T cell receptor

Abstract

The present invention relates to T cell receptors (TCRs) that bind to the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1), which is derived from the germline cancer antigen PRAME. The TCRs may comprise non-naturally occurring mutations within the alpha and / or beta variable domains relative to the native PRAME TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic agents for the treatment of malignant diseases. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to T cell receptors (TCRs) that bind to the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1), which is derived from the pro-phagocyte cancer antigen PRAME. The TCRs may comprise non-naturally occurring mutations within the α and / or β variable domains relative to the native PRAME TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic agents for the treatment of malignant diseases. [Background technology]

[0002] Background of the Invention T cell receptor (TCR) is a CD4 + and CD8 + Naturally expressed by T cells, TCRs are designed to recognize short peptide antigens displayed on the surface of antigen-presenting cells in complex with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also known as human leukocyte antigens or HLA) (Davis et al., Annu Rev Immunol, 1998;16:523-44). CD8 + T cells, also known as cytotoxic T cells, possess TCRs that specifically recognize peptides bound to MHC class I molecules. CD8+ T cells are generally responsible for detecting and mediating the destruction of diseased cells, including cancer cells and virally infected cells. The affinity of cancer-specific TCRs in the natural repertoire for their corresponding antigens is generally low as a result of thymic selection, meaning that cancer cells frequently escape detection and destruction. Novel immunotherapeutic approaches aimed at promoting cancer recognition by T cells offer highly promising strategies for the development of effective anticancer therapies.

[0003] PRAME, or preferentially expressed antigen in melanoma, was originally identified as an antigen overexpressed in melanoma (Ikeda et al., Immunity, 1997 February;6(2):199-208). It has also been referred to as CT130, MAPE, and OIP-4, and its Uniprot accession number is P78395. This protein functions as a repressor of retinoic acid receptor signaling (Epping et al., Cell, 2005 September 23;122(6):835-47). PRAME belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are attractive targets for immunotherapeutic intervention because their expression is usually restricted or absent in normal adult tissues. PRAME is expressed in many solid tumors as well as leukemias and lymphomas (Doolan et al., Breast Cancer Res Treat. 2008 May;109(2):359-65; Epping et al. Cancer Res. 2006 Nov. 15;66(22):10639-42; Ercolak et al. Breast Cancer Res Treat. 2008 May;109(2):359-65; Matsushita et al. Leuk Lymphoma. 2003 Mar.;44(3):439-44; Mitsuhashi et al. Int. J Hematol. 2014;100(1):88-95; Proto-Sequeire et al. Leuk Res. 2006 Nov.;30(11):1333-9; Szczepanski et al. Oral Oncol. 2013 Feb;49(2)::144-51; Van Baren et al. Br J Haematol. 1998 Sep;102(5):1376-9). The PRAME targeted therapy of the present invention may be particularly suitable for the treatment of cancers including, but not limited to, lung cancer (NSCLC and SCLC), breast cancer (including triple negative), ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer and head and neck cancer.

[0004] The peptide SLLQHLIGL (SEQ ID NO: 1) corresponds to amino acids 425-433 of the full-length PRAME protein and is displayed on the surface of cells in a complex with HLA-A*02 (Kessler et al., J Exp Med. 2001 Jan 1;193(1):73-88). Peptide-HLA complexes provide useful targets for TCR-based immunotherapeutic intervention.

[0005] Identification of specific TCR sequences that bind with high affinity and specificity to the SLLQHLIGL (SEQ ID NO: 1)-HLA-A*02 complex would be advantageous for the development of novel immunotherapeutic agents. Therapeutic TCRs can be used, for example, as soluble targeting agents to deliver cytotoxic agents to tumor sites or to activate immune effector functions against tumor cells (Lissin et al., "High-Affinity Monocloncal T-cell receptor (mTCR) Fusions," Fusion Protein Technologies for Biophamaceuticals: Applications and Challenges. 2013. SR Schmidt, Wiley; Boulter et al., Protein Eng. 2003 Sep;16(9):707-11; Liddy et al., Nat Med. 2012 Jun;1 8(6):980-7), or to genetically engineer T cells for adoptive therapy (Fesnak et al., Nat Rev Cancer. 2016 Aug 23;16(9):566-81).

[0006] TCRs that bind to SLLQHLIGL (SEQ ID NO: 1) in complex with HLA-A*02 have been previously reported (Amir et al., Clin Cancer Res. 2011 Sep. 1;17(17):5615-25; Griffioen et al., Clin Cancer Res. 2006 May 15;12(10):3130-6; WO2016142783). However, these TCRs have not been engineered to bind target antigens with higher affinity than native TCRs. As explained further below, supraphysiological antigen affinity is a desirable characteristic for therapeutic TCRs, which are not straightforward to produce, especially when balanced with other desirable characteristics such as specificity.

[0007] The TCR sequences described herein are described with reference to the IMGT nomenclature, which is widely known and accessible to those skilled in the art of TCRs. 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 100; and Lefranc (2003), Leukemia 17(1):260-266. Briefly, the αβ TCR consists of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains: a variable domain and a constant domain. A short connecting region connects the variable and constant domains, and this connecting region is typically considered part of the α variable region. In addition, the β chain usually contains a short diversity region next to the joining region, which is also typically considered part of the β variable region.

[0008] The variable domain of each chain is located at the N-terminus and comprises three complementarity-determining regions (CDRs) embedded in framework sequences. The CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding the α chain variable (Vα) region and several genes encoding the β chain variable (Vβ) region, which are distinguished by framework, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. The Vα and Vβ genes are designated by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature (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 joining or J genes for the α and β chains, called TRAJ or TRBJ, respectively, and a diversity or D gene for the β 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 enormous diversity of T cell receptor chains results from a combination of rearrangements between different V, J, and D genes (including allelic variants) and joining diversity (Arstila et al. (1999), Science 286(5441):958-961; Robins et al. (2009), Blood 114(19):4099-4107). The constant or C regions of the TCR α and β chains are called TRAC and TRBC, respectively (Lefranc (2001), Curr Protoc Immunol Appendix 1:Appendix 10).

[0009] The inventors of the present application have surprisingly discovered a novel TCR that can bind to the SLLQHLIGL-HLA-A*02 complex with high affinity and specificity. The TCR is engineered from a suitable scaffold sequence into which multiple mutations are introduced. The TCR of the present invention has properties that make it particularly suitable for therapeutic use. In general, identifying such TCRs is not easy and they usually have a high attrition rate.

[0010] In the first instance, a skilled artisan must identify an appropriate starting or scaffold sequence. Typically, such sequences are obtained from natural sources, e.g., antigen-responsive T cells extracted from donor blood. Given the rarity of cancer-specific T cells in the natural repertoire, it is often necessary to screen many donors, e.g., 20 or more, to find responding T cells. The selection process can take weeks or months, and even if responding T cells are found, they may not be suitable for immunotherapy. For example, the response may be too weak and / or not specific for the target antigen. Alternatively, it may not be possible to generate a clonal T cell population or expand or maintain a particular T cell line to produce enough material to identify the correct TCR chain sequence. A TCR sequence suitable as a starting or scaffold sequence should have one or more of the following properties: good affinity for the target peptide-HLA complex, e.g., 200 μM or greater; high level of target specificity, e.g., relatively weak or no binding to alternative peptide-HLA complexes; suitable for use in display libraries, such as phage display; and the ability to be refolded and purified in high yield. Given the degenerate nature of TCR recognition, determining whether a particular scaffold TCR sequence has a specificity profile that makes it eligible for genetic engineering for therapeutic use can be quite challenging, even for experienced practitioners (Wooldridge et al., J Biol Chem. 2012 Jan. 6;287(2):1168-77).

[0011] The next challenge is to genetically engineer TCRs to enhance their affinity for target antigens while retaining desirable properties such as specificity and yield. Naturally occurring TCRs have weaker affinity for target antigens (in the micromolar range) compared to antibodies, and TCRs directed against cancer antigens typically recognize them less strongly than virus-specific TCRs (Aleksic et al., Eur J Immunol. 2012 Dec;42(12):3174-9). This weak affinity, coupled with HLA downregulation on cancer cells, means that therapeutic TCRs for cancer immunotherapy typically require genetic engineering to enhance their affinity for target antigens and thus generate more potent responses. Such increased affinity is essential for soluble TCR-based reagents. In such cases, antigen-binding affinities in the nanomolar to picomolar range with binding half-lives of several hours are desirable. The improved potency generated by high-affinity antigen recognition with a low epitope number is illustrated in Figures 1e and 1f of Liddy et al. (Liddy et al., Nat Med. 2012 June;18(6):980-7). The affinity maturation process typically requires one skilled in the art to engineer specific mutations and / or combinations, including but not limited to, mutations, substitutions, insertions, and / or deletions, into the starting TCR sequence to enhance the strength of antigen recognition. Methods for engineering affinity-enhancing mutations for a given TCR are known in the art, for example, using display libraries. (Li et al., Nat Biotechnol. 2005 March;23(3):349-54; Holler et al., Proc Natl Acad Sci USA. 2000 May 9;97(10):5387-92). However, to produce a significant increase in the affinity of a given TCR for a given target, one skilled in the art may have to engineer a combination of mutations from a large pool of possible options. The specific mutations and / or combinations of mutations that produce a significant increase in affinity are unpredictable and subject to high attrition. In many cases, it is not possible to significantly improve affinity with a given TCR starting sequence.

[0012] The affinity maturation process also requires consideration of the need to maintain TCR antigen specificity. Increasing the affinity of a TCR for its target antigen increases the risk of cross-reactivity with other unintended targets as a result of the inherent degeneracy of TCR antigen recognition (Wooldridge et al., J Biol Chem. 2012 January 6;287(2):1168-77; Wilson et al., Mol Immunol. 2004 February;40(14-15):1047-55; Zhao et al., J Immunol. 2007 November 1;179(9):5845-54). At natural levels of affinity, recognition of cross-reactive antigens may be too low to generate a response. If the cross-reactive antigen is found on normal, healthy cells, the likelihood of off-target binding in vivo is high, potentially resulting in clinical toxicity. Therefore, in addition to increasing antigen binding strength, those skilled in the art must also engineer mutations and / or combinations of mutations that allow the TCR to retain high specificity for the target antigen and demonstrate a good safety profile in preclinical trials. Again, suitable mutations and / or combinations of mutations cannot be predicted. The attrition rate at this stage is even higher and may often not be achievable at all from a given TCR starting sequence.

[0013] Despite the above challenges, we have identified mutant TCRs with particularly high affinity (picomolar range) and high antigen specificity that, when prepared as soluble reagents fused to T cell redirecting moieties, demonstrate potent killing of PRAME-positive cancer cells. Summary of the Invention

[0014] In a first aspect, the present invention provides a TCR antibody having binding affinity to SLLQHLIGL (SEQ ID NO: 1) which forms a complex with HLA-A*02, the TCR antibody comprising a TCR α chain variable domain and / or a TCR β chain variable domain, each of which comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FRs are framework regions and CDRs are complementarity determining regions; (a) the sequence of the α chain CDR is: CDR1 - TISGTDY (SEQ ID NO: 39) CDR2 - GLTSN (SEQ ID NO: 40) CDR3 - CILILGHSGAGSYQLTF (SEQ ID NO: 41) optionally containing one or more mutations therein, and / or (b) the sequence of the β chain CDR is as follows: CDR1 - LNHDA (SEQ ID NO: 42) CDR2 - SQIVNDF (SEQ ID NO: 43) CDR3 - CASSPWTSGSREQYF (SEQ ID NO: 44) Optionally, include one or more mutations therein Provides T cell receptors (TCRs).

[0015] In the TCR of the first aspect, the alpha chain variable domain framework region comprises the following framework sequence: FR1 - amino acids 1-25 of SEQ ID NO:2 FR2 - amino acids 33-49 of SEQ ID NO:2 FR3 - amino acids 55-87 of SEQ ID NO:2 FR4 - amino acids 105-114 of SEQ ID NO:2 or a respective sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to said sequence; and / or The β chain variable domain framework region has the following sequence: FR1 - amino acids 1-26 of SEQ ID NO:3 FR2 - amino acids 32-48 of SEQ ID NO:3 FR3 - amino acids 56-90 of SEQ ID NO:3 FR4 - amino acids 106-114 of SEQ ID NO:3 or may comprise a respective sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to said sequence.

[0016] The term "mutation" includes substitutions, insertions, and deletions. Mutations to the parent (or wild-type, or scaffold) TCR may affect the binding affinity (k D and / or binding half-life).

[0017] Conventionally, β-strand residue F55 is considered part of framework region 3. However, for the purposes of the present invention, β-strand residue F55 is considered part of CDR2.

[0018] The α chain framework regions FR1, FR2, and FR3 can comprise amino acid sequences corresponding to those of the TRAV 26-2 chain, and / or the β chain framework regions FR1, FR2, and FR3 can comprise amino acid sequences corresponding to those of the TRBV19 chain.

[0019] The FR4 region may contain the binding regions for the α and β variable chains (TRAJ and TRBJ, respectively).

[0020] There may be at least one mutation in the TCR alpha chain variable region. There may be one, two, three, four, five or more mutations in the alpha chain CDRs. There may be one, two, three, four or five mutations in the alpha chain CDR3. With reference to the numbering of SEQ ID NO: 2, one or more of the mutations may be the following mutations: [Table 1] may be selected from: Thus, any or all of the mutations in the above table may be present, optionally in combination with other mutations.

[0021] The alpha chain CDR3 has the following mutation group (with reference to the numbering of SEQ ID NO: 2): [Table 2] may include one of: A preferred group of mutations is Group 1. Another preferred group of mutations is Group 2.

[0022] The alpha chain CDR3 is: [Table 3] It may have a sequence selected from: A preferred alpha chain CDR3 is CILILGHSRAGNYIATF (SEQ ID NO: 45). A preferred alpha chain CDR3 is CILILGHSRLGNYIATF (SEQ ID NO: 46).

[0023] There is at least one mutation in the TCR β chain variable region. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations in the CDRs of the β chain. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations in the β chain CDR3. One or more of the mutations may be the following mutations, with reference to the numbering of SEQ ID NO: 3: [Table 4] may be selected from: Thus, any or all of the mutations in the above table may be present, optionally in combination with other mutations.

[0024] The β chain CDR2 and CDR3 are mutated in the following groups (with reference to the numbering of SEQ ID NO: 3): [Table 5] may include one of: A preferred mutation group is group 1. A preferred mutation group is group 9.

[0025] The β chain CDR2 is as follows: [Table 6] It may have a sequence selected from: A preferred β chain CDR2 is SQIMGDE (SEQ ID NO: 48).

[0026] The CDR3 of the β chain is: [Table 7] It may have a sequence selected from: A preferred β chain CDR3 is CASSWWTGGASPISF (SEQ ID NO: 51). A preferred β chain CDR3 is CASSWWTGGASPIRF (SEQ ID NO: 58).

[0027] Preferred combinations of β chain CDR2 and CDR3 are shown in the table below. [Table 8] A preferred combination is Combination 1. Another preferred combination is Combination 9.

[0028] In a preferred embodiment, the TCR α and β chain CDR sequences are as follows: [Table 9] is selected from. A preferred combination is combination 1. A preferred combination is combination 17.

[0029] Mutations within the CDRs preferably improve the binding affinity of the TCR to the SLLQHLIGL-HLA-A*02 complex, but may additionally or alternatively confer other benefits, such as improved isolated form stability and improved specificity. Mutations at one or more positions may additionally or alternatively affect the interaction of adjacent positions with the cognate pMHC complex, for example, by providing a more favorable angle for the interaction. Mutations may include those that can reduce the amount of nonspecific binding, i.e., those that can reduce binding to surrogate antigens compared to SLLQHLIGL-HLA-A*02. Mutations may also include those that increase folding and / or manufacturing efficiency. Some mutations contribute to each of these properties, while others may also contribute, for example, to affinity but not specificity, or specificity but not affinity.

[0030] Generally, a total of at least 5, at least 10, at least 15, or more CDR mutations are required to obtain a TCR with pM affinity for a target antigen. A total of at least 5, at least 10, or at least 15 CDR mutations may be required to obtain a TCR with pM affinity for a target antigen. TCRs with pM affinity for a target antigen are particularly suitable as soluble therapeutics. TCRs for use in adoptive therapy applications may have lower affinity for the target antigen, i.e., fewer CDR mutations, for example, up to 1, 2, 5, or more CDR mutations in total. TCRs for use in adoptive therapy applications may have lower affinity for the target antigen, i.e., fewer CDR mutations, for example, up to 1, 2, or 5 CDR mutations in total.

[0031] Mutations may additionally or alternatively occur outside the CDRs, within the framework regions, and such mutations may improve binding, and / or specificity, and / or stability, and / or yield of purified, soluble forms of the TCR. For example, the TCRs of the present invention may additionally or alternatively comprise an α chain variable domain, wherein the α chain variable region FR1 has a G residue at position -1, i.e., inserted before position -1, using the numbering of SEQ ID NO: 2. The G at position -1 has been found to improve the efficiency of cleavage of the N-terminal methionine during production in E. coli. Inefficient cleavage may be detrimental to therapy because it may result in a heterogeneous protein product and / or because the presence of an initial methionine may be immunogenic in humans.

[0032] Preferably, the α chain variable domain of the TCR of the present invention may comprise a respective framework amino acid sequence having 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% identity to framework amino acid residues 1 to 25, 33 to 49, 55 to 87, and 105 to 114 of SEQ ID NO: 2. The β chain variable domain of the TCR of the present invention may comprise a respective framework amino acid sequence having 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% identity to framework amino acid residues 1 to 26, 32 to 48, 56 to 90, and 106 to 114 of SEQ ID NO: 3. Alternatively, the indicated percentage identity may exceed the framework sequence when considered as a whole.

[0033] The α chain variable domain may comprise any one of the amino acid sequences of SEQ ID NOs: 6-8, and the β chain variable domain may comprise any one of the amino acid sequences of SEQ ID NOs: 9-24.

[0034] For example, a TCR consists of the following alpha and beta chain pairs: [Table 10] may include: A preferred TCR chain pair is SEQ ID NO: 6 and SEQ ID NO: 9. A preferred TCR chain pair is SEQ ID NO: 7 and SEQ ID NO: 17.

[0035] Phenotypically silent variants of any of the TCRs of the invention disclosed herein are also within the scope of the present invention. As used herein, the term "phenotypically silent variant" is understood to refer to a TCR variable domain that incorporates, in addition to the mutations described above, one or more additional amino acid changes, including substitutions, insertions, and deletions, that have a phenotype similar to the corresponding TCR without said changes. For purposes of this application, TCR phenotype is defined as the binding affinity (KD and / or binding half-life) and specificity. Preferably, the phenotype of a soluble TCR associated with an immune effector includes binding affinity and specificity as well as immune activation potency and purification yield. Phenotypically silent variants have a measured K of a corresponding TCR without said alteration with respect to the SLLQHLIGL-HLA-A*02 complex when measured under identical conditions (e.g., at 25°C and / or on the same SPR chip). D and / or K within 50%, more preferably within 30%, 25%, or 20% of the binding half-life D and / or binding half-life. Suitable conditions are further provided in Example 3. As will be known to those skilled in the art, it may be possible to generate TCRs incorporating changes in the variable domain compared to those detailed above, without altering the affinity of the interaction with the SLLQHLIGL-HLA-A*02 complex and / or other functional properties. In particular, such silent mutations may be incorporated into parts of the sequence known not to be directly involved in antigen binding (e.g., framework regions and / or non-antigen-contacting parts of the CDRs). Such variants are also within the scope of the present invention.

[0036] A phenotypically silent variant may contain one or more conservative substitutions and / or one or more tolerated substitutions. A tolerated substitution refers to a substitution that does not meet the conservative definition as set forth below, but is still phenotypically silent. Those skilled in the art recognize that various amino acids have similar properties and are therefore "conservative." One or more such amino acids of a protein, polypeptide, or peptide can often be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0037] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) can often be substituted for one another. Of these possible substitutions, glycine and alanine are preferred for substitution for one another (because they have relatively short side chains), and valine, leucine, and isoleucine are preferred for substitution for one another (because they have longer, hydrophobic aliphatic side chains). Other amino acids that can often be substituted for one another include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present invention can be made with naturally occurring or non-naturally occurring amino acids. For example, herein, the methyl group of alanine may be substituted with an ethyl group and / or minor changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0038] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. Accordingly, the present invention extends to the use of any amino acid sequence which has one or more conservative substitutions and / or one or more tolerated substitutions in its sequence, such that the amino acid sequence of the TCR has at least 90% identity, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a TCR comprising amino acids 1 to 114 of SEQ ID NOs: 2, 6 to 8, and / or amino acids 1 to 114 of SEQ ID NOs: 3, 9 to 24.

[0039] "Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as appropriate, as determined by the match of their alignment. While several methods exist for measuring identity between two polypeptide sequences or two polynucleotide sequences, commonly used methods for determining identity are computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0040] Amino acid sequences can be compared using a program such as the CLUSTAL program. This program compares amino acid sequences and, if necessary, inserts spaces in either sequence to find a suitable alignment. Amino acid identity or similarity (identity + conservation of amino acid type) can be calculated for a suitable alignment. Programs such as BLASTx also align similar sequences over time and assign a value for the fit. Thus, a comparison can be made in which several similar regions are found, each with a different score. Both types of identity analysis are contemplated in the present invention.

[0041] The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for proper comparison purposes (e.g., gaps can be introduced into the first sequence for good alignment) and comparing the amino acid residues or nucleotides at corresponding positions. A "good alignment" is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions × 100).

[0042] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410 incorporate such an algorithm. The determination of percent identity between two nucleotide sequences can be performed using the BLASTn program. The determination of percent identity between two protein sequences can be performed using the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches that detect distant relatedness between molecules (supra). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs can be used (e.g., BLASTp and BLASTp). See http: / / www.ncbi.nlm.nih.gov. Common default parameters include, for example, Word Size = 3 and Expect Threshold = 10. Parameters can be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.

[0043] Other sequence analysis algorithms known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA, described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. In the present disclosure, for purposes of assessing percent identity, BLASTp with default parameters is used as the comparison methodology. Furthermore, if the stated percent identity provides a non-integer value for an amino acid (i.e., a sequence of 25 amino acids with 90% sequence identity provides a value of "22.5"), the resulting value is rounded down to the next integer, i.e., "22". Thus, in the provided example, a sequence with 22 matches out of 25 amino acids is within 90% sequence identity.

[0044] As will be apparent to those skilled in the art, the provided sequences may be truncated by 1, 2, 3, 4, 5, 6 or more residues at their C-terminus and / or N-terminus without substantially affecting the functional properties of the TCR. The provided sequences at their C-terminus and / or N-terminus may be shortened or extended by 1, 2, 3, 4 or 5 residues. All such variants are encompassed by the present invention.

[0045] Mutations, including conservative and tolerated substitutions, insertions, and deletions, can be introduced into the provided sequences using any suitable method. Such methods include, but are not limited to, polymerase chain reaction (PCR)-based methods, restriction enzyme-based cloning, or ligation-independent cloning (LIC) procedures. These methods are described in detail in many standard molecular biology textbooks. For further details regarding polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook and Russell (2001) Molecular Cloning - A Laboratory Manual (3 rd Ed.) CSHL Press. Further information on ligation-independent cloning (LIC) procedures can be found in Rashtchian (1995) Curr Opin Biotechnol 6(1):30-6. The TCR sequences provided by the present invention can be produced by solid phase synthesis or obtained from other suitable methods known in the art.

[0046] The TCR of the present invention has binding ability to the SLLQHLIGL-HLA-A*02 complex. The TCR of the present invention exhibits high specificity for the SLLQHLIGL-HLA-A*02 complex and is therefore particularly suitable for therapeutic use. The specificity relates to the ability of the TCR of the present invention to recognize antigen-positive HLA-A*02 target cells, while showing very little ability to recognize antigen-negative HLA-A*02 target cells.

[0047] Specificity can be measured in vitro, for example, in the cell assays described in Examples 6, 7, and 8. To test specificity, the TCR may be in soluble form and bound to immune effectors and / or expressed on the surface of cells such as T cells. Recognition may be determined by measuring the level of T cell activation in the presence of antibody-positive and antibody-negative target cells. Minimal recognition of antigen-negative target cells is defined as a level of T cell activation that is less than 20%, preferably less than 10%, preferably less than 5%, and more preferably less than 1% of the level that occurs in the presence of antigen-positive target cells, when measured under the same conditions and at a therapeutically relevant TCR concentration. For soluble TCRs bound to immune effectors, a therapeutically relevant concentration is less than 10 -9 This may be defined as a TCR concentration equal to or less than 100 M and / or up to 100-fold, or preferably up to 1000-fold greater than the corresponding EC50 value. Preferably, for soluble TCRs associated with immune effectors, there is at least a 100-fold difference in the concentration required for T cell activation against antigen-positive cells compared to antigen-negative cells. Antigen-positive cells may be obtained by peptide-pulsing using appropriate peptide concentrations to obtain levels of antigen presentation comparable to cancer cells (e.g., 10-fold as described in Bossi et al. (2013) Oncoimmunol. 1; 2(11):e26840). -9 The antigen-positive cells may be human cells (e.g., a peptide of M) or may naturally present the peptide. Preferably, both the antigen-positive cells and the antigen-negative cells are human cells. More preferably, the antigen-positive cells are human cancer cells. Preferably, the antigen-negative cells include those derived from healthy human tissue.

[0048] Specificity may additionally or alternatively relate to the ability of a TCR to bind to the SLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex rather than to a panel of alternative peptide-HLA complexes. This may be determined, for example, by the Biacore method of Example 3. The panel may include at least 5, preferably at least 10, alternative peptide-HLA-A*02 complexes. The alternative peptides may share a low level of sequence identity with SEQ ID NO: 1 and may be naturally presented. Preferably, the alternative peptides are derived from proteins expressed in healthy human tissue. Binding of the TCR to the SLLQHLIGL-HLA-A*02 complex may be at least 2-fold, more preferably at least 10-fold, or at least 50-fold, or at least 100-fold, and even more preferably at least 400-fold, greater than other naturally presented peptide-HLA complexes.

[0049] An alternative or additional approach to determining TCR specificity can be to identify peptide recognition motifs of the TCR using sequential mutagenesis, such as alanine scanning. Residues that form part of the binding motif are those that are not tolerated for substitution. Non-tolerant substitutions can be defined as peptide positions where the binding affinity of the TCR is reduced by at least 50%, or preferably at least 80%, relative to the binding affinity of the non-mutated peptide. Further such approaches are described in Cameron et al. (2013), Sci Transl Med. 2013 Aug. 7; 5(197):197ral03 and WO2014096803. TCR specificity in this case can be determined by identifying alternative motif-containing peptides, particularly those from the human proteome, and testing these peptides for binding to the TCR. Binding of the TCR to one or more alternative peptides may indicate a lack of specificity. In this case, further testing of TCR specificity using a cellular assay may be necessary.

[0050] The TCRs of the present invention may have an ideal safety profile for use as therapeutic agents. In this case, the TCR may be in a soluble form and preferably fused to an immune effector. Suitable immune effectors include, but are not limited to, cytokines such as IL-2 and IFN-γ; superantigens and their variants; chemokines such as IL-8, platelet factor 4, and melanoma growth stimulating protein; antibodies, including fragments, derivatives, and variants thereof, that bind to antigens on immune cells such as T cells or NK cells (e.g., anti-CD3, anti-CD28, or anti-CD16); and complement activators.

[0051] An ideal safety profile means that in addition to exhibiting good specificity, the TCRs of the present invention may have also passed preclinical safety tests, examples of such tests include whole blood assays to confirm minimal cytokine release in the presence of whole blood and therefore a low risk of causing potential cytokine release syndrome in vitro, and alloreactivity tests to confirm a low likelihood of recognition of alternative HLA types.

[0052] The TCRs of the present invention, particularly soluble forms of TCRs, may be suitable for high-yield purification. Yield can be defined based on the amount of material retained during the purification process (i.e., the amount of correctly folded material obtained at the end of the purification process relative to the amount of solubilized material obtained before refolding), and / or based on the amount of correctly folded material obtained at the end of the purification process relative to the original culture volume. High yield means a yield of 1% or more, or more preferably 5% or more, or higher. High yield means a yield of more than 1 mg / ml, or more preferably more than 3 mg / ml, or more than 5 mg / ml, or higher.

[0053] The TCRs of the invention preferably have a K greater (i.e. more potent) than non-mutated or scaffold TCRs with respect to the SLLQHLIGL-HLA-A*02 complex, for example a K in the range of 1 pM to 100 μM. DIn one aspect, the TCRs of the present invention have a K with respect to the complex of about (i.e., + / - 10%) 1 pM to about 400 nM, about 1 pM to about 1000 pM, about 1 pM to about 500 pM. D The TCR may additionally or alternatively have a binding half-life (T1 / 2) with respect to the complex ranging from about 1 minute to about 60 hours, from about 20 minutes to about 50 hours, or from about 2 hours to about 35 hours. In particularly preferred embodiments, the TCR of the present invention has a K of about 1 pM to about 500 pM with respect to the SLLQHLIGL-HLA-A*02 complex. D and / or has a binding half-life of about 2 hours to about 35 hours. Such high affinity is preferred for a soluble form of the TCR when conjugated with a therapeutic agent and / or a detectable label.

[0054] In another aspect, the TCR of the present invention has a K of about 50 nM to about 200 μM, or about 100 nM to about 1 μM, with respect to the complex. D and / or may have a binding half-life of about 3 seconds to about 12 minutes. Such TCRs may be preferred for adoptive therapy applications.

[0055] Binding affinity (equilibrium constant K D Methods for determining the binding affinity (which is inversely proportional to K) and binding half-life (denoted T1 / 2) are known to those skilled in the art. In a preferred embodiment, binding affinity and binding half-life can be determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), for example using a BIAcore instrument or an Octet instrument, respectively. A preferred method is shown in Example 3. A doubling of the affinity of a TCR results in a K D is understood to be 1 / 2. T1 / 2 is ln2 / dissociation rate (k off ) Therefore, if T1 / 2 is doubled, k off becomes 1 / 2. K of TCR D value and k offValues are typically measured for soluble forms of the TCR (i.e., truncated to remove residues of the cytoplasmic and transmembrane domains). To account for variability between independent measurements, particularly interactions with dissociation times greater than 20 hours, the binding affinity and / or binding half-life of a given TCR is measured several times, e.g., three or more times, using the same assay protocol and the results are averaged. To compare binding data for two samples (i.e., two different TCRs and / or two preparations of the same TCR), it is preferable to perform the measurements using the same assay conditions (e.g., temperature), as described in Example 3.

[0056] Certain preferred TCRs of the present invention have a substantially higher binding affinity and / or binding half-life for the SLLQHLIGL-HLA-A*02 complex than that of native TCRs. Increasing the binding affinity of a native TCR often reduces the specificity of the TCR for its peptide-MHC ligand, as demonstrated in Zhao Yangbing et al., J. Immunol, 179:9, 5845-5854. However, such TCRs of the present invention remain specific for the SLLQHLIGL-HLA-A*02 complex despite having a substantially higher binding affinity than native TCRs.

[0057] Certain preferred TCRs are capable of generating highly potent T cell responses in vitro against antigen-positive cells, particularly cells presenting low levels of antigen typical of cancer cells (i.e., in the order of 5-100, e.g., 50 antigens per cell (Bossi et al., (2013) Oncoimmunol. 1;2(11):e26840; Purbhoo et al., (2006), J Immunol 176(12):7308-7316)). Such TCRs may be in soluble form and linked to immune effectors such as anti-CD3 antibodies. The measured T cell response may be the release of T cell activation markers such as interferon-γ or granzyme B, or target cell killing, or other measures of T cell activation such as T cell proliferation. Preferably, highly potent responses have an EC50 in the pM range. 50value, most preferably a response of 100 pM or less.

[0058] The TCR of the present invention may be an αβ heterodimer. The αβ heterodimer TCR of the present invention typically comprises an α chain TRAC constant domain sequence and / or a β chain TRBC1 or TRBC2 constant domain sequence. The constant domains may be full-length, meaning that the extracellular, transmembrane, and cytoplasmic domains are present, or may be in soluble form (i.e., without the transmembrane or cytoplasmic domains). One or both of the constant domains may contain mutations, substitutions, or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. The terms TRAC and TRBC1 / 2 also encompass naturally occurring polymorphic variants, such as N to K at position 4 of TRAC (Bragado et al., International Immunology, February 1994; 6(2):223-30).

[0059] In the case of the soluble TCRs of the present invention, the constant domain sequences of the α and β chains may be modified by truncation or substitution to eliminate the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The constant domain sequences of the α and / or β chains may have a disulfide bond introduced between residues of the respective constant domains, for example, as described in WO 03 / 020763. In a preferred embodiment, the α and β constant domain sequences may be modified by substituting a cysteine residue at Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, which may form a disulfide bond between the α and β constant domains of the TCR. TRBC1 or TRBC2 may further comprise a cysteine to alanine mutation at position 75 of the constant region and an asparagine to aspartic acid mutation at position 89 of the constant region. One or both of the extracellular constant domains present in the αβ heterodimer of the present invention may have, for example, up to 15, 10, 8, or 7 or fewer amino acids deleted at one or both C-termini. One or both of the extracellular constant domains present in the αβ heterodimer of the present invention may have, for example, up to 15, 10, or 8 amino acids deleted at one or both C-termini. The C-terminus of the α chain extracellular constant region may have 8 amino acids deleted. The water-soluble TCR is preferably conjugated to a therapeutic agent and / or a detectable label.

[0060] The constant domains of an αβ heterodimeric TCR may be full-length, including both transmembrane and cytoplasmic domains. Such TCRs may contain disulfide bonds corresponding to those found in nature between the respective α and β constant domains. Additionally or alternatively, non-native disulfide bonds may be present between the extracellular constant domains. Non-native disulfide bonds are further described in WO03020763 and WO06000830. The non-native disulfide bond may be between Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2. One or both of the constant domains may contain one or more mutations, substitutions, or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. TCRs with full-length constant domains are suitable for use in adoptive therapy.

[0061] The TCRs of the present invention may be in a single chain format, including, but not limited to, αβTCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L - Vβ-Cβ type, where Vα and Vβ are the TCR α and β variable regions, respectively, Cα and Cβ are the TCR α and β constant regions, respectively, and L is a linker sequence (Weidanz et al. (1998) J Immunol Methods. December 1; 221(1-2):59-76; Epel et al. (2002) Cancer Immunol Immunother. November; 51(10):565-73; WO 2004 / 033685; WO9918129).

[0013] If present, one or both of the constant domains may be full-length, or they may be truncated and / or contain mutations as described above. Preferably, the single-chain TCR is soluble. In a specific embodiment, the single-chain TCR of the invention may have disulfide bonds introduced between residues of each constant domain, as described in WO 2004 / 033685. Single-chain TCRs are further described in WO 2004 / 033685; WO 98 / 39482; WO 01 / 62908; Weidanz et al. (1998) J Immunol Methods 221 (1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829).

[0062] The present invention also includes particles displaying the TCRs of the present invention, and the inclusion of said particles in libraries of particles. Such particles include, but are not limited to, phage, yeast cells, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (see, e.g., WO2004 / 044004; WO01 / 48145; Chervin et al. (2008) J. Immuno. Methods 339.2:175-184).

[0063] The soluble TCRs of the present invention are useful for delivering detectable labels or therapeutic agents to antigen-presenting cells and tissues containing antigen-presenting cells, and thus may be linked (covalently or otherwise) to a detectable label (for diagnostic purposes in which the TCR is used to detect the presence of cells presenting the cognate antigen) and / or a therapeutic agent and / or a PK modulating moiety.

[0064] Examples of PK modulating moieties include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci, October 28;16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J. January;5(1):113-28), PASylation (Schlapschy et al., (2013) Protein Eng Des Sel. August;26(8):489-501), albumin and albumin binding domains (Dennis et al., (2002) J Biol Chem. September 20;277(38):35035-43), and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. December;27(12):1186-90). Further PK modulating moieties include antibody Fc fragments.

[0065] Detectable labels for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes and imaging agents.

[0066] For some purposes, the TCRs of the present invention may aggregate into complexes comprising several TCRs to form multivalent TCR complexes. There are human proteins containing multimerization domains that can be used to prepare multivalent TCR complexes. For example, the tetramerization domain of p53 has been utilized to generate tetrameric scFv antibody fragments that exhibit increased serum retention and significantly reduced dissociation rates compared to monomeric scFv fragments (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392). Hemoglobin also possesses a tetramerization domain that can be used for this type of application. The multivalent TCR complexes of the present invention may have enhanced binding capabilities relative to the complex compared to non-multimeric wild-type or T cell receptor heterodimers of the present invention. Thus, multivalent complexes of the TCRs of the present invention are also encompassed by the present invention. Such multivalent TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting a particular antigen in vitro or in vivo, and are also useful as intermediates for the production of further multivalent TCR complexes having such uses.

[0067] Therapeutic agents that can be conjugated to the TCRs of the present invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxin can be present inside a liposome linked to the TCR for slow release. This prevents damaging effects during transport in the human body and ensures that the toxin has maximum effect after binding of the TCR to the appropriate antigen-presenting cell.

[0068] Examples of suitable therapeutic agents include, but are not limited to: Small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, these small molecule cytotoxic agents are also understood to include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolmide, topotecan, trimetrexate 21arbour21ate, glucuronate, auristatin E, vincristine, and doxorubicin; Peptide cytotoxins, i.e. proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNAases and RNAases; Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents may be used to facilitate the binding of these radionuclides to high-affinity TCRs or multimers thereof; Immunostimulators, i.e., immune effector molecules that stimulate the immune response, such as cytokines (e.g., IL-2 and IFN-γ); · Superantigens and their variants; · TCR-HLA fusions, e.g., fusions of peptides to peptide-HLA complexes derived from common human pathogens, e.g., Epstein-Barr virus (EBV); · Chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28 or anti-CD16); Alternative protein scaffolds with antibody-like binding properties ·Complement activators; Heterologous protein domains, homologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0069] One preferred embodiment is provided by a soluble TCR of the invention linked to an immune effector (usually by fusion to the N- or C-terminus of the α or β chain). A particularly preferred immune effector is an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody (this TCR-anti-CD3 fusion is also referred to as an ImmTAC® molecule). As used herein, the term "antibody" encompasses such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031, and 12F6. Antibody fragments and variants / analogues suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, Nanobodies® (commercially available from Ablynx (Belgium), these constructs comprise synthetic single-chain immunoglobulin variable heavy domains derived from camelid (e.g., camel or llama) antibodies), and domain antibodies (Domantis (Belgium), which comprise affinity-matured single-chain immunoglobulin variable heavy domains or immunoglobulin variable light domains) or alternative protein scaffolds that exhibit antibody-like binding properties, such as affibodies (Affibody (Sweden), which comprise an engineered Protein A scaffold) or anticalins (Pieris (Germany)), which comprise engineered anticalins), to name a few.

[0070] The TCR and anti-CD3 antibody are linked via covalent or non-covalent attachment. The covalent bond can be direct or indirect via a linker sequence. The linker sequence is typically flexible in that it is made primarily of amino acids such as glycine, alanine, and serine, which lack bulky side chains that would likely limit flexibility. Alternatively, a more rigid linker may be desirable. Suitable or optimal linker sequence lengths for use are readily determined. Linker sequences are often less than about 12 amino acids in length, e.g., less than 10 amino acids in length, or between 2 and 10 amino acids in length. Examples of suitable linkers that may be used in the TCRs of the present invention include, but are not limited to, GGGGS (SEQ ID NO: 31), GGGSG (SEQ ID NO: 32), GGSGG (SEQ ID NO: 33), GSGGG (SEQ ID NO: 34), GSGGGP (SEQ ID NO: 35), GGEPS (SEQ ID NO: 36), GGEGGGP (SEQ ID NO: 37) and GGEGGGSEGGGS (SEQ ID NO: 38) (as described in WO2010 / 133828).

[0071] Specific embodiments of the anti-CD3-TCR fusion constructs of the present invention include pairs of α and β chains, where the α chain is composed of a TCR variable domain comprising the amino acid sequence of SEQ ID NOs: 6 to 8, and / or the β chain is composed of a TCR variable domain comprising the amino acid sequence of SEQ ID NOs: 9 to 24. The α and β chains may further comprise a constant region comprising a non-native disulfide bond. The constant domain of the α chain may be deleted by 8 amino acids. The N- or C-terminus of the α or β chain may be fused to an anti-CD3 scFv antibody fragment via a linker selected from SEQ ID NOs: 31 to 38. Specific preferred embodiments of such anti-CD3-TCR fusion constructs are provided below: [Table 11]

[0072] Also included within the scope of the present invention are functional variants of the anti-CD3-TCR fusion constructs, which are functionally equivalent but preferably have at least 90% identity to the reference sequence, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0073] In a further aspect, the present invention provides a nucleic acid encoding a TCR or TCR anti-CD3 of the invention. In some embodiments, the nucleic acid is cDNA. In some embodiments, it may be mRNA. In some embodiments, the present invention provides a nucleic acid comprising a sequence encoding the α chain variable domain of a TCR of the invention. In some embodiments, the present invention provides a nucleic acid comprising a sequence encoding the β chain variable domain of a TCR of the invention. The nucleic acid may be non-naturally occurring and / or purified and / or engineered. The nucleic acid sequence may be codon optimized according to the expression system to be utilized. As known to those skilled in the art, the expression system may comprise bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or may be a cell-free expression system.

[0074] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention. Preferably, the vector is a TCR expression vector. Suitable TCR expression vectors include, for example, gamma retroviral vectors, or more preferably lentiviral vectors. Further details can be found in Zhang 2012 and references therein (Zhang et al., Adv Drug Deliv Rev. 2012 Jan. 1; 64(8):756-762).

[0075] The present invention also provides cells harboring a vector of the present invention, preferably a TCR expression vector. Suitable cells include mammalian cells, preferably immune cells, and even more preferably T cells. The vector may comprise a nucleic acid of the present invention encoding the α chain and the β chain in a single open reading frame, or may comprise a nucleic acid of the present invention encoding the α chain and the β chain, respectively, in two separate open reading frames. Another aspect provides cells harboring a first expression vector comprising a nucleic acid encoding the α chain of a TCR of the present invention and a second expression vector comprising a nucleic acid encoding the β chain of a TCR 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.

[0076] Because the TCRs of the present invention are useful in adoptive therapy, the present invention encompasses non-naturally occurring and / or purified and / or engineered cells, particularly T cells, that present the TCRs of the present invention. The present invention also provides expanded populations of T cells that present the TCRs of the present invention. Several methods exist that are suitable for transfecting T cells with nucleic acids (e.g., DNA, cDNA, or RNA) encoding the TCRs of the present invention (see, e.g., Robbins et al. (2008) J Immunol. 180:6116-6131). T cells expressing the TCRs of the present invention are suitable for use in adoptive therapy-based cancer treatment. As known to those skilled in the art, several suitable methods exist that allow for the implementation of adoptive therapy (see, e.g., Rosenberg et al. (2008) Nat Rev Cancer 8(4):299-308).

[0077] As is well known in the art, TCRs may be subject to post-translational modifications. Glycosylation is one such modification and involves the covalent attachment of oligosaccharide moieties to defined amino acids in TCR chains. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation status of a particular protein depends on several factors, including protein sequence, protein conformation, and the availability of specific enzymes. Furthermore, the glycosylation status (i.e., oligosaccharide type, covalent linkages, and total number of attachments) can affect protein function. Therefore, controlling glycosylation is often desirable when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapeutics (Jefferis et al., (2009) Nat Rev Drug Discov. March;8(3):226-34). For the soluble TCRs of the present invention, glycosylation may be controlled, for example, by using specific cell lines (including, but not limited to, mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells) or by chemical modification. Such modifications may be desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott, (2005) Pharm Sci. August;94(8):1626-35).

[0078] For administration to a patient, the TCRs of the invention (preferably linked to a detectable label or therapeutic agent, or expressed on transfected T cells), TCR-anti-CD3 fusion molecules, nucleic acids, expression vectors, or cells of the invention may be provided together with one or more pharmaceutically acceptable carriers or excipients as part of a sterile pharmaceutical composition. This pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be provided in unit dosage form, generally in a hermetically sealed container, and may be provided as part of a kit. Such kits will usually (but not necessarily) include instructions for use. The kit may contain multiple unit dosage forms.

[0079] The pharmaceutical compositions may be adapted for administration by any suitable route, for example, parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalant or intranasal. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0080] The dosage of the substances of the invention can vary widely depending on the disease or disorder being treated, the age and condition of the individual being treated, etc. Suitable dosage ranges for TCR-anti-CD3 fusion molecules can be 25 ng / kg to 50 μg / kg or 1 μg to 1 g. Ultimately, a physician will determine the appropriate dosage to be used.

[0081] The TCRs, TCR-anti-CD3 fusion molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in a substantially pure form, for example 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.

[0082] The present invention also provides: TCRs, TCR-anti-CD3 fusion molecules, nucleic acids, pharmaceutical compositions or cells of the invention for use in medicine, preferably for use in the treatment of cancer or tumors Use of a TCR, a TCR-anti-CD3 fusion molecule, a nucleic acid, a pharmaceutical composition, or a cell of the invention in the manufacture of a medicament for the treatment of cancer or tumors; a method for treating cancer or tumor in a patient, comprising administering to the patient a TCR, TCR-anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition, or cell of the invention; An injectable formulation for administration to a human subject comprising a TCR, a TCR-anti-CD3 fusion molecule, a nucleic acid, a pharmaceutical composition, or a cell.

[0083] The cancer may be a solid or liquid tumor. Preferably, the tumor expresses PRAME. The cancer may be of the breast (including triple negative), ovary, endometrium, esophagus, lung (NSCLC and SCLC), bladder, or head and neck. Additionally or alternatively, the cancer may be leukemia or malignant lymphoma. Of these cancers, breast cancer (including triple negative), ovary, and endometrium are preferred. The TCR, TCR-anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition, or cell of the present invention can be administered by injection, such as intravenously or directly into a tumor. The human subject may be of the HLA-A*02 subtype.

[0084] The method of treatment may further comprise administering additional anti-neoplastic agents separately, in combination, or sequentially. Examples of such agents are known in the art and may include immune activators and / or T cell modulators.

[0085] Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. Prior art documents referred to herein are incorporated by reference to the fullest extent permitted by law. [Brief explanation of the drawings]

[0086] [Figure 1] FIG. 1 provides the amino acid sequences of the extracellular regions of the scaffold PRAME TCR α and β chains. [Figure 2] FIG. 2 provides the amino acid sequences of the extracellular regions of the scaffold PRAME TCR α and β chains in soluble form. [Figure 3] FIG. 3 provides examples of amino acid sequences of mutant PRAME TCR α chain variable regions. [Figure 4-1] FIG. 4 provides examples of amino acid sequences of mutant PRAME TCR β chain variable regions. [Figure 4-2] FIG. 4 provides examples of amino acid sequences of mutant PRAME TCR β chain variable regions. [Figure 5-1] FIG. 5 shows the amino acid sequences of ImmTAC molecules (TCR-anti-CD3 fusions) containing the specific mutated PRAME TCR variable domains shown in FIGS. [Figure 5-2] FIG. 5 shows the amino acid sequences of ImmTAC molecules (TCR-anti-CD3 fusions) containing the specific mutated PRAME TCR variable domains shown in FIGS. [Figure 6] FIG. 6 provides cellular data showing the efficacy and specificity of the ImmTAC molecules of FIG. 5 containing the mutated PRAME TCR variable domains shown in FIGS. [Figure 7a] FIG. 7 (panels a and b) provides cellular data demonstrating the specificity of the ImmTAC molecules of FIG. 5 containing the mutated PRAME TCR variable domains shown in FIGS. [Figure 7b] FIG. 7 (panels a and b) provides cellular data demonstrating the specificity of the ImmTAC molecules of FIG. 5 containing the mutated PRAME TCR variable domains shown in FIGS. [Figure 8] FIG. 8 provides cellular data showing the killing of PRAME-positive melanoma cancer cells by the ImmTAC molecules of FIG. 5, which contain the mutated PRAME TCR variable domains shown in FIGS. [Figure 9] FIG. 9 provides cellular data showing the killing of PRAME-positive lung cancer cells by the ImmTAC molecules of FIG. 5, which contain the mutated PRAME TCR variable domains shown in FIGS.

[0087] The present invention is further illustrated by the following non-limiting examples. [Example]

[0088] Example Example 1 - Expression, refolding and purification of soluble TCRs method DNA sequences encoding the α and β extracellular regions of the soluble TCR of the present invention were separately cloned 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). The expression plasmids were separately transformed into the E. coli strain Rosetta (BL21pLysS) or the T7 expression system, and single ampicillin-resistant colonies were grown at an OD of approximately 0.6-0.8 in TYP (+ampicillin 100 μg / ml) medium at 37°C. 600 After growth to 0°C, protein expression was induced with 0.5 mM IPTG. Three hours after induction, cells were harvested by centrifugation. The cell pellet was lysed using BugBuster protein extraction reagent (Merck Millipore) according to the manufacturer's instructions. The inclusion body pellet was collected by centrifugation. The pellet was washed twice in Triton buffer (50 mM Tris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) and finally resuspended in detergent-free buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM NaEDTA). The cells were solubilized with 6 M guanidine-HCl and incubated at 0°C for 1 hour. 280 Inclusion body protein yield was quantified by measuring the extinction coefficient. Protein concentration was then calculated using the extinction coefficient. Purity of inclusion bodies was determined by solubilizing with 8 M urea and loading approximately 2 μg onto 4-20% SDS-PAGE under reducing conditions. Purity was then estimated or calculated using densitometry software (Chemidoc, Biorad). Inclusion bodies were stored at +4°C for short-term storage and at -20°C or -70°C for long-term storage.

[0089] For refolding of soluble TCR, α- and β-chain-containing inclusion bodies were first mixed and diluted in 10 ml of solubilization / denaturation buffer (6 M guanidine-HCl, 50 mM Tris HCl pH 8.1, 100 mM NaCl, 10 mM EDTA, 20 mM DTT), followed by incubation for 30 min at 37°C. Refolding was then initiated by further dilution in 1 L of refold buffer (100 mM Tris pH 8.1, 800 or 1000 mM L-arginine HCl, 2 mM EDTA, 4 M urea, 10 mM cysteamine HCl, and 2.5 mM cystamine dihydrochloride) and thorough mixing of the solution. The refolded mixture was dialyzed against 10 L of HO for 18–20 h at 5°C ± 3°C. The dialysis buffer was then exchanged twice with 10 mM Tris pH 8.1 (10 L) and dialysis was continued for another 15 h. The refold mixture was then filtered through a 0.4 μm cellulose filter.

[0090] Purification of soluble TCR was initiated by applying the dialyzed refold to a POROS® 50HQ anion exchange column and eluting bound protein with a gradient of 0 to 500 mM NaCl in 20 mM Tris pH 8.1 over 50 column volumes using an Äkta® Pure (GE Healthcare). Peak TCR fractions were identified by SDS-PAGE, then pooled and concentrated. The concentrated sample was then applied to a Superdex® 200 Increase 10 / 300 GL gel filtration column (GE Healthcare) pre-equilibrated with Dulbecco's PBS buffer. Peak TCR fractions were pooled and concentrated, and the final yield of purified material was calculated.

[0091] Example 2 - Expression, refolding and purification of ImmTAC molecules (soluble TCR-anti-CD3 fusion molecules) method ImmTAC was prepared as described in Example 1, except that the TCR β chain was fused to the anti-CD3 single-chain antibody via a linker. In addition, a cation exchange step was performed during the purification after anion exchange. In this case, the peak fractions from 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 eluted with a gradient of 0 to 500 mM NaCl in 20 mM MES. Peak ImmTAC fractions were pooled and adjusted to 50 mM Tris pH 8.1, then concentrated and applied directly to a gel filtration matrix as described in Example 1.

[0092] Example 3 - Binding Characterization Binding of purified soluble TCR and ImmTAC molecules to the appropriate peptide-HLA complexes was analyzed by surface plasmon resonance using a BIAcore 3000 or BIAcore T200 instrument or by double-layer interferometry using a ForteBio Octet instrument. Biotinylated class I HLA-A*02 molecules were refolded with the peptide of interest and purified using methods known to those skilled in the art (O'Callaghan et al. (1999), Anal Biochem 266(1):9-15; Garboczi et al. (1992), Proc Natl Acad Sci USA 89(8):3429-3433). All measurements were performed at 25°C in Dulbecco's PBS buffer supplemented with 0.005% P20.

[0093] BIAcore method Biotinylated peptide-HLA monomers were immobilized on a streptavidin-conjugated CM-5 sensor chip. Equilibrium binding constants were determined using serial dilutions of soluble TCR / ImmTAC injected at a constant flow rate of 30 μl / min over a flow cell coated with approximately 200 response units (RU) of peptide-HLA-A*02 complex. Equilibrium responses were normalized for each TCR concentration by subtracting the bulk buffer response from a control flow cell containing irrelevant peptide-HLA. K DValues were obtained by non-linear curve fitting using Prism software and the Langmuir binding isotherm: Binding = C × Max / (C + KD), where "Binding" is the equilibrium binding (RU) at the injected TCR concentration C, and Max is the maximum binding.

[0094] For high-affinity interactions, binding parameters were determined by single-cycle kinetic analysis. Five different concentrations of soluble TCR / ImmTAC were injected over a flow cell coated with approximately 100-200 RU of peptide-HLA complex at a flow rate of 50-60 μl / min. Typically, 60-120 μl of soluble TCR / ImmTAC was injected at the highest concentration between 50-100 nM, with two-fold serial dilutions used for the other four injections. The lowest concentration was injected first. To measure the dissociation phase, buffer was subsequently injected until ≥10% dissociation occurred, typically 1-3 hours later. Kinetic parameters were calculated using BIAevaluation® software. The dissociation phase was fitted to a first-order exponential decay equation to allow for calculation of the half-life. The equilibrium constant, K, was calculated. D is k off / k on was calculated from

[0095] Octet method Biotinylated peptide-HLA monomers were captured at 1 nm on a streptavidin biosensor (Pall ForteBio) pre-immobilized with streptavidin (SA). The sensor was blocked with free biotin (2 μM) for 2 min. Equilibrium binding constants were determined by immersing the loaded biosensor in serially diluted soluble TCR / ImmTAC in a 96-well or 384-well sample plate. Plate shaking was set to 1000 rpm. For low-affinity interactions (μM range), short association times (approximately 2 min) and short dissociation times (approximately 2 min) were used. Binding curves were processed by double-reference subtraction of an irrelevant pHLA-loaded reference biosensor using Octet data analysis software (Pall ForteBio). The equilibrium responses (nm) were used to obtain K from steady-state plots fitted to the equation Response = Rmax x *conc / (KD + conc), where "Response" is the equilibrium binding (nm) at each TCR concentration (conc) and Rmax is the maximum binding response at pHLA saturation. D The value was estimated.

[0096] For high affinity interactions (nM-pM range), kinetic parameters were determined from binding curves at concentrations of ≥3 TCR / ImmTAC, typically 10 nM, 5 nM, and 2.5 nM. Association times were 30 min, and dissociation times were 1-2 h. Binding curves were processed by double reference subtraction of an irrelevant pHLA-loaded, biotin-blocked reference biosensor. The kinetic parameter k on and k off was calculated by direct global fitting to the binding curves using Octet data analysis software (Pall ForteBio). D is k off / k on The dissociation half-life is calculated from t 1 / 2 =0.693 / k off was calculated from

[0097] Example 4 - Binding Characterization of Native TCRs Soluble native TCR was prepared according to the method described in Example 1 and binding to pHLA was analyzed according to Example 3. The amino acid sequences of the α and β chains corresponded to those shown in Figure 2. Soluble biotinylated HLA-A*02 was prepared using the PRAME peptide SLLQHLIGL (SEQ ID NO: 1) and immobilized on a BIAcore sensor chip.

[0098] result Binding is determined at various concentrations and the K D The value was determined to be 141 μM. Cross-reactivity (specificity) was assessed against a panel of 14 irrelevant peptide-HLA-A*02 complexes using the equilibrium BIAcore method described in Example 3. The 14 irrelevant pHLAs were divided into three groups and loaded onto one of three flow cells, providing approximately 1000 RU of each pHLA per flow cell. Thirty μL of soluble wild-type TCR was injected into all flow cells at a rate of 20 μL / min at concentrations of 130 and 488 μM. No significant binding was detected at any concentration, indicating that the native TCR was specific for the SLLQHLIGL-HLA-A*02 complex.

[0099] These data indicate that this native TCR has properties that make it suitable for use as a starting sequence for engineering high affinity therapeutic TCRs.

[0100] Example 5 - Binding characterization of certain mutant TCRs of the invention ImmTAC molecules were prepared using the amino acid sequences of the mutant TCR α and β variable domains (SEQ ID NOS: 6-24) shown in Figures 3 and 4, respectively. It was found that including a glycine residue at the beginning of the α chain (position -1 relative to the numbering of SEQ ID NOS: 2) improved the efficiency of cleavage of the N-terminal methionine during production in E. coli. Inefficient cleavage can be detrimental to therapy because it may result in a heterogeneous protein product and / or because the presence of the initial methionine may be immunogenic in humans. The full-length amino acid sequences of the ImmTAC molecules, including the α and β chains shown below, are shown in Figure 5. a28b50 - ImmTAC1 a79b74 - ImmTAC2 a79b46 - ImmTAC3

[0101] The molecules were prepared as described in Example 2 and binding to the SLLQHLIGL-HLA-A*02 complex was determined according to Example 3.

[0102] result The data in the table below demonstrate that ImmTAC molecules containing the indicated TCR variable domain sequences recognized the SLLQHLIGL-HLA-A*02 complex with particularly suitable affinity and / or half-life. [Table 12]

[0103] Example 6 - Characterization of the potency and specificity of certain mutant TCRs of the invention ImmTAC molecules containing the same TCR variable domain sequences were evaluated for their ability to mediate potent and specific redirection of CD3+ T cells against PRAME-positive cancer cells as described in Example 5. Interferon-γ (IFNγ) release was used as an indicator of T cell activation. The full-length amino acid sequence of the ImmTAC molecule, including the α and β chains, is shown in FIG. a28b50 - ImmTAC1 a79b74 - ImmTAC2 a79b46 - ImmTAC3

[0104] Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, target cells were cultured at 1 × 10 in assay medium (RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine). 6Cells were prepared at a density of 1 / ml and plated at 50,000 cells / well in a 50 μl volume. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at 50,000 cells / well in a 50 μl volume (the exact number of cells used in each experiment is donor dependent and can be adjusted to achieve a response within a range appropriate for the assay). ImmTAC molecules were titrated to final concentrations spanning the expected clinically relevant range: 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM, and added to wells in a 50 μl volume.

[0105] Plates were prepared according to the manufacturer's instructions. Target cells, effector cells, and ImmTAC molecules were added to the appropriate wells, and the final volume was adjusted to 200 μl with assay medium. All reactions were performed in triplicate. Control wells were also prepared in which ImmTAC, effector cells, or target cells were omitted. The plates were then incubated overnight (37°C / 5% CO2). The next day, the plates were washed three times with wash buffer (1x PBS containing 0.05% Tween-20 in deionized water). Primary detection antibody was then added to each well in a volume of 50 μl. The plates were incubated for 2 hours at room temperature and then washed three times again. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP to each well, incubating for 1 hour at room temperature, and repeating the washing step. Within 15 minutes before use, one drop (20 μl) of AEC chromogen was added to each 1 ml of AEC substrate, mixed, and 50 μl was added to each well. Spot color development was monitored periodically, and the plate was washed with tap water to stop the color reaction. The plate was then dried at room temperature for at least 2 hours, after which the spots were counted using a CTL analyzer (Cellular Technology Limited) equipped with Immunospot software.

[0106] In this example, the following cancer cell lines were used as target cells. Mel624 (melanoma) PRAME+ve HLA-A*02+ve Granta519 (blood lymphocytes) PRAME-ve HLA-A*02+ve ·SW620 (colon cancer) PRAME-ve HLA-A*02+ve HT144 (melanoma) PRAME+ve HLA-A*02-ve

[0107] result Each ImmTAC molecule containing the α and β variable domains shown in the table below demonstrated potent activation of redirected T cells in the presence of antigen-positive Mel624 cells. In each case, EC50 values were calculated from the data and are shown in the table below. Furthermore, each ImmTAC molecule showed minimal to no recognition of the two antigen-negative, HLA-A*02-positive cells at concentrations up to 1 nM. ImmTAC molecules also showed no recognition of PRAME-positive cells that were HLA-A*02-negative (data not shown). Figure 6 shows representative data for four ImmTAC molecules listed in the table below. [Table 13] These data demonstrate that ImmTAC molecules containing mutant TCR variable domain sequences of the present invention can mediate potent and specific T cell retargeting against PRAME-positive, HLA-A*02-positive cancer cells at concentrations that are relevant for therapeutic use.

[0108] Example 7 - Further characterization of the specificity of certain mutant TCRs of the invention To further demonstrate the specificity of ImmTAC molecules containing mutant TCR sequences, further testing was performed using the same ELISPOT methodology as described in Example 6, with a panel of normal cells derived from healthy human tissues as target cells. Normal tissues included cardiovascular, kidney, skeletal muscle, lung, vasculature, liver, and brain. In all cases, antigen-positive Mel624 cancer cells were used as a positive control.

[0109] The data presented in this example include ImmTAC molecules containing the following TCR α and β chains: a28b50 a79b74 a79b46 a79b77 The full-length amino acid sequences of the ImmTAC molecules, including a28b50, a79b74, and a79b46, are shown in Figure 5 (ImmTACs 1 to 3, respectively).

[0110] result The data shown in Figure 7 (panel a) demonstrate that ImmTAC molecules containing mutant α and β chains a28b50 and a79b46 exhibit minimal reactivity with a panel of eight normal cells compared to antigen-positive cancer cells at concentrations up to 1 nM. Similarly, the data in Figure 7 (panel b) demonstrate that ImmTAC molecules containing a28b57 and a79b46 exhibit minimal reactivity with a panel of four normal cells compared to antigen-positive cancer cells at concentrations up to 1 nM.

[0111] Example 8 - Cancer cell killing mediated by specific mutant TCRs of the invention The ability of ImmTAC molecules containing mutant TCR sequences to mediate potent killing of antigen-positive tumor cells by redirected T cells was examined using the IncuCyte platform (Essen BioScience), an assay that allows real-time microscopic detection of the release of caspase-3 / 7, a marker of apoptosis.

[0112] method The assay was performed using the CellPlayer 96-well Caspase-3 / 7 Apoptosis Assay Kit (Essen BioScience, Cat. No. 4440) according to the manufacturer's protocol. Briefly, target cells (Mel624 (PRAME+ve HLA-A*02+ve) or NCI-H1755) were plated at 10,000 cells / well and incubated overnight to allow cell adhesion. ImmTAC molecules were prepared at various concentrations, and 25 μl of each was added to the appropriate wells to achieve final concentrations between 1 pM and 100 pM. Effector cells were used at a 10:1 effector-target cell ratio (100,000 cells / well). In addition to samples containing either effector or target cells alone, a control sample without ImmTAC was also prepared. NucView assay reagent was prepared at 30 μM, and 25 μl was added to each well for a final volume of 150 μl (final concentration of 5 μM). The plates were placed in the IncuCyte instrument and imaged every 2 hours (one image per well) for 3 days. The number of apoptotic cells in each image was determined and calculated as mm 2 The number of apoptotic cells per 1000 cells was recorded. Assays were performed in triplicate. The data presented in this example include ImmTAC molecules containing the following TCR α and β chains: a28b50 a79b74 a79b46 The full-length amino acid sequences of the ImmTAC molecules, including a28b50, a79b74, and a79b46, are shown in Figure 5 (ImmTAC1, 2, and 3, respectively).

[0113] result The data shown in Figures 8 and 9 represent real-time killing of antigen-positive cancer cells (melanoma cell line Mel624 in Figure 8 and lung cancer cell line NCI-H1755 in Figure 9) in the presence of ImmTAC molecules containing mutant TCR sequences at concentrations up to 100 pM. No killing was observed in the absence of ImmTAC molecules.

Claims

1. The antibody has binding ability to the SLLQHLIGL (SEQ ID NO: 1)-HLA-A*02 complex, and comprises a TCR α chain variable domain and a TCR β chain variable domain, each of which comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR is a framework region and CDR is a complementarity determining region; The following combinations of α chain CDRs and β chain CDRs: 【Table 1】 including one of The α chain variable domain framework regions are: FR1 - amino acids 1-25 of SEQ ID NO:2 FR2 - amino acids 33-49 of SEQ ID NO:2 FR3 - amino acids 55-87 of SEQ ID NO:2 FR4 - amino acids 105-114 of SEQ ID NO:2 or a respective sequence having at least 90% identity to said sequence, and The β chain variable domain framework region comprises the following: FR1 - amino acids 1-26 of SEQ ID NO:3 FR2 - amino acids 32-48 of SEQ ID NO:3 FR3 - amino acids 56-90 of SEQ ID NO:3 FR4 - amino acids 106-114 of SEQ ID NO:3 or each sequence having at least 90% identity to said sequence T cell receptor (TCR), including:

2. The TCR of claim 1, wherein the alpha chain variable region FR1 has a G residue at position -1 using the numbering of SEQ ID NO:

2.

3. The α chain variable domain and the β chain variable domain are as follows: 【Table 2】 The TCR of claim 1 or 2, wherein the amino acid sequence is selected from the following:

4. A TCR according to any one of claims 1 to 3, which is an α-β heterodimer having an α chain TRAC constant domain sequence and a β chain TRBC1 or TRBC2 constant domain sequence.

5. The TCR of claim 4, wherein the constant domain sequences of the α chain and β chain have been modified by shortening or substitution so that the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2 is deleted.

6. A TCR as described in claim 4 or 5, in which the constant domain sequences of the α chain and / or β chain have been modified by substituting Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 with cysteine residues, and the cysteines form a non-native disulfide bond between the α constant domain and β constant domain of the TCR.

7. A TCR according to any one of claims 1 to 6, which is in a single-chain format of Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, or Vα-L-Vβ-Cβ type (wherein Vα and Vβ are TCR α and β variable regions, respectively, Cα and Cβ are TCR α and β constant regions, respectively, and L is a linker sequence).

8. A TCR according to any one of claims 1 to 7, linked to a detectable label, a therapeutic agent or a pharmacokinetics-modifying moiety.

9. The TCR of claim 8, wherein the anti-CD3 antibody is covalently linked to the C-terminus or N-terminus of the α-chain or β-chain of the TCR via a linker sequence.

10. The TCR of claim 9, wherein the linker sequence is selected from the group consisting of GGGGS (sequence number 31), GGGSG (sequence number 32), GGSGG (sequence number 33), GSGGG (sequence number 34), GSGGGP (sequence number 35), GGEPS (sequence number 36), GGEGGGP (sequence number 37) and GGEGGGSEGGGS (sequence number 38).

11. 1. A TCR-anti-CD3 fusion molecule comprising a TCR having binding to the SLLQHLIGL (SEQ ID NO: 1)-HLA-A*02 complex and an anti-CD3 antibody, The TCR comprises an α chain variable domain and a β chain variable domain, and the following combination of α chain CDRs and β chain CDRs: 【Table 3】 including one of The α chain variable domain framework regions are: FR1 - amino acids 1-25 of SEQ ID NO:2 FR2 - amino acids 33-49 of SEQ ID NO:2 FR3 - amino acids 55-87 of SEQ ID NO:2 FR4 - amino acids 105-114 of SEQ ID NO:2 or a respective sequence having at least 90% identity to said sequence, and The β chain variable domain framework region comprises the following: FR1 - amino acids 1-26 of SEQ ID NO:3 FR2 - amino acids 32-48 of SEQ ID NO:3 FR3 - amino acids 56-90 of SEQ ID NO:3 FR4 - amino acids 106-114 of SEQ ID NO:3 or each sequence having at least 90% identity to said sequence wherein the anti-CD3 antibody is covalently linked to the N-terminus or C-terminus of the TCR β chain via a linker selected from SEQ ID NOs: 31-38. TCR-anti-CD3 fusion molecule.

12. The α chain variable domain and the β chain variable domain are as follows: 【Table 4】 12. The TCR-anti-CD3 fusion molecule of claim 11, wherein the amino acid sequence is selected from the group consisting of:

13. an α chain amino acid sequence selected from SEQ ID NO: 25, 27 or 29, or an α chain amino acid sequence having at least 90% identity to the amino acid sequences set forth in SEQ ID NOs: 25, 27 and 29; and a β chain amino acid sequence selected from SEQ ID NOs: 26, 28 and 30, or a β chain amino acid sequence having at least 90% identity to the amino acid sequences set forth in SEQ ID NOs: 26, 28 and 30.

13. The TCR-anti-CD3 fusion molecule of claim 11 or 12.

14. Next: (a) an α chain amino acid sequence of SEQ ID NO: 25 and a β chain amino acid sequence of SEQ ID NO: 26; (b) an α chain amino acid sequence of SEQ ID NO: 27 and a β chain amino acid sequence of SEQ ID NO: 28, or 14. The TCR-anti-CD3 fusion molecule of claim 13, comprising (c) an alpha chain amino acid sequence of SEQ ID NO: 29 and a beta chain amino acid sequence of SEQ ID NO:

30.

15. A nucleic acid encoding the TCR α chain and TCR β chain according to any one of claims 1 to 14.

16. An expression vector comprising the nucleic acid of claim 15.

17. (a) an expression vector according to claim 16 encoding the α and β chains of a TCR according to any one of claims 1 to 14, either in a single open reading frame or in two separate open reading frames; or (b) a first expression vector comprising a nucleic acid encoding the α chain of the TCR according to any one of claims 1 to 14, and a second expression vector comprising a nucleic acid encoding the β chain of the TCR according to any one of claims 1 to 14; cell.

18. A non-naturally occurring and / or purified and / or engineered cell, in particular a T cell, presenting a TCR according to any one of claims 1 to 7.

19. 19. A pharmaceutical composition comprising a TCR according to any one of claims 1 to 10, or a TCR-anti-CD3 fusion molecule according to any one of claims 11 to 14, or a cell according to claim 17 or 18, together with one or more pharmaceutically acceptable carriers or excipients.

20. A TCR according to any one of claims 1 to 10, or a TCR-anti-CD3 fusion molecule according to any one of claims 11 to 14, or a nucleic acid according to claim 15, a pharmaceutical composition according to claim 19 or a cell according to claim 17 or 18, for use in medicine, preferably in a human subject.

21. A TCR according to any one of claims 1 to 10, or a TCR-anti-CD3 fusion molecule according to any one of claims 11 to 14, or a nucleic acid according to claim 15, a pharmaceutical composition according to claim 19 or a cell according to claim 17 or 18, for use in a method for the treatment of cancer or tumours, preferably in a human subject.

22. 22. The TCR, TCR anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition or cell for use according to claim 21, wherein the human subject has a tumor that expresses PRAME.

23. 23. The TCR, TCR anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition or cell for use according to claim 21 or 22, wherein the tumor is a solid tumor.

24. The TCR, TCR anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition or cell for use according to any one of claims 20 to 23, wherein the human subject is of the HLA-A*02 subtype.

25. The TCR, TCR-anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition or cell for use according to any one of claims 20 to 24, which is administered by injection.

26. 26. The TCR, TCR-anti-CD3 fusion molecule, nucleic acid, pharmaceutical composition or cell of claim 25, wherein the injection is intravenous injection or direct intratumoral injection.

27. An injectable formulation for administration to a human subject comprising a TCR according to any one of claims 1 to 10 or a TCR-anti-CD3 fusion molecule according to any one of claims 11 to 14.

28. 19. A method for producing a TCR according to any one of claims 1 to 10, or a TCR-anti-CD3 fusion molecule according to any one of claims 11 to 14, comprising: a) maintaining a cell according to claim 17 under conditions optimal for expression of the TCR chain; and b) isolating the TCR chain.

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