MXPA00011368A - Multivalent t cell receptor complexes - Google Patents

Abstract

The present invention relates to a synthetic multivalent T cell receptor complex for binding to an MHC-peptide complex, which multivalent T cell receptor complex comprises a plurality of T cell receptors specific for the MHC-peptide complex. It is preferred that the T cell receptors are refolded recombinant soluble T cell receptors. The synthetic multivalent T cell receptor complex can be used for delivering therapeutic agents or for detecting MHC-peptide complexes, and methods for such uses are also provided.

Description

! r < COMPLEXES OF MULTI-COLLECTED PE CELL T-RECEIVER FIELD OF THE INVENTION The present invention relates to T cell receptors (TCRs) in multivalent form and to their use to detect cells, which carry specific peptide antigens presented in the context of the majority of the histocompatibility complex (MHC) on their surfaces. The invention also relates to methods of The delivery, in particular for the delivery of therapeutic agents, to target cells using the multimeric TCRs.

General Background 1. Presentation of antigen on the cell surface 15 MHC molecules are specialized protein complexes, which present short protein fragments, peptide antigens, for recognition for the cell surface through the cell arm of the cell. adaptive immune system. MHC class I is a dimeric protein complex that consists of a variable heavy chain and a constant light chain, β2microglobulin. MHC class I presents peptides, which are processed intracellularly, loaded to a binding cleft in the MHC, and transported to the cell surface, where the complex is anchored in the membrane through the MHC heavy chain. Peptides usually have a length of 8-1 1 amino acids, depending on the degree of tonnage introduced in the peptide when bound in the MHC. The binding cleavage, which is formed through the distant membrane, domains a1 and a2 of the MHC heavy chain, has "closed" ends, imposing high restrictions on the length of the peptide that can be bound. MHC class I I is also a dimeric protein consisting of a (heavy) chain and a (light) β chain, both of which are variable glycoproteins and are anchored in the cell through transmembrane domains. Like the MHC class I, the class I I molecule forms a bond cleavage wherein peptides longer than 12-24 amino acids are inserted. The peptides are picked up from the extracellular environment through endocytosis and processed before being charged to the class I I complex, which is then transported to the cell surface. Each cell represents peptides in up to six different class I molecules and a similar number of class I molecules, the total number of MHC complexes presented being in the region of 105-106 per cell. The diversity of peptides presented in the class I molecules is typically estimated between 1, 000-10,000, with 90% of these being present in 100-1,000 copies per cell (Hunt, Michel et al., 1992; Chicz, Urban and others, 1993; Engelhard, Apella et al., 1993; Huczko, Brodnar et al., 1993). The most abundant peptides are believed to constitute between 0.4-5% of the total peptide presented which means that up to 20,000 identical complexes may be present on a single cell. An average number for the most abundant individual peptide complexes will probably be the region of 2,000-4,000 per cell, and the typical levels of recognizable T-cell epitopes are in the region of 100-500 complexes per cell (see for review (Engelhard, 1994)). 2. Recognition of antigen presenting cells A broad spectrum can present an antigen, such as the MHC peptide, and cells that have that property are known as antigen presenting cells (APCs). The type of cell that presents a particular antigen depends on how and where the antigen first encounters cells of the immune system. APCs include the interlaced dendritic cells found in the T cell areas of the lymph nodes and spleen in large numbers; Langerhan cells in the skin; follicular dendritic cells in areas of the B cell of the lymphoid tissue; monocytes, macrophages and other cells of the monocyte / macrophage lineage; B cells and T cells; and a variety of other cells such as endothelial cells and fibroblasts, which are not classical APCs but can act in the form of an APC. Antigen presenting cells are recognized by a subset of lymphocytes, which mature in the thymus (T cells), where they undergo a screening procedure designed to ensure that T cells, which correspond to peptides ks ^ M ^ é JkJ? I independent, eradicated (negative selection). In addition, T cells that do not have the ability to recognize MHC variants, which are presented (in man, HLA haplotypes), fail to mature (positive selection). The recognition of MHC peptide specific complexes by T cells is mediated by the T cell receptor (TCR), which is a heterodimeric glycoprotein consisting of a chain a and a β chain linked by a disulfide bond. Both chains are anchored in the membrane through a transmembrane domain and have a short cytoplasmic tail or tail. In a recombination process similar to that observed for antibody genes, the TCR a and ß chain genes are made available again from variable, binding, diversity or constant elements, creating an enormous diversity in the binding domains of TCR. extracellular antigen (1013 to 1015 different possibilities). The TCRs also exist in a different way with chains? and d, but these are only present in approximately 5% of the T cells. Antibodies and TCRs are the only two types of molecules that recognize antigens in a specific form. In this way, the TCR is the only specific receptor for particular peptide antigens presented in MHC, the foreign peptide usually being the only sign of an abnormality within a cell. The TCRs are expressed in a huge diversity, each TCR being specific for one or some MHC peptide complexes. The contacts between TCR and MHC peptide ligands are extremely short lived, usually with a half-life of less than 1 second. The adhesion between T cells and target cells presumably TCR / MHC-peptide, is based on the employment of multiple TCR / MHC-peptide contacts as well as a number of contacts of co-receptor-ligand. T cell recognition occurs when a T cell and an antigen presenting cell (APC) are in direct physical contact and initiated through the ligation of specific antigen-TCRs with pMHC complexes. TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily, which is associated with invariant proteins of the CD3 complex involved in the mediation of signal transduction. TCRs exist in the < ? ß and? d, which are structurally similar, but have completely different anatomical locations and probably functions. The extracellular portion of the receptor consists of two constant domains near the membrane, and two distant variable domains of the membrane that carry highly polymorphic loops analogous to the regions of complementarity determination (CDRs) of antibodies. These loops are those that form the MHC binding site of the TCR molecule and determine the specific character of the peptide. MHC ligands of class I and class II are also proteins of the immunoglobulin superfamily, but are specialized for the presentation of antigen, with a highly polymorphic peptide binding site, which allows them to present a wide array of fragments of short peptide on the cell surface of APC. Recently, examples of these interactions have been structurally characterized (Garboczi, Ghosh and others, 1996, Garcia, Degano and others, 1996, Ding, Smith and others, 1998). The crystallographic structures of murine and human class I pMHC-TCR complexes I indicate a TCR / orientation on their pMHC ligand and show poor complementarity of shape at the adjoining surface. The CDR3 loops make contact exclusively with peptide residues. Comparisons of ligand and ligand-free TCR structures also suggest that there is a degree of flexibility in CDR TCR loops (Garboczi and Biddison 1999). T cell activation models attempt to explain how such protein-protein interactions on an abutting surface between the T cell and the antigen presenting cell (APC) initiate responses such as the annihilation of a virally infected target cell. The physical properties of TCR-pMHC interactions are included as critical parameters in many of these models. For example, it has been found that quantitative changes in TCR dissociation regimes are translated into qualitative differences in the biological production of the receptor coupling, such as the total or partial activation of the i iliA.it 1 T cell, or antagonism (Matsui, Boniface and others, 1994, Rabbi itz, Beeson and others, 1996, Davis, Boniface and others, 1998). It has been shown that TCR-pMHC interactions show low affinities and relatively low kinetics. Many studies 5 have used biosensor technology, such as Biacore ™ (Willcox, Gao et al., 1999; Wyer, Willcox et al., 1999), which exploits surface plasmon resonance (SPR) and enables direct affinity and Real-time kinetic measurements of protein-protein interactions (García Scott et al., 1996, Davis, Boniface and 10 others, 1998). However, the receptors studied are either TCRs to the reactants or those that have been raised in response to an artificial immunogen. 3. TCR and CD8 Interactions with MHC-Peptide Complexes The vast majority of T cells restricted by (ie, which recognize) MHC class I-peptide complexes also require co-receptor coupling, CD8, for activation, whereas T cells restricted by M HC of class II require the coupling of CD4. The exact function of the receptors in the activation of T cells is not yet fully clarified. None is the critical mechanism and activation control parameters. However, both CD8 and CD4 have cytoplasmic domains, which are associated with the p56lck kinase, which is involved in very high tyrosine phosphorylation events. , which characterize the activation of the T cell. CD8 is - - * - »i '•» - a dimeric receptor, expressed either in the form of aa or, more commonly, in a form of aβ. CD4 is a monomer. In the CD8 receiver only the string a is associated with p56, ok. Recent determinations of the physical parameters that control TCR and CD8 to MHC binding, using soluble versions of the receptors, have shown that the TRC link dominates the recognition event. TCR has a significantly higher affinity for MHC than for co-receptors (Willcox, Gao et al., Wyer, Willcox et al., 1999). The individual interactions of MHC receptors are very short-lived at physiological temperature, that is, at 37 ° C. An approximate figure of the half-life of an MHC-peptide TCR interaction, measured with a human TRC specific for the "matrix" peptide of influenza virus presented by HLA-A * 0201 (HLA-A2) is 0.7 seconds. The half-life of the CD8aa interaction with this MHC / peptide complex is less than 0.01 seconds or at least 18 times faster. 4. Production of the MHC-peptide soluble complexes The soluble MHC-peptide complexes were first obtained through the cleavage of the surface molecules of antigen presenting cells with papain (Bjorkman, Strominger et al., 1985). Although this aspect provided a material for crystallization, it has been replaced, for class I molecules, in recent years, by individual heavy and light chain expression in E. coli followed by bending in the presence of the synthetic peptide (Garboczi, Hung et al., 1992; Garboczi, Madclen et al., 1994; Madden, Garboczi et al., 1993; Reid McAdam et al., 1996; Reid, Smith et al. , 1996, Smith, Reid and others, 1996, 1996, Smith, Reid and others, 1996, Gao, Tormo and others, 1997, Gao, Gerth and others, 1998). This aspect has several advantages over the previous methods since better production is obtained at a lower cost, the identity of the peptide can be controlled very accurately, and the final product is more homogeneous. In addition, the expression of the modified heavy or light chain, for example, fused to a protein tag, can be more easily performed.

. MHC-peptide tetramers The short life of the individual binding event between the peptide-MHC and the TCR and CD8 receptors makes this interaction unsuitable for use in the development of detection methods. This problem has been overcome through a novel technique that employs tetrameric molecules of peptide-MHC complexes (AItman and others, nineteen ninety six). The higher avidity of the multimeric interaction provides a dramatically longer half-life for molecules that bind to a T cell than could be obtained with the monomeric peptide-MCH complex binding. This technique is also described in WO 96/26962. The tetrameric peptide-MHC complex is made with a synthetic peptide, β2microglobulin (usually expressed in E. coli), and the . . . ^ M. ^ .i.A i «,« a ^ .. ^. . . soluble MHC chain weight (also expressed in E. coli). The MHC heavy chain is truncated at the start of the transmembrane domain and the transmembrane domain is replaced with a protein tag constituting a recognition sequence for the enzyme of bacterial modification BirA (Barker and Campbell, 1981; Barker and Campbell, 1981; Schatz, 1993). BirA catalyzes the biotinylation of a lysine residue in a somewhat redundant recognition sequence (Schatz, 1993), however, the specificity is high enough to ensure that the vast majority of proteins will be biotinylated only at the specific position on the label. The biotinylated protein can then be covalently linked to avidin, streptavidin or extravidin (Sigma) each of which has four binding sites for biotin, resulting in a tetrameric molecule of peptide-MHC (Aitman et al, 1996). 6. MHC-peptide tetramers and WO 96/26962 T staining and Aitman and other cells, (1996) also describe a technology for staining T cells with a particular specificity using soluble MHC-peptide complexes, made as tetrameric molecules. This technology has gained scientific importance in the detection and quantification of T cells (Callan and others, 1998, Dunbar and others, 1998, McHeyzer Williams and others, 1996, Murali Krishna and others, 1998, Ogg and others, 1998), and be potential in the diagnosis (see for review (McMichael and ^ jjí ^ g ^^ O'Callaghan, 1998)). Although the half-life of the interaction between MHC with TCR and CD8, as measured with soluble proteins, is very short, ie less than 1 second, the stable bond is achieved with the tetramer so that staining can be detected. This is due to a higher avidity of the multimeric interaction between the tetramer and the T cell. 7. Soluble TCR The production of soluble TCR has only recently been described through a number of groups. In general, all methods describe truncated forms of TCR, containing either only extracellular domains or extracellular and cytoplasmic domains. In this way, in all cases, the transmembrane domains have been eliminated from the expressed protein. Although many reports show that TCR produced according to its methods, can be recognized through TCR-specific antibodies (indicating that the part of the recombinant TCR recognized by the antibody is correctly bent), none has been able to produce soluble TCR to a good performance, which is stable at low concentrations and which can recognize MHC-peptide complexes. The first aspect to produce a crystallizable material makes use of expression in eukaryotic cells, but in materials extremely expensive to produce (García, Degano et al., 1996; García, Scott et al., 1996). Another aspect that has produced a l lilili lllll lilii I I huIL crystallizable material makes use of a blasting system of E. coli similar to that previously used for complexes M HC-peptide (Garboczi, Chosh et al, 1996; Garboczi, U ltz et al, 1996). The latter method, which involves expression of the extracellular portions of the TCR chains, truncated immediately before the cysteine residues involved in forming the disulfide bridge between chains, followed by bending ip vitro has become less generally applicable . Most heterodimeric TCRs appear to be unstable when they occur in this way due to the low affinity between the α and β chains. In addition, there are a number of other production descriptions by soluble TCR engineering. Some of these describe only the expression of either the α or β chain of the TCR, thus producing the protein that does not retain the specific MHC-peptide binding (Calaman, Carson et al, 1993, Ishii, Nakano et al., 1995). Β-chain crystals without a α-chain have been obtained, either alone or linked to a superantigen (Bentley, Boulot et al., 1995; Bentley et al., 1994; Fields, Malchiodi and Oíros, 1996). Other reports describe methods for the expression of heterodimeric TCRα / doa / β (Corr, Slanetz and others, 1994, Eliat, Kikuchi and others, 1992, Gregorie, Rebai and others, 1996, Gregoire, Malissen and others, 1991, Ishii, Nakano et al., 1995; Necker, Rebai et al., 1991; Romagne, Pyrat et al., 1996; Weber, Traunecker et al., 1992). In some cases, TCR has been expressed as an individual chain fusion protein (Broker, Peter et al., 1993; Gregoire, ^^ tog ^ í? ^ ^ ^ ^ ^ ^ ^ ^ ^ MgSá ^^^^ Malissen et al., 1996; Schluter, Schod in et al., 1996). Another strategy has been to express the TCR chains as q umeric proteins fused to the Ig hinge and constant domains (Eliat Kikuchi et al., 1992, Weber, Traunecker et al., 1992). Other chimeric TCR proteins have been expressed with designed sequences, which form coiled coils that have high affinity and specific character to each other, thus establishing a-ß contacts of TC R and increasing the solubility. It has been reported that this strategy produces soluble TCR both from the baculovirus expression system and from E. coli (Chang, Bao et al., 1994, Golden, Khandekar et al., 1992). A method for making soluble TCR, which can recognize a TCR ligand, is described herein. According to a preferred embodiment of this method, the extracellular fragments of TCR are expressed separately as fusions to the "leucine closures" of c-j un and c-fos and then re-folded in vitro. The TCR chains do not form a disulfide bond between chains since they are truncated just before the cysteine residue is involved in the formation of that link in native TCR. Rather, the heterodynamic contacts of the α and β chains are supported by the two leucine closure fragments, which mediate the heterodimerization in their native proteins. 8. Detection using TCR Peptide-specific recognition of antigen-presenting cells by T cells is based on the avidity obtained through low affinity multiple receptor / ligand interactions. These involve TCR / MHC-peptide interactions and a number of co-receptor / ligand interactions. Co-receptors, CD4 and CD8, of restricted class I I and restricted class I T cells, respectively, also have the MHC, but not the peptide, as their ligand. However, the epitopes in MHC with which CD4 and CD8 interact do not overlap with the epitope that interacts with the TCR. This recognition mechanism opens up the possibility that the specific recognition of peptide from antigen presenting cells can be mediated by soluble TCR so that the half-life of the contact can be of therapeutic use. However, it is not evident if the stability obtained through the avidity of multiple TCR / MHC-peptide interactions in the absence of co-receptor support could be sufficient for such purposes. Staining of antigen presenting cells through soluble TCR was reported by Plaksin et al. (Plaksin et al., 1997). This result was obtained with a so-called single-chain TCR, an individual protein consisting of 3 of the 4 domains of the a and ß chains from TCR. However, staining was performed by incubating antigen presenting cells with chemically modified TCR, which was then interlaced, an aspect of which might not be practicable in vivo. In addition, the method only convincingly detected levels of peptide (incubation of antigen presenting cells with approximately 1000 μM peptide), which were higher than peptide levels that could be present in vivo. The fact that cell-specific staining or T can be achieved with tetramers of M HC-peptide (AItman et al., 1996), the "reciprocal" situation can be considered to lead some support to the idea that multimeric TCR could mediate relatively stable contact to a cell that presents the important peptide antigen on the surface. However, it is not really expected to be the case, since there are three important conditions that favor the recognition of T cells through MHC-multimeric peptide on the recognition of antigen presenting cells through TCM. : i) multimeric complexes of MH C-peptide can form contacts with both TCR and co-receptors C D4 or CD8 on the surface of T cells. The multimeric TCR depends on the TCR / M HC-peptide contact only, ii) the TCR concentration on the T cell surface is significantly higher than the concentration of MHC-peptide on the surface of the antigen presenting cell (Engelhard, 1 994). iii) the antigen presenting cells present a multitude of different M HC-peptide complexes on their surface (Engelhard, 1 994), while a T cell will normally express only a combination of a / β or α / d. 9. Binding proteins to liposomes Liposomes are lipid vesicles made of double layers of lipid molecules enclosing an aqueous volume. Double layers of lipid are formed from membrane lipids, usually, but not exclusively, from phospholipids. Phospholipid molecules exhibit amphipathic properties and, therefore, are aggregated either in a crystalline state or in polar solvents to ordered structures with crystalline symmetries of typical lyotropic fluid. In aqueous solutions, the phospholipid molecules normally form spherical or oval self-closing structures, wherein one or more of the double phospholipid layers trap part of the solvent therein. The biologically active compounds trapped in liposomes are protected from the external environment and diffuse gradually to give a sustained effect. The delivery of drug through liposomes directed to specific locations through proteins on its surface has an enormous therapeutic potential (Alien, 1997; Langer, 1998). In particular, the low release of a drug at a specific location increases the efficacy of the drug, while allowing the entire amount that is administered to be reduced. The use of liposomes for such applications is developing rapidly and a large amount of data is emerging, for example, about their ability to circulate in the bloodstream (Uster et al., 1996) and their survival time (Zalipsky et al., 1996). ). A particularly useful aspect may be that drugs carried by liposome can be administered orally (Chen and Langer 1997, Chen et al., 1996, Okada et al., 1995). A number of reports describe the binding of antibodies or liposomes (Ahmad and Alien, 1992, Ahmad and others, 1993, Hansen and others, 1995). U.S. Patent 5,620,689 discloses the so-called "immunoliposomes", wherein the antibody or antibody fragments effective to bind to a selected antigen on a B lymphocyte or a T lymphocyte are bound to the distant ends of the membrane lipids in liposomes which they have a surface coating of polyethylene glycol chains. However, antibody-antigen interactions are usually of very high affinity and may not be suitable for multivalent activation for that reason.

DESCRIPTION OF THE INVENTION It is an object of the present invention to provide means for activating a specific MHC-peptide complex. It is a particular object of the present invention to provide TCR in a form that allows the detection of MHC-peptide-specific complexes, especially, although not exclusively, MHC-peptide complexes presented in vivo. It is a further object of the invention to provide an activated delivery vehicle capable of delivering reagents, in particular therapeutic agents, to expression sites of MHC-peptide-specific complexes in vivo. The inventors have now surprisingly found that TCR can be used very effectively for purposes of activation in vivo and a strategy for using TCR molecules for the purposes of activation has been successfully advised. The invention provides, in one aspect, a multivalent, synthetic T cell receptor (TCR) complex for binding to a M HC-peptide complex, said TCR complex comprising a plurality of T cell receptors specific for the MHC-peptide complex . The invention relates mainly to α-TCRs which are present in 95% of the T cells. In another aspect, the invention provides a multivalent TCR complex comprising or consisting of a multimerized recombinant T cell receptor heterodimer having improved binding capacity compared to a non-multimeric T cell receptor heterodimer. The multimeric T cell receptors may comprise two or more TCR heterodimers. In another aspect, the invention provides a method for detecting MHC-peptide complexes, the method comprising: (i) providing, (a) a T cell receptor complex A multivalent synthetic synthesis comprising a plurality of T cell receptors, and / or (b) a multivalent T cell receptor complex comprising a heterodimer of cell receptor. Multimerized recombinant T having improved lace ability compared to a non-multimeric T cell receptor heterodimer, said T cell receptors being specific for M HC-peptide complexes; (i i) contacting the mutant TC R complex with the M HC-peptide complexes; and (iii) detecting the binding of the multivalent TCR complex to the complexes of M HC-peptide. In yet another aspect, the invention provides a method for delivering a therapeutic agent to a target cell, said method comprising: (i) providing, (a) a multivalent synthetic TCR complex comprising a plurality of T cell receptors, and / or (b) a multivalent TCR complex comprising a multimerized recombinant T cell receptor heterodimer having improved binding capacity compared to a non-multimeric T cell receptor heterodimer, said T cell receptors being specific for the MHC-peptide complexes and the multivalent TCR complex having the therapeutic agent associated therewith; • A * 8 - '* - * (ii) contacting the multivalent TCR complex with potential target cells under conditions to allow binding of the T cell receptors to the target cell. Multivalent TCR complexes (or multimeric linker portions) according to the invention, are useful as such to screen or activate cells that display particular items in vitro or in vivo, and are also useful as intermediates for the production of other TCR complexes multivalencies having such uses. The multivalent TCR complex, therefore, can be provided in a pharmaceutically acceptable formulation for use in vivo. In the context of the present invention, a multivalent TCR complex is "synthetic" but can be found in, or is not native to, a living organism, for example, s i is non-metabolic and / or has no nucleus. In this way, for example, it is preferred that TCRs are in the form of multimers, or are present in a double layer of lipid, for example, in a liposome. It is also possible that the TCR complex can be formed by isolating T cells and removing the intracellular components, i.e., so that the complex has no nucleus, for example. The resulting "ghost" T cell can then simply have the cell membrane T including T cell receptors. Said ghost T cells can be formed through the lysis of T cells with a detergent, separating the intracellular components of the membrane (through centrifugation, for example) and then removing the detergent and reconstituting the membrane. In its simplest form, a multivalent TCR complex according to the invention comprises a multimer of two or three or four or more T cell receptor molecules associated (eg, covalently or otherwise linked) with one another, preferably through a linker molecule. Suitable linker molecules include multivalent binding molecules such as avidin, streptavidin and extravidin, each of which have four binding sites for biotin. In this manner, biotinylated TCR molecules can be formed in T cell receptor multimers having a plurality of TCR binding sites. The number of TCR molecules in the multimer will depend on the amount of TCR relative to the amount of linker molecule used to make the multimers, and also based on the presence or absence of any of the other biotinylated molecules. Preferred multimers are trimeric or tetrameric TCR complexes. Multivalent TCR complexes for use in screening or activation of cells expressing a specific MHC-peptide complex are preferably structures that are larger than TCR trimer or tetramers. Preferably, the structures have a diameter in the range of 10 nm to 10 μm. Each structure can display multiple TCR molecules at a sufficient distance apart to allow two or more TCR molecules ^^ on the structure are simultaneously linked to two or more MHC-peptide complexes on a cell and in this way increase the avidity of the multimeric binding portion for the cell. Suitable structures for use in the invention include membrane structures such as liposomes and solid structures, which preferably are particles such as beads, for example, latex beads. Other structures that can be externally covered with T cell receptor molecules are also suitable. Preferably, the structures are coated with multimeric T cell receptor complexes instead of with individual T cell receptor molecules. In the case of liposomes, the T cell receptor molecules can be attached to the outer part of the membrane or can be embedded within the membrane. In the latter case, the T cell receptor molecules including part or all of the transmembrane domain, can be used. In the first case, soluble T cell receptor molecules are formed. A soluble form of a T cell receptor is usually derived from the native form through the elimination of the transmembrane domain. The protein can be truncated by removing both the cytoplasmic and the transmembrane domains, or there can be an elimination only of the transmembrane domain with part or all of the cytoplasmic domain being retained. The protein can be modified to achieve the desired shape through proteolytic cleavage, or by expressing a truncated form genetically engineered or partially eliminated. In general, the soluble T cell receptor will contain the four external domains of the molecule, i.e., the variable domains a and ß and the constant domains a and ß. However, any soluble form of TCR that retains the MHC-peptide binding characteristics of the variable domains is considered. For example, it is possible to omit one or the other of the constant domains without significantly disturbing the link site. It is preferred if the multivalent TCR complex according to the invention comprises a recombinant T cell receptor heterodimer having improved binding capacity compared to a non -imeric T cell receptor heterodimeric heterodimer. The folded recombinant T cell receptor can comprise: i) an extracellular domain of α-chain? of the recombinant T-cell receptor having a first heterologous C-terminal dimerization peptide; and i) an extracellular domain of ß or d chain of recombinant T cell receptor having a second C-terminal dimerization peptide, which is specifically heterodimerized with the first dimerization peptide to form a domino of heterodimerization. Said recombinant TCR may be for recognizing MHC class I-peptide complexes and MHC class I-peptide complexes. The heterodimerization domain of the recombinant TCR ^^^ preferably is a so-called "coiled spiral" or "leucine closure". These terms are used to describe pairs of helical peptides, which interact with each other in a specific manner to form a heterodimer. The interaction occurs since there are complementary hydrophobic residues along one side of each closing peptide. The nature of the peptides is such that the formation of heterodimers is much more favorable than the formation of homodimers of the helices. Leucine closures can be synthetic or naturally occurring. Synthetic leucines can be designed to have a much higher binding affinity than naturally occurring leucine closures, which is not necessarily an advantage. In fact, the preferred leucine closures for use in the invention are naturally occurring leucine closures or leucine closures with similar binding affinity. The leucine closures of the c-jun protein and c-fos are an example of leucine closures with an appropriate binding affinity. Other suitable leucine closures include those of the myc and max proteins (Amati, Dalton and others 1992). Other leucine closures with adequate properties can be easily designed (O'Shea et al. 1993). It is preferred that the soluble TCRs in the multimeric binding portions according to the invention have leucine-closing fusions of about 40 amino acids corresponding to the heterodimerization domains of c-jun (chain) and c-fos (chain β) (O '). Shea, Rutkowski and others, 1992, Glover and Harrison, 1995). Longer leucine closures can be used. Since the specific character of the heterodimerization seems to be retained even in very short fragments of some leucine closing domains, (O'Shea, Rutkowski et al., 1992), it is possible that a similar benefit can be obtained with fragments of c- jun and c-fos shorter. Such shorter fragments can have as little as 8 amino acids, for example. In this way, the leucine closure domains can be in the range of 8 to 60 amino acids in length. The molecular principles of specific character in the pair of leucine closure are well characterized (Landschuiz, Johnson et al., 1988; McKnight, 1991) and leucine closures can be designed and engineered by those skilled in the art to form homodimers, heterodimers or trimeric complexes (Lumb and Kim, 1995, Nautiyal, Woolfson and others, 1995, Boice, Dieckmann and others, 1996, Chao Houston et al., 1996). The designed leucine closures, or other heterodimerization domains, of higher affinity than the closures of leucine c-jun and c-fos may be beneficial for the expression of soluble TCRs in some systems. However, as mentioned in detail below, when soluble TCR is doubled in vitro, a solubilizing agent is preferably included in the bending pH regulator to reduce the formation of non-productive protein aggregates. One interpretation of this phenomenon is that the fold kinetics of the leucine closure domains is faster than for the TCR chains, leading to the dimerization of the a and ß chain of unfolded TCR, at the same time causing protein aggregation . By reducing the folding process and inhibiting aggregation through the inclusion of a solubilizing agent, the protein can be maintained in solution until the folding of both fusion domains is complete. Therefore, higher affinity heterodimerization domains than c-fos and c-jun leucine closures may require higher concentrations of solubilizing agents to achieve a soluble TCRs yield comparable to that for c-jun and c -fos. Different biological systems use a variety of methods to form homo and hetero protein dimers, stable, and each of these methods in principle provides an option for the engineering design of dimerization domains in genetically modified proteins. Probably, leucine closures (Kouzarides and ZI F 1989) are the most popular dimerization modules and have been widely used for the production of genetically engineered dimeric proteins. Thus, the leucine closure of GCN4, a transcriptional activating protein of the yeast Saccharomyces cerevisiae, has been used to direct the homodimerization of a number of heterologous proteins (Hu, Newell et al., 1993, Greenfield, Montelione et al., 1988). . Therefore, the preferred strategy is to use closures that direct the formation of heterodimeric complexes such as the leucine closure pair J a / Fos (from Kruif and Logtenberg 1996, Riley, Ralston et al., 1996).

.Liii The heterodimerization domain is not limited to leucine closures. In this way, it can be provided through disulfide bridge forming elements. Alternatively, it can be provided through the SH3 domains and the hydrophobic / proline-rich contradomains, which are sensitive to the protein-protein interactions seen between proteins involved in signal transduction (reviewed by Schlessinger, (Schlessinger 1994)) . Other natural protein-protein interactions found between proteins that participate in signal transduction cascades are based on associations between post-translational modified amino acids and protein modules that specifically recognize such modified residues. Such modified amino acids after translation and protein modules can form the heterodimerization domain. An example of a protein pair of this type is provided by phosphorylated tyrosine receptors such as the Epidermal Growth Factor Receptor or Platelet Derived Growth Factor Receptor and the SO2 domain of GRB "(Lowenstein, Daly et al., 1992 Buday and Downward 1993) As in all fields of science, new dimerization modules are actively being sought (Vhevray and Nathans 1992) and the methods to engineer completely artificial modules are now being successfully developed (Zhang, Murphy and others, 1999.) In a preferred recombinant TCR, an interchain disulfide bond that is formed between two cysteine residues in the native TCR and β chains and between the native TCRα and d chains is absent. for example, by fusing the dimerization domains to the TCR receptor chains above the cysteine residues so that these residues are excluded from the recombinant oteína. In an alternative example, one or more of the cysteine residues is replaced by another amino acid residue, which is not involved in the disulfide bond formation. These cysteine residues may not be incorporated, as they can be dangerous to the in vitro folding of functional TCR. The retracted, of the chains a and ß or the chains? and d of the preferred refolded recombinant TCR of the multivalent TCR complex according to the invention takes place in vitro under conditions under suitable refolding conditions. In a particular embodiment, a recombinant TCR with the correct information is obtained through the refolding of TCR chains solubilized in a refolding pH regulator comprising a solubilizing agent, for example, urea. Advantageously, the urea may be present at a concentration of at least 0.1 M or at least 1 M or at least 2.5 M, or approximately 5 M. An alternative solubilization agent that can be used is guanidine, at a concentration of between 0.1 M and 8 M, preferably at least 1 M or at least 2.5 M. Before refolding, preferably a reducing agent is employed to ensure a complete reduction of cysteine residues. Other denaturing agents such as DTT and guanidine may be used, as necessary. Different denaturation and reduction agents can be used before the refolding step (e.g., urea, β-mercaptoethanol). Alternative redox couplings can be used during refolding, such as a redox coupling of cystamine / cysteamine, DTT or β-merc ethanol / atmospheric oxygen and cysteine in reduced and oxidized forms. Preferably, the recombinant TCR chains have a flexible linker or linker located between the TCR domain and the dimerization peptide. Suitable flexible linkers include standard glycine-containing peptide linkers, for example, linkers containing glycine and cerin. The C-terminal truncation near the cysteine residues forming the interchain disulfide bond is believed to be advantageous since the α and β chains are very close through these residues in ular TCRs. Thus, only relatively short linker sequences may be required to provide a distortionless transition from the TCR chains to the heterodimerization domain. It is preferred that the Pro-Gly-Gly or Gly-Gly linker sequences be used. However, the linker sequence can be varied. For example, the linker can be omitted completely, or reduced to an individual residue, the preferred choice in this case being an individual glycine residue. Variations of longer linkers in soluble TCR are also likely to be tolerated, provided they can be protected from protease attack, which can lead to the segregation of dimerization peptides from extracellular TCR domains ensuring loss of chain stability. a-ß. The soluble recombinant TCR is not necessarily a-BTCR. Molecules such as? -d, a-d and? -βTCR molecules, as well as TCR molecules that contain invariant chains (pre-TCR), which are only expressed early in development, are also included. Pre-TCR specifies that the lineage of the cell, which will express the T cell receptor to β, opposite those cells that will express the T cell receptor -d (for review, see (Aifantis, Azogui et al, 1998; von Boehmer, Aifantis et al., 1998; Wurch, Biro et al., 1998)). The Pre-TCR is expressed with the pairs of the ß chain of TCR with an invariant Pre-TCR α chain (Saint Ruf, Ungewiss and others, 1994, Wilson and MacDonald 1995), which appears to consign the cell to the lineage of T a-ß cell. The role of the Pre-TCR, therefore, is believed to be important during the development of thymus (Ramiro, Trigueros et al., 1996). Standard modifications to the recombinant TCR can be made as appropriate. These include, for example, altering a cysteine residue that is not in pairs in the constant region of the β chain to avoid the formation between incorrect chains or pairs between chains. The signal peptide can be omitted, as it serves no purpose in the mature receptor or its ligand binding ability, and can actually prevent the TCR from being able to recognize the ligand. In most cases, the cleavage site where the signal peptide is removed from the mature TCR chains is predicted, but not experimentally determined. The engineering design of the TCR chains expressed so that they are few, for example, up to about 10, for example, amino acids long or shorter at the N-terminus, will not matter for the functionality of the soluble TCR. Certain conditions that are not present in the original protein sequence can be added. For example, a short tag sequence, which can aid in the purification of TCR chains can be added, provided that it does not interfere with the correct structure and folding of the TCR antigen binding site. For expression in E. coli, a methionine residue can be engineered at the N-terminus starting point of the predicted mature protein sequence, in order to allow initiation of translation. Otherwise, all residues in the variable domains of the TCR chains are essential for the specificity and functionality of the antigen. In this way, a significant number of mutations can be introduced in this region without affecting the specific character and functionality of the antigen. In contrast, certain residues involved in the formation of contacts with the peptide antigen or the heavy chain polypeptide of H LA, ie the residues that constitute the CDR regions of the TCR chains, can be replaced by residues that can be improved. the affinity of the TCR for the ligand. Such substitutions, given the low affinity of most TCRs for MHS peptide ligands, may be useful to improve the specific character and functional potential of soluble TCRs. In the examples herein, the affinities of the soluble TCRs for peptide MHC ligands are determined. These measurements can be used to analyze the effects of mutations introduced in the TCR and in this way for the identification of the TCRs that contain substitutions that improve the activity of the TCR. Otherwise, all residues in the constant domains of the TCR chains are essential for the specificity and functionality of the antigen. In this way, a significant number of mutations can be introduced in this region, affecting the specific character of the antigen. In Example 17 below, it is shown that two amino acid substitutions in the constant domain of a β chain of TCR had no detectable consequences for the ability of TCR to bind an HLA-peptide ligand. ^ The TCR chain β contains a cysteine residue, which is uneven in the cellular or native TCR. The mutation of this residue improves the efficiency of the in vitro refolding of soluble TCR. Substitutions of this cysteine residue for cerin or alanine have a significant positive effect on the efficiencies of in vitro refolding. Similar positive effects, or even better effects, can be obtained with substitutions for other amino acids. As previously mentioned, it is preferred that the cysteine residues forming the interchain disulfide bond in native TCR are not present in order to avoid refolding problems. However, since the alignment of these cysteine residues is the natural design in the TCR and it has also been shown to be functional with this alignment for the leucine c-jun and c-fos closing domains (O'Shea and others, 1989), these residues d? cysteine can be included as long as the TCR can be refolded. Since the constant domains are not directly involved in contacts with peptide-MHC ligands, the c-terminal truncation point can be substantially altered without loss of functionality. For example, it should be possible to produce functional soluble TCRs excluding the entire constant domain. In principle, it may be simpler to express and fold soluble TCRs comprising only the variable regions or the variable regions and only a short fragment of the variable regions, since the polypeptides may be shorter. However, this strategy is not preferred. This is because the provision of additional stability of the pair of chain-ß through a heterodimerization domain can be complicated since the terms C designed by engineering the two chains can be spaced some distance away, requiring eslabonadoras sequences long The advantage of fusing heterodimerisation domains just prior to the cysteines forming placement link interchain disulfide, as is preferred, is that chains a and ß are kept very close to the cellular receptor. Therefore, the merger at this point is less likely to impose a distortion on the structure of the TCR. It is possible that functional soluble TCR could be produced with a larger fragment of the constant domains present than as is preferred here, ie these constant domains need not be truncated just prior to the cysteines forming the disulfide bond between chains. For example, the entire constant domain, except the transmembrane domain, can be included. It could be advantageous in this case to mutate the residues of cysteine that form in disulfide bond between chains in the cellular TCR. In addition to assisting interchain stability through a heterodimerization domain, the incorporation of cysteine residues which can form a disulfide bond between chains can be used. One possibility may be to truncate the α and β chains near the cysteine residues that form the interchain disulfide bond without removing these, so that the normal disulfide bond may occur. Another possibility may be to remove only the transmembrane domains of the α and β chains. If shorter fragments of the chains and ß are expressed, cysteine residues may be engineered, designed as substitutions at amino acid positions where the folding of the two chains can carry waste to an area suitable for the formation of disulfide bond. The purification of the TCR can be achieved through many different means. You can use alternative modes of ion exchange or other protein purification modes can also be used, such as gel filtration chromatography or affinity chromatography. In the method for producing a recombinant TCR, the folding efficiency can also be increased through the addition of certain other protein components, eg, chaperone proteins, to the refolding mixture. The improved refolding has been achieved by passing the protein through columns with immobilized minichaperones (Altamirano, Golbuk et al., 1997, Altamirano, García et al., 1999). In addition to the methods described in the examples, alternative means for the biotinylation of TCR may be possible. For example, chemical biotinylation can be used. Alternative biotinylation labels can be used, although certain amino acids in the biotin label sequence are essential (Schatz et al., 1993). The mixture used for biotinylation can also be varied. The enzyme requires Mg-ATP and a low ionic strength, although both conditions can be varied, for example, it may be possible to use a higher ionic strength and a longer reaction time. It may be possible to use a molecule other than avididine or streptavidin to form ¿TÍÍÁ ^^ i? I ^ ^ TCR multimers. Any molecule that binds biotin in a multivalent form may be adequate. Alternatively, a completely different linkage (such as polyhistidine tag for chelating nickel ions) may be advised (Quiagen Product Guide 1999, Chapter 3"Protein Expression, Purification, Detection and Assay" on page 35-37) .Preferably, the label is located to the C-terminus of the protein in order to minimize the amount of steric hindrance in the interaction with MHC peptide potential complexes To enable detection of the multivalent TCR complex, for example, for diagnostic purposes, it can be include a detectable label A suitable label can be chosen from a variety of detectable labels Suitable types of labels include fluorescent, photoactivatable, enzymatic, epitope, magnetic and particle (eg, gold) labels. suitable for use in vitro are fluorescent labels such as FITC particularly suitable for in vivo use are the brands They are suitable for the formation of external images after administration to a mammal, such as a radionuclide that emits radiation that can penetrate soft tissue. The brand can be linked to or incorporated into the multivalent TCR complex at any suitable site. In the case of liposomes it can be attached to or incorporated in the membrane, or to the inside of the membrane. In the case of particles or beads, the tag may be located in the same particle or bead, or attached to the outer part, for example, in the T-cell receptor molecules. Conveniently, the tag binds to a multivalent linker molecule , from which T-cell receptor complexes are formed. In tetrameric TCR formed using biotinylated heterodimers, fluorescent streptavidin (commercially available) can be used to provide a detectable label. A fluorescently labeled tetramer will be suitable for use in FACS analysis for example, to detect antigen presenting cells carrying the peptide for which the TCR is specific. Another way in which multivalent TCR complexes can be detected is through the use of specific antibodies in TCR, in particular monoclonal antibodies. There are many commercially available anti-TCR antibodies, such as ßFI and Fl, which recognize the constant regions of the β chain and the α chain, respectively. For therapeutic applications, a therapeutic agent is bound or incorporated into the multivalent TCR complex according to the invention. In a preferred embodiment, the multivalent TCR complex for therapeutic use is a liposome coated with T cell receptors, the therapeutic agent being trapped within the liposome. The specific character of the T-cell receptors allows the localization of drugs contained in liposome at the desired target site, such as a tumor or a cell infected with a virus. This can be useful in many situations, and in particular, against tumors since not all cells in the tumor present antigens and, therefore, not all tumor cells are detected by the immune system. With the multivalent TCR complex, a compound can be delivered so that it can exert its effect locally, but not only on the cell where it binds. In this manner, a particular strategy considers anti-tumor molecules associated with or linked to multivalent TCR complexes comprising T cell receptors specific for tumor antigens. The therapeutic agent can be, for example, a toxic moiety, for example, for use in the killing of cells, or an immunostimulating agent such as an interleukin or a cytokine. Many toxins can be used for this use, for example, radioactive compounds, enzymes (perforin for example) or chemotherapeutic agents (cis-platin, for example). An example of the multivalent TCR complex according to the invention is a tetramer that contains after TCR molecules and a peroxidase molecule. This can be achieved by mixing the TCR and the enzyme at a molar ratio of 3: 1 to generate tetrameric complexes and isolate the desired multimer from any complex that does not contain the correct ratio of molecules. Mixed molecules can contain any combination of molecules, as long as the steric hindrance does not compromise or does not significantly compromise the desired function of the molecules. The placement of the binding sites on the streptavidin molecule is suitable for mixed tetramers, since steric hindrance does not occur. Although it is an object of the invention to provide multivalent TCR complexes having a plurality of T cell receptors of identical specific character, the possibility that T cell receptors of a different specific character are also present is not excluded. In fact, there may be advantages in having two or more specific characters different from the T cell receptor, such as the possibility of activating one or more different MHC-peptide complexes at the same time. This can be useful, for example, to ensure the detection of a target antigen in different individuals having different types of H LA, since an identical foreign antigen can be processed differently and presented according to the H LA type. Similarly, the inclusion of molecules that have a different binding activity to that of the T cell receptor is also considered. Such molecules may improve the activation ability, or perform a useful function once the multivalent TCR complex has reached its goal. Examples of useful accessory molecules include CD8 to support recognition of MHC-peptide complexes through the T cell receptor, and receptors with an immunomodulatory effect. Examples of suitable MHC-p-peptide targets for the multivalent TCR complex according to the invention include, but are not limited to, viral epitopes such as HTLV-1 epitopes (eg, the Tax peptide restricted by H LA-A2; HTLV-1 is associated with leukemia), VI H epitopes, EBV epitopes, CMV epitopes; epitopes of melanoma and other cancer-specific epitopes; and epitopes associated with autoimmune disorders, for example, rheumatoid arthritis. In more detail, liposomes coated with the T cell receptor according to the invention (which can also be described as "artificial T cells") can be constructed as follows. Production of "artificial T cells" There are a number of methods for the production of liposomes. In the simplest method, dried phospholipid films are deposited in a round bottom flask with excess solvent under moderate or vigorous agitation (Bangham et al., 1965). Other methods include the application of sound to multilamellar vesicles (MLVs) (Huang, 1969), forcing a suspension of MLVs through a French press (Barenholzt et al., 1979), or through solubilization with lipid detergent. The detergent can be removed by dialysis, chromatography, adsorption, ultrafiltration or centrifugation (Brunner et al., 1976). A number of techniques for binding proteins to the surface of liposomes, usually through modified lipids, have been described. One method of these uses biotinylated lipids. Herein is described a method for producing a biotinylated T cell receptor, which can be linked to biotinylated lipid via, for example, avidin, streptavidin or extravidin. Another coupling method uses polyethylene glycol (PEG) for binding antibodies to liposomes (Hansen et al., 1995) and the use of S-succinimidyl-S-thioacetate (SATA) has also been described (Konigsberg et al., 1988). These techniques produce small unilamellar vesicles with sizes ranging from 20-100 nm. Due to stability problems and in order to allow entrapment of a larger scale of materials, preparation methods for larger unilamellar vesicles have been developed. These include dehydration-rehydration of liposomes (Tan and Gregoriadis, 1990), vesicles made through reverse phase evaporation (Szoka and Papahadjopoulos, 1978), or freeze-thaw extrusion (Mayer et al., 1985). With these methods, encapsulation efficiencies of up to 65-80% can be achieved. When initially discovered, liposomes were unstable, but in recent years, such problems have been overcome through the use of more sophisticated lipid forms and derivatized lipids (see (Alien, 1994) for review). The packaging of drugs, for example, doxorubicin (Ahmad and Alien, 1992; Ahmad et al., 1993), protein / antigens (Cohen et al., 1994; Cohen et al., 1991), or insulin (Edelman et al., 1996). be considered an established technology.

Advantages of TCR multimers linked to liposome The general advantages of this technology can be summarized in the following points: liposomes are cheap, easy to produce, easy to load using standard technology, and easy to load with a multitude of therapeutic compounds. Reagents for making liposomes, including biotinylated lipids are readily available, for example from Avanti Polar Lipids Inc., USES. the liposomes and proteins are biodegradable. TCR and lipids are non-immunogenic, therefore it is remote to evoke secondary immune responses.

Advantages of "artificial T cells" In its ability to screen antigen presenting cells and their use for this purpose, and to transport compounds to said cells, it was predicted that the TCRs bound to the liposome and the TCR multimers linked to the liposome have a number of advantages over TCR tetramers. These can be summarized in the following points: free lateral movement of TCR bound to liposome avoids any stearic impediment that could impede TCR / MHC-peptide contacts. Indeed, the lateral mobility of TCRs in lipid binding in a liposome will be reminiscent of their ability to move in the T cell membrane.

The flexibility in the liposome surface is reminiscent of the flexibility of the actual cell membrane, potentially allowing a better surface contact than that which can be obtained with a tetramer or other simple complex. - a high number of TC Rs can be linked to a liposome, therefore, a high avidity link can be ensured. With TC R tetramers, the binding will depend on the sufficient avidity that is obtained through a maximum of four TCR / MHC-peptide contacts. - both for in vitro and in vivo use, the TCR bound to liposome is less likely to lose functionality through the degradation of TC R, due to the totally high number of TCRs that can be linked to liposome than in the case of a tetramer or other complex of simple R C. - the concentration of TCR in lipids can be controlled by showing biotinylated and non-biotinylated lipids in variable relationships. Similarly, lipids with other modifications that make them useful for protein binding, for example, lipids derivatized with PEG (Alien et al., 1,995; Hansen et al., 1,995) or derivatized with SATA (Konigsber et al. 1,988) can be mixed in variable ratios. This allows the interaction resistance to the antigen presenting cell to be adapted to TCRs with different affinity or to the domino of the peptide epitope in the antigen presenting cell.

The potential to bind high numbers of molecules to the postope opens the possibility of creating liposomes with specific character of M HC-mutant peptide, using more than one TCR. For example, it can be considered that two or more TCs specific for different epitopes associated with the same disease could be linked in a liomasome giving these multiple specific characters with which cells that are affected by some disease are detected, similarly, TC R can be mixed with other molecules or proteins that can exert other desired functions near antigen presenting cells. For example, cytokines or cytokine receptors, specific antibodies, superantigens, type C co-receptors D2, DC4, C D8 or CD28, or peptides, may have properties that may be useful in this context. This application may have a very broad potential to locate reagents near certain antigen presenting cells.

Examples of drugs and diseases that can be activated with multivalent TCR complexes. A multitude of treatments for diseases can be potentially improved by locating the drug through the specific character of multivalent TCR complexes, in particular the use of TCR in liposome binding will be useful. Viral diseases for which there are drugs, for example, VI H, SIV, EBV, CMV, can benefit from the drug that is being released very close to infected cells. For cancer, localization near tumors or metastases may improve the effect of toxins or immunostimulants. In autoimmune diseases, immunosuppressive drugs can be released slowly, having a more local effect for a longer time, while minimally affecting total immunocapacity. In the prevention of graft rejection, the effect of immunosuppressive drugs can be optimized in the same way. For vaccine delivery, the vaccine antigen can be located close to the professional antigen presenting cells, thereby improving the antigen's efficacy. The method can also be applied for imaging purposes. Preferred aspects of each item of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated in their entirety by reference. The invention is further described in the following examples, which do not limit the scope of the invention in any way. Reference is made in the following to the appended drawings, in which: Figure 1 is a schematic view of a cell receptor of the leucine-binding fusion T-protein. Each chain consists of two domains of the immunoglobulin superfamily, u non-variable (V) 25 and one constant (C). The constant domains are truncated n i, 'i r i ip. -? i- i-i i-i-ii - ________________ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^ M ßm immediately n-terminal of cysteine residues between chains, and fused to a heterodimerization motif of leucine closure from c-Jun (a) or c-Fos (ß) of approximately 40 amino acids in the C-terminal through a short linker. The a-Jun and ß-Fos each contain two disulfide bonds between chains and one pair only by non-covalent contacts. The alpha chain is shorter than the beta chain due to a smaller constant domain. Figure 2 is a photograph of a reduction / nonreduction gel analysis of the heterodimeric JM22z? P receptor. Identical samples of the purified TCR closure were loaded on an SDS gel of 15% acrylamide, either under reducing conditions (lane 2) and under non-reducing conditions (lane 4). Marker proteins are shown in lanes 1 and 3. Molecular weights are shown in kilodaltons. Under both groups of conditions, the non-covalently associated heterodimer is dissociated into α and β chains. In lane 4, each chain runs with a higher mobility and as an individual band, indicating an individual species of interchain disulfide bond that is present. This is compatible with the correct disulfide bond formation. Figure 3 is a graph showing the TCR specific binding of JM22zip to the HLA-A2 Flu matrix complexes (M58-66). The complexes of H LA-A2, folded around individual and biotinylated peptides in β-2 microglobulin, were immobilized on three flow cells coated with streptavidin: 3770 resonance units (RU) of control H LA-A2 POL on the cell of flow (FC) 3, and two different levels of H LA-A2 M58-66 FLU (2970 RU in FC 1 and 4960 RU in FC2). JM22zip was injected in the soluble phase sequentially over the three flow cells at a concentration of 43 μM for 60 seconds. During the injection, an increase was observed before the antecedent in the response of both flow cells coated with HLA-A2 FLU, with approximately 1000 RU and 700 RU of specific binding of JM22zip to the flow cells 1 and 2, respectively. Figure 4 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR alpha chain of H LA-A2 matrix / soluble flu from JM22, as merged into the "leucine lock" domain of c-jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli are indicated in small letters since it is the linker sequence between the TCR and the c-jun sequences. Figure 5 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR beta-restricted chain of H LA-A2 / flu matrix from JM22, as it was fused to the domain of "leucine closure" of c-fos. The linking sequence between the TCR and the c-fos sequences is indicated in small letters. Mutation of the DNA sequence that substitutes a serine residue for a cysteine residue is indicated in bold and underlined. This mutation increases the folding efficiency of the TCR. Figure 6 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR beta chain of the H LA-A2 / soluble flu matrix from JM22, as merged into the domain of "leucine closure" of c-fos and the biotinylation label, which acts as a substrate for BrA. The linker sequence between the TCR and the c-fos sequences, and between c-fos and the biotinylation label, are indicated in small letters. Mutation of the DNA sequence that substitutes a serine residue for a cysteine residue is indicated in bold and underlined. This mutation increases the folding efficiency of the TCR. Figure 7 is a schematic diagram of the fusion protein of the TCR closing biotinylation label. Figure 8 shows the results of refolded TCR elution from the POROS 10HQ column with a sodium chloride gradient. The TCR is eluted as an individual peak at approximately 100 mM NaCl. The fractions containing the protein with an external diameter (280 nm) of more than 0.1 were combined and concentrated for biotinylation. Figure 9 shows the results of the separation of biotinylated TCR from free biotin through gel filtration on a Superdex 200H R 10/30 column (Pharmacia). The TCR-biotin is eluted around 15 ml, corresponding to a molecular weight of 69 kDa. (Standard proteins and their elution volumes: Thyroglobulin (669 kDa) 10.14 ml, Apoferritin (443 kDa) 1 1 .36 ml, beta-amylase (200 kDa) 12.72 ml, BSA dimer (134 kDa) 13.12 ml, BSA monomer (67 kDa) 14.93 ml, ovalbumin ( 43 kDa) 15.00 ml, chymotrypsinogen A (25 kDa) 18.09 ml, RNase (1 3.7 kDa) 18.91 ml). 5 Figure 10 shows the results of gel filtration d? TCR tetramers on a Superdex 200 H R 10/30 column. The peaks at 14.61 and 12.74 correspond to BSA (monomer and dimer) used to stabilize extravidin. The peak at 1 1 .59 contains TCR tetramers as judged by the presence of FITC yellow when extravidin-FITC was used to tetramerize. This peak corresponds to a molecular weight of 340 kDa, consistent with a TCR tetramer linked to extravidin. Figure 1 1 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the alpha chain of TCR restricted from HTLV-1 Tax / H LA-A2 from clone A6 (Garboczi et al., 1996; Garboczi et al., 1996), as merged into the "leucine lock" domain of c-jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli are indicated in small letters such as link sequence between TCR and the sequences c-jun. Figure 12 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR beta chain of soluble HTLV-1 -Tax / HLA-A2 from clone A6 (Garboczi and others, 1996; Garboczi et al., 1996), as merged with domain of "leucine closure" of c-gos and the label of , aaaA ^ a ^ MUi ^ rii¡ > t_ biotinylation), which acts as a substrate for BirA. The link chain between TCR and the c-fos sequences is indicated in small letters. The mutation of the DNA sequence that replaces an alanine residue with a cysteine residue is indicated in bold and underlined. Figure 13 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR alpha chain of soluble HTLV-1 Tax / HLA-A2 from clone M10B7 / D3 (Ding and others, 1998), as merged into the "leucine lock" domain of c-jun. The linking sequence between TCR and the c-jun sequences is indicated in small letters. Figure 14 shows the protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR beta chain of soluble HTLV-1 Tax / HLA-A2 from clone M10B7 / D3 (Ding et al., 1998), as merged with the "leucine lock" domain of c-fos and the biotinylation label, which acts as a substrate for BirA. The linking sequence between TCR and the c-jun sequences is indicated in small letters. The mutation of the DNA sequence that replaces an alanine residue with a cysteine residue is indicated in bold and underlined. Two silent mutations (P-G codons) introduced for cloning purposes and to remove an Xmal restriction site are also indicated in small letters. Figure 15 shows the sequences of synthetic DNA primers used for "TCR gene anchoring amplification". The recognition sites for DNA restriction enzymes used for cloning are underlined. A: "anchor initiator" poly-C. B: specific initiator of TCR alpha chain constant region. C: specific initiator of TCR beta chain constant region. Figure 16 shows the sequences of the synthetic DNA primers used for PCR amplification of DNA fragments encoding the coiled spiral ("leucine lock") regions of 40 amino acids of c-jun and c-fos. The recognition sites for the DNA restriction enzymes used for cloning are underlined. A: initiator c-jun 5 '. B: initiator c-jun 3 '. C: 5 'c-fos initiator. D: c-fos 3 'primer. Figure 17 shows the DNA and amino acid sequences respective (one letter code) of the c-fos and c-jun fragments as used for TCRs (inserts in pBJ 107 and pBJ 108). A: c-jun leucine closure as fused to TCR alpha chains. B: leucine c-fos closing as fused to TCR beta chains. Figure 18 shows the sequences of the synthetic DNA primers used for the mutation of the cysteine residue without pairs in TCR beta chains. The primers were designed for use with the "Quickchange ™" method for mutagenesis (Stratagene). A: mutation of cysteine to serine, forward primer (sense), indicating amino acid sequence and mutation. B: mutation of cysteine to serine, initiator backwards (no sense). C: mutation of cysteine to alanine, initiator forward (sense), indicating the amino acid sequence and the mutation. D: mutation of cysteine to alanine, initiator backwards (no sense). Figure 19 is a schematic representation of TCR-closing fusion protein. The four immunoglobulin 5 domains are indicated as domes, with the disulfide bridges between chains between matching pairs of cysteine residues, shown. The numbers indicate the amino acid positions in the mature T cell receptor chains; due to the slight variation in chain length after recombination, the lengths of the chains may vary slightly between the different TCRs. The residues introduced in the linking sequences are indicated in the one letter code. Figure 20 shows the sequences of synthetic DNA primers used for the PCR amplification of a and ß of TCR. The recognition sites for DNA restriction enzymes are underlined and the amino acid sequences corresponding to the respective TCR chains are indicated on the forward primer sequences. Silent DNA mutations relative to the TCR gene sequences and other DNA sequences that do not correspond to the TCR genes are shown in lowercase letters. A: 5 'PCR primer for the human Va10.2 chain of JM22-TCR influenza virus virus peptide restricted by H LA-A0202. B: 5 'PCR primer for the human VB17 chain of the influenza virus virus peptide JM22-TCR restricted by HLA-A0201. C: 5 'PCR primer for the riliU aMi Va4 mouse chain of influenza-TCR nucleoprotein peptide restricted by H2-Db. D: 5 'PCR primer for the mouse Vβ11 chain of TCR influenza nucleoprotein peptide restricted by H2-Db. E: 5 'PCR primer in human Va23 chain of peptide 003-HIV-1 Gag-TCR restricted by HLA-A0201. F: 5 'PCR primer from the human VB5.1 chain of peptide 003 HIV-1 Gag-TCR restricted by HLA-A0201. G: 5 'PCR primer from the human Va2.3 chain of the HTLV-1 Tax-TCR peptide of A6 restricted to HLA-0201. H: 5 'PCR primer from the human VB12.3 chain of the HTLV-1 Tax-TCR A6 peptide restricted by HLA-A0201. I: 5 'PCR primer from the human Va17.2 chain of the HTLV-1 Tax-TCR B7 peptide restricted by HLA-A0201. J: 5 'PCR primer from the human Vß12.3 chain of the HTLV-1 Tax-PCR B7 peptide restricted by HLA-A0201. K: 3 'PCR primer for human Ca chains, generally applicable. L: PCR 3 primer for human Cß chains generally applicable. Figure 21 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR α chain of the HLA-A2 / soluble flu matrix from JM22, as merged into the domain of "leucine closure" of c-jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli were indicated in small letters, as is the linker sequence between the TCR and c-jun sequences. Figure 22 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the restricted TCR β chain of H matrix LA-A2 / soluble flu from J M22, as fused to "leucine closure" domain of c-fos. The linker sequence between the TCR and c-fos sequences is indicated in small letters. Figure 23 shows the predicted protein sequence (one-letter code, upper) and the DNA sequence (lower) of the TCR-α restricted nucleoprotein chain of soluble influenza / H2-Db from the murine F5 receptor , according merged into the domain of "closing leucine" of c-jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli are indicated in small letters, as is the linker sequence between the TCR and c-jun sequences. 15 Figure 24 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR β-chain restrained from soluble influenza / H2-D virus nucleoprotein from the murine F5 receptor, as merged into the domain of "closure of leucine "from c-fos. Sequence The linker between the sequences of TCR and c-fos is indicated in small letters. Figure 25 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR chain a restricted by H LA-A2 / H IV-1 soluble Gag of the patient 003, as merged into the "leucine lock" domain of c- lttite ^ ÍltfHNi? ^ jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli are indicated in small letters, as is the linker sequence between the TCR and c-jun sequences. Figure 26 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR β chain restricted by soluble HLA-A2 / HIV-1Gag from patient 003, as fused to the "leucine close" domain of c-fos . The linker sequence between the TCR and c-fos sequences is indicated in small letters. Figure 27 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of clone A6 of the TCR chain a restricted by HTLV-1 Tax / soluble HLA-A2 (Garboczi, Utz and others, 1996; Garboczi, Ghosh et al., 1996), as merged into the "leucine lock" domain of c-jun. Mutations introduced at the 5 'end of the DNA sequence to improve gene expression in E. coli are indicated in small letters, as is the linker sequence between the TCR and c-jun sequences. Figure 28 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR ß chain restricted by soluble HTLV-1 Tax / HLA-A2 from clone A6 (Garboczi, Utz et al., 1996; Garboczi, Ghosh and others, 1996), as merged with the "leucine lock" domain of c-fos and the biotinylation tag, which acts as a substitute for BirA (Barker and Campbell, 1981; Barker and Campbell, 1981; Howard, Shaw and others, 1985; Schatz, 1993; O'Callaghan, Byford, 1999). The linking sequence of the TCR and c-fos sequences is indicated in small letters. The mutation of the DNA sequence that replaces the cysteine residue for an alanine residue is indicated in bold and underlined. Figure 29 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR chain a restricted by HTLV-1 Tax / soluble HLA-A2 from M10B7 / D3 (Ding and others, 1998), as merged into the "leucine lock" domain of c-jun. The linker sequence between the TCR and c-jun sequences is indicated in small letters. Figure 30 shows the predicted protein sequence (one letter code, upper) and DNA sequence (lower) of the TCR β chain restricted by soluble HTLV-1 Tax / HLA-A2 from clone M10B7 / D3 ( Ding et al., 1998) as merged with the "leucine lock" domain of c-fos and the biotinylation label that acts as a substitute for BirA. The linker sequence between the TCR and c-fos sequences is indicated in small letters. The mutation of the DNA sequence that substitutes an alanine residue for a cysteine residue is indicated in bold and underlined. Two silent mutations (P-G codons) introduced for cloning purposes and to remove an Xmal restriction site are also indicated in small letters. Figure 31 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the TCR β chain restricted by HTLV-1 Tax / H LA-A2 soluble from clone A6 (Garboczi , Utz et al., 1996; Garboczi, Ghosh et al., 1996), as merged into the domain of "leucine closure" and c-fos and the biotinylation label, which acts as a substitute for (Barker and Campbell, 1981; Barker and Campbell, 1981, Howard, Shaw, 1985, Schatz, 1993, O'Callagham, Byford, 1999). The linker sequence between the TCR and c-fos sequences is indicated in small letters. The mutation of the DNA sequence that substitutes a cysteine residue for a residue of aianine is indicated in bold and underlined. A substitution of an asparagine residue for an aspartic acid is also indicated in bold and underlined. A mutation in the constant region that had no detectable functional effect on soluble TCR. Figure 32 shows the predicted protein sequence (one letter code, upper) and the DNA sequence (lower) of the biotinylation fusion pattern) c-fos used for TCR ß chains. The recognition sites for DNA restriction enzymes are underlined and the boundaries or boundaries of the two fusion domains are indicated. The linking sequences are shown in lowercase letters. Figure 33 shows the sequence of a synthetic DNA primer used for the PCR amplification of the leucine Vß-c-fos closing fragment of human JM22 influenza matrix peptide LA-A0201.

Figure 34 is a group of photographs of gels. to. Preparation of denatured protein for TCR specific for the 003 H IV gag-HLA-A2 peptide complex analyzed by SDS-PAGE. Lane 1: large-scale molecular weight markers (Bio-Rad), lanes 2 and 3: bacteria after protein induction with 0.5 mM of IPTG, lanes 4 and 5: purified inclusion bodies solubilized in 6M pH buffer of guaniclina. b. Preparation of denatured protein for the TCR labeled with biotin specific for the HLA-A2 influenza matrix peptide complex analyzed by SDS-PAGE. Lane 1: broad-scale molecular weight markers (Bio-Rad), lanes 2 and 3: purified inclusion bodies of α-chain and β-solubilized in 6M guanidine pH buffer. c. Preparation of denatured protein for the TCR labeled with biotin specific for the peptide complex HTLV tax-H LA-A2 analyzed through SDS-PAGE. Lanes 1 and 5: large scale molecular weight markers (BioRad), lanes 2, 3 and 4: β chain expression of mutant a and β in bacteria after induction of protein expression with 0.5 mM of I PTG, lanes 6, 7 and 8: β-chain purified inclusion bodies of a, β-mutants solubilized in 6M guanidine pH buffer. Figure 35 is a chromatogram showing the elution of the JM22z heterodimer from a POROS 10HQ anion exchange column. The dotted line shows the conductivity, which is indicative of a concentration of sodium chloride, the line M ^ M ^ MMÉMihab solid shows the optical density 280 nm, which is indicative of the protein concentration of the eluate. The fractions containing the peak protein were combined for further analysis. The insert shows a chromatogram of elution of purified JM22z from a Superdex 200 HR column. The arrows indicate the caliber of the column with proteins of known molecular weight. In comparison with these proteins, the refolded JM22z protein has a molecular weight of approximately 74 kDa, which is compatible with a heterodimeric protein. Figure 36 is a photograph showing an SDS-polyacrylamide gel electrophoresis (Coomassie stained) of the purified JM22z protein. Lanes 1 and 3: standard proteins of known molecular weight (as indicated), lane 2: JM22z protein treated with pH regulator of SDS-sample containing a reducing agent (DTT) before loading the sample, lane 4 : JM22z protein with pH regulator SDS-sample in the absence of reducing agents. Figure 37. a ^ Purification of refolded biotin-labeled TCR for the LA-A2 influenza-H matrix peptide complex. i. Chromatogram of protein elution from a POROS 10HQ column. The line x indicates the absorbance 280 nm and the line and indicates the conductivity (a measure of the gradient of sodium chloride used to elute the protein). The fraction numbers are indicated by the vertical lines, ii. SDS-PAGE of the fractions eluting from the column as in i. Lane 1 contains large scale molecular weight markers (Bio-Rad) and lanes 2-13 contain 5 μl of fractions 6-15, respectively, iii. The SDS-PAGE analysis of combined fractions of i. containing TCR of flu marked with biotin. Lane 1: large scale molecular weight markers (Bio-Rad), lane 2: biotin-labeled flu-TCR protein. b. purification of TCR-labeled biotin-specific TCR for the peptide complex HTLV-tax-H LA-A2. i. Chromatogram of the elution of the protein from a POROS 10H column. The line x indicates the absorbance at 280 nm and the line indicates the conductivity (a measurement of the sodium chloride gradient used to elute the protein). The fraction numbers are indicated by the vertical lines, ii. SDS-PAGE of the fractions are eluted from the column as in i. Lane 1 contains large scale molecular weight markers (Bio-Rad) and lanes 2-10 contain 5 μl of fractions 3-1 1, respectively, iii. The SDS-PAGE analysis of combined fractions of i. of tax-TCR marked with biotin. Lane 1: broad-scale molecular weight markers (Bio-Rad), lane 2: biotin-labeled tax-TCR protein, lane 3: biotin-labeled tax-TCR protein, mutant. Figure 38 is a chromatogram showing the elution of biotinylated soluble TCR after biotinylation with the BirA enzyme from a Superdex 200 HR column equilibrated in PBS. The biotinylated TCR elutes around 15-16 minutes and the free biotin elutes in about 21 minutes. The fractions HiMMiiiiiia containing biotinylated soluble TCR are combined for future use. Figure 39 is a group of photographs of gels. The determination of biotinylation of biotinylated TCRs. to. SDS-PAGE of refolded TCRs and inclusion body preparations. Large scale molecular weight markers (Bio-Rad), lane 2: biotinylated flu-TCR, lane 3: biotinylated tax-TCR, lane 4: biotinylated mutant tax-TCR, lane 5: H IV gag-TCR (unlabeled) biotin); b. Western staining of a gel identical to a. , except that the wide scale markers were marked with biotin (Bio-Rad). The staining was with a conjugate of avidin-HRP to show the biotinylated proteins and visualization was with Opti-4CN (Bio-Rad). Figure 40 illustrates the binding of J M22z to different H LA-A2-peptide complexes (one insert). The specific character of the interaction between JM22z and H LA-A2-flu is demonstrated by comparing the SPR response by passing the TCR over a flow cell covered with 1900 RU of H LA-A2 with the responses of passing the TCR over other two flow cells covered with 4200 RU of HLA-A2, the other covered with 4300 RU of CD5. Previous responses at different concentrations of JM22z were measured at 1700 RU of H LA-A2-pol (a). The previous value was subtracted from the specific response measured at 1900 RU of H LA-A2 flu (b), and plotted against concentration (c). The Kd value of 13 μM, estimated through the non-linear curve fixation, was in accordance with the Kd value of 12 μM calculated based on a Scatchard plot of the same data. Figure 41 is a graph showing the result of the Biacore 2000 ™ analysis of biotinylated, soluble, mutant and wild type TCR taxa. 5 μl of wild-type TCR taxa were flowed at a concentration of 2.2 mg / ml and then mutant TCR tax at a concentration of 2.4 mg / ml on 4 flow cells with the following proteins bound to the following surface: A: complex tax-pMHC, B / C: flu-pMHC complex, D: OX68 control protein. Both wild-type and mutant proteins were similarly linked to the specific pMHC complex. Figure 42 shows the effect of the soluble CDßaa bond on the soluble TCR bond to the same LA-A2-flu H complex. (A) TCR or TCR plus 120 μM soluble CD8 were injected into a control flow cell covered with 4100 RU of an irrelevant protein (CD5) and a probe flow cell covered with 4700 RU of HLA-A2-flu. After subtraction of the previous value, the equilibrium response values calculated at different concentrations of TCR (open circles) or in combination with 120 μM of soluble CD8 (closed circle), are shown. The CD8 value is also displayed only (open triangles) and the difference calculated between TCR + CD8 and TCR only (open squares). (B) the time dependence of responses on 4700 RU of H LA-A2-immobilized flu of 49 μM of TCR only (open circles) or in combination with 120 μM of CD8 (closed circles) at 25 ° C and one speed flow rate of 5 μl / minute, shown (values corrected for previous contributions measured at 4100 RU of immobilized CD5); the open TCR speed is not affected by the simultaneous CD8 link. Figure 43. Tetramerization of biotinylated TCR using extravidin. Gel filtration using a Superdex 200 H R column shows that the biotinylated TCR and extravidin combine to form a higher molecular weight oligomer than any protein. Gel filtration chromatograms: A. extravidin, B. biotinylated TCR, C. TCR tetramers. Figure 44. Tetramerization of biotinylated TCR using streptavidin modified with RPE. Gel filtration using a Superdex 200 H R column shows that the biotinylated TCR and streptavidin-RPE combine to form a higher molecular weight oligomer than any protein. Gel filtration chromatograms: A. streptavidin-RPE, B. Biotinylated TCR, C. TCR-RPE tetramers. Figure 45A is a graph showing the results of the BIAcore analysis of biotinylated soluble flu-TCR. 5 μl of flu-TCR at a concentration of 1 mg / ml was flowed on three flow cells with the following obtained through streptavidin-i: non-specific control protein, ii: flu matrix pMHC, iii: tax pMHC . Figure 45B. Figure 45B is a graph showing the results of the BIAcore analysis of flu-TCR tetramers. 5 μl of the flu-TCR tetramer solution was flowed at a concentration of 0.05 mg / ml onto three flow cells with the following bound via streptavidin, i: nonspecific control protein, ii: pMHC fluid matrix , iii: tax pMHC. Figure 46A is a graph showing the results of the BIAcore analysis of biotinylated soluble tax-TCR. 5 μl of flu-TCR was flowed at a concentration of 1 mg / ml onto three flow cells with the following obtained through streptavidin-i: non-specific control protein, ii: flu matrix pMHC, iii: tax pMHC . Figure 45B is a graph showing the results of the BIAcore analysis of tax-TCR tetramers. 5 μl of the flu-TCR tetramer solution was flowed at a concentration of 0.05 mg / ml onto three flow cells with the following ones bound through streptavidin, i: nonspecific control protein, ii: pMHC fluid matrix , iii: tax pMHC. Figure 47. FACS analysis of T2 cells pulsed with varying levels of peptide and stained with specific TCR tetramers either for the influenza matrix peptide or for the HTLV tax peptide. A. Cell gate for analysis. B. T2 cell staining pulsed with "Data.001" = 0 peptide; Data.007"= 10" 4M of the peptide flu; "Data.009" = 10 5M of the peptide flu; "Data.010" = 10"6M of the peptide flu;" Data.003"= 10" 4 of the peptide tax, all stained with 5 μg of the RPE-tagged tetramers flu-TCR. C. Staining of T2 cells pulsed with: "Data.002" = 0 peptide; "Data.004" = 10_4M of the peptide tax; "Data.005" =: 10_5M of the peptide tax; "Data.006" = 10"6M of the peptide tax;" Data.008"= 10" 4 of the peptide flu, all stained with 5 μg of the tax-TCR tetramers labeled with RPE.

Figure 48. The FACS analysis of .45 cells pulsed with varying levels of peptide and stained with specific TCR tetramers either for the influenza matrix peptide or for the HTLV tax peptide. A. Cell gate for analysis. B. Staining of .45 cells pulsed with "Data.002" = 0 peptide; Data.004"= 10" 4M of the peptide flu; "Data.006" = 105M of the peptide flu; "Data.010" = 10"4 of the peptide tax, all stained with 5 μg of the flu-TCR tetramers labeled with RPE C. Staining of .45 cells pulsed with:" Data.003"= 0 peptide;" Data. 011"= 10" 4M of the peptide tax; "Data.013" = 10"5M of the peptide tax;" Data.015"= 10-6M of the peptide tax;" Data.005"= 10" 4 of the peptide flu, all stained with 5 μg of the tax-TCR tetramers marked with RPE. Figure 49. FACS analysis of T2 cells pulsed with varying levels of peptide and stained with TCR-coated latex beads ("fluospheres" - molecular probes) with a red fluorescent label. A. Gate of unstained cells for analysis. B. Gate for stained cells for analysis. Observe that the displacement is of lateral dispersion caused by the mass of the pearl bound by the cells. C. Staining of T2 cells pulsed with "Data.002" = 0 peptide; "Data.004" = 10'4M of the peptide flu; "Data.006" = 10"5M of the peptide flu;" Data.007"= 10" 6M of the peptide flu; all stained with 10 μg of beads coated with TCR. D. Staining of T2 cells pulsed with: "Data.003" = 0 peptide; "Data.009" = 10"4M of the peptide tax;" Data.010"= 105M of the peptide tax;" Data.011"= 10" 6M of the peptide tax; all stained with 10 μl of beads coated with tax-TCR.

EXAMPLES In the following examples, the general methods and materials set forth below were used.

Materials The restriction enzymes (Ndel, BamH, Hindl l, Bsu36l, Xmal) were from New England Biolabs. The Tris pH of 8.1 was made as a 2M supply solution from equal parts of Tris base and Tris HCl broth.

USB EDTA (Sigma) was made as a 0.5 M supply solution and the pH was adjusted to 8.0 using NaOH (Sigma). The glutathione in oxidized and reduced forms was sigma. Cystamine and cysteamine were from Sigma. The sodium chloride was from USB and it was made as a 4M supply solution. The minipreparation equipment for plasmid purification was from Quiagen. The PCR purification kits were from Quiagen. DTT was from Sigma. The guanidine was from Fluka.

The urea was from Sigma. The RPM I medium was from Sigma. PBS was made from Oxoide tablets. Glycerol was from BDH.

General Methods Bacterial media (TYP media) were prepared as follows: 160 g of yeast extract (Difco), 160 g of Tryptone (Difco), 50 g of NaCl (USB) and 25 g K2H were dissolved in 2 liters of demineralised water. PO4 (BDH). Aliquots of 200 ml of this solution were measured in 10 x 2 liter conical flasks and developed to 1 liter by adding 800 ml of demineralized water. The flasks were covered with 4 layers of aluminum foil, marked and autoclaved. After cooling, the flasks were stored at room temperature out of direct sunlight before use. Protein concentrations were measured using a Pierce Coomssie binding assay and BSA as a standard protein. In brief, standards of 0-25 μg BSA in a 1 ml volume of water were prepared from a 2 mg / ml BSA (Pierce) stocking material in 4 ml plastic cuvettes. Approximately 10 μg of unknown proteins was made up to 1 ml with water in the same manner. 1 ml of the Pierce Coomassie reagent was added to each cover and the contents were mixed thoroughly. The optical density was measured in 15 minutes at 595 nm using a Beckman DU-530 UV spectrophotometer. A linear regression was performed on the results of the BSA standards (the linearity was good up to 25 μg of BSA) and the unknown protein concentration was estimated through interpolation with these results. Gel filtration chromatography was performed on a Pharmacia FPLC system equipped with a computer controller. The elution of the protein was verified using a UV-M II system that measures the absorbance 280 nm wavelength. For separations On a small scale, a Superdex 200 H R 10/30 column was used and a sample was loaded using a 1 ml loop. Before carrying out the operation, the column was equilibrated with 30 ml of PBS and the sample was run at 0.5 ml / minute collecting fractions of 1 ml. For separations of large scale, a column of Superdex 75 or 200 PG 26/60 with a superlazo of 10 ml. In this case, samples of 5 or 10 ml were collected and the column was run at 4 ml / minute. All separations were carried out at room temperature. Ion exchange chromatography was performed in a system of Biocad Sprint (Perkin-Elmer). For cation exchange, a column of 20 HS or 50 HS was used. For the exchange of anions, a column of 10 HQ, 20 HQ or 50 HQ was used. The columns ran using the recommended pH regulators attached to the 6-way mixing. Samples were injected small (5-25 ml) using a 5 ml injection loop. HE ímßÉAibiÉÉÉk injected larger samples (> 100 ml) using one of the pH regulator lines. 1 ml fractions were collected during the elution phase of the column operation. Protein elution was measured through in-line absorbance at 280 nm. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a gel kit from Bio-Mini-Protean I I. The gels were emptied before use using the following procedure. The gel plate assembly was separated and checked to ensure against leakage. Then, the following mixture was prepared: 12% acrylamide / bisacrylamide (from a 30% acrylamide / 0.8% bisacrylamide supply solution (National Diagnostics)), 0.375 M Tris, pH 8.8 (from a 1.5 M supply solution with the same pH), 0.1% SDS (from a 10% SDS supply solution), 0.05% ammonium persulfate (from a 10% supply material of the same, stored at 4 ° C), and TEMED at 0.1% (Sigma). The mixture was immediately emptied into the gel plate plate assembly and a butanol plate saturated with water was placed on top to ensure a flat top surface. After fixing the gel (at least 10-15 minutes), the stacked gel was mixed as follows. 4% acrylamide (as a supply solution as before), 0.125 M Tris, pH 6.8 (from a 0.5 M supply material with the same pH), SDS ai 0.1%, 0.05% ammonium persulfate and TEMED ai 0.2%. The butanol was removed from the surface of the resolving gel via absorption on a tissue and the stacked gel mixture was emptied onto the top of the resolving gel. A gel comb was immediately inserted taking care to avoid introducing air bubbles into the gel and the stacked gel was allowed to settle for a minimum of 5 minutes. Afterwards, the gel was assembled in the gel apparatus and a pH regulator was run (3 g / l of Tris base, 14.4 g / l of glycine, 1 g / l of SDS (diluted from a supply solution concentrated 10x)) was emptied into the apparatus at the anode and the cathode. After removing the gel comb, the cavities were washed with current pH regulator to prevent the residual acrylamide mixture from attaching to the bottom of the cavities. Samples were prepared by mixing 1: 1 protein with the following mixture: 4% SDS, 0.125 M Tris, pH 6.8, 20% glycerol, 10% β-mercaptoethanol, 1% bromophenol blue (Sigma). The samples were then heated at 95 ° C for 2 minutes and cooled before loading 25 μl into the cavities in the stacked gel. Usually, approximately 1-10 μg of protein was loaded to ensure good staining and run of the gel. After charging, the gels ran at a constant voltage of 200 V during approximately 40 minutes or until the blue bromophenol dye was approximately 5 mm from the bottom of the gel. After the electrophoresis was complete, the gels were removed from the apparatus and carefully dropped into a 0.1% solution of Coomassie R-250 (Sigma) in acetic acid. 10%, 40% methanol, 50% water. Afterwards, the gels were mtm ?? t to moderately agitated for at least 30 minutes before bleaching in several changes of 10% acetic acid, 40% methanol, 50% water, until the previous one was clear. The genes were then stored in water and recorded using a UVP gel documentation system consisting of a light box, a digital camera, and a thermal printer.

Example 1 - Recombinant Soluble TCR A recombinant soluble form of the heterodimeric TCR molecule was engineered, as presented in Figure 1. Each chain consists of immunoglobulin domains distant and close to the membrane, which were fused through a short flexible linker to a coiled spiral motif, which helps to stabilize the heterodimer. The TCR constant domains were truncated immediately before the cysteine residues, which in vivo form a disulfide bond between chains. With sequentially, the two pairs of chains through the non-covalent quaternary contacts, and this is confirmed in Figure 2b. Since the Fos-Jun closing peptide heterodimers are also capable of forming an inter-chain disulfide immediately n-terminal to the linker used (O'Shea et al 1989), the alignment of the two chains relative to each other was predicted as optimal. The fusion proteins need to be linked in a way that is compatible with each of the separate components, in order to avoid the disruption of any structure. The cDNA encoding the alpha and beta chains of a TCR specific for an epitope 58-66 of influenza matrix protein in HLA-A2 was obtained from a human CTL clone VB17 + (JM22) through PCR anchored as shown in FIG. previously described (Moss and others, 1991). The alpha and beta TCR closure constructs, pJM22a-Jun and pJM22ß-Fos, were constructed separately by amplifying the variable and constant domain of each chain using standard PCR technology and dividing products on the leucine closure domains from the eukaryotic transcription factors Jun and Fos, respectively (see Figure 1). These sequences with a length of 40 amino acids have been shown to specifically heterodimerize when they are refolded from synthetic peptides, without the need for a covalent bond between chains (O'Shea et al., 1989). The primers were designed to incorporate a high AT content immediately 3 'to the initiation codon (to destabilize the secondary structure of mRNA) and using codon preference of E. coli, in order to maximize expression (Gao et al. ). Scarce cysteine in the constant TCR beta domain was mutated to cerin to ensure the prevention of the wrong disulfide bond during retraction. The DNA constructs were ligated separately to the E. coli expression vector pGMT7. The plasmid was digested and DNA sequencing confirmed that the constructions were correct. In detail, the procedures used were the following. Expression of TCR closing chains and purification of denatured inclusion antibodies: the expression plasmids GFG020 and GFG021, the pGGMT7 containing JM22a-Jun and J M22β-Fos respectively, were transformed separately to the BL21 pLyS strain of E. coli , and individual ampicillin resistant colonies were developed at 37 ° C in a TYP medium (ampicillin, 100 μg / ml) at an OD50o of 0.4 before inducing protein expression with 0.5 mM I PTG. The cells were harvested 3 hours after induction through centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B apparatus. The cell pellets were resuspended in a pH buffer containing 50 mM Tris-HCK, 25% sucrose (w / v), 1 mM NaEDTA, 0.1% (w / v) NaAzide, 10 mM DTT, pH 8.0 . After a night freeze-thaw step, the resuspended cells were applied sound in 1 minute reinforcements for a total of approximately 10 minutes in a Milsonix XL2020 sound applicator, using a standard 12 mm diameter probe. The inclusion body pellets were recovered by centrifugation for 30 minutes at 13,000 rpm in a Beckman J2-21 centrifuge. Afterwards, three washes of detergent were made to remove the cell waste and membrane components. Each time the inclusion body pellet was homogenized in a triton pH buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT, pH 8.0) before being pelleted through centrifugation for 15 minutes at 13,000 rpm in a Beckman J2-21 apparatus. Then, the detergent and salt were removed through a similar wash in the following pH buffer: 50 mM Tris-HCl, 1 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT, pH 8.0. Finally, inclusion body pellets JM22a-Jun and JM22ß-Fos were separately dissolved in a solution of urea (50 mM MES, 8 M urea, 10 mM NaEDTA, 2 mM DTT, pH 6.5) for 3 to 4 hours at 4 ° C. The insoluble material was pelleted through centrifugation for 30 minutes at 13,000 rpm in a Beckman J2-21 apparatus, and the supernatant was divided into 1 ml aliquots and frozen at -70 ° C. The inclusion bodies solubilized in urea were quantified with a Bradford-binding stain assay (Biorad). For each chain, a yield of about 100 mg of purified inclusion body was obtained from one liter of culture. Each inclusion body (JM22a-Jun, JM22B-Fos) was solubilized in a solution of urea at a concentration of approximately 200 mg / ml and was estimated through a gel analysis that was approximately 90% pure in this form ( data not revealed).

Co-folding of TCR closing fusion proteins: Initial replication experiments using a standard refolding pH regulator (100 mM Tris pH 8.5, 1 M L-Arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM glutathione oxidized, 0.2 mM PMSF) resulted in a severe protein precipitation, which was dependent on the presence of the closing domains. The fact that this phenomenon occurred at concentrations below the dimerization dissociation constant of the closure (ie, when most of the helices de cierre are expressed, as monomeric) suggested that additional forces were unfolded species of stabilization. Probably, the best explanation is that all the a-helical closing domains were first folded and that their transient heterodimerization induces aggregation between chains of partially folded intermediates of the complex immunoglobulin domains. Therefore the refolding pH regulator was altered to include 5 M urea in order to avoid hydrophobic interactions between partially folded immunoglobulin domains and allow the individual chains to fold completely before heterodimerization. This step is sufficient to prevent precipitation from occurring, and allows correctly folded TCR closure heterodimers to be assembled with acceptable productions using the following protocol. Materials for the supply of solubilized urea from TCR closure chains JM22a-Jun and JM22ß-Fos were renatured through co-dilution dilution. Approximately 30 mg (i.e., 1 μMol) of each solubilized inclusion body chain were thawed from frozen supply materials and an additional pulse of DTT (4 μmoles / ml) was added to ensure complete cysteine reduction. Then, the samples were mixed and the mixture was diluted in 15 ml of a guanidine solution (6 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA), to ensure complete denaturation of the chain. The guanidine solution containing completely reduced and denatured TCR closing chains was then injected into 1 liter of the following refluxing pH regulator: 100 mM Tris pH 8.5, 400 mM L-Arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 5 M urea, 0.2 mM PMSF. The solution was left for 24 hours. Then, the fold was dialyzed twice, first against 10 liters of 100 mM urea, secondly against 10 liters of 100 mM urea, 10 mM Tris, pH 8.0. Both the withdrawal and dialysis steps were performed at 6-8 ° C.

Purification of the refolded TCR closure: The TCR JM22zip closure was separated from the degradation products and impurities by loading the retraction performed on a POROS 10HQ analytical anion exchange column in 7 200 ml aliquots and eluting the bound protein with a gradient of 0-400 mM NaCl over 50 column volumes using a BioCad workstation (Perspeptive Biosystems). A non-covalently associated heterodimer eluted in a peak ^^ M¡ ^^ ri ^ Hiwa > individual to approximately 100 mM NaCl. The peak fractions (typically containing the heterodimer at a concentration of 100-300 μg / ml) were stored at 4 ° C before being combined and concentrated. The yield of the heterodimer is approximately 15%.

Characterization of the refolded TCR closure JM / 22zip: The JM22zip heterodimer purified through anion exchange is eluted as a protein of approximately 30 kDa from a column of Superdex 200 gel filtration dimension (Pharmacia). It is especially to include gel filtration steps prior to surface plasmon resonance binding analyzes, since the exact affinity and kinetic measurements are based on the monomeric interactions that occur. In this way, larger aggregates of the fraction of soluble protein used for analysis can be excluded. In particular, the aggregates cause slow association and dissociation rate constants to be detected. The oxidation state of each chain has been examined through reduction / no reduction gel analysis, in Figure 2. In the presence of SDS, the non-covalently associated heterodimer is dissociated into alpha and beta chains. If used in the DTT charge pH regulator, the two strands run either side of the 31 kDa marker. In the absence of said denaturing agents, both chains will behave as an individual species, but the mobility of each one increases, which suggests that each chain has formed a kind of disulfide bond, individual (Garboczi and others 1996). The reactivity of the refolded receptor antibody has been tested using surface plasmon resonance in a Biacore 2000 machine (Biacore). The TCR JM22z closure was immobilized to a dextran matrix binding surface (CM circuit) at a pH of 5.5 using standard amine coupling methods. A variable region antibody specific for the beta chain (VB17) specifically binds to the immobilized receptor, involving correct conformation.

Stability: The soluble TCRs expressed as closing fusions of leucine alfa -jun and beta-fos are stable during periods of months and, therefore, are suitable for the detection of specific antigens presented by MHC class I.

Example 2 - Kinetics and Affinity Study of Human TCR Viral Peptide-MHC Specific link of the refolded TCR closure to peptide-MHC complexes. A surface plasmon resonance biosensor was used (Biacore) to analyze in binding of a TCR closure (JM22zip, specific for the M58-66 complex of influenza matrix protein H LA-A2) to its peptide-M HC ligand (see Figure 3). This was facilitated by producing individual pMHC complexes (described below), which can be immobilized to a binding surface coated with streptavidin in a semi-oriented form, allowing efficient testing of the binding of a soluble T cell receptor of up to four different pMHC (immobilized on separate flow cells), simultaneously. Manual injection of the H LA complex allows the precise level of immobilized class I molecules to be easily manipulated. Such immobilized complexes are capable of binding both T cell receptors (see Figure 3) and the CD8aa receptor, both of which can be injected into the soluble phase. The specific binding of the TCR closure is obtained even at low concentrations (at least 40 μg / ml), implying that the TCR closure is relatively stable. The binding properties of pMHC of JM22z are observed to be qualitatively and quantitatively similar if TCR is used in either the soluble or immobilized phase. This is an important control for the partial activity of soluble species and also suggests that the biotinylated pMHC complexes are biologically as active as the non-biotinylated complexes.

Preparation of chemically biotinylated H LA complexes. Methods for the production of recombinant individual peptide class I H LA complexes have already been described (Garboczi et al., 1992). These have been modified in order to produce HLA complexes, which have chemically biotinylated β-2-microglobulin domains and, therefore, can be immobilized to a streptavidin coated binding wafer and used for plasmon binding study Of surface. The β-2-microglobulin was expressed and 40 mg were refolded in a standard refolding pH regulator (100 mM Tris pH 8.0, 400 mM L-Arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 M oxidized glutathione, 0.1 mM PMSF) essentially as described (Garboczi et al., 1992). After an optional gel filtration step, the protein was exchanged with 0.1 M sodium borate, pH 8.8, and finally concentrated to 5-10 mg / ml. The β-2-microglobulin was also quantified using a Bradford assay (Biorad). A molar excess of 5 biotin-hygroxysuccinimide (Sigma) was added from a supply material made at 10 mg / ml in DMSO. The reaction was left for 1 hour at room temperature, and stopped with 20 μl of 1 M of ammonium chloride / 250 μg of biotin ester used. The refolded HLA complex was separated from biotinylated and biotinylated free β-2-microglobulin using a Superdex 200 gel filtration size column (Pharmacia). Streptavidin was immobilized through standard amine coupling methods.

Conclusions: In this way, the protein refolding methods described in Example 1 produce a functional, correctly folded, stable recombinant receptor fusion protein, which is suitable for biophysical analysis using an optical biosensor. This has provided a reagent used to perform a detailed affinity and kinetics analysis of a human TCR-pMHC interaction. The effects of T-MHC and TCR-pMHC cell co-receptor interactions have also been studied. The recombinant techniques used are applicable in principle to both murine and human TCRs, both class I and class II, restricted, and will allow similar analyzes of a scale of TCRs. This could allow several issues to be addressed, such as the expansion of TCR affinities within an antiviral response, the properties of dominantly selected receptors and the kinetic requirements for receptor activation. The methods also provide a way to verify the specific character of the ligand of a TCR prior to crystallization assays, and may also have implications for the recombinant production of other cell surface receptors.

Example 3 - Biotinylation and tetramerization of soluble T-cell receptors. 2.5 ml of purified soluble TCR, prepared as described in Example 1, were exchanged with pH regulator. (approximately 0.2 mg / ml) to a biotinylation reaction pH regulator (10 mM Tris pH 8.0, 5 mM NaCl, 7.5 mM MgCl2), using a PD-10 column (Pharmacia). The eluted product (3.5 ml) was concentrated to 1 ml using a centricon concentrator (Amicon) with a molecular weight cut-off of 10 kDa. This formed 5 mM with added ATP from the supply material 5 solution (0.1 g / ml adjusted to a pH of 7.0). A cocktail of protease inhibitors: leupeptin, pepstatin and MPFS (0.1 mM) was added, followed by 1 mM of biotin (added as a 0.2 M supply material) and 5 μg / ml of enzyme (from a 0.5 mg / ml supply). The mixture is then incubated overnight at room temperature. Excess biotin was removed from the solution through dialysis against 10 mM Tris, pH 8.0, 5 mM NaCl (200 volumes, with 2 changes at 4 ° C). The protein was then tested for the presence of bound biotin by staining on nitrocellulose followed by blocking with 5% skim milk powder, and detection using the streptavidin-HRP conjugate (Biorad). The tetramerization of the soluble biotinylated TCR was either with the conjugate of extravidin with RPE or extravidin-FITC (Sigma). The concentration of soluble TCR-biotin was measured using a protein binding assay of Coomassie (Pierce), and a ratio of extravidin conjugate to soluble TCR of 0.224 mg / mg TCR was calculated to obtain saturation of extravidin through biotinylated TCR at a ratio of 1: 4. The extravidin conjugate was added in aliquots of 1/10 of the aggregate total, on ice, for at least 15 minutes. minutes per aliquot (to ensure saturation of extravidin). dOÍáÜÜhihi? llÉI * The soluble TCR tetramers were stored at 4 ° C in the dark. The tetramers are extremely stable over a period of months.

Example 4 - Expression, Retraction and Biotinylation of TCR Specific Site a / ß Soluble a) Design by Chain Engineering a and ß of TCR A recombinant soluble form of the heterodimeric TCR molecule was engineered as shown in Figure 7 Each chain consists of immunoglobulin domains distant and close to the membrane, which are fused through a short flexible linker to a coiled spiral motif, which helps to stabilize the heterodimer. Figures 4 to 6 and 11 to 14 show the DNA coding sequences and corresponding amino acid sequences for various TCR α and β chains from TCR having different specific characters. This example concentrates on the TCR represented by the sequences of Figures 4 to 6, but the described methods can be similarly performed using the TCRs given in Figures 1 to 14. The TCR constant domains have been truncated immediately before the cysteine residues, which in vivo form a disulfide bond between chains. Consequently, the two chains are paired through non-covalent quaternary contacts. Since the Fos-Jun closing peptide heterodimers are also capable of forming an inter-chain disulfide immediately N-terminal to the linker used (O'Shea et al., 1989), the alignment of the two chains relative to each other It was predicted as optimal. The fusion proteins need to be linked in a way that is compatible with each of the separate components, in order to avoid the disturbance of any structure. The cDNA encoding alpha and beta chains of a TCR specific for the 58-66 epitope of H-LA-A2 influenza matrix protein was obtained from a vL17 + human CTL clone (JM22) through PCR anchored as previously described (Moss and others, 1991). The CTR alpha and beta closure constructs, pJM22a-Jun and pJM22ß-Fos were constructed separately by amplifying the constant variable domain of each chain using standard PCR technology and dividing the products into the leucine closure domains from the Jun and Fos eukaryotic transcription factors, respectively. These sequences with a length of 40 amino acids have been shown to heterodimerize specifically when they replicate from synthetic peptides, without the need for a covalent chain link (O'Shea et al. 1989). Initiators were designed to incorporate a high AT content immediately 3 'to the start codon (to destabilize the secondary structure of mRNA) and using codon preferences of E. coli, in order to maximize expression (Gao et al. 1998). Scarce cysteine in the constant TCR beta domain was mutated to cerin to ensure prevention of an incorrect disulfide bond during refolding. The fused DNA and protein sequences are indicated in Figures 4 and 5. In order to allow specific biotinylation at the site of the β chain of this TCR, a DNA sequence encoding a so-called "biotin tag" It was engineered at the 3 'end of the gene expressing soluble VB17. The following PCR primers were used for engineering design of this DNA construct: 5'GCTCTAGACATATGGGCCCAGTGGATTCTGGAGTCAC-3 'and 5'-GGGGGAAGCTTAATGCCATTCGATTTTCTGAGCTTCAAAAATATCGTT CAGACCACCACCGGATCCGTAAGCTGCCAGGATGAACTCTAG-3'. The resulting PCR product was digested with restriction enzymes, Ndel and H ind l l (New England Biolabs) and ligated with T4 DNA ligand (New England Biolabs) in the pGMT7 vector (Studier et al., 1990). Figure 6 shows the DNA sequence of the insert in this construct and the deduced protein sequence. b. Expression of TCR chains Expression and refolding of a TCR with specific character for the influenza virus matrix peptide presented by H LA-A * 0201, was carried out as follows: TCR a and ß chains were expressed separately in strain BL221 DE3pLysS from E. coli, under the control of vector pGMT7 in TYP medium (1.6% bacto-tryptone, 1.6% yeast extract, 0.5% NaCl , K2HPO4 at 0.25%). The expression was induced in a phase of average recording with 0.5 mM of IPTG and, after 3-5 hours, the bacteria were harvested through centrifugation. Bacterial cells were used through resuspension in a "lysis pH regulator" (10 mM EDTA, 2 ml DTT, 10 mM Tris pH 8, 150 mM NaCl, 0.5 mM PMSF, 0.1 mg / ml lysozyme, 10 glycerol %), followed by the addition of 10 mM MgCI2 and 20 ug / ml DNasel, with a 20 minute incubation on ice, and sound application using a sound probe probe in 10x reinforcements of 30 seconds. The protein, in inclusion bodies, was then purified through several washes (usually 3) of "Triton pH regulator" (0.5% Triton X-100, 50 mM Tris pH8, 100 mM NaCl, sodium azide 0.1 %, 10 mM EDTA, 2 mM DTT) using centrifugation at 15,000 rpm for 15 minutes to form pellets of the inclusion bodies and a "dounce" homogenizer to resuspend them. The detergent was removed from the preparation with an individual wash of 50 mM Tris pH 8, 100 mM NaCl, 19 mM EDTA, 2 mM DTT, and the protein was solubilized with "pH regulator of urea" (20 mM Tris pH 8, 8 M urea, 10% glycerol, 500 mM NaCl, 10 mM EDTA, 2 mM DTT). After end-to-end mixing overnight at 4 ° C, the solution was clarified by centrifugation, and the solubilized protein was stored at -70 ° C. The protein concentration was measured through a Coomassie binding assay (Pierce). c. Refolding the TCR. The protein solubilized with urea in equal proportions was further denatured in "guanidine pH regulator" (6 M guanidine HCl, 10 mM sodium acetate, pH 5.5, 10 mM EDTA, 2 mM DTT) at 37 ° C. This solution to refold the pH regulator (5 M urea, 100 mM Tris pH 8, 400 mM L-arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 0.1 mM PMSF) on ice ensuring rapid mixing. After > 12 hours at 4 ° C, the solution was dialyzed against 10 volumes of water, then 10 volumes of 10 mM Tris, pH8, 100 mM urea. The protease inhibitor, PMSF, was added at all stages to minimize the proteolytic loss of the biotinylation label in the TCR. d. Purification of the TCR. The diluted solution of the TCR was filtered through a filter of 0. 45 microns to remove the added protein and then loaded onto a POROS 10HQ column. The refolded TCR was diluted with a gradient of sodium chloride in 10 mM Tris, pH 8, and fractions of 1 ml were collected and analyzed by SDS-PAGE. Fractions containing TCR were combined and concentrated to 1 ml using a 30 kDa cut-off centrifuge concentrator. and. Biotinylation of the TCR. The 1 ml solution of TCR was made at 7.5 mM of ATP using ATP regulated in its pH, 5 mM MgCl2, 1 mM biotin and a cocktail of protease inhibitors was added, which included PMSF, leupeptin and pepstatin. Finally, the BirA enzyme was added to a final concentration of 5 μg / ml and the reaction was allowed to proceed overnight at room temperature. Then, the TCR was separated from the free biotin through gel filtration. Fractions containing biotinylated TCR were combined and a cocktail of protease inhibitor was added. The protein concentration was also determined. Figure 7 shows a schematic diagram of the biotinylated, soluble TCR.

Example 5 - Production of TCR Tetramers and TCR Coated Pearls. In order to tetramerize the biotinylated TCR, extravidin (Sigma) was added at a molar ratio of 1: 4. Fluorescently labeled extravidin was used for cell labeling experiments. A stepwise addition was used to obtain the saturation of the extravidin, allowing something that will be incomplete in the biotinylation reaction and some inaccuracy in the protein determinations. Fifteen minutes on ice ^ AÜdhiUki allowed between each addition of extravidin for binding, followed at least overnight at 4 ° C after the final addition. Tetramerization was confirmed through gel filtration as a small sample of the solution on a calibrated column of 5 Superdex 200 (Pharmacia). The TCR tetramer solution was then stored at 4 ° C in the presence of the protease inhibitor cocktail and 0.05% sodium azide. For the production of the TCR tetramer, see Figures 4-7. A similar aspect can be used to cover several types of beads or other solid supports with soluble TCR. The beads coated with avidin / streptavidin can be obtained from commercial sources (eg, Dynabeads from DYNAL, Oslo, Norway, or MACS from Miltenyu Biotec Ltd., Bergisch Gladbach, Germany) and are commercially available in a wide scale of sizes, with a diameter of approximately 4.5 μm-65 nm. The immobilization of the MHC-peptide complexes in Dynabeads through biotin-streptavidin has previously been described (Vessey et al., 1997). The purified biotinylated protein was incubated with streptavidin-coated beads for a period of, for example, 30 minutes at 4 ° C, after which, the beads were washed to remove unbound protein. These beads coated with M HC-peptide produced a specific response in the antigen when used to stimulate a cell line expressing TCR. Similarly, TCR tetramers, or monomeric biotinylated TCR, can be immobilized on beads coated with avidin / streptavidin, or the iMHáaHÉMllH? riiil non-biotinylated TCR can be immobilized through the anti-TCR antibody coating or through chemical direct bonding or through other appropriate means.

Example 6 - Production of Liposome and Drug Packaging. The lipids and other components, sterile and endotoxin tested, are commercially available from a number of sources for example, from Sigma Chemical Company or Avanti Polar Lipids Inc., USA. Liposomes are prepared from a mixture of vesicle-forming lipids and biotinylated vesicle-forming lipids. A variety of suitable methods exist for the formation of liposomes. The biotinylated T cell receptor is then linked to the exterior of the liposomes through a suitable binding agent such as avidin, streptavidin or extravidin. Detectable labels and / or therapeutic agents are incorporated in the same membrane or trapped in the aqueous volume within the membrane.

Example 7 - Molecular Cloning of the T Cell Receptor Genes from T Cell Lines or Known T Cell Clones of Specific Character. Methods and methods for molecular cloning of TCR genes from cells are identical to all a chains and for all β chains, respectively, and therefore, are only described in this example.

An adequate number of T cells, typically 1-5 million, were used in a lysis pH regulator from the "mRNA Capture Kit" (Boehringer Mannheim). The mRNA was isolated with the reagents of the kit by hybridizing biotinylated oligo-dT to poly-A ends or tails of mRNA. The hybridized complexes were then captured by binding biotin to a PCR tube coated with streptavidin. After immobilization of the mRNA in the PCR tube, the cDNA was generated using reverse transcription of AMV (Stratagene), as described previously (Boehringer Mannheim manual for "mRNA Capture Kit"). With the cDNA still immobilized, poly-G ends or tails were generated at the 3 'ends using the terminal transferase enzyme (Boehringer Mannheim). The PCR reaction mixture was then added, including the high fidelity thermostable polymerase pfu (cloned, Stratagene), which was used in order to minimize the risk of errors in the PCR products. PCR reactions were performed using an "anchor initiator" poly-C (Figure 15A) and specific chain initiators β or a (Figures 15B and C, respectively), heating and cooling rapidly in the respective TCR constant regions. PCR reactions of 30 cycles of denaturation at 95 ° C for 1 minute, rapid heating and cooling at 50 ° C for 1 minute, and extensions at 72 ° C for 5 minutes were performed to amplify the TCR gene fragments . The PCR products were ligated to a Bluescript sequencing vector (pBluescript I I KS-, Stratagene) using the Xhol and Xmal restriction enzyme sites contained in the PCR primers (all enzymes from New England Biolabs). After transfection of the ligation mixtures in the XL-1 Blue strain of E. Coli, several clones for each chain were selected for DNA sequencing, which was performed in an automatic ABl 377 Prism sequencer using Big Dye ™ terminators ( Applied Biosystems Inc.).

Example 8 - Molecular Cloning of DNA Fragments Encoding the Coiled Spiral Regions of 40 Amino Acids ("Leucine Closure") of c-jun and c-fos. The DNA fragments encoding the 40 amino acid coiled spiral regions ("leucine lock") of c-jun and c-fos were generated through PCR reactions using human cDNA as a template and the primers shown in Figure 16 The PCR reactions were performed in a reaction pH regulator including cloned pfu polymerase (Stratagene) for 30 sites consisting of denaturation at 95 ° C for 1 minute, initiator heating and cooling rapidly at 58 ° C for 1 minute, and extension to 72 ° C for 2 minutes. Fragments of c-jun and c-fos were ligated to pBluescript I I KS- (Stratagene) using the only restriction sites Xhol and Xmal to obtain constructs pBJ 107 and pBJ 108, respectively (Figure 17). The DNA sequences of the c-jun and c-fos fragments were verified by DNA sequencing performed on an ABl 377 automatic sequencer Prism using the BigDye ™ terminators (Applied Biosystems I nc.). The sequenced c-jun and c-fos fragments were then subcloned, using the unique restriction sites Xmal and Bam H I, in the polyslinking region of the T7 polymerase expression vector, pGMT7 (Studier, Rosenberg et al., 1990).

Example 9 - Designs of TCR Fusion Protein-Leucine Closure for the Production of Stable, Soluble TCRs. Attempts to co-refold extracellular fragments of TCR α and β chains, truncated so as to contain the cysteine residue, which in vivo forms a disulfide bond, produced limited success (data not shown, see Example 12 for methods of expression and general methods and materials for refolding conditions). However, when the α and β chains of TCR were truncated immediately before, ie on the N-terminal side of, the cysteine residue forming the interchain disulfide bond, the analytical chromatography on a Superdex G-75 column ( Pharmacia) indicated that a small fraction of protein, approximately 1-2% of the amount used in the refolding reaction, was refolded to a complex of the expected molecular size for the truncated a / β heterodimer (see also (Garboczi, Utz et al. , 1996) for reference to the method).

Since incorrect disulfide bond formation can cause irreversible protein refolding during in vitro refolding, the probabilities for this to happen were reduced to the minimum by using a cysteine residue in the ß-TC constant region. R, which is uneven in the cellular TCR. The cysteine residue is replaced by a serine or an alanine residue. The synthetic DNA primers used for these mutation steps are shown in Figure 18. Co-refolding of TCR mutated α and β chains, both truncated immediately before the cysteine residue forming the interchain chain disulfide bond, showed an improvement d ramática in heterodimer productions, the protein fraction of correct molecular weight typically constituting 1 5-30 of the total protein. However, when these soluble Rs were stored overnight, the analysis of the protein showed that the fraction with a molecular weight corresponding to the heterodimeric TCR was divided into two molecular weight peaks corresponding to the monomeric TCR α and β chains. Similar observations were made after the dilution of the soluble Rs TCs, indicating that the stability of the a / β chain was low enough for analyzes that might require a longer time expansion than a mimicked number of hours od. Illution of the protein. In conclusion, these methods for producing soluble TCR only generated the receptor with extremely limited stability. To improve the stability of the a / β chain of TCR and to potentially assist the formation of the heterodimer during refolding, the TCR chains were fused to the "leucine lock" domains of c-jun and c-fos, the which are preferentially known to form heterodimers (O'Shea, Rutkowski et al., 1989; Schuermann, Hunter et al., 1991; O'Shea, Rutkowski and others, 1992; Glover and Harrison 1995). Two designs for the fusion of TCRs were tested. In one, the leucine closures were fused just after, ie c-terminal to, the cysteine residues forming the disulfide bond between chains in the a and ß chains of TCR. Since the c-jun and c-fos leucine-closing peptides are also capable of forming an inter-chain disulfide immediately N-terminal to the linker used (O'Shea, Rutkowski et al., 1989), the alignment of the two chains with relationship to one another, and to the disulfide bond between chains, was predicted as optimal. In the other design, the leucine closures were fused just before, ie, N-terminal to, the cysteine residues forming the disulfide bond between chains in the TCR chains a and β (Figure 19). Thus, in the second design, the cysteine residues are omitted from the recombinant receptor. In refolding experiments with TCR-closure chains (TCR-z) of these designs, it was found that the yield of the heterodimeric soluble receptor was better when the cysteine residues, which form the interchain disulfide bond, were omitted from the α and β chains of TCR, as in the design shown in Figure 19.

Example 10 - Construction of DNA Expression Vectors for TCR-Leucine Closure Proteins. This example describes the construction of expression vectors for the α and β chains of 5 TCRs. The strategy and design described must be adapted to any human or animal TCR gene. Although the 5 TCRs described herein are all restricted by MHC class I epitopes, the methods can be identically employed for the cloning and construction of expression vectors for MHC class I I restricted TCRs. All vectors express directed protein for the refolding of soluble TCRs, according to the design shown in Figure 19, with the exception that 2 TCRs were expressed with a tag sequence that can be biotinylated in the C-terminus (see below). and Figures 28, 29 and 30). The cloning strategies are identical for all the a and ß chains of TCR, respectively.

The degree of the leader peptide sequences, or signal from the α and β chains of TCR were predicted from analysis of the sequence data obtained from plasmids containing TCR anchor PCR products (see Example 7). On this basis, 5 primers were designed for the generation of PCR fragments for the expression of TCR chains without leader sequence (Figure 20). The 5 primers encode a methionine residue just before the mature TCR protein sequences in order to allow translation in E. coli. Silent mutations were introduced, substituting the bases C or G for A or T (Figure 20), in a number of the 5 'nearby codons of the genes in order to reduce the tendency for the formations of the secondary mRNA structure, which can adversely inhibit expression levels in E. coli (PCT / GB 98/03235; (Gao, Tomo et al., 1997; Gao, Gerth et al., 1998) .The genes encoding the Va0.2 and VB chains 17 of the JM22 influenza human peptide peptide-H LA-A0201 (peptide sequence GI LGFVFTL) TCR restricted, human Va23 and VB5.1 chains of peptide 003 H IV-1 Gag-H LA-A0201 (peptide sequence SLYNTVATL ), TCR restricted, and murine chains Va4 and VB1 1 of peptide F5 N P-H2Db (peptide sequence ASNENMDAM) were amplified through PCR using plasmids containing TCR anchoring PCR products generated as described in Example 7. Genes for the TCRs A6 (Va3.2 / Vß 12.3) and B7 (Va17.2 / VB 12.3) human, what which are specific to the HTLV-1 Tax peptide, presented by H LA-A0202 (peptide sequence LLFGYPVYV), were obtained in the form of a plasmid (Garboczi, Utz et al., 1996; Ding, Smith et al., 1998), which were used for the generation of PCR products for the construction of expression vectors for these TCR chains. The genes for these TCRs were cloned into expression vectors that contained the sequence for a biotinylated label fusion fragment of leucine c-fos (see Example 1). PCR reactions were performed with cloned pfu polymerase at standard pH regulator conditions (Stratagene) and with 25 cycles of denaturation at 95 ° C for 1 minute, rapid heating and cooling of the initiator at 60 ° C for 1 minute, and extensions at 72 ° C for 6 minutes. The PCR products were digested by restriction with Ndel and Xmal enzymes, and ligated to the pGMT7 vectors containing the c-jun inserts (a-TCR chains) and c-fos (β-chains of TC R) (see Example 8). . Figures 21 -30 show the sequences of the inserts of TCR-z and predicted protein sequences expressed by pGMT7 vectors. Figure 31 shows the sequence of the β chain of A6 TC R containing a mutation in the constant region, but which did not detectably affect the folding and function of the soluble TCR (see Examples 12 and 1 3).

Example 1 1 - Construction of DNA Vectors for Expression of TCR ß Chains Fused to a Biotinylating Fragment of Closing of Leucine c-fos. In order to allow soluble TCRs to be immobilized or allow detection or binding to the receptor, it may be useful if the protein can be produced with an additional functional fusion component. This may allow the soluble TCR to be derivatized, so that it is produced as m ultimers, or to allow detection with a high sensitivity, or to bind other functions to the receptor / receptor complexes. This example demonstrates the construction of expression vectors for TCR β chains, where a fusion polypeptide that can be specifically biotinylated in E. coli, in vivo, or with the BirA enzyme in vitro is engineered (Barker and Campbell 1981 Barker and Campbell 1981, Howard, Shaw and others, 1985, Schatz 1993, O'Callagham, Byford and others, 1999). As shown in Examples 13 and 14, these soluble TCR fusions can be expressed and refolded together with a chain a in an identical form and with yields or performances similar to the TCR β chain, which is not fused to the "ETIQU". ETA biotinylation "(BT-label). These results demonstrate that the soluble TCR described here is probably suitable for expression with a multitude of different polypeptides as fusion patterns. The β-chains of the T-cell receptor were subcloned into a pGMT7 expression vector with a C-terminal biotin-tag sequence to the leucine fos close sequence, as follows: Start-chain β of TCR-closure fos-biotin- Detention-tag The exact sequence of the ends of the constructions was as follows (also see Figure 32): Linker? BamH I | < -speaker- > | < -biotin-label The aspects were used to produce soluble TCRs with the biotin label. In the case of the human-H JM22 influenza matrix peptide restricted TCR LA-A0201, the cloned ß-chain C-fos leucine fusion fusion was modified at the 3 'end using the synthetic DNA primer shown in FIG. Figure 33 to introduce a BamH I site in place of a Hindl ll site, using a standard PCR reaction with pfu polymerase (Stratagene). The original 5 'primer (See Figure 20) containing an Ndel site was used as the forward primer. The PCR product produced was cloned into a modified pGMT7 vector containing the biotin-tag sequence (Figure 32) to form the construct presented above. This plasmid is known as JMB002. The cloned TCR specific for the epitope of HTLV-1 restricted by H LA-A0201, LLFGYPVYV, known as A6 tax TCR (Va2.3 / Vß13.2), was truncated using PCR with the forward and reverse primers shown in Figure 20. This TCR β chain was cloned at the Ndel and Xmal sites of a vector pGMT7 (JMB002) containing the c-fos-BT fragment. After construction of the fusion expression vectors, DNA sequencing was performed to ensure that no error was introduced during the subcloning procedure (all sequencing was performed in the department Biochemistry Dept. DNA Sequencing Facility, Oxford University, using ABl 377 Prism sequencer and ABL BigDye fluorescent terminators). It was observed that there were two errors in the ß tax chain TCR ß compared to the published sequence and after further investigation, it was found that both were present in the original plasmid that was received. Since both errors were 3 'from a single Bsu361 site in the TCR ß chain, this was used to clone into plasmid JMB002 (correct). Both versions of the TCR tax ß chain were expressed and refolded with a α chain and compared using Biacore. Both versions of the protein specifically bound to tax-MHC class I peptide molecules with similar apparent affinities (see Example 20). In subsequent experiments, only the correct version of the β chain was used.

Example 12 - Expression of TCR Chains in E. coli and Purification of Inclusion Bodies. The a and ß chains of TCR were expressed separately in the BL21DE3pLysS strain of E. coli under the control of the pGMT7 vector in the TPI medium, using 0.5 mM of IPTG to induce protein production when the optical density (OD) at 600 nm reached between 0.2 and 0.6. The induction was allowed to continue overnight and the bacteria were harvested through centrifugation at 4000 rpm in a Beckman J-6B centrifuge. Then, the bacterial cell pellets were resuspended in "lysis pH buffer" (10 mM Tris pH 8.1, 10 mM EDTA, 150 mM NaCl, 2 mM DTT, 10% glycerol). The mixture was chilled on ice and the following was added: 20 μg / ml lysozyme, 10 mM MgCl 2, and 20 μg / ml DNase I, followed by incubation on ice for a minimum of 1 hour. The mixture was then applied sound using a sound applicator with a 12 mM probe (Milsonix XL2020) to the total energy of 5 bursts of 30 seconds pulses at 30 second intervals to allow the mixture to cool. The temperature was maintained during this procedure through the use of an ice-water mixture. The mixture was then diluted with 5 volumes of "Triton washing pH regulator" (50 mM Tris pH 8.1, 0.5% Triton X-100, 100 mM NaCl, 0.1% sodium azide, 10 mM EDTA, 2 mM DTT) . After incubation on ice for a minimum of 1 hour, the mixture was then centrifuged at 3,500 rpm in a Beckman GS-6R centrifuge and the supernatant discarded. The pellet was resuspended in "resuspension pH regulator" (50 mM Tris pH 8.1, 100 mM NaCl, 10 mM EDTA, 2 mM DTT) using a small disposable plastic pipette. The mixture was then centrifuged at 8,000 rpm in a Beckman J2-21 centrifuge and the supernatant discarded. The pellet was then resuspended in "guanidine pH buffer" (50 mM Tris pH 8.1, 6.0 M Guanidine-HCl, 100 mM NaCl, 10 mM EDTA, 10 mM DTT) using a hand-held homogenizer. After a low speed centrifugation to remove the insoluble material, the supernatant was aliquoted and stored at -70 ° C. Routinely, an approximate yield of 100 mg per liter of bacterial culture was obtained. SDS-PAGE analysis of the purified inclusion body preparation was achieved by diluting 2 μl of the guanidine pH regulator inclusion body preparation with a SDS-PAGE sample pH regulator followed by heating at 100 ° C for 2 minutes. Samples were loaded onto the gel while heating to prevent the guanidine / SDS mixture from precipitating during loading. The inclusion body protein purified in this way was judged to be approximately 90% pure through Coomassie staining of SDS-PAGE performed in this manner (see Figure 34).

Example 13 - Refolding and Purification of the TCRz Heterodimer. Proteins solubilized with urea in equal proportions were further denatured in "guanidine pH buffer" (6 M guanidine HCl, 10 mM sodium acetate, pH 5.5, 10 mM EDTA, 2 mM DTT) at 37 ° C. The protein mixture was injected to the ice-cooled reflux pH regulator (100 mM Tris pH 8.1, 0.4 M L-arginine HCl, 5.0 M urea, 5 mM reduced glutathione, 0.5 mM oxidized glutathione) to a Total protein concentration of 60 mg / l assuring rapid mixing. After incubation on ice for at least 5 hours to allow refolding, the mixture was dialyzed against 10 volumes of demineralized water for 24 hours and then again against 10 volumes of 10 mM Tris pH 8.1 for 24 hours. The dialyzed refolded protein was then filtered to remove the aggregated protein (produced as a by-product during the retraction (through a 0.45 μ nitrocellulose membrane (Whatman).) The purification of the biotin-labeled soluble TCR was then performed by loading onto a column of POROS 20 HQ operating in a Biocad Sprint system Approximately 500 ml of the refolded protein solution could be loaded by operation and elution of the protein was achieved through a sodium chloride gradient of pH regulator of bis- tris-propane, pH 8.0. The protein was eluted at approximately 100 mM sodium chloride and the relevant fractions were immediately cooled on ice and a protease inhibitor cocktail was added. The fractions were analyzed by SDS-PAGE stained with Coomassie.

Example 14 - Refolding and Purification of the TCRz Heterodimer with a Biotinylated β Chain. Biotin labeled TCR ß chains were mixed with an equal amount of a chain expressed and purified as for the soluble T cell receptor. The heterodimeric TCRz-β-BT was refolded according to the identical procedures described in Example 13 for TCRz (see Figure 37).

Example 15 - Biotinylation of Soluble TCRz-BT Marked with Biotin. The fractions containing protein were concentrated to 2. 5 ml using 10 K cutting centrifuge concentrators (Ultrafree, Millipore). The pH regulator was exchanged using PD-10 desalting columns equilibrated with 10 mM Tris pH 8.1, 5 mM NaCl, added cocktail of additional protease inhibitor, and the protein was concentrated to approximately 1 ml again using centrifuge concentrators. To this 1 ml of biotinylated soluble TCR was added the following: 7.5 mM MgCl2, 5 mM ATP (pH 8.0), 1 mM biotin, 2.5 μg / ml biotinylation enzyme BirA. The biotinylation reaction was then allowed to proceed at room temperature (20-25 ° C) overnight. Then, the enzymatically biotinylated soluble TCR was separated from the residual unreacted biotin through gel filtration on a Superdex 200 H R column (Pharmacia) operating in a Pharmacia FPLC system (see Figure 38). The column was equilibrated with PBS and fractions of 1 ml were collected, which were immediately cooled on ice and protected with protease inhibitor cocktail again. The protein concentration was estimated using a Coomassie binding assay (Pierce) and the biotinylated protein was then allowed to store at 4 ° C for up to 1 month or at -20 ° C for longer periods. The efficiency of the biotinylation reaction was verified using Western staining of the biotinylated protein. An SDS-PAGE gel was operated using the methods described above, but instead of staining, the gel was stained on a PVDF membrane (Bio-Rad) using a semi-dry SemiPhor electrotyping device (Hoefer). The staining stack comprised six layers of filter paper (Whatman 4M) cut to the size of the gel and soaked in the transfer pH buffer (25 mM Tris base, 150 mM glycine) followed by the PVDF membrane, which was pre-wetted with methanol and then soaked in transfer pH buffer, followed by the gel, which was moderately stirred in the transfer buffer for 5 minutes, followed by six more layers of soaked filter paper. The stack was moderately compressed using a test tube to wind any air bubbles and approximately 10 ml of additional transfer pH buffer was added to aid driving. The cathode was placed on top of the stack and current was passed through the apparatus at a constant current of 50 mA for 1 hour. The membrane was incubated in a 2% solution of gelatin (BioRad) in pH buffer of PBS-T (PBS + 0.05% Tween-20) during > 1 hour at room temperature with moderate agitation. Also included were nocturnal 0.01% sodium azide incubations to inhibit the growth of bacteria. The membrane was washed with several changes (4-5) of PBS-T followed by staining with diluted avidin-H RP conjugate (Sigma), 1: 1000, in a 1% solution of gelatin in PBS-T during > 30 minutes at room temperature with moderate agitation. The membrane was then washed with several changes (4-5) of PBS-T before detection with Opti-4CN (BioRad). This is a reagent that reacts in the presence of H RP to form an insoluble blue color, which stains the membrane in its place where the relevant protein is present as indicated by the presence of bound H RP. When the RP avidin-H conjugate is used for staining, it, therefore, indicates the presence of biotin-containing protein. Figure 39 shows a graph performed in such manner in several biotinylated TCRs. The standards that operate in this stain were biotinylated broad-scale molecular weight markers (Bio-Rad). The staining clearly shows a high level of biotinylation of the TCRs containing the biotinylation label (TAG) which has reacted with the enzyme BirA.

Example 16 - Production of Soluble, Biotinylated M HC-Peptides Complexes Biotinylated soluble MHC-peptide complexes can be produced as described in Example 2.

Example 17 - Assay for the Specific Link Between Soluble TCR and MHC-flu. The soluble TCR molecule, J M22z, is specific for MH-LA-A2 MHC molecules that present an immunodominant antigen consisting of residues of 58-66 amino acid residues 58-66 (GILGFVFTL) of the influenza matrix protein. The cloning, expression and purification of JM22z is described in Examples 7, 10, 11 and 13 and in Figures 35 and 36. The interactions between JM22z and its ligand / MHC complex (HLA-A2-flu) or a combination of irrelevant HLA-A2 peptide, the production of which is described in Example 13, were analyzed in a Biacore 2000 ™ surface plasmon resonance biosensor (SPR). The SPR measures changes in the refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyze their affinity and kinetic parameters. The probe flow cells were prepared by immobilizing the individual H LA-A2 peptide complexes in separate flow cells through the linkage between the biotin entangled in ß2m and streptavidin, which had been chemically interlaced to the active surface of the cells of flow. The assay was then performed by passing JM22z over the surfaces of the different flow cells at a constant flow rate, measuring the SPR response in doing so. Initially, the specific character of the interaction was verified by passing 28 μm of JM22z at a constant flow rate of 5 μl min "1 over the three different surfaces, one coated with 2800 RU of HLA-A2-flu, the second covered with 4200 RU of H LA-A2 folded with an irrelevant peptide of reverse transcriptase of VI H (H LA-A2-poly: ILKEPVHGV), and the third covered with 4300 RU of CD5 (Figure 40, one insertion) .JM22z injections Soluble at constant flow velocity at different concentrations on H LA-A2-poly were used to define the previous resonance (Figure 40a) .The values of these control measurements were subtracted from the values obtained with H LA-A2-flu (Figure 40b) and used to calculate the binding affinities expressed as the dissociation constant, Kd (Fig. 40c) The Kd value of JM22z and the relevant MHC molecule was determined as 15 + 4 μM (n = 7) at 37 ° C and 6.6 + 2 μM (n = 14) at 25 ° C. ination using TCR immobilized in the probe flow cell and the MHC-soluble peptide complex gave a similar Kd value of 5.6 + 4 μM (n = 3) at 25 ° C. Activation activation speed was determined to be between 6.7 x 104 and 6.9 x 104M "1 s" 1 at 37 ° C, while the deactivation rate was 1 .1 s "1 (Wilcox, Gap et al., 1999). ).

Example 18 - Assay for the Specific Link between Murine Soluble TCR and MHC H2-Db-NP from Murino. In this experiment, a murine TCR, F5, specific for a peptide derived from the nucleoprotein of influenza virus (aa, 366-374; ASN ENMDAM) presented by the MHC molecule of H2-Db (H2-Db- N P). The MHC heavy chain gene used was slightly modified in the sense that it encoded only the amino acids 1 -280 of the native protein plus a 13 amino acid sequence recognized by the enzyme BirA. The resulting protein can be enzymatically biotinylated (Schatz 1993; O'Callagham, Byford et al., 1999). Analysis of SPR in the Biacore 2000 ™ SPR biosensor using this soluble TCR specific for immobilized H2-Db-N P showed that it could bind specifically to the MHC-ligand peptide combination (data not shown).

Example 19 - Comparison of the Biotinylated Soluble Tax-TCR Union with Biotinylated Soluble Mutant Tax-TCR. Biotinylated soluble tax-TCRs were prepared as in Examples 12-14 and performed in Biacore 2000 analysis as in Example 17, using biotinylated pMHC complexes refolded with either the influenza matrix peptide (GI LGFVFTL) or the peptide HTLV tax 1 1-19 (LLFGYPVYV). The biotinylated soluble TCRs flowed over all cells at 5 μl / minute for a total of 1 minute. Figure 41 shows the binding of the first biotinylated soluble tax-TCR and then the biotinylated soluble mutant tax-TCR to the complex of HTLV tax 1 1 -19 peptide MHC (A). Neither the wild-type tax-TCR nor the mutant showed binding to either the influenza-MHC-matrix peptide complex (B / C) or the monoclonal antibody control OX68 (D). Therefore, it is concluded that both wild-type and mutant biotinylated soluble TCRs clearly bind effectively and specifically to the tax-pMHC complex and show very little difference in the degree of binding.

Example 20 - TCR and CD8 Co-Receptor Linkage Analysis ai MHC-Immobilized Peptide Complex. CD8 and CD4 are surface glycoproteins that are believed to function as co-receptors for TCRs by simultaneously binding to the same MHC molecules as TCR. The CD8 is characteristic for cytotoxic T cells and binds to MHC class I molecules, while CD4 is expressed on T cells of the ancillary lineage and binds MHC class II molecules. CD8 is a dimer consisting of either two identical chains or one a chain and one ß chain. The homodimeric aa-CD8 molecule was produced as described (PCT / GB98 / 03235; (Gao, Tomo et al., 1997; Gao, Gerth et al., 1998) .In this example, the simultaneous binding of soluble TCR and molecules is described. from CD8 to the immobilized H LA-A2-flu complex As shown in Figure 42A, the binding response was simply additive by subtracting the values of the TCR response (open circles) from the values of the combined response (circle closed) values were presented (open squares) very close to the response value of 120 μM only of CD8 (open triangles) Figure 42B shows that the kinetics of the interaction of TCR-MHC-peptide was not affected by the binding The additive linkage observed indicates that TCR and CD8 bind to the MHC-peptide complex on separate intermediate surfaces.The example also illustrates that in some cases, the specific binding for one molecule will not influence the specific binding of another molecule. a, a situation that is probably different from other combinations of molecules.

Example 21 - Formation of TCR Tetramers from Biotinylated Soluble TCR. The formation of TCR tetramers was achieved using avidin or streptavidin or its derivatives. Avidin (from the egg of the hen) has an unusually high isoelectric point resulting in a high positive charge at a neutral pH, which causes a specific binding to many other proteins and surfaces. It is usually commercially modified to reduce the isoelectric point so that it behaves more like a streptavidin (from a bacterial source). In this form, it is known as extravidin (Sigma) or Neuratrividin (Molecular Probes). Any of these, or streptavidin can be modified to contain a label such as a fluorescent label for detection using FACS scanning. TCR tetramers were formed using an extravidin / streptavidin concentration of 1/4 of the total concentration of biotinylated soluble TCR. Extravidin / streptavidin was added on ice in aliquots, so that if the concentration is not totally accurate, most of the extravidin / streptavidin can be served by being saturated with TCR. The TCR tetramers were analyzed through size exclusion chromatography on a Superdex 200 HR column (Pharmacia), Figures 43 and 44. The TCR binding to avidin was confirmed by running the control samples of unbound TCR and extravidin / Streptavidin separately. The TCR tetramer was eluted from the column to a retention volume corresponding to the highest molecular weight. For unmodified extravidin, it was possible to determine the approximate molecular weight of the produced TCR tetramers (as compared to standard proteins of known molecular weight) and was approximately 285,000, compared to a calculated molecular weight for a complete TCR tetramer of 305,000 .

Example 22 - Linkage Analysis of Biotinylated Monomeric TCR and TCR Tetramers to the MHC-Peptide Complex. Peptide-MHC complexes were prepared as in Example 16 using the influenza matrix peptide (GILGFVFTL) or the HTLV tax peptide (LLFGYPVYV), recombinant HLA-A2 heavy chain and recombinant chemically biotinylated β-2 microglobulin. A Biacore 2000 ™ SPR biosensor was used to measure molecular interactions between TCRs, TCR tetramers and pMHC complexes. The biotinylated pMHC complexes were immobilized to streptavidin conjugated to the wafer surface CM-5 through coupling with amine. OX68, a biotinylated monoclonal antibody, provided by Dr. P. Anton van der Merwe of Sir William Dunn School of Pathology, was used as a non-specific control protein in one of the cells. After immobilization of the pMHC complex, the residual biotin binding sites were saturated with 10 mM biotin. This is necessary to prevent biotinylated TCRs from binding to the streptavidin-coated wafer through its biotinylation, rather than specifically through the interaction of TCR-pMHC. The soluble biotinylated TCRs were then flowed onto the wafer at a concentration of about 1 mg / ml and the TCR tetramers were flowed at a concentration of about 50 μg / ml.

Figure 45 shows the binding of the soluble biotinylated flu-TCR and flu-TCR tetramers to the pM HC complexes, Figure 46 shows the link of the tax-TCR tetramers and soluble biotinylated tax-TCR to the same complexes of pMH C. Both the biotinylated sTCRs and the TC R tetramers showed a specific full character, binding strongly to their specific MHC peptide complex, but not to the entire non-specific M HC peptide complex. The increase in affinity caused by the multimerization of the TCRs can be seen in the respective inactivation rates of the sT R and the TCR tetramer for both TCRs. The inactivation rate for both TCRs was increased for several seconds to several hours (the exact measurement of these speeds was not possible due to the effects of re-link). Some more permanent bond of biotinylated soluble TCR was observed in both cases, which was caused by the presence of ag protein watered in the preparations. In both cases, this level of strong bond was very low compared to the TCR tetramers which bear in mind that the total amount of TC R tetramer injected into the flow cell was about one-quarter of the amount of biotinylated soluble TCR injected ( 25 μl x 0.05 mg / ml compared with 5 μl x 1 mg / ml).

Example 23 - Staining of Antigen Presentation Cells with TCR Tetramers. Cell staining experiments were performed on a B-cell line called T2, which is homozygous for HLA-A2 and does not process peptide antigens that result in the presence of incomplete class I MHC molecules on the cell surface. that can be filled with a single type of external peptide. Cells were grown in an R-10 medium at 37 ° C and a 5% CO2 atmosphere. Approximately 2 million cells were taken and washed twice in the RPMI medium (centrifugation was 1,500 rpm for 5 minutes), the peptide was added at varying concentrations in 10% DMSO in RPMI. Typically, concentrations of 0, 10"4, 10" 5, and 10"6M of influenza matrix peptide (sequence: GILGFVFTL) and the peptide tax (sequence: LLFGYPVYV) were used.The cells were pulsed for 1 hour at 37 ° C. ° C to allow the peptide to bind to the MHC class I molecules on the surface of the cell, then the cells were washed twice with the RPMI medium to remove the excess peptide, the cells pulsed with peptide were stained with the TCR tetramer at room temperature with 1-10 μg of the TCR tetramer labeled with either the fluorescent marker FITC or RPE.The staining was allowed to continue for 30 minutes and the cells were then washed once more with ice-cold RPMI followed by fixation with 3% formaldehyde in the PBS solution.The stained, stained cells were then stored at 4 ° C in the dark until one week before the FACS analysis.The FACS analysis was performed using an explorer Becton-Dickinson FACS and data were recorded and analyzed using the "Cellquest" software. Figure 47 shows the specific binding of TCR tetramers made with either the flu matrix TCR or the TCR tax to their specific peptides. The antecedent and the cross reactivity were low. Interestingly, the tax TCR tetramer appears to bind better to its peptide than the TCR tetramer of flu matrix does to the peptide, although this may be an effect of variable affinities of the peptide for MHC class I molecules during peptide pulsation. Another variety of cells were also stained using TCR tetramers, .45 cells, which are a normal heterozygous B cell line for H LA-A2. The cells were prepared and the peptide was pulsed, labeled and scanned by FACS exactly as for T2 cells. Figure 48 shows the results of the TCR tetramer labeling of the pulsed peptide, .45 cells. The staining of the cells is notoriously lower than that for T2 cells, which is as expected since the .45 cells are heterozygous for HLA-A2, while the T2 cells are homozygous. In addition to this effect, it is possible that since the M HC complexes of class I on the surface of .45 cells are initially loaded with peptide, whereas the case of the T2 cells the complexes are initially empty, there could be a higher efficiency of peptide loading on the surface of T2 cells compared to the other H-LA-A2 positive cells.

Example 24 - Preparation of, and Staining with, Latex Beads Covered with TCR. In order to improve the sensitivity of the TCR voltage for antigen, beads coated with TCR labeled with a fluorescent label were made. The beads coated with fluorescently labeled neutravidin were labeled in Molecular Probes. The coat of the beads with biotinylated soluble TCR was performed by co-incubation at 4 ° C with a saturation concentration of TCR to ensure that the maximum number of binding sites on the beads was occupied with TCR. The beads were then used to label cells for pulsed peptide antigen presentation, a form similar to that described for TCR tetramers except that blocking reagents were included to reduce the background level. This strategy was not completely successful as evidenced by the high amount of staining for non-pulsed cells and for cells pulsed with irrelevant peptide. However, a substantial amount of specific labeling was also observed at the level of staining history (Figure 49). Interestingly, some tagging of the tax peptide pulsed with T2 cells was observed with beads coated with tax-TCR. This labeling is an order of magnitude less than the concentration of beads that is possible to detect using TCR tetramers indicating that the higher TCR multimerization will allow the detection and binding to lower levels of presented antigen.

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LIST OF SEQUENCES < 110 > Avidity Ltd. < 120 > Multiple T Cell Receptor Complexes < 130 > NJL / P21737WO < 140 > PCT / GB99 / 01583 < 141 > 1999-05-19 < 150 > GB 9810759.2 < 151 > 1998-05-19 < 150 > GB 9821129.5 < 151 > 1998-09-29 < 160 > 85 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 744 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene coding for TCR alpha chain JM22 restricted from human HLA-A2 / flu matrix peptide fused to the leucine closing domain c-jun. < 400 > 1 atgcaactac tagaacaaag tcctcagttt ctaagcatcc aagagggaga aaatctcact 60 gtgtactgca actcctcaag tgttttttcc agcttacaat ggtacagaca ggagcctggg 120 gaaggtcctg tcctcctggt gacagtagtt acgggtggag aagtgaagaa gctgaagaga 180 ctaacctttc agtttggtga tgcaagaaag gacagttctc tccacatcac tgcggcccag 240 caggcctcta cctggtgata cctctgtgca ggagcgggaa gccaaggaaa tctcatcttt 300 ggaaaaggca ctaaactctc tgttaaacca aatatccaga accctgaccc tgccgtgtac 360 cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420 caaacaaatg tgtcacaaag taaggattct tcacagacaa gatgtgtata aactgtgcta 480 gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540 tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600 ccagaaagtt cccccggggg tagaatcgcc cggctggagg aaaaagtgaa aaccttgaaa 660 gctcagaact cggagctggc gtccacggcc aacatgctca gggaacaggt ggcacagctt aaacagaaag tcatgaacta ctag 720 744 < 210 > 2 < 211 > 247 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: amino acid sequence of TCR alpha chain JM22 restricted from human HLA-A2 / flu matrix peptide fused to the leucine closure domain c-jun. < 400 > 2 Met Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser lie Gln Glu Gly 1 5 10 15 Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser Ser Leu 20 25 30 Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu Val Thr 35 40 45 Val Val Thr Gly Gly Val Lys Lys Leu Lys Arg Leu Thr Phe Gln 50 55 60 Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His lie Thr Ala Ala Gln 65 70 75 80 Pro Gly Asp Thr Gly Leu Tyr Leu Cys Wing Gly Wing Gly Ser Gln Gly 85 90 95 Asn Leu lie Phe Gly Lys Gly Thr Lys Leu Ser Val Lys Pro Asn lie 100 105 110 Gln Asn Pro Asp Pro Wing Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser 115 120 125 Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val 130 135 140 Ser Gln Ser Lys Asp Ser Asp Val Tyr lie Thr Asp Lys Thr Val Leu 145 150 155 160 Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Wing Val Wing Trp Ser 165 170 175 Asn Lys Ser Asp Phe Wing Cys Wing Asn Wing Phe Asn Asn Ser lie lie 180 185 190 Pro Glu Asp Thr Phe Phe Pro Ser Glu Ser Ser Pro Gly Gly Arg 195 200 205 lie Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln Asn Ser 210 215 220 Glu Leu Wing Being Thr Wing Asn Met Leu Arg Glu Gln Val Wing Gln Leu 225 230 235 240 Lys Gln Lys Val Met Asn Tyr 245 < 210 > 3 < 211 > 864 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene coding for TCR alpha chain JM22 restricted from human LA peptide LA-A2 / flu fused to the leucine closure domain c-jun. < 400 > 3 atggtggatg gtggaatcac tcagtcccca aagtacctgt tcagaaagga aggacagaat 60 gttgtgaaca gtgaccctga gaatttgaac cacgatgcca tgtactgg to ccgacaggac 120 ccagggcaag ggctgagatt gatctactac tcacagatag taaatgactt tcagaaagga 180 gatatagctg aagggtacag cgtctctcgg gagaagaagg aatcctttcc tctcactgtg 240 acatcggccc aaaagaaccc gacagctttc tatctctgtg ccagtagttc gaggagctcc 300 tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtt 360 ttcccacccg aggtcgctgt gtttgaacca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480 aggaggtgca gtgaatggga cagtggggtc agcacagacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag accccggggg tctgactgat acactccaag tcaacttgaa cggagacaga 780 ctgcgttgca gacaagaagt gaccgagatt gccaatctac tgaaagagaa ggaaaaacta 840 gagttcatcc tggcagc tta ctag 864 < 210 > 4 < 211 > 287 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Beta chain amino acid sequence of TCR JM22 restricted from human HLA-A2 / flu matrix peptide fused to the leucine c-fos closing domain. < 400 > 4 Met Val Asp Gly Gly lie Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 1 5 10 15 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 20 25 30 Wing Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu lie 35 40 45 Tyr Tyr Ser Gln lie Val Asn Asp Phe Gln Lys Gly Asp lie Wing Glu 50 55 60 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 65 70 75 80 Thr Ser Wing Gln Lys Asn Pro Thr Wing Phe Tyr Leu Cys Wing Being Ser 85 90 95 Being Arg Being Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Val Val Val Phe Wing 115 120 125 Glu Pro Ser Glu Ala Glu Lie Ser His Thr Gln Lys Wing Thr Leu Val 130 135 140 Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro 165 170 175 Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser 180 185 190 Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asn Pro Arg Asn His Phe 195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 210 215 220 Gln Asp Arg Wing Lys Pro Val Thr Gln He Val Ser Wing Glu Wing Trp 225 230 235 240 Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing Glu Thr 245 250 255 Asp Gln Leu Glu Asp Lys Lys Ser Wing Leu Gln Thr Glu He Wing Asn 260 265 270 Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Ala Tyr 275 280 285 < 210 > 5 < 211 > 918 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene encoding TCR Beta Chain JM22 Restricted Human LA-A2 / Fluid H-peptide fused to domination of leucine c-fos and biotinylation label BirA. < 400 > 5 atggtggatg gtggaatcac tcagtcccca aagtacctgt tcagaaagga aggacagaat 60 gttgtgaaca gtgaccctga gaatttgaac cacgatgcca tgtactggta ccgacaggac 120 ccagggcaag ggctgagatt gatctactac tcacagatag taaatgactt tcagaaagga 180 gatatagctg aagggtacag cgtctctcgg gagaagaagg aatcctttcc tctcactgtg 240 acatcggccc aaaagaaccc gacagctttc tatctctgtg ccagtagttc gaggagctcc 300 tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtt 360 ttcccacccg aggtcgctgt gtttgaacca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480 gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag accccggggg tctgactgat acactccaag tcaacttgaa cggagacaga 780 ctgcgttgca gacaagaagt gaccgagatt gccaatctac ggaaaaacta tgaaagagaa 840 gagttcatcc tggcagc tta cggatccggt ggtggtctga acgatatttt tgaagctcag 900 aaaatcgaat ggcattaa 918 < 210 > 6 < 211 > 305 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Beta chain amino acid sequence of TCR JM22 restricted from human HLA-A2 / flu matrix peptide fused to the leucine c-fos closing domain and BirA biotinylation label. < 400 > 6 Met Val Asp Gly Gly He Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 1 5 10 15 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 20 25 30 Wing Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu He 35 40 45 Tyr Tyr Ser Gln He Val Asn Asp Phe Gln Lys Gly Asp He Ala Glu 50 55 60 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 65 70 75 80 Thr Ser Wing Gln Lys Asn Pro Thr Wing Phe Tyr Leu Cys Wing Being Ser 85 90 95 Being Arg Being Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Val Val Val Phe Wing 115 120 125 Glu Pro Ser Glu Ala Glu He Ser His Thr Gln Lys Wing Thr Leu Val 130 135 140 Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro 165 170 175 Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser 180 185 190 Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asn Pro Arg Asn His Phe 195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 210 215 220 Gln Asp Arg Wing Lys Pro Val Thr Gln He Val Ser Wing Glu Wing Trp 225 230 235 240 Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing Glu Thr 245 250 255 Asp Gln Leu Glu Asp Lys Lys Ser Wing Leu Gln Thr Glu He Wing Asn 260 265 270 Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Wing Tyr Gly 275 280 285 Ser Gly Gly Gly Leu Asn Asp He Phe Glu Wing Gln Lys He Glu Trp 290 295 300 His 305 < 21 0 > 7 < 21 1 > 750 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene encoding for TCR alpha chain restricted from peptide H LA-A2 / HTLV-1 Tax human fused to leucine closure domain c-jun. < 400 > 7 aagtggagca atgcagaagg gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 gccctgagtt tctgggaaaa atatactcca gataatgtcc atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagggaccca cagtttggag ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttcccc cgggggtaga atcgcccggc tggaggaaaa agtgaaaacc 660 agaactcgga ttgaaagctc acggccaaca gctggcgtcc acaggtggca tgctcaggga 720 cagcttaaac agaaagtcat gaactactag 750 < 210 > 8 < 211 > 249 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Alpha chain amino acid sequence of TCR restricted peptide of H LA-A2 / HTLV-1 Tax Human of clone A6 fused to the closing domain of leucine c-jun. < 400 > 8 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Wing Wing Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln 20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu He 35 40 45 Met Ser He Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Wing 50 55 60 Gln Leu Asn Lys Wing Wing Gln Tyr Val Ser Leu Leu He Arg Asp Ser 65 70 75 80 Gln Pro Being Asp Being Wing Thr Tyr Leu Cys Wing Val Thr Thr Asp Ser 85 90 95 Trp Gly Lys Leu Gln Phe Gly Wing Gly Thr Gln Val Val Val Thr Pro 100 105 110 Asp He Gln Asn Pro Asp Pro Wing Val Tyr Gln Leu Arg Asp Ser Lys 115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr 130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr He Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Wing Val Wing 165 170 175 Trp Ser Asn Lys Ser Asp Phe Wing Cys Wing Asn Wing Phe Asn Asn Ser 180 185 190 He He Pro Glu Asp Thr Phe Phe Pro Pro Glu Ser Ser Gly 195 200 205 Gly Arg Zle Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln 210 215 220 Asn Ser Glu Leu Wing Being Thr Wing Asn Met Leu Arg Glu Gln Val Wing 225 230 235 240 Gln Leu Lys Gln Lys Val Met Asn Tyr 245 < 21 0 > 9 < 21 1 > 928 < 212 > A D N < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene coding for beta chain of the TCR restricted peptide of H LA-A2 / HTLV-1 Tax h u mana of clone A6 fused to the leucine c-fos closing domain and Bi rA biotin labeling. < 400 > 9 atgaacgctg gtgtcáctea gaccccaaaa ttccaggtcc tgaagacagg acagageatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat teattaetca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca ggacctgaaa cggtcacaga 360 aacgtgttcc cacccgaggt cgctgtgttt gagecatcag aagcagagat ctcccacacc 420 caaaaggcca cctggccaca cactggtgtg ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggggtcagca ggtgcacagt cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc agaacccccg caaccacttc cgctgtcaag tecagtteta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagaccc cgggggtctg actgatacac gacagatcaa tccaagcgga 780 cttgaagaca agaagtctgc gttgcagacc gagattgeca atctactgaa agagaaggaa 840 aaactagagt tcatcc tggc agcttacgga tccggtggtg gtctgaacga tatttttgaa 900 gctcagaaaa tcgaatggca ttaagctt 928 < 21 0 > 1 0 < 21 1 > 307 < 212 > PRT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Amino Acid Sequence Beta Chain TCR Restricted from H LA-A2 / HTLV-1 Tax peptide of human A6 clone fused to the leucine c-jfos closure domain and BirA biotinylation label. < 400 > 10 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Wing Gln Asp Met Asn His Glu Tyr 20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu He His 35 40 45 Tyr Ser Val Gly Wing Gly He Thr Asp Gln Gly Glu Val Pro Asn Gly 50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Wing Wing Pro Ser Gln Thr Ser Val Tyr Phe Cys Wing Ser Arg Pro 85 90 95 Gly Leu Wing Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Wing 115 120 125 Val Phe Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr 130 135 140 Leu Val Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 165 170 175 Gln Pro Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Wing Leu 180 185 190 Ser Ser Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asn Pro Arg Asn 195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln He Val Ser Wing Glu 225 230 235 240 Wing Trp Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing 245 250 255 Glu Thr Asp Gln Leu Glu Asp Lys Lys Ser Ala Leu Gln Thr Glu He 260 265 270 Wing Asn Leu Leu Lys Glu Lys Glu Lys Leu Glü Phe He Leu Ala Wing 275 280 285 Tyr Gly Ser Gly Gly Gly Leu Asn Asp He Phe Glu Wing Gln Lys He 290 295 300 Glu Trp His 305 < 21 0 > 1 1 < 21 1 > 765 < 212 > AD N < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene encoding for TCR alpha chain restricted from LA-A2 peptide / HTLV-1 Tax h umana of clone M 10B7 / D3 fused to the closing domain of leuci na c-jun. < 400 > 11 agaatgatga atgcaacaga ccagcaagtt aagcaaaatt caceatccct gagcgtccag 60 tttctattct gaaggaagaa tatactaaca gaactgtgac ttatttccta gcatgtttga 120 tggtacaaaa aataccctgc tgaaggtcct acattcctga tatetataag ttccattaag 180 gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 240 ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaatggag 300 ggageccaga agctggtatt tggccaagga accaggctga ctatcaaccc aaatatccag 360 ctgccgtgta aaccctgacc ccagctgaga gactctaaat ccagtgacaa gtctgtctgc 420 ctattcaccg attttgattc tcaaacaaat gtgtcacaaa gtaaggattc tgatgtgtat 480 atcacagaca aaactgtgct agacatgagg tctatggact tcaagagcaa cagtgctgtg 540 acaaatctga gcctggagca ctttgcatgt gcaaacgcct tcaacaacag cattattcca 600 gaagacacct tcttccccag cccagaaagt tcccccgggg gtagaatcgc ccggctggag 660 aaaccttgaa gaaaaagtga agctcagaac tcggagctgg cgtccacggc caacatgctc 720 agggaacagg tggcacagct taaacagaaa gtcatgaact actag 765 < 210 > 12 < 21 1 > 254 < 212 > P RT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Alpha chain amino acid sequence of the TC R restricted peptide H LA-A2 / HTLV-1 Human Tax of the clone M 1 0 B7 / D3 fused to the closing domain of leucine c-jun. < 400 > 12 Met Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser 1 5 10 15 Leu Ser Val Gln Glu Gly Arg He Ser He Leu Asn Cys Asp Tyr Thr 20 25 30 Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Glu Wing 35 40 45 Gly Pro Thr Phe Leu He Ser He Be Ser He Lys Asp Lys Asn Glu 50 55 60 Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser 65 70 75 80 Leu His He Val Pro Ser Gln Pro Gly Asp Ser Wing Val Tyr Phe Cys 85 90 95 Wing Wing Met Glu Gly Wing Gln Lys Leu Val Phe Gly Gln Gly Thr Arg 100 105 110 Leu Thr He Asn Pro Asn He Gln Asn Pro Asp Pro Wing Val Tyr Gln 115 120 125 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 130 135 140 < 21 0 > 1 3 < 21 1 > 925 < 212 > A DN < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Gene coding for beta chain of TCR restricted peptide of H LA-A2 / HTLV-1 Tax of clone M 1 0B7 / D3 h umana fused to the closing domain of leucine c-fos and biotinylation label BirA . < 400 > 13 gtgtcactca atgaacgctg gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcagttacca ggaggggggg 300 ttttacgagc agtacttcgg gccgggcacc aggctcacgg tcacagagga cctgaaaaac 360 gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420 aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 480 tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 540 cagcccgccc tcaatgactc cagatacgct ctgagcagcc gcctgagggt ctcggccacc 600 ttctggcagg acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660 aatgacgagt ggacccagga tagggccaaa cccgtcacee agatcgtcag cgccgaggcc 720 tggggtagag cagaccccgg gggtctgact gatacactcc aagcggagac agatcaactt 780 gaagacaaga agtctgcgtt gcagaccgag attgccaatc gaaggaaaaa tactgaaaga 840 ctagagttca tcctgg cagc ttacggatcc ggtggtggtc tgaacgatat ttttgaagct 900 cagaaaatcg aatggcatta agett 925 < 210 > 14 < 211 > 306 < 212 > PRT < 21 3 > Artificial Sequence l < 220 > < 223 > Description of Artificial Sequence: Amino acid sequence of beta chain of TCR restricted peptide H LA-A2 / HTLV-1 Human Tax of clone M 10B7 / D3 fused to the leucine c-fos closing domain and biotinylation label BirA. < 400 > 14 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Wing Gln Asp Met Asn His Glu Tyr 20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu He His 35 40 45 Tyr Ser Val Gly Wing Gly He Thr Asp Gln Gly Glu Val Pro Asn Gly 50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Wing Wing Pro Ser Gln Thr Ser Val Tyr Phe Cys Wing Ser Ser Tyr 85 90 95 Pro Gly Gly Ghe Phe Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Val Glu Wing Val 115 120 125 Phe Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr Leu 130 135 140 Val Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 145 150 155 160 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 165 170 175 Pro Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Wing Leu Ser 180 185 190 Ser Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asp Pro Arg Asn His 195 200 205 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 210 215 220 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln He Val Ser Wing Glu Wing 225 230 235 240 Trp Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing Glu 245 250 255 Thr Asp Gln Leu Glu Asp Lys Lys Ser Wing Leu Gln Thr Glu He Wing 260 265 270 Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Ala Tyr 275 280 285 Gly Ser Gly Gly Gly Leu Asn Asp He Phe Glu Wing Gln Lys He Glu 290 295 300 Trp His 305 < 210 > 15 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: forward poly-C "anchor" primer for PCR amplification of cDNAs extended to its 3 'terminal with a stretch of G residues using Terminal Transferase. < 400 > 15 taaatactcg aggcgcgccc cccccccccc ccc 33 < 210 > 16 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: specific PCR primer in 3 'of the human TCR alpha chain constant region. < 400 > 16 atataacccg gggaaccaga tccccacagg aactttctgg gctgggga 48 < 210 > 17 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: 3 'specific PCR primer of human TCR beta chain constant region. < 400 > 17 atataacccg gggaaccaga tccccacagt cgtctctacc ccaggcc 47 < 210 > 18 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: 5'-specific PCR primer for human c-jun leucine closure. < 400 > 18 catacaccca ggggtagaat cgcccggctg gag 33 < 210 > 19 < 211 > 50 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: specific 3 'PCR primer for human c-jun leucine closure. < 400 > 19 gtgtgtgctc gaggatccta gtagttcatg actttctgtt taagctgtgc 50 < 210 > 20 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: specific PCR primer in 5 'of human leucine c-fos close. < 400 > 20 catacacccg ggggtctgac tgatacactc caagcggag 39 < 210 > 21 < 211 > 49 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: specific PCR primer in 3 'closing of leucine c-fos human. < 400 > 21 tgtgtgctcg aggatcctag taagctgcca ggatgaactc tagtttttc 49 < 210 > 22 < 211 > 120 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > Sequence of partial human c-jun coding for the leucine closing domain as fused to alpha chains of TCR < 400 > 22 agaatcgccc ggctggagga aaaagtgaaa accttgaaag ctcagaactc ggagctggcg 60 tccacggcca acatgctcag ggaacaggtg gcacagctta aacagaaagt catgaactac 120 < 210 > 23 < 211 > 120 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > Sequence of partial human c-fos encoding the leucine closure domain as fused to TCR beta chains. < 400 > 23 ctgactgata cactccaagc ggagacagac caactagaag atgagaagtc tgctttgcag 60 accgagattg ccaacctgct gaaggagaag gaaaaactag agttcatcct ggcagcttac 120 < 210 > 24 < 211 > 40 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > Leucine c-jun close domain amino acid sequence as fused to TCR alpha chains. < 400 > 24 Arg He Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln Asn 1 5 10 15 Ser Glu Leu Wing Ser Thr Wing Asn Met Leu Arg Glu Gln Val Wing Gln 20 25 30 Leu Lys Gln Lys Val Met Asn Tyr 35 40 < 210 > 25 < 211 > 40 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > Leucine c-fos closing domain amino acid sequence as fused to TCR beta chains. < 400 > 25 Leu Thr Asp Thr Leu Gln Wing Glu Thr Asp Gln Leu Glu Asp Glu Lys 1 5 10 15 Be Ala Leu Gln Thr Glu He Ala Asn Leu Leu Lys Glu Lys Glu Lys 20 25 30 Leu Glu Phe He Leu Ala Ala Tyr 35 40 < 210 > 26 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiation of PCR of advance for the mutation of cysteine unequal of beta chains of human TCR to serine. < 400 > 26 gactccagat acagcctgag cagccg 26 < 210 > 27 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: partial amino acid sequence of the human TCR beta chain after the mutation of cysteine uneven to serine. < 400 > 27 Asp Ser Arg Tyr Ser Leu Ser Ser 1 5 < 210 > 28 < 211 > 26 < 212 > DNA < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: reverse I CRP primer for the mismatch cysteine mutation of beta chains of human R-C to serine. < 400 > 28 cggctgctca ggctgtatct ggagtc 26 < 210 > 29 < 211 > 26 < 212 > DNA < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiation of PCR for the advancement of unequal cysteine of beta chains of human TCR to alanine. < 400 > 29 gactccagat acgctctgag cagccg 26 < 210 > 30 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: partial amino acid sequence of human TCR beta chains after the mutation of cysteine uneven to alanine. < 400 > 30 Asp Ser Arg Tyr Ala Leu Ser Ser 1 5 < 210 > 31 < 21 1 > 26 < 21 2 > DNA < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: inverse PCR primer for the mismatch cysteine mutation of beta chains of human TC R to alanine. < 400 > 31 cggctgctca gagcgtatct ggagtc 26 < 210 > 32 < 211 > 57 < 212 > DNA < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: PC 5'-primer for the human alpha v 0.2 chain of TC R restricted from influenza matrix peptide JM22 / H LA-A0201. < 400 > 32 gctctagaca tatgcaacta ctagaacaaa gtcctcagtt tctaagcatc caagagg 57 < 210 > 33 < 211 > 15 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: new N-terminal amino acid sequence of the truncated Valfa10.2 chain of TCR restricted from influenza matrix protein peptide JM22 / HLA-A0201. < 400 > 33 Met Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser IleGln Glu 1 5 10 15 < 210 > 34 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: 5 'PCR primer for the amplification of the human Vbeta17 chain of TCR restriction of influenza matrix peptide JM22 / HLA-A0201. < 400 > 34 gctctagaca tatggtggat ggtggaatca ctcagtccc 39 < 210 > 35 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: New N-terminal amino acid sequence of the truncated Vbeta17 chain of TCR restricted from human JM22 influenza matrix peptide / HLA-A0201. < 400 > 35 Met Val Asp Gly Gly Lie Thr Gln Ser < 210 > 36 < 211 > 57 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: 5 'PCR primer for amplification of TCR mouse Valfa4 chain restricted from influenza virus nucleoprotein peptide / H2-Db < 400 > 36 gctctagaca tatggattct gttatcaaa tgcaaggtca agtgaccctc tcatcag 57 < 210 > 37 < 211 > 15 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: New N-terminal amino acid sequence of the truncated Valfa4 chain of TCR restricted from mouse influenza virus / H2-Db nucleoprotein peptide < 400 > 37 Met Asp Ser Val Thr Gln Met Gln Gly Gln Val Thr Leu Ser Ser 1 5 10 15 < 210 > 38 < 211 > 53 < 212 > DNA < 213 > Mus musculus < 220 > < 223 > 5 'PCR primer for the amplification of the TBE mouse Vbetall chain restricted from influenza nucleoprotein peptide / H2-Db < 400 > 38 gctctagaca tatggaacca acaaatgctg gtgttatcca aacacctagg cac 53 < 210 > 39 < 211 > 14 < 212 > PRT < 213 > Mus musculus < 220 > < 223 > New N-terminal amino acid sequence of the truncated Vbetall chain dek restricted TCR of peptide d? nucleoprotein of influenza virus / H2-Db. < 400 > 39 Met Glu Pro Thr Asn Wing Gly Val lie Gln Thr Pro Arg His 1 5, 10 < 210 > 40 < 211 > 36 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR Starter for Amplification of Human Valfa23 Chain of HIV-1 Peptide Restricted TCR Gag / HLA-A0201 < 400 > 40 ggaattccat atgaaacaag aggttacaca aattcc 36 < 210 > 41 < 211 > 8 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of truncated human Valfa23 chain of restricted TCR of HIV-1 Gag / HLA-A0201 peptide. < 400 > 41 Met Lys Gln Glu Val Thr Gln lie 1 5 < 210 > 42 < 211 > 36 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR primer for the amplification of the human Vbeta5.1 chain of the restricted TCR of HIV-1 Gag / HLA-A0201 peptide. < 400 > 42 ggaattccat atgaaagctg gagttactca aactcc 36 < 210 > 43 < 211 > 8 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of the truncated human Vbeta5.1 chain of the peptide-restricted TCR HIV-1 Gag / HLA-A0201. < 400 > 43 Met Lys Ala Gly Val Thr Gln Thr 1 5 < 210 > 44 < 211 > 33 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR primer for the amplification of the human Valfa2.3 chain of TCR A6 restricted peptide HTLV-1 Tax / H LA-A0201. < 400 > 44 cccccccata tgcagaagga agtggagcag aac 33 < 210 > 45 < 21 1 > 8 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of the chain Truncated Human Valfa2.3 of TCR A6 Restricted Peptide HTLV-1 Tax / HLA-A0201. < 400 > 45 Met Gln Lys Glu Val Glu Gln Lys 1 5 < 210 > 46 < 211 > 33 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR starter for chain amplification Human Vbeta12.3 of TCR restricted A6 of HTLV-1 peptide Tax / HLA-A0201. < 400 > 46 cccccccata tgaacgctgg tgtcactcag acc 33 < 210 > 47 < 211 > 8 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of the chain Truncated Human VCR12.3 TCR A6 Restricted from HTLV-1 Tax / HLA-A0201 Peptide. < 400 > 47 Met Lys Ala Gly Val Thr Gln Thr 1 5 < 210 > 48 < 211 > 48 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR primer for the amplification of the human Valfa 17.2 chain of TCR B7 restricted peptide HTLV-1 Tax / H LA-A0201. < 400 > 48 cccccccata tgcaacaaaa aaatgatgac cagcaagtta agcaaaat 48 < 210 > 49 < 211 > 13 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of truncated human Valfa 17.2 chain of TCR B7 restricted from HTLV-1 Tax / H peptide LA-A0201. < 400 > 49 Met Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn 1 5 10 < 210 > 50 < 211 > 45 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 5 'PCR primer for the amplification of the human Vbeta12.3 chain of TCR B7 restricted peptide HTLV-1 Tax / HLA-A0201. < 400 > 50 cccccccata tgaacgctgg tgtcactcag accccaaaat tccag 45 < 210 > 51 < 211 > 12 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > New N-terminal amino acid sequence of the chain Truncated Human VCR12.3 TCR TCR Restricted from HTLV-1 Tax / HLA-A0201 Peptide. < 400 > 51 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln 1 5 10 < 210 > 52 < 211 > 38 < 212 > DNA < 213 > Homo sapiens < 220 > < 223 > 3 'PCR primer for human Caifa chains, generally applicable. < 400 > 52 catacaaaag ggggaacttt ctgggctggg gaagaagg 38 < 21 0 > 53 < 21 1 > 33 < 21 2 > DNA < 21 3 > Homo sapiens < 220 > < 223 > PCR sensor 3 'for human Cbeta chains, generally applicable. < 400 > 53 catacacccg gggtctgctc taccccaggc ctc 33 <; 210 > 54 < 211 > 744 < 212 > DNA < 21 3 > Homo sapiens < 220 > < 223 > Mutated DNA sequence of the HR R alpha chain restricted from H LA-A2 matrix / soluble J M22 fluid, as it was fused to the human leucine c-j closing domain. < 400 > 54 atgcaactac tagaacaaag tcctcagttt ctaagcatcc aagagggaga aaatctcact 60 gtgtactgca actcctcaag tgttttttcc agcttacaat ggtacagaca ggagcctggg 120 gaaggtcctg tcctcctggt gacagtagtt acgggtggag aagtgaagaa gctgaagaga 180 ctaacctttc agtttggtga tgcaagaaag gacagttctc tccacatcac tgcggcccag 240 caggcctcta cctggtgata cctctgtgca ggagcgggaa gccaaggaaa tctcatcttt 300 ggaaaaggca ctaaactctc tgttaaacca aatatccaga accctgaccc tgccgtgtac 360 cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420 caaacaaatg tgtcacaaag taaggattct tcacagacaa gatgtgtata aactgtgcta 480 gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540 tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600 ccagaaagtt cccccggggg tagaatcgcc cggctggagg aaaaagtgaa aaccttgaaa 660 gctcagaact cggagctggc gtccacggcc aacatgctca gggaacaggt ggcacagctt aaacagaaag tcatgaacta ctag 720 744 < 21 0 > 55 < 21 1 > 247 < 21 2 > PRT < 21 3 > H omo sapiens < 220 > < 223 > A predicted amino acid sequence of the TCR alpha chain restricted from H-LA-A2 matrix / fl uble uble to part of J M22, as it was fused to the human leucine-closing domain of leucine. < 400 > 55 Met Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser He Gln Glu Gly 1 5 10 15 Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser Ser Leu 20 25 30 Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu Val Thr 35 40 45 Val Val Thr Gly Gly Val Val Lys Lys Leu Arg Leu Thr Phe Gln 50 55 60 Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His He Thr Ala Ala Gl? 65 70 75 80 Pro Gly Asp Thr Gly Leu Tyr Leu Cys Wing Gly Wing Gly Ser Gln Gly 85 90 95 Asn Leu He Phe Gly Lys Gly Thr Lys Leu Ser Val Lys Pro Asn He 100 105 110 Gln Asn Pro Asp Pro Wing Val Tyr Gln Leu Arg Asp Ser Lys Be Ser 115 120 125 Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val 130 135 140 Ser Gln Ser Lys Asp Ser Asp Val Tyr He Thr Asp Lys Thr Val Leu 145 150 155 160 Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Wing Val Wing Trp Ser 165 170 175 Asn Lys Ser Asp Phe Wing Cys Wing Asn Wing Phe Asn Asn Ser He He 180 185 190 Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Pro Gly Gly Arg 195 200 205 He Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln Asn Ser 210 215 220 Glu Leu Wing Being Thr Wing Asn Met Leu Arg Glu Gln Val Wing Gln Leu 225 230 235 240 Lys Gln Lys Val Met Asn Tyr 245 < 210 > 56 < 21 1 > 864 < 212 > A DN < 21 3 > H omo sapiens < 220 > < 223 > DNA sequence of the TC R beta chain restricted from H LA-A2 matrix / soluble J M22 fluid, as it was fused to the human c-fos leucine closing domain. < 400 > 56 atggtggatg gtggaatcac tcagtcccca aagtacctgt tcagaaagga aggacagaat 60 gttgtgaaca gtgaccctga gaatttgaac cacgatgcca tgtactggta ccgacaggac 120 ccagggcaag ggctgagatt gatctactac tcacagatag taaatgactt tcagaaagga 180 gatatagctg aagggtacag cgtctctcgg gagaagaagg aatcctttcc tctcactgtg 240 acatcggccc aaaagaaccc gacagctttc tatctctgtg ccagtagttc gaggagctcc 300 tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtt 360 ttcccacccg aggtcgctgt gtttgaacca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgectggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480 gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag accccggggg tctgactgat acactccaag tcaacttgaa cggagacaga 780 ctgcgttgca gacaagaagt gacegagatt gccaatctac ggaaaaacta tgaaagagaa 840 gagttcatcc tggcag cta 876 cta < 21 0 > 57 < 21 1 > 287 < 21 2 > PRT < 21 3 > Homo sapiens < 220 > < 223 > Predicted amino acid sequence of the restricted TCR beta chain of H LA-A2 / fl uu sol matrix from J M22, as it was fused to the human c-fos leucine closure domain. < 400 > 57 Met Val Asp Gly Gly He Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 1 5 10 15 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 20 25 30 Wing Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu He 35 40 45 Tyr Tyr Ser Gln He Val Asn Asp Phe Gln Lys Gly Asp He Wing Glu 50 55 60 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 65 70 75 80 Thr Ser Wing Gln Lys Asn Pro Thr Wing Phe Tyr Leu Cys Wing Being Ser 85 90 95 Ser Arg Ser Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 < 223 > Description of Artificial Sequence: DNA sequence of the TCR-restricted beta chain of H2-Db / nucleoprotein of soluble influenza virus from the murine F5 receptor, as it was fused to the leucine-closing domain of c-fos. < 400 > 58 atgaactatt ctccagcttt agtgactgtg atgctgtttg tgtttgggag gacccatgga 60 gactcagtaa cccagatgca aggtcaagtg accctctcag aagacgactt cctatttata 120 aactgtactt attcaaccac atggtacccg actcttttct ggtatgtcca atatcctgga 180 gaaggtccac agctcctttt acagccaaca gaaagtcaca acaagggaat cagcagaggt 240 catatgataa tttgaagcta aggaacaacg tccttccact tgcagaaagc ctcagtgcag 300 gagtcagact ctgctgtgta ctactgtgtg ctgggtgatc gacagggagg cagagctctg 360 atatttggaa caggaaccac ggtatcagtc agccccaaca tccagaaccc agaacctgct 420 gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt 480 gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaaact 540 gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag 600 cctgccaaga acaagcttca tatctccaaa gagaccaacg ccacctaccc cagttcagac 660 gttcccgggg gtagaatcgc ccggctggag gaaaaagtga aaaccttgaa agctcagaac 720 tcggagctgg cgtccacggc caacatgctc agggaacagg tggcacagct taaacagaaa 780 795 gtcatgaact actag < 210 > 59 < 211 > 264 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Predicted DNA sequence of the TCR alpha chain restricted from H2-Db / nucleoprotein of soluble influenza virus from the murine F5 receptor, as it was fused to the human c-jun leucine closure domain. < 220 > < 223 > Description of Artificial Sequence: Predicted DNA sequence of the TCR alpha chain restricted from H2-Db / nucleoprotein of soluble influenza virus from the murine F5 receptor, as it was fused to the c-jun leucine closure domain. < 400 > 59 Met Asn Tyr Ser Pro Ala Leu Val Thr Val Met Leu Phe Val Phe Gly 1 5 10 15 Arg Thr His Gly Asp Ser Val Thr Gln Met Gln Gly Gln Val Thr Leu 20 25 30 Ser Glu Asp Asp Phe Leu Phe He Asn Cys Thr Tyr Ser Thr Thr Trp 35 40 45 Tyr Pro Thr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Pro Gln 50 55 60 Leu Leu Leu Lys Val Thr Thr Wing Asn Asn Lys Gly He Ser Arg Gly 65 70 75 80 Phe Glu Wing Thr Tyr Asp Lys Gly Thr Thr Ser Phe His Leu Gln Lys 85 90 95 Wing Ser Val Gln Glu Ser Asp Ser Wing Val Tyr Tyr Cys Val Leu Gly 100 105 110 Asp Arg Gln Gly Gly Arg Ala Leu He Phe Gly Thr Gly Thr Thr Val 115 120 125 Ser Val Ser Pro Asn He Gln Asn Pro Glu Pro Wing Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln He Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe He 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Lys Wing Met Asp Ser Lys Ser Asn 180 185 190 Gly Wing Wing Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp He 195 200 205 S er Lys Glu Thr Asn Wing Thr Tyr Pro Ser Being Asp Val Pro Gly Gly 210 215 220 Arg He Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ma Gln Asn 225 230 235 240 Ser Glu Leu Wing Ser Thr Wing Asn Met Leu Arg Glu Gln Val Wing Gln 245 250 255 Leu Lys Gln Lys Val Met Asn Tyr 260 < 210 > 60 < 21 1 > 864 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: DNA sequence encoding the beta chain of restricted TCR of H2-Db / nucleoprotein of soluble influenza virus from the murine F5 receptor, as it was fused to the leucine closure of c-fos. < 400 > 60 gagttactca atgaaagctg tatctgatca aactccaaga aaacgagagg acagcaagtg 60 acactgagct gctcccctat ctctgggcat aggagtgtat acagacccca cctggtacca 120 ggacagggcc ttcagttcct ctttgaatac ttcagtgaga cacagagaaa caaaggaaac 180 ttccctggtc gattctcagg gcgccagttc tctaactctc gctctgagat gaatgtgagc 240 accttggagc tgggggactc ggccctttat ctttgcgcca gcagcttcga cagcgggaat 300 tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gaacaaggtg 360 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgectggc cacaggcttc ttccctgacc acgtggagct gagctggtgg 480 aggaggtgca gtgaatggga cagtggggtc agccaggacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atacagectg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag accccggggg tctgactgat acactccaag tcaacttgaa cggagacaga 780 ctgcgttgca gacaagaagt gacegagatt gccaatctac tgaaagagaa ggaaaaacta 840 gagtteatec tggcag cta 876 cta < 210 > 61 < 211 > 287 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Amino acid sequence of the TCR beta chain restricted from H2-Db / nucleoprotein of soluble influenza virus from the murine F5 receptor, as it was fused to the c-fos leucine lock. < 400 > 61 Met Lys Wing Gly Val Thr Gln Thr Pro Arg Tyr Leu He Lys Thr Arg 1 5 10 15 Gly Gln Gln Val Thr Leu Ser Cys Ser Pro He Ser Gly His Arg Ser 20 25 30 Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe Leu Phe 35 40 45 Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro Gly Arg 50 55 60 Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn Val Ser 65 70 75 80 Thr Leu Glu Leu Gly Asp Ser Wing Leu Tyr Leu Cys Wing Being Ser Phe 85 90 95 Asp Ser Gly Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Wing Val Phe 115 120 125 Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr Leu Val 130 135 140 Cys Leu Wing Thr Gly Phe Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Gln Asp Pro Gln Pro 165 170 175 Leu Lys Glu Gln Pro Wing Leu Asn Asp Being Arg Tyr Being Leu Being 180 185 190 Arg Leu Arg Val Being Wing Thr Phe Trp Gln Asn Pro Arg Asn His Phe 195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 210 215 220 Gln Asp Arg Ala Lys Pro Val Thr Gln He Val Ser Wing Glu Wing Trp 225 230 235 240 Gly Arg Ala Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln .Ma Glu Thr 245 250 255 Asp Gln Leu Glu Asp Lys Lys Ser Ala Leu Gln Thr Glu He Ala Asn 260 265 270 Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Ala Al to Tyr 275 280 285 < 21 0 > 62 < 21 1 > 747 < 21 2 > A DN < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: DNA sequence encoding the restricted TCR alpha chain of H LA-A2 / VI H-1 soluble Gag from patient 003, as it was fused to the dominance of human c-jun leucine closure . < 400 > 62 aagttacaca atgaaacaag gattcctgca gctctgagtg tcccagaagg agaaaacttg 60 gttctcaact gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac 120 cctgggaaag gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt 180 atgcctcgct ggaagactta tcaggacgta ggataaatca gtactttata cattgcagct 240 tctcagcctg gtgactcagc cacctacctc tgtgctgtga ccaacttcaa caaattttac 300 tttggatctg ggaccaaact caatgtaaaa ccaaatatcc agaaccctga ccctgccgtg 360 gagactctaa taccagctga atccagtgac aagtctgtct gcctattcac cgattttgat 420 tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 480 ggtctatgga ctagacatga cttcaagagc aacagtgctg tggcctggag caacaaatct 540 gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 600 agcccagaaa gttcccccgg gggtagaatc gcccggctgg aggaaaaagt gaaaaccttg 660 aaagctcaga actcggagct ggcgtccacg gccaacatgc tcagggaaca ggtggcacag 720 747 aagtcatgaa cttaaacaga ctactag < 210 > 63 < 211 > 248 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Amino acid sequence of the restricted TCR alpha chain of LA-A2 / VI H-1 soluble Gag from patient 003, as it was fused to the human c-jun leucine closure domain. < 400 > 63 Met Lys Gln Glu Val Thr Gln He Pro Ala Ala Leu Ser Val Pro Glu 1 5 10 15 Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Wing He Tyr 20 25 30 Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu 35 40 45 Leu Leu He Gln Be Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn 50 55 60 Wing Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr He Ala Wing 65 70 75 80 Ser Gln Pro Gly Asp Ser Wing Thr Tyr Leu Cys Wing Val Thr Asn Phe 85 90 95 Asn Lys Phe Tyr Phe Gly Ser Gly Thr Lys Leu Asn Val Lys Pro Asn 100 105 110 He Gln Asn Pro Asp Pro Wing Val Tyr Gln Leu Arg Asp Ser Lys Ser 115 120 125 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 130 135 140 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr He Thr Asp Lys Thr Val 145 150 155 160 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Be Wing Val Wing Trp 165 170 175 Ser Asn Lys Ser Asp Phe Wing Cys Wing Asn Wing Phe Asn Asn Be He 180 185 190 He Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Gly Gly 195 200 205 Arg He Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln Asn 210 215 220 Ser Glu Leu Wing Ser Thr Wing Asn Met Leu Arg Glu Gln Val Wing Gln 225 230 235 240 Leu Lys Gln Lys Val Met Asn Tyr 245 < 21 0 > 64 < 21 1 > 864 < 212 > A D N < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence of A DN q ue of the beta chain of TC R restricted from H LA-A2 / VI H-1 soluble Gag from patient 003, as it was fused to the leucine closure domain of c-fos human. < 400 > 64 gagttactca atgaaagctg aactccaaga tatctgatca aaacgagagg acagcaagtg 60 acactgagct gctcccctat ctctgggcat aggagtgtat cctggtacca acagacccca 120 ggacagggcc ttcagttcct ctttgaatac ttcagtgaga cacagagaaa caaaggaaac 180 ttccctggtc gattctcagg gcgccagttc tctaactctc gctctgagat gaatgtgagc 240 accttggagc tgggggactc ggccctttat ctttgcgcca gcagcttcga cagcgggaat 300 tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gaacaaggtg 360 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgectggc cacaggcttc ttccctgacc acgtggagct gagctggtgg 480 aggaggtgca gtgaatggga cagtggggtc agccaggacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atacagectg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag accccggggg tctgactgat acactccaag tcaacttgaa cggagacaga 780 ctgcgttgca gacaagaagt gacegagatt gccaatctac tgaaagagaa ggaaaaacta 840 gagtteatec tggcag cta 876 cta < 210 > 65 < 211 > 287 < 212 > PRT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Amino Acid Sequence of the Beta Chain of TC R Restricted from H LA-A2 / VI H- 1 Gag Soluble from Patient 003, as Merged to the Closing Sun of C-fos Leucine human. < 400 > 65 Met Lys Wing Gly Val Thr Gln Thr Pro Arg Tyr Leu He Lys Thr Arg 1 5 10 15 Gly Gln Gln Val Thr Leu Ser Cys Ser Pro He Ser Gly His Arg Ser 20 25 30 Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe Leu Phe 35 40 45 Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro Gly Arg 50 55 60 Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn Val Ser 65 70 75 80 Thr Leu Glu Leu Gly Asp Being Wing Leu Tyr Leu Cys Wing Being Ser Phe 85 90 95 Asp Being Gly Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Val Val Ala Val Phe 115 120 125 Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr Leu Val 130 135 140 Cys Leu Wing Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Gln Asp Pro Gln Pro 165 170 175 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ser Leu Ser 180 180 190 Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asn Pro Arg Asn His Phe 195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 210 215 220 Gln Asp Arg Wing Lys Pro Val Thr Gln He Val Wing Wing Glu Wing Trp 225 230 235 240 Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing Glu Thr 245 250 255 Asp Gln Leu Glu Asp Lys Lys Ser Wing Leu Gln Thr Glu He Wing Asn 260 265 270 Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Ala Tyr 275 280 285 < 21 0 > 66 < 21 1 > 750 < 212 > A DN < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence of A DN of the HR R alpha chain restricted from H LA-A2 / HTLV-1 Tax soluble from clone A6, as it was fused to the c-jun leucine closure domain. < 400 > 66 aagtggagca atgcagaagg gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 GTCT? Rgaaaa gccctgagtt atatactcca gataatgtcc atggtgacéia agaagatgga 180 AGGT tacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagggaccca cagtttggag ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgeaaa cgccttcaac aacagcatta ttecagaaeja caccttcttc 600 cccagcccag aaagttcccc cgggggtaga atcgcccggc tggaggaaaa agtgaaaacc 660 agaactcgga ttgaaagctc gctggcgtcc acggccaaca acaggtggca tgctcaggga 720 cagettaaac agaaagtcat gaactactag 750 < 21 0 > 67 < 21 1 > 249 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Restricted TCR alpha chain sequence of H LA-A2 / HTLV-1 soluble Tax from clone A6, as it was fused to the leucine closure domain of c-jun. < 400 > 67 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala He Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln 20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu He 35 40 45 Met Ser He Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Wing 50 55 60 Gln Leu Asn Lys Wing Wing Gln Tyr Val Ser Leu Leu He Arg Asp Ser 65 70 75 80 Gln Pro Be Asp Be Wing Thr Tyr Leu Cys Wing Val Thr Thr Asp Ser 85 90 95 Trp Gly Lys Leu Gln Phe Gly Wing Gly Thr Gln Val Val Val Thr Pro 100 105 110 Asp He Gln Asn Pro Asp Pro Wing Val Tyr Gln Leu Arg Asp Ser Lys 115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gn Thr 130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr He Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Wing Val Wing 165 170 175 Trp Ser Asn Lys Ser Asp Phe Wing Cys Wing Asn Wing Phe Asn Asn Ser 180 185 190 He He Pro Glu Asp Thr Phe Phe Pro Ser Glu Ser Ser Gly 195 200 205 G and Arg He Wing Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Wing Gln 210 215 220 Asn Ser Glu Leu Wing Ser Thr Wing Asn Met Leu Arg Glu Gln Val Wing 225 230 235 240 Gln Leu Lys Gln Lys Val Met Asn Tyr 245 < 210 > 68 < 21 1 > 928 < 212 > DNA < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: DNA sequence the beta chain of TC R restricted from H LA-A2 / HTLV-1 Tax soluble from clone A6, as it was fused to the domination of leucine c-fos and a label of biotinylation BirA. < 400 > 68 atgaacgctg gtgtcáctea gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca ggacctgaaa cggtcacaga 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cctggccaca cactggtgtg ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggggtcagca ggtgcacagt cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagaccc cgggggtctg actgatacac gacagatcaa tccaagcgga 780 cttgaagaca agaagtctgc gttgcagacc gagattgcca atctactgaa agagaaggaa 840 aaactagagt tcatcc tggc agcttacgga tccggtggtg gtctgaacga tatttttgaa 900 gctcagaaaa tcgaatggca ttaagctt 928 < 21 0 > 69 < 21 1 > 307 < 21 2 > PRT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Amino acid sequencing of the restricted TCR beta chain of H LA-A2 / HTLV-1 soluble Tax from clone A6, as it was fused to the leucine-closing domain of c-fos and a label of Bioti n BirA silage. < 400 > 69 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Wing Gln Asp Met Asn His Glu Tyr 20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu He His 35 40 45 Tyr Ser Val Gly Wing Gly He Thr Asp Gln Gly Glu Val Pro Asn Gly 50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Wing Wing Pro Ser Gln Thr Ser Val Tyr Phe Cys Wing Ser Arg Pro 85 90 95 Gly Leu Wing Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Wing 115 120 125 Val Phe Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr 130 135 140 Leu Val Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 165 170 175 Gln Pro Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Wing Leu 180 185 190 Ser Ser Arg Leu Arg Val Ser Wing Thr Phe Trp Gln Asn Pro Arg Asn 195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln He Val Ser Wing Glu 225 230 235 240 Wing Trp Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing 245 250 255 Glu Thr Asp Gln Leu Glu Asp Lys Lys Ser Ala Leu Gln Thr Glu He 260 265 270 Wing Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Ala 275 280 285 Tyr Gly Ser Gly Gly Gly Leu Asn Asp He Phe Glu Wing Gln Lys He 290 295 300 Glu Trp His 305 < 210 > 70 < 21 1 > 765 < 212 > DNA < 21 3 > Artificial Sequence l < 220 > < 223 > Description of Artificial Sequence: Restricted TCR alpha chain sequence of H LA-A2 / HTLV-1 soluble Tax from clone M 1 0B7 / D3, as it was fused to the leucine closure domain of c-jun. < 400 > 70 agaatgatga atgcaacaga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 60 tttctattct gaaggaagaa tatactaaca gaactgtgac ttatttccta gcatgtttga 120 tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 180 gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 240 ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaatggag 300 ggagcccaga agctggtatt tggecaagga accaggctga ctatcaaccc aaatatccag 360 ctgccgtgta aaccctgacc ccagctgaga gactctaaat ccagtgacaa gtctgtctgc 420 ctattcaccg attttgatte tcaaacaaat gtgtcacaaa gtaaggattc tgatgtgtat 480 atcacagaca aaactgtgct agacatgagg tctatggact tcaagagcaa cagtgctgtg 540 acaaatctga gcctggagca ctttgcatgt gcaaacgcct tcaacaacag cattattcca 600 gaagacacct tcttccccag cccagaaagt tcccccgggg gtagaatcgc ccggctggag 660 aaaccttgaa gaaaaagtga agctcagaac tcggagctgg cgtccacggc caacatgctc 720 agggaacagg tggcacagct taaacagaaa gtcatgaact actag 765 < 21 0 > 71 < 21 1 > 254 < 212 > PRT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Restricted TCR alpha chain sequence of H LA-A2 / HTLV-1 soluble Tax from clone M 1 0B7 / D3, as it was fused to the leucine closure domain of c-jun. < 400 > 71 Met Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser 1 5 10 15 Leu Ser Val Gln Glu Gly Arg He Ser He Leu Asn Cys Asp Tyr Thr 20 25 30 Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Glu Wing 35 40 45 Gly Pro Thr Phe Leu He Ser He Be Ser He Lys Asp Lys Asn Glu 50 55 60 Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser 65 70 75 80 Leu His He Val Pro Ser Gln Pro Gly Asp Ser Wing Val Tyr Phe Cys 85 90 95 Wing Wing Met Glu Gly Wing Gln Lys Leu Val Phe Gly Gln Gly Thr Arg 100 105 110 Leu Thr He Asn Pro Asn He Gln Asn Pro Asp Pro Wing Val Tyr Gln 115 120 125 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 130 135 140 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 145 150 155 160 He Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 165 170 175 Asn Ser Wing Val Wing Trp Ser Asn Lys Ser Asp Phe Wing Cys Wing Asn 180 185 190 Wing Phe Asn Asn Ser He He Pro Glu Asp Thr Phe Phe Pro Ser Pro 195 200 205 Glu Be Ser Pro Gly Gly Arg He Wing Arg Leu Glu Glu Lys Val Lys 210 215 220 Thr Leu Lys Wing Gln Asn Ser Glu Leu Wing Ser Thr Wing Asn Met Leu 225 230 235 240 Arg Glu Gln Val Wing Gln Leu Lys Gln Lys Val Met Asn Tyr 245 250 < 21 0 > 72 < 21 1 > 925 < 212 > A DN < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: DNA sequence of the restricted TCR beta chain of H LA-A2 / HTLV-1 soluble Tax from the clone M 1 0 B7, as it was fused to the leucine closing domain of c-fos and a biotinylation label Bi rA. < 400 > 72 gtgtcactca atgaacgctg gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcagttacca ggaggggggg 300 ttttacgagc agtacttcgg gccgggcacc aggctcacgg tcacagagga cctgaaaaac 360 gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420 aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 480 tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 540 cagcccgccc tcaatgactc cagatacgct ctgagcagcc gcctgagggt ctcggccacc 600 ttctggcagg acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660 aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 720 tggggtagag cagaccccgg gggtctgact gatacactcc aagcggagac agatcaactt 780 gaagacaaga agtctgcgtt gcagaccgag attgccaatc gaaggaaaaa tactgaaaga 840 ctagagttca tcctg gcagc ttacggatcc ggtggtggtc tgaacgatat ttttgaagct 900 cagaaaatcg aatggcatta agctt 925 < 21 0 > 73 < 21 1 > 306 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence of the beta chain of restricted TCR of H LA-A2 / HTLV-1 soluble Tax from the clone M 10B7 / D3, as it was fused to the leucine closing domain of c-fos and a label of BirA biotinylation. < 400 > 73 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Wing Gln Asp Met Asn His Glu Tyr 20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu He His 35 40 45 Tyr Ser Val Gly Wing Gly He Thr Asp Gln Gly Glu Val Pro Asn Gly 50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Wing Wing Pro Ser Gln Thr Ser Val Tyr Phe Cys Wing Ser Ser Tyr 85 90 95 Pro Gly Gly Ghe Phe Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Val Glu Wing Val 115 120 125 Phe Glu Pro Ser Glu Wing Glu He Ser His Thr Gln Lys Wing Thr Leu 130 135 140 Val Cys Leu Wing Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 145 150 • 155 160 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 165 170 175 Pro Leu Lys Glu Gln Pro Wing Leu Asn Asp Ser Arg Tyr Ma Leu Ser 180 185 190 Ser Arg Leu Arg Val Ser Ma Thr Phe Trp Gln Asp Pro Arg Asn His 195 200 205 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn. Asp Glu Trp 210 215 220 Thr Gln Asp Arg Ma Lys Pro Val Thr Gln He Val Ser Ma Glu Ma 225 230 235 240 Trp Gly Arg Ma Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Ma Glu 245 250 255 Thr Asp Gln Leu Glu Asp Lys Lys Ser Ma Leu Gln Thr Glu He Ma 260 265 270 Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Ma Ma Tyr 275 280 285 Gly Ser Gly Gly Gly Leu Asn Asp He Phe Glu Ma Gln Lys He Glu 290 295 300 Trp His 305 < 21 0 > 74 < 21 1 > 928 < 212 > A D N < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Mutated sequence of the restricted TCR beta chain of H LA-A2 / HTLV-1 Tax soluble from clone A6, as it was fused to the leucine closure domain of c-fos and a label of BirA biotinylation. < 400 > 74 gtgtcactca atgaacgctg gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg ccaaggagaa gtatcactga 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca ggacctgaaa cggtcacaga 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 atgggaagga tggtgggtga ggtgcacagt ggggtcagca cagaccc? ca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 agtggaccca gagaatgacg ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagaccc cgggggtctg actgatacac gacagatcaa tccaagcgga 780 cttgaagaca agaagtctgc gttgcagacc gagattgcca atctactgaa agagaaggaa 840 aaactagagt tcatcc tggc agcttacgga tccggtggtg gtctgaacga tatttttgaa 900 gctcagaaaa tcgaatggca ttaagctt 928 < 21 0 > 75 < 21 1 > 307 < 212 > PRT < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence of the restricted TCR beta chain of H LA-A2 / HTLV-1 soluble Tax mutated from clone A6, as it was fused to the c-fos leucine closure domain and a label of biotinylation BirA. < 400 > 75 Met Asn Wing Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ma Gln Asp Met Asn His Glu Tyr 20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met X? Y Leu Arg Leu He His 35 40 45 Tyr Ser Val Gly Ma Gly He Thr Asp Gln Gly Glu Val Pro Asn Gly 50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ma Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ma Ser Arg Pro 85 90 95 Gly Leu Wing Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ma 115 120 125 Val Phe Glu Pro Ser Glu Ala Glu He Ser His Thr Gln Lys Ma Thr 130 135 140 Leu Val Cys Leu Ma Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ma Leu Asn Asp Ser Arg Tyr Ma Leu 180 185 190 Ser Ser Arg Leu Arg Val Ser Ma Thr Phe Trp Gln Asp Pro Arg Asn 195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 210 215 220 Trp Thr Gln Asp Arg Ma Lys Pro Val Thr Gln He Val Ser Ma Glu 225 230 235 240 Ma Trp Gly Arg Wing Asp Pro Gly Gly Leu Thr Asp Thr Leu Gln Wing 245 250 255 Glu Thr Asp Gln Leu Glu Asp Lys Lys Ser Ma Leu Gln Thr Glu He 260 265 270 Ma Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe He Leu Wing Ala 275 280 285 Tyr Gly Ser Gly Gly Gly Leu Asn Asp He Phe Glu Ma Gln Lys He 290 295 300 Glu Trp His 305 < 21 0 > 76 < 21 1 > 1 90 < 212 > A DN < 21 3 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: DNA sequence of the biotinylation label fusion pattern c-fos / BirA used for TCR beta chains. < 400 > 76 cccgggggtc tgactgatac actccaagcg gagacagatc aacttgaaga caagaagtct 60 gcgttgcaga ccgagattgc caatctactg aaagagaagg aaaaactaga gttcatcctg 120 gcagcttacg gatccggtgg tggtctgaac gatatttttg aagctcagaa aatcgaatgg 180 cattaagctt 190 < 210 > 77 < 21 1 > 61 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Biotinylation label fusion sequence c-fos / BirA used for TCR beta chains. < 400 > 77 Pro Gly Gly Leu Thr Asp Thr Leu Gln Ma Glu Thr Asp Gln Leu Glu 1 5 10 15 Asp Lys Lys Ser Ma Leu Gln Thr Glu He Ma Asn Leu Leu Lys Glu 20 25 30 Lys Glu Lys Leu Glu Phe He Leu Ma Ma Tyr Gly Ser Gly Gly Gly 35 40 45 Leu Asn Asp He Phe Glu Ma Gln Lys He Glu Trp His 50 55 60 < 210 > 78 < 21 1 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Inverse primer used for the PCR amplification of the Vbeta-c-fos leucine closure fragment of the human TCR JM22 fusion gene restricted from influenza matrix peptide / HLA-A0201. < 400 > 78 acacacggat ccgtaagctg cgacgatgaa ctcgattttc tt 42 < 210 > 79 < 211 > 90 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Initiator for the amplification of the human Vbeta17 chain of JM22 PCR fused to the biotinylation label Bir. < 400 > 79 gggggaagct taatgccatt cgattttctg agcttcaaaa atatcgttca gaccaccaccßO ggatccgtaa gctgccagga tgaactcag 90 < 210 > 80 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Initiator for the amplification of the human Vbeta17 chain of JM22 PCR fused to the biotinylation label Bir. < 400 > 80 gctctagaca tagggccca gtggattctg gagtcac 37 < 210 > 81 < 211 > 9 < 212 > PRT < 213 > Human immunodeficiency virus < 220 > < 223 > Peptide derived from the HIV-1 Reverse Transcriptase Protein and presented as a peptide antigen by HLA-A0201. < 400 > 81 lie Leu Lys Glu Pro Val His Gly Val 1 5 < 210 > 82 < 211 > 9 < 212 > PRT < 213 > T-cell lymphotropic virus type 1 human < 220 > < 223 > Peptide derived from the HIV-1 HTLV-1 Tax protein and presented as a peptide antigen by HLA-A0201. This combination of HLA / peptide restricts the TCRs A6 and B7. < 400 > 82 Leu Leu Phe Gly Tyr Pro Val Tyr Val 1 5 < 210 > 83 < 211 > 9 < 212 > PRT < 213 > Influenza virus < 220 > < 223 > Peptide derived from the nucleoprotein of the influenza virus and presented as a peptide antigen by murine H2-Db. This MHC / peptide combination restricts the TCR of murine F5. < 400 > 83 Ala As Asn Glu Asn Met Asp Ala Met 1 5 < 210 > 84 < 211 > 9 < 212 > PRT < 213 > Influenza virus < 220 > < 223 > Peptide derived from the influenza virus matrix protein and presented as a peptide antigen by HLA-A0201. This combination of HLA / peptide restricts the JM22 TCR < 400 > 84 Gly lie Leu Gly Phe Val Phe Thr Leu 1 5 < 210 > 85 < 211 > 9 < 212 > PRT < 213 > Human immunodeficiency virus < 220 > < 223 > Peptide derived from the HIV-1 Gag protein and presented as a peptide antigen by HLA-A0201. This HLA / peptide combination restricts the cloned TCR of patient 003. < 400 > 85 Ser Leu Tyr Asn Thr Val Wing Thr Leu i 5

Claims (11)

1. - A synthetic multivalent T cell receptor (TCR) complex for binding to an MHC-peptide complex, said TCR complex comprising a plurality of T cell receptors specific for the MHC-peptide complex.

2. The TCR complex according to claim 1, wherein the T cell receptors are αß T cell receptors having a chain a and a β chain.

3. The TCR complex according to claim 2, wherein the chain a and the β chain are soluble forms of the α and β chains of the T cell receptor.

4. The TCR complex according to any of the claims precedents, wherein the T cell receptors are in the form of multimers of two or more T cell receptors.

5. The TCR complex according to claim 4, wherein the multimer is a trimer or a tetramer.

6. The TCR complex according to any of the preceding claims, wherein the T cell receptors are associated with each other through a linker molecule.

7. The TCR complex according to claim 6, wherein the linker molecule is a multivalent binding molecule such as avidin, streptavidin or extravidin.

8. The TCR complex according to claim 8, wherein at least one of the α or β chains of the T cell receptor is derived from a fusion protein comprising an amino acid recognition sequence for a modification enzyme. such as biotin.

9. The TCR complex according to claim 8, wherein the T cell receptors are biotinylated.

10. The TCR complex according to any of the preceding claims, comprising a multimerized recombinant T cell receptor heterodimer having improved binding capacity compared to a non-multimeric T cell receptor heterodimer. 1. A multivalent TCR complex comprising a multimerized recombinant T cell receptor heterodimer that has improved binding capacity compared to a non-multimeric T cell receptor heterodimer. 12. The TCR complex according to any of the preceding claims, wherein the T cell receptor is a refolded recombinant T cell receptor, which comprises: i) an extracellular domain of α-chain? of recombinant T cell receptor having a first heterologous C-terminal dimerization peptide; and ii) an extracellular β or d chain domain of recombinant T cell receptor having a second C-terminal dimerization peptide, which is specifically heterodimerized with the first dimerization peptide to form a heterodimerization domain. 13. The TCR complex according to claim 12, wherein a disulfide bond present in native T cell receptors between the α and β or β chains. and d adjacent to the cytoplasmic domain, is absent from the recombinant T cell receptor. 14. The TCR complex according to claim 12, or claim 13, wherein the heterodimerization domain is a coiled spiral domain. 15. The TCR complex according to claim 14, wherein the dimerization peptides are c-jun and c-fos dimerization peptides. 16. The TCR complex according to any of claims 12 to 15, comprising a flexible linker located between the T cell receptor chains and the heterodimerization peptides. 17. The TCR complex according to any of claims 10 to 16, wherein the T cell receptor is expressed in an E. coli expression system. 18. The TCR complex according to any of claims 10 to 17, wherein the T cell receptor is biotinylated in the C term. 19. The TCR complex according to any of the preceding claim, wherein the T cell receptors are associated with a lipid bilayer. 20. The TCR complex according to claim 19, where the lipid bilayer forms a vesicle. 21. The TCR complex according to claim 20, wherein the T cell receptors are attached to the outside of the vesicle. 22. The TCR complex according to claim 20 or claim 21, wherein the T cell receptors are attached to the vesicle through derivatized lipid components of the vesicle. 23. The TCR complex according to claim 19 or claim 20, wherein the T cell receptors are embedded in the lipid bilayer. 24. The TCR complex according to any of claims 1 to 18, wherein the T cell receptors are attached to a particle. 25. The TCR complex according to any of the preceding claims, which further comprises a detectable label. 26. The TCR complex according to any of the preceding claims, further comprising a therapeutic agent such as a cytotoxic agent or a stimulating agent. 27. The TCR complex according to any of the preceding claims, in a pharmaceutically acceptable formulation for use in vivo. 28.- A method for detecting MHC-peptide complexes, said method comprising: (i) providing, (a) a synthetic multivalent T cell receptor complex comprising a plurality of T cell receptors, and / or (b) ) a synthetic multivalent T cell receptor complex comprising a multimerized recombinant T cell receptor heterodimer having improved binding capacity compared to a non-multimeric T cell receptor heterodimer, said T cell receptors being specific for MHC- complexes peptide; (ii) contacting the multivalent TCR complex with the MHC-peptide complexes; and (iii) detecting the binding of the multivalent TCR complex to the MHC-peptide complexes. 29. The method according to claim 28, wherein the multivalent TCR complex is provided with a detectable label. 30. The method according to claim 28 or claim 29, for detecting cells that have a specific peptide antigen. The method according to any of claims 28 to 30, wherein the multivalent TCR complex is a multivalent TCR complex according to any of claims 1 to 27. 32.- A method for delivering a therapeutic agent to a target cell, said method comprises: (i) providing, (a) a synthetic multivalent TCR complex comprising a plurality of T cell receptors, and / or (b) a synthetic multivalent TCR complex comprising a heterodimer of multimerized recombinant T cell receptor having enhanced binding capacity compared to a non-multimeric T cell receptor heterodimer, said T cell receptors being specific for M HC-peptide complexes and the multivalent TCR complex having the associated therapeutic agent with the same; (ii) contacting the multivalent TCR complex with potential target cells under conditions to allow binding of the T cell receptors to the target ce

ll. 33. The method according to claim 32, wherein the multivalent TCR complex is a multivalent TCR complex according to any of claims 1 to 27.