KR20040039322A - Substances - Google Patents

Abstract

The present invention relates to (i) all or part of the T cell receptor (TCR) α chain, except for the transmembrane domain, and (ii) all or part of the TCR β chain, except for the transmembrane domain. Wherein (i) and (ii) each comprise at least a portion of a functional variable domain of the TCR chain and a constant domain of the TCR chain, and (i) and (ii) represent a constant domain residue ( soluble T cell receptor (sTCR), which is linked between residues by disulfide bonds that do not exist in native TCRs.

Description

Substance {SUBSTANCES}

The present invention relates to soluble T cell receptor (TCR).

As described in WO 99/60120, TCRs mediate the recognition of specific Major Histocompatibility Complex (MHC) -peptide complexes by T cells and, therefore, the cellular arm of the immune system. It is essential to the action of.

Antibodies and TCRs are the only two types of molecules that specifically recognize antigens, while TCRs are the only receptors for specific peptide antigens present in MHC, while foreign peptides are often the only indication of abnormality in cells. Do. T cell recognition occurs when T-cells and antigen presenting cells (APCs) are in direct physical contact and are initiated by the ligation of pMHC complexes with antigen-specific TCRs.

TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily that binds to the constant protein of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, and they are structurally similar but the T cells expressing them are anatomically quite different locations and their function also appears to be different. The extracellular portion of the receptor has two membrane-proximal constant domains and two membrane-distal variables with polymorphic loops similar to the complementarity determining regions (CDRs) of the antibody. It consists of domains. It is these loops that form the binding site of the TCR molecule and determine peptide specificity. MHC class I and class II ligands are also immunoglobulin superfamily proteins, but are specialized in antigen presentation and have polymorphic peptide binding sites to allow for a wide array of short peptide fragments on the surface of APC cells for antigen presentation.

Soluble TCR is useful not only for studying specific TCR-pMHC interactions, but also as a diagnostic tool for detecting infections or as a diagnostic tool for detecting autoimmune disease markers. Soluble TCR also applies to staining, for example, to stain cells with specific peptide antigens presented on MHC. Similarly, soluble TCRs are used to deliver therapeutic agents, such as cytotoxic compounds or immunostimulatory compounds, to cells that present specific antigens. Soluble TCR can also be used to inhibit T cells, such as those that react with autoimmune peptide antigens, for example.

Proteins composed of one or more polypeptide subunits and having transmembrane domains are difficult to produce in soluble form because in many cases the proteins are stabilized by transmembrane regions. The same is true of the TCR, which is also reflected in the scientific literature describing the truncated form of the TCR, which is a truncated TCR having only extracellular domains or having extracellular and intracellular domains. As can be recognized by a TCR specific antibody (which indicates that the recombinant TCR moiety recognized by the antibody was properly folded), the yield is poor, and is not stable at low concentrations and / or may recognize the MHC-peptide complex. Can't. This document is reviewed in WO 99/60120.

Many documents describe the production of TCR heterodimers comprising natural disulfide bridges connecting each subunit (Garboczi, et al., (1996), Nature 384 (6605): 134 -41; Garboczi, et al., (1996), J Immunol 157 (12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268 (21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; US Patent No. 6080840). However, although such TCRs could be recognized by TCR-specific antibodies, they were not aware of and / or stable in their natural ligands except at relatively high concentrations.

WO 99/60120 describes soluble TCRs which can be folded correctly to recognize natural ligands, which are stable for a significant time and can be produced in significant amounts. The TCR is a pair of C-terminal dimerisation peptides, such as leucine zippers, that bind to the TCR α or γ chain extracellular domain, which forms a dimer with the TCR β or δ chain extracellular domain, respectively. It is included. This method for producing this TCR is generally applicable to all TCRs.

Reiter et al. (Immnity, 1995.2: 281-287) describe in detail the structure of soluble molecules comprising disulfide-stabilized TCR α and β variable domains, one of which is Pseudomonas exotoxin (PE38) truncated form. Connected with Overcoming inherent instability of short-chain TCRs has been described as one of the reasons for the production of these molecules. The location of new disulfide bonds in the TCR variable domain has been confirmed through homology with the variable domains of antibodies that have already introduced disulfide bonds (eg, Brinkmann, et al. (1993), Proc. Natl. Acad. Sci. USA 90: 7538-7542, and Reiter, et al. (1994) Biochemistry 33: 5451-5459). However, since there is no such homology between the antibody and the TCR constant domain, this technique could not be used to identify sites suitable for novel interchain disulfide bonds between TCR constant domains.

In view of the importance of soluble TCRs, it would be desirable to provide other methods of producing such molecules.

In one aspect, the present invention relates to TCR β, except for (i) all or part of the T cell receptor (TCR) α chain, excluding the transmenbrane domain, and (ii) except for the transmembrane domain. Comprising all or part of the chain, (i) and (ii) each comprising a functional variable domain of the TCR chain and at least a portion of the constant domain of the TCR chain, (i) and (ii) It provides a soluble T cell receptor (sTCR), which is linked between constant domain residues by disulfide bonds that do not exist in native TCR.

In another aspect, the present invention relates to a soluble αβ-type T cell in which covalently linked disulfide bonds link residues of the immunoglobulin region of the constant domain of the α chain to residues of the immunoglobulin region of the constant domain of the β chain. Soluble αβ-form T cell receptor (sTCR).

The sTCR of the present invention has the advantage of not containing heterologous polypeptides that have immunogenicity or result in the rapid removal of sTCR from the body. In addition, the TCRs of the present invention have tertiary structures that are highly similar to the native TCRs from which they originate, and do not appear to have immunogenicity because of these structural similarities. The sTCRs according to the present invention will recognize class I MHC-peptide complexes or class II MHC-peptide complexes.

The TCR of the present invention is soluble. In connection with the present application, solubility is determined in phosphate buffered saline (PBS) (KCL 2.7 mM, KH ₂ PO ₄ 1.5 mM, NaCl 137 mM and Na ₂ PO ₄ 8 mM, pH 7.1-7.5.Life Technologies, Gibco BRL) A single dispersed heterodimer at a concentration of 1 mg / ml is defined as the ability of TCR to be purified and at least 90% of the TCR remains as a single dispersed heterodimer even after 1hr incubation at 25 ° C. To assess the solubility of the TCR, first purify the TCR as described in Example 2. After this purification, 100 μg of TCR is analyzed by analytical size exclusion chromatography such as a Pharmacia Superdex 75HR column equilibrated with PBS. Another 100 μg of TCR is incubated at 25 ° C. for 1 hour, followed by size exclusion chromatography in the same manner as before. Size exclusion results are analyzed as integrals to compare the peak areas corresponding to a single dispersed heterodimer. Each peak can be identified by comparing the elution position of the known protein standard with the mass of the molecule. Soluble TCRs in the form of a single dispersed heterodimer have a molecular weight of approximately 50 kDa. As described above, the TCR of the present invention is soluble. However, as described in more detail below, the TCR may be combined with a moiety to form an insoluble complex or provided on an insoluble solid support surface.

The numbering of TCR amino acids used herein follows the IMGT system described in The T Cell Receptor Factsbook, 2001, LeFranc & LeFranc, Academic Press. In this system, the α chain constant domain has the following notation: “TR” in TRAC * 01 indicates the T cell receptor gene; "A" represents an α chain gene, "C" represents a constant region; And “* 01” denotes allele 1. β chain constant domains have the following notation: TRBC1 * 01. In this case, there are two possible constant domain region genes "C1" and "C2". The translation domains, each encoded by an allele, can be constructed from the genetic code of several exons; As a result they are also specified. Amino acids are numbered according to the exons of the specific domain to which they belong.

The extracellular portion of the native TCR consists of two polypeptides (αβ or γδ) with respective membrane-proximal constant domains and membrane-distal variable domains (FIG. 1). Each constant and variable domain contains an intra-chain disulfide bond. The variable domain has a highly polymorphic loop similar to the complementary determining region (CDR) of the antibody. CDR3 of the TCR interacts with the peptide provided by the MHC, while CDRs 1 and 2 interact with the peptide and the MHC. Diversity of TCR sequences is produced through somatic rearrangement of linked V (varibale), D (diversity), J (joining), and C (contant) genes. But the β chain consists of the VDJC region The extracellular constant domain has a membrane contiguous region and an immunoglobulin region The membrane contiguous region consists of amino acids between the transmembrane domain and the membrane contiguous cysteine residue The constant immunoglobulin region has a membrane proximity Consisting of the remainder of the constant domain amino acid residues extending from the cysteine to the beginning of the J region, characterized by the presence of an immunoglobulin-type fold, with a single α chain constant domain known as Cα1 or TRAC * 01. There are two other β constant domains known as Cβ2 or TRBC1 * 01 and Cβ2 or TRBC2 * 01. The differences between the domains are at amino acid residues 4, 5 and 37 of exon 1, thus TRBC1 * 01 has 4N, 5K and 37 at its exon 1 and TRBC2 * 01 has 4K, 5N and at its exon 1 Has 37Y. The size of each of the TCR extracellular domains is somewhat variable.

In the present invention, disulfide bonds are introduced between residues located in the constant domain (or portion thereof) of each chain. The chain of each TCR contains enough variable domains to be able to interact with the pMHC complex. Such interaction can be measured using a BIAcore 3000 ^™ or BIAcore 2000 ^™ instrument as described in Example 3 below or WO 99/6120 of the present invention.

In one embodiment, each chain of the sTCR of the present invention also comprises an intra-chain disulfide bond. The TCRs of the present invention may comprise the entirety of the extracellular constant Ig regions of each TCR chain, or preferably the entirety of the extracellular domains of each chain, including membrane adjacent regions. Natural TCRs have disulfide bonds connecting the conserved membrane adjacent regions of each chain. In one embodiment of the invention, this disulfide bond is absent. This can be accomplished by mutating the appropriate cysteine residues (amino acids 4 of exon 2 of the TRAC * 01 gene, amino acids 2 of each of the two genes TRBC1 * 01 and TRBC2 * 01) to other amino acids or by cleaving their respective chains so that they do not contain cysteine residues. It is possible. Preferred soluble TCRs according to the present invention are residues 1, 2, 3, 4, 5, 6, 7 from which the C terminus is cleaved to remove cysteine residues that form a native TCR interchain disulfide bond, ie from the cysteine residue towards the N terminus. Natural α and β chains cut at 8, 9 or 10 sites. It should be noted, however, that natural interchain disulfide bonds may be present in the TCRs of the invention, so in some embodiments, only one of the TCR chains has a natural cysteine residue capable of forming natural interchain disulfide bonds. This cysteine can be used to attach a moiety to the TCR.

However, each TCR chain can be shorter. Because the constant domain is not directly involved in the contact of the peptide-MHC ligand, the C-terminal cleavage point can be changed without loss of function.

Alternatively, larger size constant domain fragments may be used than contemplated by the present invention, ie the constant domain need not be cleaved immediately before the cysteine to form an interchain disulfide bond. For example, all constant domains (ie, extracellular and cytoplasmic domains) other than the transmembrane domain may be included. In this case, it may be beneficial to mutate one or more of the cysteine residues that form intrachain disulfide bonds with intracellular TCRs to other amino acid residues not involved in disulfide bond formation or to delete one or more of these residues. have.

If soluble TCRs are expressed in prokaryotic cells such as E. coli, signal peptides may be omitted, which would not serve any purpose of ligand binding capacity of mature TCRs and in some circumstances This is because it prevents the formation of a functional soluble TCR. In most cases, cleavage sites at which the signal peptide is removed from the mature TCR chain are predicted, but not experimentally determined. Making the expressed TCR short or short, for example up to about 10 amino acids, at the N terminus has little effect on the function of soluble TCR (ie the ability to recognize pMHC). Any additional sequence that is not present in the original protein sequence can be added. For example, short tag sequences can be added that can aid in the purification of the TCR chain if it does not interfere with the correct structure and folding of the antigen binding site of the TCR.

When expressed in E. coli, methionine residues can be treated at the N-terminal start of the expected mature protein sequence to enable initiation of translation.

Not all residues of the variable domain of the TCR β chain are essential for antigen specificity and function. Thus, a significant number of mutations can be introduced into this domain without affecting antigen specificity and function. Not all residues of the constant domains of the TCR chain are essential for antigen specificity and function. Thus, a large number of mutations can be introduced into this region without affecting antigen specificity.

TCR β chains contain unpaired cysteine residues in cells or natural TCRs. It is more desirable to remove these cysteine residues or to mutate to other residues to avoid inaccurate intrachain or interchain pairing. Substitution of such cysteine residues, for example with other residues such as serine or alanine, can have a significantly positive effect on refolding efficiency in vitro.

Disulfide bonds can be formed by mutating non-cysteine residues in each chain with cysteine, forming disulfide bonds between the mutated residues. Each β carbon is approximately 6 kV (0.6 nm) or less in the native TCR, preferably 3.5 kPa (0.35 nm) to 5.9 kPa (0.59 nm), so that disulfide bonds can form between cysteine residues introduced at native residue positions. If the disulfide bond is between constant immunoglobulin region residues, it is preferred even if the bond may be between residues in the membrane adjacent region. Preferred which can be introduced to form a disulfide bond The sites are the following residues in exon 1 of TRAC * 01 representing the TCR α chain and TRBC1 * 01 or TRBC2 * 01 representing the TCR β chain.

Certain sTCRs of the present invention are derived from A6 Tax TCR (Garboczi et al., Nature, 1996, 384 (6605): 134-141). In one embodiment, sTCR is the entire TCR α chain, which is the N-terminus of exon 2, residue 4 of TRAC * 01 (amino acid residues 1 to 182 according to the numbering method used by Garboczi et al.) And the N-terminus of exon 2 The entire TCR β chain, residues 2 of TRBC1 * 01 and TRCB2 * 01 (numbering amino acid residues 1 to 120 according to the numbering method used by Garboczi et al.). Threonine 48 of exon 1 in TRAC * 01 (numbering used by Garboczi et al. According to the numbering method used by Garboczi et al.) In TRAC * 01 to form disulfide bonds and serine 57 of exon 1 (Garboczi et al. In TRBC1 * 01 and TRBC2 * 01) According to this numbering method, serine 172 can each be mutated to cysteine. These amino acids are located on the β strand D of the constant domains of the α and β TCR chains, respectively.

3A and 3B, residue 1 (according to the numbering method used by Garboczi et al.) Is K and N, respectively. N-terminal methionine residues are not present in native A6 Tax TCRs and, as mentioned above, are often present when each chain is produced in a bacterial expression system.

As residues of human TCRs are now identified that can be mutated to cysteine residues to form new interchain disulfide bonds, one skilled in the art can mutate any TCR to produce a soluble form of TCR with a new disulfide bond. There will be. In humans, skilled technicians only need to look for the following motifs in each TCR chain to identify the residues to be mutated (the residues that will be negatively mutated to cysteine).

In other species, the TCR chain may not have a region having 100% identity with the above motif. However, those skilled in the art will be able to use the above motif to identify the corresponding portion of the TCR α or β chain and the residues to be mutated to cysteine. In this regard, alignment techniques can be used. For example, the motif is compared with a specific TCR chain sequence to locate a TCR sequence moiety that is suitable for mutagenesis. The European Bioinformatics Institute: http://www.ebi.ac.uk/index. You can use the ClustalW available in html).

The invention includes human disulfide linked αβ TCRs as well as disulfide linked αβ TCRs of other mammals, including but not limited to mice, rats, pigs, goats and sheep. As mentioned above, those skilled in the art will be able to determine the sites corresponding to the above-mentioned human sites where cysteine residues can be introduced to form interchain disulfide bonds. For example, below are shown the amino acid sequences of the mouse Cα and Cβ soluble domains with the motif showing the rat residues corresponding to the above-mentioned human residues that can be mutated to cysteine to form TCR interchain disulfide bonds (related residues) Denoted as negative salt).

In a preferred embodiment of the invention, each of (i) and (ii) of the TCR comprises the functional variable domain of the first TCR fused to all or part of the constant domain of the second TCR, wherein the first and second TCRs are Interchain disulfide bonds, which are of the same kind, are made between residues in all or part of a constant domain that is not present in nature. In one embodiment, the first and second TCRs are human. In other words, the disulfide bond-linked constant domain acts as a framework into which the variable domain can be fused. The resulting TCR will be nearly identical to the native TCR from which the first TCR is obtained. Such a system facilitates the expression of any functional variable domain in a stable constant domain backbone.

The constant domain of the A6 Tax sTCR described above, or indeed the constant domain of the αβ TCR of a mutant with the new interchain disulfide bonds described above, can be used as a framework in which heterologous variable domains can be fused. It is desirable for the fusion protein to retain as much of the heterologous variable domain as possible. Therefore, the heterogeneous variable domain is preferably linked to the constant domain at some point of the introduced cysteine residue and the N terminus of the constant domain. In the A6 Tax TCR, the cysteine residues introduced into the α and β chains are preferably threonine 48 of exon 1 (Threonine 158 of α chain and TRBC1 * 01 according to the numbering method used by Garboczi et al.) In TRAC * 01, respectively. And serine 57 of exon 1 (serine 172 of the β chain according to the numbering method used by Garboczi et al.) In both TRBC2 * 01. Therefore, the heterologous α and β chain variable domain attachment points are respectively residues 48 (159 according to the numbering method used by Garboczi et al.) Or residues 58 (173 according to the numbering method used by Garboczi et al.) And α or β constant domains, respectively. It is preferred to be between the N terminals of.

Residues in the constant domains of the heterologous α and β chains corresponding to the point of attachment in A6 Tax TCR can be identified through sequence homology. The fusion protein is preferably configured to include all heterologous sequences on the N terminus from the point of attachment.

As will be discussed in more detail below, the sTCRs of the present invention may be derivatized or fused to a moiety at the C or N terminus. More preferred is the C terminus as it is far from the binding domain. In one embodiment, one or both of the TCR chains have cysteine residues at which the moiety can be fused at the C and / or N terminus.

Soluble TCRs (preferably human) of the present invention may be provided in generally pure form, or in purified or isolated formulations. For example, it may be provided in a form that generally does not contain other proteins.

Plurality of the soluble TCR of the present invention may be provided in a multivalent complex. Thus, in one aspect, the present invention provides a multivalent T cell receptor complex consisting of a plurality of soluble T cell receptors as described herein. Each of the plurality of soluble TCRs is preferably identical.

In another view,

(i) providing a soluble T cell receptor or multivalent T cell receptor complex as described herein;

(ii) contacting the soluble T cell receptor or multivalent TCR complex with the MHC-peptide complex; And

(iii) detecting the binding of the soluble T cell receptor or multivalent TCR complex to the MHC-peptide complex.

In the multivalent complex of the present invention, the TCR may be in the form of a multimer and / or may be present or bound in a lipid bilayer such as liposomes.

In its simplest form, the multivalent TCR complex according to the invention comprises a multimer of two or three or four or more T cell receptor molecules linked to one another (eg covalently or otherwise linked). And preferably linked through a linker moleucle. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neuravidin, and extravidin. It has four binding sites for biotin, so biotinylated TCR molecules can be formed into multimers of T cell receptors having a plurality of TCR binding sites. It will depend on the amount of TCR relative to the amount of linker molecule used to make it, and also on the presence or absence of any other biotinylated molecule Preferred multimers are dimers, trimers or tetrameric TCR complexes.

Structures significantly larger than TCR tetramers can be used for tracking or targeting cells expressing specific MHC-peptide complexes. Preferably the structure ranges from 10 nm to 10 μm in diameter. Each structure presents a multivalent TCR molecule at a sufficient distance to allow two or more TCR molecules in the structure to bind simultaneously with two or more MHC-peptide complexes in the cell and thus a multivalent binding moiety to the cell ( increase the affinity of avidity.

Suitable structures for use in the present invention include membrane structures, such as liposomes, and solid structures, which preferably include beads, such as particles, such as latex beads. Other structures that can be coated externally by T cell receptor molecules are also suitable. Preferably, the structure is coated with T cell receptor multimers rather than individual T cell receptor molecules.

In the case of liposomes, T cell receptor molecules or their multimers may be attached to the membrane or otherwise bound to the membrane. This technique is well known to those in the art.

Labels or other moieties, such as toxic or therapeutic moieties, can be included in the multivalent TCR complex of the present invention. For example, a label or other moiety can be included in the mixed molecular multimer. Examples of such multivalent molecules are tetramers comprising three TCR molecules and one peroxidase molecule. A tetramer can be obtained by mixing TCR and enzyme in a molar ratio 3: 1 to produce a complex of tetramer and separating the desired complex from another complex that does not contain the correct molecular ratio. These mixed molecules may have any combination as long as steric hindrance does not impair or significantly impair the desired function of the molecule. Positioning the binding site to the streptavidin molecule is suitable in mixed tetramers because steric hindrance does not occur.

Other methods of biotinylating TCR are possible. For example, chemical biotinylation can be used. Alternatively, biotinylation tags may be used, although certain amino acids of the biotin tag sequence are essential (Schatz, (1993). Biotechnology N Y 11 (10): 1138-43). Mixtures used for biotinylation may vary. Enzymes require Mg-ATP and low ionic strength, but these conditions can vary, for example using high ionic strength and long reaction times. It is also possible to use molecules other than avidin or streptavidin to form multimers of TCR. Any molecule that binds biotin in a multivalent manner would be suitable. Alternatively, completely different linkages can also be devised (eg poly-histidine tags for chelate nickel ions, Quiagen Product Guide 1999, Chapter 3, "Protein Expression, Purification, Detection and Assay", p. 35-37). ). Preferably, the tag is positioned towards the C-terminus of the protein to minimize the amount of steric hindrance in interacting with the peptide-MHC complex.

One or two of the TCR chains may be labeled with a detectable label, for example, with a label suitable for diagnostic purposes. Accordingly, the present invention comprises contacting an MHC-peptide complex with a TCR or multivalent TCR complex specific for the MHC-peptide complex according to the present invention and detecting that the TCR and multivalent TCR complex are bound to the MHC-peptide complex. To provide an MHC-peptide complex detection method. When tetrameric TCRs are formed using biotinylated heterodimers, fluorescent streptavidin (commercially available) can be used to provide a detectable label. Fluorescently-labeled tetramers are suitable for use in FACS analysis, eg, for detection of antigen presenting cells carrying peptides specific for TCR.

Another way to detect soluble TCRs of the invention is to use TCR-specific antibodies, in particular monoclonal antibodies. There are many commercially available anti-TCR antibodies, such as αFI and βFI, which recognize the constant regions of each of the α and β chains.

The TCR (or multivalent TCR complex) of the present invention may optionally or additionally be linked to a therapeutic agent such as a toxic moiety for use in cell death or an immune stimulant such as interleukin or cytokine (eg, covalent or Connected in other ways). The multivalent TCR complexes of the present invention may have increased binding capacity to pMHC as compared to non-multivalent T cell receptor heterodimers. Thus, the multivalent TCR complex according to the present invention is particularly useful for tracking or targeting cells presenting specific antigens in vivo or in vitro, and also as a medium for generating additional multivalent TCR complexes having such use. TCRs or multivalent TCR complexes may be provided in pharmaceutically acceptable formulations available in vivo.

The invention also provides a method of delivering a therapeutic agent to a target cell, the method comprising contacting a TCR or multivalent TCR complex according to the invention with a potential target cell under conditions that permit attachment of the TCR or multivalent TCR complex with the target cell. And the aforementioned TCR or multivalent TCR complex is specific for the MHC-peptide complex and a therapeutic agent is bound thereto.

In particular, soluble TCRs and multivalent TCR complexes can be used to deliver therapeutic agents where cells that provide specific antigens are located. This is useful in many situations, especially for tumors. The therapeutic agent is transported to exert local therapeutic effect as well as the cells to which the therapeutic agent binds. Thus, in one particular method, there are anti-tumor molecules linked to T cell receptors or multivalent TCR complexes specific for tumor antigens.

Many therapeutic agents can be used for this purpose, for example, radioactive compounds, enzymes (eg perforin) or chemotherapeutic agents (eg cis-platin). To ensure that the toxic effect is exerted at the desired location, the toxin is placed inside the liposome linked to streptavidin to allow the compound to be released slowly. This prevents the damaging effects during delivery in the body and allows the toxin to have the maximum effect after TCR binds to the appropriate antigen presenting cell.

Other suitable therapeutic agents include:

A low molecular cytotoxic agent, ie, a compound having a molecular weight less than 700 Daltons, capable of killing mammalian cells. Such compounds may also contain toxic metals that may have a cytotoxic effect. In addition, these small molecule cytotoxic agents are also understood to include pro-drugs, ie compounds that are degraded or converted to release cytotoxic agents under physiological conditions. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, Ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, Trimetreate glucuronate, auristatin E vincristine and doxorubicin.

Peptide cytotoxins, ie proteins or fragments thereof capable of killing mammalian cells. Examples include lysine, diphtheria toxin, exotoxin A, DNAase and RNAase of Pseudomonas bacteria.

Radio-nuclides, ie unstable isotopes, or γ rays, that decay by simultaneously releasing one or two or more of α or β particles, or γ rays. Examples include iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213.

Prodrugs, antibody directed enzyme pro-drugs; And immune stimulants, ie moieties that stimulate an immune response. For example, cytokines such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, other antibodies or antibody fragments, complementary Active agents, protein domains of heterogeneous individuals, allogeneic proteins, viral / bacterial protein domains and viral / bacterial peptides.

Soluble TCRs or multivalent TCR complexes of the invention can be linked with enzymes that can convert prodrugs into drags. This allows the prodrug to be converted to the drug only where it is needed (ie targeted by the sTCR).

Examples of suitable MHC-peptide targets of TCRs according to the present invention include HTLV-1 epitopes (eg, Tax peptides restricted by HLA-A2; HTLV-1 is associated with leukemia), HIV epitopes, EBV epitopes, CMV epitopes; Autoimmunity such as melanoma epitopes (eg, MAGE-1 HLA-A1 restricted epitopes) and other cancer-specific epitopes (eg, malignancies of renal cells associated with antigen G250 restricted by HLA-A2) and rheumatoid arthritis Epitopes associated with the disease, but are not limited to these. Moreover, suitable disease-associated pMHC targets for use in the present invention are listed in the HLA Factbook (Barclay (Ed) Academic Press), and many others have been identified.

Treatment of many diseases can potentially be improved by localizing the drug with the specificity of soluble TCR.

Viral diseases in which the drug is present, such as HIV, SIV, EBV, CMV, would be beneficial if the drug was released or activated near the infected cell. For cancer, placing the tumor or metastasis nearby will enhance the effects of toxins or immune stimulants. In autoimmune diseases, immunosuppressive drugs can be released slowly to have a more local effect for a long time while minimizing the overall immune capacity. In the prevention of transplant rejection, the effects of immunosuppressive drugs can be optimized in the same manner. In vaccine delivery, vaccine antigens can be placed in proximity of antigen presenting cells to enhance the efficacy of the antigen. This method can also be applied for imaging purposes.

Soluble TCRs of the invention can be used to modulate T cell activity by binding to specific pMHC and thereby inhibiting T cell activity. For example, in the case of autoimmune diseases including T cell-mediated inflammation and / or tissue damage such as type I diabetes, this method may be followed. This use requires knowledge of specific peptide epitopes provided by the appropriate pMHC.

According to the present invention the drug may usually be provided as part of a sterile pharmaceutical composition which usually comprises a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending on the method required to administer the composition to the patient). It may be provided in unit dosage form, generally in a closed container, and may be provided as part of a kit. Such kits generally (but not necessarily) include instructions for use. It may include many of the unit dosage forms mentioned above.

The pharmaceutical composition may be administered by any suitable route, eg, oral (including oral or sublingual), rectal, nasal, topical (including gut, sublingual or transdermal), vaginal or parenteral (subcutaneous, intramuscular) , Intravenous or transdermal). Such compositions can be prepared by methods well known in the art, for example by mixing the carrier (s) or additive (s) with the active ingredient under sterile conditions.

Pharmaceutical compositions suitable for oral administration include capsules or tablets; Powder or granules; Liquid; It can be provided in individual units such as syrups or suspensions (aqueous or non-aqueous solutions; or edible foams or softeners; or emulsions). Suitable additives for tablets or hard gelatin capsules include lactose, maize starch or derivatives thereof, fatty acids or fatty acid salts. Additives suitable for use in soft gelatin capsules include, for example, vegetable oils, waxes, fats, semisolid polyols, liquid polio and the like.

In the preparation of solutions and syrups, the additives that can be used include, for example, water, polyols and sugars. Suspended oil (eg vegetable oil) formulations are used to provide oil in water or water in water. Pharmaceutical compositions suitable for transdermal administration may be provided in detachable patches to bring them into close contact with the patient's envelope for extended periods of time. For example, the active ingredient may be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research 3 (6): 318 (1986). Pharmaceutical compositions suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In case of infection of the eyes or other external skin, such as the mouth and skin, the composition is preferably applied as a topical ointment or cream. When prepared as an ointment, the active ingredient will be used with a paraffinic or water soluble ointment base. Alternatively, the active ingredient may be made into a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eye include eye drops dissolved in a solvent or suspended in a suitable carrier, in particular a water soluble solvent. Pharmaceutical compositions suitable for topical administration to the oral cavity include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions suitable for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions suitable for intranasal administration in which the carrier is solid comprise coarse powders having a particle size in the range of 20 to 500 microns, for example by inhaling, ie, powder close to the nose. In a container, it is administered by rapid inhalation through the nasal route. Suitable compositions when the carrier is a liquid for administration by nasal spray or nasal solution include active ingredients that are water soluble or oily. Pharmaceutical compositions suitable for administration by inhalant include fine particle dust or fog made by various forms of means, such as metered dose pressurized aerosols, nebulizers or insulators. Pharmaceutical compositions suitable for vaginal administration may be presented as pessary, tampon, cream, gel, paste, foam or spray formulations. Pharmaceutical compositions suitable for parenteral administration include aqueous solutions containing antioxidants, buffers, bacteriostats and solutes that give the blood of the recipient and substantially an isotonic composition, and sterile injectable solutions and suspensions and thickeners of non-aqueous solutions. Contains soluble and non-aqueous sterilized suspensions containing. Additives that can be used as injectable solutions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. The compositions may be presented in unit-dose or multi-dose containers, for example, in closed ampoules and vials, and may be stored under refrigerated conditions, immediately prior to use, for example transport, such as water for injection. Only the addition of a sterilizing solution is required.

Pharmaceutical compositions include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, fragrances, salts (substrates of the invention are provided in the form of pharmaceutically acceptable salts), buffers, coatings or antioxidants. It may also contain a pharmaceutically active ingredient other than the substrate of the present invention.

The dosage of the substrate of the present invention may vary within wide ranges depending on the disease or condition to be treated, the age and conditions of the individual to be treated, and the therapist will ultimately determine the appropriate dosage to use. Dosing can be repeated as appropriate. If adverse events occur, the amount and / or frequency of medication may be reduced in accordance with standard clinical practice.

Gene cloning techniques can be used to provide sTCRs of the invention, preferably in substantially pure form. These techniques are published, for example, in J. Sambrook et al Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Thus, in a further aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding a chain of the soluble TCR of the present invention, or a sequence complementary thereto. Such nucleic acid sequences are obtained by isolating TCR encoding nucleic acids from T-cell clones and causing appropriate mutations (by insertion, deletion or substitution).

Nucleic acid molecules can be isolated or recombinant. The vector can be inserted and the vector can be inserted into a host cell. Such vectors and suitable hosts are also aspects of the present invention.

The present invention also provides a method for obtaining a TCR chain comprising culturing the host cell under conditions inducing expression of the TCR chain and then purifying the polypeptide.

Soluble TCRs of the invention can be obtained by expressing aggregates in bacteria such as E. coli and refolding in vitro.

Refolding of the TCR chains takes place in vitro under suitable refolding conditions. In a specific embodiment, the TCR with the correct structure is obtained by refolding the TCR chain dissolved in a refold buffer containing a solubilizer such as urea. Advantageously, the urea may be provided at a concentration of at least 0.1M or at least 1M or at least 2.5M or at least about 5M. Another solubilizer that may be used is guanidine, which may be used at a concentration of 0.1M to 8M, preferably at least 1M or higher or at least 2.5M or higher. Prior to refolding, it is desirable to use a reducing agent to completely reduce the cysteine residue. Furthermore, denaturing agents such as DTT and guanidine can be used as needed. Other denaturing and reducing agents may be used before the refolding step (eg urea, β-mercaptoethanol). Another redox pair may be used during refolding, such as cystamine / cysteamine redox pairs, DTT or β-mercaptoethanol / atmospheric oxygen, and reduced and oxidized cysteines.

Folding efficiency can be increased by adding any other protein component, such as chaperone protein, to the refolding mixture. Refolding has been improved by passing proteins through a column fixed with mini-chaperon (Altamirano, et al. (1999). Nature Biotechnology 17: 187-191; Altamirano, et al. (1997). Proc Natl Acad Sci USA 94 (8): 3576-8).

Alternatively, soluble TCRs of the invention can be obtained by expression in eukaryotic cell systems such as insect cells.

Purification of TCR can purify TCR in many different ways. Different ion exchange methods may be used or other protein separation methods such as gel filtration chromatography or affinity chromatography may be used.

Soluble TCRs and multivalent TCR complexes of the present invention can also be used to screen for agents such as small molecule compounds that have the ability to inhibit TCR binding to pMHC complexes. Thus, in a further aspect, the present invention comprises the steps of monitoring the binding of a peptide-MHC complex with a soluble T cell receptor of the present invention in the presence of an antigen: and selecting an agent that inhibits the binding. Provided are methods for screening agents that inhibit the binding of T cell receptors to MHC complexes.

Suitable techniques for such screening methods include methods based on Surface Plasmon Resonance described in WO 01/22084. Other known techniques that underlie this screening method include Scintillation Proximity Analysis (SPAC) and Amplified Luminescent Proximity Assay (ALPAC).

The agent selected by the screening method of the present invention may be used as a drug, or may be modified or improved to have characteristics that are more suitable for administration as a drug according to the principles of the drug development program. Such agents can be used to treat conditions involving unnecessary T cell response elements. Such conditions include cancer (eg, kidney, ovary, intestines, head and neck, testes, lungs, stomach, cervix, bladder, prostate or melanoma), autoimmune diseases, graft rejection and graft versus host disease. It includes.

The preferred features of each aspect of the invention also apply to each according to other aspects. The above-mentioned prior art documents are cited to the maximum extent permitted by law.

The invention is explained in more detail by the following examples, which are not intended to limit the scope of the invention.

1 is a schematic diagram of a soluble TCR incorporating an interchain disulfide bond according to the present invention.

2A and 2B show the nucleic acid sequences of the α and β chains of soluble A6 TCR, which were mutated to introduce cysteine codons, respectively. The shaded areas indicate the introduced cysteine codons.

3A shows the amino acid sequence of the extracellular α chain of A6 TCR where T ₄₈ is mutated (underlined) to C ₄₈ to create new disulfide interchain bonds. 3B shows the amino acid sequence of the extracellular β chain of A6 TCR in which S ₅₇ is mutated to C (underlined) to create a new disulfide interchain bond.

Figure 4 shows the protein eluate formed from the POROS 50HQ column using a 0 to 500mM NaCl gradient, as a result of anion exchange chromatography of soluble A6 TCR, and is indicated by dotted lines.

FIG. 5A shows reduced SDA-PAGE (stained with Coomassie) of the fractions obtained from the column performed in FIG. 4. FIG. 5B shows the non-reducing SDA-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 4. Peak 1 contains predominantly non-disulphide bonded β chains, Peak 2 contains predominantly interchain disulfide linked TCR heterodimers, and shoulders are contaminants of E. coli that are rarely visible in these reproductions. This is because it is mixed with the interchain disulfide linked sTCR.

FIG. 6 is a result of size exclusion chromatography of the fraction pooled at peak 1 of FIG. 5. Protein eluted with a single main peak corresponding to heterodimer.

7 is a BIAcore response curve for specific binding of HLA-A2-tax complex with disulfide linked A6 soluble TCR. Inset box shows comparative binding reaction with control when infused with disulfide linked A6 soluble TCR alone.

8A shows an A6 TCR α chain comprising new cysteine residues mutated to insert a BamHI restriction site. Shading shows mutations introduced to form BamHI restriction sites. 8B and 8C show the DNA sequences of the α and β chains of JM22 TCR mutated to include additional cysteine residues to form non-natural disulfide bonds.

9A and 9B show amino acid sequences of the extracellular portions of the α and β chains of JM22 TCR generated from the DNA sequences of FIGS. 8A and 8B, respectively.

10 is an anion exchange chromatography of soluble disulfide-linked JM22 TCR, showing the protein eluate eluted in the POROS 50HQ column using a concentration gradient of 0 to 500 mM NaCl, and is indicated by dotted lines.

FIG. 11A shows reduced SDA-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 10. FIG. 11B shows the non-reduced SDA-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 10. Peak 1 clearly contains interchain disulfide linked TCR heterodimers.

FIG. 12 shows the size exclusion chromatography of the fractions pooled from the peak 1 of FIG. 10. The protein corresponds to a heterodimer, eluted with a single main peak and yield is 80%.

FIG. 13A is a BIAcore response curve showing the specific binding of JM22 soluble TCRs with disulfide linked HLA-Flu complex. 13B is a comparative binding reaction with the control for the single injection of disulfide-linked JM22 soluble TCRs.

14A and 14B show DNA sequences of NY-ESOα and β chains mutated to include additional cysteine residues to form non-natural disulfide bonds.

15A and 15B show amino acid sequences of extracellular portions of NY-ESO TCR α and β chains formed from the DNA sequences of FIGS. 14A and 14B, respectively.

FIG. 16 is an anion exchange chromatography of soluble NY-ESO disulfide linked TCR, showing the protein eluate eluted in the POROS 50HQ column using a 0 to 500 mM NaCl concentration gradient, and is indicated by dotted lines.

FIG. 17A shows reduced SDA-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 16. FIG. 17B shows non-reduced SDA-PAGE (stained with Coomassie) of the fractions obtained from the column performed in FIG. 16. Peak 1 and Peak 2 clearly contain interchain disulfide linked TCR heterodimers.

FIG. 18 is a result of size exclusion chromatography of fractions pooled at peak 1 and peak 2 of FIG. 17. Proteins corresponded to heterodimers and eluted with a single main peak.

19 shows the BIAcore response curves for specific binding of disulfide linked NY-ESO soluble TCRs with HLA-NYESO complexes. 19A corresponds to peak 1 and FIG. 19B corresponds to peak 2. FIG.

20A and 20B show the DNA sequences of the α and β chains of the mutated NY-ESO TCR, respectively, to introduce new cysteine codons (in shaded). This sequence includes cysteines related to natural disulfide interchain bonds (in bold codons).

21A and 21B show amino acid sequences of extracellular portions of NY-ESO TCRα and β chains formed from the DNA sequences of FIGS. 20A and 21B, respectively.

22 shows soluble NY-ESO TCRα ^cys β ^cys anion exchange chromatography, showing the protein eluate eluted in the POROS 50HQ column using a gradient of 0 to 500 mM NaCl, and is indicated by dotted lines.

Figure 23 is an anion exchange chromatography of soluble NY-ESO TCRα ^cys , showing the protein eluate eluted in the POROS 50HQ column using a concentration gradient of 0 to 500mM NaCl, and is indicated by a dotted line.

FIG. 24 is an anion exchange chromatography of soluble NY-ESO TCRβ ^cys , which shows a protein eluate eluted in a POROS 50HQ column using a concentration gradient of 0 to 500 mM NaCl, and is indicated by dotted lines.

FIG. 25 shows reduced SDS-PAGE (Coomassie stained) of the NY-ESO TCR α ^cys β ^cys , TCRα ^cys and TCRβ ^cys fractions obtained in the anion exchange columns performed in FIGS. 22-24, respectively. Lanes 1 and 7 represent the molecular weight (MW) markers, lane 2 represents the NYESOdsTCR1g4α-cysβ peak (EB / 084/033), lane 3 represents the NYESOdsTCR1g4α-cys soffee (EB / 084/033), 4 represents NYESOdsTCR1g4αβ-cys (EB / 084/034), lane 5 represents NYESOdsTCR1g4α-cys β-cys soffee (EB / 084/035), lane 6 represents NYESOdsTCR1g4α-cysβ-cys peak (EB / 084) 035).

FIG. 26 shows non-reduced SDS-PAGE (Coomassie stained) of the NY-ESOTCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys fractions obtained in the anion exchange columns performed in FIGS. 22-24, respectively. Lanes 1 and 7 represent the molecular weight (MW) markers, lane 2 represents the NYESOdsTCR1g4α-cysβ small peak (EB / 084/033), lane 3 represents the NYESOdsTCR1g4α-cysβ soap peak (EB / 084/033), Line 4 shows NYESOdsTCR1g4αβ-cys (EB / 084/034), lane 5 shows NYESOdsTCR1g4α-cys β-cys small peak (EB / 084/035), lane 6 shows NYESOdsTCR1g4α-cysβ-cys peak (EB / 084) / 035).

FIG. 27 shows the results of size exclusion chromatography of soluble NY-ESO TCRα ^cys β ^cys as a protein eluate of the fractions pooled in FIG. 22. Protein eluted with a single main peak, corresponding to heterodimer.

FIG. 28 shows the results of size exclusion chromatography of soluble NY-ESO TCRα ^cys , a protein eluate of the fractions pooled in FIG. 22. The protein eluted with a single main peak corresponding to the heterodimer.

FIG. 29 shows the results of size exclusion chromatography of soluble NY-ESO TCRβ ^cys , the protein eluate of the fractions pooled in FIG. 22. Protein eluted with a single main peak corresponding to heterodimer.

30 is a BIAcore response curve showing the specific binding of NY-ESO TCRα ^cys β ^{cys to} the HLA-NY-ESO complex.

FIG. 31 is a BIAcore response curve showing the specific binding of NY-ESO TCRα ^{cys to} the HLA-NY-ESO complex.

32 is a BIAcore response curve showing the specific binding of NY-ESO TCRβ ^{cys to} the HLA-NY-ESO complex.

33A and 33B show DNA sequences of soluble AH-1.23 TCR α and β chains, respectively, mutated to introduce new cysteine codons (denoted as negative salts). This sequence includes cysteines involved in natural disulfide interchain bonds (in bold codons).

34A and 34B show amino acid sequences of the extracellular portion of AH-1.23 TCR α and β chains formed from the DNA sequences of FIGS. 33A and 33B, respectively.

35 is an anion exchange chromatography of soluble AH-1.23 TCR, which shows the protein eluate eluted from the POROS 50HQ column using a NaCl concentration gradient of 0 to 500 mM and is indicated by dotted lines.

FIG. 36 is a reduced SDS-PAGE (10% Bis-Tris gel, Coomassie stained) of the AH-1.23 TCR fraction obtained from the anion exchange column performed in FIG. The protein analyzed is the anion exchange fraction of TCR 1.23 S-S obtained from Rifold 3. Line 1 represents the molecular weight (MW) marker, line 2 represents B4, line 3 represents C2, line 4 represents C3, line 5 represents C4, line 6 represents C5, line 7 represents C6 Line 8 represents C7, line 9 represents C8 and line 10 represents C9.

FIG. 37 is a non-reduced SDS-PAGE (10% Bis-Tris gel, Coomassie stained) of the AH-1.23 TCR fraction obtained from the anion exchange column performed in FIG. The protein analyzed is the anion exchange fraction of TCR 1.23 S-S obtained from Rifold 3. Line 1 represents the molecular weight (MW) marker, line 2 represents B4, line 3 represents C2, line 4 represents C3, line 5 represents C4, line 6 represents C5, line 7 represents C6 Line 8 represents C7, line 9 represents C8 and line 10 represents C9.

FIG. 38 shows the results of size exclusion chromatography of soluble AH-1.23 TCR, a protein eluate of the fraction pooled in FIG. 35. Protein eluted with a single main peak corresponding to heterodimer.

39A and 39B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 48 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

40A and 40B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 45 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

41A and 41B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 61 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

42A and 42B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 50 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

43A and 43B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 10 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

44A and 44B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 15 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

45A and 45B show the DNA and amino acid sequences of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 12 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

46A and 46B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 22 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

47A and 47B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 52 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

48A and 48B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 43 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

49A and 49B show the DNA sequence and amino acid sequence of the soluble A6 TCR α chain mutated to introduce a new cysteine at residue 57 in exon 1 of TRAC * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

50A and 50B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 77 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

51A and 51B show DNA and amino acid sequences of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 17 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

52A and 52B show the DNA and amino acid sequences of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 13 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

53A and 53B show the DNA and amino acid sequences of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 59 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

54A and 54B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 79 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

55A and 55B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 14 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

56A and 56B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 55 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

57A and 57B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 63 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

58A and 58B show the DNA sequence and amino acid sequence of the soluble A6 TCR β chain mutated to introduce a new cysteine at residue 15 in exon 1 of TRBC2 * 01, respectively. The shaded nucleotides represent the new cysteine codons introduced and the underlined amino acids represent the introduced cysteines.

59-64 show residues 48 of exon 1 of TRAC * 01 and 57 of exon 1 of TRBC2 * 01, respectively; Residue 45 of exon 1 of TRAC * 01 and residue 77 of exon 1 of TRBC2 * 01; Residue 10 of exon 1 of TRAC * 01 and residue 17 of exon 1 of TRBC2 * 01; Residue 45 of exon 1 of TRAC * 01 and residue 59 of exon 1 of TRBC2 * 01; Residue 52 of exon 1 of TRAC * 01 and residue 55 of exon 1 of TRBC2 * 01; Anion exchange chromatography of soluble A6 TCR having a new interchain disulfide bond in residue 15 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01 resulted in an anion exchange chromatography using NaCl concentration gradient of 0 mM to 500 mM. Proteins eluted from the POROS 50 column are shown with dotted lines.

65A and 65B show reduced and non-reduced SDS-PAGE of residue 48 of exon 1 of TRAC * 01 and soluble A6 TCR having a new disulfide chain linkage in the remaining 57 of exon 1 of TRBC2 * 01, respectively. One result, electrophoretic fractions (stained with Coomassie), were collected on the anion exchange column performed in FIG. 59.

66A and 65B show reduced and non-reduced SDS-PAGE of residue 45 of exon 1 of TRAC * 01 and soluble A6 TCR with new disulfide interchain linkages in the remaining 77 of exon 1 of TRBC2 * 01, respectively. One result and electrophoretic fractions (stained with Coomassie) were collected on the anion exchange column performed in FIG.

67A and 67B show reduced and non-reduced SDS-PAGE of residue 10 of exon 1 of TRAC * 01 and soluble A6 TCR with new disulfide interchain linkages in the remaining 17 of exon 1 of TRBC2 * 01, respectively. One result, electrophoretic fractions (stained with Coomassie), were collected on the anion exchange column performed in FIG. 61.

68a and 68b show reduced and non-reduced SDS-PAGE of residue 45 of exon 1 of TRAC * 01 and soluble A6 TCR with new disulfide interchain linkages in 59 residues of exon 1 of TRBC2 * 01, respectively. One result and electrophoretic fractions (stained with Coomassie) were collected on the anion exchange column performed in FIG. 62.

69A and 69B show reduced and non-reduced SDS-PAGE of residue 52 of exon 1 of TRAC * 01 and soluble A6 TCR having a new disulfide interchain linkage in the remaining 55 of exon 1 of TRBC2 * 01, respectively. One result (electrostained with Coomassie) electrophoretic fractions were collected in an anion exchange column performed in FIG.

70A and 70B show reduced and non-reduced SDS-PAGE of residue 15 of exon 1 of TRAC * 01 and soluble A6 TCR having a new disulfide interchain linkage in the remaining 15 of exon 1 of TRBC2 * 01, respectively. One result (stained with Coomassie) electrophoretic fractions was collected in an anion exchange column performed in FIG.

71 shows the results of size exclusion chromatography, respectively, of soluble A6 TCR having residues 48 of exon 1 of TRAC * 01 and new disulfide interchain linkage at 57 residues of exon 1 of TRBC2 * 01. Superdex 200 HL gel filtration column Protein eluate. The electrophoretic fractions were collected in an anion exchange column performed in FIG.

FIG. 72 is the result of size exclusion chromatography, respectively, of soluble A6 TCR having a new disulfide interchain bond in residue 45 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01, respectively, and a Superdex 200 HL gel filtration column Protein eluate. The electrophoretic fractions were collected in an anion exchange column performed in FIG.

FIG. 73 shows the results of size exclusion chromatography, respectively, of soluble A6 TCR having residues of exon 1 of TRAC * 01 and new disulfide chain linkage at residue 17 of exon 1 of TRBC2 * 01. Superdex 200 HL gel filtration column Protein eluate. The electrophoretic fractions were collected in an anion exchange column performed in FIG.

FIG. 74 shows the results of size exclusion chromatography, respectively, of soluble A6 TCR having residues 45 of exon 1 of TRAC * 01 and new disulfide interchain bonds in 59 residues of exon 1 of TRBC2 * 01. Superdex 200 HL gel filtration column Protein eluate. The electrophoretic fractions were collected in an anion exchange column performed in FIG. 62.

FIG. 75 shows the results of size exclusion chromatography, respectively, of soluble A6 TCR having residues 52 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01 with new disulfide interchain linkages. Superdex 200 HL gel filtration column The eluate of the protein is shown. The electrophoretic fractions were collected in an anion exchange column performed in FIG.

FIG. 76 shows the results of size exclusion chromatography, respectively, of soluble A6 TCR having residue 15 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01 with a new disulfide chain linkage, and a Superdex 200 HL gel filtration column. Protein eluate. The electrophoretic fractions were collected in an anion exchange column performed in FIG.

77-80 show residues 48 of exon 1 of TRAC * 01 and residues 57 of exon 1 of TRBC2 * 01; Residue 45 of exon 1 of TRAC * 01 and residue 77 of exon 1 of TRBC2 * 01; Residue 10 of exon 1 of TRAC * 01 and residue 17 of exon 1 of TRBC2 * 01; A BIAcore response curve showing the binding of HLA-A2-tax pMHC to soluble A6 TCR with residue 45 of exon 1 of TRAC * 01 and exon 1 of exon 1 of TRBC2 * 01, respectively.

FIG. 81 shows HLA-A2-tax and HLA-A2-NY-ESO pMHC of soluble A6 TCR with new interchain disulfide bonds at residue 52 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01 BIAcore response curve showing nonspecific binding to.

FIG. 82 is a BIAcore response curve showing the binding of HLA-A2-tax pMHC with soluble A6 TCR having residue 15 of exon 1 of TRAC * 01 and exon 1 of exon 1 of TRBC2 * 01 with new interchain disulfide bond .

83A is an electron density map around the model with a 1BD2 sequence (Th Thr 164 in Chain A, Ser 174 Ser in Chain B). The map is outlined at 1.0, 2.0 and 3.0σ. 83B is an electrical density map after changing two positions of A164 and B174 to Cys and outlined at the same level as 83a.

84 compares the IBD2 TCR structure of the present invention with the NY-ESO TCR by overlaying the above structure with the above ribbon and coil depiction.

85A and 85B show DNA and amino acid sequences of the NY-ESO TCR β chain into which the biotin recognition site was inserted, respectively. Biotin recognition sites are shown prominently.

86A and 86B show DNA and amino acid sequences of NY-ESO TCR β chains in which a hexa-histidine (hexa-histidine) tag was inserted, respectively. Hexa-histidine (hexa-histidine) tags are shown prominently.

FIG. 87 shows the elution of soluble NY-ESO TCR with new disulfide bond and biotin recognition sequence in POROS 50HQ anion exchange column using NaCl concentration gradient from 0 to 500 mM.

FIG. 88 shows the elution of soluble NY-ESO TCR with new disulfide bonds and hexahydridine tags in POROS 50HQ anion exchange column using NaCl concentration gradient from 0 to 500 mM.

FIG. 89 is a protein elution profile obtained by gel filtration chromatography of an anion exchange column of an NY-ESO-biotin tag performed in 87. FIG.

FIG. 90 is a protein elution profile obtained by gel filtration chromatography of anion exchange column of NY-ESO-hexa-histidine tag performed in 88.

91A-91H show NY-ESO peptides and fluorescent NY-ESO TCR tetramers, respectively, NYESO 0 TCR 5 μg, NYESO 10 ⁻⁴ M TCR 5 μg, NYESO 10 ⁻⁵ M TCR 5 μg, NYESO 10 ⁻⁶ M TCR 5 μg, 25,000 information of HLA-A2 positive EBV transformed B cell line (PP LCL) cultured at concentrations of 10 μg NYESO 0 TCR, 10 μg NYESO 10 ^-4 M TCR, 10 μg NYESO 10 ^-5 M TCR and 10 μg NYESO 10 ^-6 M TCR FACS histogram showing staining intensity produced at.

92 shows the DNA sequence of the β-chain of soluble A6 TCR inserting the TRBC1 * 01 constant region.

FIG. 93 is shown by dotted lines as a result of anion exchange chromatography of soluble A6 TCR with the TRBC1 * 01 constant region showing protein elution on POROS 50HQ column using 0-500 mM NaCl concentration gradient.

FIG. 94A is a reduced SDS-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 93. FIG. 94B is a non-reduced SDS-PAGE (stained with Coomassie) of the fraction obtained from the column performed in FIG. 93.

FIG. 95 is a size exclusion chromatography of the fraction pooled from FIG. 93 peak 2. FIG. Peak 1 contains a TCR heterodimer with interchain disulfide bonds.

96A is a BIAcore assay for specific binding of A6 soluble TCRs that are disulfide linked with HLA-Flu complex. FIG. 96B is a comparison of the binding response of the control to infusion of disulfide linked A6 soluble TCR alone. FIG.

97 is the nucleic acid sequence of the mutated beta chain of A6 TCR with 'free' cysteine inserted.

98 Anion exchange chromatography of soluble A6 TCR with 'free' cysteine shows the protein eluate of POROS 50HQ column using NaCl concentration gradient of 0-500 mM and indicated by dotted lines.

FIG. 99A is a reduced SDS-PAGE (Coomassie stained) of the fraction obtained from the column performed in FIG. 98. FIG. FIG. 99B is a non-reduced SDS-PAGE (Coomassie stained) of the fraction obtained from the column performed in FIG. 98.

FIG. 100 is size exclusion chromatography of the pooled fractions obtained from FIG. 98 peak 2. FIG. Peak 1 contains an interchain disulfide linked TCR heterodimer.

FIG. 101A is a BIAcore assay for specific binding of disulfide linked A6 soluble TCRs with free cysteine inserted into the HLA-Flu complex. FIG. 101B is the result of comparative binding reaction with the control for the single injection of disulfide linked A6 soluble TCR.

102 shows nucleic acid sequences of mutated beta chains of A6 TCR with mutated serine residues for free cysteine.

FIG. 103 is an anion exchange chromatography of soluble A6 TCR with mutated serine residues for free cysteine showing protein eluate on POROS 50HQ column using a gradient of 0 to 500 mM NaCl.

FIG. 104A is a reduced SDS-PAGE (stained with Coomassie) of the column fraction performed in FIG. 103. FIG. 104B is a non-reduced SDS-PAGE (stained with Coomassie) of the column fractions performed in FIG. 103. Peak 2 clearly contains an interchain disulfide linked TCR heterodimer.

FIG. 105 is a size exclusion chromatography of the pooled fractions of FIG. 103 Peak 2. FIG.

The result is. Peak 1 contains an interchain disulfide linked TCR heterodimer.

106A is a BIAcore assay showing specific binding of disulfide linked A6 soluble TCRs with mutated serine residues for free cysteine in the HLA-Flu complex. 106B shows the results of comparative binding reactions with the control when injected with disulfide linked A6 soluble TCRs alone.

107 shows the nucleotide sequence of pYX112.

108 shows the nucleotide sequence of pYX122.

109 shows the DNA and protein sequences of pre-pro hybridization factor alpha fused to the TCR α chain.

110 shows the DNA and protein sequences of pre-pro hybridization factor alpha fused to TCR β chains.

111 is S. Shown is a Western blot of soluble TCR expressed in S. cerevisiae strain SEY6210. Line C contains 60 ng of purified soluble NY-ESO TCR as a control, line 1 and line 2 collected proteins from TCR transformed yeast culture.

FIG. 112 shows the nucleic acid sequence of the insert from KpnI to EcoRI of the pEX172 plasmid. The margin of plasmid is pBlueScript II KS-.

113 is a schematic of the TCR chain cloned with baculovirus.

114 shows nucleic acid sequences of BamHI insert disulfide A6αTCR constructs for insertion into pAcAB3 expression plasmids.

115 shows nucleic acid sequences of BamHI insert disulfide A6 βTCR constructs for insertion into pAcAB3 expression plasmids.

116 shows Coomassie stained gels and western blots of bacterially produced disulfide A6 TCR and insect disulfide A6 TCR.

In the examples below, unless otherwise stated, the resulting soluble TCR chain is cleaved directly at the C-terminus of the cysteine to form a native interchain disulfide bond.

Example 1- Primer design and mutagenesis of A6 Tax TCR α and β chains

In order to mutate A6 Tax threonine 48 of exon 1 of TRAC * 01 into cysteine, the following primers were prepared (mutations are shown in lowercase letters).

5'-C ACA GAC AAA tgT GTG CTA GAC AT

5'-AT GTC TAG CAC Aca TTT GTC TGT G

In order to mutate A6 Tax Serine No. 57 of exon 1 of TRBC1 * 01 and TRBC2 * 01 with cysteine, the following primers were prepared (mutations are shown in lowercase letters).

5'-C AGT GGG GTC tGC ACA GAC CC

5'-GG GTC TGT GCa GAC CCC ACT G

PCR mutations:

Expression plasmids containing genes for the A6 Tax TCR α chain and β chain were mutated by the following method using α chain primers and β chain primers, respectively. 100 ng of plasmid was mixed with 5 μl of 10 mM dNTP, 25 μl of 10 × Pfu-Stratagene, 10 units of Pfu polymerase (Stratagene) and the final volume was adjusted to H ₂ O to 240 μl. 48 μl of this mixture was mixed with primers diluted to a final concentration of 0.2 uM in a 50 μl final reaction volume. Initial denaturation at 95 ° C for 30 seconds in a Hybaid PCR express PCR machine, then the reaction mixture is denatured (95 ° C, 30 seconds), bound (55 ° C, 60 seconds), extended (73 ° C, 8 minutes) 15 times. The product was then digested with 10 units of DpnI restriction enzyme (New England Biolabs) at 37 ° C. for 5 hours. 10 μl of the cleaved reaction was transformed with competent XL1-Blue bacteria and grown 18 hours at 37 ° C. Media consisting of TYP and ampicillin (16 g / l Bacto-Tryptone, 16 g / l Yeast Extract, 5 g / l NaCl, 2.5 g / l K ₂ HPO ₄ , 100 mg / l ampicillin) after a single clone It was grown in 5 ml overnight. Plasmid DNA was purified on a Qiagen mini-prep column according to the manufacturer's instructions and the sequence was then verified by automated sequencing using a sequencing machine from Oxford University Biochemistry. Each of the mutated nucleic acids and amino acid sequences can be seen in Figures 2a and 3a for the α chain and in Figures 2b and 3b for the β chain.

Example 2 Expression, Refolding and Purification of Soluble TCR

Expression plasmids containing each of the mutated α and β chains were individually transformed into the E. coli strain BL21pLysS, and the OD ₆₀₀ value before inducing protein expression with 0.5 mM IPTG of a single clone with ampicillin resistance was determined. It was grown in TYP (ampicillin 100 μg / ml) medium at 37 ° C. until 0.4. Cells were collected by centrifugation at 4000 rpm for 30 minutes in Beckman J-6B 3 hours after induction of protein expression. Cell pellets were resuspended in a buffer of pH 8.0 containing 50 mM Tris-HCl, 25% (w / v) sucrose, 1 mM NaEDTA, 0.1% (w / v) NaAzide and 10 mM DTT. After an overnight freeze-thaw step, the resuspended cells were sonicated for a total of 10 minutes in a minute using a standard 12 mm diameter probe with a Milsonix XL2020 sonicator. Aggregate pellets were collected by centrifugation at 13000 rpm for 30 minutes in a Beckman J2-21 centrifuge. Three detergent washes were performed to remove cell debris and membrane components. Aggregate pellets at each time were treated with Triton buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCI, 10 mM NaEDTA, 0.1% (w / v) NaAzide before pelleting by centrifugation at 13000 rpm for 15 minutes in Beckman J2-21). , 2 mM DTT, pH 8.0). Detergents and salts were then removed with buffer or similar buffer, pH 8.0, 50 mM TRIS-HCl, 1 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT. Finally, aggregates were divided into 30 mg aliquots and frozen at -70 ° C. Aggregated protein yield was quantified by dissolving with 6M guanidine-HCl and measuring by Bradford dye-binding assay (PerBio). Approximately 30 mg of dissolved aggregate chain (ie 1 uM) were dissolved in a frozen stock, after which the sample was mixed and the guanidine solution (6 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA) to completely denature the mixture. ) To 15 ml. Guanidine solution containing fully reduced and denatured TCR chains was refolded with 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 and 0.2 mM PMSF. Injected in 1 L. The solution was left for 24 hours and dialyzed twice with first 10 l of 100 mM urea, second with 10 l of 100 mM urea, 10 mM Tris (pH 8.0). Both refolding and dialysis steps were performed at 6-8 ° C.

The dialysis refold solution was loaded on a POROS 50HQ anion exchange column and sTCR was separated from the degradation products and impurities as shown in Figure 4 by eluting the bound protein using a zero to 500 mM NaCl concentration gradient over 50 times the column volume. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE before pooling and concentration (FIG. 5). Finally, after pre-equilibration with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40), sTCR was purified and characterized using a Superdex 200HR gel filtration column (Figure 6). ). Peaks eluted at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

Example 3 Characterization of sTCR and Specific pMHC Using BIAcore Surface Plasmon Resonance

Surface plasmon resonance biosensors (BIAcore 3000 ^™ ) were used to analyze the binding between sTCR and its peptide MHC ligand. Binding is facilitated by creating a single pMHC complex that is immobilized on a streptavidin-coded binding surface in a semi-oriented fashion, which is up to four separate pMHC (separated flow cells) with soluble T-cell receptors. It can be used to effectively check the combination with the flow cell). Manual injection of the HLA complex allows easy manipulation of the correct amount of immobilized Class I molecules.

This immobilized complex is capable of binding both the T cell receptor and the CD8α co-receptor injected into the soluble phase. Specific binding of TCR occurs at low concentrations (at least 40 μg / ml), which means that the TCR is relatively stable. The pMHC binding characteristics of sTCR were quantitatively and qualitatively similar, even though sTCR was used as the soluble or stationary phase. This is an accurate control for the partial activity of soluble species and also suggests that biotinylated pMHC complexes are as active as complexes that are not biologically biotinylated.

Biotinylated Class IHLA-A2-peptide complexes of aggregates expressed in bacteria containing constituent subunit proteins and synthetic peptides were purified after in vitro repolling and enzymatic biotinylation in vitro (O'Callaghan et. (1999) Anal.Biochem. 266: 9-15). The HLA-heavy chain was expressed with a C-terminal biotinylation tag that replaced the protein transmembrane domain and cytoplasmic wholesaler with the appropriate structure. Aggregates were expressed at levels of about 75 mg / l cell culture. HLA light chains and β2-microglobulin were also expressed in aggregate form in the appropriate constructs in E. coli at levels of about 500 mg / l cell culture.

E. coli was dissolved and aggregates were appropriately purified to 80% purity. The protein in the aggregate was denatured with 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and the protein was denatured at 30 mg / l heavy chain and 30 mg / lβ2m at 5 ° C. It was refolded by addition to the following refold buffer, 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, mM cysteamine, 4 mg / ml peptide (eg tax 11-19). Refolding was terminated by leaving at 4 ° C. for at least 1 hour.

The buffer was exchanged with 10 mM Tris dialysis at pH 8.1 of 10 fold. Two buffer exchanges are necessary to sufficiently reduce the ionic strength of the solution. The protein solution was filtered through a 1.5 μm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 mL bed volume). Proteins were eluted using a linear 0-500 mM NaCl gradient. HLA-A2-peptide complex peak fraction eluting at about 250 mM NaCl concentration was collected and the fraction was cooled on ice by addition of protease inhibitor (Calbiochem).

The HLA complex with the biotinylated tag was buffer exchanged with 10 mM Tris pH 8.1, 5 mM NaCl using Pharmacia's fast desalting column equilibrated with 10 mM Tris pH 8.1, 5 mM NaCl buffer. Immediately after elution, the protein-containing fractions were placed on ice and proteolytic enzyme inhibitor (Calbiochem) was added. And biotinylation reagent, 1 mM biotin, 5 mM ATP (pH 8 buffered), 7.5 mM MgCl 2, and 5 g / ml BirA enzyme (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15 By the method). The mixture was left at room temperature overnight.

Biotinylated HLA complex was purified using gel filtration chromatography. The Pharmacia Superdex 75 HR 10/30 column was previously equilibrated with filtered PBS, 1 ml of the biotinylation reaction mixture was loaded and PBS was developed at a rate of 0.5 ml / min. Biotinylated HLA complex was eluted with a single peak at about 15 mL. Fractions containing protein were pooled and placed on ice and protease inhibitors (Calbiochem) were added. The biotinylated HLA complex was digested and stored frozen at -20 ° C by measuring protein concentration by Coomassie-binding assay (PerBio). Streptavidin was immobilized by standard amine coupling method.

The interactions between the A6 Tax sTCR and its ligand / MHC complex or irreversible HLA-peptide combination with new interchain bonds and the products described above were analyzed via a BIAcore 3000 ^™ surface plasmon resonance (SPR) biosensor. SPR measures the change in the regulation rate expressed in response units (RUs) near the sensor surface in a small flow cell, and in principle detects receptor-ligand interactions and detects their affinity and kinetic parameters. ) Can be used for analysis. Probes of the flow cell were prepared by immobilizing individual HLA-peptide complexes to separate flow cells through the binding of biotin crosslinked on β2m and streptavidin chemically bound to the activated surface of the flow cell. Detection is performed by passing the sTCR to the surface of a separate flow cell at a constant flow rate and thereby measuring the SPR response. First, the specificity of the interaction was sTCR with a constant flow rate of 5 μl / min, first coated with about 5000 RU of the specific peptide-HLA complex and second coated with about 5000 RU of the non-specific peptide-HLA complex (inserted in FIG. 7). ) By passing through two different surfaces. It was used to define the background resonance of soluble STCR at different concentrations of the peptide-HCA complex at a constant flow rate. These regulatory measures were obtained from specific peptide-HLA complexes and were used to determine the binding affinity expressed in dissociation constants (Kd) as shown in FIG. 7 (Price & Dwek, Principles and Problems in Physical Chemistry for Biochemists (2 ^nd Edition) 1979, Clarendon Press, Oxford).

The obtained Kd value of 1.8 μM was similar to the one that was published as the interaction value between A6 Tax sTCR and pMHC without new disulfide bonds (0.91 μM—Ding et al, 1999, Inmmunity 11: 45-56).

Example 4 Construction of Soluble JM22 TCR with a New Disulfide Bond

The soluble A6 TCR β chain prepared in Example 1 has a sequence of native BglII restriction sites (AAGCTT) suitable for use as a linking site.

PCR mutations were performed as described below to introduce BamH1 restriction sites (GGATCC) into the new cysteine codon 5 'phosphorus soluble A6 TCR α chain. The primers used as templates for this mutation are described in Figure 2a. The following primers were used.

100 ng of the plasmid was mixed with 10 mM, 5 μl of dNTP, 25 μl of 10 × Pfu-buffer (Stratagene), 10 units of Pfu polymerase (Stratagene), and adjusted to H ₂ O to a final volume of 240 μl. 48 μl of this mixture was mixed with primers diluted to a final concentration of 0.2 μM in 50 μl final reaction volume. After an initial denaturation step at 95 ° C for 30 seconds on a Hybaid PCR express PCR machine, the reaction mixture is denatured (95 ° C, 30 seconds), bound (55 ° C, 60 seconds), extended (73 ° C, 8 min.) 15 times. The product was then digested with 10 units of DpnI restriction enzyme (New England Biolabs) at 37 ° C. for 5 hours. 10 μl of the cleaved reaction was transformed with Competent XL1-Blue bacteria and grown for 18 hours at 37 ° C. TYP and ampicillin (16 g / L Bacto-Tryptone, 16 g / L yeast extract, 5 g / l NaCl, 2.5 g / l K ₂ HPO ₄ , 100 mg / l ampicillin) was grown overnight in 5 ml of medium. Plasmid DNA was purified on a Qiagen mini-prep column according to the manufacturer's instructions and the sequence was then verified by automated sequencing using a sequencing machine from Oxford University Biochemistry. The mutation introduced into the chain is 'silent' so that the amino acid sequence of this α chain remains unchanged as shown in the detailed illustration of FIG. 3A. The DNA sequence of the mutated α chain can be seen in FIG. 8A.

A6 TCR plasmids containing BamH1 in the α chain and BglII recognition sites in the β chain were used as templates to generate soluble JM22 TCRs with new disulfide bonds. The following primers were used.

JM22 TCR α and β chain structures were obtained by PCR cloning as follows. PCR reaction was performed using a template containing the primer, JM22 TCR chain. PCR products were restriction digested using the relevant restriction enzymes and cloned into pGMT7 to obtain expression plasmids. The sequence of the plasmid insert was confirmed by automated DNA sequencing. 8B and 8C show the DNA sequences of the mutated JM22 TCR α and β chains, respectively, and FIGS. 9A and 9B show the resulting amino acid sequences.

Each TCR chain was expressed, co-refolded and purified as described in Examples 1 and 2. FIG. 10 shows soluble disulfide linked JM22 TCR elution in POROS 50HQ column using 0-500 mM NaCl concentration gradient and indicated by dotted lines. FIG. 11 shows the results of reduced SDS-PAGE (coomassie stained) or non-reduced SDS-PAGE (coomassie stained) of fractions obtained from the column performed as shown in FIG. 10. FIG. 12 shows protein elution in the size exclusion column of the fraction pooled at peak 1 of FIG. 10.

BIAcore analysis for binding of JM22 TCR to pMHC was performed as described in Example 3. 13A shows BIAcore analysis for specific binding of JM22 soluble TCRs that are disulfide linked to HLA-Flu complex. 13B shows binding response compared to the control for disulfide linked JM soluble TCR. The Kd of the disulfide linked TCR for this HLA-Flu complex was determined to be 7.9 ± 0.51 uM.

Example 5 Fabrication of Soluble NY-ESO TCRs with New Disulfide Bonds

The cDNA encoding NY-ESO TCR was isolated according to known techniques from T cells provided by Enzo Cerundolo (Institute of Molecular Medicine, University of Oxford). The cDNA encoding NY-ESO TCR was generated by subjecting mRNA to reverse transcriptase.

To generate soluble NY-ESO TCRs with new disulfide bonds, an A6 TCR plasmid incorporating α chain BamHI, β chain BglII recognition sites was used as a template as described in Example 4. The following primers were used.

NY-ESO TCR α and β chain constructs were obtained by PCR cloning as follows. PCR reactions were performed using a template containing the primers presented above, NY-ESO TCR chain. PCR products were restriction digested using the relevant restriction enzymes and cloned into pGMT7 to obtain expression plasmids. The sequence of the plasmid insert was confirmed by automated DNA sequencing. 14A and 14B show DNA sequences of mutated NY-ESO TCR α and β chains, respectively, and FIGS. 15A and 15B show the resulting amino acid sequences.

Each TCR chain was expressed, co-folded and purified as described in Examples 1 and 2 except for modifications to the protocol below.

Degeneration of Soluble TCR: 30 mg of solubilized TCR β chain aggregate and 60 mg of solubilized TCR α chain aggregate were dissolved in frozen stock. The aggregates were diluted to a final concentration of 5 mg / ml in 6M guanidine solution and DTT (2M stock) was added to a final 10 mM.

Refolding of Soluble TCR: 1 L of refold solution was vigorously stirred at 5 ° C ± 3 ° C. Redox couple (2-mercaptoethylamine and cystamine, final concentrations of 6.6 mM and 3.7 mM, respectively) was added for about 5 minutes before adding the modified TCR chains. After that, the protein was refolded for about 5 hours ± 15 minutes while stirring at 5 ° C ± 3 ° C.

Dialysis of Refolded Soluble TCR: Refolded TCR was dialyzed with Spectrapor 1 membrane (Spectrum; Product No. 132670) at 10 L of 10 mM Tris (pH 8.1) at 5 ° C ± 3 ° C for 18-20 hours. It was. The buffer was then exchanged with 10 L of 10 mM Tris, pH 8.1 and dialysis continued at 5 ° C ± 3 ° C for an additional 20-22 hours.

FIG. 16 shows elution of soluble NY-ESO disulfide linked TCR protein in a POROS 50HQ column using a 0 to 500 mm NaCl concentration gradient as indicated by the dotted line. FIG. 17 shows the results of reduced SDS-PAGE (coomassie stained) or non-reduced SDS-PAGE (coomassie stained) of fractions obtained in the column performed as described in FIG. 16. Peak 1 and Peak 2 clearly include interchain disulfide linked TCR heterodimers. FIG. 18 shows size exclusion chromatography of pooled fractions obtained from peak 1 (A) and peak 2 (B) of FIG. 17. Protein eluted with the main single peak corresponding to the heterodimer.

BIAcore analysis, which is a pMHC binding with disulfide linked NY-ESO TCR, was performed as described in Example 3. FIG. 19 shows BIAcore analysis for specific binding of NY-ESO soluble TCRs disulfide-bound into HLA-NYESO complex. A is peak 1 and B is peak 2.

The Kd of the disulfide linked TCRs for the HLA-NY-ESO complex was determined to be 9.4 ± 0.84 uM.

Example 6: Construction of NY-ESO TCR comprising at least one of two cysteine residues required to form new disulfide interchain bonds and native disulfide interchain bonds

In order to generate soluble NY-ESO TCRs incorporating at least one of the cysteine residues involved in novel disulfide bonds and natural disulfide bonds, a plasmid containing α chain BamHI, β chain BglII restriction sites was described as described in Example 4. Used as a skeleton. The following primers were used.

NY-ESO TCR α and β chain constructs were obtained by PCR cloning as follows. PCR reactions were carried out using a template containing the primer, NY-ESO TCR chain, shown above. PCR products were restriction digested using the relevant restriction enzymes and cloned into pGMT7 to obtain expression plasmids. The sequence of the plasmid insert was confirmed by automated DNA sequencing. 20A and 20B show DNA sequences of mutated NY-ESO TCR α and β chains, respectively, and FIGS. 21A and 21B show the resulting amino acid sequences.

To generate soluble NY-ESO TCRs having both non-natural disulfide interchain linkages and natural disulfide interchain linkages, DNAs were isolated using all of the primers. To generate soluble NY-ESO TCRs with only one of the cysteine residues involved in non-natural disulfide interchain linkages and natural disulfide interchain linkages, DNA was isolated using one of the primers mentioned above and the appropriate primers of Example 5.

Once each TCR chain was expressed, it was co-refolded and purified as described in Example 5.

22-24 show soluble NY-ESO TCRα ^cys β ^cys (with non-natural, natural cysteine in both chains) in POROS 50HQ anion exchange column using a gradient of 0 to 500 mM NaCl, as indicated by the dashed line, TCRα ^elution of ^cys (non-natural cysteine ^branches in both chains, but only in the α chain) and NY-ESO TCRβ ^cys (non-natural cysteine ^branches in both chains, but only in the β chain). Figures 25-26 show reduced SDS-PAGE (Coomassie stained), non-reduced fractions of NY-ESO TCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys in columns performed as described above with reference to FIGS. 22-24, respectively. Type SDS-PAGE (Coomassie stained) results are shown. Figures 27 to 29 are protein elution profiles of gel filtration chromatography of fractions pooled in NY-ESO TCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys anion exchange columns performed as described in FIGS. 22-24, respectively. Protein eluted with TCR single main peak corresponding to heterodimer.

BIAcore analysis of sTCR binding to pMHC was performed as described in Example 3. 30-32 show BIAcore analysis for specific binding of NY-ESO TCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys to HLA-NYESO complexes, respectively.

TCRα ^cys β ^cys had a Kd of 18.08 ± 2.075uM, TCRα ^cys had a Kd of 19.24 ± 2.01uM, and TCRβ ^cys had a Kd of 22.5 ± 4.0692uM.

Example 7 Construction of Soluble AH-1.23 TCR with New Disulfide Interchain Bond

CDNA encoding AH-1.23 TCR was isolated according to known techniques in T cells provided by Hill Gaston (Medical School, Addenbrooke's Hospital, Cambridge). The cDNA encoding NY-ESO TCR was generated by subjecting mRNA to reverse transcriptase.

To generate soluble AH-1.23 TCRs with novel disulfide bonds, TCR plasmids containing BamHI in the α chain and BglII recognition sites in the β chain were used as the backbone, as described in Example 4. The following primers were used.

AH-1.23 TCR α and β chain structures were obtained by PCR cloning as follows. PCR reactions were performed using a template containing the primer, AH-1.23 TCR chain shown above. PCR products were restriction digested using the relevant restriction enzymes and cloned into pGMT7 to obtain expression plasmids. The sequence of the plasmid insert was confirmed by automated DNA sequencing. 33A and 33B show DNA sequences of α and β chains of the mutated AH-1.23 TCR, respectively, and FIGS. 34A and 34B show the resulting amino acid sequences.

Each TCR chain was expressed, co-refolded and purified as described in Example 5.

FIG. 35 shows the elution of TCR proteins linked by disulfide of soluble AH-1.23 in a POROS 50HQ anion exchange column using a 0 to 500 mM NaCl concentration gradient, as indicated by the dashed line. 36 and 37 show the results of the reduced SDS-PAGE (comasy stained) and non-reduced SDS-PAGE (comasy stained) fractions in the column performed as described in FIG. 35, respectively. This gel clearly shows the presence of TCR heterodimers with interchain disulfide bonds formed. FIG. 38 is an elution profile of a Superdex 75 HR gel filtration column of fractions pooled in an anion exchange column performed as described in FIG. 35. Protein elutes with a single main peak corresponding to a TCR heterodimer.

Example 8 Construction of Soluble AG TCRs with New Disulfide Interchain Bonds at Different Locations in the Immunoglobulin Region of the Constant Domain

Forming a functional soluble TCR containing a new disulfide bond in the TCR immunoglobulin region at a location other than between threonine 48 at exon 1 of TRAC * 01 and 57 serine at exon 1 of TRBC1 * 01 and TRBC2 * 01 The following experiments were conducted to investigate the possibility.

In order to mutate the A6 TCR α chain, the following primers were prepared (the numbers in the primer name indicate the position of the amino acid residue to be mutated in exon 1 of TRAC * 01 and the residue where the mutation occurred in lowercase).

T48 → C mutation

Y10 → C mutation

L12 → C mutation

S15 → C mutation

V22 → C mutation

Y43 → C mutation

T45 → C mutation

L50 → C mutation

M52 → C mutation

S61 → C mutation

To mutate the A6 TCR β chain, the following primers were prepared (the numbers in the primer name indicate the position of the amino acid residue to be mutated in exon 1 of TRBC2 * 01 and the residue where the mutation occurred in lowercase).

S57 → C mutation

V13 → C mutation

F14 → C mutation

S17 → C mutation

G55 → C mutation

D59 → C mutation

L63 → C mutation

S77 → C mutation

R79 → C mutation

E15 → C mutation

PCR mutations, amplification, linkage, and plasmid purification of PCR mutations, α and β TCR constructs through appropriate combinations of the above-mentioned primers to generate soluble TCRs comprising novel disulfide interchain linkages between the following pairs of amino acids are described in Example 1 As was done.

TCRα chain TCRβ chain Α primer used Β primer used Thr 48Thr 45Ser 61Leu 50Tyr 10Ser 15Thr 45Leu 12Ser 61Leu 12Val 22Met 52Tyr 43Ser 15 Ser 57Ser 77Ser 57Ser 57Ser 17Val 13Asp 59Ser 17Arg 79Phe 14Phe 14Gly 55Leu 63Glu 15 T48 → CT45 → CS61 → CL50 → CY10 → CS15 → CT45 → CL12 → CS61 → CL12 → CV22 → CM52 → CY43 → CS15 → C S57 → CS77 → CS57 → CS57 → CS17 → CV13 → CD59 → CS17 → CR79 → CF14 → CF14 → CG55 → CL63 → CE15 → C

39 to 58 show DNA and amino acid sequences of the A6 TCR chain mutated with the primers. The codons encoding the mutated cysteines are prominent.

Each TCR chain was expressed, co-refolded and purified as described in Example 5. The resulting product protein was also subjected to SDS-PAGE gel to determine whether soluble TCRs were correctly refolded after purification via POROS 50HQ anion exchange column. These gels were also used to observe the presence of the correct molecular weight disulfide linked protein in the purified product. TCRs with the following new disulfide chain linkages using this bacterial expression system failed to produce disulfide linked proteins of the correct molecular weight and these were no longer evaluated. However, other prokaryotic or eukaryotic expression systems are available.

TCRα chain TCRβ chain Ser 61Leu 50Ser 15Leu 12Ser 61Leu 12Val 22Tyr 43 Ser 57Ser 57Val 13Ser 17Arg 79Phe 14Phe 14Leu 63

59 to 64 are shown in the dotted line, respectively, Thr 48-Ser 57, Thr 45-Ser 77, Tyr 10-Ser 17, Thr 45- in POROS 200HQ anion exchange column using a concentration gradient of 0 to 500 mM NaCl. Asp 59, Met 52-Gly 55 and Ser 15-Glu 15 show elution of soluble TCR with new disulfide interchain linkages. Figures 65 to 70 show the results of reduced SDS-PAGE (comasy stained) and non-reduced SDS-PAGE (comasy stained) of fractions in the column performed as described in Figures 59-64, respectively. This gel clearly shows the presence of interchain disulfide linked TCR heterodimers.

71-76 are elution profiles of a Superdex 200 HR gel filtration column of the fractions pooled in an anion exchange column performed as described in FIGS. 59-64. BIAcore analysis for binding of TCR to pMHC was performed as described in Example 3. 77 to 82 show the results of demonstrating the binding ability of the purified soluble TCR to the HLA-A2 tax pMHC complex.

Thr 48-Ser 57 is K _d of 7.8 uM, Thr 45-Ser 77 is K _d of 12.7 uM, Tyr 10-Ser 17 is 34 uM K _d , and Thr 45-Asp 59 is 14.9 uM K _d and Ser 15 Glu 15 has a K _d of 6.3 uM. Met 52-Gly 55 was able to bind to its native "target" HLA-A2 tax complex, although similarly bound to the "irrelevant" target HLA-A2-NY-ESO complex (FIG. 81). ).

Example 9 X-ray crystallography of disulfide linked NY-ESO T cell receptor specific for NY-ESO-HLA-A2 complex

NY-ESO dsTCR was cloned as described in Example 5 and expressed as follows.

Expression plasmids containing the mutated α and β chains, respectively, were individually transformed into E. coli strain BL21 pLysS, and the OD ₆₀₀ value before inducing protein expression with 0.5 mM IPTG of the ampicillin resistant single clone was determined. It was grown in TYP (ampicillin 100 μg / ml) medium at 37 ° C until 0.7. Cells were collected by centrifugation at 4000 rpm for 30 minutes on Beckman J-6B 18 hours after protein expression induction.

Cell pellets were resuspended in cell lysis buffer containing 10 mM Tris-HCl, pH 8.1, 10 mM MgCl ₂ , 150 mM NaCl, 2 mM DTT, 10% glycerol. 100 μl of lysozyme (20 mg / ml) and 100 μl of Dnase I (20 μg / ml) were added per liter of bacterial culture. After 30 minutes on ice, the bacterial suspension was sonicated for a total of 10 minutes for 1 minute using a standard 12 mm diameter probe and Milsonix XL2020 sonicator. Aggregate pellets were collected by centrifugation at 13000 rpm for 30 minutes in a Beckman J2-21 centrifuge (4 ° C.). Three washes were performed with Triton wash buffer (50 mM Tris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCI, 10 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT) to remove cell debris and membrane components It was. Each aggregate aggregate pellet was then homogenized with Triton wash buffer before being pelleted by centrifugation at 13000 rpm for 15 minutes in Beckman J2-21. Detergent and salt were then removed with a similar wash buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT). Finally, the aggregates were dissolved in 6M guanidine buffer (6M guanidine-hydrochloride, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), divided into 120 mg aliquots and frozen at -70 ° C. Aggregates were quantified by dissolving with 6M guanidine-HCl and measuring by Bradford assay (PerBio).

60 mg of frozen solubilized TCR α chain aggregate (ie, 2.4 μole) and 30 mg of frozen solubilized TCR β chain aggregate (ie 1.2 μole) were mixed. The TCR mixture was diluted with 6M guanidine solution to a final concentration of 18 mL and heated at 37 ° C. for 30 minutes to complete chain denaturation. The guanidine solution containing the fully reduced and denatured TCR chains was then replaced with refolding buffer (100 mM Tris pH 8.1, 400 mM L-arginine-HCl, 2 mM EDTA, 6.6 mM 2-mercaptoethylamine, 3.7 mM cystamine, 5M urea). ) Stir to 1 l and mix. The solution was placed in a cold room (5 ° C. ± 3 ° C.) for 5 hours to allow refolding. Dialysis was carried out for 18 to 20 hours with 12 L of water followed by 18 to 20 hours (5 ° C ± 3 ° C) with 12 L of 10 mM Tris (pH 8.1). A spectrapor 1 (Spectrum Laboratories product no. 132670) dialysis membrane having a molecular weight capable of blocking 6000 to 8000 kDa was used in the dialysis process. The dialyzed protein was filtered with a 0.45 μm pore size filter (Schleicher and Schuell, Ref. Number, 10 404012) that fits perfectly into the Nalgene filtration unit.

The refolded NY-ESO TCR was separated from digests and impurities by loading the dialyzed repolling product into a POROS 50HQ (Applied Biosystems) anion exchange column using an AKTA Purifier (Amersham Biotech). POROS 50 HQ columns were previously equilibrated with 10-fold column volume of Buffer A (10 mM Tris pH 8.1) to load the proteins. Bound proteins were eluted using a NaCl concentration gradient of 0-500 mM over 7-fold column volume. Peak fractions (1 mL) were analyzed by denaturing SDS-PAGE using reduced or non-reduced sample buffer. Peak fractions containing heterodimer alpha-beta complexes were further separated using a Superdex 75HR gel filtration column previously equilibrated with 25 mM MES, pH 6.5. Protein peaks eluting at a relative molecular weight of about 50 kDa were pooled and concentrated to 42 mg / ml in an Ultrafree centrifugal concentrator (Millipore, part number UFV2BGC40) and stored at -80 ° C.

Crystallization of NY-ESO TCR was performed by a hanging drop technique using 1 μl of a 5 mM Mes (pH 6.5) protein solution (8.4 mg / ml) mixed with an equal volume of crystallization buffer at 18 ° C. Crystals also appeared in several other conditions using Crystal Screen Buffer (Hampton Research). Single volume crystals (single cubic crystal <100 μm) were grown in 30% PEG 4000, 0.1 M Na citrate, pH 5.6, 0.2 M ammonium acetate buffer and used for structure determination.

Crystals of NY-ESO TCR were quickly frozen and analyzed by diffraction of X-ray rays of Daresbury synchrotron. The crystal was diffracted at 0.25 nm (2.5 Hz) resolution. The data sets were collected and processed to give a complete set of 98.6% of the appropriate amplitude around 0.27 nm (2.7 Hz) but not to 0.25 nm (2.5 Hz). The consensus between the merging R-factor, ie the crystallographically corresponding multiple reflections, was 10.8% in all data. This is the lowest in high resolution shells. The spatial group is P21 with a cell diameter of a = 4.25 nm (42.5 A), b = 5.95 nm (59.5 A), c = 8.17 nm (81.7 A) and β = 91.5 °. Cell diameter and symmetry means that there are two copies in the cell. The asymmetric unit, au or the minimum volume required to study has only one molecule, and the other molecules in the cell are generated by 21 symmetrical operations. The position of the molecule in au is in the y direction. As long as it is in the correct position on the x-z plane, it can be moved in the y direction. This is referred to as a free parameter in the 'polar' space group.

The PBD database (DB) has only one entry, 1BD2, containing TCRs in the form of A / B heterodimers. This entry also has a co-ordinate of the HLA-cognate peptide that is complex with the TCR. TCR chain B is identical in NY-ESO, while chain A has a small difference in the C domain and a significant difference in the N domain. Molecular replacement, using the 1BD2 A / B model for MR, gives an inaccurate solution, as can be seen for a large overlap with symmetrically equivalent materials. Using the B chain alone provides a better solution that does not have large overlap with the adjacent ones. The correlation coefficient was 49%, the crystallographic R-factor was 50% and the closest approach (from the center of gravity to c-o-g) was 0.49 nm (49 μs). The rotation and movement operations necessary to transform the starting chain B model into the corresponding MR were also applied to chain A. So when a hybrid MR solution was created, the cells were well filled with minimal clash.

Electron density maps generally matched the model well, and the regulation was made to match the sequence of the NY-ESO TCR. However, the initial model has many differences, especially in the absence of side chains, which characterize the incompletely aligned parts of the model. Many of the hair-pin loops between strands with very low density were difficult to apply to the model. The crystallographic R factor of the model is 30%. The R factor has a residual, that is, the difference between the calculated amplitude and the observed amplitude.

As demonstrated in FIGS. 83A and 83B, the sequences injected in 1BD2 do not agree well with the density. A further improvement was to replace the model with Cys at No. 164 of Chain A and No. 174 of Chain B, clearly showing that this sequence alignment is more suitable for density. Since the difference is small in terms of the size of the side branches, the variation of the model will be small. The electrical density in that area changed little.

The most important aspect in this study is that it is very similar in structure to the model (1BD2) published by the new TCR. The comparison should include all of the TCRs, a small part near the constant domain or point of mutation.

The r.m.s deviation values are listed in the table below. A comparison of the structures can be seen in FIG. 84.

Chain AComplete Chain BComplete Chain AConstant Chain BConstant Shortstretch r.m.s DisplacementMean DisplacementMax Displacement 2.8312.1789.885 1.2851.0016.209 1.6581.2356.830 1.0980.8334.490 0.6130.5461.330

(All units are Å)

Short stretch refers to the outer strand of chain A (A157 to A169) and the outer strand of chain B (B170 to B183) currently connected by a disulfide bridge. Deviations were calculated only for atoms of the main chain.

These results show that the introduction of disulfide bonds has minimal effect on the partial structure of the TCR around the bonds. Some significant effects were observed when comparing the TCR with the 1BD2 structure of the published A6 TCR, but the increase in RMS displacement is mainly due to the loop structure difference (FIG. 84). This loop does not form part of the core structure of the TCR formed by the continuation of the β sheet forming the characteristic Ig fold. The RMS deviation of the entire α chain is particularly large because of the sequence difference in the variable domains between A6 (1BD2) and NY-ESO TCR. However, A6 and NY-ESO TCRs have the same variable β domain, and the RMS deviation over the entire β chain shows that the structure of this variable domain is also maintained in the TCR with the new disulfide bond. Therefore, these data demonstrate that the core structure of the TCR is also maintained in the crystal structure of the TCR with the new disulfide bond.

Example 10 Generation of Soluble NY-ESO TCR Containing Novel Disulfide Interchain Binding, C-terminal β Chain Tagging Sites

To generate soluble NY-ESO TDR with new disulfide, an A6 TCR plasmid containing BamHI restriction sites in the α chain and BglII restriction sites in the β chain was used as the backbone as described in FIG. 4.

NY-ESO TCR β-chain constructs were obtained by PCR cloning as follows. PCR reactions were performed using a template comprising the primers and NY-ESO PCR chains as shown below.

The PCR product was restriction digested with the relevant restriction enzyme and cloned into pGMT7 containing the biotin recognition sequence to obtain the expression plasmid. The sequence of the plasmid insert was confirmed by automated DNA sequencing. FIG. 85A shows the DNA sequences of NY-ESO TCR β chains each with a biotin recognition site introduced and FIG. 85B shows the resulting amino acid sequence.

The α chain structure was generated as described in Example 5. Once each TCR chain was expressed, it was co-refolded and purified as described in Example 5.

The same primers and NY-ESO templates were used to generate soluble NY-ESO TCRs containing non-natural disulfide interchain linkages and hexa-histidine tags at the C terminus of the β chain. PCR products were restriction digested with relevant restriction enzymes and cloned into pGMT7 containing hexa-histidine sequence to obtain expression plasmids. 86A shows the DNA sequences of NY-ESO TCR β chains respectively incorporating a hexa-histidine tag and FIG. 86B shows the resulting amino acid sequences.

FIG. 87 shows elution of soluble NY-ESO with new disulfide bonds and biotin recognition sequences in a POROS 50HQ column using a 0 to 500 mM NaCl concentration gradient, as indicated by dotted lines. FIG. 88 shows elution of soluble NY-ESO with new disulfide bonds and hexa histidine tags in a POROS 50HQ column using a 0 to 500 mM NaCl concentration gradient, as indicated by the dotted lines.

89 and 90 are new elution profiles of gel filtration chromatography of fractions pooled in NY-ESO-biotin and NY-ESO-hexa-histidine tagged anion exchange columns performed as shown in FIGS. 88 and 88, respectively. Protein elution was eluted with a single main peak corresponding to the TCR heterodimer.

BIAcore analysis of sTCR binding to pMHC was performed as described in Example 3. The NY-ESO-biotin TCR has a Kd of 7.5 uM and the NY-ESO-hexa-histidine-tagged TCR has a Kd of 9.6 uM.

Example 11 Cell Staining with Fluorescently Labeled Tetramers of Soluble NY-ESR TCRs with New Disulfide Interchain Bonds

TCR tetramer preparation

As prepared in Example 10, NY-ESR soluble TCRs with new disulfide bonds and biotin recognition sequences were used to form soluble TCR tetramers required for cell staining. 2.5 ml of the purified soluble TCR solution (about 0.2 mg / ml) was exchanged into the biotinylation reaction buffer (50 mM Tris pH 8.0, 10 mM MgCl ₂ ) using PD-10 column (Pharmacia). The eluate (3.5 mL) was concentrated to 1 mL using a Centricon concentrator (Amicon) while blocking the 10 kDa molecular weight. The stock was made 10 mM ATP with 0.1 g / ml pH 7.0. Add protease inhibitor cocktail Set1 (Calbiochem Biochemicals) in an amount sufficient to provide the final protease cocktail with 1/100 of the stock solution provided and add 1 mM biotin (from 0.2M stock) and 20 μg / ml enzyme (0.5). Mg / ml from stock). And incubated overnight at room temperature. Excess biotin in the solution was removed using size exclusion chromatography on an S75 HR column. The extent to which the NY-ESO TCR was biotinylated was determined by the size exclusion HPLC-based method mentioned below. 50 μl aliquots of biotinylated NY-ESO (2 mg / ml) were incubated with 50 μl of streptavidin coated agarose beads (Sigma) for 1 hour. Spin down the beads and flow 50 μL of unbound sample onto a TSK 2000 SW column (Tosoohaas) for 30 minutes at a flow rate of 0.5 ml / min (200 mM Phosphate buffer pH 7.0). The presence of biotinylated NY-ESO TCR was measured by UV spectrometer at 214 nm and 280 nm. Biotinylated NY-ESO collided with non-biotinylated NY-ESO TCR controls. The biotinylated ratio was calculated by subtracting the biotinylated peak range from the peak range of the non-biotinylated protein.

Tetrification of biotinylated soluble TCRs was obtained using the Neutravidin-Phycoerythrin conjugate (Cambridge Biosciences, UK). The concentration of biotinylated soluble TCR was determined by Coomassie Protein Assay (Pierce) and the soluble TCR 0.8 mg / ml neutravidin-phycoerythrin conjugate ratio was 1: 4 for neutravidin-PE. Calculated to saturate with biotinylated TCR. 19.5 μl of 6.15 mg / ml biotinylated NY-ESO soluble TCR solution of PBS was added slowly with gentle stirring over 1 mg / ml neutravidin. 100.5 μl of PBS was then added to this solution to give a final NY-ESO TCR tetramer concentration of 1 mg / ml.

Staining protocol

Four aliquots of HLA-A2 positive EBV transformed B cell line (PP LCL) 0.3 × 10 ⁶ in 0.5 ml of PBS were prepared at various concentrations of HLA-A2 NYESO peptide (SLLMWITQC) 0, 10 ^-4 , 10 ^-5 And 10 ⁻⁶ (M) at 37 ° C. for 2 hours. PP LCL cells were washed twice with HBSS (Hanks buffered Saline solution: Gibco, UK).

Each of these four aliquots was divided equally and stained with freshly formed biotinylated NY-ESO disulfide linked TCR with neutravidin-phycoerythrin. Cells were incubated in 5 μg or 10 μg of dsTCR complex of phycoerthrin labeled tetramer on ice for 30 minutes and washed with HBSS. The cells were washed again, resuspended in HBSS and analyzed by FACSVantage. 25,000 events were collected and the data analyzed using WinMIDI software.

result

91A to 91H show bar graphs of FACSVantage data generated in each of the prepared samples as described above. The table below lists the percentage of positively stained cells observed in each sample.

sample % Positively stained cells 0 NY-ESO Peptide, 5 μg TCR10^-4M NY-ESO Peptide, 5 μg TCR10^-5M NY-ESO Peptide, 5 μg TCR10^-6M NY-ESO Peptide, 5 μg TCR0 NY-ESO peptide, 10 μg TCR10^-4M NY-ESO peptide, 10 μg TCR10^-5M NY-ESO peptide, 10 μg TCR10^-6M NY-ESO Peptide, 10 μg TCR 0.75 84.39 35.29 7.98 0.94 88.51 8.25 3.45

These data clearly indicate that the percentage of cells labeled by the NY-ESO TCR tetramer increases in a manner correlated with the concentration of the cultured peptide (SLLMWITQC). Therefore, these NY-ESO TCR tetramers are partially suitable for specific cell labels based on the expression of the HLA-A2 NY-ESO complex.

In this example, fluorescent conjugated NY-ESO TCR tetramers were used. However, similar levels of cell binding are expected even if these labels are replaced with appropriate therapeutic moieties.

Example 12 Construction of Soluble A6 TCR with a New Disulfide Bond Inserting a Cβ1 Constant Region

All previous examples described the generation of soluble TCRs with new disulfide bonds inserting Cβ2 constant regions. This example demonstrates that soluble TCRs inserting Cβ1 constant regions can be successfully generated.

Preparation of primers for PCR stitching the A6 TCR β chain V domain with Cβ1

For the PCR construct of the A6 TCR β chain V domain, the following primers were prepared.

5'-GGAGATATACATATGAACGCTGGTGTCACT-3 '

5'-CCTTGTTCAGGTCCTCTGTGACCGTGAG-3 '

For the PCR construct of Cβ1, the following primers were prepared.

5'-CTCACGGTCACAGAGGACCTGAACAAGG-3 ’

5'-CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3 '

Beta VTCR constructs and Cβ1 constructs were each amplified using standard PCR techniques. Each was linked using stitching PCR. Plasmid DNA was purified on a Qiagen mini-prep column according to the manufacturer's instructions and the sequence was then verified by automated sequencing using a sequencing machine from Oxford University Biochemistry. The sequence of A6 + Cβ1 can be seen in FIG. 92.

In conclusion, after introducing cysteine into the C domain of both chains, the A6 + Cβ1 chain was paired with A6 alpha TCR by interchain disulfide bonds.

Soluble TCR was expressed and refolded as described in Example 2.

Purification of Refolded Soluble TCR:

sTCR was loaded from dialysis products and impurities by loading the dialyzed refold into a POROS 50HQ anion exchange column and eluting bound proteins with a gradient of 0 to 500 mM NaCl over 50 times the column volume using Akta Pharmacia as shown in FIG. Separated. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE (FIG. 94) before pooling and concentration. Finally, sTCR was isolated and characterized using a Superdex 200HR gel filtration column (FIG. 95) previously equilibrated with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). It was. Peaks released at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

BIAcore analysis of A6 TCR binding disulfide linked to pMHC was performed as described in Example 3. FIG. 96 shows BIAcore analysis of specific binding of disulfide linked A6 soluble TCRs to their cognate pMHC.

Soluble A6 TCR, which inserts a Cβ1 constant region and has a new disulfide bond, has a Kd of 2.42 ± 0.55 uM in its cognate PMHC. This value is very similar to Kd 1.8 uM which was determined in soluble A6 TCR with a new disulfide bond inserting the Cβ2 constant region as determined in Example 3.

Example 13 Construction of Soluble A6 TCR with New Disulfide Bond Inserting 'Free' Cysteine into β Chain

The β chain constant region of TCR includes cysteine residues (residue 75 of exon 1 of TRBC1 * 01 and TRBC2 * 01) that are not involved in interchain or intrachain disulfide bond formation. All of the previous examples describe the generation of soluble TCRs with new disulfide bonds between free cysteines that have been mutated with alanine to avoid inadequate disulfide bond formation that may lead to reduced yield of functional TCRs. This example demonstrates that soluble TCRs can be produced that insert 'free' cysteines.

Primer Construction and Mutation of TCR β Chain

In order to mutant TCR β-chain alanine (residue 75 of exon 1 of TRBC1 * 01 TRBC2 * 01) with cysteine, the following primers were prepared (mutations appear in lowercase).

5'-T GAC TCC AGA TAC tgT CTG AGC AGC CG

5'-CG GCT GCT CAG Aca GTA TCT GGA GTC A

PCR mutation, expression and refolding of soluble TCR were performed as described in Example 2.

Purification of Refolded Soluble TCR:

sTCR was loaded from dialysis products and impurities by loading the dialyzed refolds into a POROS 50HQ anion exchange column and eluting bound proteins with a gradient from 0 to 500 mM NaCl over 50 times the column volume using Akta Pharmacia as shown in FIG. 98. Separated. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE (FIG. 99) before pooling and concentration. Finally, sTCR was isolated and characterized using a Superdex 200HR gel filtration column (FIG. 100) pre-equilibrated with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). It was. Peaks released at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

BIAcore analysis of the binding of disulfide linked A6 TCR to pMHC was performed as described in Example 3. FIG. 101 shows BIAcore analysis of specific binding of disulfide linked A6 soluble TCRs to cognate pMHC.

Soluble A6 TCR with a new disulfide bond inserting a 'free' cysteine in the β chain has a Kd value of 21.39 ± 3.55 uM in its cognate pMHC.

Example 14 Generation of Soluble A6 TCRs with New Disulfide Bonds in Which Free Cysteine of β Chain Mutated to Serine

This example demonstrated that soluble TCRs with new disulfide bonds can be successfully generated from 'free' cysteines (residue 75 of exon 1 of TRBC1 * 01 and TRBC2 * 01) mutated to a serine of β chain.

Preparation of primers for mutation of TCR β chain

The following primers were used to mutate TCR β chain alanine that was previously substituted in native cysteine (residue 75 of exon 1 of TRBC1 * 01 and TRBC2 * 01) with serine (mutations appear lowercase).

5'-T GAC TCC AGA TAC tCT CTG AGC AGC CG

5'-CG GCT GCT CAG AGa GTA TCT GGA GTC A

PCR mutations (mutated β chains were generated as shown in FIG. 102), expression and refolding of soluble TCR were performed as described in Example 2.

Purification and Refolding of Soluble TCRs

sTCR was digested by loading the refolded dialysis into POROS 50HQ anion exchange column as shown in FIG. 103 and eluting the bound protein with a gradient of 0 to 500 mM NaCl over 50 times the column volume using Akta Purifier (Pharmacia). And from impurities. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE (FIG. 104) before pooling and concentration. Finally, sTCR was isolated and characterized using HBS-EP buffer (Superdex 200HR gel filtration05 pre-equilibrated with (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40)). . Peaks released at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

BIAcore analysis of the binding of disulfide linked A6 TCR to pMHC was performed as described in Example 3. 106 shows BIAcore analysis of specific binding of disulfide linked A6 soluble TCRs to their conjugated pMHCs.

A soluble A6 TCR with a new disulfide bond in which the 'free' cysteine of the β chain is mutated to serine has a Kd value of 2.98 ± 0.27 uM in its conjugate pMHC. This value is very similar to Kd 1.8 uM, which was determined in soluble A6 TCR with the new disulfide bond mutated with alanine to the 'free' cysteine of the β chain as determined in Example 3.

Example 15 Cloning of NY-ESO TCR α and β Chains Incorporating New Disulfide Bonds in Yeast Expression Vectors

NY-ESO TCR α and β chains were fused with the C terminus of the pre-procrossing factor alpha of Saccharomyces cerevisiae sequence and cloned into yeast expression vectors pYX122 and pYX112, respectively (FIGS. 107 and 108). ).

S. to fuse with TCR α chain. The following primers were prepared in order to PCR amplify the pre-pro hybridization factor alpha sequence of the cerevisiae strain SEY6210 (Robinson et al. (1991), Mol Cell Biol. 11 (12): 5813-24).

5'-TCT GAA TTC ATG AGA TTT CCT TCA ATT TTT AC-3 '

5'-TCA CCT CCT GGG CTT CAG CCT CTC TTT TAT C -3 '

S. to fuse with TCR β chain. The following primers were prepared in order to PCR amplify the pre-pro hybridization factor alpha sequence of the serebizae strain SEY6210.

5'-TCT GAA TTC ATG AGA TTT CCT TCA ATT TTT AC-3 '

5'-GTG TCT CGA GTT AGT CTG CTC TAC CCC AGG C-3 '

s. A clone of the serebizae strain SEY6210 was resuspended in 30 μl of distilled water added with 0.25% SDS and heated at 90 ° C. for 3 minutes to prepare yeast DNA. A pre-pro hybridization factor alpha sequence for fusion with TCR α and β chains was provided by PCR amplification of 0.25 μL of yeast gene with each primer pair mentioned above using the following PCR conditions. 12.5 pmole of each primer was mixed with 200 μM dNTP, 5 μl of 10 × Pfu buffer and 1.25 units of Pfu polymerase (Stratagene) to a final volume of 50 μl. After an initial denaturation step of 92 seconds at 92 ° C in a Hybaid PCR express PCR machine, the reaction mixture is denatured (92 ° C, 30 seconds), bound (46.9 ° C, 60 seconds), extended (72 ° C, 2 minutes) 30 times.

The following primers were prepared for PCR amplification of the TCR α chain for fusion with the pre-pro hybridization factor alpha sequence mentioned above.

5'-GGC TGA AGC CCA GGA GGT GAC ACA GAT TCC-3 '

5'-CTC CTC TCG AGT TAG GAA CTT TCT GGG CTG GG-3 ’

The following primers were prepared for PCR amplification of the TCR β chain for fusion with the pre-pro hybridization factor alpha sequence mentioned above.

5'-GGC TGA AGC CGG CGT CAC TCA GAC CCC AAA AT-3 '

5'-GTG TCT CGA GTT AGT CTG CTC TAC CCC AGG C-3

PCR conditions for amplifying the TCR α and β chains were the same as mentioned above except for the following changes: The DNA templates used to amplify the TCR α and β chains were NY-ESO TCRα and β chains ( Prepared in Example 5). The annealing temperature was 60.1 ° C.

PCR products were used for PCR stitching reactions using complementary overlapping sequences inserted into the initial PCR product to make full length chimeric genes. The resulting PCR product was digested with restriction enzymes EcoRI and XhoI and cloned into pYX122 or pYX112 digested with the same enzyme. The resulting plasmid was purified on a Qiagen ^™ mini-prep column according to the manufacturer's instructions and the sequence was then verified by automated sequencing using a sequencing machine from Genetics Ltd (Queensway, New Milton, Hampshire, United Kingdom). 109 and 110 show the DNA and protein sequences of the cloned chimeric product.

Example 16 Yeast Expression of Soluble NY-ESO TCRs with New Disulfide Bonds

Yeast expression plasmids containing the TCR α and β chains, respectively produced as described in Example 15, were prepared using Agatep et al. (Technical Tips Online (http://tto.trends.com) 1: 51: P01525). Using s. Cotransfection was performed with the cerevisiae strain SEY6210. Single clones grown in SD (synthetic dropout) agar containing histidine and uracil (Qbiogene, Illkirch, France) were incubated overnight at 30 ° C. in 10 ml of SD medium containing histidine and uracil. The overnight culture was subcultured 1: 10 with 10 ml fresh SD medium containing histidine and uracil and grown at 30 ° C. for 4 hours. The culture was centrifuged at 3800 rpm for 5 minutes in Heraeus Megafuge 2.0R (Kendro Laboratory Products Ltd, Bishop's Stortford, Hertfordshire, UK) to obtain supernatant. 5 μl of StratClean resin (Stratagene) was mixed with the supernatant and spun overnight on a blood wheel at 4 ° C. The StratClean resin was spun down at 3800 rpm on Heraeus Megafuge 2.0R to discard the medium. 25 μl of Reducing Sample Buffer (950 μl Laemmli Sample Buffer (Biorad) containing 50 μl of 2M DTT) was added to the resin, heated at 95 ° C. for 5 minutes, and the mixture was mixed with a 0.8 mA constant / cm ² gel surface. Μl was cooled on ice for 1 hour before loading on the SDS-PAGE gel. The protein of the gel was transferred to an Immuno-Blot PVDF membrane (Bio-Rad) and probed with a TCR anti α chain antibody as described in Example 17 below except for the following differences. The primary antibody (TCR anti α chain) and the secondary antibody were used in a dilution of 200 vs. 1 to 1000, respectively. 111 is a developed film photograph. These results show that culturing yeast secretes TCR at low levels in the medium.

Example 17 Expression of Disulfide A6 Tax TCR α and β Chains in Baculus

Cloning method

The α and β chains of the disulfide A6 Tax TCR were cloned into pBlueScript KS2 based vectors called pEX172 in pGMT7. Such a vector is a DRβ1 * 0101-derived leader sequence for cloning other MHC Class IIβ chains for insect cell expression, an AgeI site for insertion of other peptide coding sequences, a linker region, a DRβ in front of the Jun Leucine zipper sequence. The MluI and SalI sites were used to clone the chains. A sequence where pEX172 differs from pBlueScriptII KS-, located between KpnI and EcoRI of pBlueScriptII KS-, is shown in FIG. 112. To clone the TCR chain in insect cells, pEX172 was digested with AgeI and SalI to remove the linker region and the MluI site and allow the TCR chain to proceed where the peptide sequence begins. The TCR sequence was cloned in pGMT7 with BspEI at the 5 ′ end (having a suitable adhesion terminus with AgeI) and SalI sites at the 3 ′ end. The first three residues (GDT) of the DRβ chain were conserved to provide a cleavage site for removing the DRβ leader sequence. To prevent transcription of the Jun Leucine zipper sequence, a stop codon insertion is required before the SalI site. A schematic of this structure can be seen in FIG. 113. When the TCR chain was present in this plasmid, the BamHI fragment was cleaved and subcloned into a pAcAB3 vector having a homologous recombination site with baculovirus. The pAcAB3 vector has two promoters, one with a BamHI site and the other with a BglII cloning site. Since the A6 TCR β chain has a BglII site, the A6 TCR α chain was inserted into the BglII site and the β chain was subcloned into the BamHI site.

According to the above cloning method, the following primers were produced (the homology to the vector is shown in capital letters).

A6α: F: 5'-gtagtccggagacaccggaCAGAAGGAAGTGGAGCAGAAC

R: 5'-gtaggtcgacTAGGAACTTTCTGGGCTGGG

A6β: F: 5'-gtagtccggagacaccggaAACGCTGGTGTCACTCAGA

R: 5'-gtaggtcgacTAGTCTGCTCTACCCCAGG

PCR, cloning and subcloning

An expression plasmid containing genes for the disulfide A6 Tax TCR α chain or β chain was used as a template for the PCR reaction below. 100 ng of α plasmid is mixed with 1 μl of 10 mM dNTP, 5 μl of 10 × Pfu-Stratagene, 1.25 units of Pfu polymerase (Stratagene), 50 pmol of the A6α primer and 50 μl of final volume with H ₂ O. Similar reaction mixtures were applied to the β chain using β plasmid and β primer pairs. In the Hybaid PCR express PCR machine, the reaction mixture was denatured (95 ° C, 60 seconds), binding (50 ° C, 60 seconds), and extended (72 ° C, 8 minutes). The product was cleaved with 10 units of BspEI restriction enzyme at 37 ° C. for 2 hours and then further for 2 hours with 10 units of SalI (New England Biolabs). This cleaved reaction was linked to pEX172 digested with AgeI and SalI and transformed into competent XL1-Blue bacteria and grown at 37 ° C. for 18 hours. A single clone was picked from each α and β prep, and 5 ml of TYP and ampicillin (16 g / l Bacto-Tryptone, 16 g / l yeast extract, 5 g / l NaCl, 2.5 g / l K ₂ HPO ₄ , 100 mg / l ampicillin) overnight. The plasmids were purified on Qiagen mini-prep columns according to the manufacturer's instructions and the sequences were then verified by automated sequencing using Genetix's sequencing machine. The amino acid sequences of the α and β chains of the BamHI insert can be seen in FIGS. 114 and 115, respectively.

The α and β disulfide A6 Tax TCR chain constructs of pEX172 were cleaved for 2 hours at 37 ° C. with BamHI restriction enzyme (New England Biolabs). α-chain BamHI inserts were linked to pAcAB3 vector (Pharmingen-BD Biosciences: 21216P) digested with BglII enzyme. These were transformed with competent XL1-Blue bacteria and grown at 37 ° C. for 18 hours. Single clones were picked from the plates and grown overnight in 5 ml of TYP and ampicillin to purify the plasmid DNA as before. The plasmid was digested with BamHI, β-chain BamHI inserts were linked, transformed with competent XL1-Blue bacteria, grown overnight, grown on TYP-ampicillin medium, and miniprepping using a Qiagen mini-prep column. It was. The exact arrangement of the α chain and the β chain was confirmed by sequencing using the following sequencing primers.

pAcAB3 α anterior: 5'-gaaattatgcatttgaggatg

pAcAB3 β anterior: 5'-attaggcctctagagatccg

Expression and Analysis of A6 TCR in Transgenic, Infected, and Insect Cells

Using the Baculogold Transfection Kit (Pharmingen-BD Biosciences: 21100K) into sf9 cells grown on serum-free medium (serum free medium: Pharmingen-BD Biosciences: 551411) according to the manufacturer's instructions Heng conversion. After incubation at 27 ° C. for 5 days, 200 μl of the culture medium of these transformed cells was added to Hive Five cells 100 at 1 × 10 ⁶ cells / μl in serum free medium. After 6 more days of incubation at 27 ° C, 1 ml of medium was removed and centrifuged to pellet cell debris at 13,000 RPM for 5 minutes in a Hereus centrifuge.

10 μl of this insect A6 disulfide linked TCR supernatant was run on premade 4-20% Tris / Glycine gel (Invitrogen: EC60252) side by side with 5 μg and 10 μg positive control of bacterial A6 disulfide linked TCR. 10 μl of reduced sample buffer (950 μl of Laemmli sample buffer: Bio-Rad, 161-0737) 2 M DTT was added to prepare a reduced sample, which was heated at 95 ° C. for 5 minutes and then cooled at room temperature for 10 minutes. Μl was loaded. Non-sparked samples were prepared by adding 10 μl of Laemmli sample buffer and loaded with 20 μl.

The gel was operated at 150 volts for 1 hour in a Novex-Xcell gel tank, and the gel was stirred with Coomassie gel dye (500 ml of methanol, 1.1 g of Coomassie powder, stirred for 1 hour, 100 ml of acetic acid and 1 liter of H ₂ O). Made and stirred for 1 hour and filtered through a 0.45 mM filter) and dyed with 50 ml for 1 hour with gentle stirring. The gel was bleached three times with 30 ml of a bleaching (same as Coomassie gel dye but without Coomassie powder) solution.

Western blots were performed by transferring the protein to an Immuno-Blot PVDF membrane (Bio-Rad, 162-0174) rather than running the SDS-PAGE gel to stain the gel with Coomassie. Cut six filter papers into gel size and add Transperfer buffer (previous buffer: 2.39 g glycine, 5.81 g Tris Base, 0.77 g DTT dissolved in 500 ml H ₂ O, 200 ml methanol and 1000 ml H ₂ O). Dipped). PVDF membranes were prepared by soaking in methanol for 1 minute and soaking in transfer buffer for 2 minutes. Three sheets of filter paper are placed on the cathode side of the Immuno-blot apparatus (Pharmacia Novablot), the membrane is placed on top of the gel and finally three additional sheets of filter paper are placed on the anode side. Immuno-blot was operated at 0.8 mA constant per cm 2 of gel surface for 1 hour.

After blotting, the membranes were added to 7.5 ml of blocking buffer (Tris-buffered saline tablet (Sigma: T5030), 3 g skim milk powder (Sigma: M7409), 30 μl of Tween 20, made 30 mL with H ₂ O). Blocking for 60 minutes with gentle stirring. Membranes were washed three times for 5 minutes with TBS wash buffer (20 TBS tablets, 150 μl of Tween 20, made 300 mL with H ₂ O). Membranes were treated with 7.5 ml blocking buffer for 1 hour with gentle stirring with primary antibody diluted 50-fold of anti-TCR α-chain clone 3A8 (Serotec: MCA987) or TCR β-chain clone 8A3 (Serotec: MCA988). Membranes were washed with TBS wash buffer as before. Next, the secondary antibody treatment, which was diluted 1000-fold with HRP-labeled goat-anti mouse antibody (Santa Cruz Biotech: Sc-2005) with 7.5 ml blocking buffer, was performed for 30 minutes with gentle stirring. The membranes were washed in the same manner as the previous method and two TBS tablets were washed with 30 ml of dissolved H ₂ O.

Antibody binding was detected by Opti-4CN colorimetric detection (Biorad: 170-8235) (1.4 mL Opt-4CN dilution, 12.6 mL H ₂ O, 0.28 mL Opti-4CN substrate). The membrane was developed for 30 minutes and washed with H ₂ O for 15 minutes. The membrane was dried at room temperature and the scanned image was aligned with the image of the Coomassie stained gel (FIG. 116).

result

It can be seen in FIG. 116 that both disulfide TCRs are formed with stable heterodimers in SDS gels. When both were reduced, they were broken into α and β chains. Insect disulfide TCR heterodimers have a slightly higher molecular weight than the form produced in bacteria, probably due to polysaccharide in insect cells. In this example it was seen that the insect cells produced more than the α chain, and the free α chain was seen in the non-reduction line of the anti α Western blot.

These data clearly demonstrate that the Backlevirus expression system described above is a viable alternative to prokaryotic expression of soluble TCRs with new disulfide bonds.

Claims (32)

(i) all or part of the T cell receptor (TCR) α chain, except the transmembrane domain, and (ii) all or part of the TCR β chain, except the transmembrane domain, (i) and (ii) each comprise at least a portion of a functional variable domain of the TCR chain and a constant domain of the TCR chain, wherein (i) and (ii) are between the constant domain residues Soluble T cell receptor (sTCR), which is linked by disulfide bonds that do not exist in native TCRs. The sTCR of claim 1, wherein one or both of (i) and (ii) comprises the entirety of the extracellular constant Ig domain of the TCR chain. The sTCR of claim 1 or 2, wherein one or both of (i) and (ii) comprises the entire extracellular domain of the TCR chain. Dissolved covalent bonds soluble αβ-form T cell receptors (sTCRs) that link residues in the immunoglobulin region of the constant domain of the α chain to residues in the immunoglobulin region of the constant domain of the β chain. ). The sTCR of claim 1, wherein there is no interchain disulfide bond in the native TCR. 6. The sTCR of claim 5 wherein the native α and β TCR chains are cleaved at the C-terminus to remove cysteine residues that form native interchain disulfide. 6. The sTCR of claim 5, wherein the cysteine residue forming a native interchain disulfide bond is replaced with another residue. 8. The sTCR of claim 7, wherein the cysteine residue forming a native interchain disulfide bond is replaced with serine or alanine. 9. The sTCR of claim 1 wherein there is no unpaired cysteine residue present in the native TCR β chain. 10. 10. The sTCR of claim 1, wherein a disulfide bond not present in the native TCR is present between cysteine residues in which the β carbon atom replaces residues less than 0.6 nm in the native TCR structure. The cysteine of claim 1, wherein disulfide bonds not present in native TCR replace Thr 48 of exon 1 of TRAC * 01 and Ser 57 of exon 1 of TRBC1 * 01 or TRBC2 * 01. STCR present between residues. The cysteine of claim 1, wherein disulfide bonds not present in native TCR replace Thr 45 of exon 1 of TRAC * 01 and Ser 77 of exon 1 of TRBC1 * 01 or TRBC2 * 01. STCR present between residues. The cysteine of claim 1, wherein the disulfide bonds not present in native TCR replace Tyr 10 of exon 1 of TRAC * 01 and Ser 17 of exon 1 of TRBC1 * 01 or TRBC2 * 01. STCR present between residues. The cysteine of claim 1, wherein the disulfide bond that is not present in native TCR replaces Thr 45 of exon 1 of TRAC * 01 and Asp 59 of exon 1 of TRBC1 * 01 or TRBC2 * 01. STCR present between residues. The cysteine of claim 1, wherein the disulfide bonds not present in native TCR replace the Ser 15 of exon 1 of TRAC * 01 and the Glu 15 of exon 1 of TRBC1 * 01 or TRBC2 * 01. STCR present between residues. 16. The functional variable of any one of claims 1, 2 and 5-15, wherein (i) and (ii) are each fused to all or part of the constant domain of the second TCR. STCR comprising a domain and wherein the first and second TCRs are from the same species. The sTCR of claim 16, wherein the constant domain of the second TCR is cleaved at the N-terminus of the residue that forms a non-natural interchain disulfide bond. 18. The sTCR of any one of claims 1 to 17, wherein one or both of the α and β chains are derivatized or fused to the moiety at the C or N terminus. The sTCR of claim 1, wherein one or both of the α and β chains have cysteine residues at the C and / or N terminus to which the moiety can be fused. 20. The sTCR of any one of the preceding claims further comprising a detectable label. 21. The sTCR of any one of claims 1 to 20 combined with a therapeutic agent. 22. A multivalent T cell receptor complex comprising a plurality of sTCRs according to any one of the preceding claims. The complex of claim 22 comprising an sTCR multimer. The complex of claim 23, comprising at least two T cell receptor molecules, preferably linked with another T cell receptor molecule via a linker molecule. The complex according to any one of claims 22 to 24, wherein the sTCR or sTCR multimer is present in or attached to the lipid bilayer. (i) providing a soluble TCR according to any one of claims 1 to 21 or a multivalent T cell receptor complex according to any one of claims 22 to 25; (ii) contacting the soluble TCR or multivalent TCR complex with the MHC-peptide complex; And (iii) detecting the binding of the soluble TCR or multivalent TCR complex to the MHC-peptide complex. A pharmaceutical formulation comprising an sTCR according to any one of claims 1 to 21 and / or a multivalent TCR complex according to any one of claims 22 to 25 together with a pharmaceutically acceptable carrier. A nucleic acid molecule comprising a sequence encoding (i) or (ii) of sTCR according to claim 1 or a sequence complementary thereto. A vector comprising a nucleic acid molecule according to claim 28. A host cell comprising the vector according to claim 29. The production of (i) or (ii) according to any one of claims 1 to 21, comprising incubating the host cell according to claim 30 under conditions expressing the peptide and purifying the polypeptide. How to. 32. The method of claim 31, further comprising mixing (i) and (ii) under suitable refolding conditions.