RU2355703C2 - Single-strand recombinant t-cell receptors - Google Patents

Abstract

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

Description

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

As described, for example, in International Application WO 99/120, TCRs mediate the recognition by T cells of specific complexes of peptides of the 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 the antigens of specific peptides present in MHCs, and a foreign peptide is very often the only sign of abnormality in a cell. T-cell recognition occurs when the T cell and antigen-presenting cell (APC) are in direct physical contact, and is initiated by ligation of antigen-specific TCR with pMHC complexes.

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

Two other classes of proteins are known to function as TCR ligands. (1) CD1 antigens are I - related MHC class molecules, the genes of which are localized on a chromosome other than the chromosome of the classical MHC class I antigens of class I and class II. CD1 molecules are capable of presenting peptide and non-peptide (e.g. lipid, glycolipid) T particles to cells in a manner analogous to the methods for presenting conventional Class I and Class II complexes. See, for example, (Barclay et al, (1997) The Leucocyte Antigen Factsbook 2 ^nd Edition, Acadmeic Press) and (Bauer (1997) Eur J Immunol 27 (6) 1366-1373)). (2) Bacterial superantigens are soluble toxins capable of binding to both MHC class II molecules and a TCR subpopulation. (Fraser (1989) Nature 339221-223). Many superantigens exhibit specificity for one or two V beta segments, while in the case of others, more random (less selective) binding is observed. In any case (at any event), superantigens are able to exhibit an increased immune response due to their ability to stimulate subpopulations of T cells in a "polyclonal manner."

The extracellular portion of the native heterodimeric αβTCR consists of two polypeptides, each of which has a proximal membrane constant domain and a membrane distal variable domain (see Figure 1). Each of the constant and variable domains includes an intrachain disulfide bond. Variable domains contain highly polymorphic loops similar to the hypervariable regions (CDRs) of antibodies. T cell receptor CDR3 (TCR) interacts with peptides presented by MHC, and CDR 1 and 2 interact with peptide and MHC. The variety of TCR sequences arises from the somatic rearrangement of linked variable (V), additional (D), connective (J) and constant genes. Functional polypeptides of the α chain are formed by rearranging the VJ-C regions, while the β chains are composed of VDJ-C regions. The extracellular constant domain contains a proximal membrane region and an immunoglobulin region. There is a single constant domain of the α chain, known as TRAC, and two different β constant domains, known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acid substitutions between these β constant domains, three of which are in the domains used to produce single chain TCRs of the present invention. All these substitutions are located in exon 1 of TRBC1 and TRBC2: N ₄ K ₅ → K ₄ N ₅ and F ₃₇ → Y (nomenclature IMGT, differences TRBC1 → TRBC2), the final amino acid substitution is located between two constant regions of the β TCR chain in exon 3 of TRBC1 and TRBC2: V ₁ → E. The extent of each of the TCR extracellular domains is somewhat variable. However, one skilled in the art will determine the position of domain boundaries using the reference to The T Cell Receptor Facts Book, Lefranc & Lefranc, Publ. Academic Press 2001,

Single Chain TCR (T Cell Receptors)

Single chain TCRs (scTCRs) are artificial constructs consisting of a single amino acid chain (strand, strand) that, like native heterodermic TCRs, bind to MHC peptide complexes. Unfortunately, attempts to obtain functional alpha / beta analogues of scTCR by simply linking alpha and beta chains so that they are both expressed in a single open reading frame have been unsuccessful, apparently due to the natural instability of the combination of alpha and beta soluble domains.

Therefore, to obtain scTCR, special methods are required using various truncations of either or both alpha and beta chains. Apparently, these formats are applicable only to a very limited range of scTCR sequences. Soo Hoo et al (1992) PNAS. 89 (10): 4759-63 report the expression of murine TCR in single chain format using a clone of 2C T cells using truncated beta and alpha chains linked to a 25 amino acid linker and expression of a bacterial periplasmic protein (see also Schodin et al ( 1996) Mol. Immunol. 33 (9): 819-29). This design also forms the basis for the m6 single chain TCR reported by Holler et al (2000) PNAS. 97 (10): 5387-92, which is formed from 2C scTCR and binds to the same H2-Ld - restricted alloepitope. Shusta et al, (2000) Nature Biotechnology 18: 754-759, report the use of single chain 2 C TCR constructs in yeast imaging experiments that produce mutant TCRs with enhanced thermal stability and solubility. This post also demonstrated the ability of these visualized 2C TCRs to selectively bind to cells expressing their related pMHC. Khandekar et al (1997) J. Biol. Chem. 272 (51): 32190-7 describe the same design for mouse D 10 TCR, however, this scTCR is fused to MBP and expressed in 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 for the influenza virus-HLA-A2 matrix protein obtained using the Vα-linker-Vβ design and expressed in bacterial periplasm.

Chung et al (1994) PNAS. 91 (26) 12654-8 report the production of human scTCR using the Vα-linker-Vβ-Cβ design and expression on the surface of a mammalian cell line. This message does not contain any reference to specific TCR binding with respect to the peptide - HLA. Plaksin et al (1997) J. Immunol. 158 (5): 2218-27 describe a similar design of a Vα-linker-Vβ-Cβ to produce murine scTCR specific for the HIV epitope sp120-H-2D ^d . This scTCR is expressed as bacterial inclusion bodies and is again twisted in vitro.

Therapeutic application

There is a need to target particles that can localize on cells (conjugation of particles to cells) affected by the disease process. Such targeted particles can be used either to directly block the “erroneously directed” action of the immune system responsible for an autoimmune disease, or as a means of delivering cytotoxic agents to cancer cells.

Ideally, molecules suitable for such an application should have a specific affinity for a cell marker directly involved in a relevant disease process. For this purpose, antibodies can be used.

Screening Application

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

Therefore, the methods by which it is possible to identify compounds that bind to a certain degree of specificity with the human receptor or with ligand molecules are important, since they lead to the discovery and development of new methods of treating the disease. In particular, compounds that interfere with receptor-ligand interactions can be used directly as therapeutic agents or carriers.

Advances in combinatorial chemistry, which made it possible to obtain very large libraries of compounds relatively easily and inexpensively, dramatically increased the number of tested compounds. Currently, the limitations of screening programs are most often caused by the nature of the analysis methods that can be applied, by the preparation of suitable receptor and ligand molecules and how these analysis methods can be adapted to highly productive screening methods.

SUMMARY OF THE INVENTION

The present invention makes available a new class of alpha / beta analogs of scTCR, which are characterized by the presence of a disulfide bond between residues of a single amino acid chain, this bond contributing to the stability of the compound between the alpha and beta regions of the molecule. Such TCRs are suitable for screening or therapy purposes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a single-stranded T cell receptor (scTCR) comprising an α segment composed of the variable region of the TCR chain α region fused to the N end of the extracellular sequence of the TCR α chain region constant region, a β segment composed of the sequence of the variable region of the TCR chain β fused to N the end of the extracellular sequence of the constant region β of the TCR chain, and the linker sequence linking the C-terminus of the α segment to the N-terminus of the β segment, or vice versa, the extracellular sequences 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 are such that the sequences of the variable regions of the α and β segments are mutually oriented relative to each other almost as in native αβ T-cell receptors.

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

α and β Segments

The extracellular sequences of the constant regions of the α and β segments preferably correspond to the extracellular sequences of the constant regions of the human TCR, the same applies to the 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. Truncation of the N- or C-terminus and / or amino acid deletion and / or substitution is acceptable in comparison with the corresponding human sequences, provided that the common result is the same mutual orientation of the sequences of the variable domains of α and β segments as in native αβ T- cell receptors, and the preservation of peptide-MHC binding functionality. In particular, due to the fact that the extracellular sequences of constant regions in the α and β segments are not in direct contact with the MHC complex to which scTCR binds, they may be shorter than the extracellular sequences of the constant domain of native TCR, or may contain substitutions or deletions compared with extracellular sequences of the constant domain of native TCR.

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

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

In another embodiment, extracellular constant region sequences in the α and β segments correspond to the constant regions of α and β of the native TCR chains truncated at the C-ends so that cysteine residues that form the TCR interchain disulfide bond are excluded. Or, these cysteine residues can be replaced by another amino acid residue, such as a series or alanine, so that the native disulfide bond is lost (delegated). In addition, the native TCR β chain contains an unpaired cysteine residue and this residue can be removed (delegated) from the β scTCR sequence of the invention or replaced with a non-cysteine residue in this sequence.

In one particular embodiment of the invention, the variable region sequences of the α and β TCR chains present in the α and β segments may together correspond to the functional variable domain of the first TCR, and the extracellular sequences of the constant region of the α and β TCR chains present in the α and β segments may correspond extracellular sequences of the constant region α and β of the chains of the second TCR, while the first and second TCR belong to the same species. Thus, the sequences of the variable regions of α and β chains present in the α and β segments can correspond to the sequences of the variable regions of α and β chains of the first human TCR, and the extracellular sequences of the constant region of α and β chains can correspond to the extracellular sequences of the constant region of α and β chains second human TCR. For example, the extracellular sequences of the constant region A6 Tax sTCR can be used as a backbone along which heterologous variable domains can be fused.

In another embodiment of the invention, the variable region sequences of the α and β TCR chains present in the α and β segments may together correspond to the functional variable domain of the first TCR, and the extracellular sequences of the constant region of the α and β TCR chains present in the α and β segments may correspond to extracellular sequences of the constant region α and β of the chains of the second TCR, while the first and second TCR belong to different types. In this embodiment of the invention, it is preferable that the variable region sequences of the α and β TCR chains present in the α and β segments together correspond to the functional variable domain of the human TCR, and the extracellular sequences of the constant region of the α and β TCR chains present in the α and β segments correspond extracellular sequences of the constant region of α and β chains of murine TCR. Such embodiments of the present invention have the advantage that scTCRs contain sequences of constant regions of non-human origin that are likely to be immunogenic and are likely to thereby increase the overall immune response to TCR localized on its target cells. Thus, it is possible to enhance the immune response to aberrant cells, such as cancer cells.

Linker

In the present invention, the linker sequence binds the α and β segments to form a single polypeptide chain. The linker sequence may, for example, have the formula —P-AA-P-, where P is proline and AA is the amino acid sequence in which the amino acids are glycine and serine.

In order for scTCR to bind to the MHC peptide complex, the α and β segments must be connected so that the variable region sequences of these segments are oriented for this binding. Therefore, the linker must have a length sufficient to fill the distance between the C-end of the α segment and the N-end of the β segment, or vice versa. On the other hand, excess length should be avoided, otherwise in this case the end of the linker in the N-terminal sequence of the variable region blocks or weakens the binding of scTCR to the target peptide-MHC complex.

For example, in the case where the extracellular sequences of the constant region present in the α and β segments correspond to the constant regions of α and β of the native TCR chains truncated at the C ends so that cysteine residues that form the native TCR interchain disulfide bond are excluded, and the linker sequence connects the C-end of the α segment with the N-end of the β segment, the linker may consist of 26-41, for example, 29, 30, 31 or 32 amino acids, and the particular linker has the formula PGGG- (SGGGG) ₅ -P- where P is proline, G is gly Ying, a S represents serine.

Disulfide bond

The main characteristic of scTCR of the present invention is the disulfide bond between the extracellular sequences of the α and β segments. This bond may correspond to the native interchain disulfide bond present in native dimeric αβ TCR, or may not have an analogue in native TCR, located between cysteine residues specifically introduced into the extracellular sequences of the constant regions of α and β segments. In some cases, both native and non-native disulfide bonds may be desirable in the present scTCR.

The position of the disulfide bond obeys the requirement that the sequences of the α and β segments are mutually oriented practically as in the native αβ T-cell receptors.

A disulfide bond can be formed upon mutation of non-cysteine residues on the α and β segments into cysteine, which leads to the formation of a bond between the mutate residues. Residues are preferred whose corresponding β-carbon atoms in the native TCR are at a distance of about 6 Å (0.6 nm) or less, preferably in the range of 3.5 Å (0.35 nm) to 5.9 Å (0.59 nm), so that between cysteine residues introduced in place of native residues may form a disulfide bond. Preferred sites at which cysteine residues forming a disulfide bond can be introduced are the following residues in exon 1 of TRAC * 01 for the TCR α chain and TRBC1 * 01 or TRBC2 * 01 for the TCR β chain:

α TCR chain α TCR chain The distance between native β carbon atoms (nm) Thr 48 Ser 57 0.473 Thr 45 Ser 77 0.533 Tyr 10 Ser 17 0.359 Thr 45 Asp 59 0.560 Ser 15 Glu 15 0.59

Now that residues in human TCR have been identified that can be replaced with cysteine residues to form a new interchain disulfide bond in the scTCR of this invention, those skilled in the art will be able to mutate other types of TCR in the same way to obtain scTCR of these species. For sequences of human origin, one skilled in the art only needs to search for the following motifs in the corresponding TCR chains to identify the residue to be replaced (the highlighted residue is a cysteine-substituted residue).

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

In other types of chains, TCRs may not contain a region that is 100% identical to the above motifs. However, one skilled in the art is able to use the above motifs to identify the equivalent portion of the α or β chain of the TCR and, therefore, the residue that should be replaced with cysteine. To do this, you can use sequence alignment methods. For example, ClustalW, available online at the European Bioinformatics Institute website (http://www.ebi.ac.uk/index.html), can be used to compare the above motifs with a specific TCR chain sequence to localize a relevant portion of the TCR sequence for mutation .

The scope of the present invention includes αβ analogs of scTCR, as well as scTCR of other mammals, including, but not limited to, mouse, rat, pig, goat and sheep. Also, the present invention includes the chimeric human / non-human scTCRs discussed above. As mentioned above, one skilled in the art will be able to determine sites equivalent to the above-described sites of sequences of human origin into which cysteine residues can be introduced to form an interchain disulfide bond. For example, the amino acid sequences of murine Cα and Cβ soluble domains are shown below along with motifs showing murine residues equivalent to the residues of human origin mentioned above that can be replaced with cysteine residues to form a TCR interchain disulfide bond (in which the relevant residues are highlighted (shaded) )):

Mouse Cα soluble domain:

PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMK

AMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVP

Mouse Cβ soluble domain:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGREV

HSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDK

WPTGSPKPVTQNISAEAWGRAD

The mouse equivalent of the human α chain of Thr 48: ESGTFITDKTVLDMKAMDSK

The mouse equivalent of the human α chain of Thr 45: KTMESGTFITDKTVLDMKAM

Mouse equivalent of Tyr 10 human α chain: YIQNPEPAVYQLKDPRSQDS

The mouse equivalent of the human α chain Ser 15: AVYQLKDPRSQDSTLCLFTD

Mouse equivalent of human α chain Ser 57: NGREVHSGVSTDPQAYKESN

Mouse equivalent of human β chain Ser 77: KESNYSYCLSSRLRVSATFW

The mouse equivalent of human β chain Ser 17: PPKVSLFEPSKAEIANKQKA

The mouse equivalent of human β chain Asp 59: REVHSGVSTDPQAYKESNYS

The mouse equivalent of human β chain Glu 15: VTPPKVSLFEPSKAEIANKQ

As discussed above, A6Tax sTCR extracellular constant regions can be used as a framework through which heterologous variable domains can be fused. Preferably, the heterologous variable region sequences are linked to constant region sequences at any point between the disulfide bond and the N-ends of the constant region sequences. In the case of sequences of α and β constant regions of the A6 Tax TCR, a disulfide bond may form between cysteine residues introduced at amino acid positions 158 and 172, respectively. Therefore, it is preferable when the attachment points of the heterologous sequence of the variable region of the α and β chains are between residues 159 and 173 and the N-terminus of the sequences of α and β constant regions, respectively.

Additional aspects

The scTCR (which is preferably human) of the present invention can be obtained in substantially pure form or as a purified or isolated preparation. For example, it can be obtained practically free of other proteins.

Many scTCRs of the present invention can be prepared as a multivalent complex. Thus, the present invention in one aspect includes a multivalent T-cell receptor complex (TCR), which contains many soluble T-cell receptors as described. Preferably, each of the plurality of soluble TCRs is identical to the other TCRs.

In the polyvalent complex of the present invention, scTCRs may be in the form of multimers and / or may be present on the lipid bilayer, for example liposomes, or may be associated with this bilayer.

In its simplest form, the multivalent scTCR complex of the invention comprises a multimer of two, or three, or four, or more T cell receptor molecules associated (e.g., linked by a covalent or other bond) with each other, preferably using a linker molecule. Suitable linker molecules include, but are not limited to, multi (poly) valence binding molecules (such as avidin, streptavidin, neutravidin and extravidin), which have four biotin binding sites). Thus, biotinylated TCR molecules can form multimers of T-cell receptors having many TCR binding sites. The number of TCR molecules in a multimer depends on the number of TCRs associated with the number of linker molecules used to produce the multimer, and also on the presence or absence of any other biotinylated molecules. Preferred multimers are dimeric, makeup or tetrameric TCR complexes.

Structures much longer than TCR tetramers can be used to track or target cells expressing a specific MHC peptide 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 TCR molecules on the structure can bind simultaneously to two or more MHC peptide complexes on the cell and, thereby, increase the avidity of the multimeric binding particle with respect to the cell.

Suitable structures for use according to the invention for complexing with one or more scTCRs include membrane structures such as liposomes and solid structures, which are preferably particles, such as granules (beads, beads, spheres), for example latex granules . Other structures are also suitable on which T-cell receptor molecules can be applied on top. Preferably, the structures are coated with multimers of the T cell receptor rather than with individual T cell receptor molecules.

In the case of liposomes, T cell receptor molecules or their multimers can bind to the membrane or otherwise associate with it. Methods of binding or association with a membrane are well known to those skilled in the art.

A multivalent scTCR complex of the present invention may include a label or other particle, such as a toxic or therapeutic particle. For example, a label or other particle may be included in a mixed multimeric molecule. An example of such a multimeric molecule is a tetramer containing three scTCR molecules and one peroxidase molecule. This can be achieved by mixing TCR and the enzyme in a molar ratio of 3: 1 to obtain tetrameric 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 difficulties do not threaten or practically do not threaten the desired function of the molecules. The positioning of the binding sites on the streptavidin molecule is suitable for mixed tetramers, since there are apparently no spatial difficulties.

In another aspect, the invention includes a method for detecting MHC peptide complexes, comprising:

but. obtaining scTCR of the present invention

b. contacting scTCR with MHC peptide complexes; and

detection of scTCR binding to MHC peptide complexes.

Therapeutic application

The scTCR (or its multivalent complex) of the present invention can alternatively or additionally be associated with (for example, by covalent or other binding) a therapeutic agent, which may be, for example, a toxic particle for cell killing, or an immunostimulant such as interleukin or cytokine . The multivalent scTCR complex of the present invention may have increased TCR ligand binding ability, such as a pMHC complex or a CD1 molecule, as compared to the non-multimeric T cell receptor heterodimer. Thus, the multivalent scTCR complexes of the invention are particularly useful for monitoring cells or targeting cells presenting specific antigens in vitro or in vivo, and are also useful as intermediates for the preparation of other multivalent TCR complexes that are used for the same purpose. Therefore, scTCR or multivalent scTCR complex can be obtained in the form of a pharmaceutically acceptable preparation for in vivo use.

The invention also includes a method for delivering a therapeutic agent to a target cell, and this method comprises contacting potential target cells with a scTCR or multivalent scTCR complex of the invention under conditions allowing binding of the scTCR or multivalent scTCR complex to the target cell, said scTCR or the multivalent scTCR complex is specific for MHC-peptide complexes and contains an associated therapeutic agent.

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

Many therapeutic antigens can be used for such applications, for example, radioactive compounds, enzymes (eg, perforin), or chemotherapeutic agents (eg, cisplatin). To ensure that toxic effects occur at a given site, the toxin inside the liposome can be coupled to streptavidin so that the compound is released slowly. This prevents damage to the body during transport and ensures that the toxin exerts its maximum effect after scTCR binds to relevant antigen-presenting cells.

Other suitable therapeutic agents include:

- cytotoxic low molecular weight agents (cytotoxic agents are small molecules), for example 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 low molecular weight cytotoxic agents also include prodrugs, i.e. compounds that decompose or transform under physiological conditions with the release of cytotoxic agents. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin (rachelmycin, rachelmycin), calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer, sodium trimetremetrefetremetrinfetrite II, trimetremetremetrin II, glucuronate, auristatin E, vincristine and doxorubicin;

- peptide cytotoxins, i.e. proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, bacterial exotoxin A pseudomonas, DNAase and RNAase;

- radionuclides, i.e. unstable isotopes of elements that decay with the competitive 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, sea anemone 225 and astatin 213;

- prodrugs, such as antibody-directed enzyme prodrugs;

- immunostimulants, i.e. particles that stimulate the immune response. Examples include cytokines such as IL-2, chemokines such as IL8, platelet factor 4, protein - melanoma growth stimulator, etc., antibodies or fragments thereof, complement activators, xenogenic protein domains, allogeneic protein domains, viral domains / bacterial proteins and viral / bacterial peptides.

The soluble scTCR or multivalent scTCR complexes of the invention can be linked to an enzyme capable of converting a prodrug into a drug compound. This allows the prodrug to be converted to a drug compound only at the place where it is needed (i.e., targeted by scTCR).

Examples of suitable MHC peptide targets for scTCRs of the invention include, but are not limited to, viral epitopes such as HTLV-1 epitopes (e.g., Tax peptide cut with HLA-A2 restriction enzyme; HTLV-1 associated with leukemia), HIV epitopes , epitopes of EBV (Epstein-Barr virus, EBV), epitopes of CMV (pitomegalovirus); melanoma epitopes (for example, the MAGE epitope cut with the restriction enzyme HLA-A1) and other cancer-specific epitopes (for example, the G250 antigen associated with kidney cancer, cut with the restriction enzyme HLA-A2); and epitopes associated with autoimmune disorders, such as rheumatoid arthritis. Other disease-associated pMHC targets suitable for use in the invention are listed in the HLA Factbook (Barklay (Ed) Academic Press), and many others have been identified.

Localized drug delivery (localizing) due to the specificity of scTCR can potentially increase the number of treatments for diseases.

The treatment of viral diseases for which drugs are available, such as HIV, SIV (monkey immunodeficiency virus), EBV (EBV), CMV, will become more effective if the drug is released or activated in close proximity to infected cells. In cancer, localization near tumors or metastases will increase the effect of toxins or immunostimulants. In autoimmune diseases, immunosuppressants (immunosuppressants) can be released slowly, exerting a more local effect over a longer period of time with minimal impact on the overall immunity of the subject. To prevent transplant rejection, the effect of immunosuppressants can be optimized in the same way. For vaccine delivery, vaccine antigen can be localized near antigen-presenting cells, thereby increasing antigen efficacy. The method can also be used for image acquisition (visualization).

The scTCRs of the present invention can be used to modulate T cell activation by binding to specific ligands, such as rMHC, and thereby suppress T cell activation. Autoimmune diseases, including T cell-mediated inflammation and / or tissue damage, are susceptible to such a treatment method, for example, type I diabetes. For this application, knowledge of the specific peptide epitope provided by the relevant rMNC is required.

The medicaments of this invention are typically supplied as part of a sterile pharmaceutical composition, which typically contains a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It can be delivered in the form of a single (single) dosage form, as a rule, it is delivered in a tightly closed container and can be part of a kit. Such a set usually (although not necessarily) includes instructions for use. It may include many single dosage forms.

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

Screening Application

The scTCRs of the present invention can be used in screening methods for identifying modulators, including TCR-mediated inhibitors of the cellular immune synapse.

Systems of homogeneous amplified due to the proximity luminescent analysis (Amplified Luminescent Proximity Homogeneous Assay), such as AlphaScreen ™, are based on the use of "donor" and "acceptor" spheres (granules, beads) coated with a layer of hydrogel to which the receptor and ligand proteins. The interaction between these molecules of the receptor and ligand leads to the convergence of the spheres. When these spheres are subjected to laser irradiation, the photosensitizer in the "donor" sphere transfers ordinary oxygen to a more excited singlet state. Molecules of singlet oxygen diffuse and react with a chemiluminescent label on the "acceptor" sphere, which additionally activates fluorophores in the same sphere. The fluorophores then emit light with a wavelength of 520-620 nm, this is a signal that the interaction of the receptor with the ligand has occurred. The presence of an inhibitor of the receptor-ligand interaction leads to a weakening of the signal.

Surface Plasma Resonance Imaging (SPR) is an optical interface analysis method in which one binding partner (usually a receptor) is immobilized on a “chip” (sensory surface) and the binding of another binding partner (usually a ligand) that is soluble and forced is detected " drain "from the chip (" overflow "through the chip). Ligand binding leads to an increase in 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 composed in such a way that a change in the refractive index can be detected using surface plasma resonance, an optical phenomenon in which light incident at a certain angle on a thin metal film gives a reflected beam of lower intensity due to resonant excitation of waves with oscillating surface charge density (surface plasmons). Resonance is very sensitive to changes in the refractive index on the far side of the metal film, and this is a signal that is used to detect binding between immobilized and soluble proteins. Systems that enable convenient application of SPR molecular interaction detection and data analysis are available. Examples are Iasys ™ (Fisons) and Biacore ™ installations.

Other optical interface analysis techniques include total internal reflection fluorescence (TIFR), resonance mirror (RM), and optical grating coupler sensor (GCS), and are discussed in more detail in Woodbury and Venton (J. Chromatog. B. 725 113-137). (1999)). The proximity scintillation method (SPA) is used to screen libraries of compounds for inhibitors of low affinity interaction between CD28 and B7 (K _d value, probably 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 using the emission of beta particles from certain radioactive isotopes that transfer energy to a scintillator immobilized on the surface of the indicator. Short beta interval -particles in solution ensures that scintillation occurs only if when beta particles are emitted in the immediate vicinity of the scintillator.When used to detect protein-protein interactions, one interaction partner is labeled with a radioisotope, while the other is either bound to granules (spheres, beads) containing a scintillator or immobilized on the surface together with a scintillator. the analysis can be carried out in an optimal way, the radioisotope is placed close enough to the scintillator so that photon emission is activated only when the binding of x proteins.

Another aspect of the invention is a method for identifying an inhibitor of the interaction between scTCR and a TCR ligand selected from MHC peptide complexes, CD1 antigen complexes, superantigens and MHC peptide / superantigen complexes, which comprises contacting scTCR with a scTCR ligand binding partner in the presence or absence of of the test compound and in determining whether the presence of the test compound reduces the binding of scTCR to the ligand, such a decrease being taken as an identifying inhibitor.

A final aspect of the invention is a method for identifying a potential inhibitor of the interaction between scTCR and a TCR ligand selected from MHC peptide complexes, CD1 antigen complexes, superantigens and MHC peptide / superantigen complexes, which comprises contacting the scTCR or scTCR ligand binding partner with the test compound and determining whether a test compound actually binds to scTCR and / or a ligand, such a decrease being taken as identifying a potential inhibitor.

This aspect of the invention may find application, in particular, in assays involving an optical interface (inter-surface optical analysis), such as assays performed using a BIAcore ™ system.

Preferred features of each aspect of the invention are applicable to any other aspect, with corresponding changes. Documents of the prior art referred to in this description are introduced in the most fully permitted by law.

Examples

The invention is further described using the examples below, which in no way limit the scope of the invention.

Links are made to the following accompanying drawings, where:

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

Figure 2a shows the extracellular amino acid sequence of the α chain A6 TCR, including the mutation T ₄₈ → C (underlined), used to obtain a new disulfide interchain, and Figure 2b shows the extracellular amino acid sequence of β chain A6 TCR, including the mutation S ₅₇ → C ( underlined) used to obtain a new disulfide interchain;

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

Figure 4 summarizes the cloning strategy used to obtain scDiS A6 TCR.

Figure 5 shows the scDiS A6TCR DNA sequence.

Figure 5b shows the amino acid sequence of scDiS A6TCR.

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

Figure 7 shows the results of electrophoresis in both reducing SDS-PAGE (Coomassie stained) and non-reducing SDS-PAGE (Coomassie stained) gel for fractions A15, B10, B9 and B3, taken from the column illustrated in Figure 6. It is clearly seen that fractions B9 and B 10 contain a protein corresponding to the expected scDiS A6 TCR size.

Figure 8 illustrates elution of scDiS A6 TCR from a Superdex 200 gel filtration column for fractions B 10-B7 sampled from the ion exchange columns shown in Figure 6;

Figure 9 shows the results of electrophoresis in both reducing SDS-PAGE (Coomassie stained) and non-reducing SDS-PAGE (Coomassie stained) gel for fractions B8, B7, B3 and B2, taken from the gel filtration column, as illustrated in Figure 8 It is clearly seen that fraction B7 contains a protein corresponding to the expected scDiS A6 TCR size.

Figure 10 shows the result of the final gel filtration into the BIAcore buffer of concentrated fractions B9-B6 taken from the gel filtration column, as shown in Figure 8. scDiS A6 TCR emerges as one main peak.

Figure 11 shows BIAcore data for binding of scDiS A6 TCR to HLA-A2Tax;

Example 1 - Design of primers and mutagenesis of α and β chains of A6 Tax TCR to introduce cysteine residues necessary for the formation of a new interchain disulfide bond

To mutate the threonine 48 exon 1 A6 Tax in TRAC * 01, the following primers are created in cysteine (mutation is shown in lower case):

5 '- With ACA GAC AAA tgT GTG STA GAC AT

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

To carry out the mutation of serine 57 of exon 1 A6 Tax both in TRBC1 * 01 and and in TRBC2 * 01, the following primers are created in cysteine (the mutation is shown in lowercase letters):

5 '- With AGT GGG GTC tGC ACA GAC SS

5 '- GG GTC TGT GCa GAC CCC ACT G

PCR (PCR) mutagenesis:

Mutation of expression plasmids containing the α or β genes of the A6 Tax TCR chain is carried out using α chain primers and β chain primers, respectively, as follows. 100 ng is mixed with 5 μl of 10 mM dNTP, 25 μl of 10x Pfu buffer (Stratagene), 10 units. Pfu polymerase (Stratagene) and the final volume were adjusted to 240 μl by adding H ₂ O. 48 μl of this mixture was supplemented with primers diluted to a final concentration of 0.2 μM in 50 μl of the final volume of the reaction mixture. After the initial denaturation step of 30 seconds at 95 ° C, the reaction mixture is subjected to 15 cycles of denaturation (95 ° C, 30 sec.), Annealing (55 ° C, 60 sec.) And elongation (73 ° C, 8 min.) On a Hybaid device PCR for express PCR. The product is then digested for 5 hours at 37 ° C. using 10 DpnI restriction units (New England Biolabs). 10 μl of the digested reaction mixture was transformed into XL1 - Blue bacteria and grown for 18 hours at 37 ° C. A single colony was selected and grown overnight in 5 ml of TYP + ampicillin (16 g / L Bacto-Trypton, 16 g / L yeast extract, 5 g / L Nad, 2.5 g / L K ₂ HPO ₄ , 100 mg / L ampicillin ) Plasmid DNA was purified on a Qiagen mini-preparative column according to the manufacturer's recommendations and the sequence was confirmed by automatic sequencing at the Department of Biochemistry, Oxford University). The corresponding mutant nucleotide and amino acid sequences are shown in Figures 1a and 2a for the α chain and in Figures 1b and 2b for the β chain.

Example 2 - Design, expression and testing of a single-chain A6 TCR introducing a new disulfide interchain.

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

scDiS A6 TCR contains a 30 amino acid linker sequence between the C-terminus of the TCR α chain and the N-terminus of the β chain. Figure 3 shows the DNA sequence and amino acid sequence of this linker. The cloning strategy applied to obtain scDiS A6 TCR is summarized in Figure 4.

Briefly, the alpha and beta chains of A6 ds (double-stranded) TCR are 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

The two fragments thus obtained are crosslinked by PCR using 5 'alpha and 3' beta primers to form a single chain TCR, with a short linker containing Xmal-BamHI-Apal sites. This fragment is cloned into pGMT7. Then, a full-sized linker is introduced as an insertion in two stages, first a 42 bp fragment is introduced using the Xmal and BamHI sites:

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

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

Secondly, a fragment of 48 bp embed using the BamHI and Apal sites to create a 90 bp linker between the 3 'end of the alpha chain and the 5' end of the beta chain. Fragment 48 bp receive PCR by 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 was digested with BamHI and ApaI and ligated into a digested plasmid containing a 42 bp linker fragment.

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

Expression A6 TCR:

An expression plasmid containing single-stranded A6 TCR disulfide bond was transformed into E. coli strain BL21pLysS and single ampicillin-resistant colonies were grown at 37 ° C in TYP medium (ampicillin 100 μg / ml) to an optical density of OD ₆₀₀ 0.4 before induction protein expression using 0.5 mM IPTG. Cells are harvested three hours after induction by centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B centrifuge. The cell pellet was resuspended in buffer containing 50 mM Tris-HCl, 25% (w / v) sucrose, 1 mM NaEDTA, 0.1% (w / v) NaAzide (NaN ₃ ), 10 mM DTT, pH 8.0. After the freeze-thaw stage during the night, the resuspended cells are ultrafiltered as 1-minute “pulse beams” for 10 minutes on a Milsonix XL2020 ultrasonic disintegrator using standard 12 mm probes. The precipitate (pellets) of the inclusion bodies is regenerated by centrifugation for 30 min at a speed of 13000 rpm on a Beckman J2-21 centrifuge. Then, they are washed three times with detergent to remove cell debris and membrane components. Each time, inclusion body pellets are homogenized in Triton buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT, pH 8.0) before sediment by centrifugation for 15 minutes at 13,000 rpm in a Beckman J2-21 centrifuge. The detergent and salt are then removed by similar washing in the following buffer: 50 mM Tris-HCl, 1 mM NaEDTA, 0.1% (w / v) NaAzide, 2 mM DTT, pH 8.0. Finally, inclusion bodies are divided into 30 mg aliquots and frozen at -70 ° C. The protein yield of inclusion bodies is quantified by solubilization with 6M guanidine-HCl and measurement of binding to the dye by the Bradford method (PerBio).

About 15 mg of the solubilized chain of inclusion bodies is thawed from frozen stock solutions. The inclusion bodies are diluted to a final concentration of 5 mg / ml in a 6M guanidine solution and DTT (2 M stock solution) is added to a final concentration of 10 mM. The mixture was incubated at 37 ° C for 30 minutes. Prepare 1 liter of the following refolding buffer: 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. Redox steam (an oxidizing-reducing agent pair) (2-mercaptoethylamine and cystamine (to final concentrations of 6.6 mm and 3.7 mm, respectively) is added approximately 5 minutes before adding denatured TCR chains. Then the protein is allowed to unwind (refolding) for 5 hours ± 15 minutes with stirring at a temperature of 5 ° C ± 3 ° C. Then the product obtained by refolding is dialyzed twice, 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, both refolding and dialysis, are carried out at 6-8 ° C.

scTCRs are separated from degradation products and impurities by applying the dialyzed refolding product to a POROS 50HQ anion exchange column and eluting the bound protein in a gradient of 0-500 mM NaCl in 50 column volumes using an Akta purifier (Pharmacia) as shown in Figure 6. Peak Fractions , stored at 4 ° C and before collecting and concentrating, analyze SDS-PACE with Coomassie staining (Figure 7). Then, sTCRs were purified and characterized using a Superdex 200HR gel filtration column (Figure 8), previously equilibrated with HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% Nonidet P40) . The fractions corresponding to the peaks were stored at 4 ° C and before collecting and concentrating, analyze SDS-PACE with Coomassie staining (Figure 9). Finally, the concentrated (one-off) fractions of B9-B6 were run again on a gel chromatography column to obtain purified protein in BIAcore buffer (Figure 10). Combine the fractions corresponding to the peak with an approximate molecular weight of 50 kDa. This pool was evaporated before being characterized by surface plasma resonance on a BIAcore instrument.

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

To analyze the binding of A6 scTCR to the peptide-MHC ligand (HLA-A2 Tax), a surface plasmon resonance biosensor (BIAcore 3000 ™) is used. This analysis becomes simpler when single rMHC complexes (described below) are obtained, the semi-oriented immobilization of which on a binding surface coated with a streptavidin film makes it possible to test binding to a soluble T-cell receptor of up to four different rMHCs (immobilized on different flow cells) simultaneously. Manually injecting the HLA complex makes it easy to establish the exact level of immobilized Class A molecules.

Such immobilized complexes are capable of binding both T-cell receptors and the CD8αα coreceptor, each of which can be injected in the soluble phase.

Class I biotinylated HLA-A2-Tax complexes are refolded in vitro using expressed inclusion bodies containing subunit proteins and a synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). The HLA heavy chain is expressed with a C-terminal biotin tag; it replaces the transmembrane and cytoplasmic domains of the protein in the corresponding construct. The HLA light chain or β2-microglobulin is also expressed as inclusion bodies in E. coli using an appropriate construct, the level is ~ 500 mg / liter of bacterial culture.

E. coli cells are lysed and inclusion bodies are purified to approximately 80% purity. Protein of inclusion bodies is 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 refolded at a concentration of 30 mg / liter heavy chain, 30 mg / liter β2m in 0.4 M L - Arginine - HCl, 100 mM Tris pH 8.1, 3.7 mM cysteamine, 4 mg / ml peptide (e.g. tax 11-19), adding a single denatured protein pulse to the refolding buffer at <5 ° C. Refolding is left to completion at 4 ° C. for at least 1 hour.

The buffer is replaced with dialysis buffer - 10 volumes of 10 mm Tris pH 8.1. To sufficiently restore the ionic strength of the solution, it is necessary to replace the buffer two times. Then the protein solution is filtered through a 1.5 μm cellulose acetate filter and applied to a POROS 50HQ anion exchange column (8 ml volume of the nozzle (carrier)). The protein is eluted in a linear gradient of 0-500 mM NaCl. The HLA-A2 peptide complex was eluted at about 250 mM NaCl and the peak fractions were collected, a protease inhibitor cocktail (Calbiochem) was added, and the fractions were ice-cooled.

Buffers for biotin-labeled HLA complexes are replaced with 10 mM Tris pH 8.1, 5 mM NaCl using a Pharmacia column for fast salt removal, equilibrated in the same buffer. Immediately after elution, the fractions containing the protein are ice-cooled and a protease inhibitor cocktail (Calbiochem) is added. Biotinylation reagents are then added: 1 mM Biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl _2, and 5 μg / ml BirA enzyme (purified by O'Callaghan et at. (1999) Anal. Biochem. 266: 9 -fifteen). The mixture was then incubated at room temperature overnight.

HLA biotinylated complexes are purified by gel chromatography. The Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylated reaction mixture was applied to the column and washed with PBS at a rate of 0.5 ml / min. Biotinylated HLA complexes emerge as a single peak (approximately 15 ml). The fractions containing protein are collected, cooled with ice, and a cocktail of protease inhibitors is added. Protein concentration is determined by binding to Coomassie (PerBio) and aliquots of biotinylated HLA complexes are stored frozen at -20 ° C. Streptavidin is immobilized by standard amine binding methods.

The interaction between the A6 Tax scTCR containing the new interchain and its ligand / MHC complex or the irrelevant HLA-peptide combination, the preparation of which is described above, is analyzed using a BIAcore 3000 ™ surface plasmon resonance (SPR) biosensor. The SPR method measures changes in refractive indices, expressed in response units (RU), near the sensory surface in a small flow cell, a principle that can be used to detect receptor-ligand interactions and to analyze their affinity and kinetic parameters. Flowing cuvettes for probes are prepared by immobilizing individual HLA-peptide complexes in separate flowing cuvettes by binding between biotin crosslinked at β2m and streptavidin chemically bonded to the activated surface of the flowing cuvettes. Then the analysis is carried out by passing scTCR through the surfaces of various flow cells with a constant speed, while measuring the SPR response. The introduction (injection) of soluble sTCR into the peptide-HLA complex at a constant flow rate and at various concentrations is used to determine the background (baseline) resonance. The values of these control measurements are subtracted from the values obtained with the specific peptide-HLA complex and are used to calculate the binding affinity expressed as the dissociation constant, Kd (Price & Dwek, Principles and Problems in Physical Chemistry for Biochemists (2 ^nd Edition) 1979, Clarendon Press, Oxford).

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

Claims (3)

1. Single-stranded T-cell receptor (scTCR) containing
the α segment formed by the sequence of the variable region a of the TCR chain connected to the N-end of the extracellular sequence of the constant region of the α region of the TCR,
β segment formed by the sequence of the variable region β of the TCR chain connected to the N-terminus of the extracellular sequence of the constant region β of the TCR chain, and
a linker sequence linking the C end of the α segment to the N end of the β segment, or vice versa
wherein:
extracellular sequences of the constant regions of the α and β segments are linked by a disulfide bond, which binds cysteine residues, which are substitutions of residues selected from the group including
Thr 48 exon 1 TRAC * 01 and Ser 15 exon 1 TRBC1 * 01 or TRBC2 * 01;
Thr 45 exon 1 TRAC * 01 and Ser 77 exon 1 TRBC1 * 01 or TRBC2 * 01;
Tyr 10 exon 1 TRAC * 01 and Ser 17 exon 1 TRBC1 * 01 or TRBC2 * 01;
Thr 45 exon 1 TRAC * 01 and Asp 59 exon 1 TRBC1 * 01 or TRBC2 * 01;
and
Ser 15 exon 1 TRAC * 01 and Glu 15 exon 1 TRBC1 * 01 or TRBC2 * 01;
moreover, the distance between the β carbon atoms of these substitutions in the corresponding sequences of the extracellular constant lg domains of the α and β domains of the TCR chains is less than 0.6 nm,
it is possible that the extracellular sequences of constant regions present in the α and β segments correspond to the constant regions of α and β of the native TCR chains truncated at their C-ends so that cysteine residues that form the interchain native TCR disulfide bond are excluded, or
extracellular sequences of constant regions present in the α and β segments correspond to the constant regions of α and β of the native TCR chains in which the cysteine residues that form the native interchain disulfide bond are replaced with another amino acid residue, or
there is no unpaired cysteine residue present in the β chain of 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 corresponding to TRAC * 01, and the β segment includes a sequence corresponding to TRBC1 * 01 or TRBC2 * 01, and said non-native disulfide bond is between cysteine residues that replaced Thr 48 of exon 1 TRAC * 01 and Ser 57 of exon 1 TRBC1 * 01 or TPBC2 * 01. 3. scTCR according to claim 1 or 2, which binds covalently to a therapeutic agent.

Images