Single-strand recombinant t-cell receptors

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology. Description is given of a single-strand T-cell receptor (scTCR), containing an α segment, formed by a sequence of a variable region in a TCR chain, joined with the N end of the extracellular sequence with constant region in the TCR chain, a β segment, formed by a sequence of the variable region of the α TCR chain, joined with the N end of the extracellular sequence with constant region of the β TCR chain, and a linker sequence, joining the C end of the α segment with the N end of the β segment, or vice versa. Extracellular sequences of constant regions of α and β segments are joined by a disulphide bond. Extracellular sequences of constant regions can correspond to constant regions of α and β chains of native TCR, cut-off at their C ends such that, cysteic residues, which form the inter-chain native disulphide bond of the TCR, are excluded, or extracellular sequences of constant regions which are in the α and β segments, can correspond to constant regions of α and β chains of native TCR, in which cysteic residues, which form the native inter-chain disulphide bond, are replaced by another amino acid residue, or there is no uncoupled cysteic residue, which is in the β chain of the native TCR. This invention makes available a new class of alpha/beta analogues of scTCR, in which there is a disulphide bond between residues of a single amino acid, contributing to stability of the bond between the alpha and beta regions of the molecule.

EFFECT: such TCR are suitable for screening or for therapeutic purposes.

3 cl, 14 dwg, 3 ex

 

The present invention relates to single-chain T-cell receptors (TCR).

As described, for example, in International application WO 99/120, TCR mediates the recognition of T cells specific complexes of peptides to major histocompatibility complex (MHC, MHC) and, as such, play a significant role in the functioning of the immune system at the cellular level.

Antibodies and TCR are only two types of molecules that recognize antigens in a specific way, and, therefore, TCR is the only receptor for antigens specific peptides present in the MTL, and alien peptide is very often the only sign of the anomalous cells. T-cell recognition occurs when the T cell and antigen-presenting cell (APC) are in close physical contact, and is initiated by legirovaniem antigen-specific TCR with rmns complexes.

Native TCR is a heterodimeric protein on the cell surface belonging to the immunoglobulin superfamily, which is associated with an invariant proteins of the CD3 complex involved in mediating signal transduction. TCR exists in αβ and γδ forms, which are similar in structure but have different anatomical locations and possibly function. The ligands of MHC class I and class II I which are proteins of the superfamily of immunoglobulins, but differ in specific antigenic presentation, with the highly polymorphic binding site of the peptide, which allows them to present different sets (matrices) short peptide fragments on the cell surface of APC (antigen-presenting cells).

It is known that two other classes of proteins can function as TCR ligands. (1) CD1 antigens are I - related molecules MHC class genes are localized on chromosome other than chromosome classical antigens MHC class I and class II. The CD1 molecules capable of presenting the peptide and not the peptide (e.g., lipid, glycolipid) particles T cells in a manner analogous to the methods of presentation of regular complexes of class I and class II. See, for example, (Barclay et al, (1997) The Leucocyte Antigen Factsbook 2ndEdition, Acadmeic Press) and Bauer (1997) Eur J Immunol 27 (6) 1366-1373)). (2) Bacterial superantigen are soluble toxins that can communicate both with the molecules of class II MHC, and a subpopulation of TCR. Fraser (1989) Nature 339221-223). Many superantigen show specificity for one or two V beta segments, whereas the other is more chaotic (less selective) binding. Anyway (in any event) superantigen able to show an increased immune response due to their ability to stimulate the populatie T cell polyclonal way."

The extracellular part of the native heterodimeric αβTCR consists of two polypeptides, each of which has a membrane proximal constant domain and a membrane distal variable domain (see Figure 1). Each of the constant and variable domains includes nutricao disulfide bonds. The variable domains contain highly polymorphic loops, similar hypervariable regions (CDR) of the antibody. CDR3 T-cell receptor (TCR) interacting with peptides presented MNF, and CDR 1 and 2 interact with peptide and MHC. The diversity of TCR sequences occurs through somatic rearrangement associated variable (V), (D), junction (J) and constant genes. Functional polypeptides α chains are formed by rearrangeable V-J-C regions, whereas β chains consist of V-D-J-C regions. Extracellular constant domain contains a membrane-proximal region and the region of immunoglobulin. There is only one constant domain of the α chain, known as TRAC, and two different β constant domain known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acid substitutions between the β constant domains, three of which are in the domains used for the production of single-chain TCR according to this invention. All these substitutions are located in exon 1 of TRBC1 and TRBC2: N4 5→K4N5and F37→Y (IMGT nomenclature, the difference TRBC1→TRBC2), the final amino acid substitution is between two constant regions of β chains of the TCR in the exon 3 TRBC1 and TRBC2: V1→E. the Length of each of TCR extracellular domains is somewhat changeable. However, the specialist in the art will determine the position of the boundary of the domain, using the link to The T Cell Receptor Facts Book, Lefranc & Lefranc, Publ. Academic Press 2001,

Single-chain TCR (T cell receptor)

Single-chain TCR (scTCR) are artificial structures consisting of a single amino acid chain (strand, filament), who, like the native heterogeneously TCR bind to MHC-peptide complexes. Unfortunately, attempts to obtain functional alpha/beta analogues scTCR simple binding of alpha and beta chains so that they both expressibility in a single open reading frame, was unsuccessful, apparently because of the natural instability of the Association of alpha - and beta soluble domains.

Therefore, to obtain scTCR requires special methods using various truncations of either or both the alpha and beta chains. Apparently, these formats are applicable only to a very limited circle scTCR sequences. Soo Hoo et al (1992) PNAS. 89 (10): 4759-63 reported the expression of murine TCR single-stranded FD which mate with clone 2C T cells using a truncated beta and alpha chain, associated with the linker of 25 amino acids, and expression of bacterial periplasmatic protein (see also Schodin et al (1996) Mol. Immunol. 33 (9): 819-29). This design also forms the basis for m6 single-chain TCR reported by Holler et al (2000) PNAS. 97 (10): 5387-92, which is formed from the 2C scTCR and is associated with the same H2-Ld - restrictionenzyme yllapitoon. Shusta et al, (2000) Nature Biotechnology 18: 754-759, report on the application of single-stranded 2 With TCR structures in experiments on visualization of yeast, which receive the mutant TCR with high thermal stability and solubility. This message also demonstrated the ability of these visualized 2C TCR selectively contacted with cells expressing cognate RMS. Khandekar et al (1997) J. Biol. Chem. 272 (51): 32190-7 describe the same design for mouse D 10 TCR, but this scTCR fusion with MBP and is expressed in the bacterial cytoplasm (see also Hare et al (1999) Nat. Struct. Biol. 6 (6): 574-81). Hilyard et al (1994) PNAS. 91 (19): 9057-61 describe human scTCR specific in relation to the matrix protein of influenza virus, HLA - A2, obtained by applying design Vα-linker-Vβ and expressed in bacterial periplasm.

Chung et al (1994) PNAS. 91 (26) 12654-8 report human scTCR with application design Vα - linker - Vβ - β and expression on the surface of mammalian cell lines. This message does not contain any of the links on the specic binding of TCR against peptide - HLA. Plaksin et al (1997) J. Immunol. 158 (5): 2218-27 describe this design Vα - linker - Vβ - Cβ to get mouse scTCR, specific to the epitope of HIV sp120-H-2Dd. This scTCR is expressed in bacterial inclusion bodies, and again twisted in vitro.

Therapeutic use

There is a need for targeting of the particles which are able to be localized on the cell (conjugation of particles with cells), infected (mi) painful process. Such targeted particles can be used either for direct blocking "mistakenly directed actions of the immune system responsible for autoimmune disease, or as a means of delivery of cytotoxic agents to tumor cells.

Ideally molecules suitable for this application must have a specific affinity for a cell marker, directly participating in relevant painful process. For this purpose you can use antibodies.

Application for screening

A number of important cellular interactions and cellular responses (reactions), including TCR-mediated immune synapse is regulated by contact between cell surface receptors and ligands on the surface of other cells. These types of specific molecular contacts are crucial for proper biochemical regulation in lovecheck the body and therefore are studied very intensively. In many cases, the purpose of such research is to develop a method of modulating cellular responses to prevent diseases or with him.

Therefore, the methods by which it is possible to identify compounds that bind with some degree of specificity with human receptor or ligand molecules, important as lead to the discovery and development of new treatments for these diseases. In particular, compounds that interfere in the interaction of the receptor - ligand, can be used directly as therapeutic agents or carriers.

The success of combinatorial chemistry, which allows relatively easy and inexpensive to get a very large library of compounds that dramatically increased the number of tested compounds. Currently, the limitations of screening programs most often caused by the nature of the methods of analysis that can be applied, obtaining suitable molecules of receptor and ligand, and how these methods of analysis can be adapted to highly productive methods of screening.

Brief description of the invention

This invention makes available a new class of alpha/beta analogues scTCR, which are characterized by the presence of disulfide bonds between residues of a single amino acid chain, and this connection contributes to the stability of the connection between al the FA and beta regions of the molecule. Such TCR suitable for screening or for the purposes of therapy.

Detailed description of the invention

The present invention encompasses single-chain T-cell receptor (scTCR)containing α segment is composed of a sequence of variable region of the α chain TCR fused to the N end of the extracellular sequence of the constant region of the α chain TCR β segment is composed of a sequence of variable regions of β chains of the TCR fused to the N end of the extracellular sequence of the constant region of the β chain TCR, and the linker sequence connecting the C-end segment α N - end of the β segment, or Vice versa, and the extracellular sequence of the constant region of the α or β segments are linked by a disulfide bond, and the length of the linker sequence and the position of the disulfide bond such that the sequence of the variable regions of α and β segments mutually oriented relative to each other almost as in native αβ T-cell receptor.

In scTCR according to the invention is the requirement that the sequence of the variable regions of α and β segments are mutually oriented relative to each other almost as in native αβ T-cell receptors, check, making sure that the molecule binds to the relevant TCR ligand (complex RMS, complex CD1-antigen complex superantigen or complex is of superantigen/MTL) - if it binds, then the condition is met. Interaction with complexes RMS can be measured on the instrument BIAcore 3000™ or BIAcore 2000™. Example 3 in this description or in the International application WO 099/6120 accordingly provides a detailed description of methods for analysis of binding of the TCR with MHC - peptide complexes. These methods are equally applicable to the study of the interactions of the TCR/CD1 TCR/superantigen. In order to apply these methods to the study of the interactions of the TCR/CD1 requires a soluble form of CD1, the receipt of which is described in (Bauer (1997) Eur J Immunol 27 (6) 1366-1373).

α and β Segments

Extracellular sequences of constant regions α and β segments, preferably, correspond to the extracellular sequences of the constant regions of the human TCR, the same applies to sequences of the variable regions present in the α and β segments. However, the ratio between such sequences at the amino acid level is not necessarily 1:1. Acceptable truncation of the N - or C-end and/or amino acid deletion and/or substitution in comparison with the corresponding human sequences, provided that the overall results are the same mutual orientation of the sequences of the variable domains of the α and β segments, as in native αβ T-cell receptors, and maintaining functionality of CBE is ivania peptide - MNF. In particular, due to the fact that the extracellular sequence of the constant regions in the α and β segments are not in direct contact with the MHC complex is associated with scTCR, they can be shorter than the extracellular sequence of the constant domain of the native TCR, or may contain substitutions or deletions compared to the extracellular sequence of the constant domain of the native TCR.

The extracellular sequence of the constant region in α segment can include a sequence corresponding to the extracellular Ig constant domain of the α chain TCR, and/or extracellular sequence of the constant region in the β segment can include a sequence corresponding to the extracellular Ig constant domain of the β chain TCR.

In one embodiment of the invention, the α segment corresponds to almost the entire variable region of the α chain TCR fused to the N-end almost the entire extracellular domain of the constant region of the α chain TCR; and/or β segment corresponds to almost the entire extracellular domain of the constant region of the β chain TCR.

In another embodiment of the invention the extracellular sequence of the constant region in the α and β segments correspond to the constant regions of the α and β chains of the native TCR, truncated at the C-ends in such a manner that excluded cysteine residues, which form m is Stepnoy disulfide bonds TCR. These cysteine residues can be replaced by another amino acid residue, such as series or alanine, so lost (delegated) native disulfide bonds. In addition, native β chain TCR contains an unpaired cysteine residue and this residue can be removed (to delegate) of the β sequence scTCR according to the invention or replace with a non - cysteine residue in this sequence.

In one particular embodiment of the invention the sequence of the variable regions of α and β chains of the TCR present in α and β segments may correspond to the functional variable domain of the first TCR, and the extracellular sequence of the constant region of the α and β chains of the TCR present in α and β segments may correspond to the extracellular sequences of the constant region of the α and β chains of the second TCR, while the first and second TCR belong to the same species. Thus, the sequence of the variable regions of α and β chains that are present in α and β segments may correspond to the sequences of the variable regions of α and β chains of the first human TCR, a extracellular sequence of the constant region of the α and β chains can match the extracellular sequences of the constant region of the α and β chains of the second human TCR. For example, extracellular sequence to stantey area A6 Tax sTCR you can use as a skeleton, which can be fused heterologous variable domains.

In another embodiment of the invention the sequence of the variable regions of α and β chains of the TCR present in α and β segments may correspond to the functional variable domain of the first TCR, and the extracellular sequence of the constant region of the α and β chains of the TCR present in α and β segments may correspond to the extracellular sequences of the constant region of the α and β chains of the second TCR, while the first and second TCR belong to different species. In this embodiment of the invention it is preferable that the sequence of the variable regions of α and β chains of the TCR present in α and β segments, together corresponded to the functional variable domain of the human TCR, a extracellular sequence of the constant region of the α and β chains of the TCR present in α and β segments corresponded to the extracellular sequences of the constant region of the α and β chains of the murine TCR. Such variants of the present invention have the advantage that the scTCR contain sequences of constant regions are not of human origin, which, apparently, are immunogenic and are likely, therefore, they enhance the overall immune response to TCR, localized on its target cells. Thus, it is possible to increase the immune response to aberrant the e cells, such as cancer cells.

Linker

In the present invention the linker sequence binds α and β segments with the formation of a single polypeptide chain. The linker sequence may, for example, have the formula - R-AA-P-, where P represents Proline, and AA represents the amino acid sequence in which amino acids are glycine and serine.

In order scTCR was associated with MHC-peptide complex, α and β segments must be assembled in such a way that the sequence of the variable regions of these segments were targeted for this binding. Therefore, the linker must have a length sufficient to fill the distance between the end of the segment α and N-end of the β segment, or Vice versa. On the other hand, should avoid excessive length, or in this case, the end of the linker to the N-terminal sequence of the variable region blocks or weakens the scTCR binding with the target complex peptide - MHC.

For example, in the case when the extracellular sequence of the constant region present in α and β segments correspond to the constant regions of the α and β chains of the native TCR, truncated at the ends so that the cysteine residues that form native disulfide cross-links link TCR, are excluded, and the linker sequence binds to the-end of the segment α N-end of the β segment, the linker may consist of 26-41, for example, 29, 30, 31 or 32 amino acids, and specific linker has the formula - PGGG-(SGGGG)5-P, where P represents Proline, G represents glycine, a S is serine.

Disulfide bonds

The main characteristic of scTCR of the present invention is a disulfide linkage between the extracellular sequences α and β segments. This link may correspond to a native disulfide bond cross-links present in the native dimeric αβ TCR, or may have no equivalent in native TCR, situated between the cysteine residues, specifically entered in the extracellular sequence of the constant regions of the α and β segments. In some cases, both native and not native disulfide bonds may be desirable in the present scTCR.

The position of the disulfide bonds is subject to the requirement that the sequence α and β segments mutually oriented almost as in native αβ T-cell receptor.

Disulfide bonds can be formed by mutation of non - cysteine residues on α and β segments in cysteine, which leads to the formation of links between matitime remains. Preferred are residues corresponding to the β-carbon atoms which in native TCR is located at a distance of about 6 Å (0.6 nm) or less, preferably, in the range of 3.5 Å (0.35 nm) - .9 Å (0.59 nm), so between cysteine residues introduced in place of the native residues that can form disulfide bonds. Preferred sites, which can be introduced cysteine residues that form disulfide bonds, are the following residues in exon 1 of TRAC*01 for the α chain TCR and TRBC1*01 or TRBC2*01 for β chain TCR:

α chain TCRα chain TCRThe distance between the native β carbon atoms (nm)
Thr 48Ser 570.473
Thr 45Ser 770.533
Tyr 10Ser 170.359
Thr 45Asp 590.560
Ser 15Glu 150.59

Now, when identified residues in human TCR, which can be replaced by cysteine residues for the formation of new disulfide bond cross-links in scTCR according to this invention, specialists in the art will be able to carry mutations in the TCR of other types to get the ü scTCR these species. For sequences of human origin skilled in the art need only look for the following motifs in the respective TCR chains to identify residue, which should be replaced (the selected residue is a residue that is substituted for a cysteine).

the α Chain of Thr 48:DSDVYITDKTVLDMRSMDFK (amino acids 39-58 exon 1 of the gene TRACKS*01)
the α Chain of Thr 45:QSKDSDVYITDKTVLDMRSM (amino acids 36-55 exon 1 gene TCAS*01)
the α Chain of Tyr 10:DIQNPDPAVYQLRDSKSSDK (amino acids 1-20 of exon 1 gene TCAS*01)
α Chain Ser 15:DPAVYQLRDSKSSDKSVCLF (6-25 amino acids of exon 1 gene TCAS*01)
β Chain Ser 57:NGKEVHSGVSTDPQPLKEQP (amino acids 48-67 exon 1 gene TRBC1*01 & TRBC2*01)
β Chain Ser 77:ALNDSRYALSSRLRVSATFW (amino acids 68-87 exon 1 gene TRBC1*01 & TRBC2*01)
β Chain Ser 17:PPEVAVFEPSEAEISHTQKA (amino acids 8-27 exon 1 gene TRBC1*01 & TRBC2*01)
β Chain Asp 59:KEVHSGVSTDPQPLKEQPAL (amino acids 50-69 exon 1 of TRBC1 gene*1 & TRBC2*01)
β Chain of Glu 15:VFPPEVAVFEPSEAEISHTQ (6-25 amino acids of exon 1 gene TRBC1*01 & TRBC2*01)

In other types of chain TCR may not contain a region that is 100% identical to the above reasons. However, the specialist in the art can use the above motifs to identify equivalent parts of the α or β chain TCR and therefore the remainder, which should be replaced with cysteine. For this you can use the methods of sequence alignment. For example, ClustalW, available online on the website of the European Bioinformatics Institute (http://www.ebi.ac.uk/index.html can be used for comparison of the above motifs with the sequence chain specific TCR to localize the relevant section TCR sequences for mutations.

In the scope of the present invention includes αβ analogues scTCR and scTCR other mammals, including, but without limitation, a mouse, a rat, a pig, a goat and a sheep. The present invention also includes discussed above chimeric human/not human scTCR. As mentioned above, the specialist in the art will be able to identify sites that are equivalent to the above sites sequences of human origin, in which you can enter cysteine residues with formation of a disulfide bond cross-links. E.g. the measures below shows the amino acid sequence of murine α and β soluble domains together with motifs, showing murine residues, equivalent to the remaining sequences of human origin referred to above, which can be replaced by cysteine residues to form a TCR disulfide bond cross-links (where relevant residues highlighted (shaded)):

Mouse α soluble domain:

PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMK

AMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVP

Mouse β soluble domain:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGREV

HSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDK

WPTGSPKPVTQNISAEAWGRAD

Murine equivalent of human α chain Thr 48: ESGTFITDKTVLDMKAMDSK

Murine equivalent of human α chain of Thr 45: KTMESGTFITDKTVLDMKAM

Murine equivalent of human α chain Tyr 10: YIQNPEPAVYQLKDPRSQDS

Murine equivalent of human α chain Ser 15: AVYQLKDPRSQDSTLCLFTD

Murine equivalent of human α chain Ser 57: NGREVHSGVSTDPQAYKESN

Murine equivalent of the human β chain Ser 77: KESNYSYCLSSRLRVSATFW

Murine equivalent of the human β chain Ser 17: PPKVSLFEPSKAEIANKQKA

Murine equivalent of the human β chain Asp 59: REVHSGVSTDPQAYKESNYS

Murine equivalent of the human β-chain of Glu 15: VTPPKVSLFEPSKAEIANKQ

As discussed above, Attach sTCR extracellular constant region can be used as a framework, which can be fused heterologous variable to the ENES. Preferably, the heterologous sequence variable regions were associated with the sequences of the constant region at any point between the disulfide bond and N-ends sequences of the constant region. In the case of sequences α and β constant regions A6 Tax TCR disulfide bonds can be formed between cysteine residues introduced at amino acid position 158 and 172, respectively. Therefore, preferably, when the connecting points of the heterologous sequence variable regions of α and β chains is between residues 159 and 173 and N-end sequences α and β constant regions, respectively.

Additional aspects

scTCR (which is, preferably, a human) according to the present invention can be obtained in almost pure form or in the form of cleared or selected drug. For example, you can get practically free from other proteins.

Many scTCR of the present invention can be obtained in the form of a multivalent complex. Thus, the present invention in one aspect includes a multivalent complex of T-cell receptor (TCR), which contains a lot of soluble T-cell receptor according to this description. Preferably, each of the many soluble TCR is identical to other TCR.

In multivalent complex of the present invention scTCR can be in the form of multimers and/or may be present in the lipid bilayer, for example, liposomes, or can be associated with this Bilaam.

In its simplest form polyvalent complex scTCR according to this invention contains multimer two, or three, or four, or more molecules of T-cell receptors associated (for example, connected by covalent or other communication) with each other, preferably, by using a linker molecule. Suitable linker molecules include, but without limitation, molecules multi(poly)valent linking (such as avidin, streptavidin, neutravidin (neutravidin) and extrawide), which have four binding site with Biotin). So, biotinylated TCR molecules can form multimeric T-cell receptors with multiple TCR binding sites. The number of TCR molecules in multimer depends on the number of TCR associated with the number of linker molecules used to obtain multimer, and also from the presence or absence of any other biotinylated molecules. The preferred multimarine are dimeric, make-up or TCR tetramer complexes.

Patterns, much longer than tetramer TCR, can be used for tracking or targeting cells expressing specific MHC-Pat the command complex. Preferably, the structures have a diameter of from 10 nm to 10 μm. Each structure can visualize many scTCR molecules at a sufficient distance from each other, so that two or more molecules of the TCR on the structure were able to communicate simultaneously with two or more MHC-peptide complexes on the cell and, thereby, increase the avidity multimeric binding of particles to the cell.

Suitable structures for use according to the invention, for the formation of complexes with one or more scTCR include membrane structures, such as liposomes and solid structure, which preferably consist of particles, such as granules (beads, balls, spheres), for example latex granules. Also suitable are other structures on the top of which can be applied to molecules of the T-cell receptor. Preferably, the structure is covered with rather multimarine T-cell receptor, rather than individual molecules T-cell receptor.

In the case of liposomes molecules T-cell receptors or their multimer can contact the membrane or otherwise associated with it. Specialists in the art well-known methods of binding or Association with the membrane.

In multivalent scTCR complex of the present invention can include a label or another particle, such as toxic or therapeu the practical particle. For example, a label or other particle can be included in a mixed multimeric molecule. An example of such a multimeric molecule is a tetramer containing three scTCR molecules and one molecule peroxidase. This can be achieved by mixing TCR and the enzyme in a molar ratio of 3:1 for tetramer complexes and separating the desired complex from any complexes that do not contain the correct ratio of molecules. These mixed molecules can contain any combination of molecules, provided that spatial problems do not threaten or almost not threaten the specified function of the molecules. The positioning of the binding sites on the molecule of streptavidin suitable for mixed tetramers, as spatial difficulties, seems to be missing.

In another aspect, the invention includes a method of detecting MHC-peptide complexes, including:

A. getting scTCR according to the present invention

B. contacts scTCR with MHC-peptide complexes; and

detection of scTCR binding with MHC-peptide complexes.

Therapeutic use

scTCR (or multivalent complex of the present invention can alternatively or additionally be associated with (e.g., by covalent or other binding a therapeutic agent, which may represent, for example, toxic is th particle for killing cells, or immunostimulant, such as an interleukin or a cytokine. Multivalent scTCR complex of the present invention may have enhanced binding capacity in relation to the TCR ligand, such as complex RMS or the CD1 molecule, compared with nemotional heterodimeric T-cell receptor. Thus, multivalent scTCR complexes according to the invention is particularly applicable for tracking cells or target cells presenting specific antigens in vitro or in vivo, and also used as intermediates for other multivalent TCR complexes used for the same purpose. Therefore, scTCR or multivalent scTCR complex can be obtained in the form of a pharmaceutically acceptable formulation for use in vivo.

The invention also includes a method of delivering a therapeutic agent to the target cell, and the method includes contacting potential target cells with scTCR or multivalent scTCR complex according to the invention under conditions that allow binding of the scTCR or multivalent scTCR complex with cell-targeted and specified scTCR or multivalent scTCR complex is specific in respect of complexes of MHC-peptide and contains an associated therapeutic agent.

In particular, soluble scTCR or multivalent scTCR complex can be used to access the key therapeutic agents to the cells (to the location of the cells), presenting a specific gene. It can be used in many situations, in particular, against tumors. therapeutic agent can be delivered in such a way that its effect is manifested locally, but not only in relation to the cells with which it is associated. So, one particular method considers (outlines) anticancer molecules associated with T-cell receptors, or multivalent scTCR complexes, specific against tumor antigens.

Many therapeutic antigens can be used for this application, for example, radioactive compounds, enzymes (e.g., perforin) or chemotherapeutic agents (e.g. cisplatin). To ensure that toxic effects are carried out at the specified location, the toxin inside the liposomes can be linked with streptavidin so that the connection is released slowly. This prevents damage to the body during transport and ensures that the toxin exerts its maximum effect after binding scTCR with the relevant antigen-presenting cells.

Other suitable therapeutic agents include:

- low molecular weight cytotoxic agents (cytotoxic agents, small molecules), such as compounds capable of killing mammalian cells, having a molecular weight of less than 700 Da. With the unity may also contain toxic metals, capable of exerting a cytotoxic effect. In addition, it should be understood that these low molecular weight cytotoxic agents also include prodrugs, i.e. compounds which disintegrate or turn into physiological conditions with the release of cytotoxic agents. Examples of such agents include cisplatin, derivatives of maytansine, rahalison (racialized, rachelmycin), calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, "martimer" (sorfimer) sodium Photofrin II, temozolomide, topotecan, trimetrexate (trimetreate) glucuronate, auristatin E, vincristine, and doxorubicin;

- peptide cytotoxins, i.e. proteins or fragments thereof, capable of killing mammalian cells. Examples include ricin, diphtheria toxin, a bacterial exotoxin a of Pseudomonas, DNA-ABC and RNA-ABC;

- radionuclides, i.e. unstable isotopes of elements which disintegrate from a competitive issue to one or more of α or β particles or γ-rays. Examples include iodine-131, rhenium 186, indium 111, yttrium-90, bismuth 210 and 213, anemone 225 and astatin 213;

- prodrugs, such as antibody directed enzyme prodrug;

- Immunostimulants, i.e. particles that stimulate immune response. Examples include cytokines, such as IL-2, chemokines such as IL8, platelet factor - 4, a protein stimulating the growth of melanoma and so on, is ntotila or fragments thereof, activators of complement, domains xenogenic proteins, domains allogeneic proteins, domains, viral/bacterial proteins, and viral/bacterial peptides.

Soluble scTCR or multivalent complexes scTCR according to the invention can be linked to an enzyme capable of converting the prodrug to a drug connection. It allows to convert the prodrug into the drug connection only in the place where it is needed (i.e. aimed with scTCR).

Examples of suitable targets MHC-peptide for scTCR according to the invention include, but without limitation, viral epitopes, such as epitopes of HTLV - 1 (e.g., peptide Tax cut with restrictase HLA - A2; HTLV - 1 associated with leukemia), HIV epitopes, the epitopes of EBV (Epstein - Barr, EBV), CMV epitopes (itomegalovirus); epitopes of melanoma (e.g., epitope MAGE, cut with a restriction enzyme HLA - A1) and other specific cancer epitopes (e.g., antigen G250 associated with kidney cancer, cut with a restriction enzyme HLA - A2); and epitopes associated with autoimmune disorders such as rheumatoid arthritis. Other rmns-target, associated with diseases, suitable for use in this invention are listed in HLA Factbook (Barklay (Ed) Academic Press)identified many others.

Shipping drugs with localization (confining) at the expense of the special is licnosti scTCR can potentially increase the number of ways of treating diseases.

Treatment of viral diseases for which there are drugs, such as HIV, SIV (human immunodeficiency virus in monkeys), EBV (EBV), CMV, will be more effective if the drug substance is released or activated in the immediate vicinity of infected cells. In cancer localization near tumors or metastases will increase the effect of toxins or Immunostimulants. Autoimmune diseases immune system suppressing drugs (immunosuppressants) can be released slowly, providing more local action over a longer period of time with minimal impact on the overall immunity of the subject. To prevent transplant rejection action of immunosuppressants can be optimized in the same way. For delivery of vaccines, antigen vaccines can be localized near the antigen-presenting cells, thereby increasing the effectiveness of antigen. The method can also be used for purposes of obtaining image (visualization).

scTCR of the present invention can be used to modulate T-cell activation by binding to specific ligands, such as RMS, and thereby suppressing T-cell activation. Autoimmune diseases, including mediated T cell inflammation and/or tissue damage that is amenable to this method of treatment, such as diabetes type I. For so the th application requires knowledge of specific peptide epitope, provide relevant RMS.

Medicinal substances according to this invention, typically supplied as part of a sterile pharmaceutical composition, which usually contains a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending on the desired method of introducing it to the patient). It can be supplied as a one-time (single) dosage form, usually it comes in a tightly closed container and may be part of the set. This set is usually (though not necessarily) includes instructions for use. It can include multiple discrete dosage forms.

The pharmaceutical composition can be adapted for administration by any suitable method, such as oral (including transbukkalno or sublingual), rectal, nazalnam, local (including transbukkalno, sublingual or transdermal (transdermal)), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intracutaneous (intradermal)) method. Such compositions can be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier (carriers) or excipient (excipients) in sterile conditions.

Application for screening

scTCR of the present invention m is should be used in methods of screening, for the identification of modulators, including inhibitors mediated TCR t-cell immune synapse.

System of homogeneous amplified due to the proximity effect luminescent assay (Amplified Luminescent Proximity Homogeneous Assay), such as AlphaScreen™, based on the use of "donor" and "acceptor" spheres (pellets, beads)coated with a layer of hydrogel, which may be associated receptor and protein ligand. The interaction between these molecules and receptor ligand leads to the convergence of the fields. When these areas are exposed to laser irradiation, the photosensitizer in the "donor" area translates ordinary oxygen to a more excited singlet state. Molecules of singlet oxygen diffuse and react with the chemiluminescent label on the sphere "acceptor", which further activates fluorophores in the same field. The fluorophores then emit light with a wavelength of 520-620 nm, it is a signal of what happened interaction of the receptor with the ligand. The presence of an inhibitor of the interaction of the receptor-ligand leads to a weakening of the signal.

Method of surface plasma resonance (SPR) is a method of analysis of the optical interface in which one binding partner (usually a receptor) immobilized on the "chip" (touch surface), and detects the binding of the other binding partner (Oba is but ligand), which is soluble and is forced to "drain" from the chip ("flowing" through the chip). The binding of the ligand leads to increased protein concentration near the surface of the chip, which causes a change in the refractive index in this region. The surface of the chip is made in such a way that the change in the refractive index can be detected using surface plasma resonance, optical phenomenon in which light incident at a certain angle on a thin metal film, gives the reflected beam of lower intensity due to the resonant excitation of waves with oscillating surface charge density (surface plasmons). The resonance is very sensitive to changes of the refractive index on the far side of the metal film, and this is a signal, which is used to detect binding between the immobilized and soluble proteins. Systems that allow for convenient application of SPR detection of molecular interactions and analysis of data are available. Examples are the installation Iasys™ (Fisons) and Biacore™.

Other methods of analysis of the optical interface include fluorescence total internal reflection (TIFR indexes), resonant mirror (RM) and optical sensor (optical grating coupler sensor, GCS) and are discussed more fully in the review of Woodbury and Venton (J. Chromatog. Century 725 113-137 (1999)). Method scinti is the visible when the approximation (SPA) is used for screening libraries of compounds for inhibitors of low-affinity interaction between CD28 and B7 (K dprobably in the region of 4 μm (Van der Merwe et al J. Exp. Med. 185: 393-403 (1997), Jehn et al. Anal Biochem 165(2) 287-93 (1998). SPA is a radioactive analysis method that uses the emission of beta particles from some radioactive isotopes, which transfer energy to the scintillator, immobilized on the surface of the indicator. A short interval of beta-particles in the solution ensures that the scintillation occurs only when beta particles are emitted in close proximity to the scintillator. When applying for the detection of protein-protein interactions one partner interactions have been labelled with a radioisotope, while the other is either associated with pellets (spheres, beads)containing the scintillator, or immobilized on the surface together with the scintillator. If the analysis can be performed in an optimal way, the radioisotope is placed close enough to the scintillator in order photon emission was activated only when the process of binding of the two proteins.

Another aspect of the invention is a method for identifying an inhibitor of the interaction between the scTCR and TCR ligand selected from MHC-peptide complexes, complexes of CD1-antigen, superantigen and complexes of MHC-peptide/superantigen, which consists in contacting scTCR with your partner scTCR binding-ligand in the presence or in the absence of the test compound and determining, decreases if the presence of the tested compounds scTCR binding with the ligand, and this reduction is taken as identifying the inhibitor.

The final aspect of the invention is a method of identifying a potential inhibitor of the interaction between the scTCR and TCR ligand selected from MHC-peptide complexes, complexes of CD1-antigen, superantigen and complexes of MHC-peptide/superantigen, which consists in contacting scTCR or scTCR-landscapebased partner with a test compound and determining whether the test compound binds to scTCR and/or ligand, and the reduction is accepted as identifying a potential inhibitor.

This aspect of the invention may find application, in particular, in tests involving optical interface (megaversity optical analysis, such as analyses carried out using the BIAcore system™.

Preferred features of each aspect of the invention is applicable to any other aspect mutatis mutandis. The documents of the previous techniques mentioned in this description, are the most in its entirety permitted by law.

Examples

Hereinafter the invention is described using the following examples, which in no way limit the scope of the invention.

Links to following the existing literature accompanying drawings, where:

In Figures 1a and 1b respectively shows the nucleotide sequence of the α and β chains of soluble A6 TCR mutated in such a way as to introduce a cysteine codons;

Figure 2A shows the extracellular amino acid sequence of the α chain of A6 TCR, including mutation of T48→(Underlined)used for obtaining a new disulfide cross-links connection, and Figure 2b shows the extracellular amino acid sequence of the β chain of A6 TCR, including mutation of S57→(Underlined)used for obtaining a new disulfide cross-links telecommunications;

The Figure 3 shows the DNA and amino acid sequence of the linker Gly/Ser (30 - Mer)

The Figure 4 shows the total cloning strategy used to obtain scDiS A6 TCR.

The Figure 5 shows the DNA sequence scDiS A6TCR.

Figure 5b shows the amino acid sequence scDiS A6TCR.

The Figure 6 illustrates the elution of scDiS A6 TCR protein with ion exchange column POROS 50HQ in the gradient Nad 0-500 mm, as shown by a straight line;

The Figure 7 shows the results of electrophoresis as in pampering SDS-PAGE (colored Kumasi) and non SDS-PAGE (colored Kumasi) gel fractions A15, B10, B9 and B3 taken from the column, illustrated in Figure 6. It is clear that the fraction B9 and 10 contain protein, corresponding to oidaematophorus scDiS A6 TCR.

The Figure 8 illustrates the elution of scDiS A6 TCR with column Superdex 200 gel-filtration fractions 10-V7 selected from ion-exchange column is shown in Figure 6;

The Figure 9 shows the results of electrophoresis as in pampering SDS-PAGE (colored Kumasi) and non SDS-PAGE (colored Kumasi) gel fractions B8, B7, B3 and B2 taken with a column for gel filtration, as illustrated in Figure 8. It is clear that the fraction V7 contains protein corresponding to the expected size scDiS A6 TCR.

The Figure 10 shows the result of the final gel filtration in buffer BIAcore concentrated fractions B9-B6, taken with a column for gel filtration, as shown in Figure 8. scDiS A6 TCR comes in the form of one main peak.

The Figure 11 shows the data BIAcore for binding scDiS A6 TCR to HLA-Atah;

Example 1 - Design of primers and mutagenesis of the α and β chains A6 Tax TCR with the aim of introducing cysteine residue required for the formation of a new disulfide bond cross-links

To implement the mutation of threonine 48 of exon 1 A6 Tax in TRAC*01 cysteine create the following primers (mutations are shown in lowercase letters):

5' - ACA GAC AAA tgT GTG HUNDRED GAC AT

5' - ATA GTC TAG CAC ACA TTT GTC TGT G

To implement the mutation of serine 57 of exon 1 A6 Tax in TRBC1*01 and and TRBC2*01 cysteine create the following primers (mutations are shown in lowercase letters):

5' - AGT GGG GTCtGC ACA GAC SS

5' - GG GTC TGT GCa GAC CCC ACT G

PCR (PCR) mutagenesis:

Mutation of the expression plasmids containing the genes for the α or β chain A6 Tax TCR carried out using primers α chain and primers β chain, respectively, as follows. 100 ng mixed with 5 μl of 10 mm dNTP, 25 µl buffer 10x Pfu (Stratagene), 10 units of Pfu polymerase (Stratagene) and the final volume was adjusted to 240 ál by adding N2O. 48 ál of this mixture complementary primers diluted to final concentration of 0.2 μm in 50 μl final volume of the reaction mixture. After the initial denaturation of 30 seconds at 95°C, the reaction mixture was subjected to 15 cycles of denaturation (95°C, 30 sec.), annealing (55°C, 60 sec.) and elongation (73°C, 8 minutes) on the device Hybaid PCR Express - PCR. The product is then digested for 5 hours at 37°C with 10 Units of restrictase DpnI (New England Biolabs). 10 μl of the split reaction mixture transformed into XL1 - Blue bacteria and grown for 18 hours at 37°C. Pick a single colony and grown overnight in 5 ml TYP + ampicillin (16 g/l Bacto-Tipton, 16 g/l yeast extract, 5 g/l Nad, 2.5 g/l K2HPO4, 100 mg/l ampicillin). Plasmid DNA purified on minipreparation column (Qiagen according to the manufacturer's recommendations and the sequence confirmed by automated sequencing at the facility Department of Biochemistry (Department of biochemistry), oxford University). The corresponding mutant n is kleitinna and amino acid sequences shown in Figures 1A and 2A for the α chain and Figures 1b and 2b for the β chain.

Example 2 - Design, expression and testing of single-stranded A6 TCR, to introduce a new disulfide cross-links connection.

The expression vectors containing the DNA sequence of the α and β chains of the mutant A6 TCR, introducing additional cysteine residues required for the formation of a new disulfide bond, obtained in Example 1 and shown in Figures 1A and 1b, is used as a basis for obtaining single-stranded A6 TCR except that removes the stop codon (TAA) at the end of the sequence α chain as follows:

scDiS A6 TCR contains a linker sequence of 30 amino acids between the end of the α chain TCR and N - end of the β chain. The Figure 3 shows the DNA sequence and amino acid sequence of this linker. The cloning strategy used to obtain scDiS A6 TCR, total are shown in Figure 4.

In short, the alpha and beta chain A6 ds (double-stranded) TCR amplified by PCR using primers containing restriction sites, as shown in Figure 4, i.e:

Alpha 5': ccaaggccatatgcagaaggaagtggagcagaactct

Alpha 3' primer: ttgggcccgccggatccgcccccgggggaactttctgggctgggg

Beta 5' primer: tcccccgggggcggatccggcgggcccaacgctggtgtactcag

Beta 3' primer: gggaagcttagtctgctctaccccaggcctcg

Two fragments thus obtained, sew by PCR (PCR)using 5' - alpha and 3' beta primers for the formation of single-chain TCR, with a short linker, the content is relevant sites Xmal - BamHI - Apal. This fragment clone in pGMT7. Then, full-linker is injected in the form of insertions in two stages, first enter the fragment 42 BP using Xmal sites and BamHI:

5' - CC GGG GGT GGC TST GGC GGT GGC GGT TCA GGC GGT GGC G -3'

3' - CCA CCG AGA CCG CCA CCG CCA AGT CCG CCA CCG CCT AC -5'

Secondly, fragment 48 P.O. embed using sites BamHI and Apal for creating linker 90 BP between the 3' end of the alpha chain and the 5' end of the beta chain. Fragment 48 BP PCR receive the elongation of a mixture of the following oligomers:

5' - GC GGA TCC GGC GGT GGC GGT TCG GGT GGC GGT GGC TC - 3'

3' - CCA AGC CCA CCG CCA CCG AGT CCG CCA CCG CCC GGG TG - 5'

The product of this extension were cleaved with BamHI and ApaI and are ligated into the cleaved plasmid containing the fragment linker 42 BP

The complete DNA sequence and the complete amino acid sequence scDiS A6 TCR shown in Figures 5A and 5b, respectively.

The expression of A6 TCR:

The expression plasmids containing single-chain linked by a disulfide bond A6 TCR, transformed into a strain of E. coli BL21pLysS and single resistant to ampicillin colony grown at 37°C in an environment TYP (ampicillin 100 μg/ml) to an optical density OD6000.4 before inducing protein expression with 0.5 mM IPTG. Cells are harvested three hours after induction by centrifugation for 30 minutes at 4000 rpm in a centrifuge Beckman J-6B. Cellular precipitate resuspended in buffer containing 50 mm Tris - HCl, 2% (wt/V) sucrose, 1 mm NaEDTA, 0.1% (wt/V) NaAzide (NaN3), 10 mm DTT, pH 8.0. After the stage of freezing and thawing during the night resuspendable cells subjected to ultrafiltration in a 1-minute "beam pulses" within 10 minutes by ultrasonic disintegrator Milsonix XL2020, using standard probes with a diameter of 12 mm of the Sediment (pellets) of inclusion bodies recovered by centrifugation for 30 min at a speed of 13000 rpm in a centrifuge Beckman J2 - 21. Then thrice washed with detergent to remove cell debris and membrane components. Each time the pellet of inclusion bodies homogenized in Triton buffer (50 mm Tris - HCl, 0.5% Triton - X100, 200 mm NaCl, 10 mm NaEDTA, 0.1% (wt/V) NaAzide, 2 mm DTT, pH 8.0) before precipitated by centrifugation for 15 minutes at 13000 rpm in a centrifuge Beckman J2-21. The detergent and salt then remove the same shaded in the following buffer: 50 mm Tris - HCl, 1 mm NaEDTA, 0.1% (wt/V) NaAzide, 2 mm DTT, pH 8.0. Finally, bullock inclusion divided into aliquots of 30 mg and frozen at -70°C. the protein Yield of inclusion bodies quantify the solubilization using 6M guanidine - HCl and measuring the binding of the dye according to the method of Bradford (PerBio).

About 15 mg solubilizing chain tel enable thawed from frozen initial solutions. The inclusion body was diluted to a final concentration of 5 mg/ml in 6M solution of guanidine and DTT (2 M source solution) was added to ejnoy concentration of 10 mm. The mixture is incubated at 37°C for 30 minutes to Prepare 1 liter of the next buffer for refolding: 100 mm Tris pH 8.5, 400 mm L - arginine, 2 mm EDTA, 5 mm reduced glutathione, 0.5 mm oxidized glutathione, 5 M urea, 0.2 mm PMSF and vigorously stirred at 5°C.±3°C. the Redox couple (a couple of oxidant - reductant) (2-mercaptoethylamine and tsistamin (to final concentration of 6.6 mm and 3.7 mm, respectively) was added over 5 minutes before adding denaturirovannykh TCR chains. Then the squirrels leave unwind (refolding) for 5 hours ±15 minutes under stirring at a temperature of 5°C±3°C. Then the product obtained by refolding, double-dialist, first against 10 liters of 100 mm urea, then against 10 liters of 100 mm urea, 10 mm Tris (Tris) pH 8.0. Both stages, as refolding, and the dialysis is carried out at 6-8°C.

scTCR separated from degradation products and impurities, causing cialisovernight product refolding on anion-exchange column POROS 50HQ and elwira associated protein in the gradient of 0-500 mm NaCl in 50 volumes column using Akta purifier (Pharmacia), as shown in figure 6. Fractions corresponding to the peaks, stored at 4°C and before to collect and concentrate, analyze SDS-RACE with staining of Kumasi (Figure 7). Then sTCR purify and characterize, using a column for gel filtration (gel chromatography) Superdex 200HR (Figure 8), the pre-urav beshennuyu buffer HBS-EP (10 mm HEPES pH 7.4, 150 mm NaCl, 3.5 mm EDTA, 0.05% Nonidet R40). Fractions corresponding to the peaks, stored at 4°C and before to collect and concentrate, analyze SDS-RACE with staining of Kumasi (Figure 9). Finally, concentrated (one stripped off) fractions B9-B6 forth again on the column for gel chromatography to obtain purified protein in BIAcore buffer (Figure 10). Combine the fractions corresponding to the peak with an estimated molecular mass of 50 kDa. This pool is evaporated before be characterized by means of surface plasma resonance on a BIAcore instrument.

Example 3 - Characteristics of the scTCR binding with HLA-A2 Tax by means of surface plasma resonance with a Biacore biosensor

For analysis of binding A6 scTCR with ligand peptide-MHC (HLA - A2 Tax) use biosensor surface plasmon resonance (BIAcore 3000™). This analysis becomes simpler when you get a single complexes RMS (described below), palouriotissa immobilization on the surface of the binding, film-coated streptavidin, makes possible the testing of binding to soluble T-cell receptor to four different RMS (immobilized on different flow cells) simultaneously. Injection of HLA complex manually allows you to easily set the exact level of immobilized molecules of class A.

Such immobilized complexes capable of CBE is Ivate as T-cell receptors, and coreceptor CD8αα, each of which can be injected in the soluble phase.

Biotinylated HLA-A2 - Tax complexes of class I is subjected to refolding in vitro, using downregulation of inclusion body containing a compound of the subunits proteins and synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'callaghan et al. (1999) Anal. Biochem. 266: 9-15). Heavy chain HLA Express with C-terminal bitenova tag, it replaces the transmembrane and cytoplasmic domains of the protein in the corresponding design. Light chain of HLA or β2-microglobulin also Express in the form of inclusion bodies in E. coli using a matched design, the level is ~500 mg/liter of bacterial culture.

The E. coli cells are lysed and inclusion body cleanse to approximate purity of 80%. Protein inclusion bodies are denatured in a solution of 6 M guanidine - HCl, 50 mm Tris pH 8.1, 100 mm NaCl, 10 mm DTT, 10 mm EDTA and subjected to refolding at a concentration of 30 mg/liter heavy chain, 30 mg/liter of β2m in 0.4 M L - Arginine - HCl, 100 mm Tris pH 8.1, 3.7 mm group probably facilitates, 4 mg/ml peptide (for example, tax 11-19), adding a single pulse of denatured protein in the refolding buffer for at <5°C. Refolding leave before completing at 4°With at least 1 hour.

The replace buffer buffer for dialysis in 10 volumes of 10 mm Tris pH 8.1. For sufficient recovery of the ionic strength of the races is the thief should be twice to replace the buffer. Then the protein solution was filtered through a 1.5 micron filter of cellulose acetate and applied to the anion exchange column POROS 50HQ (volume nozzles (medium) 8 ml). Protein elute in a linear gradient of 0-500 mm NaCl. HLA - A2 - peptide complex elute approximately at 250 mm NaCl, and fractions corresponding to the peak, collect, add a cocktail of protease inhibitors (Calbiochem) and fraction is cooled with ice.

Buffers for complexes of HLA labeled bitenova label, replace with 10 mm Tris pH 8.1, 5 mm NaCl, using a Pharmacia column for quick removal of salts, equilibrated in the same buffer. Immediately after the elution fractions containing the protein, cooled with ice, and add a cocktail of protease inhibitors (Calbiochem). Then add the reagents to biotinylate: 1 mm Biotin, 5 mm ATP (buffered to pH 8), 7.5 mm MgCl2and 5 μg/ml of the enzyme BirA (cleaned by O'callaghan et at. (1999) Anal. Biochem. 266: 9-15). The mixture is then incubated at room temperature overnight.

Biotinylated HLA complexes purified by gel-chromatography. Pre-column Pharmacia Superdex 75 HR 10/30 balance filtered PBS and 1 ml biotinylating the reaction mixture is applied on the column and wash with PBS rate of 0.5 ml/min Biotinylated complexes of HLA come in the form of a single peak (approximately 15 ml). Collect fractions containing protein, cool them with ice, and add a cocktail of protease inhibitors. To the protein concentrations determined by the method of binding Kumasi (PerBio) and aliquots of biotinylated HLA complexes stored frozen at - 20°C. Streptavidin immobilized standard methods of bonding with the amine.

The interaction between Tax A6 scTCR containing the new cross-links connection, and its complex ligand/MHC or irrelevant by a combination of HLA-peptide, the receipt of which is described above, analyzed by the biosensor BIAcore 3000™ surface plasmon resonance (SPR). Method SPR measures changes in refractive index expressed in response units (RU), near the touch surface in a small flow cell, a principle that can be used to detect the interaction of the receptor - ligand and to analyze their affinity and kinetic parameters. Flow-through cuvette for probes prepared by immobilization of individual complexes of HLA-peptide in separate flow cells by binding between Biotin and sewn on β2m, and streptavidin, chemically associated with the activated surface of the flow-through cuvettes. Then the analysis is carried out, skipping scTCR through the surface of various flow-through cuvette with constant speed, while measuring this SPR response. Introduction (injection) soluble in sTCR complex peptide-HLA with a constant flow rate and in different concentrations are used to determine the background (base) resonance. The values of these control measurements is subtracted from values obtained with the specific complex of the peptide-HLA, and used to calculate affin the spine of the binding, expressed as dissociation constants, Kd (Price & Dwek, Principles and Problems in Physical Chemistry for Biochemists (2ndEdition) 1979, Clarendon Press, Oxford).

BIAcore analysis scDiS A6 TCR shows that this molecule specifically binds to a cognate ligand (HLA-A2 Tax) with kd 12.4±1.62 ám.

1. Single-chain T-cell receptor (scTCR), containing
α segment, formed by a sequence of variable regions and chain TCR connected to the N-end of the extracellular sequence of the constant region of the α chain TCR,
β segment, formed by a sequence of variable regions of β chains of the TCR, which is connected with the N-end of the extracellular sequence of the constant region of the β chain TCR, and
the linker sequence connecting With the end of the segment α with N end β of the segment, or Vice versa,
when you do this:
extracellular sequences of constant regions α and β segments are linked by a disulfide bond, which binds to cysteine residues, which are substitutions of residues selected from the group including
Thr 48 of exon 1 of TRAC*01 and Ser 15 of exon 1 of TRBC1*01 or TRBC2*01;
Thr 45 of exon 1 of TRAC*01 and Ser 77 of exon 1 of TRBC1*01 or TRBC2*01;
Tyr 10 of exon 1 of TRAC*01 and Ser 17 of exon 1 of TRBC1*01 or TRBC2*01;
Thr 45 of exon 1 of TRAC*01 and Asp 59 of exon 1 of TRBC1*01 or TRBC2*01;
and
Ser 15 of exon 1 of TRAC*01 and Glu 15 of exon 1 of TRBC1*01 or TRBC2*01;
the distance between the β carbon atoms of these substitutions in the corresponding sequences of the extracellular intercept ntih lg domains of the α and β chains of the TCR is less than 0.6 nm,
it is possible that the extracellular sequences of constant regions present in α and β segments correspond to the constant regions of the α and β chains of the native TCR, truncated at their C-ends so that cysteine residues, which form cross-links of native disulfide bonds TCR, excluded, or
extracellular sequences of constant regions present in α and β segments correspond to the constant regions of the α and β chains of the native TCR, in which cysteine residues that form native disulfide cross-links connection, are replaced with another amino acid residue, or
there is no unpaired cysteine residue present in the β chain of the native TCR.

2. scTCR according to claim 1, characterized in that the extracellular sequence of the constant domain of the α segment includes a sequence that corresponds to TRAC*01 and β segment includes a sequence that corresponds to TRBC1*01 or TRBC2*01, and not specified native disulfide bond is between cysteine residues substituted Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TPBC2*01.

3. scTCR according to claim 1 or 2, which binds covalent bond with a therapeutic agent.



 

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6 cl, 4 dwg, 5 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: adhesive protein containing protein and peptide repeated at least once, which is combined to carboxy- and/or amino-ends of the protein, is described. Polynucleotide containing nucleotide sequence, which encodes the adhesive protein, is represented. Expression vector containing functionally incorporated nucleotide sequence, which encodes the adhesive protein, is described. Transformant, which is transformed by the vector selected from the group, which contains prokaryote, eukaryote and cells, derived from eukaryotes, is represented. The methods of producing and purification of the adhesive protein are represented. Adhesive containing the adhesive protein as an active component in efficient number is offered. The method for regulation of adhesive strength of the present adhesive, including treatment with the substance selected from the group, which contains oxidising agent, filler and surfactant, or regulation of concentration of the adhesive protein, which is the surfactant of the adhesive, is described. Covering agent containing the adhesive protein as an active component in efficient number is offered.

EFFECT: present invention allows producing adhesive protein, which can be efficiently produced in volume to use them instead of chemical adhesive.

42 cl, 33 dwg, 1 tbl, 17 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of gene engineering and biotechnology and can be used for labeling biological objects. The nucleic acid molecule was extracted that encoded the fluorescing protein selected from fluorescing proteins represented by the Copepoda Crustacea biological kinds and fluorescing mutants of the aforesaid proteins. The aforesaid nucleic acid was functionally bonded to appropriate elements of expression regulation to be used in the method of producing aforementioned fluorescing protein. On the basis of the aforesaid extracted nucleic acid cloning and expressing vectors are produced as well as the expressing cassette. The cell and a stable cellular lineage, containing the aforesaid expressing cassette produce the fluorescing protein. Note that the said fluorescing protein, the nucleic acid encoding the protein above and expressive genetic structures containing the said nucleic acid, are used incorporated with the set designed to label a biological molecule. The fluorescing protein is also used in the methods of labeling a biological molecule, a cell or a cellular organella.

EFFECT: expanded biological objects labeling means.

12 cl, 9 dwg, 1 tbl, 7 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to gene engineering and biotechnology and can be used in a pharmaceutical industry. Cleared and extracted polynucleotide encodes protein with functional activity of a glucose carrier and the GLUT4 protein amino acid lineage wherein valine in position 85 is replaced by methionine. The protein under consideration features biological activity in yeast strains S. cerevisiae deprived of functional carriers of glucose and Erg4 protein.

EFFECT: production of protein with functional activity of glucose carrier in yeasty expressing system.

5 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: medicine; biotechnologies.

SUBSTANCE: DNA-vaccine is intended for activation of the immune response against the cancer cells, containing the design of DNA operably coding survivin protein and cytokine CCL21, built in attenuated vector Salmonella typhimurium. The way of inhibition of growth of a tumour by vaccine introduction under the invention to a mammal is opened. The product containing the described vaccine is presented. The way of vaccination of a mammal against a cancer where the way includes a stage of introduction to a mammal of effective quantity of the described DNA-vaccine causing the immune response is offered.

EFFECT: expansion of assortment of DNA-vaccines causing the immune response and unexpectedly high level of cytotoxicity against tumoral cells.

21 cl, 42 dwg, 6 tbl, 22 ex

FIELD: medicine; biotechnologies.

SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.

EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and concerns obtaining factor VII protein by method of recombinant DNA. Recombinant plasmid DNA was constructed for expression of blood clotting factor VII in mammalian cells, which is product of ligating of fragment of cDNA of human factor VII gene, flanked by sites of restrictases Xhol and BamHI recognising, with large XhoI/BglII fragment of vector pEFZeo, including genes of resistance to ampicillin and zeocin. As result of BHK cell transformation with new recombinant plasmid, cell line BHK/F7 was obtained, which produces recombinant protein of factor VII with output of up to 40 mkg/ml.

EFFECT: obtaining cell line producing recombinant protein of factor VII.

2 cl, 4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns medicine, particularly immunotherapy. Invention claims bispecific antibody and its application in tumour treatment. New antibody can link to EGF receptor and includes two antigene-binding sites linking to different epitopes of indicated receptor, thus ensuring higher receptor engagement by antibodies at the same dosage. It allows for higher antibody density on receptor and significant inhibition of tumour growth and/or enhancement of solid tumour or metastasis apoptosis.

EFFECT: higher receptor engagement by antibodies at the same dosage.

16 cl, 2 ex

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