-1~ 3.2 × 10^-Fours^-1Was in the range. this By using these values and keeping the total number of binding sites constant, a two-site model is used. To fit all sensorgrams. As a result, the association of highly associative components K_aThe value is 1.2 × 10^FourM^-1s^-1~ 0.6 × 10^FourM^-1s^-1And the resulting K_D The values were all very similar to 53, 56, 60 and 51 nM. Low affinity components Could not be interpreted. However, the sensorgram data The contribution of this component is small and accounts for 3-18% of the total signal at the end of the meeting. Was.   The removal of unbound ligand initiates the dissociation phase, which is used to determine the dissociation rate. Number k_dIs 5.6 × 10^-Fours^-1(T_1/2Which is equivalent to about 21 minutes). k_dTo k_aDivided by Equilibrium dissociation constant K calculated as_DIs about 56 nM, which was observed in FIG. 2C Correlates well with the extent of inhibition. Recently, using BIAcore analysis, two different types of T cell Kinetic indicators and affinities of the body have been published (Corr et al., 1994; Matsui et al., 1995). Association rate constant k_aIs 10^ThreeAnd 10^FiveM^-1s^-1And the dissociation rate constant k_dIs 0.06 And 0.2s^-1(T_1/2Is equivalent to 12 to 27 seconds). Separation Constant K_DIs 10^-5~Ten^-7M is calculated. In comparison, antibodies generally interact with T cells They associate at about the same rate, but dissociation is orders of magnitude slower (Pellequer et al., 1993). Typical Has an overall affinity of 10^-7~Ten^-TenM range. Fab 13.4.1 affinity And kinetic indicators are typical for antibodies, despite having T-cell-like specificity It was a typical thing. Example 4 Specific inhibition of antigen-specific and MHC-restricted T cell responses.   Given the specificity and affinity of Fab13.4.1, this is_255-262Specific and K^kTo Inhibits restricted T-cell hybridomas but has different peptide specificities Have K^kIs not expected to inhibit T cell hybridomas restricted by You. In fact, Fab13.4.1 is Ha_255-262Specific and K^kTypes of T cells restricted by It was able to inhibit the hybridomas HK8.3-5H3 and HK8.3-6F8 (Fig. 4 ) But NP_50-57Specific and K^kT cell hybridomas restricted by No effect on HK9.5-24 and HK9.5-162 was observed (Fig. 4, insertion part). Therefore, Fab 13.4.1 is not affected by T-cell hybridomas HK8.3-5H3 and HK8.3-6F8. Ha spatially or allosterically related to the epitope recognized as_{255-26 Two} -K^kRecognize dependent epitopes. Ha_255-262Specific and K^k2 species restricted by The concentration of Fab 13.4.1 required to obtain inhibition of a class of T-cell hybridomas K measured by e-analysis_DIt was predicted that the value approximated. Example 5 Use for antigen-specific and MHC-restricted antibody targeting   Interestingly, these antibodies can be used to control specific genes, such as viruses or mutated oncogenes. Directs the toxin to cells with intracellular targets (tumor cells), thereby By killing such cells, the function of killer T cells can be mimicked. These antibodies can be raised in laboratory animals and subsequently "humanized" Recombinants can be produced in large quantities upon transfer to isotype constructs. Also Administration and control compared to T cells currently used for adoptive immunotherapy It is considered easy.

────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. ⁶ identifications FI A61K 31/00 617 A61K 31/00 617E 621 621D 625 625 629 629A 629 631 631H 631C 633 633 635 635 637 637A 39/395 39/395 LTU C07K 14/74 C07K 14/74 16/00 16/00 C12P 21/08 C12P 21/08 G01N 33/53 G01N 33/53 D 33/531 33/531 A // (C12P 21/08 C12R 1 : 19) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (K E, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AT, AU, AZ, BB, BG , BR, BY, CA, CH, CN, CZ, CZ, DE, DE, DK, DK, EE, EE, ES, FI, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Engberg Jean Denmark Copenhagen OH. C. Rambies Gade 43 (72) Inventor Fugar Lads Denmark Copenhagen Auf Rederic Vise Ve 13 4s (72) Inventor Stryn Annette Denmark Bronshoi Stenmag Rebay 29

Claims (1)

[Claims] 1. Method for producing antibody or antibody fragment that specifically recognizes peptide-MHC complex And (a) Phage library expressing nucleic acid encoding antibody or antibody fragment Production of, (b) expressing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex Phage selection, and (c) for producing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex. Transfer of the nucleic acid encoding the antibody or antibody fragment of the phage to appropriate cells for Methods that include 2. Method for producing antibody or antibody fragment that specifically recognizes peptide-MHC complex And (a) isolation of RNA from an animal immunized with the peptide-MHC complex, (b) amplification of the nucleic acid encoding the antibody or antibody fragment, and expression of the nucleic acid Production of phage libraries, (c) expressing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex Phage selection, and (d) for producing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex. Transfer of the nucleic acid encoding the antibody or antibody fragment of the phage to appropriate cells for Methods that include 3. Expressing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex Phage selection depends on whether the cells expressing the peptide-MHC complex and the phage library 3. The method according to claim 1 or 2, wherein the method is performed by incubation with lee. 4. Expressing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex Phage selection was performed by binding the phage library to the peptide-MHC complex. 3. The method according to claim 1 or 2, wherein the method is performed by incubation with a dose. 5. Expressing an antibody or antibody fragment that specifically recognizes the peptide-MHC complex Selection of phage allows for alternate matrices that retain the peptide-MHC complex as a common factor Of a library of phage expressing antibodies or antibody fragments D 5. The method according to claims 3 and 4, wherein the method is performed by ringing. 6. Alternating matrices contain cells and peptides expressing the peptide-MHC complex. 6. The method according to claim 5, wherein the beads are beads to which a tide-MHC complex is bound. 7. 2. Use of purified MHC molecules enriched in one particular peptide. The method according to any one of claims 1 to 6. 8. A peptide-MHC complex prepared by the method according to any one of claims 1 to 7. An antibody or antibody fragment that specifically recognizes the union. 9. Non-glycosylated antibodies that specifically recognize the peptide-MHC complex Or antibody fragment. Ten. Selected from the group consisting of antibiotics, cytotoxic drugs and antineoplastic drugs 10. The antibody or antibody fragment according to claim 8, which is conjugated to a pharmaceutical. 11． Is bound to a superantigen capable of activating T lymphocytes, or Is associated with a polymerized carbohydrate having the ability to activate complement. An antibody or antibody fragment according to any of the preceding claims. 12． The presence of a peptide-MHC complex comprising the antibody or the antibody fragment according to claim 8 or 9. A diagnostic composition for detecting a. 13. Contacting a cell or tissue sample from an individual with the diagnostic composition of claim 12; And the antibody or antibody fragment according to claim 8 or 9 in a cell or tissue sample. Peptide-M in an individual, including determining whether it is bound to the peptide-MHC complex A method for determining the presence of an HC complex in vitro. 14. A pharmaceutically acceptable antibody or antibody fragment according to any one of claims 8 to 11. A pharmaceutical composition for blocking an inappropriate T cell response, comprising: 15． Eradicate intracellular pathogens selected from the group consisting of viruses, bacteria and parasites An antibody or antibody fragment according to any one of claims 8 to 11 for killing A pharmaceutical composition comprising a pharmaceutically acceptable excipient. 16． HLA class I-related diseases (ankylosing myelitis, Reiter's disease, psoriatic spondylitis, idiopathic Mchromatosis, psoriasis vulgaris and Behcet's disease) and HLA class II related Diseases (rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus Disease, Sjögren's disease, IDDM, Addison's disease, Graves' disease, Hashimoto's disease, steatosis in children , Primary biliary cirrhosis, pemphigus vulgaris, acquired epidermolysis bullosa, Hodgkin's disease, cervix Squamous cell carcinoma, multiple sclerosis, optic neuritis, narcolepsy, myasthenia gravis, Good Prevention of diseases selected from the group consisting of pasture syndrome and alopecia areata. Use of a pharmaceutical composition according to any one of claims 14 to 16 for or treatment. 17． Consists of viruses, bacteria and parasites by directing complement lysis A method according to any one of claims 14 to 16 for combating a pathogen in a cell selected from the group. Use of a pharmaceutical composition as described in paragraph.