KR100945977B1 - Soluble t cell receptor - 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.

Trax membrane, soluble T cell receptor (TCR), cysteine, disulfide bond

Description

SOLUBLE T CELL RECEPTOR

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 a polymorphic loop similar to the complementarity determining region (CDR) 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 T cell receptor (sTCR) is useful not only for studying specific TCR-pMHC interactions but also as a diagnostic tool for detecting infection 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), yields are not good, are not stable at low concentrations and / or can recognize an MHC-peptide complex. none. This document has been 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 did not recognize and / or stabilize 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. This TCR is comprised of a pair of C-terminal dimerisation peptides, such as leucine zippers, which comprise a TCR α or γ chain extracellular domain that forms a dimer with the TCR β or δ chain extracellular domain, respectively. Doing. This method for producing a TCR is generally applicable to all TCRs.

Reiter et al. (Immunity, 1995.2: 281-287) describe in detail the structure of soluble molecules comprising disulfide-stabilized TCR α and β variable domains, one of the α and β variable domains being Pseudomonas exotoxin (PE38). ) It is connected to the cut form. Overcoming the inherent instability of short-chain TCRs is one of the reasons for producing 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 new 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 invention includes (i) all or part of the T cell receptor α chain, excluding the transmembrane domain, and (ii) all or part of the TCR β chain, excluding the transmembrane domain, and (i And (ii) each comprise a functional variable domain of the TCR chain and at least a constant domain portion of the TCR chain, wherein (i) and (ii) are naturally present between the constant domain residues. It provides a soluble T cell receptor, which is linked by disulfide bonds that do not exist in TCR.

In another aspect, the 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. Provide a receptor.

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 are unlikely to be immunogenic due to this structural similarity. The sTCRs according to the present invention may 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) TCR is defined as the ability of TCR to be purified to a single dispersed heterodimer at a concentration of 1 mg / ml 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 and then analyzed 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 as 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 native TCR consists of two polypeptides (αβ or γδ), each with a membrane-proximal constant domain and a membrane-distal variable domain (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 created through somatic rearrangement of linked V (varibale), D (diversity), J (joining), and C (contant) genes.The functional α chain polypeptide is a rearranged VJC region. The β chain consists of the VDJC region, but the extracellular constant domain has a membrane-adjacent region and an immunoglobulin region The membrane-adjacent region consists of amino acids between the transmembrane domain and the membrane-adjacent cysteine residue. The globulin region consists of the remainder of the constant domain amino acid residues, extending from the membrane-adjacent cysteine to the beginning of the J region, characterized by the presence of an immunoglobulin-type fold, known as Cα1 or TRAC * 01. There is a single α chain constant domain and two other β constant domains known as Cβ1 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 37F 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. Each chain of 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 sTCRs of the invention also comprises an intra-chain disulfide bond. The TCRs of the invention may comprise the entirety of the extracellular constant Ig regions of each TCR chain, or may preferably comprise 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, such disulfide bonds are not present. This can be accomplished by mutating a suitable cysteine residue (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 another amino acid or by cleaving each chain so that no cysteine residues are included. 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 present invention, and 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. Since the constant domain is not directly involved in the contact of the peptide-MHC ligand, the C-terminal cleavage point can be altered without substantial loss of function.

Alternatively, larger domain constant domain fragments may be used than would be desirable in 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 cysteine residues that form intrachain TCR and intrachain disulfide bonds 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 situations 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 combined. 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 engineered at the N terminal starting point of the expected mature protein sequence to enable initiation of translation.

Not all residues of the variable domain of the TCR 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 native TCRs. It is more desirable to remove this cysteine residue or to mutate with another residue in order to avoid incorrect 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. Disulfide bonds may also be between residues of the membrane-adjacent region, but it is preferred that disulfide bonds be between constant immunoglobulin region residues. Preferred sites are the following residues in exon 1 of TRAC * 01 representing the TCR α chain and exon 1 of TRBC1 * 01 or TRBC2 * 01 representing the TCR β chain.

One sTCR of the present invention was derived from A6 Tax TCR (Garboczi et al., Nature, 1996, 384 (6605): 134-141). In one embodiment, sTCR is the entire TCR α chain (exchange 2 of TRAC * 01, the N-terminus of residue 4, according to the numbering method used by Garboczi et al., Amino acid residues 1 to 182 of α chain) and TRBC1 * 01 and TRCB2 The entirety of the TCR β chain (the numbering method used by Garboczi et al., According to the numbering method used by Garboczi et al.) Of exon 2 of * 01 and residue 2 of * 2 is included. Threonine 48 of exon 1 in TRAC * 01 to form disulfide bonds (Threonine 158 in α chain according to the numbering method used by Garboczi et al.) And serine 57 of exon 1 in both TRBC1 * 01 and TRBC2 * 01 (Garboczi et al. According to this numbering method, the serine 172 of the β chain can be mutated to cysteine, respectively. 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 currently 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 mutated with cysteines).

In other species, the TCR chain may not have a region having 100% identity with the above motif. However, one 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 to a specific TCR chain sequence to locate the appropriate TCR sequence portion for mutating the European Bioinformatics Institute (http://www.ebi.ac.uk/index. You can use the ClustalW available in html).

The present 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, the amino acid sequences of the mouse Cα and Cβ soluble domains are shown below with motifs showing murine residues corresponding to the above-mentioned human residues that can be mutated to cysteine to form a TCR interchain disulfide bond. (Related residues are indicated as negative salts).

In a preferred embodiment of the invention, each of (i) and (ii) of the TCR comprises a functional variable domain of the first TCR fused to all or part of the constant domain of the second TCR, wherein the first TCR and the second TCR Silver is derived from the same species and interchain disulfide bonds 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 from humans. 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 substantially the same as 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 the constant domain of the αβ TCR of a mutant that actually has the novel interchain disulfide bonds described above, can also 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 the 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 towards 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 substantially pure form or in purified or isolated formulations. For example, it may be provided in a form that does not substantially contain other proteins.

The plurality of soluble TCRs 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 comprising a plurality of soluble T cell receptors described herein. Each of the plurality of soluble TCRs is preferably the same.

In another aspect, the present invention

(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, neutravidin, and extravidin. 4 biotinylated TCR molecules can thus be formed as a multimer of T cell receptors having multiple 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 other biotinylated molecules, 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 has a diameter in the range of 10 nm to 10 μm. Each structure is sufficiently separated 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 increase the affinity of the multimeric binding moiety for the cell. It can represent a number of TCR molecules.

Suitable structures for use in the present invention include membrane structures such as liposomes, and solid structures, preferably particles such as beads, for example 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 may 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. An example of such a multimeric molecule is a tetramer 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 can 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. Although specific amino acids of the biotin tag sequence are essential, other biotinylation tags may be used (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 chelated nickel ions: Quiagen Product Guide 1999, Chapter 3, "Protein Expression, Purification, Detection and Assay", p. 35). -37). Preferably, the tag is located 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 includes contacting an MHC-peptide complex with a TCR or multimeric TCR complex specific for an MHC-peptide complex according to the present invention and detecting that the TCR or multimeric TCR complex is 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 Can be connected in other ways). The multivalent TCR complexes of the present invention may have increased binding capacity to pMHC as compared to non-multimeric 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 is also useful as an intermediate for generating additional multivalent TCR complexes having such use. TCRs or multivalent TCR complexes may be provided in pharmaceutically acceptable formulations for use in vivo.

The present invention also provides a method of delivering a therapeutic agent to a target cell, wherein the method provides for the attachment of a potential target cell to a TCR or multivalent TCR complex according to the invention under conditions that permit attachment of a TCR or multivalent TCR complex to the target cell. Including contacting, 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 delivered to exert a therapeutic effect locally 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;

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

Prodrugs such as antibody directed enzyme pro-drugs; And
Immune stimulant, ie a moiety that stimulates an immune response. For example, antibodies or antibody fragments, complements such as cytokines such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein Active agents, xenogeneic protein domains, allogeneic proteins, viral / bacterial protein domains and viral / bacterial peptides;

Soluble TCRs or multivalent TCR complexes of the invention may be linked with enzymes capable of converting prodrugs into drugs. This allows the pro-drug to be converted to the drug only where the drug is needed (ie targeted by 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. Additional disease-associated pMHC targets suitable 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 enhanced 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 effect 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 the specific peptide epitope provided by the appropriate pMHC.

In accordance with the present invention, the drug may usually be provided as part of a sterile pharmaceutical composition which usually comprises a pharmaceutically acceptable carrier. The 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.

Pharmaceutical compositions may be administered by any suitable route, eg, oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual or transdermal), vaginal or parenteral (subcutaneous, intramuscular, Intravenous or intradermal). Such compositions may be prepared by methods well known in the art of pharmacy, such as by mixing the carrier (s) or excipient (s) with the active ingredient under sterile conditions.

Pharmaceutical compositions suitable for oral administration include capsules or tablets; Powders or granules; Liquid formulations; It may be provided in individual units such as syrups or suspensions (aqueous or non-aqueous liquids; or edible foams or whippers; or emulsions). Suitable additives for use in tablets or hard gelatin capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Excipients suitable for use in soft gelatin capsules include, for example, vegetable oils, waxes, fats, semisolid polyols, liquid polyols and the like.

In the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols and sugars. Suspension oil (eg vegetable oil) formulations are used to provide oil-in-water or water-in-oil suspensions. 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 can be used with paraffinic bases or water-miscible ointment bases. 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 for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially a water-soluble solvent. Pharmaceutical compositions for topical administration to the oral cavity include lozenges, pastilles and mouthwashes.

Suitable pharmaceutical compositions for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions for nasal 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 the powder, In any container, it is administered by rapid inhalation through the nasal route. Suitable compositions include aqueous or oil solutions of the active ingredient when the carrier is a liquid for administration by nasal spray or nasal drop. Pharmaceutical compositions for administration by inhalant include fine particle dust or fog generated by various forms of means, such as metered dose pressurized aerosols, nebulizers or insulators. Suitable pharmaceutical compositions for vaginal administration may be pessary, tampon, cream, gel, paste, foam or spray formulations. Pharmaceutical compositions for parenteral administration may include aqueous and non-aqueous sterile injectable solutions and suspending agents and thickeners containing antioxidants, buffers, bacteriostats and solutes that substantially give the blood of the recipient and the composition of the isotonic solution. Aqueous and non-aqueous sterile suspensions. Excipients that can be used as injection solutions include, for example, water, alcohols, polyols, glycerin and vegetable oils. The composition may be provided in unit-dose or several-dose containers, eg, in closed ampoules and vials, and may be stored under refrigerated conditions, just prior to use, for example, in a sterile solution such as water for injection. Only addition is required.

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

The dosage of a substance of the present invention may vary within wide ranges depending on the disease or condition to be treated, the age and condition of the individual to be treated, and the physician 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 present invention, preferably sTCRs 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 a soluble TCR of the present invention, or a sequence complementary thereto. Such nucleic acid sequences can be obtained by separating TCR encoding nucleic acids from T-cell clones (by insertion, deletion or substitution) and inducing appropriate mutations.

Nucleic acid molecules may be in isolated or recombinant form. The vector can be inserted and the vector can be inserted into a host cell. Such vectors and suitable hosts are also an aspect 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 can be performed in vitro under suitable refolding conditions. In a specific embodiment, the TCR with the correct shape is obtained by refolding the dissolved TCR chain 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 preferred 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 can be used before the refolding step (eg urea, β-mercaptoethanol). Other redox pairs can be used during the 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 certain other protein components, such as chaperone protein, to the refolding mixture. Refolding has been improved by passing proteins through a column with immobilized mini-chaperone (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 be accomplished 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 this screening method include methods based on Surface Plasmon Resonance described in WO 01/22084. Other known techniques that underlie this screening method include SPA (Scintillation Proximity Analysis) and APA (Amplified Luminescent Proximity Assay).

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 a drug development program. Such agents can be used to treat conditions involving unnecessary T cell response elements. Such conditions include cancer (eg, kidney, ovary, intestine, head and neck, testes, lungs, stomach, cervix, bladder, prostate or melanoma), autoimmune diseases, graft rejection and graft versus host disease. It includes.

Preferred features of each aspect of the invention apply as appropriate to each of the 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 A6 TCR α chain extracellular amino acid sequence in which T ₄₈ is mutated to C (underlined) to create a new disulfide interchain bond. 3B shows the amino acid sequence of the β chain cells of A6 TCR where S ₅₇ is mutated to C (underlined) to create a new disulfide interchain bond.

FIG. 4 shows the protein eluate from the POROS 50HQ column using an anion exchange chromatography of soluble A6 TCR with 0 to 500 mM NaCl concentration gradient (indicated by dashed 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 mainly contains non-disulphide bonded β chains, peak 2 mainly contains interchain disulfide-linked TCR heterodimers and shoulders are rarely visible in these reproductions. This is due to the contamination of E. coli mixed with the connected 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 graph shows comparative binding reaction with control when disulfide-linked A6 soluble TCR was injected alone.

8A shows an A6 TCR α chain comprising new cysteine residues mutated to insert a BamHI restriction site. Shading indicates 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.

FIG. 10 shows the protein eluate eluted from the POROS 50HQ column using an anion exchange chromatography of soluble disulfide-linked JM22 TCR, using a 0-500 mM NaCl concentration gradient (indicated by dashed 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 surely contains interchain disulfide-linked TCR heterodimer.

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%.

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

14A and 14B show the DNA sequences of the α and β chains of NY-ESO 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 shows the protein eluate eluted from the POROS 50HQ column using an anion exchange chromatography of soluble NY-ESO disulfide-linked TCR, using a 0-500 mM NaCl concentration gradient (indicated by dashed lines).

FIG. 17A shows reduced SDA-PAGE (stained with Coomassie) of the fractions 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 the fractions pooled at peak 1 (A) and peak 2 (B) of FIG. 17. Protein eluted with a single main peak corresponding to heterodimer.

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

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

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

22 shows soluble NY-ESO TCRα ^cys β ^cys As a result of the anion exchange chromatography, the protein eluate eluted on the POROS 50HQ column using a gradient of 0 to 500 mM NaCl (indicated by the point islands) is shown.

FIG. 23 shows the protein eluate eluted from the POROS 50HQ column using an anion exchange chromatography of soluble NY-ESO TCRα ^cys using a 0-500 mM NaCl concentration gradient (indicated by dashed lines).

FIG. 24 shows the protein eluate eluted from the POROS 50HQ column using an anion exchange chromatography of soluble NY-ESO TCRβ ^cys with a gradient of 0 to 500 mM NaCl (indicated by dashed 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-cysβ soffee (EB / 084/033), and lanes 4 represents NYESOdsTCR1g4αβ-cys (EB / 084/034), lane 5 represents NYESOdsTCR1g4α-cys β-cys soffee (EB / 084/035), and 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 peak (EB / 084/033), lane 3 represents the NYESOdsTCR1g4α-cysβ soffee (EB / 084/033) 4 shows NYESOdsTCR1g4αβ-cys (EB / 084/034), lane 5 shows NYESOdsTCR1g4α-cys β-cys small peak (EB / 084/035), and lane 6 shows NYESOdsTCR1g4α-cysβ-cys peak (EB / 084 / 035).

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

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

FIG. 30 is a BIAcore response curve showing specific binding of HLA-NY-ESO complex and NY-ESO TCRα ^cys β ^cys .

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

FIG. 32 is a BIAcore response curve showing specific binding of HLA-NY-ESO complex and NY-ESO TCRβ ^cys .

33A and 33B show DNA sequences of soluble AH-1.23 TCR α and β chains mutated to introduce new cysteine codons (shown in shades), respectively. 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.

FIG. 35 shows the protein eluate eluted from the POROS 50HQ column using NaCl concentration gradient (indicated by the dot) of 0 to 500 mM as a result of anion exchange chromatography of soluble AH-1.23 TCR.

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. Lane 1 represents the molecular weight (MW) marker, lane 2 represents B4, lane 3 represents C2, lane 4 represents C3, lane 5 represents C4, lane 6 represents C5, lane 7 represents C6 Lane 8 represents C7, lane 9 represents C8 and lane 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. 35. The protein analyzed is the anion exchange fraction of TCR 1.23 S-S obtained from Rifold 3. Lane 1 represents the molecular weight (MW) marker, lane 2 represents B4, lane 3 represents C2, lane 4 represents C3, lane 5 represents C4, lane 6 represents C5, lane 7 represents C6 Lane 8 represents C7, lane 9 represents C8 and lane 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 sequence and amino acid sequence 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 shaded 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 the DNA sequence and amino acid sequence 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 sequence and amino acid sequence 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 sequence and amino acid sequence 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 at 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 new interchain disulfide bonds at residue 15 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01 resulted in an anion exchange chromatography with a NaCl concentration gradient of 0 mM to 500 mM. Show the protein eluted from the POROS 50 column.

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 and electrophoretic fractions (stained with Coomassie) were collected on the anion exchange column performed in FIG.

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 and electrophoretic fractions (stained with Coomassie) were collected on the 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. Fractions were collected on the anion exchange column performed in FIG. 59.

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. Fractions were collected on the 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. Fractions were collected on the anion exchange column performed in FIG. 61.

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. Fractions were collected on the 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 Protein eluate. Fractions were collected on the 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. Fractions were collected on the anion exchange column performed in FIG. 64.

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 soluble A6 TCR to HLA-A2-tax pMHC with new interchain disulfide bonds at residue 15 of exon 1 of TRAC * 01 and exon 1 of TRBC2 * 01. to be.

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 contoured at 1.0, 2.0 and 3.0σ. 83B is an electron density map after changing two positions of A164 and B174 to Cys and outlined at the same sigma level as 83a.

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

85A and 85B show the 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 the DNA and amino acid sequences of the NY-ESO TCR β chain into which a hexa-histidine (hexa-histidine) tag was inserted, respectively. Hexa-histidine (hexa-histidine) tags are shown prominently.

FIG. 87 shows elution of soluble NY-ESO TCR with new disulfide bonds and biotin recognition sequence in a POROS 50HQ anion exchange column using a NaCl concentration gradient of 0 to 500 mM (indicated by dashed lines).

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 of 0 to 500 mM (indicated by dashed lines) and indicated by dotted lines.

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 FIG. 87.

FIG. 90 is a protein elution profile obtained by gel filtration chromatography of an anion exchange column of NY-ESO-hexa-histidine tag performed in FIG. 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) incubated at concentrations of 10 μg of NYESO 0 TCR, 10 μg of NYESO 10 ^-4 M TCR, 10 μg of NYESO 10 ^-5 M TCR, and 10 μg of NYESO 10 ^-6 M TCR FACS histogram showing staining intensity produced at.

Figure 92 shows the DNA sequence of the β-chain of soluble A6 TCR into which the TRBC1 * 01 constant region was inserted.

FIG. 93 shows the protein eluate eluted on POROS 50HQ column using soluble A6 TCR with TRBC1 * 01 constant region as anion exchange chromatography, using 0-500 mM NaCl gradient (indicated by dashed line).

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 peak 2 of FIG. 93. Peak 1 contains a TCR heterodimer with interchain disulfide bonds.

96A is a BIAcore assay for specific binding of HLA-Flu complex with disulfide-linked A6 soluble TCR. FIG. 96B is the result of comparative binding with control for 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.                 

FIG. 98 shows the protein eluate eluted from a POROS 50HQ column using a NaCl concentration gradient (indicated by dashed line) of 0-500 mM NaCl as anion exchange chromatography of soluble A6 TCR with 'free' cysteine.

FIG. 99A is reduced SDS-PAGE (Coomassie stained) of fractions 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 fractions pooled from peak 2 of FIG. 98. Peak 1 contains interchain disulfide-linked TCR heterodimers.

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 β chains of A6 TCR with mutated serine residues for free cysteine.

FIG. 103 shows an anion exchange chromatography of soluble A6 TCR with mutated serine residues for free cysteine, showing protein eluate eluted from POROS 50HQ column using 0-500 mM NaCl gradient (indicated by dashed lines). .

FIG. 104A is a reduced SDS-PAGE (stained with Coomassie) of the column fractions 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 certainly contains interchain disulfide-linked TCR heterodimers.

FIG. 105 is Size Exclusion Chromatography of Fractions Pooled from FIG. 103 Peak 2

The result is. Peak 1 contains interchain disulfide-linked TCR heterodimers.

106A is a BIAcore assay showing the specific binding of disulfide-linked A6 soluble TCRs with mutated serine residues for free cysteine in the HLA-Flu complex. 106B is the result of comparative binding reaction with the control for the single injection of disulfide-linked A6 soluble TCRs.

107 shows the nucleotide sequence of pYX112.

108 shows the nucleotide sequence of pYX122.

109 shows DNA and protein sequences of pre-pro mating factor alpha fused to TCR α chains.

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. Lane C contains 60 ng of purified soluble NY-ESO TCR as a control and lanes 1 and 2 contain proteins collected in TCR transformed yeast culture.

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

113 is a schematic of the TCR chain for cloning into baculovirus.

114 shows nucleic acid sequences of disulfide A6 αTCR constructs as BamHI inserts for insertion into pAcAB3 expression plasmids.

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

116 shows Coomassie stained gels and western blots of disulfide A6 TCR produced by bacteria and disulfide A6 TCR produced by insects.

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 Mutation of A6 Tax TCR α and β Chains

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In order to mutate A6 Tax threonine No. 48 of exon 1 of TRAC * 01 to cysteine, the following primer was 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 to 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 mutagenesis:

Expression plasmids containing genes for the α and β chains of A6 Tax TCR were mutated in the following manner 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 supplemented with primers diluted to a final concentration of 0.2 uM in 50 μl final reaction volume. After initial degeneration at 95 ° C for 30 seconds in a Hybaid PCR express PCR machine, the reaction mixture is denatured (95 ° C, 30 seconds), annealed (55 ° C, 60 seconds), extended (73 ° C, 8 minutes) Was performed 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. Pick up a single clone to include 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) Incubated 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. Each mutated nucleic acid and amino acid sequence is shown 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 the mutated α and β chains were individually transformed into the E. coli strain BL21pLysS, and the OD ₆₀₀ value was 0.4 before inducing protein expression with 0.5 mM IPTG for a single clone with ampicillin resistance. Were incubated in TYP (ampicillin 100 μg / ml) medium at 37 ° C. Cells were collected by centrifugation at 4000 rpm for 30 minutes in Beckman J-6B 3 hours after protein expression induction. 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 Triton buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCl, 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). The detergent and the salt were similarly removed by washing with a buffer of pH 8.0 (50 mM Tris-HCl, 1 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT). Finally, the aggregates were divided into 30 mg aliquots and frozen at -70 ° C. Aggregate protein yield was quantified by dissolving with 6M guanidine-HCl and measuring by Bradford dye-binding assay (PerBio).
Approximately 30 mg of each dissolved aggregate chain (i.e. 1 umole) was thawed from the frozen stock, the samples were then mixed and the mixture was completely guanidine solution (6 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA) 15 Dilution with mL dilutes the chain completely. Guanidine solution containing fully reduced and denatured TCR chains was converted into refolding buffer (100 mM Tris, pH 8.5), 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 5M 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 refolding solution was loaded onto a POROS 50HQ anion exchange column and sTCR was removed from the degradation products and impurities by eluting the bound protein using a concentration gradient from 0 to 500 mM NaCl over 50 times the column volume using Akta Pharmacia. Isolated (FIG. 4). Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE before pooling and concentration (FIG. 5). Finally, sTCR was purified and characterized using a Superdex 200HR gel filtration column previously equilibrated with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40) (FIG. 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 Using BIAcore Surface Plasmon Resonance for Binding of sTCR to Specific pMHC

Surface plasmon resonance biosensors (BIAcore 3000 ^™ ) were used to analyze the binding between sTCR and its peptide MHC ligand. This is facilitated by creating a single pMHC complex immobilized on a streptavidin-coated binding surface in a semi-oriented fashion, with soluble T-cell receptors and up to four different pMHC (individual flow cells). immobilized in the cell) can be effectively tested simultaneously. Manual injection of the HLA complex allows the exact amount of fixed Class I molecules to be easily manipulated.

This immobilized complex is capable of binding to both T cell receptors and CD8α coreceptors that can be 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 when sTCR was used as a 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 were purified after in vitro repolling from aggregates expressed in bacteria containing constituent subunit proteins and synthetic peptides and subjected to enzymatic biotinylation in vitro (O'Callaghan). et al. (1999) Anal. Biochem. 266: 9-15). The HLA-heavy chain was expressed with a C terminal biotinylation tag that placed the transmembrane domain and cytoplasmic domain of the protein in the appropriate construct. Aggregates were expressed at about 75 mg / l cell culture level. HLA light chains and β2-microglobulins were also expressed in E. coli at levels of about 500 mg / l cell culture in aggregate form from appropriate constructs.

E. coli cells were digested and the aggregates were purified to about 80% purity. Proteins from the aggregates were denatured with 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and a single pulse of denatured protein at 30 mg / l heavy chain and 30 mg / lβ2m concentration. Add to refold buffer below 5 ° C., 0.4 M L-arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM β-cysteamine, 4 mg / ml peptide (e.g. tax 11-19) By repolling. Refolding was completed by placing at 4 ° C. for at least 1 hour.

The buffer was exchanged with 10 mM Tris dialysis at pH 8.1 of 10 times volume. 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 gradient of NaCl of 0-500 mM. The HLA-A2-peptide complex peak fraction eluting at about 250 mM NaCl concentration was collected and the protease inhibitor (Calbiochem) was added and the fraction was cooled on ice.

The buffer of the HLA complex with the biotinylated tag was 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 cooled on ice and proteolytic enzyme inhibitor (Calbiochem) was added. And biotinylation reagents: 1 mM biotin, 5 mM ATP (pH 8 buffered), 7.5 mM MgCl 2, and 5 μg / ml BirA enzyme (O, Callaghan et al. (1999) Anal. Biochem. 266: 9-15 Purified)) was added. 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, loaded with 1 ml of the biotinylation reaction mixture and developed at 0.5 ml / min with PBS. Biotinylated HLA complex was eluted with a single peak at about 15 mL. Fractions containing protein were pooled, cooled on ice and protease inhibitor (Calbiochem) was added. Protein concentration was measured by Coomassie-binding assay (PerBio) and aliquoted biotinylated HLA complex was stored frozen at -20 ° C. Streptavidin was immobilized by standard amine coupling method.

The interaction of A6 Tax sTCR with a new interchain bond with its ligand / MHC complex or irreversible HLA-peptide combination (preparation thereof described above) was analyzed via a BIAcore 3000 ^™ surface plasmon resonance (SPR) biosensor. SPR measures the change in the rate of regulation in terms of response unit (RU) near the sensor surface in a small flow cell, and its principle is to detect receptor-ligand interactions and their affinity and kinetic parameters. ) Can be used for analysis. Probes of flow cells were prepared by immobilizing individual HLA-peptide complexes into separate flow cells through a bond between biotin crosslinked on β2m and streptavidin chemically crosslinked to the activated surface of the flow cell. Detection was performed by passing the sTCR over the surface of the different flow cells at a constant flow rate while measuring the SPR response. First, the specificity of the interaction was determined by applying a sTCR at 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 (FIG. 7 insert). This was verified by passing over another surface. Injection of soluble sTCR at a constant flow rate and different concentration onto the peptide-HLA complex was used to define background resonance. These regulatory measurements were subtracted from the values obtained for specific peptide-HLA complexes and used to calculate the binding affinity expressed as 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 that reported for the interaction 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 BglII restriction site (AAGCTT) suitable for use as a linking site in its native sequence.

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

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100 ng of the plasmid was mixed with 5 μl of 10 mM dNTP, 25 μl of 10 × Pfu-buffer (Stratagene), and 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 supplemented with primers diluted to a final concentration of 0.2 uM in 50 μl final reaction volume. After an initial denaturation step at 95 ° C. for 30 seconds in a Hybaid PCR express PCR machine, the reaction mixture is denatured (95 ° C., 30 seconds), annealing (55 ° C., 60 seconds), extended (73 ° C., 8 minutes) Was performed 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 at 37 ° C. for 18 hours. Pick up a single clone and include 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) Incubated 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. Since the mutations introduced into the chain are 'silent' the amino acid sequence of this chain is not different from that described in Figure 3a. The DNA sequence of the mutated α chain can be seen in FIG. 8A.

A6 TCR plasmid containing BamH1 in the α chain and BglII recognition site in the β chain was used as a template to generate soluble JM22 TCR incorporating a new disulfide bond. 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 DNA sequences of 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. 10 shows soluble disulfide-linked JM22 TCR elution from a POROS 50HQ column using a 0-500 mM NaCl concentration gradient (indicated by dashed 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 from the size exclusion column of the fraction pooled at peak 1 of FIG. 10.

BIAcore analysis for binding of pMHC and JM22 TCR was performed as described in Example 3. 13A shows BIAcore analysis for specific binding of HLA-Flu complex with disulfide-linked JM22 soluble TCR. 13B shows the binding response compared to the control for disulfide-linked JM22 soluble TCRs. The Kd of the disulfide-linked TCRs 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

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

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

NY-ESO TCR α chain and β chain structures 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-refolded and purified as described in Examples 1 and 2 except for the following modifications in the protocol:

Degradation of Soluble TCR: Thaw 30 mg of solubilized TCR β chain aggregate and 60 mg of solubilized TCR α chain aggregate from 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. The mixture was incubated at 37 ° C. for 30 minutes.

Refolding of Soluble TCR: 1 L of refold buffer was vigorously agitated at 5 ° C ± 3 ° C. Redox couples (2-mercaptoethylamine and cystamine, final concentrations of 6.6 mM and 3.7 mM, respectively) were added about 5 minutes before adding the modified TCR chains. The protein was then refolded for about 5 hours ± 15 minutes with stirring at 5 ° C ± 3 ° C.

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

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

BIAcore analysis for binding of disulfide-linked NY-ESO TCR with pMHC was performed as described in Example 3. 19 shows BIAcore analysis for specific binding of HLA-NYESO complexes with disulfide-linked NY-ESO soluble TCRs. 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 TCRs Comprising at least One of Two Cysteine Residues Required to Form New Disulfide Interchain Bond and Natural Disulfide Interchain Bond

In order to generate soluble NY-ESO TCRs inserted with at least one of the cysteine residues involved in the new disulfide bonds and the natural disulfide bonds, a plasmid comprising BamHI in the α chain and BglII restriction sites in the β chain was described in Example 4. As used, it was used as a framework. The following primers were used.

NY-ESO TCR α chain and β chain structures were obtained by PCR cloning as follows. PCR reaction was carried out using the template shown above, a template containing the 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. 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, DNA isolated using all of the primers was used. In order 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, isolated DNA was used using one of the aforementioned primers together with the appropriate primers of Example 5. It was.

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

22-24 show soluble NY-ESO TCRα ^cys β ^cys (with non-natural and natural cysteine in the chain), TCRα ^cys (sheep) from a POROS 50HQ anion exchange column using a 0 to 500 mM NaCl concentration gradient (indicated by dashed lines). Non-natural cysteine branches in the chain but natural cysteine has only the α chain) and TCRβ ^cys (both non-natural cysteine branches but the natural cysteine has only the β chain). Figures 25-26 show reduced SDS-PAGE (Coomassie staining), non-for fractionation on NY-ESO TCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys columns, performed as described in FIGS. 22-24, respectively. Show reduced SDS-PAGE (Coomassie staining) results. Figures 27-29 are protein elution profiles of gel filtration chromatography of fractions pooled from 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 a single main peak corresponding to heterodimer.

BIAcore analysis for binding of pMHC and sTCR was performed as described in Example 3. 30-32 show BIAcore analysis for specific binding of HLA-NYESO complex with NY-ESO TCRα ^cys β ^cys , TCRα ^cys and TCRβ ^cys, 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 from known T cells from Hill Gaston (Medical School, Addenbrooke's Hospital, Cambridge) according to known techniques. The cDNA encoding NY-ESO TCR was generated by treating mRNA with reverse transcriptase.

To generate soluble AH-1.23 TCR with a new disulfide bond, a TCR plasmid containing BamHI in the α chain and BglII recognition site in the β chain was 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 comprising 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 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 elution of disulfide-linked TCR protein of soluble AH-1.23 from POROS 50HQ anion exchange column using 0-500 mM NaCl concentration gradient (indicated by dashed line). 36 and 37 show the results of reduced SDS-PAGE (comasy staining) and non-reducing SDS-PAGE (comasy staining), respectively, for fractions from the column performed as described in FIG. These gels clearly show the presence of TCR heterodimers with interchain disulfide bonds formed. FIG. 38 is an elution profile from Superdex 75 HR gel filtration column for fractions pooled in an anion exchange column performed as described in FIG. 35. Protein eluted with a single main peak corresponding to a TCR heterodimer.

Example 8 Construction of Soluble A6 TCRs with New Disulfide Interchain Bonds at Different Locations in the Immunoglobulin Region of the Constant Domain

Is it possible to form a functional soluble TCR containing a new disulfide bond in the TCR immunoglobulin region at a position other than between threonine 48 of exon 1 of TRAC * 01 and 57 serine of exon 1 of TRBC1 * 01 / TRBC2 * 01? The following experiments were conducted to investigate.

To mutate the A6 TCR α chain, the following primers were prepared (numbers in primer names indicate the position of the amino acid residues to be mutated in exon 1 of TRAC * 01 and mutated residues 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 (numbers in primer names indicate the position of the amino acid residues to be mutated in exon 1 of TRBC2 * 01 and mutated residues 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 mutagenesis, amplification, ligation, and plasmid purification of the α and β TCR constructs through appropriate combinations of the above-mentioned primers to generate soluble TCRs containing new disulfide interchain linkages between the following amino acid pairs are described in Example 1 As was done.

TCRα chain TCRβ chain Α primer used Β primer used Thr 48 Thr 45 Ser 61 Leu 50 Tyr 10 Ser 15 Thr 45 Leu 12 Ser 61 Leu 12 Val 22 Met 52 Tyr 43 Ser 15 Ser 57 Ser 77 Ser 57 Ser 57 Ser 17 Val 13 Asp 59 Ser 17 Arg 79 Phe 14 Phe 14 Gly 55 Leu 63 Glu 15 T48 → C T45 → C S61 → C L50 → C Y10 → C S15 → C T45 → C L12 → C S61 → C L12 → C V22 → C M52 → C Y43 → C S15 → C S57 → C S77 → C S57 → C S57 → C S17 → C V13 → C D59 → C S17 → C R79 → C F14 → C F14 → C G55 → C L63 → C E15 → C
39 to 58 show DNA and amino acid sequences of the A6 TCR chain mutated with the primers. Codons encoding mutated cysteines were highlighted.

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

TCRα chain TCRβ chain Ser 61 Leu 50 Ser 15 Leu 12 Ser 61 Leu 12 Val 22 Tyr 43 Ser 57 Ser 57 Val 13 Ser 17 Arg 79 Phe 14 Phe 14 Leu 63
59-64 show Thr 48-Ser 57, Thr 45-Ser 77, Tyr 10-Ser 17, Thr 45-Asp 59, from POROS 200HQ anion exchange column using 0-500 mM NaCl concentration gradient (indicated by dotted lines), respectively. Elution of soluble TCR with new disulfide interchain linkages in the Met 52-Gly 55 and Ser 15-Glu 15 residues. Figures 65-70 show the results of reduced SDS-PAGE (Coomassie stain) and non-reduced SDS-PAGE (Coomassie stain), respectively, for fractions from the columns performed as described in Figures 59-64. This gel clearly shows the presence of interchain disulfide-linked TCR heterodimers.

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71-76 are elution profiles from Superdex 200 HR gel filtration columns for fractions pooled from anion exchange columns performed as described in FIGS. 59-64. BIAcore analysis for binding of TCR to pMHC was performed as described in Example 3. 77-82 are BIAcore results demonstrating the binding capacity of purified soluble TCR to 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 similarly binds to the HLA-A2-NY-ESO complex, an "unrelated target", but was able to bind to the HLA-A2 tax complex, a "native target" (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 the induction of protein expression with 0.5 mM IPTG of the ampicillin resistant single clone was 0.7. Incubated in TYP (ampicillin 100 μg / ml) medium at 37 ° C. Cells were collected by centrifugation for 30 minutes at 4000 rpm in Beckman J-6B 18 hours after protein expression induction.

The cell pellet was resuspended in cytolysis 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 ultrasonically pulverized for a total of about 10 minutes using a Milsonix XL2020 sonicator equipped with a standard 12 mm diameter probe. 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 NaCl, 10 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT) to remove cell debris and membrane components It was. Aggregate pellets were homogenized each time in Triton wash buffer before being pelleted by centrifugation at 13000 rpm for 15 minutes in Beckman J2-21. Detergent and salt were then removed by similar washing in resuspension 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 dye-binding assay (PerBio).

60 mg of frozen solubilized TCR α chain (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 chain was then added to the 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). 1) and mix with stirring. The solution was placed in a cold room (5 ° C. ± 3 ° C.) for 5 hours to allow refolding to occur. Subsequently, dialysis was carried out with 12 L of water for 18 to 20 hours, followed by dialysis with 12 L of 10 mM Tris (pH 8.1) for 18 to 20 hours (5 ° C. ± 3 ° C.). A spectrapor 1 (Spectrum Laboratories product no. 132670) dialysis membrane having a molecular weight capable of blocking a molecular weight of 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) mounted on a Nalgene filtration unit.

The refolded NY-ESO TCR was separated from digests and impurities by loading the dialysed repolling product onto a POROS 50HQ (Applied Biosystems) anion exchange column using an AKTA Purifier (Amersham Biotech). The POROS 50 HQ column was previously equilibrated with 10-fold column volume of Buffer A (10 mM Tris pH 8.1) before loading the protein. Bound proteins were eluted using a NaCl concentration gradient of 0-500 mM exceeding 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 purified 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 carried out by a hanging drop technique using 1 μl of a 5 mM Mes (pH 6.5) protein solution (8.4 mg / ml) mixed with the same volume of crystallization buffer at 18 ° C. Crystals also appeared in several other conditions using Crystal Screen Buffer (Hampton Research). Single cubic crystals (<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 flash-frozen and analyzed by diffraction of X-ray beams of Daresbury synchrotron. The crystal was diffracted at 0.25 nm (2.5 Hz) resolution. One data set was collected and processed to give an appropriate 98.6% complete set up to about 0.27 nm (2.7 μs) but not to 0.25 nm (2.5 μs). The agreement between the merging R-factor, ie, multiple measures for crystallographically identical reflection, was 10.8% in all data. This is the lowest in high resolution shells. The spatial group is P2 ₁ with cell size 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. Cell diameter and symmetry means that there are two copies in the cell. The asymmetric unit, au or minimum volume required for the study has only one molecule and the other molecules in the cell are generated by 2 ₁ symmetrical operation. The position of the molecule in au is arbitrary in the y direction. As long as it is in the correct position on the xz 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 HLA-cognate peptides that forms a 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. The use of the 1BD2 A / B model in MR for molecular replacement (MR) provides an inaccurate solution, as is seen in the extensive overlap with symmetric equivalent molecules. Using the B chain alone has no large overlap with the neighbors, providing a better solution. 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 required to transform the initial chain B model into MR equivalents were applied to chain A. The hybrid MR solution, generated as follows, was well packed into the cell with minimal clash.

Electron density maps were generally consistent with the model, allowing for adjustments consistent with the sequence of the NY-ESO TCR. However, the first model has many gaps, and in particular loses the side chains, which characterizes the misaligned parts of the model. Many hair-pin loops between very low density strands were difficult to apply to the model. The crystallographic R factor of the model is 30%. The R factor is residual, which is the difference between the calculated amplitude and the observed amplitude.

As demonstrated in FIGS. 83A and 83B, the injected sequence from 1BD2 does not match the density well. The model was replaced with Cys at No. 164 in Chain A and No. 174 in Chain B and further refinement showed that this sequence alignment is more suitable for density. Since the difference is small in terms of the side chain size, there is little variation in the model. The electrical density in that area hardly changed.

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 may include a small portion of all of the TCR, 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 A Complete Chain B Complete Chain A Constant Chain B Constant Short stretch r.m.s Displacement Mean Displacement Max Displacement 2.831 2.178 9.885 1.285 1.001 6.209 1.658 1.235 6.830 1.098 0.833 4.490 0.613 0.546 1.330

(All units are Å)

Short stretches represent a single strand from chain A (A157 to A169) and a single strand of chain B (B170 to B183) currently linked 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 local structure of the TCR around the bond. Some great effects were observed when comparing TCR with the published 1BD2 structure of A6 TCR, but the increase in RMS displacement is mainly due to the difference in loop structure (FIG. 84). This loop does not form part of the core structure of the TCR formed by a series of β sheets forming characteristic Ig folds. 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 TCRs with new disulfide bonds. Therefore, these data indicate that the core structure of the TCR is maintained in the crystal structure of the TCR with the new disulfide bond.

Example 10 Construction of Soluble NY-ESO TCRs Comprising New Disulfide Interchain Bond and C-Term β Chain Tagging Sites

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

NY-ESO TCR β-chain constructs were obtained by PCR cloning as follows. PCR reactions were performed using a template comprising 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 an expression plasmid. The sequence of the plasmid insert was confirmed by automated DNA sequencing. FIG. 85A shows the DNA sequence of the NY-ESO TCR β chain incorporating a biotin recognition site and FIG. 85B shows the resulting amino acid sequence.

The α chain structure was generated as described in Example 5. Each TCR chain was expressed, co-refolded and purified as described in Example 5.
To generate soluble NY-ESO TCRs comprising non-natural disulfide interchain linkages and hexa-histidine tags at the C terminus of the β chain, primers and NY-ESO templates as mentioned above were used. The PCR product was restriction digested with the relevant restriction enzyme and cloned into pGMT7 containing the hexa-histidine sequence to obtain an expression plasmid. 86A shows the DNA sequences of NY-ESO TCR β chains each inserted with a hexa-histidine tag and FIG. 86B shows the resulting amino acid sequence.

delete

FIG. 87 shows elution of soluble NY-ESO TCR containing new disulfide bonds and biotin recognition sequences from POROS 50HQ anion columns with 0 to 500 mM NaCl concentration gradient (indicated by dashed lines). FIG. 88 shows elution of soluble NY-ESO TCR containing new disulfide bonds and hexa histidine tags from a POROS 50HQ anion exchange column using a 0-500 mM NaCl concentration gradient (indicated by dashed lines).

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

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

NY-ESR soluble TCR with disulfide bond and biotin recognition sequence prepared as in Example 10 was used to form soluble TCR tetramers required for cell staining. 2.5 ml of purified soluble TCR solution (about 0.2 mg / ml) was exchanged into 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) that blocks 10 kDa molecular weight. This was made 10 mM with ATP added from stock (0.1 g / ml, adjusted to pH 7.0). Add 1 volume of protease inhibitor cocktail Set 1 (Calbiochem Biochemicals) in an amount sufficient to provide the final protease cocktail with 1/100 of the stock solution provided, followed by 1 mM biotin (0.2M stock). ) And 20 μg / ml enzyme (added from 0.5 mg / ml stock). The mixture was then incubated overnight at room temperature. Excess biotin was removed from the solution using size exclusion chromatography on an S75 HR column. The level of biotinylated NY-ESO TCR was determined by the size exclusion HPLC-based method mentioned below. 50 μl aliquots of biotinylated NY-ESO TCR (2 mg / ml) were incubated with 50 μl of streptavidin coated agarose beads (Sigma) for 1 hour. The beads were spun down and 50 μl of unbound sample was run on 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 detected by UV spectrometer at 214 nm and 280 nm. Biotinylated NY-ESO was performed in contrast to non-biotinylated NY-ESO TCR controls. The percentage of biotinylation was calculated by subtracting the biotinylated peak area from the peak area of the non-biotinylated protein.

Tetrification of biotinylated soluble TCRs was performed using the Neutravidin-Phycoerythrin conjugate (Cambridge Biosciences, UK). The concentration of biotinylated soluble TCR was determined by Coomassie Protein Assay (Pierce), and the ratio of soluble TCR 0.8 mg / ml neutravidin-phycoerythrin conjugate was 1: 4 ratio of neutravidin-PE. Calculated to be saturated with biotinylated TCR. 19.5 μl of 6.15 mg / ml biotinylated NY-ESO soluble TCR solution in PBS was slowly added to 150 μl of 1 mg / ml Neutravidin on ice with gentle shaking. 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 0.3 × 10 ⁶ HLA-A2 positive EBV transformed B cell lanes (PP LCL) in 0.5 ml of PBS were subjected to HLA at various concentrations (0, 10 ⁻⁴ , 10 ^−5, and 10 ⁻⁶ M). Incubated with A2 NYESO peptide (SLLMWITQC) at 37 ° C. for 2 hours. PP LCL cells were then washed twice with HBSS (Hanks buffered Saline solution: Gibco, UK).

Each of the four aliquots was divided equally and stained with newly tetramerized biotinylated NY-ESO disulfide-linked TCR with neutravidin-phycoerythrin. Cells were incubated with 5 μg or 10 μg of phycoerythrin labeled tetrameric dsTCR complex on ice for 30 minutes and washed with HBSS. 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-91H show FACSVantage data generated in each of the samples prepared as described above in a bar graph. The table below shows the percentage of positively stained cells observed in each sample.

sample % Positively stained cells 0 NY-ESO Peptide, 5 μg TCR 10^-4 M NY-ESO Peptide, 5 μg TCR 10^-5 M NY-ESO Peptide, 5 μg TCR 10^-6 M NY-ESO Peptide, 5 μg TCR 0 NY-ESO peptide, 10 μg TCR 10^-4   M NY-ESO Peptide, 10 μg TCR 10^-5  M NY-ESO Peptide, 10 μg TCR 10^-6  M 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 way that correlates with the concentration of the peptides they were cultured (SLLMWITQC). Therefore, these NY-ESO TCR tetramers are suitable moieties for specific cell labels based on the expression of the HLA-A2 NY-ESO complex.

delete

In this example, fluorescent conjugated NY-ESO TCR tetramers were used. However, similar levels of cell binding are expected even if such 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 to Cβ1

For PCR preparation of 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 is shown 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:

The sTCR was degraded by loading the dialysed refolding material onto 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. 93. And from impurities. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE (FIG. 94) before pooling and concentration. Finally, sTCR was purified 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 eluting at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

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

Soluble A6 TCR with a new disulfide bond inserting a Cβ1 constant region has a Kd of 2.42 ± 0.55 uM for its cognate pMHC. This value is very similar to Kd 1.8 uM, which was determined for 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 the formation of intrachain or intrachain disulfide bonds. All previous examples describe the generation of soluble TCRs with new disulfide bonds in which free cysteine was mutated to alanine to avoid inadequate disulfide bond formation that could 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 Chains

To mutate TCR β-chain alanine (residue 75 of exon 1 of TRBC1 * 01 and TRBC2 * 01) into cysteine, the following primers were prepared (mutations are shown 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 mutagenesis, expression and refolding of soluble TCR were performed as described in Example 2.

Purification of Refolded Soluble TCR:

The dialysis refold material was loaded onto a POROS 50HQ anion exchange column and sTCR was digested by eluting the bound protein with a gradient of 0 to 500 mM NaCl over 50 times the column volume using Akta Pharmacia as shown in FIG. 98. Isolate from impurities. Peak fractions were stored at 4 ° C. before pooling and concentration and analyzed by Coomassie stained SDS-PAGE (FIG. 99). Finally, sTCR was purified and characterized using a Superdex 200HR gel filtration column (FIG. 100) 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 eluting at a relative molecular weight of about 50 kDa were pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

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

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

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

This example demonstrates that soluble TCRs with new disulfide bonds in which “free” cysteines (residue 75 of exon 1 of TRBC1 * 01 and TRBC2 * 01) are mutated to β-serine can be successfully produced.

Design of primers and mutagenesis of TCR β chains

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

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

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

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

Purification of Refolded Soluble TCR

As shown in FIG. 103, sTCR was digested by loading the dialyzed refold material 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 Purifier (Pharmacia). Isolate from product and impurities. Peak fractions were stored at 4 ° C. and analyzed by Coomassie stained SDS-PAGE (FIG. 104) before pooling and concentration. Finally, sTCR was purified and characterized using a Superdex 200HR gel filtration column (FIG. 105) previously equilibrated with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). The peak eluting at a relative molecular weight of about 50 kDa was pooled and concentrated prior to characterization by BIAcore surface plasmon resonance analysis.

BIAcore analysis for binding of disulfide-linked A6 TCR to pMHC was performed as described in Example 3. FIG. 106 shows BIAcore analysis for specific binding of disulfide-linked A6 soluble TCRs to its cognate pMHC.

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 for its cognate pMHC. This value is very similar to Kd 1.8 uM for soluble A6 TCR with a new disulfide bond in which the “free” cysteine of β chain is mutated to alanine as measured 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 are fused with the C terminus of the pre-pro mating factor alpha sequence from Saccharomyces cerevisiae and with yeast expression vectors pYX122 and pYX112, respectively. Cloning was done (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 from the Cerevisiae 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. The yeast DNA was prepared by resuspending the colonies of cerevisiae strain SEY6210 in 30 μl of 0.25% SDS in water and heating at 90 ° C. for 3 minutes. Pre-pro hybridization factor alpha sequences for fusion with TCR α and β chains were generated by PCR amplification of 0.25 μL of yeast DNA 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 30 seconds at 92 ° C. on a Hybaid PCR express PCR machine, the reaction mixture was denatured (92 ° C., 30 seconds), annealing (46.9 ° C., 60 seconds), and extended (72 ° C., 2 minutes) 30 times. Was performed.

The following primers were designed to PCR amplify 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 (executed). Prepared in Example 5). Annealing temperature was 60.1 degreeC.

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 with 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 cloned chimeric products.

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

Yeast expression plasmids each comprising a TCR α chain and a β chain generated as described in Example 15 were subjected to the protocol of Agatep et al. (Technical Tips Online (http://tto.trends.com) 1: 51: P01525). Using s. Co-transformation with the Selevisia strain SEY6210. Single clones grown in synthetic dropout (SD) agar containing histidine and uracil (Qbiogene, Illkirch, France) were incubated overnight at 30 ° C. in 10 ml of SD medium containing histidine and uracil. Overnight cultures were sub-cultured 1: 10 in 10 mL fresh SD medium containing histidine and uracil and incubated at 30 ° C. for 4 hours. Cultures were 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 at 4 ° with a blood wheel. StratClean resin was spun down at 3800 rpm in Heraeus Megafuge 2.0R and discarded 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, cooled on ice, and then 20 μl of the mixture was 0.8 for 1 hour. The SDS-PAGE gel was loaded with mA constant / cm ² gel surface. 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 except for the following differences. The primary antibody (TCR anti α chain) and the secondary antibody were diluted 1: 200 and 1: 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 chain and β chain of the disulfide A6 Tax TCR were cloned from pGMT7 into a pBlueScript KS2 family vector called pEX172. This vector uses a leader sequence derived from DRB1 * 0101, an AgeI site for insertion of other peptide coding sequences, a linker region, and a MluI and SalI site for cloning the DRβ chain in front of the Jun Leucine zipper sequence, thereby allowing for different expression of insect cells. It was designed to clone the MHC class IIβ chain. 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 cleaved with AgeI and SalI to remove the linker region and MluI site and allow the TCR chain to proceed where the peptide sequence begins. The TCR sequence was cloned from pGMT7 with BspEI (having AgeI and a suitable adhesive end) at the 5 'end and SalI site 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. In order to prevent transcription of the Jun Leucine zipper sequence, insertion of a stop codon before the SalI site is required. A schematic of this structure is shown in FIG. 113. If a 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 different 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.

In accordance with the cloning method described above, the following primers were designed (showing homology to the vector 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 the α plasmid was mixed with 1 μl of 10 mM dNTP, 5 μl of 10 × Pfu-Stratagene, 1.25 units of Pfu polymerase (Stratagene), and 50 pmol of the A6α primer and 50 μl of the final volume with H ₂ O. Similar reaction mixtures were applied to the β chain using β plasmid and β primer pairs. The reaction mixture was denatured (95 ° C., 60 seconds), annealed (50 ° C., 60 seconds), extended (72 ° C., 8 minutes) in a Hybaid PCR express PCR machine. The product was cleaved with 10 units of BspEI restriction enzyme at 37 ° C. for 2 hours and then further further with 10 units of SalI (New England Biolabs) for 2 hours. This cleaved reaction was linked to pEX172 digested with AgeI and SalI, transformed into competent XL1-Blue bacteria and incubated at 37 ° C. for 18 hours. Single clones from each α and β prep were picked up and 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) 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 Genetix's sequencing machine. The amino acid sequences of the α and β chains of the BamHI insert are shown in FIGS. 114 and 115, respectively.

α and β disulfide A6 Tax TCR chain constructs of pEX172 were digested with BamHI restriction enzyme (New England Biolabs) at 37 ° C. for 2 hours. 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 incubated at 37 ° C. for 18 hours. Single clones were picked from the plates and grown overnight in 5 ml of TYP and ampicillin and plasmid DNA was purified as described above. The plasmid was digested with BamHI and the β-chain BamHI insert was linked, transformed into competent XL1-Blue bacteria, grown overnight, grown in TYP-ampicillin medium, and then miniprepping using a Qiagen mini-prep column. ). 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

Transfection, Infection, Expression and Analysis of A6 TCR in Insect Cells

Expression plasmids with α and β chains were transferred to sf9 cells grown in serum free medium (Pharmingen-BD Biosciences: 551411) using Baculogold transfection kit (Pharmingen-BD Biosciences: 21100K) according to the manufacturer's instructions. Infected. After incubation at 27 ° C. for 5 days, 200 μl of the culture medium of these transfected cells was added to 100 mL of Hive Five cells at 1 × 10 ⁶ cells / μl in serum free medium. After further incubation at 27 ° C. for 6 days, 1 ml of medium was removed and the cell debris was pelleted by centrifugation at 13,000 RPM for 5 minutes in a Hereus centrifuge.

10 μl of this insect A6 disulfide-linked TCR supernatant was developed on pre-made 4-20% Tris / Glycine gel (Invitrogen: EC60252) alongside 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, heated at 95 ° C. for 5 minutes, and then cooled at room temperature for 10 minutes. 20 μl was loaded. 10 μl of Laemmli sample buffer was added to prepare a non-reducing sample and 20 μl was loaded.

The gel was developed at 150 volt for 1 hour in a Novex-Xcell gel tank and the gel was coated with Coomassie gel dye (500 g of methanol, 1.1 g of Coomassie powder, stirred for 1 hour, 100 ml of acetic acid was added and 1 L with H ₂ O). After stirring for 1 hour and filtered with a 0.45mM filter) was dyed with 50ml light stirring for 1 hour. The gel was decolorized three times for 30 minutes with gentle shaking with 50 ml of a bleaching solution (same as Coomassie gel dye but without Coomassie powder).

Rather than staining the gel with Coomassie, Western blots were performed by SDS-PAGE gels as before except that proteins were transferred to Immuno-Blot PVDF membranes (Bio-Rad: 162-0174). Six filter papers were cut to gel size and immersed in transfer 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). PVDF membranes were prepared by soaking in methanol for 1 minute and soaking in transfer buffer for 2 minutes. Three filter papers were placed on the anode surface of the Pharmacia Novablot and the membrane was placed on top of the gel and finally three additional filter papers were placed on the cathode side. Immunoblotting was performed at 0.8 mA constant per cm 2 of gel surface for 1 hour.

After blotting, the membranes were lightly diluted in 7.5 ml of blocking buffer (Tris-buffered saline tablet (Sigma: T5030), 3 g skim milk powder (Sigma: M7409), 30 μl of Tween 20 and made 30 ml with H ₂ O). Shake and block for 60 minutes. Membranes were washed three times with TBS wash buffer for 5 minutes (20 TBS tablets, 150 μl of Tween 20, made 300 mL with H ₂ O). Membranes were treated in 7.5 ml of blocking buffer for 1 hour with gentle shaking with a primary antibody diluted 50-fold dilution 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, a secondary antibody treatment, which was diluted 1000-fold with HRP-labeled goat anti-mouse antibody (Santa Cruz Biotech: Sc-2005) in 7.5 ml of blocking buffer, was performed for 30 minutes with gentle shaking. 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 of Opt-4CN dilution, 12.6 ml of H ₂ O, 0.28 ml of Opti-4CN substrate). The membrane was colored 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. Both were divided into α chain and β chain when reduced. 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 excessive α chains, and the free α chains were seen in the non-reducing lanes of the anti-α Western blot.

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

<110> Avidex Ltd <120> Substances <130> P325660WO / NJL <140> PCT / GB02 / 03986 <141> 2002-08-30 <150> GB 0121187.9 <151> 2001-08-31 <150> GB0219146.8 <151> 2002-08-16 <150> US60 / 404182 <151> 2002-08-16 <160> 183 <170> PatentIn version 3.1 <210> 1 <211> 20 <212> PRT <213> Homo sapiens <400> 1 Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser 1 5 10 15 Met Asp Phe Lys             20 <210> 2 <211> 20 <212> PRT <213> Homo sapiens <400> 2 Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp 1 5 10 15 Met Arg Ser Met             20 <210> 3 <211> 20 <212> PRT <213> Homo sapiens <400> 3 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys 1 5 10 15 Ser Ser Asp Lys             20 <210> 4 <211> 20 <212> PRT <213> Homo sapiens <400> 4 Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser 1 5 10 15 Val Cys Leu Phe             20 <210> 5 <211> 20 <212> PRT <213> Homo sapiens <400> 5 Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu 1 5 10 15 Lys Glu Gln Pro             20 <210> 6 <211> 20 <212> PRT <213> Homo sapiens <400> 6 Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser 1 5 10 15 Ala Thr Phe Trp             20 <210> 7 <211> 20 <212> PRT <213> Homo sapiens <400> 7 Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His 1 5 10 15 Thr Gln Lys Ala             20 <210> 8 <211> 20 <212> PRT <213> Homo sapiens <400> 8 Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 1 5 10 15 Gln Pro Ala Leu             20 <210> 9 <211> 20 <212> PRT <213> Homo sapiens <400> 9 Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile 1 5 10 15 Ser His Thr Gln             20 <210> 10 <211> 91 <212> PRT <213> Mus musculus <400> 10 Pro Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro 1 5 10 15 Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln             20 25 30 Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys         35 40 45 Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile     50 55 60 Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu 65 70 75 80 Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro                 85 90 <210> 11 <211> 126 <212> PRT <213> Mus musculus <400> 11 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu             20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn         35 40 45 Gly Arg Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys     50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe                 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro             100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp         115 120 125 <210> 12 <211> 20 <212> PRT <213> Mus musculus <400> 12 Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala 1 5 10 15 Met asp ser lys             20 <210> 13 <211> 20 <212> PRT <213> Mus musculus <400> 13 Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp 1 5 10 15 Met Lys Ala Met             20 <210> 14 <211> 20 <212> PRT <213> Mus musculus <400> 14 Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg 1 5 10 15 Ser Gln Asp Ser             20 <210> 15 <211> 20 <212> PRT <213> Mus musculus <400> 15 Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys 1 5 10 15 Leu Phe Thr Asp             20 <210> 16 <211> 20 <212> PRT <213> Mus musculus <400> 16 Asn Gly Arg Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr 1 5 10 15 Lys Glu Ser Asn             20 <210> 17 <211> 20 <212> PRT <213> Mus musculus <400> 17 Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser 1 5 10 15 Ala Thr Phe Trp             20 <210> 18 <211> 20 <212> PRT <213> Mus musculus <400> 18 Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 1 5 10 15 Lys Gln Lys Ala             20 <210> 19 <211> 20 <212> PRT <213> Mus musculus <400> 19 Arg Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu 1 5 10 15 Ser Asn Tyr Ser             20 <210> 20 <211> 20 <212> PRT <213> Mus musculus <400> 20 Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile 1 5 10 15 Ala Asn Lys Gln             20 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 cacagacaaa tgtgtgctag acat 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 atgtctagca cacatttgtc tgtg 24 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 cagtggggtc tgcacagacc c 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 gggtctgtgc agaccccact g 21 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 atatccagaa cccggatcct gccgtgta 28 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 tacacggcag gaatccgggt tctggatat 29 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 ggagatatac atatgcaact actagaacaa 30 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 tacacggcag gatccgggtt ctggatatt 29 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 29 ggagatatac atatggtgga tggtggaatc 30 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 30 cccaagctta gtctgctcta ccccaggcct cggc 34 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 ggagatatac atatgcagga ggtgacacag 30 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 tacacggcag gatccgggtt ctggatatt 29 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 ggagatatac atatgggtgt cactcagacc 30 <210> 34 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 cccaagctta gtctgctcta ccccaggcct cggc 34 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 35 ggagatatac atatgcagga ggtgacacag 30 <210> 36 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 36 cccaagctta acaggaactt tctgggctgg ggaagaa 37 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 37 ggagatatac atatgggtgt cactcagacc 30 <210> 38 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 38 cccaagctta acagtctgct ctaccccagg cctcggc 37 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 39 gggaagctta catatgaagg aggtggagca gaattctgg 39 <210> 40 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 40 tacacggcag gatccgggtt ctggatatt 29 <210> 41 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 41 ttggaattca catatgggcg tcatgcagaa cccaagacac 40 <210> 42 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 42 cccaagctta gtctgctcta ccccaggcct cggc 34 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 43 cacagacaaa tgtgtgctag acat 24 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 44 atgtctagca cacatttgtc tgtg 24 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 45 ccctgccgtg tgccagctga gag 23 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 46 ctctcagctg gcacacggca ggg 23 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 47 ccgtgtacca gtgcagagac tctaaatc 28 <210> 48 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 48 gatttagagt ctctgcactg gtacacgg 28 <210> 49 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 49 cagctgagag actgtaaatc cagtgac 27 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 50 gtcactggat ttacagtctc tcagctg 27 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 51 cagtgacaag tcttgctgcc tattcac 27 <210> 52 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 gtgaataggc agcaagactt gtcactg 27 <210> 53 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 gattctgatg tgtgtatcac agacaaat 28 <210> 54 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 atttgtctgt gatacacaca tcagaatc 28 <210> 55 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 55 ctgatgtgta tatctgtgac aaaactgtgc 30 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 56 gcacagtttt gtcacagata tacacatcag 30 <210> 57 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 57 agacaaaact gtgtgtgaca tgaggtct 28 <210> 58 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 58 agacctcatg tcacacacag ttttgtct 28 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 59 actgtgctag actgtaggtc tatggac 27 <210> 60 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 60 gtccatagac ctacagtcta gcacagt 27 <210> 61 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 61 cttcaagagc aactgtgctg tggcc 25 <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 62 ggccacagca cagttgctct tgaag 25 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 63 cagtggggtc tgcacagacc c 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 64 gggtctgtgc agaccccact g 21 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 65 ccgaggtcgc ttgttttgag ccatcag 27 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 66 ctgatggctc aaaacaagcg acctcgg 27 <210> 67 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 67 ggtcgctgtg tgtgagccat caga 24 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 68 tctgatggct cacacacagc gacc 24 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 69 gtgtttgagc catgtgaagc agagatc 27 <210> 70 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 70 gatctctgct tcacatggct caaacac 27 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 71 gaggtgcaca gttgtgtcag cacagac 27 <210> 72 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 72 gtctgtgctg acacaactgt gcacctc 27 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 73 gggtcagcac atgcccgcag ccc 23 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 74 gggctgcggg catgtgctga ccc 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 75 cccgcagccc tgcaaggagc agc 23 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 76 gctgctcctt gcagggctgc ggg 23 <210> 77 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 77 agatacgctc tgtgcagccg cct 23 <210> 78 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 78 aggcggctgc acagagcgta tct 23 <210> 79 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 79 ctctgagcag ctgcctgagg gtc 23 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 80 gaccctcagg cagctgctca gag 23 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 81 gctgtgtttt gtccatcaga a 21 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 82 ttctgatgga caaaacacag c 21 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 83 ggagatatac atatgggtgt cactcagaac 30 <210> 84 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 84 ccaccggatc cgtctgctct accccaggcg gagatataca tatgaacgct ggtgtcact 59 <210> 85 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 85 ccttgttcag gtcctctgtg accgtgag 28 <210> 86 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 86 ctcacggtca cagaggacct gaacaagg 28 <210> 87 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 87 cccaagctta gtctgctcta ccccaggcct cggc 34 <210> 88 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 88 tgactccaga tactgtctga gcagccg 27 <210> 89 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 89 cggctgctca gacagtatct ggagtca 27 <210> 90 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 90 tgactccaga tactctctga gcagccg 27 <210> 91 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 91 cggctgctca gagagtatct ggagtca 27 <210> 92 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 92 tctgaattca tgagatttcc ttcaattttt ac 32 <210> 93 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 93 tcacctcctg ggcttcagcc tctcttttat c 31 <210> 94 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 94 tctgaattca tgagatttcc ttcaattttt ac 32 <210> 95 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 95 gtgtctcgag ttagtctgct ctaccccagg c 31 <210> 96 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 96 ggctgaagcc caggaggtga cacagattcc 30 <210> 97 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 97 ctcctctcga gttaggaact ttctgggctg gg 32 <210> 98 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 98 ggctgaagcc ggcgtcactc agaccccaaa at 32 <210> 99 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 99 gtgtctcgag ttagtctgct ctaccccagg c 31 <210> 100 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 100 gtagtccgga gacaccggac agaaggaagt ggagcagaac 40 <210> 101 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 101 gtaggtcgac taggaacttt ctgggctggg 30 <210> 102 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 102 gtagtccgga gacaccggaa acgctggtgt cactcaga 38 <210> 103 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 103 gtaggtcgac tagtctgctc taccccagg 29 <210> 104 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 104 gaaattatgc atttgaggat g 21 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 105 attaggcctc tagagatccg 20 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HLA-A2 NYESO peptide <400> 106 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 <210> 107 <211> 621 <212> DNA <213> Homo sapiens <400> 107 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaatgt 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 108 <211> 741 <212> DNA <213> Homo sapiens <400> 108 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 109 <211> 245 <212> PRT <213> Homo sapiens <400> 109 Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met             20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr         35 40 45 Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr     50 55 60 Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro Gly                 85 90 95 Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu             100 105 110 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val         115 120 125 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu     130 135 140 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 145 150 155 160 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln                 165 170 175 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser             180 185 190 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His         195 200 205 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp     210 215 220 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 225 230 235 240 Trp Gly Arg Ala Asp                 245 <210> 110 <211> 621 <212> DNA <213> Homo sapiens <400> 110 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc ggatcctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaatgt 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 111 <211> 615 <212> DNA <213> Homo sapiens <400> 111 atgcaactac tagaacaaag tcctcagttt ctaagcatcc aagagggaga aaatctcact 60 gtgtactgca actcctcaag tgttttttcc agcttacaat ggtacagaca ggagcctggg 120 gaaggtcctg tcctcctggt gacagtagtt acgggtggag aagtgaagaa gctgaagaga 180 ctaacctttc agtttggtga tgcaagaaag gacagttctc tccacatcac tgcggcccag 240 cctggtgata caggcctcta cctctgtgca ggagcgggaa gccaaggaaa tctcatcttt 300 ggaaaaggca ctaaactctc tgttaaacca aatatccaga acccggatcc tgccgtgtac 360 cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420 caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa atgtgtgcta 480 gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540 tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600 ccagaaagtt cctaa 615 <210> 112 <211> 735 <212> DNA <213> Homo sapiens <400> 112 atggtggatg gtggaatcac tcagtcccca aagtacctgt tcagaaagga aggacagaat 60 gtgaccctga gttgtgaaca gaatttgaac cacgatgcca tgtactggta ccgacaggac 120 ccagggcaag ggctgagatt gatctactac tcacagatag taaatgactt tcagaaagga 180 gatatagctg aagggtacag cgtctctcgg gagaagaagg aatcctttcc tctcactgtg 240 acatcggccc aaaagaaccc gacagctttc tatctctgtg ccagtagttc gaggagctcc 300 tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480 gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atacagcctg agcagccgcc tgagggtctc ggccaccttc 600 tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaacct gtcacccaga ttgtcagcgc cgaggcctgg 720 ggtagagcag actaa 735 <210> 113 <211> 204 <212> PRT <213> Homo sapiens <400> 113 Met Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln Glu Gly 1 5 10 15 Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser Ser Leu             20 25 30 Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu Val Thr         35 40 45 Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr Phe Gln     50 55 60 Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His Ile Thr Ala Ala Gln 65 70 75 80 Pro Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Ala Gly Ser Gln Gly                 85 90 95 Asn Leu Ile Phe Gly Lys Gly Thr Lys Leu Ser Val Lys Pro Asn Ile             100 105 110 Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser         115 120 125 Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val     130 135 140 Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu 145 150 155 160 Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser                 165 170 175 Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile             180 185 190 Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 <210> 114 <211> 244 <212> PRT <213> Homo sapiens <400> 114 Met Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 1 5 10 15 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp             20 25 30 Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile         35 40 45 Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu     50 55 60 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 65 70 75 80 Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser                 85 90 95 Ser Arg Ser Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr             100 105 110 Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe         115 120 125 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val     130 135 140 Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro                 165 170 175 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ser Leu Ser Ser             180 185 190 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe         195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr     210 215 220 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 225 230 235 240 Gly Arg Ala Asp <210> 115 <211> 627 <212> DNA <213> Homo sapiens <400> 115 atgcaggagg ygacacagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60 ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120 gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180 agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240 cagcctggtg actcagccac ctacctctgt gctgtgaggc ccacatcagg aggaagctac 300 atacctacat ttggaagagg aaccagcctt attgttcatc cgtatatcca gaaccctgac 360 cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 420 gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480 aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540 aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600 ttcttcccca gcccagaaag ttcctaa 627 <210> 116 <211> 729 <212> DNA <213> Homo sapiens <400> 116 atgggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240 gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 300 ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360 cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420 ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480 gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540 ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600 gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660 tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720 gcagactaa 729 <210> 117 <211> 208 <212> PRT <213> Homo sapiens <220> <221> misc_feature (222) (4) .. (4) <223> X is any amino acid <400> 117 Met Gln Glu Xaa Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn             20 25 30 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu         35 40 45 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala     50 55 60 Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser 65 70 75 80 Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Pro Thr Ser                 85 90 95 Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val             100 105 110 His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp         115 120 125 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser     130 135 140 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 145 150 155 160 Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala                 165 170 175 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn             180 185 190 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 118 <211> 244 <212> PRT <213> Homo sapiens <400> 118 Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln 1 5 10 15 Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser             20 25 30 Trp Tyr Arg Gln Asp Pro Gly Met Glu Thr Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr                 85 90 95 Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr             100 105 110 Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe         115 120 125 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val     130 135 140 Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro                 165 170 175 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser             180 185 190 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe         195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr     210 215 220 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 225 230 235 240 Gly Arg Ala Asp <210> 119 <211> 630 <212> DNA <213> Homo sapiens <400> 119 atgcaggagg ygacacagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60 ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120 gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180 agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240 cagcctggtg actcagccac ctacctctgt gctgtgaggc ccacatcagg aggaagctac 300 atacctacat ttggaagagg aaccagcctt attgttcatc cgtatatcca gaaccctgac 360 cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 420 gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480 aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540 aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600 ttcttcccca gcccagaaag ttcctgttaa 630 <210> 120 <211> 732 <212> DNA <213> Homo sapiens <400> 120 atgggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240 gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 300 ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360 cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420 ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480 gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540 ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600 gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660 tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720 gcagactgtt aa 732 <210> 121 <211> 209 <212> PRT <213> Homo sapiens <220> <221> misc_feature (222) (4) .. (4) <223> X is any amino acid <400> 121 Met Gln Glu Xaa Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn             20 25 30 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu         35 40 45 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala     50 55 60 Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser 65 70 75 80 Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Pro Thr Ser                 85 90 95 Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val             100 105 110 His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp         115 120 125 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser     130 135 140 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 145 150 155 160 Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala                 165 170 175 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn             180 185 190 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 Cys <210> 122 <211> 243 <212> PRT <213> Homo sapiens <400> 122 Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln 1 5 10 15 Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser             20 25 30 Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser         35 40 45 Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn     50 55 60 Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala 65 70 75 80 Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly                 85 90 95 Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu             100 105 110 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro         115 120 125 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu     130 135 140 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 145 150 155 160 Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys                 165 170 175 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu             180 185 190 Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys         195 200 205 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp     210 215 220 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 225 230 235 240 Ala Asp Cys <210> 123 <211> 624 <212> DNA <213> Homo sapiens <400> 123 atgaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60 ctcaactgca cttacagtga ccgaggttcc cagtccttct tctggtacag acaatattct 120 gggaaaagcc ctgagttgat aatgttcata tactccaatg gtgacaaaga agatggaagg 180 tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240 cccagtgatt cagccaccta cctctgtgcc gtgaaggggg ggtctggggg ttaccagaaa 300 gttacctttg gaactggaac aaagctccaa gtcatcccaa atatccagaa cccggatcct 360 gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420 tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480 tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540 aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600 ttccccagcc cagaaagttc ctaa 624 <210> 124 <211> 735 <212> DNA <213> Homo sapiens <400> 124 atgggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 60 agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 120 ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 180 aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 240 gtagtgcgag gagattcggc agcttatttc tgtgccagct caccacagac agggggcaca 300 gatacgcagt attttggccc aggcacccgg ctgacagtgc tcgaggacct gaaaaacgtg 360 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420 gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480 gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540 cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600 tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660 gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720 ggtagagcag actaa 735 <210> 125 <211> 207 <212> PRT <213> Homo sapiens <400> 125 Met Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser             20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met         35 40 45 Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln     50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Gly Gly Ser Gly                 85 90 95 Gly Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile             100 105 110 Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser         115 120 125 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln     130 135 140 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 145 150 155 160 Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val                 165 170 175 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn             180 185 190 Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 126 <211> 244 <212> PRT <213> Homo sapiens <400> 126 Met Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly Gln 1 5 10 15 Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val Tyr             20 25 30 Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr Leu         35 40 45 Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg Phe     50 55 60 Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln Gln 65 70 75 80 Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro Gln                 85 90 95 Thr Gly Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr             100 105 110 Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe         115 120 125 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val     130 135 140 Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro                 165 170 175 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser             180 185 190 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe         195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr     210 215 220 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 225 230 235 240 Gly Arg Ala Asp <210> 127 <211> 621 <212> DNA <213> Homo sapiens <400> 127 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaatgt 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 128 <211> 206 <212> PRT <213> Homo sapiens <400> 128 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 129 <211> 621 <212> DNA <213> Homo sapiens <400> 129 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatctg tgacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 130 <211> 206 <212> PRT <213> Homo sapiens <400> 130 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Cys Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 131 <211> 621 <212> DNA <213> Homo sapiens <400> 131 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaactgtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 132 <211> 206 <212> PRT <213> Homo sapiens <400> 132 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Cys Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 133 <211> 621 <212> DNA <213> Homo sapiens <133> 133 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgtgtgaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 134 <211> 206 <212> PRT <213> Homo sapiens <400> 134 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Cys Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 135 <211> 621 <212> DNA <213> Homo sapiens <400> 135 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtgccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 136 <211> 206 <212> PRT <213> Homo sapiens <400> 136 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Cys Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 137 <211> 621 <212> DNA <213> Homo sapiens <400> 137 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactg taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 138 <211> 206 <212> PRT <213> Homo sapiens <400> 138 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Cys Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <139> <211> 621 <212> DNA <213> Homo sapiens <400> 139 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagt gcagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 140 <211> 206 <212> PRT <213> Homo sapiens <400> 140 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Cys Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 141 <211> 621 <212> DNA <213> Homo sapiens <400> 141 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctt gctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 142 <211> 206 <212> PRT <213> Homo sapiens <400> 142 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Cys Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 143 <211> 621 <212> DNA <213> Homo sapiens <400> 143 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480 gtgctagact gtaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 144 <211> 206 <212> PRT <213> Homo sapiens <400> 144 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Cys Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 145 <211> 621 <212> DNA <213> Homo sapiens <400> 145 atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120 tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240 cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300 cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtgtatcac agacaaaact 480 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600 cccagcccag aaagttccta a 621 <210> 146 <211> 206 <212> PRT <213> Homo sapiens <400> 146 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Cys Ile Thr Asp Lys Thr 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205 <210> 147 <211> 741 <212> DNA <213> Homo sapiens <400> 147 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 148 <211> 246 <212> PRT <213> Homo sapiens <400> 148 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 149 <211> 741 <212> DNA <213> Homo sapiens <400> 149 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgtgta gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 150 <211> 246 <212> PRT <213> Homo sapiens <400> 150 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Cys Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 151 <211> 741 <212> DNA <213> Homo sapiens <400> 151 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatgtg aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 152 <211> 246 <212> PRT <213> Homo sapiens <400> 152 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Cys Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 153 <211> 741 <212> DNA <213> Homo sapiens <400> 153 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgcttgtttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 154 <211> 246 <212> PRT <213> Homo sapiens <400> 154 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Cys Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 155 <211> 741 <212> DNA <213> Homo sapiens <400> 155 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca catgcccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 156 <211> 246 <212> PRT <213> Homo sapiens <400> 156 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Cys Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 157 <211> 741 <212> DNA <213> Homo sapiens <400> 157 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gctgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 158 <211> 246 <212> PRT <213> Homo sapiens <400> 158 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Cys Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 159 <211> 741 <212> DNA <213> Homo sapiens <400> 159 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgtgt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 160 <211> 246 <212> PRT <213> Homo sapiens <400> 160 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Cys Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 161 <211> 741 <212> DNA <213> Homo sapiens <400> 161 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt tgtgtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 162 <211> 246 <212> PRT <213> Homo sapiens <400> 162 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Cys Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 163 <211> 741 <212> DNA <213> Homo sapiens <400> 163 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gccctgcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 164 <211> 246 <212> PRT <213> Homo sapiens <400> 164 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Cys Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 165 <211> 741 <212> DNA <213> Homo sapiens <400> 165 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt tgtccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 166 <211> 246 <212> PRT <213> Homo sapiens <400> 166 Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr 1 5 10 15 Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr             20 25 30 Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His         35 40 45 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly     50 55 60 Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu 65 70 75 80 Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Arg Pro                 85 90 95 Gly Leu Ala Gly Gly Arg Pro Glu Gln Tyr Phe Gly Pro Gly Thr Arg             100 105 110 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala         115 120 125 Val Phe Cys Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr     130 135 140 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 145 150 155 160 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro                 165 170 175 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu             180 185 190 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn         195 200 205 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu     210 215 220 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 225 230 235 240 Ala Trp Gly Arg Ala Asp                 245 <210> 167 <211> 783 <212> DNA <213> Homo sapiens <400> 167 atgggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240 gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 300 ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360 cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420 ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480 gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540 ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600 gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660 tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720 gcagacggat ccggtggtgg tctgaacgat atttttgaag ctcagaaaat cgaatggcat 780 taa 783 <210> 168 <211> 260 <212> PRT <213> Homo sapiens <400> 168 Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln 1 5 10 15 Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser             20 25 30 Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser         35 40 45 Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn     50 55 60 Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala 65 70 75 80 Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly                 85 90 95 Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu             100 105 110 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro         115 120 125 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu     130 135 140 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 145 150 155 160 Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys                 165 170 175 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu             180 185 190 Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys         195 200 205 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp     210 215 220 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 225 230 235 240 Ala Asp Gly Ser Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys                 245 250 255 Ile Glu Trp His             260 <210> 169 <211> 762 <212> DNA <213> Homo sapiens <400> 169 atgggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240 gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 300 ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360 cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420 ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480 gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540 ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600 gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660 tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720 gcagacggat ccggtggtgg tcatcatcac catcatcact aa 762 <210> 170 <211> 253 <212> PRT <213> Homo sapiens <400> 170 Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln 1 5 10 15 Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser             20 25 30 Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser         35 40 45 Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn     50 55 60 Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala 65 70 75 80 Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly                 85 90 95 Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu             100 105 110 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro         115 120 125 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu     130 135 140 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 145 150 155 160 Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys                 165 170 175 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu             180 185 190 Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys         195 200 205 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp     210 215 220 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 225 230 235 240 Ala Asp Gly Ser Gly Gly Gly His His His His His His                 245 250 <210> 171 <211> 741 <212> DNA <213> Homo sapiens <400> 171 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaac 360 aaggtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttcttcc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac tctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 172 <211> 741 <212> DNA <213> Homo sapiens <400> 172 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac tgtctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 173 <211> 741 <212> DNA <213> Homo sapiens <400> 173 atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60 acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120 ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180 gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240 tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300 gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480 tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540 gagcagcccg ccctcaatga ctccagatac tctctgagca gccgcctgag ggtctcggcc 600 accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720 gcctggggta gagcagacta a 741 <210> 174 <211> 6671 <212> DNA <213> Expression vector pYX112 <400> 174 gaattcacca tggatcctag ggcccacaag cttacgcgtc gacccgggta tccgtatgat 60 gtgcctgact acgcatgata tctcgagctc agctagctaa ctgaataagg aacaatgaac 120 gtttttcctt tctcttgttc ctagtattaa tgactgaccg atacatccct tttttttttt 180 gtctttgtct agctccagct tttgttccct ttagtgaggg ttaattcaat tcactggccg 240 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 300 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 360 aacagttgcg cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg 420 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 480 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 540 taaatcgggg gctcccttta gggttccgat ttagtggttt acggcacctc gaccccaaaa 600 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 660 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 720 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 780 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 840 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 900 agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat 960 ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc 1020 ctgcttttct gtaacgttca ccctgtacct tagcatccct tccctttgca aatagtcctc 1080 ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt 1140 gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt 1200 aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt 1260 cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc 1320 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct 1380 gcttcaaacc gctaacaata cctggcccca gcacaccgtg tgcattcgta atgtctgccc 1440 attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag 1500 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct 1560 taactgtgcc ctccatcgaa aaatcagtca atatatccac atgtgttttt agtaaacaaa 1620 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg 1680 aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac 1740 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc 1800 tgcaggtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa 1860 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct 1920 gttcggagat taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa 1980 aaaaaaatga tgaattgaat tgaaaagctg tggtatggtg cactctcagt acaatctgct 2040 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 2100 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 2160 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 2220 gcctattttt ataggttaat gtcatgataa taatggtttc ttaggacgga tcgcttgcct 2280 gtaacttaca cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt 2340 ttatttattt ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt 2400 tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt 2460 tacatatata tttattagac aagaaaagca gattaaatag atatacattc gattaacgat 2520 aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac 2580 aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga 2640 tccccctaga gtcttttaca tcttcggaaa acaaaaacta ttttttcttt aatttctttt 2700 tttactttct atttttaatt tatatattta tattaaaaaa tttaaattat aattattttt 2760 atagcacgtg atgaaaagga cccaggtggc acttttcggg gaaatgtgcg cggaacccct 2820 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccgtga 2880 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2940 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3000 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3060 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3120 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3180 gctcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3240 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3300 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3360 ttggacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 3420 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 3480 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3540 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3600 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3660 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3720 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3780 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3840 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3900 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3960 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4020 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4080 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4140 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4200 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4260 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4320 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4380 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4440 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4500 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4560 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 4620 gtggataacc gtattaccgc ctttgagtga gctgataccg gtcgccgcag ccgaacgacc 4680 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 4740 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 4800 ggcagtgagc gcaacgcaat taatgtgagt tacctcactc attaggcacc ccaggcttta 4860 cactttatgc ttccggctcc tatgttgtgt ggaattgtga gcggataaca atttcacaca 4920 ggaaacagct atgaccatga ttacgccaag ctcgaaatac gactcactat agggcgaatt 4980 gggtaccggg ccggccgtcg agcttgatgg catcgtggtg tcacgctcgt cgtttggtat 5040 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 5100 aaaaaaagcg gttagctctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5160 ttatcactca tggttatggc aggaactgca taattctctt actgtcatgc catccgtaag 5220 atgcttttct gtgactggtg tactcaacca agtcattctg agaatagtgt atgcggcgac 5280 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa 5340 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 5400 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 5460 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 5520 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 5580 atcagggtta ttgtctcatg agcgatacat atttgaatgt atttagaaaa ataaacaaat 5640 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 5700 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattggg 5760 gatctacgta tggtcattct tcttcagatt ccctcatgga gaagtgcggc agatgtatat 5820 gacagagtcg ccagtttcca agagacttta ttcaggcact tccatgatag gcaagagaga 5880 agacccagag atgttgttgt cctagttaca catggtattt attccagagt attcctgatg 5940 aaatggttta gatggacata cgaagagttt gaatcgttta ccaatgttcc taacgggagc 6000 gtaatggtga tggaactgga cgaatccatc aatagatacg tcctgaggac cgtgctaccc 6060 aaatggactg attgtgaggg agacctaact acatagtgtt taaagattac ggatatttaa 6120 cttacttaga ataatgccat ttttttgagt tataataatc ctacgttagt gtgagcggga 6180 tttaaactgt gaggacctca atacattcag acacttctga cggtatcacc ctacttattc 6240 ccttcgagat tatatctagg aacccatcag gttggtggaa gattacccgt tctaagactt 6300 ttcagcttcc tctattgatg ttacactcgg acaccccttt tctggcatcc agtttttaat 6360 cttcagtggc atgtgagatt ctccgaaatt aattaaagca atcacacaat tctctcggat 6420 accacctcgg ttgaaactga caggtggttt gttacgcatg ctaatgcaaa ggagcctata 6480 tacctttggc tcggctgctg taacagggaa tataaagggc agcataattt aggagtttag 6540 tgaacttgca acatttacta ttttcccttc ttacgtaaat atttttcttt ttaattctaa 6600 atcaatcttt ttcaattttt tgtttgtatt cttttcttgc ttaaatctat aactacaaaa 6660 aacacataca g 6671 <175> 175 <211> 6743 <212> DNA <213> Expression vector pYX122 <400> 175 gaattcacca tggatcctag ggcccacaag cttacgcgtc gacccgggta tccgtatgat 60 gtgcctgact acgcatgata tctcgagctc agctagctaa ctgaataagg aacaatgaac 120 gtttttcctt tctcttgttc ctagtattaa tgactgaccg atacatccct tttttttttt 180 gtctttgtct agctccagct tttgttccct ttagtgaggg ttaattcaat tcactggccg 240 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 300 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 360 aacagttgcg cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg 420 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 480 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 540 taaatcgggg gctcccttta gggttccgat ttagtggttt acggcacctc gaccccaaaa 600 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 660 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 720 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 780 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 840 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 900 agatccgtcg agttcaagag aaaaaaaaag aaaaagcaaa aagaaaaaag gaaagcgcgc 960 ctcgttcaga atgacacgta tagaatgatg cattaccttg tcatcttcag tatcatactg 1020 ttcgtataca tacttactga cattcatagg tatacatata tacacatgta tatatatcgt 1080 atgctgcagc tttaaataat cggtgtcact acataagaac acctttggtg gagggaacat 1140 cgttggttcc attgggcgag gtggcttctc ttatggcaac cgcaagagcc ttgaacgcac 1200 tctcactacg gtgatgatca ttcttgcctc gcagacaatc aacgtggagg gtaattctgc 1260 ttgcctctgc aaaactttca agaaaatgcg ggatcatctc gcaagagaga tctcctactt 1320 tctccctttg caaaccaagt tcgacaactg cgtacggcct gttcgaaaga tctaccaccg 1380 ctctggaaag tgcctcatcc aaaggcgcaa atcctgatcc aaaccttttt actccacgcg 1440 ccagtagggc ctctttaaat gcttgaccga gagcaatccc gcagtcttca gtggtgtgat 1500 ggtcgtctat gtgtaagtca ccaatgcact caacgattag cgaccagccg gaatgcttgg 1560 ccagagcatg tatcatatgg tccagaaacc ctatacctgt gtggacgtta atcacttgcg 1620 attgtgtggc ctgttctgct actggttctg cctctttttc tgggaagatc gagtgctcta 1680 tcgctagggg accagccttt aaagagatcg caatctgaat cttggtttca tttgtaatac 1740 gctttactag ggctttctgc tctgtcatct ttgccttcgt ttatcttgcc tgctcatttt 1800 ttagtatatt cttcgaagaa atcacattac tttatataat gtataattca ttatgtgata 1860 atgccaatcg ctaagaaaaa aaaagagtca tccgctaggg gaaaaaaaaa aatgaaaatc 1920 attaccgagg cataaaaaaa tatagagtgt actagaggag gccaagagta atagaaaaag 1980 aaaattgcgg gaaaggactg tgttatgact tccctgacta atgccgtgtt caaacgatac 2040 ctggcagtga ctcctagcgc tcaccaagct cttaaaacgg gaatttatgg tgcactctca 2100 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 2160 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 2220 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 2280 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttaggacg 2340 gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat aatttgggaa 2400 tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag taaataaaga 2460 aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa aatttcaaca 2520 aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat agatatacat 2580 tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc ttctacacag 2640 acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat aaaaggtagt 2700 atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac tattttttct 2760 ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa aatttaaatt 2820 ataattattt ttatagcacg tgatgaaaag gacccaggtg gcacttttcg gggaaatgtg 2880 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 2940 caataaccgt gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 3000 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 3060 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 3120 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 3180 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 3240 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 3300 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 3360 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 3420 ctaaccgctt ttttggacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 3480 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 3540 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 3600 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 3660 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 3720 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 3780 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 3840 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 3900 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 3960 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4020 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4080 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4140 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4200 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4260 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4320 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4380 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4440 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4500 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4560 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4620 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 4680 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 4740 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 4800 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 4860 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttacctcac tcattaggca 4920 ccccaggctt tacactttat gcttccggct cctatgttgt gtggaattgt gagcggataa 4980 caatttcaca caggaaacag ctatgaccat gattacgcca agctcgaaat acgactcact 5040 atagggcgaa ttgggtaccg ggccggccgt cgagcttgat ggcatcgtgg tgtcacgctc 5100 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 5160 ccccatgttg tgaaaaaaag cggttagctc ttcggtcctc cgatcgttgt cagaagtaag 5220 ttggccgcag tgttatcact catggttatg gcaggaactg cataattctc ttactgtcat 5280 gccatccgta agatgctttt ctgtgactgg tgtactcaac caagtcattc tgagaatagt 5340 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata 5400 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5460 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5520 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5580 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5640 attgaagcat ttatcagggt tattgtctca tgagcgatac atatttgaat gtatttagaa 5700 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 5760 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 5820 tcaagaattg gggatctacg tatggtcatt cttcttcaga ttccctcatg gagaagtgcg 5880 gcagatgtat atgacagagt cgccagtttc caagagactt tattcaggca cttccatgat 5940 aggcaagaga gaagacccag agatgttgtt gtcctagtta cacatggtat ttattccaga 6000 gtattcctga tgaaatggtt tagatggaca tacgaagagt ttgaatcgtt taccaatgtt 6060 cctaacggga gcgtaatggt gatggaactg gacgaatcca tcaatagata cgtcctgagg 6120 accgtgctac ccaaatggac tgattgtgag ggagacctaa ctacatagtg tttaaagatt 6180 acggatattt aacttactta gaataatgcc atttttttga gttataataa tcctacgtta 6240 gtgtgagcgg gatttaaact gtgaggacct caatacattc agacacttct gacggtatca 6300 ccctacttat tcccttcgag attatatcta ggaacccatc aggttggtgg aagattaccc 6360 gttctaagac ttttcagctt cctctattga tgttacactc ggacacccct tttctggcat 6420 ccagttttta atcttcagtg gcatgtgaga ttctccgaaa ttaattaaag caatcacaca 6480 attctctcgg ataccacctc ggttgaaact gacaggtggt ttgttacgca tgctaatgca 6540 aaggagccta tatacctttg gctcggctgc tgtaacaggg aatataaagg gcagcataat 6600 ttaggagttt agtgaacttg caacatttac tattttccct tcttacgtaa atatttttct 6660 ttttaattct aaatcaatct ttttcaattt tttgtttgta ttcttttctt gcttaaatct 6720 ataactacaa aaaacacata cag 6743 <210> 176 <211> 296 <212> PRT <213> Homo sapiens <400> 176 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Thr Glu Asp Glu Thr Ala Gln             20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe         35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu     50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Ala Glu Ala Gln Glu Val Thr Gln Ile Pro                 85 90 95 Ala Ala Leu Ser Val Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser             100 105 110 Phe Thr Asp Ser Ala Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro         115 120 125 Gly Lys Gly Leu Thr Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu     130 135 140 Gln Thr Ser Gly Arg Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg 145 150 155 160 Ser Thr Leu Tyr Ile Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr                 165 170 175 Leu Cys Ala Val Arg Pro Thr Ser Gly Gly Ser Tyr Ile Pro Thr Phe             180 185 190 Gly Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp         195 200 205 Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val     210 215 220 Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys 225 230 235 240 Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser                 245 250 255 Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp             260 265 270 Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr         275 280 285 Phe Phe Pro Ser Pro Glu Ser Ser     290 295 <210> 177 <211> 903 <212> DNA <213> Homo sapiens <400> 177 gaattcatga gatttccttc aatttttact gcagttttat tcgcagcatc ctccgcatta 60 gctgctccag tcaacactac aacagaagat gaaacggcac aaattccggc tgaagctgtc 120 atcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt ttccaacagc 180 acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc taaagaagaa 240 ggggtatctt tggataaaag agaggctgaa gcccaggagg tgacacagat tcctgcagct 300 ctgagtgtcc cagaaggaga aaacttggtt ctcaactgca gtttcactga tagcgctatt 360 tacaacctcc agtggtttag gcaggaccct gggaaaggtc tcacatctct gttgcttatt 420 cagtcaagtc agagagagca aacaagtgga agacttaatg cctcgctgga taaatcatca 480 ggacgtagta ctttatacat tgcagcttct cagcctggtg actcagccac ctacctctgt 540 gctgtgaggc ccacatcagg aggaagctac atacctacat ttggaagagg aaccagcctt 600 attgttcatc cgtatatcca gaacccggat cctgccgtgt accagctgag agactctaaa 660 tccagtgaca agtctgtctg cctattcacc gattttgatt ctcaaacaaa tgtgtcacaa 720 agtaaggatt ctgatgtgta tatcacagac aaatgtgtgc tagacatgag gtctatggac 780 ttcaagagca acagtgctgt ggcctggagc aacaaatctg actttgcatg tgcaaacgcc 840 ttcaacaaca gcattattcc agaagacacc ttcttcccca gcccagaaag ttcctaactc 900 gag 903 <210> 178 <211> 330 <212> PRT <213> Homo sapiens <400> 178 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Thr Glu Asp Glu Thr Ala Gln             20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe         35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu     50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Ala Glu Ala Gly Val Thr Gln Thr Pro Lys                 85 90 95 Phe Gln Val Leu Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln             100 105 110 Asp Met Asn His Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met         115 120 125 Gly Leu Arg Leu Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln     130 135 140 Gly Glu Val Pro Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp 145 150 155 160 Phe Pro Leu Arg Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr                 165 170 175 Phe Cys Ala Ser Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly             180 185 190 Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro         195 200 205 Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr     210 215 220 Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His 225 230 235 240 Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val                 245 250 255 Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser             260 265 270 Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln         275 280 285 Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser     290 295 300 Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile 305 310 315 320 Val Ser Ala Glu Ala Trp Gly Arg Ala Asp                 325 330 <210> 179 <211> 1005 <212> DNA <213> Homo sapiens <400> 179 gaattcatga gatttccttc aatttttact gcagttttat tcgcagcatc ctccgcatta 60 gctgctccag tcaacactac aacagaagat gaaacggcac aaattccggc tgaagctgtc 120 atcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt ttccaacagc 180 acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc taaagaagaa 240 ggggtatctt tggataaaag agaggctgaa gccggcgtca ctcagacccc aaaattccag 300 gtcctgaaga caggacagag catgacactg cagtgtgccc aggatatgaa ccatgaatac 360 atgtcctggt atcgacaaga cccaggcatg gggctgaggc tgattcatta ctcagttggt 420 gctggtatca ctgaccaagg agaagtcccc aatggctaca atgtctccag atcaaccaca 480 gaggatttcc cgctcaggct gctgtcggct gctccctccc agacatctgt gtacttctgt 540 gccagcagtt acgtcgggaa caccggggag ctgttttttg gagaaggctc taggctgacc 600 gtactggagg acctgaaaaa cgtgttccca cccgaggtcg ctgtgtttga gccatcagaa 660 gcagagatct cccacaccca aaaggccaca ctggtgtgcc tggccacagg cttctacccc 720 gaccacgtgg agctgagctg gtgggtgaat gggaaggagg tgcacagtgg ggtctgcaca 780 gacccgcagc ccctcaagga gcagcccgcc ctcaatgact ccagatacgc tctgagcagc 840 cgcctgaggg tctcggccac cttctggcag gacccccgca accacttccg ctgtcaagtc 900 cagttctacg ggctctcgga gaatgacgag tggacccagg atagggccaa acccgtcacc 960 cagatcgtca gcgccgaggc ctggggtaga gcagactaac tcgag 1005 <210> 180 <211> 317 <212> DNA <213> pEX172 Plasmid <400> 180 ggatccagca tggtgtgtct gaagctccct ggaggctcct gcatgacagc gctgacagtg 60 acactgatgg tgctgagctc cccactggct ttgtccggag acaccggtgg cggatctcta 120 gttccacgcg gtagtggagg cggtggttcc ggagacacgc gttagtaggt cgacggaggc 180 ggtgggggta gaatcgcccg gctggaggaa aaagtgaaaa ccttgaaagc tcagaactcg 240 gagctggcgt ccacggccaa catgctcagg gaacaggtgg cacagcttaa acagaaagtc 300 atgaactact aggatcc 317 <210> 181 <211> 884 <212> DNA <213> Homo sapiens <400> 181 ggatccagca tggtgtgtct gaagctccct ggaggctcct gcatgacagc gctgacagtg 60 acactgatgg tgctgagctc cccactggct ttgtccggag acaccggaga caccggacag 120 aaggaagtgg agcagaactc tggacccctc agtgttccag agggagccat tgcctctctc 180 aactgcactt acagtgaccg aggttcccag tccttcttct ggtacagaca atattctggg 240 aaaagccctg agttgataat gtccatatac tccaatggtg acaaagaaga tggaaggttt 300 acagcacagc tcaataaagc cagccagtat gtttctctgc tcatcagaga ctcccagccc 360 agtgattcag ccacctacct ctgtgccgtt acaactgaca gctgggggaa attgcagttt 420 ggagcaggga cccaggttgt ggtcacccca gatatccaga accctgaccc tgccgtgtac 480 cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 540 caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa atgtgtgcta 600 gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 660 tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 720 ccagaaagtt cctaagtcga cggaggcggt gggggtagaa tcgcccggct ggaggaaaaa 780 gtgaaaacct tgaaagctca gaactcggag ctggcgtcca cggccaacat gctcagggaa 840 caggtggcac agcttaaaca gaaagtcatg aactactagg atcc 884 <210> 182 <211> 1004 <212> DNA <213> Homo sapiens <400> 182 ggatccagca tggtgtgtct gaagctccct ggaggctcct gcatgacagc gctgacagtg 60 acactgatgg tgctgagctc cccactggct ttgtccggag acaccggaga caccggaaac 120 gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 180 cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 240 gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 300 aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 360 gctccctccc agacatctgt gtacttctgt gccagcaggc cgggactagc gggagggcga 420 ccagagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 480 ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 540 gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 600 gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 660 cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 720 tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 780 gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 840 ggtagagcag actaagtcga cggaggcggt gggggtagaa tcgcccggct ggaggaaaaa 900 gtgaaaacct tgaaagctca gaactcggag ctggcgtcca cggccaacat gctcagggaa 960 caggtggcac agcttaaaca gaaagtcatg aactactagg atcc 1004 <210> 183 <211> 206 <212> PRT <213> Homo sapiens <400> 183 Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu 1 5 10 15 Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln             20 25 30 Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile         35 40 45 Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala     50 55 60 Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser 65 70 75 80 Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Thr Asp Ser                 85 90 95 Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val Thr Pro             100 105 110 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys         115 120 125 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr     130 135 140 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys 145 150 155 160 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala                 165 170 175 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser             180 185 190 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser         195 200 205

Claims (67)

(i) a T cell receptor (TCR) α chain, excluding the transmembrane domain, and (ii) a TCR β chain, excluding the transmembrane domain, (i) and (ii), respectively By disulfide bonds between cysteine residues comprising a functional variable domain of this TCR chain and a constant domain of the TCR chain, wherein (i) and (ii) have replaced the following residues: Soluble T cell receptor (sTCR), characterized in that linked: Thr 48 of exon 1 of TRAC ^* 01 and Ser 57 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; Thr 45 of exon 1 of TRAC ^* 01 and Ser 77 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; Tyr 10 of exon 1 of TRAC ^* 01 and Ser 17 of exon 1 of TRBC1 * 01 or TRBC2 ^* 01; Thr 45 of exon 1 of TRAC ^* 01 and Asp 59 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; or Ser 15 of exon 1 of TRAC ^* 01 and Glu 15 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. The soluble T cell receptor 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 soluble T cell receptor of claim 1, wherein one or both of (i) and (ii) comprises the entirety of the extracellular domain of the TCR chain. Soluble αβ-form T cell receptor (sTCR), in which disulfide covalent linkages cysteine residues replaced the following residues: Thr 48 of exon 1 of TRAC ^* 01 and Ser 57 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; Thr 45 of exon 1 of TRAC ^* 01 and Ser 77 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; Tyr 10 of exon 1 of TRAC ^* 01 and Ser17 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; Thr 45 of exon 1 of TRAC ^* 01 and Asp 59 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01; or Ser 15 of exon 1 of TRAC ^* 01 and Glu 15 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. The soluble T according to claim 1, wherein (i) and (ii) are connected by newly formed disulfide bonds between the constant domain residues, and the interchain disulfide bonds present in the native TCR are removed or mutated. Cellular receptors. delete delete Disulfide covalent linkages 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, and interchain disulfide bonds present in the native TCR are removed or mutated. Soluble αβ-type T cell receptor. 6. The soluble T cell receptor of claim 5, wherein said newly formed disulfide bond is between cysteine residues substituted at residues wherein the β carbon atom of the residue to be replaced in the native TCR structure is less than 0.6 nm apart. 6. The soluble T cell receptor of claim 5, wherein said newly formed disulfide bond is present between a Thr 48 of exon 1 of TRAC ^* 01 and a cysteine residue that replaces Ser 57 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. delete delete delete delete delete 6. The soluble T cell receptor according to claim 5, wherein the soluble T cell receptor is free of cysteine residues in which the natural α and β TCR chains are cleaved at the C terminus to form native interchain disulfide bonds. 6. The soluble T cell receptor of claim 5, wherein the cysteine residue forming said native interchain disulfide bond is replaced with serine or alanine. delete 9. Soluble T cell receptor according to any one of claims 1, 4 and 8, characterized in that unpaired cysteine residues present in the native TCR β chain have been removed or mutated. 18. The method according to any one of claims 1, 2, 5, 9, 10, 16 and 17, wherein each of (i) and (ii) is a constant domain of a second TCR. And a functional variable domain of a first TCR fused to said first TCR and said second TCR, wherein said first TCR and said second TCR are from the same species. 21. The soluble T cell receptor of claim 20, wherein said constant domain of said second TCR is cleaved at the N terminus of a residue forming a non-natural interchain disulfide bond. The method of claim 1, wherein one or both of the α and β chains are fused to a multimeric binding moiety at its C or N terminus, a toxic moiety for use in apoptosis, or a therapeutic moiety. Soluble T cell receptor. The soluble T cell of claim 22, wherein one or both of the α and β chains have a cysteine residue to which the moiety can be fused at its C, N or C and N terminus. Receptors. The soluble T cell receptor according to any one of claims 1, 4 and 8, wherein one or both of the α and β chains further comprise the detectable label. The soluble T cell receptor according to any one of claims 1, 4 and 8, wherein the soluble T cell receptor is associated with a therapeutic agent. Multivalent T cell receptor complex comprising a plurality of soluble T cell receptors according to claim 1. 27. The multivalent T cell receptor complex of claim 26, wherein said multivalent T cell receptor complex comprises a soluble T cell receptor multimer. The multivalent T cell receptor complex of claim 27, wherein the multivalent T cell receptor complex comprises two or more T cell receptor molecules joined with another T cell receptor molecule via a linker molecule. The multivalent T cell receptor complex of claim 27, wherein the soluble T cell receptor or soluble T cell receptor multimer is present in or attached to a particle in the lipid bilayer. (i) providing a soluble T cell receptor according to any one of claims 1, 4 and 8 or a multivalent T cell receptor complex according to any one of claims 26 to 29; (ii) contacting the soluble T cell receptor or multivalent T cell receptor complex with an MHC-peptide complex; And (iii) detecting binding of said soluble T cell receptor or multivalent T cell receptor complex to an MHC-peptide complex. A soluble T cell receptor according to any one of claims 1, 4 and 8, a multivalent T cell receptor complex according to any one of claims 26 to 29 or both are pharmaceutically acceptable. A pharmaceutical formulation for treating cancer, autoimmune disease, transplant rejection or graft-versus-host disease, characterized in that it comprises a carrier. A nucleic acid molecule comprising a sequence encoding (i) or (ii) of the soluble T cell receptor according to claim 1 or a sequence complementary thereto. A vector comprising the nucleic acid molecule of claim 32. A host cell comprising the vector according to claim 33. 35. A method of producing (i) or (ii) according to claim 1, comprising culturing the host cell according to claim 34 under conditions expressing the peptide and purifying the polypeptide. 36. The method of claim 35, further comprising mixing (i) and (ii). A host cell having a vector comprising a nucleic acid molecule encoding a T cell receptor α chain (i) comprising a functional variable domain and a constant domain of the TCR chain and excluding a transmembrane domain, and a functional variable domain and a constant of the TCR chain Culturing a host cell having a vector comprising a nucleic acid molecule encoding a TCR β chain (ii) excluding a transmembrane domain, under a condition inducing expression of (i) and (ii); Purifying the above (i) and (ii); And Mixing (i) and (ii) such that they are linked by a newly formed disulfide bond between constant domain residues. 38. The method of claim 37, wherein one or both of (i) and (ii) comprises the entirety of the extracellular constant Ig domain of the TCR chain. The method of claim 37 or 38, wherein one or both of (i) and (ii) comprises the entirety of the extracellular domain of the TCR chain. Culturing a host cell having a vector comprising a nucleic acid molecule encoding a TCR α chain, and a host cell having a vector comprising a nucleic acid molecule encoding a TCR β chain under conditions that induce expression of each TCR chain; Purifying each TCR chain; And Soluble αβ-type T comprising mixing each of the TCR chains by disulfide covalent linkage so that the residues of the immunoglobulin region of the constant domain of the α chain are linked to the residues of the immunoglobulin region of the constant domain of the β chain How to produce cell receptors. 41. The method of any one of claims 37, 38, and 40, wherein the soluble T cell receptor has been removed or mutated between the interchain disulfide bonds present in the native TCR. 42. The method of claim 41, wherein said soluble T cell receptor is free of cysteine residues in which native α and β TCR chains are cleaved at the C terminus to form native interchain disulfides. 42. The method of claim 41, wherein the soluble T cell receptor is replaced with a serine or alanine cysteine residue that forms a native interchain disulfide bond. delete 41. The method of any one of claims 37, 38 and 40, wherein the soluble T cell receptor has been removed or mutated with unpaired cysteine residues present in the native TCR β chain. 39. The disulfide bond substituted for said newly formed residue is between said cysteine residues substituted for residues wherein the β carbon atom of the residue to be replaced in said native TCR structure is less than 0.6 nm. How to. 39. The method of claim 37 or 38 wherein the newly formed disulfide bond is between a Thr 48 of exon 1 of TRAC ^* 01 and a cysteine residue that replaces Ser 57 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. How to. 39. The method of claim 37 or 38 wherein the newly formed disulfide bond is between a Thr 45 of exon 1 of TRAC ^* 01 and a cysteine residue that replaces Ser 77 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. How to. 39. The method of claim 37 or 38, wherein the newly formed disulfide bond is between a Tyr 10 of exon 1 of TRAC ^* 01 and a cysteine residue that replaces Ser 17 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. How to. 39. The method of claim 37 or 38, wherein the newly formed disulfide bond is present between a Thr 45 of exon 1 of TRAC ^* 01 and a cysteine residue that replaces Asp 59 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. How to. The method of claim 37 or 38, wherein the newly formed disulfide bond is present between a cysteine residue that replaces Ser 15 of exon 1 of TRAC ^* 01 and Glu 15 of exon 1 of TRBC1 ^* 01 or TRBC2 ^* 01. How to. The method according to claim 37 or 38, wherein (i) and (ii) each comprise a functional variable domain of a first TCR fused to a constant domain of a second TCR, wherein the first TCR and the second TCR are A method characterized by derived from the same species. The method of claim 52, wherein the constant domain of the second TCR is cleaved at the N terminus of a residue that forms a non-natural interchain disulfide bond. The method of claim 37 or 38, wherein one or both of the α and β chains are fused to a multimeric binding moiety at its C or N terminus, a toxic moiety for use in apoptosis, or a therapeutic moiety. Characterized in that the method. 55. The method of claim 54, wherein one or both of the α and β chains have a cysteine residue to which the moiety can be fused at its C, N, or both C and N ends. 41. The method of any one of claims 37, 38 and 40, wherein the soluble T cell receptor further comprises a detectable label. 41. The method of any one of claims 37, 38 and 40, wherein the soluble T cell receptor is associated with a therapeutic agent. 38. The method of claim 37, further comprising combining a plurality of soluble T cell receptors to form a multivalent T cell receptor complex. 59. The method of claim 58, wherein the soluble T cell receptors combine to form a soluble T cell receptor multimer. 60. The method of claim 59, wherein the soluble T cell receptor multimer is characterized in that two or more T cell receptor molecules are coupled to another T cell receptor molecule via a linker molecule. 60. The method of claim 59, wherein the soluble T cell receptor or soluble T cell receptor multimer is combined within a lipid bilayer or attached to a particle. (i) providing a soluble T cell receptor produced according to the method of any one of claims 37, 38 and 40 or a multivalent T cell receptor complex produced according to the method according to claim 58; (ii) contacting said soluble T cell receptor or multivalent T cell receptor complex with an MHC-peptide complex; And (iii) detecting binding of said soluble T cell receptor or multivalent T cell receptor complex to an MHC-peptide complex. 41. A soluble T cell receptor produced according to the method of any one of claims 37, 38 and 40, a multivalent T cell receptor complex produced according to the method of claim 58 or both with a pharmaceutically acceptable carrier. A pharmaceutical agent for treating cancer, autoimmune disease, transplant rejection or graft-versus-host disease, characterized in that it comprises together. 23. The soluble T cell receptor of claim 22, wherein said multimeric binding moiety is a linker molecule selected from the group consisting of avidin, streptavidin, neutravidin and extravidin. The method of claim 22, wherein the therapeutic moiety is from a group consisting of cytokines, chemokines, antibodies or antibody fragments, complement activators, heterologous protein domains, homologous protein domains, viral protein domains and viral peptides. Soluble T cell receptor, characterized in that the selected immunostimulant. 55. The method of claim 54, wherein said multimer binding moiety is a linker molecule selected from the group consisting of avidin, streptavidin, neutravidin and extravidin. 55. The therapeutic moiety of claim 54, wherein the therapeutic moiety is selected from the group consisting of cytokines, chemokines, antibodies or antibody fragments, complement activators, heterologous protein domains, homologous proteins, viral protein domains and viral peptides. A method of immunostimulating.

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