RU2775394C2 - T-cell receptors specific relatively to complex of tumor antigen ny-eso-1/hla-a*02 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: T-cell receptor (hereinafter – TCL) is proposed that binds to SLLMWITQC peptide restricted by HLA-A*02, originating from cancer antigen NY-ESO-1. Fused protein TCR/anti-CD3 is also proposed, containing the specified TCR and an antibody against CD3. In addition, the invention relates to nucleic acid and an expression vector for the production of TCR or fused protein TCR/anti-CD3, to cells expressing TCR or fused protein.

EFFECT: fused proteins and cells, according to the present invention, are suitable for use as new immune-therapeutic reagents for the treatment of malignant disease.

44 cl, 12 dwg, 12 tbl, 8 ex

Description

The present invention provides T cell receptors (TCRs) that bind to the HLA-A*02 restricted peptide SLLMWITQC derived from the cancer antigen NY-ESO-1. These TCRs may contain mutations within the variable domains of their alpha and/or beta chains compared to native TCR specific for NY-ESO-1. 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 (TCRs) are naturally expressed by CD4 ⁺ and CD8 ⁺ T cells. TCRs are designed to recognize short peptide antigens presented on the surface of antigen-presenting cells in complex with molecules of the major histocompatibility complex - MHC (in humans, MHC molecules are also known as human leukocyte antigens, or HLA, Davis, et al., (1998), Annu Rev Immunol 16: 523-544.). CD8 ⁺ T cells, also referred to as cytotoxic T cells, specifically recognize class I MHC-associated peptides and are generally responsible for detecting and assisting in the destruction of infected or cancerous cells.

The NY-ESO-1 antigen belongs to the family of cancer antigens encoded by germline cells (Chen, et al., (1997), Cytogenet Cell Genet 79(3-4): 237-240; international application publication WO 9814464) and has the accession number in the Uniprot P78358 database. It has been found that such fetal antigens (germline antigens) are often expressed in various types of cancer, while their expression in normal tissues is limited to the testicles of adult males and other immunologically privileged areas. The cancer specificity of these antigens makes them ideal targets for anticancer therapeutics. The exact function of NY-ESO-1 remains unknown, but its expression has been found in testes of embryonic and adult males (Satie, et al., (2002), Lab Invest 82(6): 775-780), ovarian and uterine myometrium, and also in a wide variety of cancers, including myeloma (Andrade, et al., (2008), Cancer Immun 8: 2), ovarian cancer (Odunsi, et al., (2003), Cancer Res 63(18): 6076- 6083), non-small cell lung cancer (Konishi, et al., (2004), Oncol Rep 11(5): 1063-1067), and melanoma (Barrow, et al., (2006), Clin Cancer Res 12(3 Pt 1) : 764-771). The 9-mer SLLMWITQC peptide (SEQ ID NO 1) corresponds to amino acids 157-165 of the full length NY-ESO-1 protein. The same peptide is also found in LAGE-1A (accession number 075638-2) another cancer antigen (Lethe, et al., (1998) Int J Cancer 76(6): 903-908). This peptide binds to HLA-A*02, and the peptide-HLA complex is able to stimulate cytotoxic T cells, leading to the lysis of NY-ESO-1 ⁺ HLA-A*02 ⁺ tumor cells (Duffour, et al., (1999), Eur J Immunol 29(10): 3329-3337 and International Application Publication WO2000020445). The SLLMWITQC/HLA-A*02 complex is thus a target antigen suitable for immunotherapeutic intervention.

The identification of specific TCR sequences that bind to the SLLMWITQC/HLA-A*02 complex holds promise for the development of new immunotherapeutic agents. Such therapeutic TCRs can be used, for example, as soluble targeting agents to deliver cytotoxic or immune effector agents to tumors (Lissin, et al., (2013). "High-Affmity 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 Immunother 63(9): 969-975). for immunotherapeutic use are capable of actively recognizing the target antigen, which means that said TCR must have high affinity and/or a long binding half-life to the target antigen in order to induce a strong response. against the target antigen (low micromolar concentration range), thus it is often necessary to identify mutations, including but not limited to substitutions, insertions and/or deletions, that can be introduced into a given TCR sequence to improve antigen binding. For use as soluble targeting agents, a TCR binding affinity for antigen in the nanomolar-picomolar range and a binding half-life of several hours is preferred. It is also desirable that therapeutic TCRs exhibit a high level of specificity for the target antigen in order to reduce the risk of toxicity resulting from off-target binding in clinical use. Achieving such high specificity can be particularly challenging given the natural 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). Finally, it is desirable that these therapeutic TCRs be able to be expressed and purified in a highly stable form.

The TCR sequences defined in this application are described in accordance with the IMGT nomenclature, which is widely known and available to those skilled in the art of TCR. See, for example: 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 1O; Lefranc, (2003), Leukemia 17(1): 260-266. **-TCRs consist of two chains linked by disulfide bonds. In general, each chain (alpha and beta) is considered to contain two domains, namely a variable and a constant domain. The short binding region connects the variable and constant domains and is generally considered to be part of the variable region. In addition, the beta chain usually contains a short region of variability between the variable and connecting region.

The variable domain of each strand is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in a framework sequence. The CDRs contain a peptide-MHC binding recognition site.

Several genes encode alpha chain variable regions (Va) and several genes encode beta chain variable regions (VP). These genes differ in their CDR1 and CDR2 framework sequences and in a partially defined CDR3 sequence. The Va and VP genes are designated by IMGT nomenclature with 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). Similarly, there are several junction or J genes called 'TRAJ' or 'TRBJ' for the alpha and beta chains respectively, and a variability or D gene for the beta chain 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 tremendous diversity in alpha and beta chain variable region sequences is the result of combinatorial rearrangements of various V, J, and D genes that include allelic variants and additional diversity in J segments (Arstila, et al., (1999), Science 286(5441) : 958-961; Robins et al., (2009), Blood 114(19): 4099-4107. (2001), Curr Protoc Immunol Appendix 1: Appendix 1O).

In the present application and claims, the term "variable domain of the TCR alpha chain (or a chain)" refers to the connected regions of TRAV and TRAJ; only the TRAV region or the TRAV region and part of the TRAJ region, and the term "constant domain of the TCR alpha chain (or a chain)" refers to the extracellular TRAC region or to the C-terminal truncated or full-length TRAC sequence. Similarly, the term "variable domain of the TCR beta chain (or P chain)" may refer to the connected regions of TRBV and TRBD/TRBJ; only to TRBV and TRBD sections; only to the TRBV and TRBJ regions, or to the TRBV region and partially to the TRBD and/or TRBJ regions, and the term "TCR beta-chain (or p-chain) constant domain" refers to the extracellular region of TRBC or the C-terminal truncated or full-length TRBC sequence.

TCRs that target NY-ESO-1 have been previously described (International Publications WO05113595 and WO08039818; source McCormack, et al., (2013), Cancer Immunol Immunother 62(4): 773-785).

The present inventors have identified alternative TCR alpha and beta variable domain sequences that bind to the NY-ESO-1/HLA-A*02 complex. Such sequences are particularly suitable for use as therapeutic TCRs for targeted immunotherapy of cancer types in which the SLLMWITQC/HLA-A*02 complex is presented.

The inventors have identified a native TCR comprising the use

the following circuits:

Alpha chain: TRAV3*01/TRAJ28*01

Beta chain: TRBV29-1*01 /TRBD2*01 /TRBJ2-3*01

(Note: the designation '*01' refers to the allelic variant for the specified sequence, as designated in accordance with the IMGT nomenclature) The specified native TCR was used as a template, on the basis of which the mutant sequences according to the present invention were obtained.

Brief description of the invention

The present invention according to a first aspect provides a T cell receptor (TCR) having the property of binding to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex and comprising a TCR alpha chain variable domain and/or a TCR beta chain variable domain , where

said alpha chain variable domain contains an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 1-117 of SEQ ID NO: 2, and/or

said beta chain variable domain contains an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 1-115 of SEQ ID NO: 3.

According to a second aspect, the invention provides a TCR that binds to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex with an affinity greater than 50 μM, where: alpha chain CDRs 1, 2 and 3 contain the sequences of SEQ ID NO: 41 , 42 and 43, respectively, and/or the CDRs of beta chain 1, 2, and 3 comprise the sequences of SEQ ID NOs: 44, 45, and 46, respectively, and/or at least one of the CDRs contains one or more conservative substitutions with respect to the sequences SEQ ID NO: 41-46; and/or at least one of the CDRs contains up to three valid substitutions with respect to the sequences of SEQ ID NOs: 41-46.

The alpha chain variable domain according to the first or second aspect may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO: 2):

and/or the beta chain variable domain according to the first or second aspect may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO: 3):

The alpha chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO:2):

and/or the beta chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO:3):

The variable domain of the alpha chain may contain from 2 to 6 of the following mutations (according to the sequence numbering of SEQ ID NO: 2):

and/or the beta chain variable domain may contain from 3 to 9 of the following mutations (according to SEQ ID NO: 3):

The alpha chain variable domain may comprise at least one of

the following groups of mutations:

Group 1: I51L, T52G, G53D, D54S, N55A

Group 2: I51L, T52G, G53D, D54S, N55A, N97D

Group 3: I51L, T52G, G53D, D54S, N55A, N97R

Group 4: I96S, N97D, S98Q, G99H

Group 5: D95S, I96S, N97R, S98Q, G99H

Group 6: I51L, G53D,

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

Group 1: N50W, Q51T, S53G

Group 2: S27K, Q28R, V29L, T30A, M31L, N50W, Q51T, S53G

Group 3: S27K, Q28R, V29L, T30A, M31L, N50W, Q51T, S53G, G100A

Group 4: S27K, Q28R, V29L, T30A, N50W, Q51T

Group 5: S27K, Q28R, V29L, T30A, N50W, Q51T S53G

Group 6: S27K, Q28R, V29L, T30A, N50W, Q51T S53G, G100A

Group 7: S27K, Q28R, V29L, T30A, M31L, N50W, Q51T

Group 8: T30A, N50W, G100A.

The alpha chain variable domain and the beta chain variable domain may contain the following groups of mutations, respectively:

The TCR according to the present invention may contain an alpha chain variable domain that contains the following mutation (according to the sequence numbering of SEQ ID NO: 2):

and/or the beta chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO: 3):

In the variable domain of the alpha chain, the sequence of amino acid residues 27-32, 50-57 and 91-107 is selected from the following sequences:

The TCR alpha chain variable domain may comprise an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the sequence of SEQ ID NO: 12.

In the variable domain of the beta chain, the sequence of amino acid residues 27-31, 49-55 and 93-106 is selected from the following sequences:

The TCR beta chain variable domain may comprise an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the sequence of SEQ ID NO: 15.

The alpha chain variable domain may comprise the sequence of amino acid residues 27-32, 50-57 and 91-107, and the beta chain variable domain may comprise the sequence of amino acid residues 27-31, 49-55 and 93-106 selected from the following sequences:

The alpha chain variable domain may comprise an amino acid sequence that is at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% is identical to the sequence of SEQ ID NO: 12, and the beta chain variable domain may contain an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 15.

The alpha chain variable domain may be selected from the amino acid sequence of SEQ ID NO: 6-12 or 51, and the beta chain variable domain may be selected from the amino acid sequence of SEQ ID NO: 13-23 or 52.

The alpha chain variable domain and the beta chain variable domain can be selected from the following amino acid sequences:

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

Alpha and beta chain constant domain sequences can be modified by truncation or replacement to remove the native disulfide bond between TRAC exon 2 Cys4 and TRBC1 or TRBC2 exon 2 Cys2, and/or the alpha and/or beta constant domain sequence(s) -chains can be modified by replacing Thr 48 TRAC and Ser 57 TRBC1 or TRBC2 with cysteine residues, where these cysteine residues form a non-native disulfide bond between the TCR alpha and beta chain constant domains.

The TCR of the present invention may be in single chain form of the type Va-L-Vp, Vp-L-Va, Va-Ca-L-Vp, Va-L-Vp-Cp, where Va and VP are the variable regions of a- and of the TCR P chains, respectively, Ca and Cp are the constant regions of the TCR a and P chains, respectively, and L is the linker sequence.

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

The invention also provides a TCR/anti-CD3 hybrid comprising a TCR of the present invention and an anti-CD3 antibody covalently linked to the C- or N-terminus of the alpha or beta chain of said TCR, and such TCR/anti-CD3 hybrid may contain a variable domain alpha chain selected from any of SEQ ID NOs: 6-12 or 51 and a beta chain variable domain selected from any of SEQ ID NOs: 13-23 or 52. The beta chain may be linked to an antibody sequence against CD3 via a linker sequence. Said linker sequence may be selected from the group consisting of GGGGS (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGSGG (SEQ ID NO: 26), GSGGG (SEQ ID NO: 27), GSGGGP (SEQ ID NO: 28), GGEPS (SEQ ID NO: 29), GGEGGGP (SEQ ID NO: 30), and GGEGGGSEGGGS (SEQ ID NO: 31).

The TCR/anti-CD3 hybrid of the present invention may comprise pairs of an alpha chain amino acid sequence and a beta chain amino acid sequence that are at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence pairs shown in the Table below.

The invention also provides a nucleic acid encoding a TCR or TCR/anti-CD3 fusion according to the present invention.

Also provided is a non-naturally occurring and/or purified and/or engineered cell presenting a TCR or TCR/anti-CD3 fusion of the present invention, comprising (a) a TCR expression vector that contains a nucleic acid of the present invention in a single open reading frame, or two separate open reading frames encoding the alpha chain and the beta chain, respectively; or (b) a first expression vector that contains a nucleic acid encoding a TCR alpha chain or TCR/anti-CD3 fusion of the present invention and a second expression vector that contains a nucleic acid encoding a TCR beta chain or TCR/anti-CD3 fusion. CD3 according to the present invention.

A cell presenting a TCR or a TCR/anti-CD3 fusion or a cell containing the expression vector(s) may be a T cell.

The invention also provides a pharmaceutical composition comprising a TCR or TCR/anti-CD3 hybrid of the present invention, or a cell of the present invention, together with one or more pharmaceutically acceptable carriers or excipients.

The invention also provides a TCR, a TCR/anti-CD3 hybrid, or a nucleic acid or cell of the present invention for use in medicine. A TCR, TCR/anti-CD3 hybrid, cell, or nucleic acid may be provided for use in a method of treating cancer in a human subject.

A human subject may have a tumor that expresses NY-ESO-1 and/or LAGE-1A, and said tumor may be a solid tumor and be selected from synovial sarcoma, non-small cell lung carcinoma (NSCLC), bladder cancer, gastric cancer, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, melanoma, multiple myeloma, hepatocellular carcinoma, and head and neck cancer. A human subject may be an HLA-A*02 subtype subject.

Detailed description of the invention

According to a first aspect, the present invention provides a T cell receptor (TCR) having the property of binding to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex and comprising a TCR alpha chain variable domain and/or a TCR beta chain variable domain , where the specified variable domain of the alpha chain contains an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 1-117 of SEQ ID NO: 2, and/or the beta chain variable domain contains an amino acid sequence that is at least 90%, e.g., 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1-115 of SEQ ID NO: 3. The TCR may be isolated, cell-free, and/or soluble, i.e. . it may not be a TCR that occurs naturally in a T cell in the human body.

The invention also provides a TCR that binds to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex with an affinity greater than 50 μM, where:

Alpha chain CDRs 1, 2 and 3 contain the sequences SEQ ID NOs: 41, 42 and 43, respectively, and/or Beta chain CDRs 1, 2 and 3 contain the sequences SEQ ID NOs: 44, 45 and 46, respectively; and/or at least one of the CDRs contains one or more conservative substitutions with respect to the sequences of SEQ ID NO: 41 - 46; and/or at least one of the CDRs contains up to three valid substitutions with respect to the sequences of SEQ ID NOs: 41-46. The affinity of the TCR for the SLLMWITQC/HLA-A*02 complex can range from 50 μM to 100 nM. Preferably, these substitutions change the binding affinity by no more than +/- 50%, or more preferably, no more than +/-20% compared to unsubstituted TCR.

To obtain stable soluble TCRs according to the present invention, a soluble native TCR variant was used as the starting sequence having the sequence shown in Figure 2. For this purpose, cysteine substitutions were introduced into the TRAC and TRBC regions so that a non-native interchain disulfide bond could be formed. Suitable positions for placing these cysteine substitutions are described in WO 03020763. Figure 2 shows the wild-type extracellular sequences of the TCR alpha and beta chains in soluble form, respectively. The sequence of SEQ ID NO: 4 is identical to the native extracellular sequence of the alpha chain of SEQ ID NO: 2, except for the replacement of Thr48 TRAC with Cys. Similarly, the sequence of SEQ ID NO: 5 is identical to the native extracellular sequence of the beta chain of SEQ ID NO: 3, except for the replacement of Ser57 TRBC with Cys, Cys75 with Ala, and Asn201 with Asp. The wild-type soluble TCR described above can be used to provide a reference sample against which the binding profile of mutant TCRs can be compared. The TCR according to the first aspect may also be the TCR according to the second aspect.

TCR receptors according to any or both aspects of the invention may be non-naturally occurring and/or purified and/or engineered. TCR according to the present invention may contain more than one mutation,

present in the variable domain of the alpha chain and/or the variable domain of the beta chain, in relation to the native TCR NY-ESO-1.

The terms "engineered TCR" and "mutated TCR" are used interchangeably in this application and generally mean a TCR that contains one or more introduced mutations with respect to the wild-type NY-ESO-1 TCR, in particular in the alpha chain variable domain and/or the variable domain of the beta chain. Mutations are preferably introduced within the CDR. These mutations (mutation) generally result in improved binding affinity of the TCR to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex. The alpha chain variable domain may contain at least one, two, three, four, five or six of the following mutations (according to the sequence numbering of SEQ ID NO: 2):

and/or the beta chain variable domain may contain at least one, two, three, four, five, six, seven, eight or nine of the following mutations (according to the sequence numbering of SEQ ID NO: 3):

The alpha chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO:

one) :

and/or the beta chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO:

one) :

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

Group 1: I51L, T52G, G53D, D54S, N55A

Group 2: I51L, T52G, G53D, D54S, N55A, N97D

Group 3: I51L, T52G, G53D, D54S, N55A, N97R

Group 4: I96S, N97D, S98Q, G99H

Group 5: D95S, I96S, N97R, S98Q, G99H

Group 6: I51L, G53D;

and/or the beta chain variable domain contains at least one of the following

mutation groups:

Group 1: N50W, Q51T, S53G

Group 2: S27K, Q28R, V29L, T30A, M31L, N50W, Q51T, S53G

Group 3: S27K, Q28R, V29L, T30A, M31L, N50W, Q51T, S53G, G100A

Group 4: S27K, Q28R, V29L, T30A, N50W, Q51T

Group 5: S27K, Q28R, V29L, T30A, N50W, Q51T S53G

Group 6: S27K, Q28R, V29L, T30A, N50W, Q51T S53G, G100A

Group 7: S27K, Q28R, V29L, T30A, M3IL, N50W, Q51T

Group 8: T30A, N50W, G100A.

Specific combinations of mutation groups can be as shown in the table below:

Specific combinations of mutations may be present in the TCR of the present invention, as shown in the tables below:

In particular, the alpha and beta chain variable domains may contain the following combinations of amino acid sequences:

According to specific embodiments of the invention, there are 1, 2, 3, 4, 5, 6, 7 or 8 mutations in the CDR of the alpha chain, for example, 2-6 mutations, and/or in the CDR

the beta chain has 1, 2, 3, 4, 5, 6, 7, 8 or 9 mutations, eg 3-9 mutations. In some embodiments, the 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 1-117 of SEQ ID NO: 2, provided that the α chain variable domain contains at least one of the mutations described above. In some embodiments, the TCR P 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 1-115 of SEQ ID NO: 3, provided that the p-chain variable domain contains at least one of the mutations described above. The alpha chain variable domain may comprise the amino acid sequence of any of SEQ ID NOs: 6-12 or 51. The beta chain variable domain may comprise the amino acid sequence of any of SEQ ID NOs: 13-23 or 52.

Mutations can also be introduced outside of the CDRs. Such mutations may improve binding, but preferably increase purified product yield and stability. Examples of such mutations in the variable domain of the alpha chain (according to the sequence numbering of SEQ ID NO: 2) can be the following mutations:

and/or the beta chain variable domain may contain at least one of the following mutations (according to the sequence numbering of SEQ ID NO: 3):

Mutations in the original TCR may include mutations capable of increasing binding affinity (k_D and/or binding half-life) TCR with SLLMWITQC. Mutations may include those that are capable of reducing the frequency of non-specific binding, ie. reduce binding to antigens other than binding to SLLMWITQC, or increase the specificity of TCR binding to SLLMWITQC. Mutations may include those mutations that increase the efficiency of folding and/or production. Some mutations may affect each of these characteristics, others may affect, for example, affinity but not specificity, or specificity but not affinity, etc.

Phenotypically non-expressing ("silent") variants of any TCR according to the present invention described in this application are included in the scope of the invention. As used herein, the term “phenotypically silent (“silent”) variant refers to a TCR that contains one or more additional amino acid substitutions in addition to the substitutions listed above, where the phenotype of said TCR is similar to that of the corresponding TCR that does not contain the specified substitution (substitutions ). As used herein, the TCR phenotype includes antigen binding affinity (K _D and/or binding half-life) and antigen specificity. The K _D and/or binding half-life to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex of said phenotypically silent variant may be within 20% of the measured K _D and/or binding half-life of the corresponding TCR , which does not contain the specified substitution(s), when measured under identical conditions (for example, at a temperature of 25°C and on the same SPR matrix). Suitable conditions are further defined in Example 3. Antigenic Specificity

further defined below. As known to those skilled in the art, it is possible to produce TCRs containing substitutions in the variable domains for the TCRs detailed above without changing the affinity of the interaction with the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex. In particular, such "silent" mutations can be included in parts of the sequence that are not known to be directly involved in antigen binding (eg, parts outside of the CDR or parts of the CDR not in contact with the peptide antigen). Such conventional options are included in the scope of the present invention. TCRs in which one or more conservative and/or acceptable substitutions have been made also form part of the present invention. Permissible substitutions are also phenotypically "silent" but may not be conservative, as defined below. Allowed substitutions may result in reduced affinity for the SLLMWITQC/HLA-A*02 complex compared to a TCR that does not contain these allowable substitutions. The affinity may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50% compared to the affinity of a TCR that does not contain the specified allowable substitution. The decrease in affinity does not result in an affinity less than (ie, less than) 50 μM.

The TCRs of the present invention may also contain one or more conservative substitutions that have a similar amino acid sequence and/or that retain the same function. One skilled in the art will recognize that different amino acids have similar properties and are thus "conservative". One or more such amino acids of a protein, polypeptide or peptide can often be substituted for one or more other such amino acids without inhibiting the desired activity of said protein, polypeptide or peptide.

Thus, the amino acids glycine, alanine, valine, leucine and isoleucine often substitute for each other (amino acids containing aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used to replace each other (because they have relatively short side chains), and valine, leucine and isoleucine are used to replace each other (because they

have bulkier 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).

Substitutions of this type are often referred to as "conservative" or "semi-conservative" amino acid substitutions. The present invention thus extends to the use of a TCR comprising the amino acid sequence described above but containing one or more conservative substitutions in the sequence such that the amino acid sequence of said TCR is at least 90%, e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a TCR containing amino acids 1-117 of SEQ ID NO: 2, 6-12 or 51 and/ or amino acids 1-115 of SEQ ID NO: 3, 13-23 or 52.

Specific combinations of alpha chain variable domain and beta chain variable domain may be as follows:

Amino acid substitutions with respect to the sequences listed above can be performed using a suitable method, for example, using site-directed mutagenesis or solid phase synthesis.

It should be understood that amino acid substitutions using natural or non-natural amino acids are possible within the scope of the present invention. For example, this application contemplates that the methyl group of alanine can be replaced by an ethyl group, and/or that minor changes can be made to the peptide backbone. Regardless of the use of natural or synthetic amino acids, it is preferred that only L-amino acids be present.

"Identity", as known in the art, is the similarity between two or more polypeptide sequences or two or more polynucleotide sequences based on the comparison of these sequences. In the art, identity also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences (as the case may be) as determined by strand matches of such sequences. While there are a number of methods for measuring identity between two polypeptide or two polynucleotide sequences, the methods that are commonly used to determine identity are coded into 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)).

You can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by introducing gaps in any sequence if necessary. It is possible to calculate amino acid identity or similarity (identity plus amino acid type conservatism) for optimal alignment. A program like BLASTx aligns similar sequences with maximum

length and assigns a value to the match. Thus, it is possible to obtain comparison results in which several areas of similarity are found, all of which have a different score. Both types of identity analysis are considered according to the present invention.

Percent identity between two amino acid sequences or two nucleic acid sequences is determined by aligning sequences for optimal comparison (eg, gaps can be introduced in the first sequence for best sequence alignment) and comparing amino acid residues or nucleotides at the appropriate positions. The "best alignment" is the alignment of two sequences that results in the maximum percent identity. Percent identity is determined based on the number of identical amino acid residues or nucleotides in the compared sequences (ie % identity = number of identical positions/total number of positions x100).

Determination of percent identity between two sequences can be performed using a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 algorithm, modified according to Karlin and Altschul (1993) Proc. Natl. Acad. sci. USA 90:5873-5877). The NBLAST and XBLAST programs of Altschul et al. (Altschul, et al. (1990) J. Mol. Biol. 215:403-410) include this algorithm. Nucleotide searches by BLAST analysis can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to nucleic acid molecules. BLAST protein searches can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to protein molecules for use in the present invention. To get aligned sequences with

gaps, Gapped BLAST can be used to compare them, 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 reveals distant similarities between molecules (same source). When using the BLAST, Gapped BLAST and PSI-Blast programs, you can use the default settings in 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, CABIOS (1989) algorithm. The ALIGN program (version 2.0), which is part of the CGC sequence alignment package, includes such an algorithm. Others Algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput Appl Biosci., 10:3-5 and FASTA as described in Pearson and Lipman (1988). ) Proc. Natl. Acad. Sci. 85:2444-8 In the case of the FASTA algorithm, ktup is a control parameter that sets the search sensitivity and speed.

Mutations can be carried out using a suitable method, including, but not limited to, methods based on polymerase chain reaction (PCR), enzyme-based restriction cloning, or ligation-independent cloning (NLK). These methods are described in detail in many standard molecular biology texts. Additional details regarding polymerase chain reaction (PCR) and enzyme-based restriction cloning can be found in Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) ^CSHL Press. Additional information on ligation-independent cloning (LIC) techniques can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6.

The TCR according to the second aspect may comprise an alpha chain framework region 2 (FM2) and an alpha chain framework region 3 (FM3), wherein the FM2 and FM3 regions comprise SEQ

ID NOs: 47 and 48, respectively, and/or contain one or more conservative substitutions and/or up to three valid substitutions.

A TCR according to a second aspect may comprise an FM2 beta chain region and an FM3 beta chain region, wherein said FM2 and FM3 regions comprise the sequences of SEQ ID NO:49 and 50, respectively, and/or contain one or more conservative substitutions and/or up to three allowed substitutions.

The TCR according to the second aspect may comprise amino acids 1-117 of SEQ ID NO: 2 and/or amino acids 1-115 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 defined in Table 1 or Table 2.

The TCRs of the present invention have the property to bind to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex. The TCRs of the present invention have been found to be highly specific for said epitope compared to other, irrelevant epitopes, and are thus particularly suitable for use as targeting vectors for the delivery of therapeutic agents or detectable labels to cells and tissues presenting said epitopes. Specificity in the context of the TCRs of the present invention refers to their ability to recognize HLA-A*02 expressing target cells containing the SLLMWITQC peptide, with minimal ability to recognize HLA-A*02 expressing target cells not containing said peptide. To assess specificity, TCRs may be present in soluble form and/or may be expressed on the surface of T cells. Recognition can be determined by measuring the level of T cell activation in the presence of the TCR and the target cell. In this case, the minimum recognition of peptide-free target cells is defined as the level of T cell activation less than 20%, preferably less than 10%, more preferably less than 5%, of the level of T cell activation observed in the presence of cells containing the peptide. -targets, when measured in

the same conditions. Soluble TCR specificity according to the present invention can be defined as the therapeutically relevant concentration of TCR. A therapeutically significant concentration can be defined as a TCR concentration of 10 ⁻⁹ M or less and/or a TCR concentration of up to 100 or up to 1000 times the corresponding EC50 value. Cells containing the peptide can be obtained by priming with said peptide or, more preferably, said cells can naturally present said peptide. Preferably, both the peptide-containing and non-peptide-containing cells are human cells. Specificity can be measured, for example, in cellular assays such as those described in Examples 6-8. Specificity may also refer to the ability to bind to the NY-ESO-1/HLA-A*02 complex and not bind to multiple naturally occurring peptide/HLA complexes as determined by Biacore, for example. Preferably, binding to the NY-ESO-1/HLA-A*02 complex is at least 400 times greater than binding to other naturally presented peptide/HLA complexes.

TCR согласно настоящему изобретению с комплексом может составлять в диапазоне от примерно 1 сек до примерно 60 ч или более, от примерно 30 мин до примерно 60 ч или более или от примерно 6 ч до примерно 60 ч или более. K_D TCR, предназначенных для применения в качестве растворимых терапевтических средств и/или диагностических средств с сочетании с поддающейся выявлению меткой или терапевтическим агентом, для комплекса предпочтительно составляет от примерно 1 пкМ до примерно 200 пкМ или от примерно 20 пкМ до примерно 100 пкМ по результатам определения с использованием способа BIAcore Примера 3, и/или полупериод связывания указанных TCR для комплекса может составлять от примерно 2 ч до 60 ч или более или от примерно 20 ч до примерно 60 ч или более по результатам The K _D TCR of the present invention for the NY-ESO-1/HLA-A*02 complex may be greater than (ie, greater than) 50 μM, eg between 50 μM and 1 pM. The K _D of specific TCRs of the present invention for said complex may be from about 1 pM to about 50 nM, from about 1 pM to about 400 pM, from about 20 pM to about 200 pM. Binding Half Time

The TCR of the present invention with the complex can range from about 1 second to about 60 hours or more, from about 30 minutes to about 60 hours or more, or from about 6 hours to about 60 hours or more. K _D TCR intended for use as soluble therapeutics and/or diagnostics in combination with a detectable label or therapeutic agent for the complex is preferably from about 1 pM to about 200 pM or from about 20 pM to about 100 pM according to the results determination using the BIAcore method of Example 3, and/or the binding half-life of said TCRs for the complex may be from about 2 hours to 60 hours or more, or from about 20 hours to about 60 hours or more, according to the results

determinations using the BIAcore method of Example 3. Specific TCRs of the present invention may be suitable for use in adoptive therapy; The K _D of such TCRs for the complex may be from about 50 nM to about 50 μM, or from about 100 nM to about 1 μM, and/or the binding half-life of such TCRs for the complex may be from about 3 seconds to about 12 minutes.

The binding affinity and/or binding half-life of specific TCRs according to the present invention for the SLLMWITQC/HLA-A*02 complex is substantially greater than the binding affinity of native TCR. An increase in the binding affinity of a native TCR often results in a decrease in the specificity of said TCR for its peptide/MHC ligand, as shown in Zhao Yangbing et al., The Journal of Immunology, The American Association of Immunologists, US, vol. 179, No. 9, 1 November 2007, 5845-5854. However, the TCRs of the present invention can retain specificity for the SLLMWITQC/HLA-A*02 complex despite a significantly higher binding affinity compared to native TCR.

Binding affinity (inversely proportional to the equilibrium constant K _D ) and binding half-life (expressed as T ¹ / ₂ ) can be determined using surface plasmon resonance (BIAcore) and/or the Octet method of Example 3 in this application. It should be understood that doubling the TCR affinity results in a halving of K _D . T ¹ / ₂ is calculated as ln2 divided by the dissociation rate (k _off ). Thus, doubling T' ¹ / ₂ 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. for TCR forms truncated with the removal of cytoplasmic and transmembrane domain residues. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, eg 3 or more times, using the same analytical protocol and the average of the results is taken.

The TCR for use as a targeting agent for delivering therapeutic agents to an antigen presenting cell may be in soluble form (ie, not contain transmembrane or cytoplasmic domains). In the TCRs according to the present invention, preferably soluble **-heterodimeric TCRs, for their stability, a disulfide bond between the residues of the respective constant domains can be inserted, as described, for example, in the publication of international application WO 03/020763. One or two of the constant domains present in the aP heterodimer of the present invention may be truncated at the C-terminus or C-terminus, for example, truncated by up to 15 or 10 or 8 or fewer amino acids. The C-terminus of the extracellular constant region of the alpha chain may be shortened by 8 amino acids. For use in adoptive therapy, the off-heterodimeric TCR can, for example, be transfected as full-length chains containing both cytoplasmic and transmembrane domains. TCRs for use in adoptive therapy may contain a disulfide bond corresponding to a naturally occurring disulfide bond between the respective alpha and beta chain constant domains. Additionally or alternatively, a non-native disulfide bond may be present.

TCR according to the present invention may be sp-heterodimers or may be single-stranded. Single chain forms include ap-TCR polypeptides of the Va-L-Vp, Vp-L-Va, Va-Ca-L-Vp or Va-L-Vp-Cp types, where Va and Vp are the a- and p-chain variable regions TCR, respectively, Ca and Cp are constant regions of the a- and p-chain of TCR, respectively, and L is a linker sequence. According to specific embodiments of the invention, single-stranded TCRs according to the present invention may contain a disulfide bond inserted between the residues of the respective constant domains, as described in the publication of international application WO 2004/033685. Single chain TCRs are further described in international application publications WO2004/033685; W098/39482; WO01/62908 and 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).

One skilled in the art will appreciate that it is possible to shorten the proposed sequences at the C-terminus and/or N-terminus by 1, 2, 3,4, 5 or more residues without substantially affecting the TCR binding characteristics. All such standard options are included in 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 also be modified by replacing Thr 48 TRAC and Ser 57 TRBC1 or TRBC2 with cysteine residues, where said cysteine residues form a disulfide bond between the alpha and beta chain constant domains of the TCR .

According to a further aspect, the present invention provides a nucleic acid encoding a TCR according to the first and/or second aspect of the invention. In some embodiments, the nucleic acid is cDNA. In some embodiments, the invention provides a nucleic acid comprising a sequence encoding a TCR α chain variable domain of the present invention. In some embodiments, the invention provides a nucleic acid comprising a sequence encoding a TCR P chain variable domain of the present invention. The nucleic acid may be non-naturally occurring and/or purified and/or engineered.

According to another aspect, the invention provides a vector that contains a nucleic acid according to the present invention. Preferably, the vector is a TCR expression vector.

The invention also provides a cell containing a vector according to the present invention, preferably a TCR expression vector. The vector may contain a nucleic acid of the present invention encoding in one open reading frame or two separate open reading frames an alpha chain and a beta chain, respectively. According to another aspect, the invention provides a cell comprising 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 particularly useful for 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 a non-naturally occurring and/or purified and/or engineered cell, in particular a T cell presenting the TCR of the present invention. The invention also provides a proliferating population of T cells presenting the TCRs of the present invention. There are a number of methods available 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 are suitable for use in cancer treatment based on adoptive therapy. 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 said antigen presenting cells. Thus, these soluble TCRs can be linked (covalently or otherwise) to a detectable label (for

diagnostic purposes, where TCR is used to detect the presence of cells presenting the SLLMWITQC/HLA-A*02 complex); a therapeutic agent or a pharmacokinetic (PK) modifying moiety (eg, by PEGylation).

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 effect develops in the desired area, the toxin can be placed inside the TCR-bound liposome so that the compound is released slowly, thus preventing the damaging effect during transport in the body and ensuring that said toxin has minimal exposure after TCR binding to relevant antigen-presenting cells.

Other suitable therapeutic agents include:

• low molecular weight cytotoxic agents, ie. compounds having the ability to kill mammalian cells and having a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals capable of exerting a cytotoxic effect. Moreover, it is to be understood that these small molecular weight cytotoxic agents also include pro-drugs, ie. compounds that are degraded or converted under physiological conditions to release cytotoxic agents. Examples of such agents include cisplatin, maytansine derivatives, rachelmicin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer

sodium (Photofrin II), temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine and doxorubicin;

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

• radionuclides, ie. unstable isotopes of elements whose decay is accompanied by the emission of one or more a- or p-particles or y-rays. 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 said radionuclides with high affinity TCRs or their multimers;

• immunostimulants, ie. effector molecules of the immune system that stimulate the immune response. For example, cytokines such as IL-2 and IFN-γ;

• superantigens and their mutant forms;

• TCR/HLA hybrids, eg, a fusion into a peptide/HLA complex, where 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 stimulating protein, etc.;

• antibodies or fragments thereof, including antibodies against a T cell or NK cell determinant (eg, antibodies against CD3, CD28, or CD 16);

• alternative protein scaffolds having antibody-like binding characteristics;

• complement activators;

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

One preferred embodiment provides a TCR/aHTH-CD3 fusion comprising a TCR of the present invention linked (generally by fusion at the N- or C-terminus of the alpha or beta chain) to an anti-CD3 antibody or

a functional fragment or variant (such TCR/aHTH-CD3 hybrids may be referred to as ImmTAC™ molecules). As used in this application, the term TCR includes TCR hybrids such as a TCR/aHTH-CD3 hybrid. When used in this application, the term "antibody" includes the indicated fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OCTH, UCHT-1, BMA-031, and 12F6. Antibody fragments and variants/analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, nanobodies (nanobodies™ - constructs, which are supplied by Ablynx (Belgium) and contain a single synthetic immunoglobulin heavy chain variable domain derived from an antibody of camelids, for example, a camel or a llama) and domain antibodies (Domantis, Belgium) containing a single immunoglobulin heavy chain variable domain or an immunoglobulin light chain variable domain affinity matured), or alternative protein scaffolds having antibody-like binding characteristics, such as affitels (Affibody, Sweden, containing engineered protein A scaffold) or anticalins (Pieris, Germany, containing engineered ancalins), among others.

The binding of the TCR and the CD3 antibody may or may not be direct through a linker sequence. Linker sequences are generally flexible because they consist primarily of amino acids such as glycine, alanine, and serine, which do not have bulky side chains likely to limit mobility. Applicable or optimal length of linker sequences is easy to determine. The length of the linker sequence is often less than about 12, such as less than 10 or 5 to 10 amino acids. Suitable linkers that can be used in the TCR/aHTH-CD3 hybrids of the present invention include, but are not limited to: GGGGS (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGSGG (SEQ ID NO: 26) , GSGGG (SEQ ID NO: 27), GSGGGP (SEQ ID NO: 28), GGEPS (SEQ ID NO: 29), GGEGGGP (SEQ ID NO: 30) and GGEGGGSEGGGS (SEQ ID NO: 31), as described in the publication international application WO2010/133828.

Specific embodiments of the aHTH-CD3/TCR fusion constructs of the present invention include alpha and beta chain pairs as shown in the table below.

A particularly preferred aHTH-CD3/TCR fusion contains the alpha chain of SEQ ID NO: 37 and the beta chain of SEQ ID NO: 38.

For some purposes, the TCRs of the present invention may be combined into a multi-TCR complex to form a multivalent TCR complex. There are a number of human proteins containing a multimerization domain that can be used in the production of multivalent TCR complexes, such as the tetramerization domain of p53, which has been used to generate tetramers of scFv antibody fragments that have increased serum stability and a significantly reduced dissociation rate compared to 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 type of application. The multivalent TCR complex of the present invention may have an increased ability to bind the SLLMWITQC/HLA-A*02 complex compared to a non-multimeric wild-type TCR or a T-cell receptor heterodimer of the present invention. Thus, the multivalent TCR complexes of the present invention are also included in the present invention. Such multivalent TCR complexes of the present invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo and are also useful as intermediates.

compounds to obtain additional multivalent TCR complexes that can be used in this way.

As is well known in the art, TCRs can be subject to post-translational modifications. Glycosylation is one such modification that involves the covalent attachment of oligosaccharide moieties to a specific amino acid in the TCR chain. For example, aspartic residues or serine/threonine residues are well-known oligosaccharide attachment positions. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation, and the availability of particular enzymes. Moreover, glycosylation status (ie, oligosaccharide type, covalent bond, and total number of attachments) can affect protein function. Thus, when making recombinant proteins, it is often desirable to control glycosylation. Controlled glycosylation has been used to improve antibody therapeutics (Jefferis R., Nat Rev Drug Discov. 2009 Mar;8(3):226-34.). For soluble TCRs of the present invention, glycosylation can be controlled in vivo, for example using specific cell lines, or in vitro by chemical modification. Such modifications are desirable because glycosylation can improve pharmacokinetic properties, reduce immunogenicity, and allow more accurate mimicry of native human protein (Sinclair AM and Elliott S., Pharm Sci. 2005 Aug;94(8):1626-35).

TCR or TCR/aHTH-CD3 fusions of the present invention (preferably linked to a detectable label or therapeutic agent or expressed on a transfected T cell) or cells of the present invention for administration to patients may be presented in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. Therapeutic or imaging TCRs, TCR hybrids or cells according to the invention are typically provided as part of a sterile pharmaceutical composition which typically contains

a pharmaceutically acceptable carrier. Said pharmaceutical composition may be presented in any suitable form (depending on the desired route of administration to the patient). Said composition may be provided in unit dosage form and generally provided in a sealed container and may be presented as part of a kit. Such a kit usually (but not necessarily) contains instructions for use. Said kit may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any suitable route such as parenteral (including subcutaneous, intramuscular or intravenous), enteric (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 subject being treated, and the like. A suitable dosage range for the soluble TCR of the present invention associated with an anti-CD3 antibody may be between 25 ng/kg and 50 μg/kg. Ultimately, the appropriate dosage for use is determined by the physician.

TCRs, pharmaceutical compositions, vectors, nucleic acids, and cells of the present invention may be present in substantially pure form, e.g., at least 80%, at least 85%, 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 %, at least 99% or 100% pure form.

The invention also provides:

• TCR, TCR/aHTH-CD3 hybrid, nucleic acid or cell of the present invention for use in medicine, preferably for use in a method of treating cancer.

• the use of a TCR, TCR/aHTH-CD3 fusion, nucleic acid or cell of the present invention in the manufacture of a medicament for the treatment of cancer;

• a method for treating cancer in a patient, comprising administering to said patient a TCR, a TCR/aHTH-CD3 hybrid, a nucleic acid, or a cell according to the present invention.

The cancer to be treated may be synovial sarcoma, non-small cell lung carcinoma (NSCLC), bladder cancer, gastric cancer, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, melanoma, multiple myeloma, hepatocellular carcinoma and head and neck cancer.

The preferred features of each aspect of the invention correspond to the preferred features of 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.

The invention is described below with reference to the following non-limiting figures and examples, where:

The Figure 1 shows the amino acid sequences of the extracellular regions of the native variable domains of the alpha and beta chains according to the present invention;

The Figure 2 shows the amino acid sequences of soluble extracellular regions of native alpha and beta chains according to the present invention;

Figure 3 shows the amino acid sequences of the mutant TCR alpha chain variable regions of the present invention;

Figure 4 shows the amino acid sequences of the mutant TCR beta chain variable regions of the present invention;

Figure 5 shows the amino acid sequences of ImmTAC molecules containing the TCR sequences of the present invention;

Figure 6 shows the amino acid sequences of ImmTAC molecules containing the TCR sequences of the present invention;

Figure 7 shows the amino acid sequences of ImmTAC molecules containing the TCR sequences of the present invention;

Figure 8 shows the amino acid sequences of ImmTAC molecules containing the TCR sequences of the present invention;

Figure 9 shows the active and specific binding of ImmTAC molecules according to the present invention;

Figure 10 shows an additional activity study for 2 ImmTAC molecules according to the present invention;

Figure 11 shows an additional specificity study for 3 ImmTAC molecules according to the present invention; and

Figure 12 shows the killing of tumor cells by ImmTAC-redirected T cells.

Examples

Example 1 Expression, refolding and purification of soluble TCRs

The DNA sequences encoding the extracellular regions of the alpha and beta chains of the 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 the E. coli Rosetta (BL21pLysS) strain and single ampicillin-resistant colonies were grown at 37°C in TYP medium (+ ampicillin, 100 μg/ml) until CC -0.6-0.8 was reached, followed by induction of protein expression with 0.5 mM IPTG. Cells were harvested three hours after induction by centrifugation. Cell pellets were lysed with BugBuster Protein Extraction Reagent (Merck Millipore) according to the manufacturer's instructions. Precipitates containing inclusion bodies were recovered by centrifugation. The pellets were washed twice in Triton-containing buffer (50 mM Tris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM Na-EDTA) and finally resuspended in detergent-free buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM Na-EDTA). The protein yield of inclusion bodies was quantified by solubilization with 6 M guanidine-HCl and measuring OD280. The protein concentration was then calculated using the extinction coefficient. The purity of inclusion bodies was measured by solubilizing with 8 M urea and applying ~2 μg to 4-20% SDS-PAGE under reducing conditions. Purity was then evaluated or calculated using a densitometric program (Chemidoc, Biorad). The inclusion bodies were stored at +4°C for short term storage and at -20°C or -70°C for longer storage.

For refolding soluble TCRs containing α- and β-chain inclusion bodies were first mixed and diluted in 10 ml of 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 an additional dilution in 1 L of buffer for

refolding (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 mixed well. The refolded mixture was dialyzed against 10 l of H ₂ O for 18-20 hours at 5 °C ± 3 °C. Thereafter, the dialysis buffer was replaced twice with 10 mM Tris (pH 8.1, 10 L) and dialysis continued for an additional 15 hours. The refolded mixture was then filtered through cellulose filters (0.45 µm).

Purification of soluble TCRs was initiated by loading the dialyzed refold product 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 using 50 column volumes using Akta® Purifier (GE Healthcare). Peak TCR fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by pooling and concentration. The concentrated sample was then applied to a Superdex® 75HR gel filtration column (GE Healthcare) pre-equilibrated in Dulbecco's PBS buffer. The TCR peak fractions were pooled and concentrated, and the final yield of purified material was calculated.

Example 2 Expression, Refolding and Purification of ImmTAC Molecules (TCR/aHTH-CD3 Soluble Hybrids)

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, the cation exchange step was carried out during purification after the anion exchange. In this case, the peak fractions resulting from the anion exchange were diluted 20-fold in 20 mM MES (pH 6.5) and applied to a POROS® 50HS cation exchange column. Bound protein was washed out with a gradient of 0-500 mM NaCl in 20 mM MES. ImmTAC peak fractions were pooled and adjusted to pH 8.1 with 50 mM Tris, then concentrated and applied directly to a gel filtration matrix as described in Example 1.

Example 3 - Binding Characterization

Binding analysis of purified soluble TCRs and ImmTAC molecules to the relevant peptide/HLA complex was performed by surface plasmon resonance using the BIAcore 3000 or BIAcore T200 instrument or by biolayer interferometry using the 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 carried out at 25°C in Dulbecco's phosphate-buffered saline (PBS) with the addition of 0.005% P20.

BIAcore Analysis

Biotinylated peptide-HLA monomers were immobilized on streptavidin-coupled CM-5 sensor chips. Equilibrium binding constants were determined by passing serial dilutions of soluble TCR/ImmTAC at a constant flow rate of 30 μl/min through a peptide/HEA-A*02 complex coated flow cell (-200 response units, RU). Steady-state responses were normalized for each TCR concentration by subtracting the volume buffer response in a control flow cell containing the irrelevant HLA peptide. The Kd value was obtained by non-linear curve fitting using the Prism program and the Langmuir binding isotherm: binding = C*Max/(C + KD), where "binding" is the equilibrium binding in RU, C is the concentration of TCR administered, and Max is maximum binding.

For high affinity interactions, binding parameters were determined using single cycle kinetic analysis. Soluble TCR/ImmTAC at five different concentrations was passed through a flow cell coated with peptide-HLA complex (-100-200 RU) at a flow rate of 50-60 μl/min. Typically, 60-120 µl of soluble TCR/ImmTAC was injected at the maximum

concentration of 100-200 nm, followed by 2-fold dilutions, which were used for the other four injections. The minimum concentration was administered first. Dissociation phase buffer was then added until >10% dissociation occurred, typically after 1-3 hours. Kinetic parameters were calculated using the BIAevaluation® software. The dissociation phase was brought to a single exponential decay equation, providing a half-cycle calculation. The equilibrium constant Kd was calculated from k _off /k _on .

Octet Analysis

Biotinylated peptide/HLA monomers were captured on 1 nm streptavidin (SA) biosensors (Pall ForteBio) preliminarily immobilized with streptavidin. The sensors were blocked with free biotin (2 μM) for 2 minutes. Equilibrium binding constants were determined by placing loaded biosensors in serial dilutions of soluble TCR/ImmTAC in a 96-well or 384-well sample plate. The plate agitation speed was set to 1000 rpm. For low affinity interactions (μM range), a short association time (~2 minutes) and a short dissociation time (~2 minutes) were used. Binding curves were processed by subtracting twice the reference value for reference biosensors loaded with irrelevant pHLA using Octet data analysis software (Pall ForteBio). Steady state responses (nm) were used to estimate the Kd value from the resting state plots adjusted to the equation: answer = Rmax*conc/(KD + conc) where "response" is the equilibrium binding in nm for each TCR concentration (conc.), and Rmax is the response corresponding to the maximum binding, when saturated with pHLA.

For high affinity interactions (nM-pcm range), kinetic parameters were determined from binding curves at concentrations >3 TCR/ImmTAC, 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 subtracting twice the reference values for reference biosensors loaded with irrelevant pHLA and blocked with biotin. Kinetic

k _on and k _off parameters were calculated by general fit directly to the binding curves using the Octet data analysis program (Pall ForteBio). Kd was calculated from k _off /k _on and the dissociation half-life was calculated from the equation t _1/2 = 0.693/k _off .

Example 4 Binding Characterization of Soluble Wild TCR According to the Invention

Soluble wild-type 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 corresponded to those shown in Figure 2. Soluble biotinylated HLA-A*02 was prepared either wild-type NY-ESO-1 peptide (SLLMWITQC) or NY-ESO-1 heteroclitic peptide (SLLMWITQV SEQ ID NO: 34) and immobilized on a BIAcore sensor chip. The determined Kd values were 5.2 μM and 4.3 μM, respectively. No significant binding was found to 15 irrelevant HLA-A*02 peptide complexes. The data obtained indicate that the TCR binds to the target with suitable affinity and specificity. These TCR chains thus provide a scaffold suitable for identifying additional TCRs according to the present invention.

Example 5 Binding Characterization of Soluble High Affinity TCRs and ImmTAC Molecules of the Invention

Soluble mutant TCRs and ImmTAC molecules were prepared as described in Examples 1 and 2 and binding characteristics were determined according to Example 3. TCR alpha and beta chains contained mutations in at least one CDR relative to the CDR sequences shown in Figure 2 (SEQ ID N0:41-46). The amino acid sequences of specific variable regions of the alpha and beta chains of the mutant TCRs of the present invention are shown in Figures 4 and 5, respectively. The table below shows the characteristics

binding for soluble TCR and/or ImmTAC molecules containing the indicated alpha and beta chain variable regions. Binding was measured using heteroclitic peptide NY-ESO-1 (SLLMWITQV).

BUT = not defined

¹ Corresponds to ImmTAC 1 from Example 6, full length alpha and beta chain sequences are shown in Figure 5

² Corresponds to ImmTAC2 from Example 6, full length alpha and beta chain sequences are shown in Figure 6

³ Corresponds to ImmTAC3 from Example 6, full length alpha and beta chain sequences are shown in Figure 7

⁴ Corresponds to ImmTAC4 from Example 6, full length alpha and beta chain sequences are shown in Figure 8

These data demonstrate that the TCR alpha and beta chain sequences of the present invention lead to the production of soluble TCRs and ImmTAC molecules with binding characteristics suitable for the development of immunotherapeutic reagents.

Example 6 Active and Specific Targeting of T Cells by ImmTAC Molecules of the Present Invention

The ability of ImmTAC molecules containing the alpha and beta chain variable region sequences of the present invention to mediate active and specific redirection of CD3+ T cells was studied by an ELISPOT assay using interferon-γ (IFN-γ) secretion as a measure of T-activation. cells.

The sequences of the alpha and beta chains of the four studied ImmTAC molecules are presented in Figures 5-8.

Analyzes were performed using a human IFN-y ELISPOT assay kit (BD Biosciences). Target cells were prepared at a density of 1 x 10 ⁶ /ml in assay medium (RPMI 1640 containing 10% heat-inactivated PBS and 1% penicillin-streptomycin-L-glutamine mixture) and plated at a density of 50,000 cells per well in a volume of 50 μl. In the present example, the following target cell lines were used:

• human lung cancer cell line NCI-H1755 (NY-ESO-l ^+ve ; HLA-A*02 ^+ve ) (obtained from the American Type Culture Collection, ATCC, catalog number: CRL-5892)

• HAo5 human heart cells (NY-ESO-l"^ve; HLA-A*02 ^+ve ) (obtained from Promocell, catalog number: C-12271)

Peripheral blood mononuclear cells (PBMC) isolated from fresh donor blood were used as effector cells and plated at a concentration of 40,000 cells per well in a volume of 50 μl. Various concentrations of ImmTAC were used, covering the expected clinically significant range, which were added to the well in a volume of 50 μl.

The plates were prepared according to the manufacturer's instructions. Target cells, effector cells, and ImmTAC molecules were added to the appropriate wells and made up to a final volume of 200 μl of assay medium. All reactions were performed in triplicate. Also prepared control wells with no 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 (lxPBS prep sachet,

containing 0.05% P20 diluted in deionized water). Then, primary antibodies were added to each well for detection in a volume of 50 μl. The plates were incubated at room temperature for 2 hours before washing again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP conjugate to each well and incubating at room temperature for 1 hour and repeating the washing step. The plates were then washed twice with 200 μl PBS (pH 7.4). Not more than 15 min prior to use, for every 1 ml of AEC substrate, one drop (20 μl) of AEC chromogen was added and mixed. 50 μl of the resulting mixture was added to each well. The appearance of dots was constantly monitored and the plates were washed in tap water to stop development. The plates were dried at room temperature for at least 2 hours, followed by counting points using a CTL analyzer with Immunospot software (Cellular Technology Limited).

The data presented in Figure 9 shows that ImmTAC molecules containing the alpha and beta chain variable sequences of the present invention are able to mediate active T cell redirection against HLA-A*02 ^+ve cancer cells expressing the target antigen. No activity was observed against antigen-negative cells expressing HLA-A*02 ^+ve . The data obtained indicate that ImmTAC molecules are specific for target cells within the range of clinically relevant concentrations (< 1 nM).

An additional evaluation of the activity of the ImmTAC 3 and 4 molecules was carried out to determine the EC50 values. ELISpot analyzes were performed as described above using ^ImmTAC concentrations ranging from 10"13M to 10" ^8M . Data were analyzed using Prism 5.0 software (GraphPad) to calculate EC50 values. The determined values were 95 pM and 121 pM for ImmTAC 3 and 4 molecules, respectively (Figure 10). These data support the ability of these ImmTAC molecules to mediate active redirection of the T cell response.

Example 7 - Additional study of the specificity of ImmTAC molecules according to the present invention

Conducted an additional study of the specificity of ImmTAC molecules on a panel of normal cells. In this example, ImmTAC 2-4 molecules were used (Figures 6-8). The secretion of interferon-γ (IFN-γ) was used as a recordable indicator of T-cell activation.

Analyzes were performed using the DuoSet ELISA kit for IFN-γ (R&D Systems, catalog number: DY285) according to the manufacturer's instructions. Briefly, IFN-y was diluted to 10,000 pg/ml and 2-fold dilutions were prepared to obtain a standard curve. Target cells were counted and plated at a concentration of 10,000 cells per well in 10 µl of assay medium. ImmTAC molecules were diluted to give a final concentration of 2 nM and 1 nM in 10 μl per well. A control sample without ImmTAC was also prepared. Effector PBMCs were thawed and plated at 10,000 cells per well in 10 μl. The plates were incubated for 48 hours, after which they were developed and analyzed.

In this example, NCl-H1755 cells were used as antigen positive control and HTC-116 cells were used as antigen negative control. The cell panel included cardiomyocytes (CM12, CM5 and CM10), aortic endothelial cells (HAo5), respiratory tract epithelial cells (HCAEC2 and HCAEC5), skeletal muscle tissue myoblasts (HSkMM3), and HPF9 cells. All cell lines were HLA-A*02 ^+ve cells. Analyzes were performed in three repetitions.

The data presented in Figure 11 demonstrates minimal production of IFN-γ in the presence of cell lines derived from normal tissues, compared with antigen-positive cancer cell lines, within the range of therapeutically relevant concentrations of ImmTAC. These data indicate

that the ImmTAC molecules of the present invention have a high level of specificity and thus are particularly suitable for therapeutic use.

Example 8 - Active killing of tumor cells by ImmTAC-redirected T cells

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

Analyzes were performed using the caspase-3/7 CellPlayer 96-well plate apoptosis assay kit (Essen Bioscience, cat. no.: 4440) according to the manufacturer's protocol. Briefly, target cells (NCl-H1755 - NYESO ^+ve HLA A*02 ^+ve or HCT-116 - NYESO" ^ve HLA A*02 ^+ve ) were seeded at a concentration of 5000 cells per well and incubated overnight to allow them ImmTAC solutions were prepared at concentrations from 2.16 nM to 8.8 pM 25 µl of solution at each concentration was added to the appropriate well PBMCs were used as effector cells and seeded at a concentration of 50,000 per well in 50 µl A control sample was also prepared No ImmTAC NucView Assay Reagent prepared at a concentration of 30 μM in 25 μl was added to each well (to give a final concentration of 5 μM) The plate was placed in an IncuCyte instrument and images were acquired every 2 hours (1 image per well) in within 3 days The number of apoptotic cells in each image was determined and recorded as the number of objects per mm ^2. Analyzes were performed in triplicate.

The data presented in Figure 12 demonstrates real-time killing of tumor cells by ImmTAC-redirected T cells. The results are presented for ImmTAC3 and ImmTAC4 molecules. For both ImmTAC molecules, there were

shows redirected destruction of antigen-positive tumor cells by T-cells at a concentration of only 26 pM. ImmTAC3 has been shown to redirect T-cell killing of antigen-positive tumor cells at a concentration of less than 10 pM. A low level of destruction of antigen-negative cells was observed only at the highest concentration (2.16 nm).

These data confirm that ImmTAC3 and ImmTAC4 mediate active retargeted killing of antigen-positive tumor cells by T cells within a therapeutically relevant range of concentrations.

--->

SEQUENCE LIST

<110> Immunocore Limited

<120> COMPLEX SPECIFIC T-CELL RECEPTORS

TUMOR ANTIGEN NY-ESO-1/HLA-A*02

<130> P100579WO

<150> 1522592.3

<151> 2015-12-22

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Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

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Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

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Asn Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

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Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Cys Gln Arg Gly

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Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr

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Ala Asn Gln Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

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Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

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Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

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Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

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

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

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

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Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln

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Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

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Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

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Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ser

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Asp Gln His Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

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Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

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

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Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

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Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

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Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Ser Ser

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Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

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Leu Ser Val Ile Pro

115

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

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20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

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Arg Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

Leu Ser Val Ile Pro

115

<210> 12

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Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

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20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

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Arg Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

Leu Ser Val Ile Pro

115

<210> 13

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Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

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Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

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Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

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Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

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Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

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Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 15

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<213> Artificial sequence

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<223> Mutant beta chain (b52)

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Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Leu Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 16

<211> 115

<212> Protein

<213> Artificial sequence

<220>

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<400> 16

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 17

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<212> Protein

<213> Artificial sequence

<220>

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Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 18

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b65)

<400> 18

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Leu Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 19

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b65l)

<400> 19

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Leu Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 20

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b67)

<400> 20

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 21

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b67l)

<400> 21

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 22

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b68)

<400> 22

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 23

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b68l)

<400> 23

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr

35 40 45

Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<210> 24

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 24

Gly Gly Gly Gly Ser

fifteen

<210> 25

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 25

Gly Gly Gly Ser Gly

fifteen

<210> 26

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 26

Gly Gly Ser Gly Gly

fifteen

<210> 27

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 27

Gly Ser Gly Gly Gly

fifteen

<210> 28

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 28

Gly Ser Gly Gly Gly Pro

fifteen

<210> 29

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 29

Gly Gly Glu Pro Ser

fifteen

<210> 30

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 30

Gly Gly Glu Gly Gly Gly Pro

fifteen

<210> 31

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 31

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

1 5 10

<210> 32

<211> 203

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC alpha chain (containing SEQ ID NO:

7(a24) and a constant domain having the sequence of SEQ ID NO: 4,

shortened by 8 amino acids)

<400> 32

Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

1 5 10 15

Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

85 90 95

Asn Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

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

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr

195 200

<210> 33

<211> 503

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC beta chain (containing an anti-CD3 antibody scFv fragment,

fused via a linker to a TCR beta chain containing SEQ ID NO: 15(b52) and

constant domain having the sequence of SEQ ID NO: 3)

<400> 33

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

260 265 270

Arg Gly Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala

275 280 285

Leu Met Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile

290 295 300

Ala Thr Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val

305 310 315 320

Ile Asp Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu

325 330 335

Thr Val Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser

340 345 350

Val Gly Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Ala Trp Gly Arg Ala Asp

500

<210> 34

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> NY-ESO-1 heteroclitic peptide

<400> 34

Ser Leu Leu Met Trp Ile Thr Gln Val

fifteen

<210> 35

<211> 203

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC alpha chain (containing SEQ ID NO:

7(a24) and a constant domain having the sequence

SEQ ID NO: 4, shortened by 8 amino acids)

<400> 35

Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

1 5 10 15

Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

85 90 95

Asn Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

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

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr

195 200

<210> 36

<211> 503

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC beta chain (containing an anti-CD3 antibody scFv fragment,

fused via a linker to a TCR beta chain containing the SEQ sequence

ID NO: 18(b65) and a constant domain having the sequence

SEQ ID NO: 5)

<400> 36

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

260 265 270

Arg Gly Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala

275 280 285

Leu Met Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile

290 295 300

Ala Thr Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val

305 310 315 320

Ile Asp Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu

325 330 335

Thr Val Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser

340 345 350

Val Gly Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Ala Trp Gly Arg Ala Asp

500

<210> 37

<211> 203

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC alpha chain (containing SEQ ID NO:

12(a82) and a constant domain having the sequence of SEQ ID NO: 4,

shortened by 8 amino acids)

<400> 37

Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

1 5 10 15

Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

85 90 95

Arg Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

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

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr

195 200

<210> 38

<211> 503

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC beta chain (containing an anti-CD3 antibody scFv fragment,

fused via a linker to a TCR beta chain containing the SEQ sequence

ID NO: 15(b52) and constant domain having the sequence of SEQ ID

NO: 5)

<400> 38

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

260 265 270

Arg Gly Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala

275 280 285

Leu Met Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile

290 295 300

Ala Thr Ala Trp Thr Gly Gly Glu Ala Thr Tyr Glu Ser Gly Phe Val

305 310 315 320

Ile Asp Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu

325 330 335

Thr Val Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser

340 345 350

Val Gly Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Ala Trp Gly Arg Ala Asp

500

<210> 39

<211> 203

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC alpha chain (containing SEQ ID NO:

12(a82) and a constant domain having the sequence of SEQ ID NO: 4,

shortened by 8 amino acids)

<400> 39

Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

1 5 10 15

Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Gly Asp Ser Ala Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

85 90 95

Arg Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

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

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr

195 200

<210> 40

<211> 503

<212> Protein

<213> Artificial sequence

<220>

<223> ImmTAC beta chain (containing an anti-CD3 antibody scFv fragment,

fused via a linker to a TCR beta chain containing the SEQ sequence

ID NO: 18(b65) and constant domain having the sequence of SEQ ID

NO: 5)

<400> 40

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

260 265 270

Arg Gly Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Lys Arg Leu Ala

275 280 285

Leu Met Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile

290 295 300

Ala Thr Ala Trp Thr Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val

305 310 315 320

Ile Asp Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu

325 330 335

Thr Val Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser

340 345 350

Val Gly Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Ala Trp Gly Arg Ala Asp

500

<210> 41

<211> 6

<212> Protein

<213> Homo sapiens

<400> 41

Val Ser Gly Asn Pro Tyr

fifteen

<210> 42

<211> 8

<212> Protein

<213> Homo sapiens

<400> 42

Tyr Ile Thr Gly Asp Asn Leu Val

fifteen

<210> 43

<211> 17

<212> Protein

<213> Homo sapiens

<400> 43

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

1 5 10 15

Phe

<210> 44

<211> 5

<212> Protein

<213> Homo sapiens

<400> 44

Ser Gln Val Thr Met

fifteen

<210> 45

<211> 7

<212> Protein

<213> Homo sapiens

<400> 45

Ala Asn Gln Gly Ser Glu Ala

fifteen

<210> 46

<211> 14

<212> Protein

<213> Homo sapiens

<400> 46

Cys Ser Val Gly Gly Ser Gly Gly Ala Asp Thr Gln Tyr Phe

1 5 10

<210> 47

<211> 17

<212> Protein

<213> Homo sapiens

<400> 47

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

1 5 10 15

Lys

<210> 48

<211> 33

<212> Protein

<213> Homo sapiens

<400> 48

Lys Gly Ser Tyr Gly Phe Glu Ala Glu Phe Asn Lys Ser Gln Thr Ser

1 5 10 15

Phe His Leu Lys Lys Pro Ser Ala Leu Val Ser Asp Ser Ala Leu Tyr

20 25 30

Phe

<210> 49

<211> 17

<212> Protein

<213> Homo sapiens

<400> 49

Met Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala

1 5 10 15

Thr

<210> 50

<211> 37

<212> Protein

<213> Homo sapiens

<400> 50

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

1 5 10 15

Asn Leu Thr Phe Ser Thr Leu Thr Val Ser Asn Met Ser Pro Glu Asp

20 25 30

Ser Ser Ile Tyr Leu

35

<210> 51

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant alpha chain (a86)

<400> 51

Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly

1 5 10 15

Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu

35 40 45

Lys Tyr Leu Thr Asp Asp Asn Leu Val Lys Gly Ser Tyr Gly Phe Glu

50 55 60

Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser

65 70 75 80

Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ile

85 90 95

Asn Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys

100 105 110

Leu Ser Val Ile Pro

115

<210> 52

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant beta chain (b71)

<400> 52

Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Lys Gln Arg Gly

1 5 10 15

Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Ser Gln Val Ala Met Met

20 25 30

Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Pro Thr Leu Ile Ala Thr

35 40 45

Ala Trp Gln Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp

50 55 60

Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val

65 70 75 80

Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly

85 90 95

Gly Ser Gly Ala Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu

100 105 110

Thr Val Leu

115

<---

Claims (53)

1. A T cell receptor (TCR) having the property of binding to the SLLMWITQC complex (SEQ ID NO: 1)/HLA-A*02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, where the specified alpha chain variable domain contains an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to the sequence of SEQ ID NO: 12, said beta chain variable domain contains an amino acid sequence that is at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 15, and where the sequence of amino acid residues 27-32, 50-57 and 91-107 of the alpha chain variable domain and the sequence of amino acid residues 27-31, 49-55 and 93-106 of the beta variable domain -chain is selected from the following sequences: Alpha chain beta chain 27-32 50-57 91-107 27-31 49-55 93-106 VSGNPY YLGDSALV CAVRDINSGAGSYQLTF KRLAL AWTGGEA CSVGGSGAADTQYF VSGNPY YLGDSALV CAVRDINSGAGSYQLTF KRLAL AWTGSE CSVGGSGGADTQYF VSGNPY YLGDSALV CAVRDIRSGAGSYQLTF KRLAL AWTGGEA CSVGGSGAADTQYF VSGNPY YLGDSALV CAVRDIRSGAGSYQLTF KRLAL AWTGSE CSVGGSGGADTQYF 2. TCR according to claim. 1, characterized in that the sequence of the variable domain of the alpha chain and the sequence of the variable domain of the beta chain are selected from the following amino acid sequences: alpha chain variable domain Beta chain variable domain SEQ ID NO: 7 SEQ ID NO: 15 SEQ ID NO: 7 SEQ ID NO: 18 SEQ ID NO: 12 SEQ ID NO: 15 SEQ ID NO: 12 SEQ ID NO: 18 3. A TCR according to claim 1 or 2, which is an alpha/beta heterodimer containing a TRAC alpha chain constant domain sequence and a TRBC1 or TRBC2 beta chain constant domain sequence. 4. TCR according to claim 3, characterized in that the sequences of the constant domain of the alpha and beta chains are modified by shortening or replacing with the removal of the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. 5. TCR according to claim 3 or 4, characterized in that the sequence (s) of the constant domain of the alpha and / or beta chain is modified by replacing Thr 48 TRAC and Ser 57 TRBC1 or TRBC2 with cysteine residues, where these cysteine residues form a non-native a disulfide bond between the constant domains of the alpha and beta chains of the TCR. 6. TCR according to any one of the preceding claims, presented in a single chain form of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, where Vα and Vβ are variable regions TCR α and β chains, respectively, Cα and Cβ are constant regions of the TCR α and β chains, respectively, and L is a linker sequence. 7. A TCR according to any one of the preceding claims associated with a detectable label, therapeutic agent or pharmacokinetic (PK) modifying moiety. 8. TCR that binds to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex with an affinity greater than 50 μM, where alpha chain CDRs 1, 2 and 3 and beta chain CDRs 1, 2 and 3 are selected from: Alpha chain beta chain 27-32 50-57 91-107 27-31 49-55 93-106 VSGNPY YLGDSALV CAVRDINSGAGSYQLTF KRLAL AWTGGEA CSVGGSGAADTQYF VSGNPY YLGDSALV CAVRDINSGAGSYQLTF KRLAL AWTGSE CSVGGSGGADTQYF VSGNPY YLGDSALV CAVRDIRSGAGSYQLTF KRLAL AWTGGEA CSVGGSGAADTQYF VSGNPY YLGDSALV CAVRDIRSGAGSYQLTF KRLAL AWTGSE CSVGGSGGADTQYF 9. TCR according to claim 8, characterized in that the variable domain of the alpha chain contains an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 100%, identical to the sequence of SEQ ID NO: 12, and the variable domain of the beta chain contains an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to the sequence of SEQ ID NO: 15. 10. TCR according to any one of paragraphs. 8, 9, characterized in that the variable domain of the alpha chain is selected from the amino acid sequence of SEQ ID NO: 6-12 or 51 and the variable domain of the beta chain is selected from the amino acid sequence of SEQ ID NO: 13-23 or 52. 11. TCR according to any one of paragraphs. 8-10, characterized in that the sequence of the variable domain of the alpha chain and the sequence of the variable domain of the beta chain are selected from the following amino acid sequences: alpha chain variable domain Beta chain variable domain SEQ ID NO: 7 SEQ ID NO: 15 SEQ ID NO: 7 SEQ ID NO: 18 SEQ ID NO: 12 SEQ ID NO: 15 SEQ ID NO: 12 SEQ ID NO: 18 12. TCR according to any one of paragraphs. 8-11, which is an alpha/beta heterodimer containing a TRAC alpha chain constant domain sequence and a TRBC1 or TRBC2 beta chain constant domain sequence. 13. TCR according to claim 12, characterized in that the sequences of the constant domain of the alpha and beta chains are modified by shortening or replacing with the removal of the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. 14. TCR according to claim 12 or 13, characterized in that the sequence (s) of the constant domain of the alpha and / or beta chain is modified by replacing Thr 48 TRAC and Ser 57 TRBC1 or TRBC2 with cysteine residues, where these cysteine residues form a non-native a disulfide bond between the constant domains of the alpha and beta chains of the TCR. 15. TCR according to any one of paragraphs. 8-14 presented in a single chain form of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, where Vα and Vβ are the variable regions of α- and β- TCR chains, respectively, Cα and Cβ are constant regions of TCR α- and β-chains, respectively, and L is a linker sequence. 16. TCR according to any one of paragraphs. 8-15 associated with a detectable label, therapeutic agent, or pharmacokinetic (PK) modifying moiety. 17. A TCR/anti-CD3 fusion protein comprising a TCR according to any one of the preceding claims and an anti-CD3 antibody covalently linked to the C or N terminus of the alpha or beta chain of said TCR having the property to bind to the SLLMWITQC complex (SEQ ID NO : 1)/HLA-A*02. 18. The TCR/anti-CD3 fusion protein of claim 17, comprising an alpha chain variable domain selected from SEQ ID NOs: 6-12 or 51 and a beta chain variable domain selected from SEQ ID NOs: 13- 23 or 52. 19. The TCR/anti-CD3 fusion protein of claim 18, wherein the beta chain is linked to an anti-CD3 antibody sequence via a linker sequence. 20. TCR/anti-CD3 fusion protein according to claim 19, characterized in that said linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGSGG (SEQ ID NO: : 26), GSGGG (SEQ ID NO: 27), GSGGGP (SEQ ID NO: 28), GGEPS (SEQ ID NO: 29), GGEGGGP (SEQ ID NO: 30), and GGEGGGSEGGGS (SEQ ID NO: 31). 21. The TCR/anti-CD3 fusion protein according to claim 19, containing an alpha and beta chain that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences shown in the Table below: Alpha chain
SEQID NO: beta chain
SEQID NO: Shown 32 33 Figure 5 35 36 Figure 6 37 38 Figure 7 39 40 Figure 8 22. TCR/anti-CD3 fusion protein according to claim 21, containing an alpha chain consisting of the sequence of SEQ ID NO: 37 and a beta chain consisting of the sequence of SEQ ID NO: 38. 23. Fusion protein TCR/anti-CD3 according to any one of paragraphs. 17-20, containing the variable domain of the alpha chain, consisting of the amino acid sequence of SEQ ID NO: 12, and the variable domain of the beta chain, consisting of the amino acid sequence of SEQ ID NO: 15, where the specified beta chain is connected to the antibody against CD3 through a linker sequences. 24. Fusion protein TCR/anti-CD3 according to any one of paragraphs. 17-20, containing the variable domain of the alpha chain, consisting of the amino acid sequence of SEQ ID NO: 12, and the variable domain of the beta chain, consisting of the amino acid sequence of SEQ ID NO: 15, where the specified alpha chain is linked to the antibody against CD3 through a linker sequences. 25. TCR/anti-CD3 fusion protein according to claim 24, characterized in that the variable domain of the alpha chain consists of the amino acid sequence of SEQ ID NO: 12 and the variable domain of the beta chain consists of the amino acid sequence of SEQ ID NO: 15, where the specified the beta chain is linked to the anti-CD3 antibody via a linker sequence of SEQ ID NO: 24. 26. A nucleic acid encoding a TCR or TCR/anti-CD3 fusion protein according to any one of the preceding claims. 27. An expression vector containing a nucleic acid according to claim 26. 28. A non-naturally occurring and/or purified and/or engineered cell for treating a tumor whose cells express NY-ESO-1 and/or LAGE-1A in a human subject with the HLA-A*02 subtype, where said cell presents a TCR according to any one of paragraphs. 1-16 or a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25, said cell being a cytotoxic cell. 29. The cell according to claim 28, which is a T-cell. 30. Cell for expression of TCR or TCR/anti-CD3 fusion protein, containing: (a) the TCR expression vector of claim 27 in one open reading frame or two different open reading frames encoding an alpha chain and a beta chain, respectively; or (b) a first expression vector that contains a nucleic acid encoding a TCR alpha chain according to any one of paragraphs. 1-16 or a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25, and a second expression vector that contains a nucleic acid encoding a TCR beta chain according to any one of paragraphs. 1-16 or a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25, while these vectors are capable of providing the expression of TCR according to any one of paragraphs. 1-16 or a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25 having the property to bind to the SLLMWITQC (SEQ ID NO: 1)/HLA-A*02 complex. 31. The cell according to claim 30, which is a T-cell. 32. A pharmaceutical composition for the treatment of a tumor whose cells express NY-ESO-1 and/or LAGE-1A in a human subject with the HLA-A*02 subtype, comprising a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25, or a cage according to any one of paragraphs. 28-31, wherein said cell is a cytotoxic T cell, together with one or more pharmaceutically acceptable carriers or excipients. 33. A method for treating a tumor whose cells express NY-ESO-1 and/or LAGE-1A in a human subject with the HLA-A*02 subtype in need of said treatment, comprising administering to said subject a pharmaceutically effective dose of a pharmaceutical composition according to claim 33. 32. 34. The method of treating a human subject according to claim 33, further comprising separate, combined, or sequential administration of an antineoplastic agent. 35. A dosage form for the treatment of a tumor whose cells express NY-ESO-1 and/or LAGE-1A, for injection to be administered to a human subject with an HLA-A*02 subtype, containing a TCR/anti-CD3 fusion protein according to any from paragraphs. 17-25, a cage according to any one of paragraphs. 28–31, wherein said cell is a cytotoxic T cell, or a pharmaceutical composition according to claim 32. 36. The use of a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25 for treating a tumor whose cells express NY-ESO-1 and/or LAGE-1A in a human subject with the HLA-A*02 subtype. 37. Use according to claim 36, characterized in that said tumor is a solid tumor. 38. Use according to claim 36 or 37, characterized in that said tumor is selected from synovial sarcoma, non-small cell lung carcinoma (NSCCL), bladder cancer, gastric cancer, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cancer esophagus, melanoma, multiple myeloma, hepatocellular carcinoma, and head and neck cancer. 39. Application according to any one of paragraphs. 36-38, characterized in that said TCR/anti-CD3 fusion protein is administered by injection, for example intravenous injection or direct injection into the tumor. 40. The use of cells according to any one of paragraphs. 28-31 for treating a tumor whose cells express NY-ESO-1 and/or LAGE-1A in a human subject with the HLA-A*02 subtype, said cell being a cytotoxic T cell. 41. Use according to claim 40, wherein said tumor is a solid tumor. 42. Use according to claim 40 or 41, characterized in that said tumor is selected from synovial sarcoma, non-small cell lung carcinoma (NSCCL), bladder cancer, gastric cancer, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cancer esophagus, melanoma, multiple myeloma, hepatocellular carcinoma, and head and neck cancer. 43. Application according to any one of paragraphs. 40-42, characterized in that said cell is administered by injection, such as intravenous injection or direct injection into the tumor. 44. The method of obtaining TCR according to any one of paragraphs. 1-16 or a TCR/anti-CD3 fusion protein according to any one of paragraphs. 17-25, comprising a) maintaining the host cell of claim 30 under optimal conditions for nucleic acid expression, and b) isolating the TCR or TCR/anti-CD3 fusion protein encoded by said nucleic acid.

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