RU2775623C2 - T-cell receptors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to T-cell receptors (hereinafter – TCR), which bind to HLA-A*02 complex with a cancer antigen MAGE A4 GVYDGREHTV (SEQ ID NO: 1); it can be used in medicine.

EFFECT: obtained TCR can be part of a hybrid TCR-anti-CD3 molecule containing TCR and an antibody against CD3 for the redirection of T-cells to cells representing antigen MAGE-A4, and they can be used for the effective therapy of MAGE A4-expressing tumors.

45 cl, 11 dwg, 12 tbl, 7 ex

Description

The present invention provides T cell receptors (TCRs) that bind HLA-A*02 restricted peptide GVYDGREHTV (SEQ ID NO: 1) derived from germline MAGE A4 cancer antigen. These TCRs may contain non-natural mutations in the variable domains of the alpha and/or beta chains relative to the native MAGE A4 TCR. The TCRs of the present invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of cancer.

State of the art

T cell receptors (TCR) are naturally expressed by CD4 ⁺ and CD8 ⁺ T cells. TCRs are designed to recognize short peptide antigens that are located on the surface of antigen-presenting cells in complex with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also called human leukocyte antigens or HLA) (Davis, et at., (1998), Annu Rev Immunol 16: 523-544.). CD8 ⁺ T cells, also referred to as cytotoxic T cells, specifically recognize MHC class I-associated peptides and are generally responsible for detecting and participating in the destruction of diseased cells. CD8 ⁺ T cells are capable of destroying cancerous as well as virus-infected cells; however, the affinity of the natural repertoire of TCRs expressed by tumor-specific T cells is usually low as a result of selection in the thymus, which means that malignant cells often escape detection and destruction. New immunotherapeutic approaches to promote T-cell recognition of cancer provide a promising strategy for the development of effective anti-cancer agents.

MAGE A4 belongs to the MAGE family of cancer antigens encoded in germline cells (De Plaen, et al., (1994), Immunogenetics 40 (5): 360-369) and has Uniprot accession number P43358. It has been found that such antigens are frequently expressed in many cancerous tissues, and in normal tissues their expression is limited to adult testes and other immunologically "privileged" areas such as the placenta. The tumor-specific nature of these genes makes them ideal targets for cancer therapy. The exact function of MAGE A4 remains unknown, but it is thought to play a role in embryonic development. High expression of MAGE A4 has been reported in several tumor types, including melanoma, carcinoma of the esophagus, head and neck, lung, breast, and bladder (Bergeron, (2009), Int J Cancer 125(6): 1365-1371; Cabezon, et al., (2013), Mol Cell Proteomics 12(2): 381-394 Cuffel, et al., (2011), Int J Cancer 128(11): 2625-2634 Forghanifard, et al., (2011 ), Cancer Biol Ther 12(3): 191-197; Karimi, et al., (2012), din Lung Cancer 13(3): 214-219; Svobodova, et al., (2011), Eur J Cancer 47 (3): 460-469). Said 10-amino acid (10-mer) peptide GVYDGREHTV (SEQ ID NO 1) corresponds to amino acids 230-239 of the full length MAGE A4 protein. This peptide binds to HLA-A*02, and the peptide-HLA complex has been shown to stimulate cytotoxic T cells, leading to the lysis of MAGE A4 positive, HLA-A*02 positive tumor cells (Duffour, et al., ( 1999), Eur J Immunol 29 (10): 3329-3337 and WO 2000020445). Thus, the GVYDGREHTV HLA-A*02 complex is a target antigen that can be used for immunotherapeutic intervention.

The identification of specific TCR sequences that bind to the GVYDGREHTV-HLA-A*02 complex with high specificity is an effective approach for the development of new immunotherapeutic agents. Therapeutic TCRs can be used, for example, as soluble targeting agents to deliver cytotoxic or immune effector agents to a tumor (Lissin, et al., (2013). "High-Affinity Monocloncal T-cell receptor (mTCR) Fusions. Fusion Protein Technologies for Biophamaceuticals: Applications and Challenges". S. R. Schmidt, Wiley; Boulter, et al., (2003), Protein Eng 16(9): 707-711; Liddy, et al., (2012), Nat Med 8: 980 -987), or alternatively, they can be used to construct T cells for adoptive therapy (June, et al., (2014), Cancer Immunol lmmunother 63(9): 969-975). However, such TCR sequences are not yet known in the art, and methods for detecting TCRs with specific characteristics suitable for therapeutic use have a high percentage of negative results and therefore do not give the skilled person sufficient reason to hope for success.

The skilled artisan must first identify a suitable parent or framework sequence. Typically, such sequences are obtained from natural sources, for example, from antigen-responsive T cells isolated from donated blood. Given the rarity of tumor-specific T cells in the natural repertoire, it is often necessary to screen many donors, such as 20 or more, before finding a responding (reactive) T cell. The screening process can take weeks or months, and even if a responding T cell is found, it may not be suitable for immunotherapeutic use. For example, the response may be too weak and/or not specific for the target antigen, or alternatively, it may not be possible to create a clonal population of T cells and to expand or maintain a given T cell line to obtain sufficient material to identify the correct sequences of the TCR chain. Furthermore, since TCRs are confluent and have been predicted to be able to bind approximately one million different HLA peptides (Wooldridge, et al. it is difficult to determine whether a particular TCR has a specificity profile that would enable it to be used to design a molecule for therapeutic use.

TCR sequences that are suitable as parent or framework sequences should have good affinity for the target peptide-HLA complex, e.g., 200 μM or greater affinity, show a high level of specificity for target recognition, e.g., relatively little or no binding to alternative peptide-HLA complexes, be modifiable for use in display libraries such as phage display, and allow refolding and purification in high yield.

Naturally occurring TCRs have poor affinity for the target antigen (low micromolar range) compared to antibodies, and TCRs against cancer antigens are generally capable of poorer antigen recognition than virus-specific TCRs (Aleksic, et al. (2012) Eu J Immunol., 42 (12), 3174-3179). This weak affinity, combined with the reduction in HLA on cancer cells, means that therapeutic TCRs for cancer immunotherapy require genetic modification to increase their affinity for the target antigen and thus provide a stronger response. Desirable for soluble target agents based on TCR is the binding affinity of the TCR antigen in the range from nanomolar to picomolar, with a bound half-life of several hours. This increased activity generated by high affinity antigen recognition at low epitope numbers is illustrated in Figures 1e and 1f by Liddy et al. (Liddy, et al. (2012), Nat Med, 18 (6), 980-987). In affinity maturation, the skilled artisan will typically identify specific mutations and/or combinations of mutations, including but not limited to substitutions, insertions and/or deletions, that can be introduced into the original TCR sequence to increase the strength of antigen recognition. Techniques are known in the art to identify mutations of any given TCR that increase affinity, such as using display libraries. (Li et al., (2005) Nat Biotechnol. 23(3):349-354; Holler et al., (2000). Proc Natl Acad Sci USA; 97(10): 5387-5392). However, to provide a significant increase in the affinity of a given TCR for a given target, one skilled in the art must select specific mutations and/or combinations of mutations from a large number of possible alternatives. Specific mutations and/or combinations of mutations that will result in a significant increase in affinity are unpredictable and there is a high rate of selection failures. In many cases it may not be possible to achieve a significant increase in affinity with a given initial TCR sequence.

The affinity maturation process should also include the need to maintain the specificity of the TCR antigen. Increasing the affinity of a TCR for its target antigen results in a significant risk of cross-reactivity with other unintended targets as a result of the inherent degeneracy of TCR antigen recognition. (Wooldridge, et al., (2012), J Biol Chem 287(2): 1168-1177; Wilson, et al., (2004), Mol Immunol 40(14-15): 1047-1055; Zhao et al. , (2007) J. Immunol, 179;9, 5845-5854). At natural levels of affinity, cross-reactive antigen recognition may be too low to elicit a response. If the cross-reactive antigen is presented on normal healthy cells, there is a high possibility of off-target binding in vivo, which can be clinically toxic. Thus, in addition to increasing the strength of antigen binding, one of skill in the art should also select mutations and/or combinations of mutations that allow the TCR to retain high specificity for the target antigen and show a good safety profile in preclinical testing. In addition, such mutations and/or combinations of mutations are not predictable. The failure rate at this stage is even higher, and in many cases obtaining a target molecule from a given initial TCR sequence may not be possible at all.

The mutations needed to provide high affinity and high specificity should also provide a TCR that can be expressed, that can be refolded and purified in reasonable yield, and that is highly stable in the purified form.

Despite the difficulties described above in identifying TCR sequences with suitable characteristics for therapeutic use, the authors of the present invention surprisingly found a TCR sequence that represents an ideal starting point or framework for obtaining therapeutic TCRs. In addition, the present inventors have unexpectedly identified suitable mutations that can be introduced into the variable domains of the alpha and beta chains of this framework to obtain TCR sequences with ideal characteristics for TCR-based targeted immunotherapy of cancers in which MAGE A4 is expressed.

Brief description of the invention

In a first aspect, the present invention provides a T cell receptor (TCR) having GVYDGREHTV binding property (SEQ ID NO: 1) in complex with HLA-A*02 and comprising a TCR alpha chain variable domain and/or a beta chain variable domain. TCR chains, while:

said alpha chain variable domain contains an amino acid sequence that is at least 90% identical to the sequence of amino acid residues 1-113 of SEQ ID NO: 2, and/or

said beta chain variable domain contains an amino acid sequence that is at least 90% identical to the sequence of amino acid residues 1-116 of SEQ ID NO: 3.

The alpha chain variable domain may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1-113 of SEQ ID NO: 2 and/or the beta chain variable domain may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 1-116 of SEQ ID NO: 3.

In a second aspect, the invention relates to a TCR that binds to the HLA-A*02 GVYDGREHTV complex (SEQ ID NO: 1) with an affinity greater than 200 μM, wherein: CDRs 1, 2 and 3 of the alpha chain contain SEQ ID NOs 6, 7 and 8 respectively and/or CDR 1, 2 and 3 beta chains contain SEQ ID NOS 11, 12 and 13 respectively; and/or at least one of the CDRs contains one or more conservative substitutions relative to SEQ ID NOs: 6-8 and 11-13; and/or at least one of the CDRs contains up to three valid substitutions with respect to SEQ ID NOs: 6-8 and 11-13.

The affinity of the TCR of the present invention for the GVYDGREHTV HLA-A*02 complex can range from 200 μM to 1 pM. Preferably, said substitutions do not change the binding affinity by more than +/- 50%, or more preferably by no more than +/- 20% relative to TCR without substitutions. Preferably, said substitutions do not increase binding affinity for alternative peptide-HLA complexes.

The scaffold TCR has the following uses for alpha and beta chain variable domains:

Alpha chain: TRAV10*01/TRAJ6*01

Beta chain: TRBV28*01/TRBD1*01/TRBJ2-7*01/ (Note that the element "*01" denotes the allelic variant for this sequence, according to IMGT nomenclature.) and the following CDR3 alpha and beta sequences -chains:

Alpha chain: WNHSGGSYIPTF (SEQ ID NO: 8)

Beta strand: ASSFLMTSGDPYEQYF (SEQ ID NO: 13)

The term "framework TCR" or "original TCR" is used in this application as a synonym for the terms "wild-type TCR" or "WT TCR", or "wild-type (non-mutated) TCR", or "native TCR", or "parental TCR", and means a TCR having an alpha chain variable domain containing residues 1-113 of SEQ ID NO: 2 and a beta chain variable domain containing residues 1-116 of SEQ ID NO: 3. The TCR WT constant domain may be full-length or may be truncated and/or mutated to produce soluble TCR. In either case, cysteine residues can be introduced into the TRAC and TRBC regions so that a non-native interchain disulfide bond can be formed. Suitable positions for the location of these cysteine regions are described in WO 03020763. Figure 2 of the accompanying drawings shows the extracellular sequences of the alpha and beta chains of wild-type TCRs, respectively, in soluble format. SEQ ID NO: 4 is identical to the extracellular sequence of the native alpha chain of SEQ ID NO: 2, except that the cysteine at position 48 of the constant domain has been replaced by threonine. Similarly, SEQ ID NO: 5 is identical to the extracellular sequence of the native beta chain of SEQ ID NO: 3, except that the cysteine at position 57 of the constant domain was replaced by serine, the cysteine at position 75 of the constant domain was replaced by alanine, and the asparagine at position 89 the constant domain was replaced by aspartic acid. Soluble wild-type TCR can be used as a reference to compare against it the binding profile of the mutated TCRs of the present invention.

The TCR sequences defined herein are described with reference to the IMGT nomenclature, which is widely known and available to those working in the TCR field. For example, see: LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10O; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, αβ TCRs consist of two chains linked by disulfide bonds. Each chain (alpha and beta) is generally considered to have two domains, namely a variable and a constant domain. The short J segment connects the variable and constant domains and is generally considered to be part of the alpha variable region. In addition, the beta chain usually contains a short D segment next to the J segment, which is also usually considered part of the beta variable region.

The variable domain of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in a framework sequence. The CDRs contain a recognition site for peptide-MHC binding. There are several genes encoding alpha chain variable regions (Vα) and several genes encoding beta chain variable regions (Vβ) that differ in their framework, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. The Vα and Vβ genes are designated in IMGT nomenclature by the prefix TRAV and TRBV, respectively (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Likewise, there are several junction or J genes called TRAJ or TRBJ for the alpha and beta chain respectively, and for the beta chain diversity or D genes called TRBD. (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2): 107-114; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 97-106; LeFranc and LeFranc, (2001), " T cell Receptor Factsbook", Academic Press). The vast diversity of T cell receptor circuits is the result of combinatorial rearrangements between different V, J, and D genes that include allelic variants and J segment diversity (Arstila, et al., (1999), Science 286(5441): 958-961 ; Robins et al., (2009), Blood 114(19): 4099-4107.) The constant or C regions of the TCR alpha and beta chains are referred to as TRAC and TRBC, respectively. (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

The alpha chain variable domain of the first or second aspect may contain a mutation in at least one of the following positions relative to residue positions 1-113 of SEQ ID NO: 2: M50, T51, F52, S53, E54, H94, S95, G96 , S98. Mutations can be selected from the following amino acids with respect to residue positions 1-113 of SEQ ID NO: 2:

Additionally or alternatively, the variable domain of the beta chain according to the first or second aspect may contain a mutation in at least one of the following positions relative to the positions of residues 1-116 of the sequence of SEQ ID NO: 3: M27; D28; H29, E30, N31, Y50, D51, V52, K53, M54, F95, L96, M97, T98. Mutations can be selected from the following amino acids with respect to residue positions 1-116 of SEQ ID NO: 3:

The alpha chain variable domain may contain 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the mutations shown in Table 1 and/or the beta chain variable domain may contain 1, 2, 3, 4, 5 6, 7, 8, 9 or 10 mutations shown in Table 2.

The alpha chain variable domain may contain at least one of the following groups of mutations:

Group 1: M50L, T51D, F52Y, S53A, E54I

Group 2: H94S, S95A, G96N, S98L

Group 3: H94R S95A, G96D, S98L

Group 4: M50L, T51D, F52Y, S53A, E54I, H94R S95A, G96D, S98L

Group 5: M50L, H94S, S95A, G96S, S98L

Group 6: M50L, H94S, S95A, G96Q, S98L

and/or the beta chain variable domain may contain at least one of the following groups of mutations:

Group 1: M27A, D28P, H29L, E30S, N31K F95S, L96D, M97Q, T98N

Group 2: Y50R, D51F, V52A, K53T, M54G, F95S, L96D, M97Q, T98N

Group 3: F95S, L96D, M97Q, T98N

Group 4: M27L, Y50R, D51F, V52A, K53T, M54G, F95S, L96D, M97Q, T98N

Group 5: M27A, D28P, H29L, E30S, N31K, M54L, F95S, L96D, M97Q, T98N

For example, an alpha chain variable domain may contain group 4 mutations, and a beta chain variable domain may contain group 1 mutations; the alpha chain variable domain may contain group 4 mutations and the beta chain variable domain may contain group 5 mutations; the alpha chain variable domain may contain group 2 mutations and the beta chain variable domain may contain group 2 mutations; the alpha chain variable domain may contain group 6 mutations and the beta chain variable domain may contain group 4 mutations; the alpha chain variable domain may contain group 5 mutations, and the beta chain variable domain may contain group 4 mutations.

Mutations may additionally or alternatively be performed outside of the CDR; such mutations may improve binding and/or specificity and/or stability and/or useful yield of the purified soluble form of TCR. For example, the TCR of the present invention may additionally or alternatively contain an alpha chain variable domain having the following mutations with respect to residue positions 1-113 of SEQ ID NO: 2:

In the variable domain of the alpha chain, the sequence of amino acid residues 27-32, 50-56 and 91-103 can be selected from the following sequences:

The TCR alpha chain variable domain may comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any from the sequences of SEQ ID NO: 16-24 or 46-64.

In the variable domain of the beta chain, the sequence of amino acid residues 27-31, 49-54 and 92-107 can be selected from the following sequences:

The TCR beta chain variable domain may comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any from the sequences of SEQ ID NO: 25-29 or 65-81.

The alpha chain variable domain sequence of amino acid residues 27-32, 50-56 and 91-103 and the beta chain variable domain sequence of amino acid residues 27-31, 49-54 and 92-107 may be selected from the following:

The alpha chain variable domain may comprise the amino acid sequence of any of SEQ ID NOs: 16-24 or 46-64 and/or the beta chain variable domain may comprise the amino acid sequence of any of SEQ ID NOs: 25-29 or 65-81.

For example, an alpha chain variable domain may comprise the amino acid sequence of SEQ ID NO: 22, and a beta chain variable domain may comprise the amino acid sequence of SEQ ID NO: 26; the alpha chain variable domain may comprise the amino acid sequence of SEQ ID NO: 22, and the beta chain variable domain may comprise the amino acid sequence of SEQ ID NO: 27; the alpha chain variable domain may comprise the amino acid sequence of SEQ ID NO: 20, and the beta chain variable domain may comprise the amino acid sequence of SEQ ID NO: 28; the alpha chain variable domain may comprise the amino acid sequence of SEQ ID NO: 24, and the beta chain variable domain may comprise the amino acid sequence of SEQ ID NO: 29; the alpha chain variable domain may comprise the amino acid sequence of SEQ ID NO: 23, and the beta chain variable domain may comprise the amino acid sequence of SEQ ID NO: 29.

The TCR of the present invention may be an alpha-beta heterodimer having an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence.

The TCR of the present invention may be in single chain format, including but not limited to Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L -Vβ-Cβ, where Vα and Vβ are the TCR α and β chain variable regions, respectively, Cα and Cβ are the TCR α and β chain constant regions, respectively, and L is the linker sequence.

The TCR of the present invention may be associated with a detectable label, a therapeutic agent, or a pharmacokinetic modifier (PK modifying moiety).

The TCR of the present invention may comprise an anti-CD3 antibody covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR. Such a TCR may comprise an alpha chain variable domain selected from any of SEQ ID NOs: 16-24 or 46-64 and a beta chain variable domain selected from any of SEQ ID NOs: 25-29 or 65-81 , hybridized with an anti-CD3 antibody. The beta chain can be linked to the anti-CD3 antibody sequence using a linker sequence; such a linker sequence may be selected from the group consisting of GGGGS (SEQ ID NO: 30), GGGSG (SEQ ID NO: 31), GGSGG (SEQ ID NO: 32), GSGGG (SEQ ID NO: 33), GSGGGP (SEQ ID NO: 34), GGEPS (SEQ ID NO: 35), GGEGGGP (SEQ ID NO: 36), and GGEGGGSEGGGS (SEQ ID NO: 37).

Preferred TCR-anti-CD3 fusion molecules comprise an alpha chain amino acid sequence selected from SEQ ID NOs: 38-41 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 38-41 and the beta chain amino acid sequence selected from SEQ ID NO: 42-45, or the sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NOs: 42-45 .

The TCR of the present invention may be included in a particle library. For such purposes, the TCR can be presented, for example, on the surface of a bacteriophage, yeast cell, mammalian cell, or ribosome. The TCR may be isolated, cell-free and/or soluble, i.e. it may not occur naturally in a human T cell.

The TCRs of the present invention may be non-naturally occurring and/or purified and/or engineered. The TCRs of the present invention may have more than one mutation present in the alpha chain variable domain and/or the beta chain variable domain relative to the native MAGE A4 TCR.

The TCR of the present invention may comprise an alpha chain framework 2 (FR2) and an alpha chain framework 3 (FR3), wherein the FR2 and FR3 regions comprise SEQ ID NOs: 9 and 10, respectively, and/or contain one or more, for example, one , two or three conservative substitutions and/or up to three valid substitutions.

The TCR of the present invention may comprise a beta chain FR2 region and a beta chain FR3 region, wherein the FR2 and FR3 regions comprise SEQ ID NOs: 14 and 15, respectively, and/or contain one or more, for example one, two or three, conservative substitutions and /or up to three valid substitutions.

The TCR of the present invention may comprise amino acids 1-113 of SEQ ID NO: 2 and/or amino acids 1-116 of SEQ ID NO: 3, each of which may contain one or more conservative substitutions and/or up to three allowable mutations and/or one or more of the mutations listed in Tables 1, 2 and 3.

"Engineered TCR" and "mutated TCR" are used interchangeably herein to refer to a TCR having one or more introduced mutations relative to the native MAGE A4 TCR, in particular in the alpha chain variable domain and/or in the beta chain variable domain. The mutation(s) generally improves the binding affinity of the TCR to the HLA-A*02 GVYDGREHTV complex (SEQ ID NO: 1), but may additionally or alternatively confer other advantages such as improved stability in isolated form and improved specificity. Mutations at one or more positions may additionally or alternatively affect the interaction of the adjacent position with the pMHC cognate complex, for example by providing a more favorable angle for interaction. In order to improve TCR binding to the HLA-A*02 GVYDGREHTV complex (SEQ ID NO: 1), mutations are preferably carried out in one or more regions of the CDR.

In some embodiments, there are 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations in the alpha chain CDR, e.g., 4, 5, or 9 mutations, and/or 1, 2, 3, 4, 5 6, 7, 8, 9 or 10 mutations in the beta chain CDR, eg 4, 9 or 10 mutations.

In some embodiments, a TCR α-chain variable domain of the present invention may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of amino acid residues 1-113 of SEQ ID NO: 2, provided that the α chain variable domain has at least one of the mutations described above, for example, in Table 1 or in Table 3. In some embodiments, the TCR B chain variable domain of the present invention may comprise an amino acid sequence, which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98% or less at least 99% identical to the sequence of amino acid residues 1-116 of the sequence of SEQ ID NO: 3, provided that the variable domain of the B chain has at least one of the mutations described above, for example, in Table 2.

Mutations in the parental (or wild-type) TCR may include mutations that are capable of increasing the binding affinity (Kd and/or binding half-life) of the TCR to GVYDGREHTV. Mutations may include mutations that are capable of reducing the amount of non-specific binding, ie, reducing binding to antigens other than binding to GVYDGREHTV. Mutations may include mutations that increase the efficiency of folding and/or receiving. Some mutations may contribute to each of these characteristics, others may promote, for example, affinity but not specificity, or promote specificity but not affinity, and so on.

The scope of the invention includes phenotypically silent variants of any TCR of the present invention disclosed in this application. As used herein, the term "phenotypically silent variants" is intended to mean a TCR that includes one or more additional amino acid changes, including substitutions, insertions, and deletions, in addition to those set forth above, wherein the TCR has a phenotype similar to the corresponding TCR without being specified. changes. For the purposes of this application, the TCR phenotype includes antigen-binding affinity (Ko and/or binding half-life) and antigen specificity. A phenotypically silent variant may have a K _D and/or binding half-life for the HVA-A*02 GVYDGREHTV complex (SEQ ID NO: 1) within 50% or more preferably within 20% of the measured K _D and/or binding half-life of the corresponding TCR without the indicated change(s) when measured under identical conditions (eg, at 25°C and/or on the same SPR chip). Suitable conditions are further provided in Example 3. Antigenic specificity is further defined below. As known to those skilled in the art, it is possible to generate TCRs that include changes in their variable domains from the TCRs detailed above without changing the affinity of interaction with the GVYDGREHTV (SEQ ID NO: 1) HLA-A*02 complex. In particular, such silent mutations may be included in parts of the sequence known not to be directly involved in antigen binding (eg, CDRs or parts of CDRs that do not come into contact with the peptidic antigen). Such trivial variations are included within the scope of this invention.

Phenotypically silent variants may contain one or more conservative substitutions and/or one or more valid substitutions. Acceptable and conservative substitutions may result in a change in K _D and/or binding half-life for the HVA-A*02 GVYDGREHTV complex (SEQ ID NO: 1) within 50% or more preferably within 20%, even more preferably within within 10% of the measured K _D and/or binding half-life of the corresponding TCR without specified conservative and/or acceptable substitution(s), when measured under identical conditions (e.g., at 25°C and/or on the same SPR chip ) provided that this change in K _D does not cause the affinity to be less than (i.e., weaker than) 200 μM. By acceptable substitutions are meant those substitutions that do not fall within the definition of conservative as defined below, but are nonetheless phenotypically silent.

The TCRs of the present invention may include one or more conservative substitutions that have a similar amino acid sequence and/or that retain the same function (ie, are phenotypically silent as defined above). The skilled artisan will recognize that different amino acids have similar properties and are therefore "conservative". One or more such amino acids of a protein, polypeptide or peptide can often be replaced by one or more other such amino acids without losing the desired activity of that protein, polypeptide or peptide.

Accordingly, the amino acids glycine, alanine, valine, leucine and isoleucine can often replace each other (amino acids having aliphatic side chains). Of these possible substitutions, it is preferable that glycine and alanine are used to replace each other (because they have relatively short side chains), and that valine, leucine and isoleucine be used to replace each other (because they have larger aliphatic side chains that are hydrophobic ). Other amino acids that can often substitute for each other include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulfur-containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention may be made using naturally occurring or non-naturally occurring amino acids. For example, this application contemplates that the methyl group on alanine can be replaced with an ethyl group, and/or that minor changes can be made to the peptide backbone. Regardless of whether natural or synthetic amino acids are used, it is preferred that only L-amino acids be present.

Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. Therefore, the present invention extends to the use of a TCR comprising the amino acid sequence described above, but with one or more conservative substitutions and/or one or more allowable substitutions in the sequence such that the amino acid sequence of the TCR is at least 90% identical, e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% TCR containing amino acids 1-113 SEQ ID NO: 2, 16-24 or 46 -64 and/or amino acids 1-116 SEQ ID NO: 3, 25-29 or 65-81.

The term "identity" as used in the art refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, respectively, as determined by the match between strings of such sequences. Although there are a number of methods for measuring identity between two polypeptide or two polynucleotide sequences, the methods commonly used to determine identity are codified in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG software package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec Biol 215, 403 (1990)).

A program such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and selects the optimal alignment by inserting gaps in any sequence if necessary. For optimal alignment, amino acid identity or similarity (identity plus amino acid type conservatism) can be calculated. A program like BLASTx is able to align the longest stretch of similar sequences and assign a score to that fit. Thus, it is possible to obtain a comparison in which several areas of similarity are found, each of which has a different score. The present invention provides for both types of identity analysis.

Percent identity between two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (e.g., gaps may be introduced into the first sequence to better align with the given sequence) and comparing amino acid residues or nucleotides at the appropriate positions. The "best alignment" is the alignment of two sequences that results in the highest percentage of identity. Percent identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (ie % identity = number of identical positions/total number of positions ×100).

Determination of percent identity between two sequences can be performed using a mathematical algorithm known to those skilled in the art. An example of such a mathematical algorithm for comparing two sequences is the algorithm of Carlin and Altshul (1990) Proc. Natl. Acad. sci. USA 87:2264-2268, modified by Karlin and Altschul (1993) Proc. Natl. Acad. sci. USA 90:5873-5877. The NBLAST and XBLAST programs described by Altschul, et al. (1990) J. Mol. Biol. 215:403-410 included such an algorithm. To obtain nucleotide sequences homologous to nucleic acid molecules, nucleotide searches can be performed according to BLAST algorithms using the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to protein molecules for use in the invention, protein searches according to BLAST algorithms can be done with the XBLAST program, score = 50, word length = 3 words. To obtain an alignment with gaps for comparison, the Gapped BLAST algorithm can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search that finds distant matches between molecules (ibid.). When using the BLAST, Gapped BLAST and PSI-Blast programs, you can use the default settings of the respective programs (eg XBLAST and NBLAST). See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm, Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment package, contains such an algorithm. Other sequence analysis algorithms known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. sci. 85:2444-8. In the FASTA program, the control parameter ktup sets the sensitivity and speed of the search.

Mutations, including conservative and acceptable substitutions, insertions and deletions, may be introduced into the sequences of the present invention by any suitable method, including, but not limited to, methods based on polymerase chain reaction (PCR), restriction enzyme cloning, or ligation-free procedures. cloning (LIC). These methods are detailed in many standard molecular biology texts. For more information on polymerase chain reaction (PCR) and restriction enzyme cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) CSHL Press. Further information on ligation-free cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The TCR sequences of the present invention can be obtained using solid phase synthesis or any other suitable method known in the art.

The TCRs of the present invention have the property to bind the GVYDGREHTV (SEQ ID NO: 1) HLA-A*02 complex. The TCRs of the present invention have been found to strongly recognize this epitope over other inappropriate epitopes and thus are particularly useful as targeting vectors for delivering therapeutic agents or detectable labels to cells and tissues presenting these epitopes. The specificity for the TCRs of the present invention is related to their ability to recognize HLA-A*02 target cells that are antigen positive while having minimal ability to recognize HLA-A*02 target cells that are antigen negative.

Specificity can be measured in vitro, for example, in cellular assays such as those described in Example 6. To test specificity, TCRs can be in soluble form and/or can be hybridized to an immune effector and/or can be expressed on the surface of cells, such like T cells. Recognition can be determined by measuring the level of T cell activation in the presence of the TCR of the present invention and target cells. The minimum recognition of antigen-negative target cells is defined as the level of T-cell activation less than 20%, preferably less than 10%, preferably less than 5% and more preferably less than 1% of the level obtained in the presence of antigen-positive target cells when measured. under the same conditions and at a therapeutically significant concentration of TCR. For the soluble TCRs of the present invention, a therapeutically relevant concentration can be defined as a TCR concentration of 10 ^-9 M or less and/or a concentration of 100, preferably up to 1000 times the corresponding EC 50 value. priming using an appropriate concentration of peptide to obtain a level of antigen presentation comparable to cancer cells (e.g. 10 ^-9 M peptide as described in Bossi et al., (2013) Oncoimmunol. 1; 2 (11): e26840) or they may inherently present said peptide. Preferably, both the antigen positive and antigen negative cells are human cells. Preferably, the antigen positive cells are human cancer cells. Antigen negative cells preferably include cells derived from healthy human tissues.

Specificity may additionally or alternatively refer to the ability of the TCR to bind to the HLA-A*02 GVYDGREHTV complex (SEQ ID NO: 1) rather than to a panel of alternative peptide-HLA complexes. It can, for example, be determined by the Biacore method of Example 3. Said panel may contain at least 5 and preferably at least 10 alternative peptide-HLA-A*02 complexes. Alternative peptides may have a low level of sequence identity to SEQ ID NO: 1 and may be presented naturally. Alternative peptides may be derived from proteins expressed in healthy human tissues. Binding to the GVYDGREHTV-HLA-A*02 complex may be at least 2-fold greater than other naturally occurring HLA-peptide complexes, more preferably at least 10-fold, or at least 50-fold or at least 100 times greater, or even more preferably at least 400 times greater.

An alternative or complementary approach to determine TCR specificity would be to identify the TCR peptide recognition motif using sequential mutagenesis, such as alanine scanning. Residues that are part of the binding motif are residues that are not eligible for replacement. Invalid substitutions can be defined as those peptide positions at which the TCR binding affinity is reduced by at least 50%, or preferably at least 80%, relative to the binding affinity for the wild-type peptide. This approach is described in more detail in Cameron et al., (2013), Sci Transl Med. 2013 Aug 7; 5 (197): 197ra103 and WO 2014096803. TCR specificity in this case can be determined by identifying alternative peptide-containing motifs, in particular alternative peptide-containing motifs in the human proteome, and testing these peptides for TCR binding. TCR binding to one or more alternative peptides may indicate a lack of specificity. In this case, further testing of TCR specificity with cellular assays may be required.

As is known to those skilled in the art, peptides derived from members of the MAGE family can have a high level of sequence identity with peptides derived from other members of the MAGE family. For example, there are peptides derived from MAGE-A8 and MAGE-B2 that differ by only two residues from SEQ ID NO 1 (GVYDGREHTV). Said peptides and cells expressing said MAGE family members may be excluded from the above definition of specificity, especially if said MAGE family members are known to be cancer antigens such as MAGE-A8 and MAGE-B2. Therefore, the TCRs of the present invention can recognize peptides with high percent sequence identity that are derived from other members of the MAGE family, including MAGE-A8 and MAGE-B2, and presented in the context of HLA A*02. The recognition of these peptides by the TCR of the present invention may be at the same or lower level than the recognition of GVYDGREHTV (SEQ ID NO: 1) HLA-A*02.

Some of the TCRs of the present invention may have an ideal safety profile for use as therapeutic agents. In this case, the TCRs may be in soluble form and may preferably be hybridized to an immune effector. The ideal safety profile means that, in addition to demonstrating good specificity, the TCRs of the present invention may have been subject to additional pre-clinical safety testing. Examples of such tests include whole blood tests to confirm minimal cytokine release in the presence of whole blood and thus low risk of potential in vivo cytokine release syndrome, and alloreactivity tests to confirm low potential for recognition of alternative HLA types.

Some soluble TCRs of the present invention can be purified in high yield. "High yield" means a yield of more than 1% or more, preferably more than 10% or more.

для комплекса в диапазоне от приблизительно 1 с до приблизительно 60 ч, от 1 мин до приблизительно 60 ч, от приблизительно 20 мин до приблизительно 50 ч или от приблизительно 2 до приблизительно 35 часов. Определенные TCR по настоящему изобретению могут иметь

для комплекса от приблизительно 8 до приблизительно 35 часов. TCR, которые предназначены для применения в виде растворимых терапевтических средств и/или средств для диагностики, когда они связаны с детектируемой меткой или терапевтическим агентом, предпочтительно имеют K_D для этого комплекса от приблизительно 1 до приблизительно 100 пМ или от приблизительно 20 до приблизительно 80 пМ, и/или период полужизни связывания для данного комплекса от приблизительно 2 ч до приблизительно 60 ч или от приблизительно 8 ч до приблизительно 35 ч. Некоторые TCR по настоящему изобретению могут быть пригодны для применения в адоптивной терапии; такие TCR могут иметь Kd для данного комплекса от приблизительно 50 нМ до приблизительно 200 мкМ или от приблизительно 100 нМ до приблизительно 1 мкМ и/или период полужизни связывания для комплекса от приблизительно 3 с до приблизительно 12 мин.The TCRs of the present invention may have a K _D for the GVYDGREHTV-HLA-A*02 complex greater than (ie, corresponding to stronger binding) 200 μM, for example, from 1 pM to 200 μM. Some TCRs of the present invention may have a K _D for this complex from about 1 to about 400 nM, from about 1 to about 200 pM, from about 1 to about 100 pM. Some TCRs of the present invention may have a K _D for this complex of approximately 20-80 pM. The TCRs of the present invention may have a binding half-life

for the complex in the range of from about 1 second to about 60 hours, from 1 minute to about 60 hours, from about 20 minutes to about 50 hours, or from about 2 to about 35 hours. Certain TCRs of the present invention may have

for the complex from about 8 to about 35 hours. TCRs that are intended for use as soluble therapeutics and/or diagnostics when associated with a detectable label or therapeutic agent preferably have a K _D for this complex of about 1 to about 100 pM, or about 20 to about 80 pM , and/or a binding half-life for a given complex from about 2 hours to about 60 hours, or from about 8 hours to about 35 hours. Some TCRs of the present invention may be suitable for use in adoptive therapy; such TCRs may have a Kd for a given complex from about 50 nM to about 200 μM, or from about 100 nM to about 1 μM, and/or a binding half-life for the complex from about 3 s to about 12 minutes.

Some preferred TCRs are capable of generating a highly efficient in vitro T cell response against antigen positive cells, in particular cells that have low antigen levels typical of cancer cells (i.e., approximately 50 antigens per cell (Bossi et al., ( 2013) Oncoimmunol) 1; 2 (11): c. 26840; Purbhoo et al. (2006) J Immunol 176 (12): 7308-7316.)). Such TCRs may be in soluble form and associated with an immune effector such as an anti-CD3 antibody. The measured T cell response can be the release of markers of T cell activation, such as interferon y or granzyme B, or cell killing, or other measure of T cell activation. Preferably, a high potency response is one with an EC50 value in the pM range, eg, 100 pM or below.

Some preferred TCRs of the present invention have a binding affinity and/or binding half-life for the GVYDGREHTV-HLA-A*02 complex substantially higher than that of the native TCR. Increasing the binding affinity of a native TCR often reduces the specificity of that TCR for its peptide-MHC ligand, and this is demonstrated by Zhao et al., (2007) J. Immunol, 179: 9, 5845-5854. However, such TCRs of the present invention remain specific for the GVYDGREHTV-HLA-A*02 complex despite having a substantially higher binding affinity than the native TCR.

) могут быть определены с использованием поверхностного плазмонного резонанса (BIAcore) и/или метода Octet из Примера 3 в настоящем документе. Понятно, что удвоение аффинности TCR приводит к уменьшению K_d вдвое.

рассчитывается как In2, деленный на скорость диссоциации (k_off). Следовательно, удвоение

приводит к уменьшению k_off вдвое. Значения K_d и k_off для TCR обычно измеряют для растворимых форм TCR, то есть тех форм, которые укорочены для удаления остатков цитоплазматического и трансмембранного доменов. Предпочтительно аффинность связывания или период полужизни связывания TCR измеряют несколько раз, например, 3 или более раз, используя один и тот же протокол анализа, и получают среднее значение результатов.Binding affinity (inversely proportional to the equilibrium constant K _D ) and binding half-life (expressed

) can be determined using surface plasmon resonance (BIAcore) and/or the Octet method from Example 3 herein. It is clear that doubling the TCR affinity leads to a halving of K _d .

calculated as In2 divided by the dissociation rate (k _off ). Therefore, doubling

leads to a decrease in k _off by half. The K _d and k _off values for TCR are usually measured for soluble forms of TCR, ie those forms that are truncated to remove remnants of the cytoplasmic and transmembrane domains. Preferably, the binding affinity or binding half-life of the TCR is measured several times, eg 3 or more times, using the same assay protocol and the average of the results is obtained.

For use as a targeting agent for delivering therapeutic agents to an antigen-presenting cell, the TCR may be in soluble form (ie, lack transmembrane or cytoplasmic domains). For the stability of the TCRs of the present invention and preferably soluble ap heterodimeric TCRs may contain an introduced disulfide bond between the residues of the respective constant domains, as described, for example, in WO 03/020763. One or both of the extracellular constant domains present in the α-heterodimer of the present invention may be truncated at the C-terminus or C-terminus, for example up to 15 or 10 or 8 or fewer amino acids. The C-terminus of the extracellular constant domain of the alpha chain may be shortened by 8 amino acids. One or both extracellular constant domains may contain one or more mutations. The extracellular alpha chain constant may contain an aspartic (N) or lysine (K) residue at position 4 due to natural polymorphism. For use in adoptive therapy, αβ heterodimeric TCR can, for example, be transfected as full length chains having both cytoplasmic and transmembrane domains. TCRs for use in adoptive therapy may contain a disulfide bond corresponding to that which occurs in nature between the respective alpha and beta constant domains; additionally or alternatively, a non-native disulfide bond may be present.

The TCRs of the present invention may be αβ heterodimers. The TCRs of the present invention may be in single chain format. Single chain formats include, but are not limited to, αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ types. where Vα and Vβ are TCRα and β variable regions, respectively, Cα and Cβ are TCR α and β constant regions, respectively, and L is the linker sequence (Weidanz et al., (1998) J. Immunol Methods. Dec 1; 221 (1-2): 59-76; Epel et al., (2002), Cancer Immunol Immunother Nov; 51 (10): 565-73; WO 2004/033685; WO 9918129). One or both constant domains may be full length, or they may be truncated as described above and/or contain mutations. The extracellular alpha chain constant may contain an aspartic (N) or lysine (K) residue at position 4 due to natural polymorphism. In some embodiments, the single chain TCRs of the present invention may have an introduced disulfide bond between the residues of the respective constant domains, as described in WO 2004/033685. Single chain TCRs are further described in WO 2004/033685; W098/39482; WO 01/62908; Weidanz et al. (1998) J Immunol Methods 221(1-2): 59-76; Hoo et al. (1992) Proc Natl Acad Sci U S A 89(10): 4759-4763; Schodin (1996) Mol Immunol 33(9): 819-829).

As will be appreciated by one of ordinary skill in the art, it is possible to truncate the presented sequences at the C-terminus and/or N-terminus by 1, 2, 3, 4, 5 or more residues without significantly affecting the TCR binding characteristics. All such trivial variations are covered by the present invention.

The alpha-beta-heterodimeric TCRs of the present invention typically comprise a TRAC alpha chain constant domain sequence and/or a TRBC1 or TRBC2 beta chain constant domain sequence. The alpha and beta chain constant domain sequences can be modified by truncation or substitution to remove the native disulfide bond between TRAC exon 2 Cys4 and TRBC1 or TRBC2 exon 2 Cys2. The sequence(s) of the alpha and/or beta chain constant domain can be modified by replacing the Thr 48 TRAC and Ser 57 residues of TRBC1 or TRBC2 with a cysteine, said cysteines forming a disulfide bond between the alpha and beta chain constant domains of the TCR. TRBC1 or TRBC2 may further include a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. The constant domain may additionally or alternatively contain additional mutations, substitutions or deletions relative to native TRAC and/or TRBC1/2 sequences. The terms TRAC and TRBC1/2 encompass natural polymorphic variants, eg N to K at position 4 of the TRAC domain (Bragado et al Int Immunol. 1994 Feb; 6(2):223-30).

Also included within the scope of the invention are variants, fragments and derivatives of the TCRs of the invention.

The invention also includes particles presenting the TCRs of the present invention and the inclusion of said particles in a particle library. Such particles include, but are not limited to, phage, yeast ribosomes, or mammalian cells. The method of obtaining such particles and libraries is known in the art (for example, see WO 2004/044004; WO 01/48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

In a further aspect, the present invention relates to a nucleic acid encoding a TCR of the present invention. In some embodiments, the nucleic acid is cDNA. In some embodiments, the invention relates to a nucleic acid containing a sequence encoding the variable domain of the TCR α chain of the present invention. In some embodiments, the invention relates to a nucleic acid containing a sequence encoding the TCR β chain variable domain of the present invention. The nucleic acid may be non-naturally occurring and/or purified and/or engineered. The nucleic acid sequence may be codon-optimized according to the expression system used.

In another aspect, the invention relates to a vector containing the nucleic acid of the present invention. Preferably, such a vector is a TCR expression vector.

The invention also relates to a cell carrying the vector of the present invention, preferably a vector for TCR expression. Such a vector may contain a nucleic acid of the present invention encoding a single open reading frame or two separate open reading frames encoding an alpha chain and a beta chain, respectively. In another aspect, there is provided a cell carrying a first expression vector that contains a nucleic acid encoding a TCR alpha chain of the present invention and a second expression vector that contains a nucleic acid encoding a TCR beta chain of the present invention. Such cells are especially useful in adoptive therapy. The cells of the present invention may be isolated and/or recombinant and/or non-naturally occurring and/or engineered.

Since the TCRs of the present invention are useful in adoptive therapy, the invention includes non-naturally occurring and/or purified and/or engineered cells, in particular T cells presenting the TCRs of the present invention. The invention also relates to a propagated population of T cells presenting the TCR of the present invention. There are a number of methods suitable for transfecting T cells with a nucleic acid (such as DNA, cDNA, or RNA) encoding a TCR of the present invention (see, for example, Robbins et al., (2008) J Immunol. 180: 6116-6131) . T cells expressing the TCR of the present invention will be suitable for use in adoptive therapy-based cancer treatment. As known to those skilled in the art, there are a number of suitable methods by which adoptive therapy can be performed (see, for example, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

The soluble TCRs of the present invention are useful for delivering detectable labels or therapeutic agents to antigen-presenting cells and tissues containing antigen-presenting cells. Therefore, they can be associated (covalently or otherwise) with a detectable label (for diagnostic purposes when the TCR is used to detect the presence of cells presenting the GVYDGREHTV-HLA-A*02 complex); a therapeutic agent; or a pharmacokinetic (PK) modifying group.

Examples of PK modifying groups include, but are not limited to, PEG

(Dozier et al., (2015) Int J Mol Sci. Oct 28; 16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J. Jan; 5(1):113-28), PASylation (Schlapschy et al., (2013) Protein Eng Des Sel. Aug; 26(8):489-501), albumin (Dennis et al., (2002) J Biol Chem. Sep 20; 277(38):35035- 43) and/or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12): 1186-90).

Detectable labels for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

Therapeutic agents that may be associated with the TCR of the present invention include immunomodulators, radioactive compounds, enzymes (eg perforin) or chemotherapeutic agents (eg cisplatin). To ensure that the toxic effects occur at the desired site, the toxin can be contained within a TCR-bound liposome so that the compound is released slowly. This will prevent damaging effects during transport in the body and maximize the effect of the toxin once the TCR has bound to the appropriate antigen-presenting cells.

Other suitable therapeutic agents include:

• low molecular weight cytotoxic agents, ie compounds that have the ability to kill mammalian cells having a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals capable of exerting a cytotoxic effect. In addition, it should be understood that these small molecular weight cytotoxic agents also include prodrugs, that is, compounds that undergo degradation or transformation under physiological conditions to release cytotoxic agents. Examples of such agents include cisplatin, maytansin derivatives, rachelmicin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, photofrin II sorfimer, temozolomide, topotecan, trimethreate glucuronate, auristatin E, vincristine, and doxorubicin;

• peptide cytotoxins, ie proteins or their fragments capable of killing mammalian cells. For example, ricin, diphtheria toxin, Pseudomonas, Pseudomonas aeruginosa exotoxin A, DNase and RNase;

• radionuclides, ie unstable isotopes of elements that decay when one or more α- or β-particles or γ-rays are simultaneously emitted. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents can be used to facilitate the association of these radionuclides with high affinity TCRs or their multimers;

• Immunostimulants, ie immune effector molecules that stimulate an immune response. For example, cytokines such as IL-2 and IFN-γ,

• Superantigens and their mutants;

• TCR-HLA fusion molecules, eg a peptide-HLA complex fusion, wherein said peptide is derived from a common human pathogen such as Epstein-Barr virus (EBV);

• chemokines such as IL-8, platelet factor 4, melanoma growth promoting protein, etc.;

• antibodies or fragments thereof, including determinant antibodies against T cells or NK cells (eg anti-CD3, anti-CD28 or anti-CD16);

• alternative protein scaffolds with antibody-like binding characteristics

• complement activators;

• xenogenic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides.

One preferred embodiment includes a TCR of the present invention associated (typically by hybridization to the N- or C-terminus of an alpha or beta chain) with an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody (such TCR-anti- -CD3 can be called ImmTACTM molecules). Used in this application, the term "antibody" covers such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031, and 12F6. Antibody fragments and variants/analogs that are suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab') ₂ fragments, dsFv and scFv fragments, Nanobodies™ nanobodies (these constructs , marketed by Ablynx (Belgium), contain a synthetic single variable domain of an immunoglobulin heavy chain derived from camelid antibodies (for example, camelid or llama) and Domain Antibodies antibodies (Domantis (Belgium)), containing a single variable domain of an immunoglobulin heavy chain with affinity matured or immunoglobulin light chain variable domain, or alternative protein scaffolds that exhibit antibody-like binding characteristics such as Affibodies (Affibody (Sweden) containing engineered protein A backbone) or Anticalins (Pieris (Germany) containing engineered anticalins), among many others.

The binding of the TCR and the anti-CD3 antibody can be via covalent or non-covalent attachment. Covalent attachment may be direct or indirect through a linker sequence. Linker sequences are generally flexible because they are composed primarily of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that can limit flexibility. Suitable or optimal lengths of linker sequences are easily determined. Often the linker sequence will be less than 12 in length, such as less than 10 or 2-10 amino acids. Suitable linkers that can be used in the TCR of the present invention include, but are not limited to: GGGGS (SEQ ID NO: 30), GGGSG (SEQ ID NO: 31), GGSGG (SEQ ID NO: 32), GSGGG (SEQ ID NO: 32), NO: 33), GSGGGP (SEQ ID NO: 34), GGEPS (SEQ ID NO: 35), GGEGGGP (SEQ ID NO: 36) and GGEGGGSEGGGS (SEQ ID NO: 37) (as described in WO 2010/133828).

Representative variants of the anti-CD3-TCR fusion constructs of the present invention include alpha and beta chain pairs wherein the alpha chain consists of a variable domain containing the amino acid sequence of SEQ ID NOs: 16-24 or 46-64 and/or the beta chain consists of a variable domain containing the amino acid sequence of SEQ ID NO: 25-29 or 65-81. Said alpha and beta chains may further comprise a constant region containing a non-native disulfide bond. The N- or C-terminus of the alpha and/or beta chain can be hybridized to the scFv anti-CD3 antibody fragment via a linker selected from SEQ ID NOs: 30-37. Some preferred embodiments of such anti-CD3-TCR hybrid constructs are presented below:

Each linker with the sequence of SEQ ID NO: 30-37 can be used with each or any of the preferred CD3-TCR hybrid constructs. For example, included in the invention is a TCR-CD3 fusion comprising the alpha chain of SEQ ID NO: 24 and the beta chain of SEQ ID NO: 29, wherein the beta chain is hybridized to an anti-CD3 scFv through a linker of either of SEQ ID NO: 31- 37.

For some purposes, the TCRs of the present invention may be aggregated into a multi-TCR complex to form a polyvalent TCR complex. There are a number of human proteins containing a multimerization domain that can be used in the preparation of polyvalent TCR complexes. For example, the tetramerization domain of the p53 gene, which was used to generate tetramers of scFv antibody fragments that showed increased serum stability and a significantly reduced dissociation rate compared to the monomeric scFv fragment (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392). Hemoglobin also contains a tetramerization domain which can be used for this application. The polyvalent TCR complex of the present invention may have improved binding capacity to the GVYDGREHTV-HLA-A*02 complex compared to the wild type or T cell receptor non-multimeric heterodimer of the present invention. Thus, the polyvalent TCR complexes of the present invention are also included in the invention. Such multivalent TCR complexes of the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for making further multivalent TCR complexes having such applications.

As is well known in the art, TCRs can be subject to post-translational modifications. One such modification is lycosylation, which involves the covalent attachment of oligosaccharide moieties to specific amino acids in the TCR chain. For example, well-known oligosaccharide attachment sites are asparagine residues or serine/threonine residues. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation, and the presence of certain enzymes. In addition, glycosylation status (i.e. oligosaccharide type, covalent bond, and total number of attachment points) can affect protein function. Therefore, when obtaining recombinant proteins, it is often desirable to control glycosylation. Controlled glycosylation has already been used to improve antibody-based therapy. (Jefferis et al., (2009) Nat Rev Drug Discov Mar; 8 (3): 226-34.). For the soluble TCRs of the present invention, glycosylation can be controlled in vivo using, for example, specific cell lines, or in vitro by chemical modification. Such modifications are desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human protein (Sinclair and Elliott, (2005) Pharm Sci. Aug; 94(8): 1626-35).

For administration to patients, the TCRs of the present invention (preferably associated with a detectable label or therapeutic agent or expressed on transfected T cells), TCR-anti-CD3 fusion molecules, nucleic acids, expression vectors, or cells of the present invention may be provided in a pharmaceutical composition together. with one or more pharmaceutically acceptable carriers or excipients. Therapeutic or imaging TCRs or cells of the invention will typically be provided in a sterile pharmaceutical composition, which will typically include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending on the desired route of administration to the patient). It may be provided in unit dosage form, will typically be provided in a sealed container, and may be provided as part of a kit. Such a kit will usually (though not necessarily) include instructions for use. It may contain a plurality of said unit dosage forms.

Such a pharmaceutical composition may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation or intranasal administration. Such compositions may be prepared by any method known in the pharmaceutical art, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

The dosages of the substances of the present invention may vary widely, depending on the disease or disorder being treated, the age and condition of the person being treated, etc.: a suitable dosage range for the soluble TCR of the present invention associated with anti-CD3 antibody, may range from 25 ng/kg to 50 μg/kg. Ultimately, the appropriate doses to use will be determined by the physician.

The TCRs, pharmaceutical compositions, vectors, nucleic acids, and cells of the present invention may be provided in substantially pure form, e.g., at least 80% pure, at least 85% pure, at least 90% pure, at least 91% pure. , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 %.

The invention also provides:

• TCR, TCR-anti-CD3 hybrid molecule, nucleic acid, pharmaceutical composition or cell of the present invention for use in medicine, preferably for use in a method of treating cancer or tumors;

• the use of a TCR, TCR-anti-CB3 fusion molecule, nucleic acid, pharmaceutical composition or cell of the present invention in the manufacture of a medicament for the treatment of cancer or tumor;

• a method of treating a cancer or tumor in a patient, comprising administering to the patient a TCR, a TCR-anti-CB3 fusion molecule, a nucleic acid, a pharmaceutical composition, or a cell of the present invention;

• an injectable composition for administration to a human, comprising a TCR, a TCR-anti-CB3 fusion molecule, a nucleic acid, a pharmaceutical composition, or a cell of the present invention.

Said cancer may be breast, esophagus, head and neck, lung, ovarian, or bladder cancer. The tumor may express MAGE A4 and/or may be a solid tumor. The TCR, TCR-anti-CB3 fusion molecule, nucleic acid, pharmaceutical composition, or cell of the present invention may be administered by injection, such as intravenous or direct intratumoral injection. A human subject may have the HLA-A*02 subtype.

The preferred features of each aspect of the invention are the same as for each of the other aspects, mutatis mutandis. The prior art documents referred to in this application are included to the maximum extent permitted by law.

Description of drawings

Figure 1: Amino acid sequence of the extracellular regions of the alpha and beta chains of native MAGE A4 TCR.

Figure 2: Amino acid sequence of native extracellular regions of the alpha and beta chains of soluble MAGE A4 TCR.

Figure 3: exemplary amino acid sequences of mutated variable regions of the MAGE A4 TCR alpha chain.

Figure 4: exemplary amino acid sequences of mutated MAGE A4 TCR beta chain variable regions.

Figure 5: exemplary alpha chain amino acid sequences of MAGE A4TCR-anti-CD3 fusion molecules.

Figure 6: exemplary amino acid sequences of the beta chain of MAGE A4TCR-anti-CD3 fusion molecules.

Figure 7: Cellular data showing the activity and specificity of MAGE A4 TCR-anti-CD3 fusion molecules.

Figure 8: Cellular data showing the activity and specificity of additional MAGE A4 TCR-anti-CD3 fusion molecules.

Figure 9: Additional evidence for the specificity of MAGE A4 TCR-anti-CD3 fusion molecules

Figure 10: Additional specificity data for A4 TCR-anti-CD3 MCR fusion molecules

Figure 11: Evidence that MAGE A4 TCR-anti-CD3 fusion molecules lead to cancer cell death

The invention is further described in the following non-limiting examples.

Examples

Example 1 Expression, refolding and purification of soluble TCRs

Methodology

The DNA sequences encoding the extracellular regions of the alpha and beta soluble TCRs of the present invention were cloned separately into pGMT7-based expression plasmids using standard methods (as described in Sambrook et al. Molecular cloning. Vol. 2. (1989) New York: Cold spring harbor laboratory press). Expression plasmids were transformed separately into E. coli Rosetta (BL21pLysS), and individual ampicillin-resistant colonies were grown at 37°C in TYP medium (+ampicillin 100 μg/ml) until an OD ₆₀₀ density of ~0.6-0.8 was reached before induction of protein expression using 0.5 mm IPTG. Cells were harvested three hours after induction by centrifugation. The cell pellet was lysed with BugBuster protein extraction reagent (Merck Millipore) according to the manufacturer's instructions. Granules of intracellular bodies were removed by centrifugation. The beads were washed twice in Triton buffer (50 mM Tris-HCl, pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) and finally resuspended in detergent-free buffer (50 mM Tris-HCl , pH 8.1, 100 mM NaCl, 10 mM NaEDTA). The protein yield of intracellular bodies was quantified by solubilization with 6 M guanidine-HCl and measuring OD ₂₈₀ . The protein concentration was then calculated using the extinction coefficient. The purity of the intracellular bodies was measured by solubilization with 8 M urea and loading ~2 μg into a 4-20% DNS-PAGE gel under reducing conditions. Purity was then evaluated or calculated using densitometry software (Chemidoc, Biorad). Intracellular bodies were stored at +4°C for short-term storage and at -20°C or -70°C for long-term storage.

For refolding soluble TCR, containing a and B chains, intracellular bodies were first mixed and diluted in 10 ml solubilization/denaturation buffer (6 M guanidine hydrochloride, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA, 20 mM DTT) followed by incubation for 30 min at 37°C. Refolding was then initiated by further dilution in 1 L of refolding buffer (100 mM Tris, pH 8.1, 400 mM L-arginine HCL, 2 mM EDTA, 4 M urea, 10 mM cysteamine hydrochloride and 2.5 mM cystamine dihydrochloride), and the solution was well mixed. The refolded mixture was dialyzed with 10 l H ₂ O for 18-20 hours at 5±3°C. After this time, the dialysis buffer was replaced twice with 10 mM Tris, pH 8.1 (10 L) and dialysis continued for another 15 hours. The refolded mixture was then filtered through 0.45 µm cellulose filters.

Soluble TCR purification was initiated by loading a dialyzed refold onto a POROS® 50HQ anion exchange column and eluting the bound protein with a gradient of 0-500 mM NaCl in 20 mM Tris pH 8.1 in 50 column volumes using an Akta® purifier (GE Healthcare). Peak TCR fractions were identified by DNS-PAGE before pooling and concentration. The concentrated sample was then applied to a Superdex® 75HR gel filtration column (GE Healthcare) pre-equilibrated in Dulbecco's phosphate-buffered saline. The TCR peak fractions were pooled and concentrated, then the final yield of purified material was calculated.

Example 2 Expression, Refolding and Purification of ImmTAC Molecules (TCR-anti-CD3 Soluble Hybrid Molecules)

Methodology

ImmTAC was prepared as described in Example 1, except that the TCR beta chain was hybridized through a linker with a single chain anti-CD3 antibody. In addition, during purification after anion exchange, a cation exchange step was performed. In this case, peak fractions from anion exchange were diluted 20-fold in 20 mM MES (pH 6.5) and loaded onto a POROS® 50HS cation exchange column. Bound protein was eluted with a gradient of 0-500 mM NaCl in 20 mM MES. ImmTAC peak fractions were pooled and adjusted to 50 mM Tris pH 8.1 and then concentrated and applied directly to a gel filtration matrix as described in Example 1.

Example 3 Binding Study

Binding analysis of purified soluble TCRs and ImmTAC molecules to the corresponding peptide-HLA complex was performed by surface plasmon resonance using a BIAcore 3000 or BIAcore T200 instrument, or by biolayer interferometry using a ForteBio Octet instrument. Biotinylated class I HLA-A*02 molecules were refolded with the peptide of interest and purified using methods known to those skilled in the art (O'Callaghan et al. (1999). Anal Biochem 266(1): 9-15; Garboczi, et al (1992) Proc Natl Acad Sci USA 89(8): 3429-3433). All measurements were performed at 25°C in Dulbecco's phosphate-buffered saline supplemented with 0.005% P20.

BIAcore methodology

Biotinylated peptide-HLA monomers were immobilized on streptavidin-coated CM-5 sensor chips. Equilibrium binding constants were determined by serial dilutions of soluble TCR/ImmTAC injected at a constant flow rate of 30 μl/min and passed over a flow cell coated with ~200 response units (RU) of the peptide-HLA-A*02 complex. Steady-state responses were normalized for each TCR concentration by subtracting the buffer response on a control flow cell containing a foreign peptide-HLA. The K _D value was obtained by fitting a non-linear curve using the Prism software and the Langmuir binding isotherm, bound = C * Max/(C+K _D ), where "bound" is the equilibrium binding in RU at the C concentration of the injected TCR, and Max is the maximum binding.

For high affinity interactions, binding parameters were determined by analyzing single cycle kinetics. Five different concentrations of soluble TCR/ImmTAC were injected through a flow cell coated with ~100-200 RU of peptide-HLA complex at a flow rate of 50-60 μl/min. Typically, 60-120 µl of soluble TCR/ImmTAC was injected at a maximum concentration of 100-200 nM, followed by 2-fold dilutions for the other four injections. The lowest concentration was administered first. The buffer was then injected to measure the phase of dissociation until a dissociation of ≥ 10% was reached, usually after 1-3 hours. Kinetic parameters were calculated using BIAevaluation® software. The dissociation phase was approximated by a single exponential decay equation allowing calculation of the binding half-life. The equilibrium constant K _D was calculated from k _off //k _on .

Octet Method

Biotinylated peptide-HLA monomers were captured up to 1 nM on streptavidin-based biosensors (SA) (Pall ForteBio) previously immobilized with streptavidin. The sensors were blocked with free biotin (2 μM) for 2 minutes. Equilibrium binding constants were determined by immersing loaded biosensors in soluble TCR/ImmTAC serially diluted in a 96-well or 384-well sample plate. The shake of the tablet was set to 1000 rpm. For low coaffinity interactions (µM range), short association times (~2 minutes) and dissociation times (~2 minutes) were used. Binding curves were processed by double subtraction of reference biosensors loaded with foreign pHLA using Octet data analysis software (Pall ForteBio). Equilibrium responses (nm) were used to estimate the K _D value from steady state plots fitted to the equation Response = Rmax * conc/(K _D + conc) where "response" is the equilibrium binding in nm at each concentration of TCR (conc) , and Rmax is the maximum binding response at pHLA saturation.

For high affinity interactions (in the nM-pM range), kinetic parameters were determined from binding curves at ≥ 3 TCR/ImmTAC concentrations, typically 10 nM, 5 nM, and 2.5 nM. The association time was 30 minutes and the dissociation time was 1-2 hours. Binding curves were processed by double subtraction of reference biosensors loaded with foreign pHLA and blocked with biotin. Kinetic parameters k _on and k _off were calculated by global fitting directly to binding curves using Octet data analysis software (Pall ForteBio). K _D was calculated from k _off /k _on , and the half-dissociation period was calculated from t _1/2 = 0.693/k _off .

Example 4 Native TCR Binding Assay

Soluble native TCR was prepared according to the methods described in Example 1 and pHLA binding was analyzed according to Example 3. The amino acid sequences of the alpha and beta chains were as shown in FIG. 2. Soluble biotinylated HLA-A*02 was generated using the MAGE A4 GVYDGREHTV peptide and immobilized on a BIAcore sensor chip.

results

Binding was determined at various concentrations and the K _D value for the interaction was 142 μM. Cross-reactivity (specificity) was assessed against a panel of 15 HLA-A*02 foreign peptide complexes using the BIAcore equilibrium binding method from Example 3. Fifteen pHLA foreigners were pooled into three groups and loaded into one of three flow cells, yielding approximately 1000 RU each. pHLA per flow cell. 20 μl of wild-type soluble TCR at a concentration of 73 μM was injected into all flow cells at a rate of 20 μl/min. At both concentrations, no significant binding was found, indicating that the native TCR is specific to the GVYDGREHTV-HLA-A*02 complex.

The data obtained show that this TCR binds to the target with suitable affinity and specificity and therefore provides a useful starting sequence for the production of therapeutic TCRs.

Example 5 Binding Study of Soluble Mutated TCRs and ImmTAC Molecules of the Invention

Soluble mutated TCRs and ImmTAC molecules were generated based on the sequences shown in FIG. 2. Samples were prepared as described in Examples 1 and 2, and binding characteristics were determined in accordance with Example 3.

results

A single point mutation from cysteine to valine at position 19 of the alpha chain (SEQ ID NO: 6) was found to improve refolding and purification yield without affecting affinity or specificity (a K _D value of 145 μM was reported, and cross-reactivity with the same panel of 15 alternative HLA peptide complexes that were tested with WT were not observed).

TCR alpha and/or beta chains have been identified that contain mutations in at least one CDR region relative to the CDR sequences shown in FIG. 2 (SEQ ID NOs: 4 and 5). These TCR sequences recognized the GVYDGREHTV HLA-A*02 complex with the most appropriate affinity and/or binding half-life. In some cases, additional mutations have been identified that improve TCR stability and/or yield, including mutation of the K1A alpha chain (relative to the positions of SEQ ID NO: 4). The amino acid sequences of some of the mutated TCR alpha and beta chain variable regions of the present invention are shown in FIG. 4 and 5, respectively. The table below shows the binding characteristics for soluble TCRs or ImmTAC molecules (soluble anti-CD3 TCR fusion molecules) containing the indicated alpha and beta variable regions.

nd = undefined

^a Corresponding to ImmTAC3 from Example 6, the full alpha and beta chain sequences are shown in SEQ ID NO: 39 and SEQ ID NO: 44, respectively. Values are based on the average of 7 independent measurements

^b Corresponds to ImmTAC1 from Example 6, the full alpha and beta chain sequences are shown in SEQ ID NO: 38 and SEQ ID NO: 42, respectively. Values are based on the average of 7 independent measurements

^c Corresponds to ImmTAC2 from Example 6, full alpha and beta chain sequences are shown in SEQ ID NO: 38 and SEQ ID NO: 43, respectively

^d Corresponds to ImmTAC4 from Example 6, full alpha and beta chain sequences are shown in SEQ ID NO: 40 and SEQ ID NO: 45, respectively. Values are based on the average of 4 independent measurements

^e Corresponds to lmmTAC5 from Example 6, full alpha and beta chain sequences are shown in SEQ ID NO: 41 and SEQ ID NO: 45, respectively. Other combinations of alpha and beta variable regions were also tested for binding to the GVYDGREHTV HLA-A*02 complex. chains containing the mutations of the present invention. The data presented in the table below was obtained using the Biacore method as described above. These alpha and beta chain variable domain sequences were obtained as ImmTAC molecules.

nd - undefined

The data presented in Tables 9 and 10 show that some of the TCR variable sequences of the present invention have high binding affinity and long binding half-life for the GVYDGREHTV HLA-A*02 complex and are therefore particularly suitable for use as soluble therapeutic reagents.

In addition to binding the GVYDGREHTV HLA-A*02 cognate complex, the TCRs of the present invention were also evaluated for binding to similar peptides derived from MAGE A8 and MAGE B2 and presented by HLA-A*02. The table below shows Biacore binding data for three ImmTAC molecules containing the indicated alpha and beta variable domain sequences. All three ImmTAC molecules recognize the MAGE-A8 peptide at the same level as the cognate peptide and the MAGE-B2 peptide at a weaker level.

^a Corresponding to ImmTAC1 of Example 6, the full alpha and beta chain sequences are shown in SEQ ID NO: 38 and SEQ ID NO: 42, respectively.

^b Corresponds to ImmTAC3 from of Example 6, full alpha and beta chain sequences are shown in SEQ ID NO: 39 and SEQ ID NO: 44, respectively.

^c Corresponds to ImmTAC4 of Example 6, the full alpha and beta chain sequences are shown in SEQ ID NO: 40 and SEQ ID NO: 45, respectively.

Example 6 Efficient and Specific Targeting of T Cells by ImmTAC Molecules

ImmTAC molecules containing mutated alpha and beta chain variable region sequences with particularly high affinity for the target antigen were tested for their ability to mediate efficient and specific targeting of CD3+ T cells by an ELISPOT assay using interferon-γ (IFN- γ) as a recordable indicator of T-cell activation.

In this example, the alpha chain variable region sequence was selected from SEQ ID NOs: 20-24 and the beta chain variable region sequence was selected from SEQ ID NOs: 26-29. The variable domain sequences were hybridized to the corresponding alpha or beta extracellular constant domain sequences and contained a non-native disulfide bond. In each case, the beta chain was hybridized through a linker with anti-CD3 scFv; the linker was selected from SEQ ID NO: 30-37. The complete sequences of the tested ImmTAC molecules are presented in SEQ ID NOs, shown in the following table:

Methodology

Analyzes were performed using the human IFN-γ ELISPOT kit (BD Biosciences). Target cells were prepared at a density of 1×10 ⁶ /ml in assay medium (RPM 11640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine) and seeded at a density of 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donated blood were used as effector cells and seeded at a density of 10,000-50,000 cells per well in a volume of 50 μl (the exact number of cells used in each experiment depends on the donor and can be adjusted to obtain a response in the appropriate range for analysis). Various concentrations of ImmTAC were used, covering the expected clinically significant range, and added to the well in a volume of 50 μl.

The plates were pre-prepared according to the manufacturer's instructions. Target cells, effector cells, and ImmTAC molecules were added to appropriate wells and made up to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Also prepared control wells, excluding ImmTAC, effector cells or target cells. The plates were then incubated overnight (37°C/5% CO ₂ ). The next day, the plates were washed three times with wash buffer (1 sachet of phosphate-buffered saline containing 0.05% P20 diluted in deionized water). Then, the primary detection antibody was added to each well in a volume of 50 μl. The plates were incubated at room temperature for 2 hours and then washed again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin horseradish peroxidase conjugate to each well and incubating at room temperature for 1 hour, and repeating the washing step. Not more than 15 minutes prior to use, one drop (20 μl) of AEC chromogen was added to each 1 ml of AEC substrate and mixed, and 50 μl was added to each well. The development of the stains was monitored regularly and the plates were washed in tap water to stop the development reaction. The plates were then allowed to dry at room temperature for at least 2 hours before counting spots using a CTL analyzer with Immunospot software (Cellular Technology Limited).

results

The data presented in Figures 7 and 8, upper panels, show that ImmTAC molecules 1-2 and 3-5, respectively, are able to mediate efficient (i.e., with an EC ₅₀ less than 100 pM) redirection of T cells against cancer cells expressing the target antigen (NCl-H1703 - human lung cancer cell line). T cell activation against antigen-negative cancer cells (human papillary adenocarcinoma cell line NCl-H441 for ImmTAC molecules 1-2 and human breast cancer cell line CAMA-1 for ImmTAC molecules 3-5) within the clinically relevant concentration range of 1 nM ) was not found, demonstrating the specificity of the response.

ImmTAC molecules were tested for specificity using cells derived from normal healthy human tissues as target cells. The bottom panel in Fig. 7 shows that ImmTAC 1-2 molecules have minimal reactivity at a clinically relevant concentration against human skin vasculature cells. Likewise, the bottom panels in FIG. 8 show that 3-5 ImmTAC molecules have minimal reactivity at a clinically significant concentration against human skin vasculature and human kidney cells.

ImmTAC 1 and 3 molecules were further tested for specificity against a panel of human cells derived from normal healthy tissues using the same ELISPOT methodology described above. The data shown in Figure 9 shows limited T cell activation within the clinically relevant concentration range of 1 nM) for healthy tissues, including cells in the vasculature of the skin, heart, skeleton, liver, and lungs.

ImmTAC 1 and 4 molecules were further tested for reactivity against an expanded panel of >10 normal cell types using the same ELISPOT methodology described above and with finer ImmTAC concentration range steps (0.01 nM, 0.1 nM, 0 .2 nM, 0.3 nM, 0.5 nM and 1 nM). Figure 10 shows representative data from skeletal, cardiac and renal cells. In each case, antigen positive cells (NCl-H1703) and antigen negative cells (NCl-H441) were included as controls. The data obtained demonstrate negligible reactivity with normal cells relative to antigen-positive cells within the clinically relevant concentration range (< 1 nM).

These data show that these ImmTAC molecules exhibit a high level of activity and specificity and are therefore particularly suitable for therapeutic applications.

Example 7 Efficient killing of tumor cells by redirected ImmTAC T cells

The ability of the ImmTAC molecules of the present invention to mediate efficient redirected killing of antigen-positive tumor cells by T cells was examined using the IncuCyte platform (Essen Bioscience). This assay allows real-time microscopic detection of the release of caspase-3/7, a marker of apoptosis.

Methodology

Analyzes were performed using the Apoptosis Assay Kit with Caspase-3/7 reagent and a 96-well CellPlayer plate (Essen Bioscience, Cat. No. 4440) and performed according to the manufacturer's protocol. Briefly, target cells (NCl-H1703 antigen positive HLA A*02 positive and NCl-H441 antigen negative HLA A*02 positive) were plated at 5000 cells per well and incubated overnight to give them attach. ImmTAC solutions were prepared at concentrations from 0.5 nM to 0.01 nM, and 25 μl of each concentration was added to the appropriate well. Effector cells were used at a ratio of effector:target cells of 10:1 (50,000 cells per well). A control sample without ImmTAC was also prepared. The NucView assay reagent was prepared at a concentration of 30 μM and 25 μl was added to each well and the final volume was adjusted to 150 μl (resulting in a final concentration of 5 μM). The plate was placed in the IncuCyte instrument and images were recorded every 2 hours (1 image per well) for 3 days. The number of apoptotic cells in each image was then determined and expressed as the number of apoptotic cells per mm ² . Analyzes were performed in triplicate.

results

The data presented in Figure 11 shows the killing of tumor cells in real time by T cells redirected by ImmTAC molecules. The results are presented for ImmTAC1 and ImmTAC4. Both ImmTAC molecules demonstrate redirected T-cell killing of antigen-positive tumor cells at a concentration of 0.01 nM. Destruction of antigen-negative cells is not observed even at the highest concentration (0.5 nm).

These data confirm that ImmTAC1 and immTAC4 mediate efficient redirected killing of antigen-positive tumor cells by T cells.

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SEQUENCE LIST

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<213> Homo sapiens

<400> 10

Lys Ser Asn Gly Arg Tyr Thr Ala Thr Leu Asp Ala Asp Thr Lys Gln

1 5 10 15

Ser Ser Leu His Ile Thr Ala Ser Gln Leu Ser Asp Ser Ala Ser Tyr

20 25 30

Ile Cys

<210> 11

<211> 5

<212> PROTEIN

<213> Homo sapiens

<400> 11

Met Asp His Glu Asn

fifteen

<210> 12

<211> 6

<212> PROTEIN

<213> Homo sapiens

<400> 12

Ser Tyr Asp Val Lys Met

fifteen

<210> 13

<211> 16

<212> PROTEIN

<213> Homo sapiens

<400> 13

Ala Ser Ser Phe Leu Met Thr Ser Gly Asp Pro Tyr Glu Gln Tyr Phe

1 5 10 15

<210> 14

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 14

Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr

1 5 10 15

Phe

<210> 15

<211> 37

<212> PROTEIN

<213> Homo sapiens

<400> 15

Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr Ser Val Ser Arg Glu Lys

1 5 10 15

Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser Ala Ser Thr Asn Gln Thr

20 25 30

Ser Met Tyr Leu Cys

35

<210> 16

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (c19v)

<400> 16

Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ser Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 17

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a7)

<400> 17

Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ser Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 18

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a12)

<400> 18

Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 19

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a13)

<400> 19

Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Asn

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 20

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a13ka)

<400> 20

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Asn

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 21

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a19)

<400> 21

Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 22

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a19ka)

<400> 22

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 23

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a13kaLS)

<400> 23

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Ser

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 24

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a13kaLQ)

<400> 24

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 25

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b1)

<400> 25

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 26

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b14)

<400> 26

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 27

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b14L)

<400> 27

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Leu Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 28

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b21)

<400> 28

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 29

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b21L)

<400> 29

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 30

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 30

Gly Gly Gly Gly Ser

fifteen

<210> 31

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 31

Gly Gly Gly Ser Gly

fifteen

<210> 32

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 32

Gly Gly Ser Gly Gly

fifteen

<210> 33

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 33

Gly Ser Gly Gly Gly

fifteen

<210> 34

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 34

Gly Ser Gly Gly Gly Pro

fifteen

<210> 35

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 35

Gly Gly Glu Pro Ser

fifteen

<210> 36

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 36

Gly Gly Glu Gly Gly Gly Pro

fifteen

<210> 37

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 37

Gly Gly Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

1 5 10

<210> 38

<211> 199

<212> PROTEIN

<213> Artificial sequence

<220>

<223> alpha chain (a19ka)

<400> 38

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro Tyr Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr

195

<210> 39

<211> 199

<212> PROTEIN

<213> Artificial sequence

<220>

<223> alpha chain (a13ka)

<400> 39

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Asn

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro Tyr Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr

195

<210> 40

<211> 199

<212> PROTEIN

<213> Artificial sequence

<220>

<223> alpha chain (a13kaLQ)

<400> 40

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro Tyr Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr

195

<210> 41

<211> 199

<212> PROTEIN

<213> Artificial sequence

<220>

<223> alpha chain (a13kaLS)

<400> 41

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Ser

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro Tyr Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr

195

<210> 42

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> beta chain (b14)

<400> 42

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

145 150 155 160

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn

180 185 190

Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

195 200 205

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg

260 265 270

Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser

275 280 285

Lys Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile

290 295 300

Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu

305 310 315 320

Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu

325 330 335

Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser

340 345 350

Ser Asp Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly

355 360 365

Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

370 375 380

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

385 390 395 400

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

405 410 415

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

420 425 430

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

435 440 445

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro

450 455 460

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

465 470 475 480

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

485 490 495

Ala Glu Ala Trp Gly Arg Ala Asp

500

<210> 43

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> beta chain (b14L)

<400> 43

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

145 150 155 160

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn

180 185 190

Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

195 200 205

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg

260 265 270

Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser

275 280 285

Lys Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile

290 295 300

Tyr Phe Ser Tyr Asp Val Lys Leu Lys Glu Lys Gly Asp Ile Pro Glu

305 310 315 320

Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu

325 330 335

Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser

340 345 350

Ser Asp Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly

355 360 365

Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

370 375 380

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

385 390 395 400

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

405 410 415

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

420 425 430

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

435 440 445

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro

450 455 460

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

465 470 475 480

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

485 490 495

Ala Glu Ala Trp Gly Arg Ala Asp

500

<210> 44

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> beta chain (b21)

<400> 44

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

145 150 155 160

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn

180 185 190

Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

195 200 205

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg

260 265 270

Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu

275 280 285

Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile

290 295 300

Tyr Phe Ser Arg Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu

305 310 315 320

Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu

325 330 335

Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser

340 345 350

Ser Asp Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly

355 360 365

Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

370 375 380

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

385 390 395 400

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

405 410 415

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

420 425 430

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

435 440 445

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro

450 455 460

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

465 470 475 480

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

485 490 495

Ala Glu Ala Trp Gly Arg Ala Asp

500

<210> 45

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> beta chain (b21L)

<400> 45

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

130 135 140

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe

145 150 155 160

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn

180 185 190

Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn

195 200 205

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg

260 265 270

Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu

275 280 285

Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile

290 295 300

Tyr Phe Ser Arg Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu

305 310 315 320

Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu

325 330 335

Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser

340 345 350

Ser Asp Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly

355 360 365

Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

370 375 380

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

385 390 395 400

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

405 410 415

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

420 425 430

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

435 440 445

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro

450 455 460

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

465 470 475 480

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

485 490 495

Ala Glu Ala Trp Gly Arg Ala Asp

500

<210> 46

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a36)

<400> 46

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 47

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a37)

<400> 47

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Tyr Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 48

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a38)

<400> 48

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ala Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 49

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (a39)

<400> 49

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ser Gln

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 50

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a40)

<400> 50

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gly

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 51

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a41)

<400> 51

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Ser Ala Gln

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 52

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a30)

<400> 52

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 53

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a42)

<400> 53

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 54

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a31)

<400> 54

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Phe Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 55

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a43)

<400> 55

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ser Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 56

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a32)

<400> 56

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 57

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a44)

<400> 57

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ala Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 58

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a33)

<400> 58

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ser Asp

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 59

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a45)

<400> 59

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Gly

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 60

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain(a34)

<400> 60

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Asp Tyr Ala Ile Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn Arg Ala Asp

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 61

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (aWTka)

<400> 61

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ser Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 62

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (aM50L)

<400> 62

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Leu Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ser Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 63

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (aS95A)

<400> 63

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ala Gly

85 90 95

Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 64

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated alpha chain (aS98L)

<400> 64

Ala Asn Gln Val Glu Gln Ser Pro Gln Ser Leu Ile Ile Leu Glu Gly

1 5 10 15

Lys Asn Val Thr Leu Gln Cys Asn Tyr Thr Val Ser Pro Phe Ser Asn

20 25 30

Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly Pro Val Ser Leu Thr

35 40 45

Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr Ala

50 55 60

Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala Ser

65 70 75 80

Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Asn His Ser Gly

85 90 95

Gly Leu Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His

100 105 110

Pro

<210> 65

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b41)

<400> 65

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 66

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b42)

<400> 66

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Phe Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 67

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b43)

<400> 67

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Asp Ala Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 68

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b44)

<400> 68

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Val Thr Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 69

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b45)

<400> 69

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Ala Lys Gly Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 70

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b46)

<400> 70

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Leu Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Arg Phe Ala Thr Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 71

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b32)

<400> 71

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 72

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b33)

<400> 72

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Asp Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 73

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b34)

<400> 73

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro His Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 74

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b35)

<400> 74

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Glu Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 75

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b36)

<400> 75

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 76

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b37)

<400> 76

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Phe Asp

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 77

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b38)

<400> 77

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Leu

85 90 95

Gln Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 78

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b39)

<400> 78

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Met Asn Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 79

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (b40)

<400> 79

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Ala Pro Leu Ser Lys Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Ser Asp

85 90 95

Gln Thr Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 80

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (bL96D)

<400> 80

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Phe Asp

85 90 95

Met Thr Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<210> 81

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mutated beta chain (bM97Q)

<400> 81

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Phe Leu

85 90 95

Gln Thr Ser Gly Asp Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Thr

115

<---

Claims (136)

1. T-cell receptor (TCR), which has the ability to bind to the HLA-A*02 complex with the MAGE A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) and contains a heterodimer of alpha and beta chains, where the specified heterodimer includes an alpha chain containing the variable domain of the TCR alpha chain, and the beta chain containing the variable domain of the TCR beta chain, wherein: (a) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 17; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (b) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 25; (c) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (d) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 20; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (e) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (f) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 27; (g) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (h) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 23; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (i) the TCR alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 46; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (j) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 47; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (k) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 48; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (l) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 49; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (m) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 50; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (n) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 51; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (o) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 20; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 65; (p) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 66; (q) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 67; (r) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 68; (s) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 69; (t) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 70; (u) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 52; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (v) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 53; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (w) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 54; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (x) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 55; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (y) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 56; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (z) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 57; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (aa) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 58; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (bb) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 59; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (cc) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 60; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (dd) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 71; (ee) the TCR alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 72; (ff) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 73; (gg) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 74; (hh) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 75; (ii) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 76; (jj) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 77; (kk) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the TCR beta chain variable domain contains the amino acid sequence of SEQ ID NO: 78; or (ll) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the TCR beta chain variable domain comprises the amino acid sequence of SEQ ID NO: 79. 2. TCR according to claim 1, characterized in that said TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24, and said TCR beta chain variable domain contains the amino acid sequence of SEQ ID NO: 29. 3. TCR according to claim 1, characterized in that said TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22, and said TCR beta chain variable domain contains the amino acid sequence of SEQ ID NO: 26. 4. A TCR according to any one of the preceding claims, which is an alpha-beta heterodimer having a TRAC alpha chain constant domain sequence and a TRBC1 or TRBC2 beta chain constant domain sequence. 5. TCR according to claim 4, characterized in that the alpha and beta chain constant domain sequences are modified by truncation or substitution to remove the native disulfide bond between TRAC exon 2 Cys4 and TRBC1 or TRBC2 exon 2 Cys2. 6. TCR according to claim 4 or 5, characterized in that the sequence(s) of the constant domain of the alpha and/or beta chain is modified(s) by replacing the residues Thr 48 TRAC and Ser 57 TRBC1 or TRBC2 with cysteine, and these cysteines form a non-native disulfide bond between the alpha and beta chain constant domains of the TCR. 7. A hybrid TCR molecule for targeting an agent to cells that present the MAGE A4 antigen, or to tissue that contains cells that present the MAGE A4 antigen, comprising a TCR according to any of the preceding claims that is covalently linked to said agent. 8. A TCR hybrid molecule according to claim 7, characterized in that said agent is a detectable label, therapeutic agent, or pharmacokinetic (PK) modifying group, wherein said detectable label is preferably selected from a fluorescent label, a radioactive label, an enzyme, a nucleic acid probe, or a contrast agent; wherein said therapeutic agent is preferably selected from a low molecular weight cytotoxic agent, a peptide cytotoxin, a radionuclide, a cytokine, a superantigen or a mutant form thereof, a peptide-HLA complex, a chemokine, an antibody or an antigen-binding fragment thereof, a protein scaffold, a complement activator, or a viral or bacterial protein domain, or peptide; and/or wherein said FA-modifying group is preferably selected from PEG, PAS or albumin. 9. A hybrid TCR molecule according to claim 8, characterized in that said agent is an anti-CD3 antibody covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR. 10. A hybrid TCR molecule according to claim 9, characterized in that said agent is an anti-CD3 antibody covalently linked to the C- or N-terminus of the TCR beta chain via a linker sequence. 11. The hybrid TCR molecule according to claim 10, characterized in that said linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 30), GGGSG (SEQ ID NO: 31), GGSGG (SEQ ID NO: 32), GSGGG (SEQ ID NO: 33), GSGGGP (SEQ ID NO: 34), GGEPS (SEQ ID NO: 35), GGEGGGP (SEQ ID NO: 36) and GGEGGGSEGGGS (SEQ ID NO: 37). 12. Hybrid TCR molecule according to any one of paragraphs. 9-11, characterized in that said anti-CD3 antibody is an antibody in the format of a single-chain variable fragment (scFv). 13. A TCR-anti-CD3 fusion molecule containing a TCR having the property of binding to the HLA-A*02 complex with the MAGE-A4 cancer antigen GVYDGREHTV (SEQ ID NO: 1) and an anti-CD3 antibody for redirecting T cells to cells, presenting the MAGE-A4 antigen, or a tissue that contains cells that present the MAGE A4 antigen, where the specified TCR contains a heterodimer of alpha and beta chains, including an alpha chain that contains the variable domain of the TCR alpha chain, and a beta chain, which contains the TCR beta chain variable domain, wherein said anti-CD3 antibody is covalently linked to the N-terminus or C-terminus of the TCR beta chain, and wherein (a) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 17; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (b) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 25; (c) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (d) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 20; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 28; (e) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (f) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 27; (g) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (h) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 23; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (i) the TCR alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 46; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (j) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 47; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (k) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 48; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (l) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 49; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (m) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 50; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (n) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 51; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 29; (o) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 20; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 65; (p) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 66; (q) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 67; (r) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 68; (s) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 69; (t) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 24; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 70; (u) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 52; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (v) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 53; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (w) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 54; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (x) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 55; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (y) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 56; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (z) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 57; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (aa) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 58; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (bb) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 59; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (cc) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 60; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 26; (dd) the variable domain of the TCR alpha chain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 71; (ee) the TCR alpha chain variable domain comprises the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 72; (ff) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 73; (gg) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 74; (hh) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 75; (ii) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 76; (jj) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 77; (kk) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the TCR beta chain variable domain contains the amino acid sequence of SEQ ID NO: 78; or (II) the TCR alpha chain variable domain contains the amino acid sequence of SEQ ID NO: 22; and the variable domain of the TCR beta chain contains the amino acid sequence of SEQ ID NO: 79; wherein said anti-CD3 antibody is covalently linked to the N-terminus or C-terminus of the TCR beta chain via a linker sequence selected from SEQ ID NOs: 30-37. 14. TCR-anti-CD3 hybrid molecule according to claim 13, containing (a) the alpha chain amino acid sequence corresponding to SEQ ID NO: 38 and the beta chain amino acid sequence corresponding to SEQ ID NO: 42; (b) an alpha chain amino acid sequence corresponding to SEQ ID NO: 38 and a beta chain amino acid sequence corresponding to SEQ ID NO: 43; (c) the alpha chain amino acid sequence corresponding to SEQ ID NO: 39 and the beta chain amino acid sequence corresponding to SEQ ID NO: 44; (d) the alpha chain amino acid sequence corresponding to SEQ ID NO: 40 and the beta chain amino acid sequence corresponding to SEQ ID NO: 45; or (e) the alpha chain amino acid sequence corresponding to SEQ ID NO: 41 and the beta chain amino acid sequence corresponding to SEQ ID NO: 45. 15. The TCR-anti-CD3 hybrid molecule according to claim 14, comprising the alpha chain amino acid sequence corresponding to SEQ ID NO: 40 and the beta chain amino acid sequence corresponding to SEQ ID NO: 45. 16. The TCR-anti-CD3 hybrid molecule according to claim 14, comprising the alpha chain amino acid sequence corresponding to SEQ ID NO: 38 and the beta chain amino acid sequence corresponding to SEQ ID NO: 42. 17. Nucleic acid encoding a TCR that has the ability to bind to the HLA-A*02 complex with the MAGE A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) and contains a heterodimer of alpha and beta chains, where the specified nucleic acid encodes an alpha chain TCR and TCR beta chain according to any one of paragraphs. 1-6. 18. A nucleic acid encoding a TCR fusion molecule for delivering an agent to cells that present the MAGE A4 cancer antigen or to tissue that contains cells that present the MAGE A4 antigen, wherein said nucleic acid encodes a TCR fusion molecule as claimed in any from paragraphs. 7-12, wherein said agent is a peptide or polypeptide. 19. A nucleic acid encoding a TCR-anti-CD3 hybrid molecule comprising a TCR having the property of binding to the HLA-A*02 complex with the MAGE-A4 cancer antigen GVYDGREHTV (SEQ ID NO: 1) and an anti-CD3 antibody, wherein said nucleic acid the acid encodes the alpha chain and the beta chain of the TCR-anti-CD3 hybrid molecule as claimed in any one of claims. 13-16, wherein said nucleic acid further encodes an anti-CD3 antibody covalently linked to the N-terminus or C-terminus of the TCR beta chain via a linker sequence selected from SEQ ID NOs: 30-37. 20. An expression vector encoding a TCR that has the ability to bind to the HLA-A*02 complex with the MAGE A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) and contains a heterodimer of alpha and beta chains, where the expression vector contains the nucleic acid according to p .17. 21. An expression vector according to claim 20, characterized in that said vector contains a first open reading frame encoding the TCR alpha chain and a second open reading frame encoding the TCR beta chain. 22. An expression vector encoding a hybrid TCR molecule for delivering an agent to cells that present the MAGE A4 cancer antigen or to tissue that contains cells that present the MAGE A4 cancer antigen, said expression vector containing the nucleic acid according to claim 18. 23. The expression vector of claim 22, wherein said vector contains a first open reading frame encoding the TCR alpha chain and a second open reading frame encoding the TCR beta chain. 24. An expression vector encoding a TCR-anti-CD3 fusion molecule comprising a TCR capable of binding to the HLA-A*02 complex with the MAGE-A4 cancer antigen GVYDGREHTV (SEQ ID NO: 1) and an anti-CD3 antibody, wherein said vector expression contains a nucleic acid according to claim 19. 25. The expression vector of claim 24, wherein said vector contains a first open reading frame encoding the TCR alpha chain and a second open reading frame encoding the TCR beta chain. 26. Cell for the expression of TCR, which has the ability to bind to the HLA-A*02 complex with the cancer antigen MAGE A4 GVYDGREHTV (SEQ ID NO: 1) and contains a heterodimer of alpha and beta chains, carrying (a) an expression vector according to claim 20 containing one open reading frame or an expression vector according to claim 21 containing two different open reading frames; or (b) a first expression vector that contains a nucleic acid encoding a TCR alpha chain according to any one of paragraphs. 1-6, and a second expression vector that contains a nucleic acid encoding a TCR beta chain according to any one of paragraphs. 1-6. 27. A cell for expressing a hybrid TCR molecule for delivering an agent to cells that present the MAGE A4 antigen, or to tissue that contains cells that present the MAGE A4 antigen, carrying (a) an expression vector according to claim 22 containing one open reading frame or an expression vector according to claim 23 containing two different open reading frames; or (b) a first expression vector that contains a nucleic acid encoding the alpha chain of the hybrid TCR molecule as claimed in any one of paragraphs. 9-12, and a second expression vector that contains a nucleic acid encoding the beta chain of the hybrid TCR molecule, as stated in any one of paragraphs. 9-12. 28. A cell for expressing a TCR-anti-CD3 fusion molecule containing a TCR having the ability to bind to the HLA-A*02 complex with the MAGE-A4 cancer antigen GVYDGREHTV (SEQ ID NO: 1) and an anti-CD3 antibody carrying (a) an expression vector according to claim 24 containing one open reading frame or an expression vector according to claim 25 containing two different open reading frames; or (b) a first expression vector that contains a nucleic acid encoding the alpha chain of a TCR-anti-CD3 fusion molecule as claimed in any one of paragraphs. 13-16, and a second expression vector that contains a nucleic acid encoding the beta chain of a TCR-anti-CD3 hybrid molecule as claimed in any one of paragraphs. 13-16. 29. Pharmaceutical composition for the diagnosis or treatment of cancer or tumors, expressing the complex HLA-A*02 c cancer antigen MAGE A4 GVYDGREHTV (SEQ ID NO: 1), containing an effective amount of TCR according to any one of paragraphs. 1-6, a hybrid TCR molecule according to any one of paragraphs. 7-12 or a TCR-anti-CD3 hybrid molecule according to any one of paragraphs. 13-16 together with one or more pharmaceutically acceptable carriers or excipients. 30. TCR according to any one of paragraphs. 1-6 or a pharmaceutical composition according to claim 29, which contains a TCR, for use in medicine, preferably in a human subject. 31. Hybrid TCR molecule according to any one of paragraphs. 7-12 or a pharmaceutical composition according to claim 29, which contains a hybrid TCR molecule, for use in medicine, preferably in a human subject. 32. Hybrid molecule TCR-anti-CD3 according to any one of paragraphs. 13-16 or a pharmaceutical composition according to claim 29, which contains a TCR-anti-CD3 hybrid molecule for use in medicine, preferably in a human subject. 33. TCR according to any one of paragraphs. 1-6 or a pharmaceutical composition according to claim 29, which contains a TCR, for use in a method of treating cancer or tumor, preferably in a human subject. 34. Hybrid TCR molecule according to any one of paragraphs. 7-12 or a pharmaceutical composition according to claim 29, which contains a hybrid TCR molecule, for use in a method of treating cancer or tumor, preferably in a human subject. 35. Hybrid molecule TCR-anti-CD3 according to any one of paragraphs. 13-16 or a pharmaceutical composition according to claim 29 which contains a TCR-anti-CD3 fusion molecule for use in a method of treating cancer or tumor, preferably in a human subject. 36. A TCR-anti-CD3 fusion molecule or pharmaceutical composition for use according to claim 35, wherein said human subject has a tumor expressing MAGE A4. 37. Hybrid molecule TCR-anti-CD3 or pharmaceutical composition for use according to paragraphs. 35,36, said tumor being a solid tumor. 38. Hybrid molecule TCR-anti-CD3 or pharmaceutical composition for use according to any one of paragraphs. 35-37, wherein said human subject belongs to the HLA-A*02 subtype. 39. Hybrid molecule TCR-anti-CD3 or pharmaceutical composition for use according to any one of paragraphs. 35-38, which are administered by injection, such as intravenous injection or injection directly into the tumor. 40. A method of treating a human subject in need, comprising administering to said subject a pharmaceutically effective dose of the pharmaceutical composition of claim 29, wherein said human subject has a tumor that expresses MAGE A4. 41. A method of treating a human subject in need, comprising administering to said subject a pharmaceutically effective dose of the pharmaceutical composition of claim 29 and an antineoplastic agent, wherein said human subject has a tumor that expresses MAGE A4, wherein said the composition and agent are administered singly, in combination, or sequentially. 42. Composition for diagnosing or treating cancer or tumor expressing the HLA-A*02 complex with the MAGE A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) for injection into a human subject, containing an effective amount of TCR according to any one of paragraphs . 1-6, a hybrid TCR molecule according to any one of paragraphs. 7-12 or a TCR-anti-CD3 hybrid molecule according to any one of paragraphs. 13-16. 43. A method for producing a TCR that has the ability to bind to the HLA-A * 02 complex with the MAGE A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) and contains a heterodimer of alpha and beta chains, including: a) maintaining the cell according to item 26 in optimal conditions for the expression of TCR chains, and b) isolating said TCR chains. 44. A method for producing a hybrid TCR molecule for delivering an agent to cells that present the MAGE A4 antigen, or to a tissue that contains cells that present the MAGE A4 antigen, including: a) maintaining the cell according to claim 27 under optimal conditions for the expression of the specified hybrid TCR molecules, and b) isolating said TCR hybrid molecule. 45. A method for producing a TCR-anti-CD3 hybrid molecule containing a TCR having the property of binding to the HLA-A*02 complex with the MAGE-A4 GVYDGREHTV cancer antigen (SEQ ID NO: 1) and an anti-CD3 antibody, comprising: a) maintaining cells according to claim 28 under optimal conditions for expression of said TCR-anti-CD3 fusion molecule, and b) isolating said TCR-anti-CD3 fusion molecule.

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