EA006601B1 - Soluble t cell receptor - Google Patents

Abstract

The present invention provides a soluble T cell receptor (sTCR), which comprises (i) all for part of a TCR alpha chain, except the transmembrane domain thereof, and (ii) all or part of a TCR beta chain, except the transmembrane domain thereof. (i) and (ii) each comprise a functional variable domain and at least a part of the constant domain of the TCR chain, and are linked by a disulphide bond between constant domain residues which is not present in native TCR.

Description

As indicated in the patent application \ ¥ 0 99/60120, TSCs mediate the recognition of specific complexes. The main histocompatibility complex (Mart H181 ocotrayU1yu Sotr1ekh, MHC) peptide with lymphocytes and in this capacity are important for the functioning of the cellular branch of the immune system.

Only two types of molecules - antibodies and TSC - can recognize antigens in a specific way, therefore, TSC is the only receptor for recognition of specific peptide antigens presented in the MHC, while a foreign peptide is often the only sign of cell abnormality. T-lymphocyte recognition occurs when a T-lymphocyte and an antigen-presenting cell (apdep rhehebbpd ce11, APC) are in direct physical contact and is initiated by ligation of antigen-specific TSC with rMHC complexes (peptide-MHC).

TSC is a heterodimeric protein on the cell surface of the immunoglobulin superfamily, which is associated with invariant proteins of the SEZ complex involved in mediating signal transduction. TSCs exist in the forms of αβ and γδ, which are similar in structure, however, the T-lymphocytes expressing them have completely different anatomical locations and, probably, functions. The extracellular part of the receptor consists of two constant domains proximal relative to the membrane and two variable domains distal relative to the membrane, containing polymorphic loops similar to the hypervariable regions (elliptic 4e1epshpd testidon RNA) of antibodies. It is these loops that form the binding site of the TSC molecule and determine peptide specificity. MHC class I and class II ligands also belong to the immunoglobulin superfamily proteins, however, they specialize in presenting antigens and possess a polymorphic part that binds peptides and allows them to represent different sets of short peptide fragments on the surface of APC cells.

Soluble TSCs are useful not only for studying the specific interactions of TSCrMNS, but also as a potential diagnostic tool for detecting infection or markers of autoimmune disease. Soluble TSCs are also used for staining, for example, for staining cells to determine the presence of a particular peptide antigen presented in the context of MHC. Similarly, soluble TSCs can be used to deliver a therapeutic agent, for example a cytotoxic compound or an immunostimulating compound, to cells representing a particular antigen. Soluble TSCs can also be used to suppress T-lymphocytes, for example, reacting with an autoimmune peptide antigen.

When obtaining a soluble form of proteins formed by several polypetid subunits and containing a transmembrane domain, difficulties may arise, since in many cases the protein is stabilized by its transmembrane region. This also applies to TSCs, and is reflected in the scientific literature, which describes truncated forms of TSCs containing only extracellular domains or extracellular and cytoplasmic domains that can be recognized by antibodies specific to this TSC (indicating that part of the recombinant TSC recognized it was laid accordingly), but which could not be obtained in good yield, stable at low concentrations and / or capable of recognizing peptide MHC complexes. A review of such literature is given in \ UO 99/60120.

A number of studies have described the preparation of TSC heterodimers that contain a native disulfide bridge that binds the corresponding subunits (Cactus ek1., (1996), Icig 384 (6605): 134-41; baclose, e! A1., (1996) , I. 1ttypo1. 157 (12): 5403-10; Syapd e! A1., (1994), ΡΝΑ8 I8A 91: 11408-11412; Eauobey e! A1., (1993), I. Bü1. Set. 268 ( 21): 15455-15460; Oo14ep e! A1., (1997), I. 1 vol. Me! 206: 163-169; I8 Ra! Ep! Io. 6080840). However, although such TSCs can be recognized by TSC-specific antibodies, they have not been shown to recognize their native ligand, except at relatively high concentrations and / or they were unstable.

In UO 99/60120, a soluble TSC is described, which is correctly positioned and therefore can recognize its native ligand, is stable for a certain period of time, and can be obtained in sufficient quantities. This TSC contains the extracellular domain of the TSC α or γ chain dimerized with the extracellular domain of the TSC β or δ chain, respectively, using a pair of dimerization peptides of the C-terminal region, in particular peptides with leucine zippers. This strategy for obtaining TSCs is generally applicable to all TSCs.

Kayet e! A1., 1 TIPIU, 1995, 2: 281-287, describe in detail the construction of a soluble molecule containing disulfide-stabilized variable α- and β-domains of TSC, one of which is associated with the truncated form of Rkeibotopac exooxox (PE38). One of the reasons for obtaining such a molecule was to overcome the instability inherent in single-stranded TSC. The position of the new disulfide bond in the variable domains of TSC was identified by homology with the variable domains of antibodies into which they were previously introduced (see, for example, Vptiktapi, e! A1. (1993), Prog.

-1006601 No. 111. Asab. 8οί. I8A 90: 7538-7542, and the Web, e! a1. (1994) WusNetNetMgu 33: 5451-5459). However, there is no such homology between the antibody and constant domains. Therefore, the use of this method in order to identify the corresponding sites for new interchain disulfide bonds located between the constant domains of TCB was impossible.

Given the importance of soluble TSB, it would be desirable to provide an alternative way to obtain such molecules.

According to a first objective, the present invention provides a soluble T-lymphocyte receptor (co1b1e T ce11 geserog, kTSB), which contains (I) all or part of the TCB α-chain, except for its transmembrane domain and (II) the entire TCB β-chain or its part, with the exception of its transmembrane domain, each of components (I) and (II) containing a functional variable domain and at least part of the constant domain of the TCB chain, and they are connected by a disulfide bond between the (amino acid) residues of the constant domain, which is absent in native Mr. TSV.

According to another object of the invention, the invention provides a soluble T-lymphocyte receptor (kTSB) in αβ form in which a covalent disulfide bond connects the (amino acid) residue from the immunoglobulin region of the constant domain of the α chain with the residue from the immunoglobulin region of the constant domain of the β chain.

The advantage of kTSV according to the invention lies in the fact that they do not contain heterologous polypeptides, which can be immunogenic or which can lead to the rapid elimination of kTSV from the body. In addition, the TCBCs of the present invention have a three-dimensional structure that is highly similar to the native TSCs from which they were derived. Due to this structural similarity, they are unlikely to be immunogenic. KTSV in accordance with the invention can be used to recognize complexes of MHC class I - peptide or complexes of MHC class II - peptide.

TSCs according to the present invention are soluble. In the context of this application, solubility is defined as the ability of TSCs to be purified in the form of a monodispersed heterodimer in phosphate buffered saline (pHcohRa1e LiGGegebache, PB8) (KCb 2.7 mM, KH ₂ PO ₄ 1.5 mM, No. 1C1 137 mM and Ν α ₂ ΡΟ _{April 8} mM, pH 7.1-7.5. faire Tes1to1osche5. 01so DDL) at a concentration of 1 mg / ml, with> 90% yield ECT remains in the form of monodisperse heterodimer after incubation at 25 ° C for 1 hour. To determine the solubility of the TSB, it is first purified as described in Example 2. After this purification, 100 μg of TSC is analyzed by gel chromatography using, for example, a Ryatshaaa 8iregbeh 75 NK column balanced with PB8. Another 100 μg TC K were incubated at 25 ° C for 1 h and then analyzed by gel chromatography as described above. Then, using the integration, the chromatogram is analyzed and the areas under the peaks corresponding to the monodisperse heterodimer are compared. The corresponding peaks can be identified by comparison with the position of the elution of standard proteins with known molecular weight. Monodispersed heterodimeric soluble TCB has a molecular weight of about 50 kDa. As indicated above, TSCs according to the present invention are soluble. However, as explained in more detail below, TSC can be connected to the group so that the resulting complex is insoluble, or they can be presented on the surface of an insoluble solid substrate.

The numbering of TSC amino acids used in this description corresponds to the immunogenetic database system ([ttypedebeps ba! Bake,! MST) described in the work of Tne T Ce11 Veserug Rask! Bok, 2001, Bertais & Bertaps, Asabetk Rheck. In this system, the α-chain constant domain has the following designation: TBAC * 01, where TB is the T-lymphocyte receptor gene, A is the α-chain gene, C is the constant region and * 01 is the allele 1. The β-chain constant domain has the following designation: TVVS1 * 01. In this case, there are two possible genes for the constant region C1 and C2. The translated domain encoded by each allele can be formed by the genetic code of several exons, therefore they are also specific. Amino acids are numbered according to the exon of the particular domain in which they are present.

The extracellular part of native TSC consists of two polypeptides (αβ or γδ), each of which has a constant domain proximal to the membrane and a variable domain distal to the membrane (see Fig. 1). Each of the constant and variable domains contains an intrachain disulfide bond. Variable domains contain highly polymorphic loops similar to the hypervariable regions (antithelium backbone hepatic, hepatic, COB) antibodies. SEV3 TSV interacts with the peptide represented by MHC, and SEV 1 and 2 interact with the peptide and MHC. The variety of TSC sequences is formed by somatic rearrangement of the connected variable (Yapae, V), diverse (OschetkNu, Ό), connective (1o1n1pd, 1) and constant (Sopkaii, C) genes. Functional polypeptides of α-chains consist of rearranged U-1-C sites, while β-chains consist of U-E-1-C sites. The extracellular constant domain contains a proximal membrane region and an immunoglobulin region. The proximal to the membrane region contains amino acids between the transmembrane domain and the cysteine residue proximal to the membrane. The constant domain of immunoglobulin consists of the rest of the residues of amino acids of the constant domain, occupying a section from proximal to the cysteine membrane to the beginning of the connective region, and is characterized by the presence of an immunoglobulin-type folding. There is one constant domain of the α chain, known as Ca1 or TNAC * 01, and two different constant β domains, known as Cb 1 or TNBS1 * 01 and Cv2 or TNBS2 * 01. The difference between these constant β-domains is in amino acid residues 4, 5, and 37 of exon 1. Thus, TNBS1 * 01 contains 1,4Ν, 5K, and 37P in its exon, and TNBS2 * 01 contains 14K, 5Ν, and 37Υ in its exon. The sizes of all extracellular domains of TSN vary within certain limits.

According to the present invention, a disulfide bond is introduced between residues located in the constant domains (or parts thereof) of the corresponding chains. The corresponding TSN chains contain a sufficient number of variable domains to interact with its rMNS complex. Such an interaction can be measured using a B1Acoge 3000 ™ or a B1Acoge 2000 ™ device, as described further in Example 3 or in patent application \ ¥ 0 99/6120, respectively.

In one embodiment of the invention, the corresponding §TCN chains according to the invention also contain intrachain disulfide bonds. TCHs according to the present invention may comprise the entire extracellular constant region 1 e of the corresponding TCH chains, and preferably the entire extracellular domain of the corresponding chains, i.e. including the region proximal to the membrane. In native TSN there is a disulfide bond that connects the conserved regions of the corresponding chains proximal to the membrane. In one embodiment of the present invention, this disulfide bond is absent. This can be achieved by mutating the corresponding cysteine residues (aminoxylot 4, exon 2 of the TNAC * 01 gene and amino acid 2 of both genes TNBS1 * 01 and TNBS2 * 01, respectively) into another amino acid or truncation of the corresponding chains in such a way as to exclude cysteine residues. A preferred soluble TCH according to the invention contains native TCH α and β chains truncated in the C-terminal region so as to exclude cysteine residues that form a native interchain disulfide bond, i.e. truncated in residues 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the Ν-terminal region with cysteine residues. However, it should be noted that a native interchain disulfide bond may be present in TCH according to the present invention and that in some embodiments only one of the TCH chains has a native cysteine residue that forms a native interchain disulfide bond. This cysteine can be used to attach various groups to TSN.

However, the corresponding TSN chains may be shorter. Since the constant domains do not have direct contacts with the peptide-MHC ligands, the position of the truncation point in the C-region can be substantially changed without loss of functionality.

Alternatively, a larger fragment of constant domains may be presented than in the preferred case, i.e. constant domains can not be processed immediately before cysteine residues, which form an interchain disulfide bond. For example, you can include the full constant domain with the exception of the transmembrane domain (i.e., extracellular and cytoplasmic domains). In this case, it may be preferable to mutate one or more cysteine residues forming an interchain disulfide bond in cellular TCH into another amino acid residue that is not involved in the formation of a disulfide bond, or to remove one or more of these residues.

A signal peptide can be excluded if soluble TSN is to be expressed in prokaryotic cells, for example, E. soi, since it does not perform any task in mature TSN while ensuring its ability to bind to the ligand and, under certain circumstances, can inhibit the formation of functional soluble TSN. In most cases, the cleavage site by which the signal peptide is removed from mature TCH chains can be predicted, but cannot be determined experimentally. In this case, modification of the expressed TSN chains so that they are several, for example, up to 10 amino acids, longer or shorter in the Ν-terminal region, may not be significant for the functionality (i.e., ability to recognize rMNC) of soluble TSN. You can enter certain additives that are not in the sequence of the original proteins. So, for example, you can add a short label sequence, which can help clean the TSN chains, provided that it is not an obstacle to the correct structure and placement of the TSN antigen-binding site.

For expression in E. soi, the methionine residue can be introduced into the starting point of the кон-terminal region of the predicted mature protein sequence to ensure translation initiation is possible.

Not all residues in the variable domains of TSN chains are essential for antigenic specificity and functionality. Therefore, a significant number of mutations can be introduced into this domain without affecting antigenic specificity and functionality. Also, far from all residues in the constant domains of TSN chains are essential for antigenic specificity and functionality. Therefore, a significant number of mutations can be introduced into this area without affecting antigenic specificity.

The TSC β-chain contains a cysteine residue that is unpaired in the cellular or native TSC. It is preferable to remove this cysteine group or mutate it into another group in order to avoid incorrect interchain or intrachain pairing. Substituents of this cysteine group, for example, serine or alanine, can have a significant positive effect on the stacking efficiency of ΐπ νΐΐΓΟ.

A disulfide bond can be obtained by mutating non-cysteine residues in the corresponding chains into cysteine and initiating the formation of bonds between the mutated groups. Preferred are groups in which the distance between the corresponding β-carbon atoms in the native TSC is about 6 A (0.6 nm) or less, and preferably in the range from 3.5 A (0.35 nm) to 5.9 A (0 , 59 nm), while a disulfide bond can be obtained between cysteine residues introduced instead of native groups. Preferably, the disulfide bond is between residues in the constant region of the immunoglobulin, however, it can also be between the residues in the proximal region of the membrane. The preferred sites on which cysteine residues can be introduced to produce a disulfide bond are the following residues in exon 1 TKAC * 01 for the TSC α chain and TKBC1 * 01 or TKBC2 * 01 for the TSC β chain: ________________________

α chain TSC. B-chain TSK The distance between the native atoms β- carbon (nm) Thr 48 8eg57 0.473 Thr 45 8eg77 0.533 Tug 10 8eg 17 0.359 Thr 45 Azr 59 0.560 8eg 15 011115 0.59

One of the SSTCs according to the present invention is obtained from TSC A6 Tax (OaBoe / 1 e! A1., LaShge, 1996, 384 (6605): 134-141). In one implementation variant, Т TSC contains the entire region of the TSC α-chain in the direction of the конца-terminus from exon 2, the remainder is 4 TKAC * 01 (residues 1-182 amino acids of the α-chain according to the numbering used in the work of Oaboe / 1 e! A1. ) and the entire region of the TSC β-chain in the direction of the Ν-end from exon 2, residue 2 TKAC * 01 and TKSB2 * 01 (residues 1–210 amino acids of the β-chain according to the numbering used in the work of Ogboe / 1 e! a1.). To obtain a disulfide bond, exon 1 threonine 48 in TCAS * 01 (158 α-chain threonine according to the numbering used in Oagoe / 1 e! A1.) And exon 1 series 57 in TKVS1 * 01 and TKVS2 * 01 (172 β series -chains according to the numbering used in the work of Cactus-ζ.ί e! a1.) can be mutated into cysteine. These amino acids are located in the β-chain Ό of the constant domain of α- and β-chains of TSC, respectively.

It should be noted that in FIG. 3a and 3b, group 1 (according to the numbering used in the work CagLoc-ζ.ί e! A1.) Represents K and N, respectively. The methionine residue in the кон-terminal region is not present in the native TSC A6 Tax and, as indicated above, is sometimes present if the corresponding chains are produced in bacterial expression systems.

Now that residues in human TSCs that can be mutated into cysteine groups are identified to obtain a new interchain disulfide bond, those skilled in the art can mutate any TSC so as to obtain a soluble form of said TSC containing a new interchain disulfide bond. In the case of the human body, specialists only need to find the following motifs in the corresponding TSC chains to determine the group to be mutated (the shaded group is a group for mutation into cysteine).

-4006601

α chain Tbr 48: O8OUU1TOK | U1T) MK.8MORK (amino acids 39-58 of exon 1 gene TKAS * 01) a-chain Tb 45: 98CO8OUU1 | OKTYOMK. 8M (amino acids 36-55 of exon 1 of the TKAC * 01 gene) a-chain Tug 10: ϋΐρΝΡϋΡΑν | ρΕΚΟ8Κ88ϋΚ (amino acids 1-20 of exon 1 gene TKAS * 01) a-chain 8eg 15: ^ ΡΑVΥ ^^ Κ ^ | Κ88 ^ Κ8VС ^ Ρ (amino acids 6-25 exon 1 gene TKAS * 01) 0-chain 8eg 57: Ν0ΚΕνΗ80ν @ ΤϋΡ9ΡΕΚΕ9Ρ (amino acids 48-67 exon 1 of the TKVS1 * 01 and TKVS2 * 01 genes) 0-chain 8eg 77: ΑΕΝϋ8ΚΥΑΕ | 8Κ.ΕΚ.ν8ΑΤΡΨ (amino acids 68-87 of exon 1 of the TKVS1 * 01 and TKVS2 * 01 genes) 0-chain 8eg 17: RREUAURER | EAE18HT (/) KA (amino acids 8-27 exon 1 of the TKBC1 * 01 and TKBC2 * 01 genes) 0-chain Azr 59: КЕУН8ОУ8Т | ррРЬКЕРРЬ (amino acids 50-69 exon 1 of the TKBC1 * 01 and TKBC2 * 01 genes)

0-chain O1 and 15:

URRREUAUR | P8EAA18NTR (amino acids 6-25 exon 1 of the TKBC1 * 01 and TKBC2 * 01 genes)

In other types of chains, TSCs may not have a region that is 100% identical to the above configurations. However, those skilled in the art will be able to use the above configurations to identify the equivalent portion of the TSC α or β chain and, therefore, the group to be mutated into cysteine. For this purpose, you can use the method of comparative analysis of the primary structure. So, for example, you can use the C1i81a1 ^ system, presented on the website of the European Institute of Bioinformatics (yyr: //^^^.ЕЫ.ас.ик/1пбех.й1ш1), to compare the above configurations with the sequence of circuits of a particular TSC to determine the position of the corresponding part of the TSC sequence for mutation.

The present invention includes human disulfide-linked αβ TSCs, as well as disulfide-linked αβ TSCs of other mammals, in particular, but not limited to, mice, rats, pigs, goats and sheep. As indicated above, those skilled in the art will be able to identify sites equivalent to the human sites described above into which cysteine groups can be introduced to obtain an interchain disulfide bond. So, for example, the amino acid sequences of the soluble mouse Ca and S domains are presented together with motifs showing mouse amino acid residues equivalent to the above human amino acid residues, which can be mutated into cysteines with the formation of TSC interchain disulfide bonds (corresponding groups are shaded)

-5006601

Samysha's soluble domain:

ΡΥΙ0ΝΡΕΡΑνΥΰΕΚΟΡΚ50Ο3ΤΕ0ΕΓΤΟΕΟ3αΐΝνΡΚΤΜΕ36ΤΓΙΤΟΚΤνΒΟΜΚΑΜΟ3Κ3Ν

6ΑΙΑΜ3Νζ) Τ3ΓΊΌζ) 0ΙΕΚΕΤΝΑΤΥΡ330νΡ

Soluble mouse Οβ domain:

EOKMUTRRKUZERZAKETAMKOKUSYUSARSGERONUEZIIUUMSKUUNZSUZTORO

ΑΥΚΕ3ΝΥ3Υ0Ε33ΚΕΚν3ΑΤΓΝΗΝΡΚΝΗΕΚ0ζ) ν <2ΕΗ0Ε3ΕΕΕΚνίΡΕ63ΡΚΡνΤ (2ΝΙ3ΑΕΑΜ

ΟΡΑϋ a-chain of the mouse, equivalent to the α-chain of Thr 48 of the person: E8CrTP1TOK | UOMKAMO8K a-chain of the mouse, equivalent to the α-chain of Thr 45 of man: ΚΤΜΕ8ΟΤΕΙ (ϋΚΤΥΕϋΜΚΑΜ a-chain of the mouse, equivalent to the α-chain of Tug 10 of man: ΥΙ0ΝΡΕΡΑν || 0ΕΚΟΡΚ8 (2ϋ8 а- mouse chain, equivalent to the α-chain of Zeg 15 people: ΑνΥ () ΕΚ1) ΡΚ | () 08ΤΕΟΕΡΤΟ β-chain of the mouse, equivalent to the β-chain Zeg of 57 people: ΝΟΚΕνΗδθν | ΤΟΡ0ΑΥΚΕ8Ν mouse β-chain, equivalent to β-chain 8eg 77 people: ΚΕ8ΝΥ8ΥΟΕ§8ΚΕΚΥ8ΑΤΡΨ mouse β-chain, equivalent to β-chain Zeg 17 people: ΡΡΚΥ8ΕΡΕΡ | ΚΑΕΙΑΝΚ0ΚΑ mouse β-chain, equivalent to β-chain Azr 59 people: ΚΕνΗ8ΟΥ8Τ | | Ρ0ΑΥΚΕ8ΝΥ8 mouse β-chain, equivalent to the human β-chain O1and 15: νΤΡΡΚν8ΕΓ | Ρ8ΚΑΕΙΑΝΚ0

In a preferred embodiment of the present invention, both TSCs (I) and (II) contain the functional variable domain of the first TSC fused to the full constant domain or part of the second TSC, the first and second TSCs being of the same species and the interchain is disulfide the relationship between residues in the indicated corresponding full constant domain or part thereof is absent in the native TSC. In one embodiment, the first and second TSCs are human TSCs. In other words, constant domains with a disulfide bond play the role of a framework to which variable domains can join. The resulting TSC will be almost identical to the native TSC, from which the first TSC is obtained. Such a system makes it easy to express any functional variable domain on a stable framework from a constant domain.

The constant domains of zTSC A6 Tax described above, or the constant domains of any αβ-mutants of zTSC containing the new interchain disulfide bond described above, can be used as a framework to which heterologous variable domains can be joined (merged). Preferably, the fusion proteins retain the conformation of heterologous variable domains as much as possible. Therefore, it is preferable that the heterologous variable domains be associated with constant domains at any point between the introduced cysteine groups and the кон-terminal region of the constant domain. For TSC A6 Tax, the cysteine groups in the α and β chains are preferably located at the position of threonine 48 of exon 1 in TKAC * 01 (threonine 158 α chain according to the numbering used in the work ΟηγΕοοζι е! А1.) And serine 57 of exon 1 in TKVS1 * 01 and TKVS2 * 01 (serine 172 β-chains according to the numbering used in the work ΟοτΕοοζί е! А1.) Respectively. Therefore, it is preferable that the points of attachment of the heterologous variable domains of the α and β chains lie between groups 48 (159 according to the numbering used in the work ΟπτΕοοζί е! А1.) Or 58 (173 according to the numbering used in the work of СапЬос / ί с1 а1.) and the Ν-terminal region of the constant domains α or β, respectively.

Groups in the constant domains of heterologous α and β chains corresponding to the attachment points in TSN A6 Tax can be identified by sequence homology. The fusion protein is preferably designed so that it includes the entire heterologous sequence in the direction of the конца-end to the point of attachment.

As described in more detail below, one can obtain derivatives of §TCN according to the present invention with some group in its C- or--terminal region or merge with this group. A C-terminal region is preferred because it is distal with respect to the binding domain. In one embodiment, one or both TSN chains contain a cysteine residue in its C- and / or цевой -terminal region, with which the corresponding group can be fused.

The soluble TCH (which is preferably human TCH) according to the present invention can be obtained in substantially pure form or in the form of a purified or isolated preparation. So, for example, it can be obtained in a form that practically does not contain other proteins.

Many soluble TCHs according to the present invention can be obtained in the multivalent complex. Thus, one of the objectives of the present invention is a multivalent complex of T-lymphocyte receptors (T ce11 tester, TSH), which contains many soluble T-lymphocyte receptors as described herein. All soluble TSNs in this set are preferably identical.

Another object of the invention is a method for detecting peptide-MHC complexes, which comprises

1) obtaining a soluble T-lymphocyte receptor or multivalent complex of lymphocyte receptors, according to the present description,

2) contacting the soluble T-lymphocyte receptor or polyvalent complex of TCH with peptide-MHC complexes and

3) determination (detection) of binding of a soluble T-lymphocyte receptor or a multivalent complex of TSN with complexes of MHC-peptide.

In the multivalent complex according to the present invention, TCHs may be in the form of multimers and / or may be present in or associated with a lipid double layer, for example, in the form of a liposome.

In its simplest form, the multivalent TCH complex according to the invention comprises a multimer of two, three, four or more T-lymphocyte receptor molecules linked (for example, by covalent or another bond) to another molecule, preferably via a linker molecule. Suitable linker molecules include, but are not limited to, polyvalent attachment molecules, for example avidin, streptavidin, neutravidin, and extravidin, each of which has four biotin binding sites. Thus, biotinylated TSN molecules can be obtained in multimers of T-lymphocyte receptors having many TSN binding sites. The number of TSN molecules in the multimer depends on the ratio of the number of TSN to the number of linker molecules used to obtain multimers, as well as the presence or absence of other biotinylated molecules. Preferred multimers are dimeric, trimeric or tetrameric complexes of TSN.

Structures much larger than TCH tetramers can be used to track or identify cells expressing a specific MHC peptide complex. Preferred structures have a diameter in the range of 10 nm to 10 μm. Each structure can contain many TSN molecules with a sufficient distance between them, so that two or more TSN molecules in the structure can simultaneously combine with two or more peptide-MHC complexes in the cell and thereby increase the affinity of the multidimensional linking group for the specified cell.

Suitable structures for use according to the invention include membrane structures, in particular liposomes, and solid structures, which are preferably particles, for example spheres, in particular latex spheres. Other structures that may have an outer coating with T-lymphocyte receptor molecules are also suitable. The structures are preferably coated with multimers of T-lymphocyte receptors, rather than with individual Trimphocyte receptor molecules.

In the case of liposomes, T-lymphocyte receptor molecules or their isomers can be attached to the membrane or otherwise linked to it. Appropriate methods are well known in the art.

A marker or other group, in particular a toxic or therapeutic group, can be included in the multivalent TSN complex of the present invention. For example, a marker or other group may be included in a mixed molecular multimer. An example of such a multimeric molecule is a tetramer containing three TSN molecules and one peroxidase molecule. It can be obtained by mixing TSN and the enzyme in a molar ratio of 3: 1 to form tetrameric complexes, and to separate the desired complex from complexes that do not contain the desired molecular ratio. These mixtures of molecules may contain any combination of molecules, provided that the steric hindrance does not impair or does not substantially impair the desired function of the molecules. The positioning of the binding sites on the streptavidin molecule is suitable for mixed tetramers, since the appearance of a steric hindrance is not likely.

Alternative biotinylation agents for TSC are also possible. So, for example, chemical biotinylation can be used. Alternative biotin labels can be used, however, certain amino acids in the sequence of the biotin label are essential (δοϊιαίζ. (1993). Vu1ec1to1od N Υ 11 (10): 1138-43). The mixture used for biotinylation can also be changed. The enzyme requires MD-ATP and low ionic strength, however, both of these conditions can be changed, for example, it may be possible to use a higher ionic strength and a longer reaction time. It is possible to use a molecule other than avidin or streptavidin to obtain multimers of TSC. Suitable is any molecule that binds biotin to a polyvalent compound. Alternatively, a completely different relationship may be used, in particular, a polyhistidine tag with a chelated nickel ion (Oshadsp Rgois! Szys 1999, Sarjug 3 Prgosch Exrgekkίοη, Rsgysa Noi, Ee1es1yup and Akkau p. 35-37). The label is preferably positioned in the direction of the Cone region of the protein in order to minimize the amount of steric hindrance when interacting with peptide-MHC complexes.

One or both TSC circuits can be labeled with a detectable (detectable) label, for example a label that is suitable for diagnostic purposes. Thus, the invention provides a method for detecting peptide-MHC complexes, said method comprising contacting peptide-MHC complexes with TSC. or a multimeric complex of TSC according to the invention, which is specific for the peptide-MHC complex, and determining the binding of TSC or multimeric complex of TSC to the peptide-MHC complex. In tetrameric TSCs formed with biotinylated heterodimers, fluorescent streptavidin (commercially available) can be used to obtain a detectable label. A tetramer with a fluorescent label can be used for analysis by a cell sorter method with excitation of fluorescence (Diogeksei 1-asyuyoy se11 koyeg, EAC8), for example, to detect antigen-presenting cells containing a peptide for which TSC is specific.

Another way that soluble TSCs of the present invention can be detected is by using TSC-specific antibodies, in particular monoclonal antibodies. There are many commercially available anti-TSC antibodies, in particular αΕ1 and βΕ1, which recognize the constant regions of the α and β chains, respectively.

The TSC (or its polyvalent complex) according to the present invention can alternatively or additionally be combined (for example, by covalent or another bond) with a therapeutic agent, which may be, for example, a toxic group used to kill cells, or an immunostimulating agent, in particular interleukin or cytokine. The multivalent complex of TSC according to the present invention may have increased binding ability for rMNC compared to the multimeric heterodimer of the T-lymphocyte receptor. Thus, the multivalent TSC complexes according to the invention are particularly useful for tracking or detecting (1) where cells representing certain antigens ίη νίΙΐΌ or ίη у1уо, and are also useful as intermediates for the preparation of other multivalent TSC complexes having the same use. In this case, TSC or the multivalent complex of TSC can be obtained in a pharmaceutically acceptable composition for the use of ίη у1уо.

The invention also provides a method for delivering a therapeutic agent to a target cell, which comprises contacting potential target cells with a TSC or a multivalent complex of a TSC in accordance with the invention under conditions that allow for the attachment of a TSC or a multivalent complex of a TSC to a target cell, wherein said TSC or the multivalent complex TSCs are specific for peptide-MHC complexes and contain an associated therapeutic agent.

A soluble TSC or polyvalent complex of TSC can be used, in particular, for delivery of therapeutic agents to cells representing a specific antigen. This is useful in many cases, and especially in the treatment of tumors. The therapeutic agent can be delivered in such a way that it will exert its influence in a specific area, and not only in those cells with which it is associated. Thus, a special strategy involves the use of antitumor molecules associated with T-lymphocyte receptors or multivalent complexes of TSCs specific for tumor antigens.

For this purpose, for example, a variety of therapeutic agents can be used, in particular radioactive compounds, enzymes (e.g. perforin) or chemotherapeutic agents (e.g. cis-platinum). In order to provide toxic effects in the desired region, the toxin inside the liposome can be coupled to streptavidin to slowly release the compound. This will prevent damage during transport in the body and provide the maximum toxin effect after connecting TSN with the corresponding antigen-presenting cells.

Other suitable therapeutic agents include small cytotoxic agents, i.e. compounds capable of killing mammalian cells having a molecular weight of less than 700 Da. Such compounds may also contain toxic metals capable of exerting a cytotoxic effect. In addition, it should be understood that these cytotoxic agents for small molecules also include prodrugs, i.e., compounds that decompose or transform under physiological conditions, releasing cytotoxic agents. Examples of such agents include cis-platinum, maytansine derivatives, raschelmitsin, calischamycin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer-sodium-photofrin II, temozolmide, topotecan, trimethacrycinnitrocinucleotin glucinate and dicinotin glucinate glucinate, , i.e., proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNase and RNase, radionuclides, i.e. unstable isotopes of elements that decompose with the parallel emission of one or more α- or β-particles or γ-rays. Examples include iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213, prodrugs (drug precursors), in particular enzyme prodrugs directed to antibodies, immunostimulants, i.e. groups that stimulate the immune response. Examples include cytokines, in particular 1b-2, chemokines, in particular 1b-8, platelet factor 4, melanoma growth stimulating protein, etc., antibodies or fragments thereof, complementary activators, xenogenic protein domains, allogeneic protein domains, viral / bacterial protein domains and viral / bacterial peptides.

Soluble TCH or polyvalent TCH complexes according to the invention may be associated with an enzyme capable of transforming a prodrug into a drug. This allows you to transform the prodrug into a drug only in the area where it is required (i.e., the targeted action of kTsN).

Examples of suitable peptide-MHC targets for TSN according to the invention include, in particular, but not limited to, viral epitopes, for example, epitopes of NTLU (human T cell leukemia virus) -1 (in particular, the Pex peptide limited by HLA-A2; HTLV-1 - associated with leukemia), HIV epitopes, epitopes of EVU, epitopes of SMU, epitopes of melanoma and other epitopes specific to cancer (for example, the associated C250 antigen limited by HLA-A2), as well as epitopes associated with autoimmune disorders, in particular with rheumatoid arthritis. In addition, disease-related pMHC targets suitable for use in accordance with the present invention are listed in HbA Ea1bOoc (Wag1au (Eb) Asabepis Rheck), and many other targets have also been identified.

In many cases, treatment of the disease can be made much more effective due to the localization of the drug, due to the specificity of soluble TSN.

For the treatment of viral diseases for which there are drugs, for example, HIV, B1U (§1t1ai and 1tipobeDeps Upik, monkey immunodeficiency virus), EVU (Erk1esh-Watg Uttik, Epstein-Barr virus), SMU (Cytotomy1ou | gik. Cytomegalovirus), it is useful to isolate or in close proximity to infected cells. In the case of cancer, localization in the vicinity of tumors or metastases enhances the effectiveness of toxins or immunostimulants. In autoimmune diseases, the possibility of slow release of immunosuppressive drugs provides an increased local effect for a longer period of time with minimal impact on the patient's overall immune activity. To prevent transplant rejection in the same way, the effect of immunosuppressive drugs can be optimized. To deliver the vaccine, the antigen used to prepare the vaccine can be localized close to the antigen presenting cells, thereby increasing the effectiveness of the antigen. The method can also be used to visualize images (tissues).

The soluble TSNs of the present invention can be used to modulate plateau activation by binding to specific pMHCs and thereby inhibit T-lymphocyte activation. This approach is applicable to autoimmune diseases, including T-lymphocyte-mediated inflammation and / or tissue damage, such as type I diabetes. For this application, knowledge of the specific peptide epitope represented by the corresponding rMNC is required.

The medicaments of the invention are usually supplied as part of a sterile pharmaceutical formulation, which typically contains a pharmaceutically acceptable carrier. This pharmaceutical composition, as a rule, may have any suitable form (depending on the desired method of administration to the patient). It can be supplied in unit dosage form, usually in an airtight container, and can be part of a kit. Such a kit usually (but not necessarily) contains instructions for use. It may include many of these standard doses.

-9006601

The pharmaceutical composition can be adapted for administration in any suitable way, for example, orally (including buccally or sublingually), rectally, nasally, locally (including buccally, sublingually or transdermally), vaginally or parenterally (including subcutaneously, intramuscularly, intravenously or intradermally). Such formulations can be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with one or more carriers or excipients under sterile conditions.

Pharmaceutical formulations intended for oral administration can be presented in the form of discrete doses, for example capsules or tablets, as well as powders, granules, solutions, syrups or suspensions (in aqueous or non-aqueous liquids, or in the form of edible foam, cream or emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, as well as stearic acid or its salts. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols.

Examples of fillers that can be used to prepare solutions and syrups include water, polyols, and sugars. To obtain oily suspensions (for example, vegetable oils), suspensions of the type oil-in-water or water-in-oil can be used. Pharmaceutical formulations for transdermal administration may be presented in the form of discrete patches designed to provide close contact with the epidermis of the recipient over a long period of time. The active ingredient can be delivered, in particular, from the lining by iontophoresis, as described in the General case in Riagtaseis 1 Neckerbs, 3 (6): 318 (1986). Pharmaceutical formulations for topical administration can be prepared in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In case of infection of the eye or other external tissues, for example the oral cavity and skin, the compositions are preferably applied in the form of a local ointment or cream. For the preparation of ointments, the active ingredient can be used with a paraffin or water-miscible oil base. Alternatively, the active ingredient may be incorporated into a cream with an emulsion base such as oil in water or water in oil. Pharmaceutical formulations for topical administration to the eye include eye drops in which the active ingredient is dissolved or suspended in an appropriate carrier, in particular in an aqueous solvent. Pharmaceutical formulations for topical administration to the oral cavity include tablets, lozenges, and oral rinses.

Pharmaceutical formulations for rectal administration may be presented in the form of suppositories or enemas. Pharmaceutical formulations for nasal administration in which the carrier is a solid include large powders with a particle size of, for example, from 20 to 500 microns, which are also administered as snuff, i.e. by rapid inhalation through the nasal passage from a powder container, held close to the nose. Suitable formulations in which the carrier is a liquid and which are intended to be administered as nasal sprays or drops include aqueous or oily solutions of the active ingredient. Pharmaceutical formulations for administration by inhalation include fine dust or mist, which can be prepared using various types of metered, pressurized aerosols, nebulizers or insufflators. Pharmaceutical formulations for vaginal administration may be presented in the form of pessaries, tampons, creams, gels, pastes, foams or sprays. Pharmaceutical solutions for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats and dissolved substances that make the solution practically isotonic with the blood of the patient concerned, as well as aqueous and non-aqueous suspensions, which may include suspended agents and thickeners . Excipients that can be used in injection solutions include, for example, water, alcohols, polyols, glycerin and vegetable oils. The compositions can be in a container with one or more doses, for example, in sealed ampoules and vials, they can be stored in a freeze-dried (lyophilized) state and immediately before use they require only the addition of sterile liquid, for example, water for injection. Injectable solutions and suspensions for immediate administration can be prepared from sterile powders, granules and tablets.

Pharmaceutical formulations may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavors, salts (the substances of the present invention may themselves be presented in salt form), buffers, coating agents or antioxidants. They may also contain other therapeutic active agents other than the substances of the present invention.

The dosage of the substances according to the present invention can vary widely depending on the disease or disorder to be treated, the age and condition of the patient who needs such treatment, etc., therefore, the therapist must finally determine the dose for admission. Dosage can be repeated as needed. In case of side effects, the dose and frequency of administration can be reduced in accordance with normal clinical practice.

To obtain §TCN according to the invention, preferably in substantially pure form, gene cloning methods can be used. These methods are described in the literature, for example, 1. 8th.

-10006601 e! a1. Mölösi1ag Clööpd 2n6 Ebüyup, Co16 8rppd Nagyog Láboguyu Rgeck (1989). Thus, according to a further object, the present invention provides a nucleic acid molecule that comprises a sequence encoding the soluble TSC chain of the present invention, or a complementary sequence. Such nucleic acid sequences can be obtained by isolating a nucleic acid encoding TSC from T lymphocyte clones and creating the corresponding mutations (by insertion, deletion or substitution).

An acid molecule may be present in isolated or recombinant form. It can be introduced into a vector, which, in turn, can be introduced into the host cell. Such vectors and corresponding hosts are other objects of the present invention.

The invention also provides a method for producing a TSC chain, comprising incubating said host cell under conditions causing expression of the TSC chain, and subsequent purification of the polypeptide.

The soluble TSCs of the present invention can be obtained by expression in bacteria, in particular in E. co11, in the form of inclusion bodies, followed by re-folding ίη νίΐΐΌ.

The re-laying of the TSC chains can occur ίη νίίΐΌ in modes suitable for re-laying. In one embodiment, the implementation of the TSC with the correct conformation is obtained by re-stacking the soluble chains of the TSC in a re-stacking buffer containing a solubilizer, such as urea. Urea may be present in a concentration of preferably at least 0.1 M, or at least 1 M, or at least 2.5 M, or about 5 M. Alternatively, guanidine with a concentration of from 1 to 8 M, preferably at least 1 M or at least 2.5 M, can be used as a solubilizer. A reducing agent is preferably used before re-laying to ensure complete restoration of the cysteine groups. In addition, denaturing agents, in particular OTT and guanidine, can be used if necessary. Various denaturants and reducing agents can be used before the re-styling operation (e.g. urea, β-mercaptoethanol). Alternatively, redox pairs, in particular redox pairs cystamine / cysteamine, OTT or β-mercaptoethanol / atmospheric oxygen and cysteine in reduced and oxidized forms, can be used during re-laying.

Stacking efficiency can also be increased by adding some other protein components, such as chaperone proteins, to the re-stackable mixture. Improvements in re-folding were achieved by passing the protein through columns with immobilized mini-chaperones (AIatgapo, e! A1. (1999). No. 11ige Vu! Espododu 17: 187-191; AIatgapo, e! A1. (1997). Proc. No. 11. Asab. 8c1. I8A 94 (8): 3576-8).

The soluble TSCs of the present invention can be obtained alternatively by expression in a system of eukaryotic cells, in particular insect cells.

TSK can be cleaned in many different ways. So, for example, you can use various options for ion exchange or other methods of protein purification, in particular gel chromatography or affinity chromatography.

The soluble TSCs and multivalent TSC complexes according to the present invention also find use in screening agents, in particular small chemical compounds capable of inhibiting the binding of TSC to its pMHC complex. Thus, according to a further object, the present invention provides a method for screening an agent that inhibits binding of a T-lymphocyte receptor to a peptide-MHC complex, said method comprising monitoring the binding of a soluble T-lymphocyte receptor according to the present invention to a peptide-MHC complex in the presence of an appropriate agent and selection of agents that inhibit said binding.

Suitable methods for such screening include a method based on surface plasma resonance and described in AO 01/22084. Other well-known methods that can form the basis for this screening method are express scintillation analysis (8CM! 111a1up Rhoh1Sh11u Apa1uk1k, 8PA) and express analysis with enhanced luminescence (Atryleb Lyshesep! Rokh1tyu Akkau).

Agents selected by the screening methods of the invention can be used as drugs or as the basis for a drug development program, being modified or otherwise improved to obtain characteristics that will make them more suitable for administration as a medicament. Such medications can be used to treat conditions that include an undesirable component of the lymphocyte response. These conditions include cancer (e.g., kidney, ovary, intestines, head and neck, testis, lung, stomach, cervix, bladder, prostate, or melanoma skin cancer), autoimmune diseases, transplant rejection, and a transplant against the host disease.

The preferred characteristics of each of the specified objectives of the invention, as well as other tasks, are ty1a11k ti! Apb1k (can be obtained by making the appropriate changes). Known documents referred to in this description are included in their entirety, permitted by law.

-11006601

Examples

The following are examples of the implementation of the invention, in no way limiting the scope of the invention.

Links are made to the attached figures, which show the following.

FIG. 1 is a schematic illustration of the structure of soluble TCH with an introduced interchain disulfide bond according to the invention;

FIG. 2a and 2b are nucleic acid sequences of α and β chains, respectively, of soluble TSH A6 mutated in such a way as to introduce a cysteine codon. Shading indicates the introduced cysteine codon;

FIG. 3a is the amino acid sequence of the extracellular part of the α-chain of TSN A6, including the T ₄₈ ^ C mutation (underlined), which is used to obtain a new disulfide interchain;

FIG. 3b is the amino acid sequence of the extracellular part of the β chain of TSN A6, including the mutation 8 ₅₇ ^ C (underlined), which is used to obtain a new disulfide interchain;

FIG. 4 is a curve obtained by anion exchange chromatography of soluble TSN A6, showing elution of the protein from a PONO 50H0 column using a gradient of No. 1C1 0-500 mM, as indicated by a dashed line;

FIG. 5 - A. Reducing electrophoresis in a polyacrylamide gel with sodium dodecisulfate (8O1ish Eobesu! 8i1Ga1e - Po1uasgu1ash1be Ce1 E1es1gor1yuge515. 8O8-PACE) (with Coomassie staining) of the eluate fractions from the column shown. 4.

B. Non-reducing 808-PACE (Coomassie stained) eluate fractions from the column shown in FIG. 4. Peak 1 definitely contains mainly a β-chain without a disulfide bond, peak 2 contains a TSN heterodimer with an interchain disulfide bond, and the shoulder is caused by contaminants of E.co11 mixed with §TCN containing an interchain disulfide bond and is poorly visible on this reproduction;

FIG. 6 is a curve obtained by gel chromatography of the combined fractions of peak 1 in FIG.

5. The protein elutes as a single major peak corresponding to a heterodimer;

FIG. 7 is a reaction curve of B1Acoge for specific binding of soluble TSH A6 with a disulfide bond and the HLA-A2-1ax complex. The inset shows the binding reaction compared to a control sample for one injection of soluble TSH A6 with a disulfide bond;

FIG. 8a is a TCH α chain sequence containing a new cysteine residue mutated to be included in the WatH1 restriction site. Shading indicates mutations introduced to obtain a WatH1 restriction site;

FIG. 8b and 8c show the DNA sequence of the α and β chains of TSN ΙΜ 22 mutated to include additional cysteine residues in order to obtain a non-native disulfide bond;

FIG. 9a and 9b are the amino acid sequences of the extracellular part of the α and β chains, respectively, of TSN ΙΜ 22 obtained from the DNA sequences of FIG. 8a and 8b;

FIG. 10 is a curve obtained by anion exchange chromatography of soluble TCH ΙΜ22 with a disulfide bond, showing protein elution from a PONO 50 50NO column using a 0-500 mM gradient No. 1 Cl, as indicated by a dashed line;

FIG. 11a is a reducing 808-PACE (with Coomassie staining) fractions of the eluate from the column shown in FIG. 10;

FIG. 11b is a non-reducing 8O8-PACE (with Coomassie staining) fractions of the eluate from the column shown in FIG. 10. Peak 1 definitely contains a TSN heterodimer that is linked by an interchain disulfide bond;

FIG. 12 is a curve obtained by gel chromatography of the combined fractions of peak 1 in FIG. 10. The protein elutes as a single major peak corresponding to the heterodimer. The yield is 80%;

FIG. 13 - A. The reaction curve of B1Acoge for specific binding of soluble TCH 1–22 with a disulfide bond and the HLA-E1i complex.

B. Binding reaction compared to a control sample for a single injection of soluble TSH No. 22 with a disulfide bond;

FIG. 14a and 14b show the DNA sequence of the HU-E8O α- and β-chain mutated to include additional cysteine residues in order to obtain a non-native disulfide bond;

FIG. 15a and 15b are the amino acid sequences of the extracellular part of the α and β chains, respectively, of TCH HU-E8O obtained from the DNA sequences of FIG. 14a and 14b;

FIG. 16 is a curve obtained by anion exchange chromatography of soluble TCH ΝΥE8O with a disulfide bond, showing protein elution from a PONO 50 50NO column using a 0-500 mM gradient No. C1, as indicated by a dashed line;

-12006601 Fig. 17 - A. BOS-RACE reducing (Coomassie stained) eluate fractions from the column shown in FIG. sixteen.

B. Non-reducing 8O8-PACE (Coomassie stained) fractions of the eluate from the column shown in FIG. 10. Peaks 1 and 2 definitely contain a HSC heterodimer that is linked by an interchain disulfide bond;

FIG. 18 is a curve obtained by gel chromatography of the combined fractions of peak 1 (A) and peak 2 (B) in FIG. 17. The protein elutes as a single major peak corresponding to a heterodimer;

FIG. 19 is a reaction curve of B1Acoge for specific binding of soluble TSC ΝΥ-EZO to a disulfide bond and the HLA-ΝΥ-EZO complex. A - peak 1. B - peak 2;

FIG. 20a and 20b are DNA sequences of the α and β chains of soluble TSC ΝΥ ^^ mutated in such a way as to introduce a new cysteine codon (shown by shading). Sequences include cysteine contained in the native disulfide interchain (codon is shown in bold);

FIG. 21a and 21b are the amino acid sequences of the extracellular part of the α and β chains, respectively, of TSC CT-E8O obtained from the DNA sequences of FIG. 20a and 20b;

FIG. 22 is a curve obtained by anion-exchange chromatography of soluble ΝΥ-ЕЗО, showing elution of the protein from a ROCO8 50NO column using a gradient No. 1C1 0-500 mM, as indicated by the dashed line;

FIG. 23 is a curve obtained by anion exchange chromatography of soluble TSCx /. ⁴ '' NΥ-E8O showing elution of the protein from a POCO8 50NO column using a gradient of No. 1C1 0500 mM, as indicated by the dotted line;

FIG. 24 is a curve obtained by anion-exchange chromatography of soluble TSCD ⁴ '' Е-EZO, showing protein elution from a POCO8 50NO column using a gradient of 1C1 0500 mM, as indicated by a dashed line;

FIG. 25 - reducing 8O8-PACE (with Coomassie staining) TSCα ^female β ^female , TSC /. ⁴ and TSCβ ^females ΝΥ-EZO fractions from the anion exchange column eluates shown in FIG. 22-24 respectively. Lanes 1 and 7: M ^ markers, lane 2: NΥE8OάkTSK1d4 α-bitch β peak (EB / 084/033); lane 3: small peak NΥE8OάkTSK1d4 α-bit β (ЕВ / 084/033), lane 4: \ УЕ8Ос1кТСК1у4 α β-bit (ЕВ / 084/034), lane 5: small peak KUE8O4kTSK1d4 α-bit β-bit (Е 084/035) and lane 6: peak KUE8O4kTSK1d4 α-female β-female (EB / 084/035);

FIG. 26 - reducing 8O8-PACE (with Coomassie staining) TSC NΥ-E8Oα ^female β- ^female , TSCx /. ⁴ and TSCβ ^{bitches of} eluate fractions from the anion exchange column shown in FIG. 22-24 respectively. Lanes 1 and 7: markers M ^, lane 2: peak KUE8O4kTSK1d4 α-bitch 13 (EB / 084/033); lane 3: small peak KUE8O4kTSK1d4 α-female β (EB / 084/033), lane 4: KUE8O4kTSK1d4 α β female (EB / 084/034), lane 5: small peak KUE8O4kTSK1d4 female β-female (EB / 035) and lane 6: peak KUE8O4kTSK1d4 α-bitch β-bitch (EB / 084/035);

FIG. 27 is a curve obtained by exchange gel chromatography of soluble TSC ΝΥE8O / ^female βΉ showing elution of the combined fractions of FIG. 22. The protein elutes as a single base peak corresponding to a heterodimer;

FIG. 28 is a curve obtained by exchange gel chromatography of soluble TSC ΝΥE8O / ^female , showing elution of the combined fractions of FIG. 22. The protein elutes as a single base peak corresponding to a heterodimer;

FIG. 29 is a curve obtained by exchange gel chromatography of soluble TSC ΝΥE8Oβ ^bitches , showing elution of the combined fractions of FIG. 22. The protein elutes as a single base peak corresponding to a heterodimer;

FIG. 30 — B1Acoge reaction of specific binding of TSC NΥ-E8Oα ^female β ^ to the HLANА-E8O complex;

FIG. 31 — B1Acoge reaction of specific binding of TSC NΥ-E8Oα ^bitches to the HLA-ΝΥE8O complex;

FIG. 32 - B1Acoge reaction of specific binding of TSC NΥ-E8Oβ ^bitches to the HLA-ΝΥE8O complex;

FIG. 33a and 33b are the DNA sequences of the α and β chains, respectively, of the soluble TSC AN-1.23 mutated in such a way as to introduce a new cysteine codon (shown by shading). Sequences include cysteine contained in the native disulfide interchain (codon is shown in bold);

FIG. 34a and 34b are the extracellular amino acid sequences of the α and β chains of TSC AN-1.23 obtained from the DNA sequences of FIG. 33a and 33b;

FIG. 35 is a curve obtained by anion-exchange chromatography of soluble TSC AN1.23, showing elution from a POCO8 50NO column using a gradient of 0-500 mM No. C1, as indicated by a dashed line;

-13006601 Fig. 36 — reducing 8E8-PACE (10% bis-Tris gel, with Coomassie staining) fractions of TSC AN-1.23 from the eluate of the anion exchange column of FIG. 35. The studied proteins are TSK 1.23 8-8 anion-exchange fractions from re-stacking 3. Lane 1: markers Μ \ ν, lane 2: B4, lane 3: C2, lane 4: C3, lane 5: C4, lane 6: C5 , lane 7: C6, lane 8: C7, lane 9: C8 and lane 10: C9;

FIG. 37 - non-reducing 8Ό8-ΡΑΟΕ (10% bis-Tris gel, with Coomassie staining) fractions TSK AN-1.23 from the eluate of the anion exchange column of FIG. 35. The studied proteins are TSK 1.23 8-8 anion-exchange fractions from re-stacking 3. Lane 1: markers Μν, lane 2: B4, lane 3: C2, lane 4: C3, lane 5: C4, lane 6: C5, lane 7: C6, lane 8: C7, lane 9: C8 and lane 10: C9;

FIG. 38 is a curve obtained by exchange gel chromatography of soluble TSC AN-1.23, showing elution of the combined fractions of FIG. 35. The protein elutes as a single base peak corresponding to a heterodimer;

FIG. 39a and 39b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 48 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 40a and 40b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 45 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 41a and 41b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 61 in exon 1, TCAC * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 42a and 42b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC Α6 mutated in such a way as to introduce new cysteine at position 50 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 43a and 43b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 10 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 44a and 44b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 15 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 45a and 45b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC mutated in such a way as to introduce new cysteine at position 12 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 46a and 46b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 22 in exon 1 of TCAS * 01. The shaded nucleotides show the introduced new cysteine codon, and the shaded amino acid shows the introduced cysteine;

FIG. 47a and 47b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 52 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 48a and 48b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 43 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 49a and 49b are, respectively, the DNA and amino acid sequences of the α-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 57 in exon 1 of TCAS * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 50a and 50b are, respectively, the DNA and amino acid sequences of the β chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 77 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

-14006601 Fig. 51a and 51b are, respectively, the DNA and amino acid sequences of the β chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 17 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 52a and 52b are, respectively, the DNA and amino acid sequences of the β-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 13 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 53a and 53b are, respectively, the DNA and amino acid sequences of the β chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 59 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 54a and 54b are, respectively, the DNA and amino acid sequences of the β chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 79 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 55a and 55b are, respectively, the DNA and amino acid sequences of the β-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 14 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 56a and 56b are, respectively, the DNA and amino acid sequences of the β-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 55 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 57a and 57b are, respectively, the DNA and amino acid sequences of the β-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 63 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 58a and 58b are, respectively, the DNA and amino acid sequences of the β-chain of soluble TSC A6 mutated in such a way as to introduce new cysteine at position 15 in exon 1 of TKBC2 * 01. Shaded nucleotides indicate the introduced new cysteine codon, and the underlined amino acid shows the introduced cysteine;

FIG. 59-64 are the curves obtained by anion exchange chromatography of soluble TSC A6 containing a new disulfide interchain bond between residues 48 of exon 1 TKAC * 01 and 57 exon 1 TKVS2 * 01; residues of 45 exon 1 TKAS * 01 and 77 exon 1 TKVS2 * 01; residues of 10 exon 1 TKAS * 01 and 17 exon 1 TKVS2 * 01; residues of 45 exon 1 TKAS * 01 and 59 exon 1 TKVS2 * 01; residues of 52 exon 1 TKAS * 01 and 55 exon 1 TKVS2 * 01; residues of 15 exon 1 TKAC * 01 and 15 exon 1 TKVS2 * 01, respectively, when the protein is eluted from the POCO8 50 column using a gradient of 0-500 mM NaCl, as indicated by the dashed line;

FIG. 65a and 65b, respectively, the results of the reducing and non-reducing 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain between residues 48 of exon 1 TKAC * 01 and 57 exon 1 TKBC2 * 01, the eluate of fractions was taken from the anion exchange column eluate, shown in FIG. 59;

FIG. 66a and 66b, respectively, the results of the reductive and nonreduction 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain between residues 45 of exon 1 TKAC * 01 and 77 exon 1 TKBC2 * 01, the eluate of fractions was taken from the anion exchange column eluate, shown in FIG. 60;

FIG. 67a and 67b are, respectively, the results of the reducing and non-reducing 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain between residues 10 of exon 1 TKAC * 01 and 17 exon 1 TKBC2 * 01, the eluate of the fractions was taken from the anion exchange column eluate, shown in FIG. 61;

FIG. 68a and 68b, respectively, the results of the reducing and non-reducing 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain between residues 45 of exon 1 TKAC * 01 and 59 exon 1 TKBC2 * 01, the eluate of the fractions was taken from the anion exchange column eluate, shown in FIG. 62;

FIG. 69a and 69b, respectively, the results of the reductive and nonreduction 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain between residues 52 of exon 1 TKAC * 01 and 55 exon 1 TKBC2 * 01, the eluate of the fractions was taken from the anion exchange column eluate, shown in FIG. 63;

FIG. 70a and 70b, respectively, the results of reductive and nonreduction 8-8PACE (with Coomassie staining) of soluble TSC A6 containing a new disulfide interchain bond between residues 15 of exon 1 of TBAC * 01 and 15 of exon 1 of TBBC2 * 01, the eluate of fractions was taken from the anion exchange column eluate, shown in FIG. 64;

FIG. 71 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 48 of exon 1 of TBAC * 01 and 57 of exon 1 of TBBC2 * 01, showing protein elution from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 59;

FIG. 72 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 45 of exon 1 of TBAC * 01 and 77 of exon 1 of TBBC2 * 01, showing protein elution from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 60;

FIG. 73 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 10 of exon 1 TBAC * 01 and 17 exon 1 of TBBC2 * 01, showing elution of the protein from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 61;

FIG. 74 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 45 of exon 1 of TBAC * 01 and 59 of exon 1 of TBBC2 * 01, showing elution of the protein from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 62;

FIG. 75 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 52 of exon 1 of TBAC * 01 and 55 of exon 1 of TBBC2 * 01, showing protein elution from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 63;

FIG. 76 is a curve obtained by gel chromatography of soluble TSB A6 containing a new disulfide interchain between residues 15 of exon 1 of TBAC * 01 and 15 of exon 1 of TBBC2 * 01, showing protein elution from column 8 and blockbe 200 Hb gel filtration. The eluate fractions were taken from the eluate of the anion exchange column shown in FIG. 64;

FIG. 77-80 are VMsohe reaction curves showing, respectively, the compound of soluble TSB A6 containing a new disulfide interchain between residues 48 of exon 1 TBAC * 01 and 57 exon 1 TBBC2 * 01, residues 45 exon 1 TBAC * 01 and 77 exon 1 TBBC2 * 01, residues 10 exon 1 TBAC * 01 and 17 exon 1 TBBC2 * 01 and residues 45 exon 1 TBAC * 01 and 59 exon 1 TBBC2 * 01 with rMNS HLA-A2-1ax;

FIG. 81 is a BMsoge curve showing non-specific binding of soluble TSB A6 containing a new disulfide interchain between residues 52 of exon 1 of TBAC * 01 and 55 of exon 1 of TBBC2 * 01 with HLA-A2-1ax and with pMHC 1ΙΕΛ-Λ2-ΝΥ-Ε8Ο;

FIG. 82 is a reaction height curve showing the binding of soluble TCB A6 containing a new disulfide interchain between residues 15 of exon 1 of TBAC * 01 and 15 of exon 1 of TBBC2 * 01 with rMNS HLA-A2-1ax;

FIG. 83a is an electron density map of a model with the sequence 1ВЭ2 (circuit A TNG164, circuit B 8eg 174). Contours are plotted on the map for σ 1.0, 2.0, and 3.0;

FIG. 83b is an electron density map after improving the model by introducing Suk into two positions A 164 and B 174. Contours are plotted on the map for the same values of σ as in FIG. 83a;

FIG. 84 is a comparison of the structures TSV 1VE2 and TSV ΝΥ-Ε8Ο according to the present invention by superimposing these structures in the image in the form of a tape and a spiral;

FIG. 85a and 85b are the DNA and amino acid sequences, respectively, of the TCB ΝΥ-Ε8Ο β-chain, including the biotin recognition site. Biotin recognition site highlighted;

FIG. 86a and 86b are the DNA and amino acid sequences, respectively, of the β-chain of TCB ΝΥ-Ε8Ο, including a hexahistidine tag. The hexahistidine label is saturated;

FIG. 87 - elution of soluble TSB ΝΥ-Ε8Ο containing a new disulfide bond and a sequence for recognizing biotin from an anion-exchange column PΟBΟ8 50NO using a gradient of 0-500 mM No. C1, as indicated by a dashed line;

FIG. 88 - elution of soluble TSB ΝΥ-Ε8Ο containing a new disulfide bond and hexahistidine tag from an anion-exchange column of ΟBΟ8 50NO using a gradient of 0-500 mM NaCl, as indicated by the dashed line;

FIG. 89 is a protein elution profile obtained by gel chromatography of pooled biotin-labeled ΝΥ-Ε8Ο fractions from the eluate of the anion exchange column shown in FIG. 87;

FIG. 90 is a protein elution profile obtained by gel chromatography of pooled fractions labeled with hexahistidine ΝΥ-Ε8Ο from the eluate of the anion exchange column shown in FIG. 88;

FIG. 91a-H are PAC8 histograms illustrating the staining intensity obtained in 25,000 cases for the line (PP ECB) of B cells transformed with positive ΕBV NBA-A2 and cultured at the following concentrations of ΝΥ-Ε8Ο peptide and TCB fluorescent tetramers

-16006601

ХУ-ЕБСЕ, respectively, ХУЕБО 0 - ТСН 5 μg, ХУЕБО 10 ^-4 М - ТСН 5 μg, ХУЕБО 10 ^-5 М - ТСН 5 μg, НУЕБО 10 ^-6 М - ТСН 5 μg, ХУЕБО 0 - ТСН 10 μg, ХУЕБО 10 ^-4 M - TSN 10 μg, HUEBO 10 ^-5 M - TSN 10 μg, HUEBO 10 ^-6 M - TSN 10 μg;

FIG. 92 is a DNA sequence of the β chain of TSN A6 containing the constant region of TNBC1 * 01;

FIG. 93 is an anion exchange chromatography curve of soluble TSH A6 containing the constant region THBC1 * 01, showing elution from the RONOB 50HO column using a gradient of 0-500 mM BaCl, as indicated by the dashed line;

FIG. 94 - A. Reducing δΌδ-PAOE (Coomassie stained) fractions from the column eluate, as shown in FIG. 93.

B. Non-reducing δΌδ-PAOE (with Coomassie staining) fractions from the eluate from the column, as shown in phi. 93;

FIG. 95 is a gel chromatogram of the combined fractions of peak 2 of FIG. 93. Peak 1 contains a TSN heterodimer that is linked by an interchain disulfide bond;

FIG. 96 - A. Analysis of B1Acoge of a specific compound of soluble TSH A6 containing a disulfide bond with the HLA-II complex.

B. Comparison of the binding reaction with a control for one injection of soluble TSH A6 with a disulfide bond;

FIG. 97 is a nucleic acid sequence of a mutated TSN A6 β chain containing free cysteine;

FIG. 98 is an anion exchange chromatogram of soluble TSN A6 containing free cysteine, showing elution from a RONOB 50NO column using a gradient of 0-500 mM NaCl, as indicated by a dashed line;

FIG. 99 - A. Restoring BEB-PACE (with Coomassie staining) fractions from the eluate from the column shown in FIG. 98.

B. Non-reducing BEB-PACE (with Coomassie staining) fractions from the eluate from the column shown in FIG. 98;

FIG. 100 is a gel chromatogram of the pooled fractions represented by peak 2 in FIG. 98. Peak 1 contains a TSN heterodimer that is linked by an interchain disulfide bond;

FIG. 101 - A. Analysis of B1Acoge of a specific compound of soluble TSH A6 containing a disulfide bond with the HLA-II complex.

B. Comparison of the binding reaction with a control for one injection of soluble TSH A6 with a disulfide bond;

FIG. 102 is a nucleic acid sequence of a mutated TSN A6 β chain containing a serine residue mutated into free cysteine;

FIG. 103 is an anion exchange chromatogram of soluble TSN A6 containing a serine residue mutated into free cysteine, showing elution from a PONO 50 50NO column using a gradient of 0-500 mM NaCl, as indicated by a dashed line;

FIG. 104 - A. Restorative BB-RLSE (with Coomassie staining) of eluate fractions from the column shown in FIG. 103.

B. Non-reducing δΌδ-PAOE (Coomassie stained) fractions from the eluate from the column shown in FIG. 103. Peak 2 definitely contains a TSN heterodimer that is linked by an interchain disulfide bond;

FIG. 105 is a gel chromatogram of the pooled fractions represented by peak 2 in FIG. 103. Peak 1 contains a TSN heterodimer that is linked by an interchain disulfide bond;

FIG. 106 - A. Analysis of B1Acoge of a specific compound of soluble TSH A6 containing a serine residue. mutated into free cysteine, with the HLA-II complex.

B. Comparison of the binding reaction with a control for one injection of soluble TSH A6 with a disulfide bond;

FIG. 107 — nucleotide sequence ρΥΧ112;

FIG. 108 - nucleotide sequence ρΥΧ122;

FIG. 109 — DNA and protein sequences of the rge-rego typd factor alpha fused to the TSN α-chain; FIG. 110 — DNA and protein sequences of the rge-rego typd factor alpha fused to the TSN β-chain; FIG. 111 - Western blotting of soluble TSN expressed in strain δEΥ6210 δ. now. Lane C contains 60 ng of purified soluble TSH NΥ-EδO used as a control sample. Lanes 1 and 2 contain proteins collected from two separate transformed TSN yeast cultures;

FIG. 112 is the nucleic acid sequence of Krp1 to the EcoH1 insert of plasmid pEX172. The remainder of the plasmid is pB1ieBspr1 II KB-;

FIG. 113 is a schematic representation of TSN chains for cloning into baculovirus;

FIG. 114 is a nucleic acid sequence of a TSH α A6 disulfide construct in the form of a WatCh insert for insertion into the plasmid of expression of pACAB3;

-17006601 Fig. 115 - disulfide construct TSC β A6 in the form of WatH1 for insertion into the plasmid expression of pACAB3;

FIG. 116 — Coomassie stained gel and Western blot for the insect disulfide TSC A6 and disulfide TSC A6 obtained by the bacterium.

In all of the following examples, unless otherwise indicated, the resulting soluble TSC chains are truncated immediately before the C-terminal region of the cysteine groups forming the native interchain disulfide bond.

Example 1. Design of primers and mutagenesis of α and β chains of TSC A6 Tax.

For the threonine 48 A6 Tax exon 1 mutation in TCAS * 01, the following primers were designed in cysteine (the mutation is shown in lower case):

5’-C ASA OAS AAA 1§T OTO STA OAS AT

5’-AT OTS TAO SAS Asa TTT OTS TOT O

The following primers were designed for cysteine mutation 57 A6 Tax exon 1 in TKVS1 * 01 and TKVS2 * 01 in cysteine (mutation is shown in lowercase letters):

5’-C AOT LLC OTS US ASA OAS SS

5’-00 OTS TOT OSA OAS SSS AST O

Mutagenesis of CSCs (RoNTgas scat ι ^ Ή ^ η, polymerase chain reaction).

Expression plasmids containing genes for α or β chains of TSC A6 were mutated using α chain primers or β chain primers, respectively, as follows. 100 ng of the plasmid was mixed with 5 μl of 10 mM 4XGR, 25 μl of 10xP1i buffer (8ΐ ^ aΐadeie), 10 units of P1i polymerase (8ΐ ^ aΐadeie) and the final volume was adjusted to 240 μl with water. Primers were added to 48 μl of this mixture and diluted to obtain a final concentration of 0.2 μmol in 50 μl of the final reagent volume. After the initial stage of denaturation for 30 s at 95 ° С, 15 cycles of denaturation of the mixture (95 ° С, 30 s), annealing (55 ° С, 60 s) and elongation (73 ° С, 8 min) were carried out in an RSK express installation - NLA14 RSK. Then the product was fermented for 5 hours at 37 ° C with 10 units of the restriction enzyme Ori! (Ν + \ ν Ед1аиά Вю1абз). 10 μl of the product of the fermentation reaction was transformed into competent bacteria Xb 1-B1ie and grown for 18 h at 37 ° C. A single colony was taken and grown overnight in 5 ml TΥP + ampicillin (bactotriptone 16 g / l, yeast extract 16 g / l, No. C1 5 g / l, K ₂ NRA ₄ 2.5 g / l, ampicillin 100 mg / l). Plasmid DNA was purified in a mini-preparation column ^^ adei according to the manufacturer's instructions and the sequence was checked using automatic sequencing on the equipment for sequencing of the Department of Biochemistry, University of Oxford. The corresponding mutated nucleic acid and amino acid sequences are shown in FIG. 2a and 3a for the α chain and FIG. 2b and 3b for the β chain.

Example 2. Expression, re-styling and purification of soluble TSC.

Expression plasmids containing the mutated α-chain and β-chain, respectively, were transformed separately into the E. colH BT21bb8 strain, and individual ampicillin-resistant colonies were grown at 37 ° C in TΥP medium (ampicillin 100 μg / ml) to OE ₆₀₀ 0, 4 before induced protein expression at 0.5 mM 1PTO. Cells were taken 3 hours after induction by centrifugation for 30 min at a speed of 4000 rpm./min in a Vesstai 1-6V centrifuge. The pellet containing cells was resuspended in a buffer containing 50 mM TPZ-HC1, 25% (w / v) sucrose, 1 mM Ν / ιΙΆΓΛ (sodium ethylene diamine tetraacetic acid), 0.1% (w / v) azide sodium, 10 mM ETT (dithiotrentol), pH 8.0. After cyclic freezing and thawing overnight, the resuspended cells were treated with ultrasonic pulses for 1 minute for about 10 minutes in a Myzoshx X2020 ultrasonic unit using a standard probe with a diameter of 12 mm. Precipitated inclusion bodies were recovered by centrifugation for 30 minutes at 13,000 rpm in a Vesstai 12-21 centrifuge. Then, to remove debris and membrane components, the precipitate was washed with three portions of detergent. Each time, the pellet of inclusion bodies was homogenized in a buffer of TgIoi (500 mM Tpz-HC1, 0.5% TgLoy-X100, 200 mM No. C1, 10 mM No. EETA, 0.1% (w / v) sodium azide, 2 mM MA, pH 8.0) before sedimentation by centrifugation for 15 min at 13000 rpm./minutes in a Vesstai 12-21 centrifuge. After that, the detergent and salt were removed by similar washing with the following buffer: 50 mM TpnSH1, 1 mM NOEETA, 0.1% (w / v) sodium azide, 2 mM 1) 1) 1 / pH 8.0. And, finally, inclusion bodies were divided into 30 mg aliquots and frozen at -70 ° C. The yield of the inclusion bodies protein was quantified by dissolving in a 6 M solution of guanidine-HCl and binding analysis with the dye Bga4 £ og4 (RegVyu).

Thawed approximately 30 mg (i.e. 1 μmol) of the dissolved inclusion bodies of each chain from frozen aliquots, then mixed the samples and diluted a mixture of 15 ml of guanidine solution (6 M guanidine chloride, 10 mM sodium acetate 10 mM EETA) to ensure complete denaturation chains. Then a guanidine solution was introduced containing completely reduced and denatured TSC chains in 1 L of the following re-folding buffer: 100 mM Tp pH 8.5, 400 mM L-arginine, 2 mM EETA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 5 M urea, 0.2 mM PM8G (phenylmethanesulfonyl fluoride). The solution was kept for 24 hours. After that, the re-styling mixture was dialyzed twice, first against 10 l of 100 mM urea, and then against 10 l of 100 mM urea and 10 mM Tp8 pH 8.0. Re-styling and dialysis operations were performed at 6-8 ° C.

The HAZB was separated from the decomposition and contamination products by loading the dialyzed re-laid product into the POW8 50NO anion exchange column and eluting the bound protein with a gradient of 0-400 mM # 1C1 in 50 column volumes using the Ak1a (P1agtac1a) purification system as shown in FIG. 4. Peak fractions were stored at 4 ° C until combined and concentrated and analyzed using 8-8-ΡΑΟΕ with Coomassie staining (Fig. 5). Finally, the HFCB was purified and analyzed using a column 8 andregbeh 200НВ gel filtration (Fig. 6), after balancing in HB8-EP buffer (10 mM ΗΕΡΕ8 pH 7.4, 150 mM NaCl, 3.5 mM ΕΌΤΑ, 0, 05% off! P40). Elution peak fractions with a relative molecular weight of about 50 kDa were pooled and concentrated before analysis using surface plasma resonance on a B1Asohe apparatus.

Example 3. The study of the binding of zTSV with specific rMNS using surface plasma resonance on the installation B1Asohe.

A surface plasma resonance biosensor (B1Acoge 3000 ™) was used to analyze the binding of zTSB to its peptide MHC ligand. This was ensured by creating single rMNC complexes (described below), which were immobilized on a binding surface coated with streptavidin in a semi-oriented form, which made it possible to effectively control the binding of a soluble T-lymphocyte receptor simultaneously with four different rMNCs (immobilized in separate flow cells). The manual introduction of the HLA complex (Pitan 1eikosu! E apbdepb, the main human histocompatibility complex) makes it easy to regulate the exact level of immobilized class I molecules.

Such immobilized complexes are capable of binding both T-lymphocyte receptors and the co-receptor CE8aa, all of which can be introduced into the soluble phase. Specific binding of TCB was obtained even at low concentrations (at least 40 μg / ml), which indicated that TCB was relatively stable. Observations showed that the properties of zTSV to bind rMRS are similar from a quantitative and qualitative point of view, if TSW is used in a soluble or immobilized phase. This is an important means of controlling the partial activity of soluble forms, and also indicates that biotinylated complexes of pMHC are as biologically active as non-biotinylated complexes.

Class I NBA-A2 biotinylated peptide complexes re-stacked ίη ugo from bacterially expressed inclusion bodies containing constitutive subunits of proteins and a synthetic peptide, followed by purification and enzymatic biotinylation of ίη ugo (O'Ca11ad1ap e! A1. (1999) Apa1. Vusyet 266. : 9-15). The HLA heavy chain, which replaces the transmembrane and cytoplasmic domains of the protein in the corresponding construct, was expressed with a biotinylated label in the C-terminal region. Received expression levels of inclusion bodies of about 75 mg / L bacterial culture. The HLA light chain or b2-microglobulin was also expressed in the form of inclusion bodies in Ε. ^ 1ί from the corresponding construct at the level of about 500 mg / l of the bacterial culture.

E. cells were lysed and the inclusion bodies were purified to a purity of approximately 80%. Protein from inclusion bodies was denatured in 6 M ganidin-HC1, 50 mM Tp8 pH 8.1, 100 mM NaCl, 10 mM ETT, 10 mM Ε ^ ΤΑ and re-laid at a concentration of 30 mg / L heavy chain, 30 mg / L β2ιη in 0.4 M L-arginine-HC1, 100 mM TPZ pH 8.1, 3.7 mM ciastamine, 6.6 mM β-cysteamine, 4 mg / ml peptide (e.g.! ax 11-19), by adding one portion of the denatured protein to the re-folding buffer at a temperature below 5 ° C. The re-laying was completed at 4 ° C for at least 1 hour.

The buffer was replaced by dialysis in 10 volumes of 10 mm TPZ pH 8.1. To significantly reduce the ionic strength of the solution, it was necessary to replace the buffer twice. Then, the protein solution was filtered through a 1.5 μm filter from cellulose acetate and loaded into a POW8 50NO anion exchange column (layer volume 8 ml). The protein was eluted with a linear gradient of 0-500 mM No. 1C1. The NEA-A2 peptide complex was eluted at approximately 250 mM No. 1C1. Peak fractions were collected, a mixture of protease inhibitors (Ca1 / u2) was added, and the fractions were cooled on ice.

In biotinylation-labeled HLA complexes, the buffer was replaced in 10 mM Tp pH 8.1, 5 mM No. 1C1 using a fast desalination column (Ryagtaaa) balanced in the same buffer. Immediately after elution, the fractions containing the protein were cooled on ice and a mixture of protease inhibitors (CaCluset) was added. Then biotinylation agents were added: 1 mM Biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MdCl ₂ and 5 μg / ml B1gA enzyme (purified according to O'Ca11ad1ap e! A1. (1999) Apa1. Vusyet. 266: 9-15). After that, the mixture was kept at room temperature overnight.

Biotinylated HLA complexes were purified by gel chromatography. Column 8iregbech 75 HB 10/30 (Ryagtasha) was pre-balanced with filtered phosphate-buffered saline (p1u5p11a1e-LiGgegezalpe, PB8) and 1 ml of the reaction mixture for biotinylation and the PB8 column was treated at 0.5 ml / min. Biotinylated HLA complexes were eluted to form one peak at approximately 15 ml. The fractions containing protein were combined, cooled on ice and a mixture of protease inhibitors was added. Protein concentration was determined by Coomassie binding method (Regby). Aliquots of biotinylated HLA complexes were stored frozen at -20 ° C. Streptavidin was immobilized by standard methods by forming an amine bond.

The interactions between the zTCH Tax A6 containing a new interchain bond and its ligand / MHC complex or an extraneous HLA-peptide combination, the preparation of which is described above, were analyzed using surface plasma resonance (8PH) on a B1asage 3000 ™ biosensor. 8RN measurements are based on a change in the refractive index, expressed in reactive units (CI), near the sensor surface in a miniature flow cell — a principle that can be used to determine receptor ligand interactions and analyze their affinity and kinetic parameters. Test flow cells were prepared by immobilizing individual HLA-peptide complexes in separate flow cells using binding between biotin crosslinked with β2ηι and streptavidin, which was chemically bonded to the activated surface of the flow cells. Then an analysis was carried out by passing zTSN over the surface of various flow cells at a constant flow rate, while measuring the reaction 8RN. At first, the interaction specificity was checked by passing zTSN at a constant flow rate of 5 μl min ^-1 over two different surfaces: one with a coating of about 5000 NI of the specific peptide-HLA complex, the second with a coating of about 5000 NI of the non-specific peptide-HLA complex (Fig. 7, insertion) The introduction of soluble zTSN over the peptide-HLA complex at a constant flow rate and various concentrations was used to determine the background resonance. The results of these control measurements subtracted from the values obtained with specific peptide-hemoglobin and used to calculate the affinity of the compound expressed by the dissociation constant, Kd (Rpse & E ^ ek Rpps1r1ez and RgoYetz ίη Ryuz1sa1 Syet1z! Iy £ og ByusepiLz (2 ^ηά Eyyyup ) 1979, Claupyop Przegs, Ox £ oj), as shown in FIG. 7.

The obtained Kd value (1.8 μmol) is close to that published in the literature for the interaction between TSN Tax A6 without a new disulfide bond and pMHC (0.91 μmol - ϋίη§ е! А1., 1999, 1ptipyu 11: 45-56).

Example 4. Obtaining soluble TSN TM22 containing a new disulfide bond.

The β-chain of soluble TSN A6 obtained in Example 1 contains in its native sequence the Bd111 restriction site (AACSTT) suitable for use as a ligation site.

Mutagenesis of PCH was performed, as described in detail below, to introduce the BatH1 restriction site (CCATCC) into the α-chain of soluble TSH A6, 5 'of the new cysteine codon. The sequence shown in FIG. 2a was used as a matrix for this mutagenesis. The following primers were used:

IВАТН1 |

5'-ATATSSASAASSSdsASASSTSSSSTSTA-3 '

5'-TASASSSSASSASaTSsSSSTTSTSSATAT-Z '

100 ng of the plasmid was mixed with 5 μl of 10 mM NTP, 25 μl of 10xP buffer and (8! Ha! Apepe), 10 units of P £ and polymerase (8! Ha! Apepe) and the final volume was made up to 240 μl with water . Primers were added to 48 μl of this mixture and diluted to obtain a final concentration of 0.2 μmol in 50 μl of the final reagent volume. After the initial stage of denaturation for 30 s at 95 ° С, 15 cycles of denaturation of the mixture (95 ° С, 30 s), annealing (55 ° С, 60 s), and elongation (73 ° С, 8 min) were carried out in an RSN express installation - Nuam RSN. Then, the product was fermented for 5 hours at 37 ° C with 10 units of the restriction enzyme 1) pn1 (\ e \\ - EpDapy B1a3b). 10 μl of the fermentation reaction product was transformed into competent XY-B1ie bacteria and grown for 18 h at 37 ° C. One colony was selected and grown overnight in 5 ml TΥP + ampicillin (bactotryptone 16 g / l, yeast extract 16 g / l, No. C1 5 g / l, K ₂ NRA ₄ 2.5 g / l, ampicillin 100 mg / l). The plasmid DNA was purified on a P1aep min mini-preparation column according to the manufacturer's instructions and the sequence was checked using automatic sequencing on the sequencing equipment of the Department of Biochemistry, University of Oxford. The mutations introduced into the α chain were silent; therefore, the amino acid sequence of this chain remained the same as shown in FIG. 3a. The DNA sequence for the mutated α chain is shown in FIG. 8a.

To obtain soluble TSH 1M22 containing a new disulfide bond, TSH A6 plasmids containing the restriction sites of the BatH1 α-chain and Bd111 β-chain were used as a matrix. The following primers were used:

-20006601 | Νάθΐ]

5'-ZCASATATASATATSSAASTASTASAASAA-Z '

5'-TASAS6SSASSATSSSSSSTTSTSSATATT-3 '

I WatN1 |

ΙΝάθΙ |

5'-SSASATATASATATSSTSSATSSTSSAATS-Z '

5'-SSSAA6STTASTSTST6STSTASSSSSSASSSSSTSS6S-3 '| Vd111 |

Constructs of the α and β chains of TSC 1M22 were cloned as follows. CSC reactions were performed using the above primers and matrices containing TSC 1M22 chains. CSC products were restriction fermented and cloned into pCMT7 to obtain expression plasmids. The sequence of plasmid inserts was confirmed by automatic DNA sequencing. FIG. 8b and 8c show the DNA sequence of the mutated α and β chains of TSC 1M22, respectively, and FIG. 9a and 9b are the resulting amino acid sequences.

The corresponding TSC chains were expressed, re-stacked and purified as described in Examples 1 and 2. FIG. 10 shows elution of a soluble TSC 1M22 protein with a disulfide bond from a POCO 50HP column using a gradient of 0-500 mM CaCl, as shown by the dashed line. In FIG. 11 shows the results of reducing 8P8-PACE (with Coomassie staining) and non-reducing 8O8-PACE (with Coomassie staining) gel fractions from the eluate from the column shown in FIG. 10. Peak 1 definitely contains a TSC heterodimer that is linked by an interchain disulfide bond. In FIG. 12 shows elution of the protein from the combined fractions of peak 1 of FIG. 10.

The B1A assay of the binding of TSC 1M22 to rMHC was performed as described in Example 3. FIG. 13a shows the result of the analysis by the B1A-specific method of specific binding of soluble TSC 1M22 with a disulfide bond to the HEA-E1i complex. FIG. 13b shows a binding reaction compared to a control sample for a single injection of soluble TSC 1M22 with a disulfide bond. The Kd value of this TSC with a disulfide bond for the NEA-Pi complex was 7.9 ± 0.51 μmol.

Example 5. Obtaining soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond.

cDNA encoding TSC ΝΥ-Ε8Ο was isolated from T-lymphocytes obtained from Εηζο Segipbo1o (Institute of Molecular Medicine, University of Oxford), in accordance with a known method. cDNA encoding TSC ΝΥ -8Ε was obtained by treating mRNA with reverse transcriptase.

To obtain soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond, plasmids TSC A6 containing the restriction sites of the α-chain of BhH1 and β-chain of Bd111 were used as a matrix, as described in Example 4. The following primers were used:

I ΝάθΙ |

5'-SSASATATASATATSSASSASSSTSASASAS-3 '

5'-TASASSSSASSASSATSSSSSTTSTSSATATT-Z '

I WatN1 |

ΙΝάθΙ I

5'-SSASATATASATATSSSTSTSASSASASS-3 '

5'-SSSAASSTTASTST6STSTASSSSSSAS6SSTSSSS -3 ’

I Vd1P |

Constructs of α and β chains of TSC ΝΥ-Ε8Ο were obtained by cloning of CSC as follows. CSC reactions were carried out using the above primers and matrices containing TSC chains ΝΥ-Ε8Ο. CSC products were restriction fermented and cloned into pCMT7 to obtain expression plasmids. The sequence of plasmid inserts was confirmed by automatic DNA sequencing. FIG. 14a and 14b show the DNA sequence of the mutated α and β chains of TSC ΝΥΕ8Ο, respectively, and FIG. 15a and 15b are the resulting amino acid sequences.

Appropriate TSC chains were expressed, re-stacked and purified as described in Examples 1 and 2 with the following protocol changes.

Denaturation of soluble TSCs: Thawed 30 mg of the dissolved inclusion body of the β chain of TSC and 60 mg of the dissolved Taurus of inclusion of the α chain of TSC from frozen aliquots. Inclusion bodies were diluted to a final concentration of 5 mg / ml in 6 M guanidine solution and ITT (2 M mother liquor) was added to a final concentration of 10 mM. The mixture was kept at 37 ° C for 30 minutes. Re-laying of soluble TSCs: 1 L of re-laying buffer was intensively mixed at 5 ° C ± 3 ° C. About 5 minutes before the introduction of the denatured TSC chains, a redox pair of 2-mercaptoethylamine and cystamine was added (to final concentrations of 6.6 mM and 3.7 mM, respectively). The composition was then kept under stirring for about 15 hours ± 15 minutes at 5 ± 3 ° C to allow for protein folding. Dialysis of re-laid soluble TSCs. The re-laid TSC was dialyzed in a 8resarog 1 membrane (8resigit; product No. 132670) against 10 L of 10 mM Tg18 pH 8.1 at 5 ± 3 ° C for 18-20 hours. After this time, the dialysis buffer was replaced fresh 10 mM TPZ pH 8.1 (10 L) and continued dialysis at 5 ± 3 ° C for an additional 20-22 hours

FIG. 16 illustrates the elution of the soluble TSC ΝΥ-Ε8Ο protein coupled with a disulfide bond from a ΡΟΚΟ8 50HP column using a 0-500 mM No. C1 gradient, as indicated by a dashed line. In FIG. 17 shows the results of reducing 8Ό8-ΡΑΟΕ (with Coomassie staining) and non-reducing 8Ό8-ΡΑΟΕ (with Coomassie staining) gel fractions from the eluate from the column shown in Figure 16. Peaks 1 and 2 definitely contain a TSK heterodimer that is linked by an interchain disulfide bond. FIG. 18 shows gel chromatography results of the combined fractions represented by peak 1 (A) and peak 2 (B) in FIG. 17. The protein elutes, forming a single main peak corresponding to the heterodimer.

The B1Asoge assay for determining the binding of TSC ΝΥ-Ε8Ο attached by a disulfide bond to rMHC was performed as described in Example 3. FIG. Figure 19 shows the result of B1Assoge analysis of the specific binding of soluble TSC ΝΥ-Ε8 присоедин attached by a disulfide bond to the ΗΕΑ-ΝΥΕ8Ο complex. A. peak 1, B. peak 2.

The CD value of this TSC attached by a disulfide bond for the ΗΕΑ-ΝΥ-Ε8Ο complex was 9.4 ± 0.84 μmol.

Example 6. Obtaining soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond and at least one of two cysteine groups necessary for the formation of a native disulfide interchain.

To obtain soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond and at least one of the two cysteine groups involved in the formation of a native disulfide interchain, plasmids containing the restriction sites of the BatH1 α chain and the Bd111 β chain were used as a base, as described in example 4. Used the following primers:

I ΝάθΙ I

5'-SSASATATASATATSSASSASSTSASASAS-Z '

5'-SSSAASSTTAASASSASSAASTTTSTSSSSSTSSSSAASAA-Z '| ΗϊηάΙΙΙΙ

I ΝάθΙ |

5'-SSASATATASATATSSSTSTSASSASASS-3 '

5'-SSSAASSTTAASASTSTSSTSTASSSSSSASSSSSTSSSS -3 '| Vd111 I

Constructs of a and β chains of TSC ΝΥ-Ε8Ο were obtained by cloned DSC as follows. CSC reactions were carried out using the above primers and matrices containing TSC chains ΝΥ-Ε8Ο. CSC products were restriction fermented and cloned into pOMT7 to obtain expression plasmids. The sequence of plasmid inserts was confirmed by automatic DNA sequencing. FIG. 20a and 20b show the DNA sequence of the mutated a and β chains of TSC ΝΥ-Ε8Ο, respectively, and FIG. 21a and 21b are the resulting amino acid sequences.

To obtain soluble TSC ΝΥ-Ε8Ο containing a non-native disulfide interchain bond and a native disulfide interchain bond, DNA isolated using both of the above primers was used. To obtain soluble TSC ΝΥ-Ε8Ο with a non-native disulfide interchain and only one of the cysteine groups involved in the formation of a native disulfide interchain, DNA was isolated using one of the above primers together with the corresponding primer from Example 5.

Appropriate TSC chains were expressed, re-stacked and purified as described in Example 5.

FIG. 22-24 show elution of the protein of soluble TSC ΝΥ-Ε8Οα ^ίΎ8 β ^ ' ⁸ (i.e., non-native and native cysteine groups in both chains), TSCa ^ss8 (with non-native cysteine groups in both chains and native cysteine group only in α-chain ), and TSCβ ^cy8 (with non-native cysteine groups in both chains and a native cysteine group only in the β-chain) from ΡΟKΟ8 50HP anion exchange columns using a 0-500 mM No. C1 gradient, as shown by the dashed line. FIG. 25 and 26 show, respectively, the results of reducing 8Ό8-ΡΑΟΕ (with Coomassie staining) and non-reducing 8Ό8-ΡΑΟΕ (with Coomassie staining) gels of the TSC ΝΥ-Ε8Οα ^ίΎ8 β ^ίΎ8 , TSCα ^su8 and TSCβ ^{su8 fractions} from the eluates from the column shown in FIG. . 22-24. They clearly show the formation of TSC heterodimers, which are joined by an interchain disulfide bond. FIG. 27-29 show protein elution profiles obtained by gel chromatography of the combined fractions of TSC ΝΥ

-22006601

Ε80α ° ^γδ TSCα ^suδ and TSCr ^su8 from the eluates of the ion exchange columns shown in FIG. 22-24 respectively. The protein elutes as a single major peak, corresponding to the HSC heterodimer.

An analysis of the B1Aase binding of zTSC to rMHC was performed as described in Example 3. FIG. Figures 30-32 show the results of the B1A-analysis of the specific binding of TSCi ^ ⁸ in ^cy8 , TSCα ^cyδ and TSCv ^cy8 ΝΥ-Ε80, respectively, with the complex H ^ Α -NΥΕ80.

TSKα ^{suδ su8} had a Kd 18.08 ± 2.075 micromol, TSKi ^{05 '8} had a Kd 19.24 ± 2.01 pmol and TSKv ^su8 Kd was 22.5 ± 4.0692 micromolar.

Example 7. Obtaining soluble TSC AN-1.23 containing a new disulfide interchain.

cDNA encoding TSC AN-1.23 was isolated from T-lymphocytes obtained from H111 Oaz1op (medical school, hospital A44epbooke, Cambridge), in accordance with a known method. cDNA encoding TSC ΝΥ -Ε80 was obtained by treatment of mRNA with reverse transcriptase.

To obtain soluble TSC AN-1.23 containing a new disulfide bond, TSC plasmids containing the restriction sites of the WatH1 α chain and the Bd111 β chain were used as a scaffold, as described in Example 4. The following primers were used:

I ΝάθΙ |

5'-SSSAASSTSTASATATSAASSAS6TSSASSASSASAATTSTSS-3 '

5'-TASASSSSASSASSATSSSSSTTSTSSATATT-3 '| WatN1 | | ΝάθΙ I

5'-TTSSAATTSASATATSSSSSTSATSSASAASSSSAASASAS-Z '

5'-SSSAASSTTASTSTSSTSTASSSSSSASSSSSTSSSS-3 '

IВд111 |

The constructs of the α and β chains of TSK AN-1.23 were obtained by cloning of RSK as follows. RSK reactions were carried out using the above primers and matrices containing TSC chain AN-1.23. CSC products were restriction fermented and cloned into pOMT7 to obtain expression plasmids. The sequence of plasmid inserts was confirmed by automatic DNA sequencing. FIG. 33a and 33b show the DNA sequence of the mutated α and β chains of TSC AN-1.23, respectively, and FIG. 34a and 34b are the resulting amino acid sequences.

Appropriate TSC chains were expressed, re-stacked and purified as described in Example 5.

FIG. 35 illustrates the elution of the soluble TSC AN-1.23 protein attached by a disulfide bond from a P0K08 50H0 column using a 0-500 mM No. C1 gradient, as indicated by a dashed line. In FIG. 36 and 37 show the results of reducing 8O8-PAOE (with Coomassie staining) and non-reducing 8O8-PAOE (with Coomassie staining) gels, respectively, of the eluate fractions from the column shown in FIG. 35. These gels definitely indicate the presence of a TSC heterodimer that is linked by an interchain disulfide bond. FIG. 38 shows the elution profile from column 8 and 4Ex 75 NK gel chromatography of pooled fractions from the eluate of the anion exchange column shown in FIG. 35. The protein elutes, forming a single main peak corresponding to the heterodimer.

Example 8. Obtaining soluble TSC A6 containing a new disulfide interchain in alternative positions in the immunoglobulin region of the constant domain.

The following experiments were performed to clarify the possibility of obtaining functional soluble TSCs containing a new disulfide bond in the mmunoglobulin TSC region at a position different from the intermediate position between threonine 48 of exon 1 in TKAS * 01 and serine 57 of exon 1 in TKVS1 * 01 / TKVS2 * 01.

The following primers were designed for mutation of the α-chain of TSC A6 (the numbers in the primer names refer to the position of the amino acid residue to be mutated in exon 1 of TCAS * 01, the mutated residues are indicated in lowercase letters)

-23006601

T48-> C MUTATION '-C AC AOAC AAA1 _ё TSTOSTAS AC AT-3' -ATOTSTAOOSASASATTTOTOTSTO-Z '

ΥΙΟ-es MUTATION ’-SSSTOSSOTST ^ SSAOOSTAOAO-Z’ ’

5’-STSTSASSTOOsASASOOOOOOOO-3 ’

Y2-> C MUTATION '-COTOTASSAO1 _ё сАООАСТАААТС-3''-ОАТТТА6АОТСТСТ _е сАСССОТАСАСОО-3'

815—> C MUTATION '-CA6SG0AS ASASTdTAAATSSAOOTAS-Z ’’ О OTSASTSOATTTAsAOOTSTSTSASOSTO-Z ’

Y22 ^ C MUTATION

5'-SAOTOASAAOOTST1 _e STOSSTATTSAS-3 '

5’-OTOAATAAOOSAOosaAOASTTOTSASTO-3 ’

U43-> С MUTATION ’-ОАТТСО АТАТО§ТАТС АСО ОААААА-3’ ’-АТТСТСОТАТААТАСАСАСАТСАААТС-З’

T45-ES MUTATION ’-STO ASTSTATATATADU AS A A AASTSTOS-3’

Z'-OSASAOOTTTTOTSASOATATASASATSAO-W ¹

T50- ”C MUTATION’ - AS AS AA A ASTSTS1d1SAS ATOAOOTS T-3 JSC

5’-ASASSTSATOTASasASASAOOTTTTOTTST-3 ’

M52-> C MUTATION ’-ASTOTOSTAO AS1d1 AOOTSTATOOOOS-3’ ’-OTOTSATAOASSTASOOTSTAOOSASAOOT-Z’

861-> C MUTATION ’-STTSAAOAOOSAYASUTOSTOTOOSS-Z’ ’СО -COSSASAOOSASAOTOTOSTSTTOAAO-Z’

The following primers were designed for the mutation of the β chain of TSN A6 (the numbers in the primer names refer to the position of the amino acid residue to be mutated in exon 1 of TNBC2 * 01. The mutated residues are indicated in lower case):

857-> C MUTATION ’-C AOTOOOOTSYUSASAOAOSSS-3’ ’’ -OOTSTOTOSAOASSASSASTO-Z ’

Y13- + C MUTATION '-CCOA6HOTSOST! _ё 1TTTOTOOSSATSAO-3 '' -STATOOOOSTSAAAASAOOSASSTSOO-3 '

P14—> C MUTATION ’-OOTSOSTO1d! OJSCSSATSAOA-3’ ’-STOATOOSTSasASASAOOSOASS-Z’

-24006601

817-> C MUTATION ’-OTOTTTOAOOSSAT§1OAAOOSAOAOATS-3’ ’-OATSTSTSTTSasATOOSTSAAASAS-Z’

055-> С MUTATION ’-ОАОООСОСАВАСОТУЮТСАСОСАААС-З’ ’“ ОТТОТОСОСАСААТАТОСАСССТС-З ’

О59 ^ С MUTATION ’-ООСОСАСАСА1§СССОСАСССС-3’ ’’-ООСОСОООСОТАТОСОСС-З ’

6363-> C MUTATION ’—CSSOSAOOSSS1vSAAOOAOOSAOOS-3’ ’—OSTOSTSTSTOSAOOOSTOSOOO-Z’

877—> C MUTATION ’—AOATASOSTOSTOYUSAOOSSOSST-Z’ ’—AOOOOOOSTASAAOAOOSOTATST-Z’

K79—> C MUTATION ’-STSTOAOOS AOSYUSSTOOOOOOTS-Z’ ’’-OASSTSAOOSaOSTOSTOSTAOAO-Z ’

E15-> MUTATION

5 '-ССТ0Т0ТТТ1 _ё 1ССАТСА6АА- 3'

5 '-TTSTOATOOasAAASASASAOOS-3'

MSC mutagenesis, amplification, ligation and plasmid purification of α and β TSC constructs were performed as described in Example 1 using an appropriate combination of the above primers to obtain soluble TSCs containing new disulfide interchain bonds between the following pairs of amino acids:

TSC α-chain β chain TSC Used a is an example Used β primer Thr 48 Zeg 57 T48-> C 857—> C Thr 45 8eg 77 T45—> C 877—> C 8eg61 Zeg 57 861-> C 857-> C Ley 50 Zeg 57 B50-> c 857 ^ C Tug 10 8eg 17 Υ10-Η3 817—> C 8eg 15 Wa113 815—> C U13-> C Thr 45 Azr 59 T45-> C 059> C Ley 12 8eg 17 S2-> C 817—> C 8eg61 Agd 79 861 ^ C K79—> C Ley 12 Pb 14 N2—> C P14—> C Wa1 22 Pb 14 U22—> C G14 ^ C Me! 52 C1 55 M52-> C C55-> C Tug 43 Ley 63 Υ43—> C B63-> c 8eg 15 C1 and 15 815-> C E15-> C

In FIG. 39-58 show the DNA and amino acid sequences of the mutated DNA chains and the amino acid sequences of the mutated TSC A6 chains amplified by the above primers. Codons encoding mutated cysteine groups are indicated by isolation.

-25006601

The corresponding TSC chains were expressed, re-stacked and purified as described in Example 5. After purification in a POCO 50110 anion-exchange column), the obtained proteins were passed on 81) 8-Pare gels to verify the formation of soluble protein with proper re-folding. These gels were also analyzed to determine the presence or absence of a protein containing a disulfide bond and having the desired molecular weight in the purified material. When using this bacterial expression system, the studied TSCs containing the following new disulfide interchain bonds did not form a protein containing a disulfide bond with the corresponding molecular weight; therefore, they were excluded from further experiments. However, there are alternative prokaryotic or eukaryotic expression systems.

TSC α-chain β chain TSC Zeg 61 8eg57 Ley 50 8eg57 Zeg 15 Wa113 Ley 12 8eg 17 Zeg 61 Agd 79 Ley 12 Pb 14 Wa1 22 Pb 14 Tug 43 Ley 63

FIG. 59-64 show elution of soluble TSCs containing new disulfide interchain bonds between the following groups: Tg 48-8g 57, Tg 45-8g 77, Tg 10-8g 17, Tg 45-Acre 59, Me! 52-O1u 55 and 8eg 15-O1i 15, respectively, from the anion-exchange column ROCO8 200110) using a gradient of 0-500 mM No. C1, as shown by the dashed line. FIG. 65-70 show the results of reducing 808-PACE (with Coomassie staining) and non-reducing 8O8-PA6E (with Coomassie staining) gels, respectively, of the eluate fractions from the column shown in FIG. 59-64. The gels definitely indicate the presence of TSC heterodimers that are linked by an interchain disulfide bond.

FIG. 71-76 show elution profiles from column 8 and 200 of NK gel filtration of the combined fractions from the eluates of the anion exchange column shown in FIG. 59-64.

An analysis of B1Acase of binding of TSC to rMHC was performed as described in Example 3. FIG. 77-82 show B1Acoge curves demonstrating the ability of purified soluble TSCs to bind to the pMHA NEA-A2! Ax complexes.

Tg 48-8g 57 had a Cd equal to 7.8 μmol, Tg 45-8g 77 had a Cd equal to 12.7 μmol, Tg 10-8g 17 had a Cd equal to 34 μmol, Tg 45-Acre 59 had a Cd equal to 14 , 9 μmol and 8eg 15-C1i 15 had a CD equal to 6.3 μmol. Me! 52-C1u 55 could bind to its natural target - the HEA-A2! Ax complex, however, it also contacted in the same way with an extraneous target - the H ^ Α-Α2-NΥ-E8O complex (see Fig. 81).

Example 9. X-ray diffraction analysis of the T-lymphocyte receptor NΥ-E8O attached by a disulfide bond and specific for the XU-E8O-1 complex P.A-A2.

bcTSC NΥ-E8O was cloned as described in Example 5 and expressed as follows.

Expression plasmids containing the mutated α-chain and β-chain, respectively, were transformed separately into E. coli strain BP21rPuk8 and individual ampicillin-resistant colonies were grown at 37 ° C in TUR medium (ampicillin 100 μg / ml) to OO ₆₀₀ 0.7 before induced protein expression at 0.5 mM 1PTO. Cells were selected 18 hours after induction by centrifugation for 30 min at a speed of 4000 rpm./min in a Vesktap 1-6V centrifuge. The cell pellet was resuspended in lysis buffer containing 10 mM Tpc-HCl pH 8.1, 10 mM MdC1 ₂ , 150 mM No. C1, 2 mM OTT, 10% glycerol. For each liter of the bacterial figure, 100 μl of lysozyme (20 mg / ml) and 100 μl of deoxyribonuclease I (20 μg / ml) were added. After exposure to ice for 30 minutes, the bacterial suspension was treated with ultrasonic pulses of 1 minute duration for 10 minutes in an ultrasonic setup MPkoshkh XE2020 using a standard probe with a diameter of 12 mm. Precipitated inclusion bodies were recovered by centrifugation for 30 minutes at 13,000 rpm in a Wescapup 12-21 centrifuge (4 ° C). Then, to remove debris and membrane components, the precipitate was washed with three portions of washing buffer Thyop (50 mM Tpc-HC1 pH 8.1, 0.5% Tp! Op-X100, 100 mM No. 01, 10 mM SHEOTA, 0.1% (wt. ./vol.), 2 mM OOT). Each time, the inclusion Taurus precipitate was homogenized in Tp! Op wash buffer before sedimentation by centrifugation for 15 min at 13000 rpm in a Weststap 12-21 centrifuge. After that, the detergent and salt were removed by similar washing in a re-suspension buffer (50 mM Tpc-HCl pH 8.1, 100 mM No. C1, 100 mm No. C1, 10 mm HaEOTA, 0.1% (w / v) sodium azide, 2 mM OOT). And finally, inclusion bodies were dissolved in 6 M guanidine buffer (6 M guanidine hydrochloride, 50 mM Tpc pH 8.1,100 mM No. 0, 10 mM EOTA, 10 mM OPT), 120 mg aliquots and frozen at -70 ° C. Quantification of inclusion bodies was carried out by dissolving in a 6 M solution of guanidine-HC1 and analyzing the compound with the dye VgabGogb (RetVyu).

About 60 mg (i.e., 2.4 μmol) of the frozen dissolved α-chain was mixed with 30 mg (i.e., 1.2 μmol) of the frozen dissolved β-chain. The TSN mixture was diluted to a final volume of 18 ml with 6 M guanidine buffer and heated to 37 ° C for 30 minutes to ensure complete chain denaturation. Then, a solution of guanidine containing completely reduced and denatured TSN chains was introduced in 1 L of cold re-stacking buffer (100 mM Tpv pH 8.1, 400 mM L-arginine-HC1, 2 mM ΕΌΤΆ, 6.6 mM 2-mercaptoethylamine, 3 , 7 mm cystamine, 5 M urea) with stirring. The solution was kept for 5 hours in a cold room (5 ± 3 ° C) to allow for repeated styling. After that, the re-laid product was dialyzed against 12 L of water for 18–20 h, and then against 12 L of 10 mM Tpv pH 8.1 for 18–20 h (5 ± 3 ° C). For the indicated dialysis process, a 8res1tarot 1 dialysis membrane was used (article No. 132670, 8res1tish baobaFpev), which provides a molecular weight cut-off of 6-8000 kDa. The dialyzed protein was filtered through filters with a pore size of 0.45 μm (8cBe1set apb 8c1sche11, No. 10 404012) installed on a filtration unit No. 11gepe.

The re-laid TSN ΝΥ-Ε8Θ was separated from the decomposition and contamination products by loading the dialyzed re-laid product into the RONO 50 50NO anion exchange column (Arrieb Vu1esy). Before loading the protein column PONO 50 50NO balanced 10 column volumes of buffer A (10 mm TP8 pH 8.1). The bound protein was eluted with a gradient of 0-400 mM ΝηΟΙ in 7 column volumes. Peak fractions (1 ml) were analyzed using denaturing 8E8-PACE. using reducing and non-reducing sample buffer. Then, the peak fractions containing the heterodimeric alpha-beta complex were purified using an 8iretech 75NH gel filtration column previously balanced in 25 mM ME8, pH 6.5. Peak protein eluting with a relative molecular weight of about 50 kDa was combined, concentrated to 42 mg / ml in INTAGTEE centrifugal concentrators (Mbyrote, article No. IRU2BOC40) and stored at -80 ° C.

TSN Е -EZO was crystallized by hanging drop method at 18 ° С using 1 μl of a protein solution (8.4 mg / ml) in 5 mM Me§ at pH 6.5 mixed with an equivalent volume of crystallization buffer. The crystals appeared in several different modes when using buffers Stu81a1 8step (Nashrop Neveatsy). Single cubic crystals (<100 μm) were grown in a buffer containing 30% REO 4000, 0.1 M sodium citrate with a pH of 5.6, and 0.2 M ammonium acetate, and used to determine the structure.

ТСН ΝΥ-ЕЗО crystals were subjected to ultrafast freezing and X-ray diffraction analysis of the crystals was carried out on an Oagewu synchrotron. The crystals were diffracted to a resolution of 0.25 nm (2.5 A). The obtained data set was processed and received 98.6% of the full set of amplitudes, acceptable up to about 0.27 nm (2.7 A), but suitable for use (calculation) up to 0.25 nm (2.5 A). The combined H-factor, i.e. the consistency between many changes in crystallographically equivalent reflections was 10.8% for all data. This is the minimum for the highest resolution area. We found that the space group corresponds to P2! with a cell size of a = 4.25 nm (42.5 A), b = 5.95 nm (59.5 A), c = 8.17 nm (81.7 A), β = 91.5 °. Cell dimensions and symmetry indicate the presence of two copies in the cell. An asymmetric element, ai or the minimum volume that is to be studied, contains only one molecule, and the other molecule in the cell is formed as a result of symmetry 2 !. The positioning of the molecule in the direction of the y axis in ai is arbitrary. As long as it is in the correct position in the x-z plane, it can be arbitrarily translated in the direction of the y axis. This is called a free parameter in a given polar spatial group.

The REV database has only one position containing heterodimeric TSN A / B, namely 1–2. This position also has the coordinates of the HLA-related peptide in complex with TSN. Chain B TSN was the same as in No. D-E8O, while chain A had slight differences in domain C and significant differences in domain Ν. When using the 1ВЭ2 А / В model for molecular substitution (sho1esi1at ter1asessep1, MN), incorrect resolution was obtained, which showed extensive overlap of molecules with equivalent symmetry. Using only chain B gave the best resolution, in which there were no significant conflicts with neighbors. The correlation coefficient was 49%, the crystallographic H-factor was 50%, and the minimum approximation (center of gravity to center of gravity) was 0.49 nm (49 A). The rotation and translation operations necessary for transforming the initial model of chain B into the equivalent MN were applied to circuit A. The resulting hybrid resolution of the MN fit well into the cell with minimal collisions.

The electron density maps were generally consistent with the model and made it possible to adjust it to match the sequence of TSN Е-EZO. However, the original model had many gaps, in particular, it lacked side chains, which characterizes poor ordering of parts of the model. Many hairpin loops in the spaces between the threads had a low density and were difficult to model. The crystallographic H-factor mo-27 of Delhi was 30%. This K-factor is residual, i.e. represents the difference between the calculated and observed amplitudes.

As shown in FIG. 83a and 83b, the input sequence from 1BE2 does not match density very well. Changing the model to Suz at positions 164 in chain A and 174 in chain B, with subsequent further improvement, clearly showed that such a job sequence is much better consistent with the density. In this case, differences in side chain sizes are minimal, which led to small perturbations in the model. The electron density in this region also had small changes.

The most important aspect of this work is that the new TSC is very similar in structure to the published model (1VI2). The comparison included the entire TSC, constant domains, or small fragments near the point of mutation.

Deviations of the values of g.t.z. parameters are presented in the table below. A comparison of structures is shown in FIG. 84.

Complete circuit A Full circuit B The constant domain of chain A The constant domain of chain B Short section Displacement g. T. 3 2.831 1.285 1.658 1.098 0.613 Average displacement 2.178 1.001 1.235 0.833 0.546 Maximum offset 9.885 6.209 6.830 4.490 1.330

(All items are indicated in A)

A short section means a single strand of chain A (A157 to A169) and a single strand of chain B (B 170 to B 183), which are connected by a disulfide bridge. Deviations are calculated only for the main atoms of the chains.

These results confirm that the introduction of a disulfide bond has a minimal effect on the local structure in the bond zone. A slightly more significant effect is observed when comparing TSC with the published structure (1VI2) of TSC A6, however, an increase in the KM8 displacement is largely due to differences in loop conformations (see Fig. 84). These loops do not form part of the structure of the TSC core, which is formed by a sequence of β-folds that create a characteristic folding of immunoglobulin. The deviation of KM8 for the whole α-chain is especially large, which is caused by the difference in the sequence of variable domains in A6 (1VI2) and TSC ΝΥ-Ε8Ο. However, A6 and TSC ΝΥ-Ε8Ο have the same variable β-domain, and KM8 deviations for the whole β-chain show that the structure of this variable domain is also preserved in TSC with a new disulfide bond. Thus, the obtained data show that the structure of the TSC core is retained in the crystal structure of the TSC with a new disulfide bond.

Example 10. Obtaining soluble TSC ΝΥ-Ε8Ο containing a new disulfide interchain bond and labeled sites in the C-terminal region of the β-chain.

To obtain soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond, plasmids TSC A6 containing the restriction sites of the WatH1 α chain and the Bd111 β chain were used as the basis, as described in Example 4.

The β-chain constructs of TSC ΝΥ-Ε8Ο were obtained by cloning of CSCs as follows. CSC reactions were performed using the primers and matrices listed below containing TSC chains ΝΥ-Ε8Ο.

I ΝάθΙ |

Γ “ά5 '-САСАТАТАСАТАТСССТСТСАССАСААС-3'

Κθν5'-SSASSSSSATSSSTSTSTSTASSSSSSASSSS-3 '| YourH1 |

CSC products were restriction fermented and cloned into pCMT7 containing a recognition biotin sequence to obtain expression plasmids. The sequence of plasmid inserts was confirmed by automatic DNA sequencing. FIG. 85a shows a TSC ΝΥ-Ε8Ο β chain DNA sequence containing a biotnin recognition site, and FIG. 85b is the resulting amino acid sequence.

The α-chain construct was obtained as described in Example 5. The corresponding TSC chains were expressed, re-stacked, and purified as described in Example 5.

To obtain a soluble TSC ΝΥ-Ε8Ο containing a non-native disulfide interchain bond and a hexahistidine tag in the C-terminal region of the β chain, the same ΝΥ-Ε8Ο primers and matrices were used, as described above. CSC products were restriction fermented and cloned into pCMT7 containing a hexahistidine sequence to obtain expression plasmids. FIG. 86a shows a TSC ΝΥ-Ε8Ο β chain DNA sequence containing a hexahistidine tag, and FIG. 86b is the resulting amino acid sequence.

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FIG. 87 shows the elution of soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond and a sequence for recognizing biotin from an anion exchange column ROCO8 50HC. using a gradient of 0-500 mm No. C1, as indicated by the dashed line. FIG. 88 illustrates the elution of soluble СК-Ε8Ο TSC containing a new disulfide bond and hexahistidine tag from ROCO 50NC anion exchange columns using a 0-500 mM No. C1 gradient, as indicated by the dotted line.

FIG. 89 and 90 are gel chromatography profiles of the pooled ΝΥ-Ε8Ο eluates fractions labeled with biotin and hexahistidine from the anion exchange column and shown in FIG. 87 and 88 respectively. The protein elutes, forming a single main peak, corresponding to the hetero-dimer TSC.

The analysis of B1Asogue of kTSC associated with rMNS was performed as described in Example 3. TSC ΝΥ-Т8СК labeled with biotin had a Cd of 7.5 μM, TSC ΝΥ-Ε8Ο labeled with hexahistidine had a Cd of 9.6 μM.

Example 11. Staining of cells using fluorescent labeled tetramers of soluble TSC ΝΥ-Ε8Ο containing a new disulfide interchain.

Preparation of TSC Tetramer

ΝΥ-Ε8Ο soluble TSCs containing a new disulfide bond and a biotin recognition sequence obtained as described in Example 10 were used to prepare soluble TSC tetramers required for staining cells. A 2.5 ml buffer solution of purified soluble TSC (~ 0.2 mg / ml) was made in the biotinylation reaction buffer (50 mM Tp5 pH 8.0, 10 mM MdC1 ₂ ) using a 10-10 column (Ryagtaaa). The eluate (3.5 ml) was concentrated to 1 ml using a Se1psoi (At1soi) concentrator with a molecular weight cut-off of 10 kDa. This operation was performed until 10 mM was obtained by adding ATP from the mother liquor (0.1 g / ml, adjusted pH 7.0). Then a mixture of protease inhibitors was added (set 1 of a mixture of protease inhibitors, Ca1Osiet Vusjetkak) in a volume sufficient to obtain a final concentration of a mixture of protease inhibitors equal to 1/100 of the mother liquor in the delivery state, and then 1 mM biotin (from 0.2 M stock solution) and 20 μg / ml of enzyme (from 0.5 mg / ml of stock solution). The mixture was allowed to stand overnight at room temperature. The excess biotin was removed from the solution by gel chromatography in a column of 875 NK. The biotinylation level of TSC ΝΥ-Ε8Ο was determined by high performance liquid chromatography (HPLC) as described below. An aliquot of 50 μl of biotinylated TSC ΝΥ-Ε8Ο (2 mg / ml) was kept with 50 μl of streptovidin-coated agarose granules (81 dtA) for 1 hour. Then, the granules were pelleted by centrifugation and 50 μl of an unbound sample was passed through a T8K 2000 8 XV column (Tokoyaak ) at a flow rate of 0.5 ml / min (200 mm phosphate buffer with a pH of 7.0) for 30 minutes The presence of biotinylated TSC ΝΥ-Ε8Ο was determined using an ultraviolet spectrophotometer at a wavelength of 214 and 280 nm. Biotinylated ΝΥΕ8Ο was passed against a control sample of non-biotinylated TSC ΝΥ-Ε8Ο. The biotinylation percentage was calculated as the difference in the peak areas of the biotinylated and non-biotinylated protein.

Tetramerization of biotinylated soluble TSC was performed using a neutravidin phycoerythrin (PV) conjugate (Satybde Vukaiseisek, IR). The concentration of biotinylated soluble TSC was determined by the method of staining the Coomassie protein (P1egse), and the content of the neutravidin-phycoerythrin conjugate of 0.8 mg / mg of soluble TSC was calculated to ensure saturation of the neutravidin-PE with biotinylated TSC in the ratio of 1: 4. 19.5 μl of a solution with a concentration of 6.15 mg / ml of biotinylated soluble TSC ΝΥ-Ε8Ο in phosphate-buffered saline (pHdegree LiuHegebay, PB8) was slowly added to 150 μl of soluble neutravidin-FE conjugate on ice with gentle stirring. Then, 100.5 μl of PB8 was added to this solution to obtain a final concentration of TSC ΝΥ-Ε8Ο 1 mg / ml tetramer.

Staining protocol

Four aliquots of 0.3 × ^{10 6} NBA-A2 positive B-cells of the line (PP ECb) transformed with ΕΒν in 0.5 ml of PB8 were cultured with different concentrations (0, 10 ^-4 , 10 ^-5 and 10 ^-6 M) of the peptide (8 ^^ МВІТ ^ С) NBA-A2 ΝΥΕ8Ο for 2 hours at 37 ° С. Then these cells were washed twice with PP LBP in Hanks balanced salt solution (Naik'k Lighegeb 8a1e 8o1iuyu, HB88) (C1bco, IR).

Each of the four aliquots was divided into equal parts and stained with TSC свеж-Ε8Ο biotinylated, freshly tetramerized neutravidin-phycoerythrin coupled with a disulfide bond. Cells were incubated with 5 or 10 μg of phycoerythrin labeled with tetrameric bkTSK complexes on ice for 30 minutes and washed with HB88. Then the cells were washed again, resuspended in HB88 and analyzed using the EAC8 Vaΐΐade system. 25,000 units were selected and data analyzed using νίηΜΙΌΙ software.

results

In FIG. 91a-11 show histograms of data obtained by the analysis of ΕAC8Vaηΐade for each sample prepared as described above. The following table shows the percentage of positively stained cells observed in each of the samples.

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Sample

Positive peptide ΧΥ-Ε8Ο, 5 μg TSC.

10 ' ⁴ M д-Ε8Ο peptide, 5 μg TSC. 10 ' ⁵ M д-Ε8Ο peptide, 5 μg TSC ΙΟ' ⁶ M ΝΥ-Ε8Ο peptide, 5 μg TSC. 0 peptide ΝΥ-Ε8Ο, 10 μg TSC.

ΙΟ ⁴ M peptide ΝΥ-Ε8Ο, 10 TSK.

ΙΟ ' ⁵ M peptide ΝΥ-Ε8д, 10 TSK.

mcg stained cells (%)

0/75

84.39

35.29

7.98

0.94

88.51

8.25 mcg

These data definitely show that the increase in the proportion of cells labeled with HUEBO TSN tetramers correlates with the concentration of the peptide (BccMHHGOS) in which they were cultured. Thus, these TSN XU-EBO tetramers are components suitable for specific labeling of cells based on the expression of the HEA-A2 XU-EBO complex.

In this example, a fluorescent conjugated tetramer TSN XU-EBO was used. However, similar levels of cell binding can be expected if this label is replaced with a suitable therapeutic component.

Example 12. Obtaining soluble TSN A6 with a new disulfide bond containing a constant region of Sv1.

All the preceding examples describe the preparation of soluble TSN with a new disulfide bond containing constant region C [32. The present example shows that soluble TSNs containing the constant region Cb 1 can also be successfully obtained.

Design primers for splicing using RSN variable domain β-chain TSN A6 with SV 1

For the construct RSN of the variable domain of the β-chain TSN A6 created the following primers:

’-OSAOATATASATATOAASOSTOOTOTSAST-Z’ ’-SSTTOTTSAOOTSSTSTOTbASSOTOAO-Z’

For construct RSN S [31 created the following primers:

’-SSASASSTS ASASASASOASSTOAAS AASZS-3’ ’’ -SSS AAOOSTTAOTOT6STSTASSSSASASOSSTSSOSOS-3 ’

The β-UTSN construct and construct C [31 were obtained separately by amplification of the variable and constant domains of each chain using standard RSN technology. They were interconnected using PCN splicing (kysypd-RSN). The DNA plasmid was purified in a ^^ aden mini-preparation column according to the manufacturer's instructions and the sequence was checked by automatic sequencing on the sequencing equipment of the Department of Biochemistry, University of Oxford. The sequence A6 + C31 is shown in FIG. 92.

Then, the A6 + C31 chain was paired with the TSH A6 α-chain using an interchain disulfide bond after cysteine was introduced into the C-region of both chains.

Soluble TCH was expressed and re-laid as described in Example 2.

The purification of the re-laid soluble TSN kTSN was separated from the decomposition and contamination products by loading the dialyzed re-laid product into an anion exchange column RONOB 5011C) and eluting the bound protein with a gradient of 0-400 mM XaCl in 50 column volumes using the Ak1a (Pagtac1a) purification system, as shown in FIG. 4. Peak fractions until combined and concentrated were stored at 4 ° C and analyzed using BBB-RAOE with Coomassie staining (Fig. 94). And finally, the KTSN was purified and analyzed using a Biregbeh 200NH gel filtration column (Fig. 95), after balancing it in HBB-EP buffer (10 mM NEREB pH 7.4, 150 mM HaCl, 3.5 mM EOTA, 0 , 05% off! P40). Elution peak fractions with a relative molecular weight of about 50 kDa were pooled and concentrated before analysis using surface plasma resonance using a Schasogeg apparatus

A Schasoge analysis of the TSH A6 compound with a disulfide bond with rMHC was performed as described in Example 3. FIG. 96 shows the result of a Schasoge analysis of the specific binding of soluble TSH A6 with a disulfide bond to its related rMHC.

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The soluble TSB A6 with a new disulfide bond containing the constant region Cβ1 had a CD equal to 2.42 ± 0.55 μmol for its related rMNC. This value is very close to the CD, equal to 1.8 μmol, defined for soluble TCB A6 with a new disulfide bond containing a constant region of Cβ2, as determined in example 3.

Example 13. Obtaining soluble TCB A6 with a new disulfide bond containing free cysteine in the β-chain.

The constant regions of the TCB β-chain include a cysteine residue (residue 75 in exon 1 of TBBC1 * 01 and TBBC2 * 01), which is not involved in the formation of interchain or intrachain disulfide bonds. All the preceding examples describe the preparation of soluble TSBs with a new disulfide bond, in which this free cysteine is mutated into alanine to avoid the possible formation of unnecessary disulfide bonds, which can lead to a decrease in the yield of functional TSB. This example shows the possibility of obtaining soluble TCB containing the specified free cysteine.

Primer Design and TSB β-Chain Mutagenesis

The following primers were designed for cysteine for the alanine mutation of the TCB β chain of TCB (residue 75 in exon 1 of TBBC1 * 01 and TBBC2 * 01) (the mutation is shown in lowercase letters):

5’-T OAS TSS ASA TAS ΐ§Τ STS ASS ASS ASS

5’-SS OST OST S AS Asa STA TST OSA STS A

Mutagenesis of RSV, expression and re-folding of soluble TSB was performed as described in Example 2.

The purification of the re-laid soluble TSB KTSB was separated from the decomposition and contamination products by loading the dialyzed re-laid product into the PΟBΟ8 50HP anion-exchange column and eluting the bound protein with a gradient of 0-500 mM \ aC1 in 50 column volumes using the Ak! A purification system (Ryagtas1a), as shown in FIG. 98. The peak fractions were stored at 4 ° C until combined and concentrated and analyzed using 8 ^ 8-PAΘΕ with Coomassie staining (Fig. 90). Finally, the CTSB was purified and analyzed using column 8 and 200hB gel filtration column 8 (Fig. 100), after balancing in HB8-BR buffer (10 mM HΕPΕ8 pH 7.4, 150 mM No. C1, 3.5 mM Ε ^ TA, 0 , 05% off! P40). Elution peak fractions with a relative molecular weight of about 50 kDa were pooled and concentrated prior to analysis using surface plasma resonance on a Height setup.

Analysis The binding height of TSB A6 with a disulfide bond and rMHC was performed as described in Example 3. FIG. 101 shows the result of a Schasoge analysis of the specific binding of soluble TCB A6 with a disulfide bond to its related rMHC.

The soluble TSB A6 with a new disulfide bond containing free cysteine in the β-chain had a CD equal to 21.39 ± 3.55 μmol for its related rMNC.

Example 14. Obtaining soluble TCB A6 with a new disulfide bond in which free cysteine in the β chain is mutated into serine.

This example shows the possibility of successfully producing soluble TSB with a new disulfide bond in which the free cysteine in the β-chain (residue 75 in exon 1 TEBC 1 * 01 and TBBC2 * 01) is mutated into serine.

Primer Design and TSB β-Chain Mutagenesis

For the alanine mutation of the TCB β-chain, which was previously replaced by native cysteine (residue 75 in exon 1 of TBBC1 * 01 and TBBS2 * 01), the following primers were constructed in the series (mutation is shown in lowercase letters):

5’-T OAS TSS ASA TAS UT STS ASS ASS ASS

5’-SS SST SST SAS ASa STA TST SOA STS A

Mutagenesis of RSV (as a result of which a mutated β-chain was obtained, as shown in Fig. 102), expression and re-folding of soluble TSB was performed as described in example 2.

The purification of the re-laid soluble TSB KTSB was separated from the decomposition and contamination products by loading the dialyzed re-laid product into the PΟBΟ8 50HP anion-exchange column and eluting the bound protein with a gradient of 0-500 mM \ aC1 in 50 column volumes using the Ak! A purification system (Ryagtas1a), as shown in FIG. 103. The peak fractions were stored at 4 ° C until combined and concentrated and analyzed using 8 ^ 8-PAΘΕ with Coomassie staining (Fig. 104). Finally, the CTSB was purified and analyzed by column 8 andregbeh 200HV gel filtration (Fig. 105), after balancing in buffer 11138-1: 1 '(10 mM HΕPΕ8 pH 7.4, 150 mM No. C1, 3.5 mM Ε ^ TA, 0.05% off! P40). Elution peak fractions with a relative molecular weight of about 50 kDa were pooled and concentrated prior to analysis using surface plasma resonance on a Height setup.

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The B1A assay of the binding of TSC A6 to a disulfide bond and pMHC was performed as described in Example 3. FIG. 106 shows the result of B1Acoge analysis of the specific binding of soluble TSC A6 with a disulfide bond to its related pMHC.

A soluble TSC A6 with a new disulfide bond in which the free cysteine in the β chain was mutated into serine had a CD equal to 2.98 ± 0.27 μmol for its related rMNC. This value is very close to Kd = 1.8 μmol obtained for soluble TSC A6 with a new disulfide bond in which free cysteine in the β chain was mutated into alanine, as defined in Example 3.

Example 15. Cloning of α and β chains of TSC ΝΥ-Ε8Ο containing a new disulfide bond into yeast expression vectors.

The α- and β-chains of TSC ΝΥ-Ε8лияние were fused with the C-terminal region of the alpha sequence of the regergo pipe-factor from Sassyagosus seseu181ae and cloned into the yeast expression vectors ρΥΧ122 and ρΥΧ112, respectively (see Figs. 107 and 108).

The following primers were designed to amplify the CSC of the alpha sequence of the rge-pgco Schepd factor from strain 8–6210 8. cepu181ae (Klössop e! A1. (1991), Mo1. For fusion with the TSC α-chain.

5'-TST SAA TTS ATS ASA TTT SST TSA ATT TTT

Ce11. Vu1. 11 (12): 5813-24)

AC-3 '

5'-TSA SST SST SSS SST STT SAS SST SST TTT TAT S -3 '

The following primers were designed to amplify the DSC of the alpha sequence of the rge-rego Schepd factor from strain 8–6210 8. segeu181ae for fusion with the TSC β-chain.

5'-TST S7XA TTS ATS ASA TTT SST TSA ATT TTT AS-3 '

5'-STS TST SSA STT AST STS STS TAS SSS SSS SSS-3 '

Yeast DNA was obtained by re-suspending a colony of strain 8–6210 8. cegeu181ae in 30 μl 0.25% 8–8 in water and heating for 3 min at 90 ° C. Alpha sequences of the rge-rgo shieldpd factor for fusion with the TSC α and β chains were obtained by DSC amplification of 0.25 μl of yeast DNA with the corresponding primer pairs using the following DSC modes. 12.5 ml of each primer was mixed with 200 μM Sr, 5 μl of 10 × Pb buffer and 1.25 units of polymerase Pb and (81α1αβ) in a final volume of 50 μl. After the initial stage of denaturation for 30 s at 92 ° C, 30 cycles of denaturation of the reaction mixture (92 ° C, 30 s), annealing (46.9 ° C, 60 s) and elongation (72 ° C, 2 min) were carried out in express -installation of RSK - NLa14 RSK.

The following primers were designed for CSC amplification of the α chain of TSC in order to fuse with the above alpha sequence of the rge-rego shape factor.

5'-CCC TSA ACC CCA CCA CCT CAC ASA SAT TSS-3 '

5'-STS STS TSS AST TAS SAA STT TST SSS STS SS-3 '

The following primers were designed for CSC amplification of the TSC β-chain in order to fuse with the above alpha sequence of the rge-rgo shape factor.

5'-SSS TSA SSS SSS SST SAS SAS TSA SAS SSS AAA AT-3 '

5'-STS TST SSA STT AST STS STS TAS SSS SSS SSS-3 '

The CSC modes for amplification of α and β chains of TSCs were the same as described above, with the following changes: the матрицыΛ matrices used to amplify α and β chains of TSCs were α and β chains of TSC ΝΥ-Ε8Ο respectively (obtained according to example 5), and the applied annealing temperature was 60.1 ° C.

After that, the RAC products were used in the cross-linking reaction of RAC (sRcSypd RAC) using complementary overlapping sequences introduced into the starting RAC products to obtain the full length chimeric gene. The resulting CSC products were fermented with the restriction enzymes EcoC I and XHO I and cloned into ρ X122 or ρΥX112, fermented with the same enzymes. The resulting plasmids were purified in an O | adep ™ mini-prep column according to the manufacturer's instructions and the sequence was monitored using automatic sequencing on sequencing equipment Oepez L14., Oyepz ^ ay, \ e \\ - M111op, Nashrzyge, ipeb Kshdbosh. FIG. 109 and 110 show the DNA and protein sequences of cloned chimeric products.

Example 16. Expression in yeast of soluble TSC ΝΥ-Ε8Ο containing a new disulfide bond.

Yeast expression plasmids containing α and β chains of TSC, respectively, and obtained according to Example 15, were jointly transformed into strain 8ΕΥ6210 8. segeu181ae using the Ada! Ee protocol! a1. (1998) (Tessysa1 T1p3 () n11pe (L1p: / L1oTgep48.soosh) 1: 51: P01525). One colony grown on synthetic isolation agar (zupFace 4horoi1, 8Ώ) containing histidine and uracil (Oodepe, 111knsN, Rgapsa) was cultured overnight at 30 ° C in 10 ml of 8Ώ medium containing histidine and uracil. The resulting culture was inoculated at a ratio of 1:10 in 10 ml of fresh 8Ώ medium containing histidine and uracil and grown for 4 hours at 30 ° C. The culture was centrifuged for 5 min at

3800 rpm in a centrifuge Negayiz MedaGide 2.0K (KeBoBaGoYuu Prgos1is1z βΐά., B1zNor'z 81og1Gogd, Neshogdzyge, ϋΚ) and the supernatant was taken. 5 μl of the 8ΐ ^ aΐC1еаη resin (8ΐ ^ аΐadeηе) was mixed with the supernatant and left overnight while rotating in L1 odekee1 at 4 ° С. The 8ΐ ^ aΐaC1eaη resin was precipitated by centrifugation at 3800 rpm in a Negaeiz Meda Guide 2.0K centrifuge and the medium was removed. 25 μl of sample recovery buffer (950 μl Liemmi sample buffer (Wughaf containing 50 μl of 2M ETT) was added to the resin and the samples were heated at 95 ° C for 5 min and then cooled on ice, after which 20 μl of the mixture was loaded onto gel 8E8-PAOE at a constant current density of 0.8 tA / cm ^{2 of} the gel surface for 1 h. Proteins in the gel were transferred to membranes for immunoblotting 1ttiηο-B1οΐ RUEB (polyvinylidene fluoride) (Vu-Kaf and tested with an antibody against TSC α-chain, as described below in Example 17, with the following modifications: Primary antibodies (against the α chain TSC) and secondary antibodies were used at dilutions of 1 in 200 and 1 in 1000, respectively. An image of the developed membrane is shown in Fig. 111. The result shows a low level of TSC secretion by yeast culture on Wednesday.

Example 17. Expression of α - and β-chains of disulfide TSC A6 Tax in baculovirus.

The cloning strategy of the α and β chains of the disulfide TSC A6 Tax was cloned from pOMT7 into a vector based on pB1ie8spr! K82, called pEX172. This vector was designed for cloning various β-chains of MHC class II, for expression of insect cells using the leader sequence from the EQI 1 * 0101 of the Ade! to insert various sequences encoding the peptides, the linker region and then the sites M1i1 and 8a11 for cloning βΕβ chains before the leucine zipper sequence Ήη. The sequence in which pEX 172 is different from pB1ie8spr! II K8-, located between the sites of KrY and EcoSh rV1ie8spr! II K8- and shown in FIG. 112. For cloning of TSC chains into insect cells, the indicated pEX172 was cut off with AdeI and 8aI to remove the linker region and the сайт1Ή site and to ensure the entry of TSC chains at the beginning of the peptide sequence. The TSC sequences were cloned from pOMT7 with the BpREI site at the end of 5 '(it had sticky ends compatible with AdeC and the 8aP site at the end of 3'. To obtain the cleavage site to remove the βΕβ leader sequence, the first three groups of the βΕβ chain (OOT) were retained. to prevent the transcription of the Ήη leucine zipper sequence, it was necessary to insert a termination codon in front of site 8aI. A diagram of this construct is shown in Fig. 113. After the TSC chains appeared in this plasmid, the BAT fragment was excised and were cloned into the pACAB3 vector, which has homologous recombinant sites for baculovirus. The pACAB3 vector contains two divergent promoters - one with the BAT site and one with the VdB cloning site. The TSC A6 β-chain contains the VdS site, so the TSC A6 α-chain was inserted into the site WBS, and then the β chain was subcloned into the WAT site.

In accordance with the above cloning strategy, the following primers were designed (vector homology is shown in capital letters):

Aba: G: 5'-d'adGssddadasassddaASAASASSAASTSSASSASSAAS

K: 5'-dSaddksdasTASSAASTTTSTSSSSSTSSS

Α6β: E: 5'-dkad GssddadasassddaAASSSTSTSTSASTSASAS

B: 5'-dGaddGsdasTASTSTSSTSTASSSSSSASS

DSC, cloning and subcloning

Expression plasmids containing the genes for the α or β chain of the disulfide TSC A6 Tax were used as matrices in the following CSC reactions. 100 ng of the α-plasmid was mixed with 1 μl of 10 mM NTP, 5 μl of 10xPGi buffer (8ΐ ^ aΐadeηе), 1.25 units of Rgi polymerase (8ΐ ^ aΐadeηе), 50 rt1 of the above A6α primers and, adding water, the final volume was adjusted to 50 μl . A similar reaction mixture was made for the β chain using a β plasmid and a pair of β primers. 35 cycles of denaturation (95 ° С, 60 s), annealing (50 ° С, 60 s) and elongation (72 ° С, 8 min) were carried out with the reaction mixtures in the express installation RSC-НуЬаШ РСК. Then, the product was fermented for 25 hours at 37 ° C with 10 units of the restriction enzyme BpE ^ and then for 2 hours with 10 units of 8AI (Ά \ ν Еηд1аηά Вю1абз). These products of the fermentation reaction were ligated in pEX172, which was previously fermented with AdeI and 8aP, and then transformed into competent XY-B1ie bacteria and grown for 18 h at 37 ° C. One colony was selected from each α- and β-preparation and grown overnight in 5 ml of ТΥР + ampicillin (bactotriptone 16 g / l, yeast extract 16 g / l, No. Сl 5 g / l, K ₂ NRA ₄ 2.5 g / l, ampicillin 100 mg / l). The plasmid DNA was purified on a ^^ aden mini-preparation column according to the manufacturer's instructions and the sequence was monitored by automatic sequencing on Oe ^^ x sequencing equipment. The amino acid sequences of the Watts inserts are shown in FIG. 114 and 115 for the α chain and β chain, respectively.

These α- and β-constructs of the chains of disulfide TSC A6 Tax in pEX172 were fermented for 2 hours at 37 ° C with the restriction enzyme Bacterium (Ά \ ν Еηд1аηά Вю1абз). An insertion of the WbT α-chain was ligated into the pACav3 vector (Pka ^ t ^ ηdeη-B ^ B ^^ e ^ ez: 21216Р), previously fermented by the enzyme

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Bd111. It was transformed into competent X1-B1ie bacteria and grown for 18 h at 37 ° C. One colony was taken from this plate and grown in 5 ml of ΣΥΓ + ampicillin, and the plasmid DNA was purified as described above. Then this plasmid was fermented with BatH1 and the BatH1 β-chain insert was ligated, transformed into competent XB1-B1ie bacteria, grown overnight, placed in TΥP-ampicillin medium and grown until mini-preparation, as described above, using a P1Adep mini-preparation column. The correct orientation of the α and β chains was confirmed by sequencing using the following sequencing primers:

RASAVZ а in the forward direction: 5’-yesааЕЕаЕдса> Еддда1: д рАСАВЗ β in the forward direction: З’-аББаддссСсБададаБссд

Transfection, infection, expression and analysis of TSH A6 in insect cells

An expression plasmid containing the α-chain and β-chain was transfected into cells of z9 (Reagentdep BP Buzz1epsept: 551411), grown using serum-free medium (Pagtdep-BP Buzz1epsez: 551411), using the Basi1odepsept transfectional kit 21100K) according to the manufacturer's instructions. After 5 days, 200 μl of the medium in which the indicated transfected cells were grown was added at 27 ° C to 100 ml of H1bb E1ue cells containing 1 x 10 ⁶ cells / ml in serum-free medium. After another 6 days at 27 ° C, 1 ml of this medium was removed and centrifuged at 13,000 rpm in a Negeiz microcentrifuge for 5 min to precipitate cell debris.

μl of the obtained supernatant A6 disulfide bound TSH insect was run in parallel with the positive control samples 5 μg and 10 μg of bacterial A6 disulfide bound TSN on a pre-prepared 4-20% Tp / glycine gel (1nu11godep: EC60252). Reconstituted samples were obtained by adding 10 μl of sample recovery buffer (950 μl of LaetMN sample buffer (Boo-Nad: 161-0737) 50 μl of 2M ETT), heating at 95 ° C for 5 min, cooling at room temperature for 10 min and subsequent loading of 20 μl. Unreduced samples were obtained by adding 10 μl of Laemmli buffer for imaging and loading 20 μl.

The gel was passed at 150 V for 1 h on a Nomex-Xse11 installation, after which the gel was stained in 50 ml of Coomassie gel dye for 1 h with gentle stirring (1.1 g of Coomassie powder was mixed in 500 ml for 1 h, 100 were added ml of acetic acid, the volume was adjusted to 1 L by adding H ₂ O and stirred for 1 h, and then filtered through a 0.45 μm filter). The gel was discolored 3 times for 30 min with gentle stirring in 50 ml in a decolorizing composition (corresponding to the composition of Coomassie for staining the gel, but without dye powder).

Western blot analysis was performed by passing 8E8-PACE gels as described above, however, the proteins were transferred onto 1ttipo-B1o1 RUNE membranes (Bu-Nad: 162-0174) instead of staining the gels with Coomassie dye. Six paper filters were cut to the size of the gel and impregnated with blotting buffer (2.39 g of glycine, 5.81 g of TPZ base and 0.77 g of ETT were dissolved in 500 ml of H ₂ O, 200 ml of methanol was added, and then the volume was adjusted to 1000 ml by adding H ₂ O). The RUEE membrane was prepared by impregnation with methanol for 1 min, and then with a blotting buffer for 2 min. Three paper filters were laid on the surface of the anode of the immunoblotting device (Reagtash - \ o \ 'ab1o1), then a membrane, gel on top of it, and finally three more paper filters on the cathode side. Immunoblotting was performed for 1 h at 0.8 mA DC / cm gel surface.

After blotting, the membrane was blocked in 7.5 ml of blocking buffer (4 tablets of physiological saline, buffered with TPZ (81 dt: T5030), 3 g of non-skimmed milk powder (81 dt: M7409), 30 μl of T \\ eUp 20 with the addition of Н ₂ О to a final volume of 30 ml) for 60 minutes with gentle stirring. Then, the membrane was washed three times for 5 min with TB8 wash buffer (20 tablets of TB8, 150 μl of T ^ un 20 with the addition of H ₂ O to a final volume of 300 ml). After that, the membrane was incubated with primary antibodies at a dilution of 1:50 of clone 3A8 of the anti-TCH α chain (8go1ec: MCA987) or of clone 3A8 of the anti-TCH α chain (8go1ec: MCA988) in 7.5 ml of blocking buffer for 1 h with light stirring. Washed the membrane, as described above, in TB8 buffer. Then, secondary antibodies of HHP-labeled goat antibodies against mouse antibodies were incubated (8AP! А О БУ1есЕ: 8с2005) at a dilution of 1: 1000 in 7.5 ml of blocking buffer for 30 min with gentle stirring. The membrane was washed as described above, and then washed in 30 ml of H ₂ O with 2 TB8 tablets.

Antibody binding was determined by the calorimetric method using Opΐ-4CN (Bugad: 1708235) (1.4 ml of diluent Or1-4C> 12.6 ml of H ₂ O, 0.28 ml of substrate Ory-4SK). Membranes were stained for 30 minutes and then washed in H2O for 15 minutes. The membranes were dried at room temperature and the scanned images were combined with the image of a gel stained with Coomassie (Fig. 116).

results

As seen in FIG. 116, both disulfide TSNs are in the form of a heterodimer that is stable in 8-8 gel. Upon restoration, both TSNs decompose into α and β chains. The insect TSN disulfide heterodimer has a slightly higher molecular weight than the bacterially produced variant, apparently due to glycosylation from insect cells. It can be seen that in this case the insect cells produce an excess α-chain, and the free α-chain is visible on the unrepaired track of the anti-α western blot.

These data clearly show that the baculovirus expression system described above is an effective alternative to the prokaryotic expression of soluble TSCs containing new disulfide bonds.

Claims (60)

1. A soluble T-lymphocyte receptor (Z1B1E T1111 Geser, ZTSC), which contains (I) the entire TSC α-chain or its part, except for its transmembrane domain, and (II) the entire TSC-chain or its part, for with the exception of its transmembrane domain, wherein each of components (I) and (II) contains a functional variable domain and at least a part of the constant domain of the TSC chain, while components (I) and (II) are connected by a disulfide bond located between cysteine residues replaced by Tig 48 exon 1 TKAS * 01 and 8eg 57 exon 1 TKVS1 * 01 or TKVS2 * 01; Tig 45 exon 1 TKAS * 01 and 8eg 77 exon 1 TKVSR01 or TKVS2 * 01; Tug 10 exon 1 Thr 45 exon 1 TKAS * 01 and 8eg 17 exon 1 TKVS1 * 01 or TKVS2 * 01; 8th 15 exon 1 TKAS * 01 and Azr 59 exon 1 TKAS * 01 and O1 and 15 exon 1 TKVS1 * 01 or TKVS2 * 01; or TKVS1 * 01 or TKVS2 * 01. 2. zTSC according to claim 1, characterized in that one or both components (I) and (II) contain the entire extracellular constant domain домен of the TSC chain. 3. zTSC according to claim 1 or 2, characterized in that one or both of the components (I) and (II) contain the entire extracellular domain of the TSC chain. 4. Soluble T-lymphocyte receptor αβ-form (zTSC), characterized in that the covalent disulfide bond connects the cysteine residues, which are replaced TKVS1 * 01 or TKVS2 * 01; TKVS1 * 01 or TKVS2 * 01; TKVS1 * 01 or TKVS2 * 01; TKVS1 * 01 or TKVS2 * 01; or TKVS1 * 01 or TKVS2 * 01. Thr 48 exon 1 Tig 45 exon 1 Tug 10 exon 1 Tig 45 exon 1 TKAS * 01 and 8eg 57 exon 1 TKAS * 01 and 8eg 77 exon 1 TKAS * 01 and Zeg 17 exon 1 TKAS * 01 and Azr 59 exon 1 TKAS * 01 and O1 and 15 exon 1 8th 15 exon 1 5. A soluble T-lymphocyte receptor (zo1b1e T1111 geser1og, zTSC), which contains (I) the entire α-chain of TSC or its part, with the exception of its transmembrane domain, and (II) the entire β-chain of TSC or its part, with the exception of its transmembrane domain, each of components (I) and (II) containing a functional variable domain and at least a part of the constant domain of the TSC chain, and at the same time they are connected by a disulfide bond located between amino acid residues of the constant domains, which is absent in the native TSC moreover, the native TSK does not contain ezhtsepochechnuyu disulfide bond. 6. zTSC according to claim 5, characterized in that one or both components (I) and (II) contain the entire extracellular constant domain Ή of the TSC chain. 7. zTSC according to claim 5 or 6, characterized in that one or both components (I) and (II) contain the entire extracellular domain of the TSC chain. 8. A soluble T-lymphocyte receptor of the β-form (zTSC), characterized in that the covalent disulfide bond connects the amino acid residue in the immunoglobulin region of the constant domain of the α chain with the residue of the immunoglobulin region of the constant domain of the β chain, and the native TSC does not contain an interchain disulfide bond . 9. zTSC according to claims 5-8, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues, which are replaced by amino acid residues, the distance between the β-carbon atoms of which is less than 0.6 nm in the native structure of TSC . 10. zTSC according to claims 5 to 9, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues that replace the Tb1 48 exon 1 TKAC * 01 and 8g 57 exon 1 TKVS1 * 01 or TKVS2 * 01 residues. 11. zTSC according to claims 5 to 9, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues that replace the Tb1 45 exon 1 TKAC * 01 residues and 8g 77 exon 1 TKBC1 * 01 or TKBC1 * 01. 12. zTSC according to claims 5 to 9, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues that replace the residues Tug 10 of exon 1 TKAS * 01 and 8g 17 of exon 1 TKVS1 * 01 or TKVS2-01. -35006601 13. zTSC according to claims 5 to 9, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues, which replace Tyg 45 exon 1 TKAC * 01 and Azr 59 exon 1 TKVS1 * 01 or TKVS2-01. 14. zTSC according to claims 5 to 9, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues, which replace the residues 8e15 exon 1 TKAS * 01 and C1i 15 exon 1 TKVS1 * 01 or TKVS2 * 01. 15. zTSC according to claims 1 to 4, characterized in that the native TSC does not contain an interchain disulfide bond. 16. zTSC according to claims 5-15, characterized in that the native α and β chains of TSC are truncated in the C-terminal region so that cysteine residues that form the native interchain disulfide bond are excluded. 17. zTSC according to claims 5-15, characterized in that the cysteine residues that form the native interchain disulfide bond are replaced by a residue of another amino acid. 18. zTSC according to claim 17, wherein the cysteine residues that form the native interchain disulfide bond are replaced with serine or alanine. 19. zTSC according to claims 1-18, characterized in that the unpaired cysteine residue present in the native β-chain of TSC is absent. 20. zTSC according to claims 1, 2, 5, 6 and 9-19, characterized in that each of the components (I) and (II) contains a functional variable domain of the first TSC fused to the full constant domain or part of the second TSC, wherein the first and second TSCs come from the same species (animals). 21. zTSC according to claim 20, characterized in that the constant domains of the second TSC are truncated in the direction of the Ν-end from residues that form a non-native interchain disulfide bond. 22. zTSC according to claims 1-21, characterized in that one or both chains are derivatized by any group in their C- or Ν-terminal region or merged with this group. 23. zTSC according to claims 1 to 22, characterized in that one or both chains contain a cysteine residue in their Strength of the Ν-terminal region with which any group can be fused. 24. zTSK according to claims 1 to 23, characterized in that it also contains a detectable label. 25. zTSC according to claims 1-24, characterized in that it is associated with a therapeutic agent. 26. A multivalent complex of a T-lymphocyte receptor (T ce11 gesyug. TSC), containing many zTSC according to claims 1-25. 27. The complex according to p, characterized in that it contains a multimer zTSK. 28. The complex according to p. 27, characterized in that it contains two, three, four or more molecules of the lymphocyte receptor, bound to one another, preferably by means of a linker molecule. 29. The complex according to claims 26, 27 or 28, characterized in that the soluble T-lymphocyte receptors or their multimers are in the lipid double layer (bilayer) or attached to any particle. 30. A method for detecting MHC-peptide complexes, comprising (1) obtaining soluble TSC according to claims 1-25 or a multivalent T-lymphocyte receptor complex according to claims 26-29, (2) contacting the soluble TSC or multivalent complex TSC with MHC-complexes a peptide; and (3) detecting binding of a soluble TSC or multivalent complex of TSC to MHC-peptide complexes. 31. A pharmaceutical composition containing zTSC according to claims 1-25 and / or a multivalent complex of TSC according to claims 26-29 together with a pharmaceutically acceptable carrier. 32. A nucleic acid molecule containing a sequence encoding components (I) or (II) of zTSC according to claims 1 to 25, or a sequence complementary to it. 33. A vector containing the nucleic acid molecule of claim 32. 34. A host cell containing the vector of claim 33. 35. The method of obtaining components (I) or (II) according to claims 1 to 25, including incubating the host cell according to clause 34 under conditions causing expression of the peptide, and subsequent purification of the polypeptide. 36. The method according to clause 35, characterized in that it also includes mixing the components (I) and (II) under conditions suitable for re-laying. 37. A method of obtaining a soluble T-lymphocyte receptor (zTSC), comprising incubating a host cell that contains a vector containing a nucleic acid molecule encoding (I) all or part of the TSC α-chain, except for its transmembrane domain, and the host cell, which contains a vector containing a nucleic acid molecule encoding (II) all or part of the TSC β chain except its transmembrane domain, under conditions causing expression of components (I) and (II), each of components (I) and (II) contains functional vari abel domain and at least part of the constant domain of the TSC chain, purification of components (I) and (II) and -36006601 mixing components (I) and (II) under conditions for re-folding, such that they turn out to be connected by a disulfide bond, which is located between amino acid residues of the constant domain and which is absent in the native TSC. 38. The method according to clause 37, wherein one or both of the components (I) and (II) contain the entire extracellular constant domain C of the TSC chain. 39. The method according to clause 37 or 38, characterized in that one or both of the components (I) and (II) contain the entire extracellular domain of the TSC chain. 40. A method of obtaining an αβ-form of a soluble T-lymphocyte receptor (8TSK), comprising incubating a host cell that contains a vector containing a nucleic acid molecule encoding a TSC α-chain and a host cell that contains a vector containing a nucleic acid molecule encoding the TSC β-chain, under conditions causing expression of the corresponding TSC chains, purification of the corresponding TSC chains and mixing of the corresponding TSC chains under re-folding conditions so that the covalent disulfide bond connects the backbone the amino acid current in the immunoglobulin region of the constant domain of the α chain with the remainder in the immunoglobulin region of the constant domain of the β chain. 41. The method according to PP.37-40, characterized in that the native TSC does not contain an interchain disulfide bond. 42. The method according to paragraph 41, wherein the native α and β chains of TSCs are truncated in the C-terminal region so that cysteine residues that form the native interchain disulfide bond are excluded. 43. The method according to paragraph 41, wherein the cysteine residues that form the native interchain disulfide bond are replaced by a residue of another amino acid. 44. The method according to item 43, wherein the cysteine residues that form the native interchain disulfide bond are replaced by serine or alanine. 45. The method according to claims 37-44, wherein the unpaired cysteine residue present in the native β-chain of TSC is absent. 46. The method according to claims 37-45, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues, which are replaced by amino acid residues, the distance between the β-carbon atoms of which is less than 0.6 nm in the native structure of TSC . 47. The method according to claims 37-46, characterized in that the disulfide bond that is absent in the native TSC is located between cysteine residues that replace the Tb1 48 exon 1 TKAC * 01 and 8g 57 exon 1 TKBC1 * 01 or TKBC1 * 01 residues. 48. The method according to claims 37-46, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues that replace the Tb1 45 exon 1 TKAC * 01 and 8g 77 exon 1 TKBC1 * 01 or TKBC1 * 01 residues. 49. The method according to claims 37-46, characterized in that the disulfide bond absent in the native TSC is located between cysteine residues that replace the residues Tug 10 of exon 1 TKAC * 01 and 8g 17 of exon 1 TKVS1 * 01 or TKVS2-01. 50. The method according to claims 37-46, characterized in that the disulfide bond that is absent in the native TSC is located between cysteine residues that replace the Tb1 45 exon 1 TKAC * 01 and A8p 59 exon 1 TKBC1 * 01 or TKBC1 * 01 residues. 51. The method according to claims 37-46, characterized in that the disulfide bond, which is absent in the native TSC, is located between cysteine residues, which replace the residues 8eg 15 of exon 1 TKAC * 01 and C1 and 15 of exon 1 TKVS1 * 01 or TKVS2 * 01. 52. The method according to claims 37, 38 and 41-51, characterized in that each of the components (I) and (II) contains a functional variable domain of the first TSC, fused with the full constant domain or part of the second TSC, the first and the second TSCs come from the same species (animals). 53. The method according to paragraph 52, wherein the constant domains of the second TSC are truncated in the direction of the Ν-end from residues that form a non-native interchain disulfide bond. 54. The method according to claims 37-53, characterized in that one or both chains are derivatized by any group in their C- or Ν-terminal region or fused to the specified group. 55. The method according to claims 37-54, characterized in that one or both chains contain a cysteine residue in their C- or Ν-terminal region, with which any group can be fused. 56. The method according to PP.37-55, characterized in that 8TSK also contains a detectable label. 57. The method according to claims 37-56, wherein 8TSK is associated with a therapeutic agent. 58. The method according to any of paragraphs.30, 35-57, characterized in that it also includes combining a variety of 8TSK to form a multivalent complex of the T-lymphocyte receptor (TSC). 59. The method according to p, characterized in that 8TSK combined with the formation of multimer 8TSK. 60. The method according to p, characterized in that two, three, four or more molecules of the T-lymphocyte receptor bind to one another, preferably through a linker molecule.