Treatment of hematological malignancies

Abstract

Provided are novel nucleic acid sequences, vectors, modified cells, peptides and pharmaceutical compositions useful for the treatment of human subjects suffering from [delta] NPM1-positive hematological malignancies. Corresponding methods and uses are also provided.

Description

Technical Field

[0001] Provided are novel nucleic acid sequences, vectors, modified cells, peptides and pharmaceutical compositions useful for treating human subjects with ΔNPM1-positive hematological malignancies, and corresponding methods and uses. Background Art

[0002] Hematologic malignancies are cancers that affect the blood and lymphatic system. The cancer may begin in blood-forming tissues (such as the bone marrow) or in cells of the immune system. Examples of hematologic malignancies include myeloid malignancies, such as acute myeloid leukemia (AML).

[0003] Acute myeloid leukemia is a bone marrow malignancy characterized by the accumulation of myeloid precursor cells that are arrested in differentiation. Currently, standard therapy consists of induction chemotherapy followed by intensive consolidation chemotherapy or high-dose therapy combined with autologous or allogeneic hematopoietic stem cell transplantation (alloSCT), resulting in a 5-year survival rate of 40-45% for patients under 65 years of age and only 10% for patients older than 65 years. 1-2 AlloSCT is associated with a low relapse rate, but high toxicity limits this benefit. Therefore, alloSCT treatment is limited to patients with a good performance status but a poor prognosis based on adverse cytogenetic or molecular abnormalities or detectable persistent or recurrent disease after chemotherapy. In the majority of patients, relapse occurs within 3 years after the start of chemotherapy, indicating that new targeted therapies that are highly effective and have no or limited toxicity are urgently needed to treat patients with AML and improve their survival. 2

[0004] The molecular characterization of AML has accelerated in recent decades. Whole-genome and exome sequencing have shown that AML has a low mutational burden, with an average of 13 coding mutations per patient. For cancer types with a higher mutational burden, such as melanoma and lung cancer, a small fraction of somatic mutations have been shown to encode neoantigens. 3 Neoantigens are peptides derived from tumor-specific DNA mutations that can be recognized by specific T cells when presented in the context of HLA on tumor cells. The formation of these antigens is a probabilistic process in which each additional mutation increases the chance of generating a neoantigen. Due to the low mutation load in AML, the number of neoantigens is expected to be limited. 3

[0005] Neoantigens can act as cancer rejection antigens in vivo following treatment with checkpoint inhibitors or adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TILs). 3-4Checkpoint inhibitors are antibodies that block inhibitory signals on T cells mediated by CTLA-4 (ipilimumab) or PD-1 (pembrolizumab and nivolumab), thereby stimulating the immune system to target neoantigens. Checkpoint inhibitors and TIL therapy have been shown to be effective against tumors with high mutational burden, but not against tumors with low mutational burden. However, despite the low overall mutational burden in AML, somatic abnormalities frequently occur in a limited number of driver genes that are recurrently mutated in multiple patients. 5 Therefore, neoantigens arising from recurrent mutations in AML are relevant for the development of targeted immunotherapies.

[0006] New immunotherapies are needed to treat hematologic malignancies, including myeloid malignancies such as AML. Summary of the invention

[0007] The inventors have recognized that mutant forms of nucleolar phosphatase (ΔNPM1 or NPM1 mut ) is an ideal target for immunotherapy of hematological malignancies, such as myeloid malignancies (particularly AML), because the formation of the mutant protein is restricted to malignant hematopoietic cells.

[0008] Nucleolar phosphatase 1 (NPM1) is a driver gene that is frequently mutated in approximately 30% of AML patients. 5 Mutated NPM1 is also observed in other types of hematologic malignancies (e.g., other myeloid malignancies), although at a much lower frequency in tumors other than AML. mut ) carry a characteristic 4-base pair (4-bp) frameshift insertion in exon 12 of the gene. The resulting ΔNPM1 protein is 4 amino acids (AA) longer than the wild-type protein, and its C-terminal 11AA is shifted to an alternative reading frame (CLAVEEVSLRK (SEQ ID NO: 27)). As a result, the ΔNPM1 protein is translocated from the nucleolus (where it functions as a nucleocytoplasmic shuttling protein) to the cytoplasm. 6 Therefore, the ΔNPM1 protein is located intracellularly. However, HLA-restricted ΔNPM1-derived peptides are accessible to T cell receptors on the cell surface and can therefore be recognized by T cells.

[0009] By studying the HLA class I ligand set of primary AML, the inventors have identified five peptides encoded by alternative reading frames of ΔNPM1 that are presented by HLA class I. The five identified peptides are CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28), and AVEEVSLR (SEQ ID NO: 29). These peptides can be used as therapeutic agents (e.g., vaccines) to treat or prevent ΔNPM1-positive AML. 23 Alternatively, they can be used as target antigens for treating such patients with modified cells described herein (e.g., peripheral blood lymphocytes or tumor infiltrating lymphocytes (TILs)), wherein the modified cells have a T cell receptor that specifically recognizes one of the specified peptides.

[0010] Advantageously, T cells expressing TCRs specific for peptides selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29) can be used as effective immunotherapy for treating ΔNPM1-positive AML. Therefore, TCR gene transfer methods using these peptide-specific TCRs can bring new treatment options for ΔNPM1-positive AML patients.

[0011] The present inventors have demonstrated for the first time that CLAVEEVSL (SEQ ID NO: 1) is present on the surface of primary AML cells isolated from HLA-A*02:01-positive AML patients. Advantageously, the peptide can therefore be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in HLA-A*02:01-positive human patients. Alternatively, it can be used as a target antigen for treating such patients using modified cells described herein (e.g., peripheral blood lymphocytes or tumor infiltrating lymphocytes (TILs)) having a T cell receptor that specifically recognizes CLAVEEVSL (SEQ ID NO: 1).

[0012] To investigate whether T cells with T cell receptors (TCRs) specific for CLAVEEVSL (SEQ ID NO: 1) presented in the context of HLA-A*02:01 are present in the T cell repertoire from healthy individuals, HLA-A*02:01 tetramers were generated against CLAVEEVSL (SEQ ID NO: 1) and its cysteine ​​variants, and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. Multiple tetramer-positive T cell clones were tested, and only two showed specific binding to CLAVEEVSL (SEQ ID NO: 1) and recognition of HLA-A*02:01 and ΔNPM1-positive AML. The T cell receptor of the strongest reactive clone (1A2) was sequenced and introduced into the CD8 + and CD4 + In T cells, we showed that ΔNPM1 specifically recognized and cleaved HLA-A*02:01-positive primary AML in a co-receptor-independent manner.

[0013] Thus, the inventors have identified a TCR that specifically binds to the neoantigen CLAVEEVSL (SEQ ID NO: 1).

[0014] Advantageously, T cells expressing TCRs specific for CLAVEEVSL (SEQ ID NO: 1) can therefore be used as an effective immunotherapy for treating HLA-A*02:01-positive patients with ΔNPM1-positive AML. Therefore, the TCR gene transfer method using CLAVEEVSL (SEQ ID NO: 1)-specific TCR can bring new treatment options for HLA-A*02:01-positive patients with ΔNPM1-positive AML.

[0015] In addition, the peptide CLAVEEVSL (SEQ ID NO: 1) (and in particular its cysteine ​​form, i.e., C*LAVEEVSL) can be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in HLA-A*02:01-positive patients. Thus, the peptide itself also has utility, for example in isolated form, or when formulated into a pharmaceutical composition.

[0016] By studying the HLA class I ligand set of primary AML, the inventors also identified unique 9-mer and 11-mer peptides encoded by alternative reading frames of ΔNPM1 (AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27), respectively). Binding of AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) to HLA-A*03:01 and HLA-A*11:01, respectively, was confirmed by monomer folding to produce tetramers (as described in detail herein for the CLAVEEVSL (SEQ ID NO: 1) peptide). Therefore, each of the AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) peptides can be used as therapeutic agents (e.g., vaccines) to treat or prevent ΔNPM1-positive AML in HLA-A*03:01 or HLA-A*11:01-positive human patients. Alternatively, each of these peptides can be used as a target antigen for treating such patients using the modified cells described herein (e.g., peripheral blood lymphocytes or tumor infiltrating lymphocytes (TILs)), which have T cell receptors that specifically recognize AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), respectively.

[0017] For AVEEVSLRK (SEQ ID NO: 26) with HLA-A * 01: 01, monomer folding to produce a tetramer has also been successfully demonstrated (as described in detail herein for the CLAVEEVSL (SEQ ID NO: 1) peptide). Therefore, the ability of AVEEVSLRK (SEQ ID NO: 26) to bind to HLA-A * 01: 01 has been confirmed. AVEEVSLRK (SEQ ID NO: 26) was also identified in the HLA class I ligand panel from an HLA-A * 01: 01-positive AML subject (AML4443) lacking HLA-A * 03: 01 and HLA-A * 11: 01 (see Figure 2). Therefore, the AVEEVSLRK (SEQ ID NO: 26) peptide can also be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in HLA-A * 01: 01-positive human patients. Alternatively, the peptide can be used as a target antigen for treating such patients with the modified cells described herein (e.g., peripheral blood lymphocytes or tumor infiltrating lymphocytes (TILs)) that have a T cell receptor that specifically recognizes AVEEVSLRK (SEQ ID NO: 26).

[0018] To investigate whether T cells with T cell receptors (TCRs) specific for AVEEVSLRK (SEQ ID NO: 26) presented in the context of HLA-A*03:01 or HLA-A*11:01 are present in the T cell repertoire from healthy individuals, HLA-A*03:01 tetramers and HLA-A*11:01 tetramers were generated against AVEEVSLRK (SEQ ID NO: 26), and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. Multiple tetramer-positive T cell clones were tested, and two with specific binding to AVEEVSLRK (SEQ ID NO: 26) in the context of HLA-A*03:01 were identified (reactive clone (3B3) and reactive clone (31.3.F1); Fig.37 and Fig.40 In addition, two T cell clones were identified that specifically bound to AVEEVSLRK (SEQ ID NO: 26) in the context of HLA-A*11:01 (reactive clone (6F11) and reactive clone (26.2.D6; Fig.37 and 40 Each of these clones (6F11, 26.2.D6, 31.3.F1, and 3B3) was also tested for reactivity ( Fig.38 , 39 and 40 ), in which each clone exhibited cytokine release when the ΔNPM1 peptide was presented only in the appropriate HLA-A context.

[0019] Thus, the inventors have identified four TCRs that specifically bind to the neoantigen AVEEVSLRK (SEQ ID NO: 26) in the context of HLA-A*03:01 (TCR from clone 3B3, and TCR from clone 31.3.F1) or HLA-A*11:01 (TCR from 6F11, and TCR from clone 26.2.D6).

[0020] The T cell receptors of clones 26.2.D6 and 6F11 were sequenced and introduced into the CD8 + T cells. The TCR-T cells showed specific binding to PE-labeled pHLA-A*11:01-AVEEVSLRK tetramers. In IFN-γ ELISA, the TCR-T cells specifically reacted with HLA-A*11:01-positive AML with ΔNPM1.

[0021] The T cell receptor of clone 31.3.F1 was also sequenced and introduced into the CD8 +T cells. The TCR-T cells showed specific binding to PE-labeled pHLA-A*03:01-AVEEVSLRK tetramers. In IFN-γ ELISA, the TCR-T cells specifically reacted with HLA-A*03:01-positive AML with ΔNPM1.

[0022] Thus, the inventors have identified and sequenced three TCRs that specifically bind to AVEEVSLRK (SEQ ID NO: 26).

[0023] Advantageously, T cells expressing TCRs specific for AVEEVSLRK (SEQ ID NO: 26) can therefore be used as an effective immunotherapy for treating HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01-positive patients with ΔNPM1-positive AML. Therefore, the TCR gene transfer method using AVEEVSLRK (SEQ ID NO: 26)-specific TCR can bring new treatment options for HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01-positive patients with ΔNPM1-positive AML.

[0024] The inventors also identified TCRs from clones 6F11 and 26.2.D6 that strongly bind to AVEEVSLRK (SEQ ID NO: 26) and to a lesser extent CLAVEEVSLRK (SEQ ID NO: 27) when CLAVEEVSLRK (SEQ ID NO: 27) is presented by HLA-A*11:01. Since both AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) contain the core sequence AVEEVSLRK (SEQ ID NO: 26), these clones specifically bind to neoantigens containing this core sequence. Therefore, the TCR described herein as "specifically binding to AVEEVSLRK (SEQ ID NO:26)" encompasses TCRs that bind to AVEEVSLRK (SEQ ID NO:26) and CLAVEEVSLRK (SEQ ID NO:27), but does not encompass TCRs that bind only to CLAVEEVSLRK (SEQ ID NO:27) (the latter TCR would be described as being specific only to CLAVEEVSLRK (SEQ ID NO:27)).

[0025] In addition, the peptide AVEEVSLRK (SEQ ID NO: 26) can be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in HLA-A*03:01, HLA-A*11:01, or HLA-A*01:01-positive patients. Thus, the peptide itself also has utility, for example in isolated form, or when formulated into a pharmaceutical composition.

[0026] The inventors have demonstrated that the peptide CLAVEEVSLRK (SEQ ID NO: 27) is presented by HLA-A*03:01 or HLA-A*11:01. Specific binding to CLAVEEVSLRK (SEQ ID NO: 27) may occur in the appropriate HLA context (i.e., specific binding to the peptide will only occur when presented by the appropriate HLA, as described above).

[0027] To investigate whether T cells with T cell receptors (TCRs) specific for CLAVEEVSLRK (SEQ ID NO:27) presented in the context of HLA-A*03:01 are present in the T cell repertoire from healthy individuals, HLA-A*03:01 tetramers were generated against CLAVEEVSLRK (SEQ ID NO:27) and its cysteine ​​variants, and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. Multiple tetramer-positive T cell clones were tested, and one with specific binding to C*LAVEEVSLRK in the context of HLA-A*03:01 was identified (reactive clone (1F2); Fig.37 ).

[0028] Thus, the inventors have identified a TCR (TCR from clone 1F2) that specifically binds to the neoantigen C*LAVEEVSLRK in the context of HLA-A*03:01.

[0029] Advantageously, T cells expressing TCRs specific for CLAVEEVSLRK (SEQ ID NO: 27) (and in particular for the cysteine ​​form of SEQ ID NO: 27, i.e., C*LAVEEVSLRK) can be used as effective immunotherapy in the treatment of HLA-A*03:01 or HLA-A*11:01-positive patients with ΔNPM1-positive AML. Therefore, TCR gene transfer methods using CLAVEEVSLRK (SEQ ID NO: 27)-specific TCRs (particularly C*LAVEEVSLRK) can bring new treatment options for HLA-A*03:01 and HLA-A*11:01-positive patients with ΔNPM1-positive AML.

[0030] In addition, the peptide CLAVEEVSLRK (and in particular its cysteine ​​form, i.e., C*LAVEEVSLRK) can be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in HLA-A*03:01 or HLA-A*11:01-positive patients. Thus, the peptide itself also has utility, for example in isolated form, or when formulated into a pharmaceutical composition.

[0031] The present invention is particularly suitable for treating patients with ΔNPM1-positive AML. However, ΔNPM1 is also present in a subset of patients with other forms of hematological malignancies, particularly myeloid malignancies. Therefore, the present invention is also suitable for treating patients with ΔNPM1-positive hematological malignancies, such as, but not limited to, myeloid malignancies (e.g., AML).

[0032] Thus, in one aspect, the present invention provides an isolated nucleic acid sequence encoding:

[0033] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to a peptide selected from the group consisting of CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29); and / or

[0034] (b) a polypeptide comprising the CDR3 of a TCR β chain polypeptide, which TCR β chain polypeptide specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0035] The nucleic acid sequence may encode (a) and (b), wherein (a) and (b) together specifically bind to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0036] The encoded polypeptide can specifically bind to CLAVEEVSL (SEQ ID NO: 1). The peptide can be in a cysteine-containing form. Thus, the encoded polypeptide can specifically bind only to C*LAVEEVSL (a cysteine-containing form of SEQ ID NO: 1).

[0037] Alternatively, the encoded polypeptide may specifically bind to AVEEVSLRK (SEQ ID NO: 26).

[0038] Alternatively, the encoded polypeptide may specifically bind to CLAVEEVSLRK (SEQ ID NO: 27). The peptide may be in a cysteine-form. Thus, the encoded polypeptide may specifically bind only to C*LAVEEVSLRK (cysteine-form SEQ ID NO: 27).

[0039] In one example, the isolated nucleic acid sequence can comprise one or more features of the TCR of clone 1A2 described herein.

[0040] For example, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with CAVTGARLMF (SEQ ID NO: 2). Alternatively, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0041] The CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity with CASSPGGLSNEQF (SEQ ID NO: 5). Alternatively, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0042] The CDR3 of (a) may be located within the TCR alpha chain variable region that specifically binds to the selected peptide (ie, SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29).

[0043] (a) may further comprise a TCR α chain constant region. In other words, the polypeptide of (a) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to a selected peptide.

[0044] The TCR α chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8. Alternatively, the TCR α chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0045] The CDR3 of (b) may be located within the TCR β chain variable region that specifically binds to the selected peptide (ie, SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29).

[0046] (b) may further comprise a TCR β chain constant region. In other words, the polypeptide of (b) may comprise a full-length TCR β chain variable region and a full-length TCR β chain constant region that specifically binds to a selected peptide.

[0047] The TCR β chain variable region of (b) may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. Alternatively, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0048] The CDR3 of (a) may be located within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 8, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 2. Alternatively, (a) comprises a TCR alpha chain constant region.

[0049] In any of the embodiments described herein, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:14, and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:15.

[0050] The CDR3 of (b) may be located within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 11, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 5. Alternatively, (b) comprises a TCR beta chain constant region.

[0051] In any of the embodiments described herein, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:16, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:17.

[0052] In one example, the isolated nucleic acid sequence can comprise one or more features of the TCR of clone 26.2.D6 described herein.

[0053] For example, the CDR3 of (a) may have an amino acid sequence having at least 90% sequence identity with CAESKGQNFVF (SEQ ID NO: 35). Alternatively, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 36 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0054] The CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity with CASTTWGTGGHEQYF (SEQ ID NO: 43). Alternatively, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 44 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0055] The CDR3 of (a) may be located within the TCR alpha chain variable region that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0056] (a) may further comprise a TCR α chain constant region. In other words, the polypeptide of (a) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to a selected peptide.

[0057] The TCR α chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37. Alternatively, the TCR α chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 38 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0058] The CDR3 of (b) may be located within the variable region of the TCR β chain that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0059] (b) may further comprise a TCR β chain constant region. In other words, the polypeptide of (b) may comprise a full-length TCR β chain variable region and a full-length TCR β chain constant region that specifically binds to a selected peptide.

[0060] The TCR β chain variable region of (b) may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45. Alternatively, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 46 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0061] The CDR3 of (a) may be located within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 37, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 35. Alternatively, (a) comprises a TCR alpha chain constant region.

[0062] In any of the embodiments described herein, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:39, and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:41.

[0063] The CDR3 of (b) may be located within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 45, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 43. Alternatively, (b) comprises a TCR beta chain constant region.

[0064] In any of the embodiments described herein, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:47, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:49.

[0065] In one example, the isolated nucleic acid sequence can comprise one or more features of the TCR of clone 6F11 described herein.

[0066] For example, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with CAVSPAGNQFYF (SEQ ID NO: 51). Alternatively, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 52 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0067] The CDR3 of (b) may have an amino acid sequence that has at least 90% sequence identity with CASSLGSNQPQHF (SEQ ID NO: 59). Alternatively, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 60 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0068] The CDR3 of (a) may be located within the TCR alpha chain variable region that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0069] (a) may further comprise a TCR α chain constant region. In other words, the polypeptide of (a) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to a selected peptide.

[0070] The TCR α chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 53. Alternatively, the TCR α chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 54 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0071] The CDR3 of (b) may be located within the variable region of the TCR β chain that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0072] (b) may further comprise a TCR β chain constant region. In other words, the polypeptide of (b) may comprise a full-length TCR β chain variable region and a full-length TCR β chain constant region that specifically binds to a selected peptide.

[0073] The TCR β chain variable region of (b) may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 61. Alternatively, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 62 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0074] The CDR3 of (a) may be located within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 53, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 51. Alternatively, (a) comprises a TCR alpha chain constant region.

[0075] In any of the embodiments described herein, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:55, and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:57.

[0076] The CDR3 of (b) may be located within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 61, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 59. Alternatively, (b) comprises a TCR beta chain constant region.

[0077] In any of the embodiments described herein, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:63, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:65.

[0078] In one example, the isolated nucleic acid sequence can comprise one or more features of the TCR of clone 31.3.F1 described herein.

[0079] For example, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with CALSGGGQNFVF (SEQ ID NO: 67). Alternatively, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 68 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0080] The CDR3 of (b) may have an amino acid sequence that has at least 90% sequence identity with CASSQGSGFRHF (SEQ ID NO: 75). Alternatively, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 76 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0081] The CDR3 of (a) may be located within the TCR alpha chain variable region that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0082] (a) may further comprise a TCR α chain constant region. In other words, the polypeptide of (a) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to a selected peptide.

[0083] The TCR α chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 69. Alternatively, the TCR α chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 70 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0084] The CDR3 of (b) may be located within the variable region of the TCR β chain that specifically binds to a selected peptide (eg, SEQ ID NO: 26).

[0085] (b) may further comprise a TCR β chain constant region. In other words, the polypeptide of (b) may comprise a full-length TCR β chain variable region and a full-length TCR β chain constant region that specifically binds to a selected peptide.

[0086] The TCR β chain variable region of (b) may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 77. Alternatively, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 78 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0087] The CDR3 of (a) may be located within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 69, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 67. Alternatively, (a) comprises a TCR alpha chain constant region.

[0088] In any of the embodiments described herein, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:71, and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:73.

[0089] The CDR3 of (b) may be located within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 77, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 75. Alternatively, (b) comprises a TCR beta chain constant region.

[0090] In any of the embodiments described herein, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:79, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:81.

[0091] The selected peptide CLAVEEVSL (SEQ ID NO: 1) may be cysteine-ylated.

[0092] The selected peptide CLAVEEVSLRK (SEQ ID NO: 27) may be cysteine-ylated.

[0093] The nucleic acid sequence may encode a T cell receptor.

[0094] For the avoidance of doubt, the inventors have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) is presented by HLA-A*02:01 (i.e. is HLA-A*02:01 restricted). In addition, the inventors have determined that the peptides AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) are presented by HLA-A*03:01 or HLA-A*11:01, respectively, and that AVEEVSLRK (SEQ ID NO: 26) is also presented by HLA-A*01:01. Therefore, specific binding to any of these peptides may occur in the appropriate HLA background (i.e. specific binding to the peptide may only occur if the peptide is presented by the appropriate HLA, as described above).

[0095] The nucleic acid sequence of the present invention can be a non-naturally occurring nucleic acid sequence (e.g., its entire sequence may not exist intact in nature). For example, the nucleic acid sequence of the present invention can be operably linked to a promoter, wherein the promoter is not naturally associated with an equivalent human nucleic acid sequence (e.g., a human TCR sequence or a fragment thereof) in nature; that is, it is not the entire promoter naturally associated with a nucleic acid in a natural environment. In this context, such promoters can be considered exogenous promoters. Examples of suitable promoters are described elsewhere.

[0096] In a further aspect, the present invention provides a vector comprising the nucleic acid sequence of the present invention.

[0097] The vector may be a plasmid or a viral vector. Optionally, the vector is selected from adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, herpes virus, minicircle vector and synthetic DNA or RNA. Optionally, the vector comprises a promoter, to which the nucleic acid sequence is operably linked, as described above.

[0098] In a further aspect, the present invention provides a modified cell transfected or transduced with the nucleic acid sequence of the present invention or the vector of the present invention.

[0099] The transfected or transduced nucleic acid sequence of the invention or the vector of the invention may be operably linked to a promoter, as described above.

[0100] The modified cells may be selected from CD8 T cells, CD4 T cells, NK cells, NKT cells, γ-δ T cells, hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines.

[0101] The modified cell may be a human cell.

[0102] In a further aspect, the present invention provides an isolated peptide comprising an amino acid sequence selected from the group consisting of:

[0103] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated;

[0104] (ii) AVEEVSLRK (SEQ ID NO: 26);

[0105] (iii) CLAVEEVSLRK (SEQ ID NO: 27); wherein the cysteine ​​amino acid may or may not be cystylated;

[0106] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0107] (v) AVEEVSLR (SEQ ID NO: 29).

[0108] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0109] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0110] The peptide may have no more than 20 amino acids.

[0111] The peptide may consist of a sequence selected from the group consisting of:

[0112] (i) SEQ ID NO: 1, wherein the cysteine ​​amino acid may or may not be cystylated;

[0113] (ii) SEQ ID NO: 26;

[0114] (iii) SEQ ID NO: 27; wherein the cysteine ​​amino acid may or may not be cystylated;

[0115] (iv) SEQ ID NO:28; and

[0116] (v) SEQ ID NO:29.

[0117] In a specific embodiment, the peptide may consist of the sequence of SEQ ID NO: 1, wherein the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinated.

[0118] In a specific embodiment, the peptide may consist of the sequence of SEQ ID NO: 27, wherein the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0119] In a further aspect, the present invention provides an isolated nucleic acid sequence encoding a peptide of the present invention.

[0120] In a further aspect, the present invention provides a vector comprising the nucleic acid sequence of the present invention.

[0121] In a further aspect, the present invention provides a pharmaceutical composition comprising: a nucleic acid sequence of the present invention, a vector of the present invention, a modified cell of the present invention, or an isolated peptide of the present invention, and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0122] When the pharmaceutical composition comprises an isolated peptide according to the invention (or a nucleic acid or vector encoding the isolated peptide), the composition may be formulated as a vaccine. Vaccine formulations suitable for peptides and nucleic acids are well known in the art.

[0123] In a further aspect, the present invention provides a method of treating or preventing ΔNPM1 -positive hematological malignancies in a human subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0124] A method of treating or preventing a ΔNPM1-positive hematological malignancy in a human subject may comprise administering to the subject a therapeutically effective amount of a peptide described herein (or a nucleic acid (eg, RNA or DNA) or vector encoding the peptide).

[0125] As an example, the isolated peptide comprises or consists of an amino acid sequence selected from the group consisting of:

[0126] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated;

[0127] (ii) AVEEVSLRK (SEQ ID NO: 26);

[0128] (iii) CLAVEEVSLRK (SEQ ID NO: 27); wherein the cysteine ​​amino acid may or may not be cystylated;

[0129] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0130] (v) AVEEVSLR (SEQ ID NO: 29).

[0131] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0132] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated. The hematological malignancy may be a myeloid malignancy.

[0133] The myeloid malignancy may be acute myeloid leukemia.

[0134] The methods can induce or enhance a cell-mediated immune response in a subject.

[0135] For the avoidance of doubt, the inventors have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) is HLA-A*02:01 restricted. In addition, the inventors have determined that the peptides AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) are each presented by either HLA-A*03:01 and HLA-A*11:01, and AVEEVSLRK (SEQ ID NO: 26) is also presented by HLA-A*01:01. CLAVEEVSL (SEQ ID NO: 1) and / or CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-restricted form.

[0136] Thus, methods for treating or preventing ΔNPM1-positive hematological malignancies in HLA-A*02:01-positive human subjects may preferentially use a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to CLAVEEVSL (SEQ ID NO: 1), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding CLAVEEVSL (SEQ ID NO: 1), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence CLAVEEVSL (SEQ ID NO: 1) (all of which are described in more detail elsewhere herein). CLAVEEVSL (SEQ ID NO: 1) may specifically be in a cysteine-ylated form.

[0137] Similarly, methods for treating or preventing ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*03:01 or HLA-A*11:01 may preferentially use a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27) (all of which are described in more detail elsewhere herein).

[0138] In addition, methods for treating or preventing ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*01:01 may preferentially use a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQ ID NO:26), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding AVEEVSLRK (SEQ ID NO:26), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK (SEQ ID NO:26) (all of which are described in more detail elsewhere herein).

[0139] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-ylated form.

[0140] In a further aspect, the present invention provides a pharmaceutical composition of the present invention for use in treating or preventing a ΔNPM1 -positive hematological malignancy in a human subject.

[0141] The pharmaceutical composition of the present invention for treating or preventing ΔNPM1-positive hematological malignancies in human subjects may comprise a therapeutically effective amount of a peptide described herein (or a nucleic acid (eg, RNA or DNA) or vector encoding the peptide).

[0142] As an example, the pharmaceutical composition may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0143] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated;

[0144] (ii)AVEEVSLRK (SEQ ID NO:26);

[0145] (iii) CLAVEEVSLRK (SEQ ID NO: 27); wherein the cysteine ​​amino acid may or may not be cystylated;

[0146] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0147] (v) AVEEVSLR (SEQ ID NO: 29).

[0148] The pharmaceutical composition may be used as an immunotherapy (eg as a vaccine).

[0149] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0150] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0151] The hematological malignancy may be a myeloid malignancy.

[0152] The myeloid malignancy may be acute myeloid leukemia.

[0153] The pharmaceutical composition can be used to induce or enhance a cell-mediated immune response in a subject.

[0154] As previously described, the inventors have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) is HLA-A*02:01 restricted. In addition, the inventors have determined that the peptides AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) are presented by HLA-A*03:01 and HLA-A*11:01, respectively, and AVEEVSLRK (SEQ ID NO: 26) is also presented by HLA-A*01:01.

[0155] CLAVEEVSL (SEQ ID NO: 1) and / or CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-ylated form.

[0156] Thus, in the treatment or prevention of ΔNPM1-positive hematological malignancies in HLA-A*02:01-positive human subjects, a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to CLAVEEVSL (SEQ ID NO: 1), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding CLAVEEVSL (SEQ ID NO: 1), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence CLAVEEVSL (SEQ ID NO: 1) (all of which are described in more detail elsewhere herein) may be preferentially used. CLAVEEVSL (SEQ ID NO: 1) may specifically be in a cysteine-ylated form.

[0157] Similarly, in the treatment or prevention of ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*03:01 or HLA-A*11:01, a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27) (all of which are described in more detail elsewhere herein) may be preferentially used.

[0158] In addition, when treating or preventing ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*01:01, a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQID NO:26), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or a nucleic acid encoding AVEEVSLRK (SEQ ID NO:26), a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK (SEQ ID NO:26) (all of which are described in more detail elsewhere herein) can be preferentially used.

[0159] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-ylated form.

[0160] In a further aspect, the present invention provides use of a pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing a ΔNPM1 -positive hematological malignancy in a human subject.

[0161] The pharmaceutical composition may comprise a therapeutically effective amount of a peptide described herein (or a nucleic acid (eg, RNA or DNA) or vector encoding the peptide).

[0162] As an example, the pharmaceutical composition may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0163] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated;

[0164] (ii)AVEEVSLRK (SEQ ID NO:26);

[0165] (iii) CLAVEEVSLRK (SEQ ID NO: 27); wherein the cysteine ​​amino acid may or may not be cystylated;

[0166] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0167] (v) AVEEVSLR (SEQ ID NO: 29).

[0168] The pharmaceutical composition may be used as an immunotherapy (eg as a vaccine).

[0169] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0170] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0171] The hematological malignancy may be a myeloid malignancy.

[0172] The myeloid malignancy may be acute myeloid leukemia.

[0173] As mentioned elsewhere herein, the inventors have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) is HLA-A*02:01 restricted. In addition, the inventors have determined that the peptides AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) are each presented by HLA-A*03:01 and HLA-A*11:01, and AVEEVSLRK (SEQ ID NO: 26) is also presented by HLA-A*01:01. CLAVEEVSL (SEQ ID NO: 1) and / or CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-ylated form.

[0174] Thus, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to CLAVEEVSL (SEQ ID NO: 1), vectors encoding such nucleic acid sequences, modified cells comprising such nucleic acid sequences or vectors, or isolated peptides comprising the sequence CLAVEEVSL (SEQ ID NO: 1), all of which are described in more detail elsewhere herein, can be preferentially used in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in HLA-A*02:01-positive human subjects. CLAVEEVSL (SEQ ID NO: 1) can specifically be in a cysteine-ylated form.

[0175] Similarly, pharmaceutical compositions comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or an isolated peptide comprising the sequence AVEEVSLRK (SEQ ID NO:26) or CLAVEEVSLRK (SEQ ID NO:27) (all of which are described in more detail elsewhere herein) can be preferentially used in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*03:01 or HLA-A*11:01.

[0176] In addition, pharmaceutical compositions comprising a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK (SEQ ID NO:26), a vector encoding such a nucleic acid sequence, a modified cell comprising such a nucleic acid sequence or vector, or an isolated peptide comprising the sequence AVEEVSLRK (SEQID NO:26) (all of which are described in more detail elsewhere herein) can be preferentially used in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in human subjects who are positive for HLA-A*01:01.

[0177] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteine-ylated form.

[0178] In a further aspect, the present invention provides a method for producing a T cell receptor, comprising contacting a nucleic acid sequence of the present invention with a cell under conditions wherein the nucleic acid sequence is incorporated into the cell and expressed by the cell to produce a T cell receptor that specifically binds to a peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0179] The method may be ex vivo.

[0180] As described above, specific binding to any of CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) can be performed in the context of the appropriate HLA (e.g., specific binding to the peptide will only occur when presented by the appropriate HLA, as described above).

[0181] In a further aspect, the present invention provides the use of a peptide as a biomarker for ΔNPM1-positive hematological malignancies in a human subject, wherein the peptide is selected from:

[0182] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated;

[0183] (ii)AVEEVSLRK (SEQ ID NO:26);

[0184] (iii) CLAVEEVSLRK (SEQ ID NO: 27); wherein the cysteine ​​amino acid may or may not be cystylated;

[0185] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0186] (v) AVEEVSLR (SEQ ID NO: 29).

[0187] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0188] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0189] In a further aspect, the present invention provides a method for diagnosing a ΔNPM1-positive hematological malignancy in a human subject, comprising:

[0190] Determining the presence of a peptide in a sample isolated from a subject, wherein the peptide is selected from: (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cystylated; (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​amino acid may or may not be cystylated; (iv) VEEVSLRK (SEQ ID NO: 28); and AVEEVSLR (SEQ ID NO: 29),

[0191] wherein the presence of the peptide in the sample identifies the subject as having a ΔNPM1 -positive hematological malignancy, and wherein the absence of the peptide in the sample identifies the subject as not having a ΔNPM1 -positive hematological malignancy.

[0192] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0193] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0194] In a further aspect, the present invention provides a method of treating or preventing a ΔNPM1-positive hematological malignancy in a human subject, the method comprising:

[0195] (i) determining the presence of a peptide in a sample isolated from a subject, wherein the peptide is selected from CLAVEEVSL (SEQ ID NO: 1) (wherein the cysteine ​​amino acid may or may not be cystylated), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27) (wherein the cysteine ​​amino acid may or may not be cystylated), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0196] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0197] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0198] The statements provided previously regarding each peptide and its HLA-restricted properties (and particularly in the context of suitable pharmaceutical compositions for use in methods of treating or preventing ΔNPM1-positive hematological malignancies in subjects with a particular HLA status) apply equally here.

[0199] In a further aspect, the present invention provides a pharmaceutical composition of the present invention for use in treating or preventing a ΔNPM1-positive hematological malignancy in a human subject, wherein the subject has been identified as suffering from a ΔNPM1-positive hematological malignancy by the presence of a peptide in a sample isolated from the subject, wherein the peptide is selected from:

[0200] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​residue may or may not be cystylated;

[0201] (ii) AVEEVSLRK (SEQ ID NO: 26);

[0202] (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​residue may or may not be cystylated;

[0203] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0204] (v) AVEEVSLR (SEQ ID NO: 29).

[0205] In this aspect, a subject identified as having a ΔNPM1-positive hematological malignancy is a subject who, prior to treatment, has been diagnosed as having a ΔNPM1-positive hematological malignancy due to the presence of a peptide in a sample isolated from the subject, wherein the peptide is selected from:

[0206] (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​residue may or may not be cystylated;

[0207] (ii) AVEEVSLRK (SEQ ID NO: 26);

[0208] (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​residue may or may not be cystylated;

[0209] (iv) VEEVSLRK (SEQ ID NO: 28); and

[0210] (v) AVEEVSLR (SEQ ID NO: 29).

[0211] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 1 is cystylated.

[0212] In a specific embodiment, the cysteine ​​amino acid of SEQ ID NO: 27 is cystylated.

[0213] The statements provided previously regarding each peptide and its HLA-restricted properties (and particularly in the context of appropriate pharmaceutical compositions for use in treating or preventing ΔNPM1-positive hematological malignancies in subjects with a particular HLA status) apply equally here.

[0214] Throughout the description and claims of this specification, the words “comprise” and “comprising” and variations thereof mean “including but not limited to”, and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0215] Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0216] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0217] The patents, scientific and technical literature cited herein establish the knowledge available to those skilled in the art at the time the application was filed. The entire disclosures of issued patents, published and pending patent applications, and other publications cited herein are incorporated herein by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the event of any inconsistency, the present disclosure controls.

[0218] Various aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0220] Figure 1Validation of C*LAVEEVSL (a cysteine ​​version of SEQ ID NO: 1) as a peptide eluted from HLA-A*02:01 positive AML with ΔNPM1 is provided. The mass spectrum of the eluted peptide from HLA-A*02:01 positive AML10197 with ΔNPM1 (upper panel) and the mass spectrum of the synthetic peptide C*LAVEEVSL (a cysteine ​​version of SEQ ID NO: 1) after cysteineization of the first residue (lower panel) are shown. The data show a perfect match between the mass spectra of the two peptides.

[0221] Figure 2 provides validation of ΔNPM1 peptides eluted from primary AML. Tandem mass spectra of eluted peptides from AML (upper panel) and synthetic peptides (lower panel) are shown. C*=Cysteination of Cys-residues. (A) Tandem mass spectra of eluted peptides from AML10197 (upper panel) and AML 3361 (middle panel) and synthetic peptide C*LAVEEVSL (a cysteine ​​version of SEQ ID NO: 1), and eluted peptides from AML3361 and synthetic peptide AVEEVSLRK (lower panel). (B) Tandem mass spectra of eluted peptides from AML9448 (upper panel), AML5444 (middle panel) and AML5518 (lower panel) and synthetic peptide AVEEVSLRK (SEQ ID NO: 26). (C) Tandem mass spectra of the eluted peptide and synthetic peptide AVEEVSLRK (SEQ ID NO: 26) from AML6498 (upper panel) and AML4443 (middle panel), and the eluted peptide and synthetic peptide C*LAVEEVSLRK (cystylated SEQ ID NO: 27) from AML9448 (lower panel). (D) Tandem mass spectra of the eluted peptide and synthetic peptide C*LAVEEVSLRK (cystylated form of SEQ ID NO: 27) from AML6498 (upper panel), the eluted peptide and synthetic peptide VEEVSLRK (SEQ ID NO: 28) from AML3361 (middle panel), and the eluted peptide and synthetic peptide AVEEVSLR (SEQ ID NO: 29) from AML5518 (lower panel).

[0222] Figure 3CD8 cells of ΔNPM1 are shown as single cells isolated from PBMCs from HLA-A*02:01 positive healthy individuals using a mixture of ΔNPM1-CLA and ΔNPM1-C*LA pMHC tetramers. A. Growing T cell clones were tested for staining with pMHC tetramers. Both T cell clones 1A2 (top) and 4A8 (bottom) were positive for ΔNPM1-CLA tetramers, while only clone 1A2 was stained with ΔNPM1-C*LA. B. Tetramer-positive T cell clones 1A2 (upper panel) and 4A8 (lower panel) were tested by IFN-γ ELISA for reactivity against HLA-A*02:01-positive T2 cells exogenously loaded with titrated concentrations of the non-cystylated ΔNPM1 peptide CLAVEEVSL (SEQ ID NO: 1) (circles), the cystylated ΔNPM1 peptide C*LAVEEVSL (cystylated form of SEQ ID NO: 1) (squares), or the irrelevant HLA-A*02:01-restricted CMV peptide NLVPMVATV (SEQ ID NO: 31) (triangles). Only clone 1A2 showed recognition of both the cystylated and non-cystylated ΔNPM1 peptides. No reactivity was observed against the irrelevant NLVPMVATV (SEQ ID NO: 31) peptide. The average release of IFN-γ in duplicate wells (ng / ml) is shown. C. Clones 1A2 (upper panel) and 4A8 (lower panel) were tested for reactivity against five HLA-A*02:01-positive primary AMLs by IFN-γ ELISA. The panel included three AMLs with ΔNPM1 and two AMLs with wtNPM1. T cell clone 1A2 reacted to varying degrees against the three AMLs with ΔNPM1, whereas clone 4A8 recognized only two of the three AMLs. Both T cell clones failed to recognize AMLs with wtNPM1. An HLA-A*02:01-specific alloreactive T cell clone (Allo-A2 clone; lower panel) was included as a positive control. The average release of IFN-γ (ng / ml) from duplicate wells is shown.

[0223] Figure 4Specificity for ΔNPM1 after TCR gene transfer is shown. The genes for the ΔNPM1-specific TCR α and β chains of clone 1A2 were cloned into a modified MP71-TCR-flex retroviral vector for TCR gene transfer. CD8 and CD4 cells isolated from HLA-A*02:01-positive healthy individuals were retrovirally transduced with TCR for ΔNPM1 and TCR for the HLA-A*02:01-restricted CMV peptide NLVPMVATV (SEQ ID NO:31) as a control. On day 6 after transduction, TCR-transduced T cells were purified using an APC-coupled antibody against mouse TCR-Cβ and magnetic anti-APC beads. A. Flow cytometric analysis of TCR-transduced T cells at day 7 post-transduction using antibodies against CD8 or CD4 and pMHC tetramers to detect CLAVEEVSL (SEQ ID NO: 1) (ΔNPM1-CLA; left) or NLVPMVATV (SEQ ID NO: 31) (CMV-NLV; right). and CD4 Cells were stained with ΔNPM1-CLA tetramers but not with CMV-NLV tetramers. In contrast, CD8 and CD4 T cells showed binding to CMV-NLV tetramers, but not to ΔNPM1-CLA tetramers. Results for donor 1 are shown, but results for donor 2 were similar. B. Reactivity of TCR-transduced T cells to their target peptides was analyzed by IFN-γ ELISA. TCR-transduced CD8 and CD4 cells were co-incubated with T2 cells exogenously loaded with titrated concentrations of the ΔNPM1 peptide CLAVEEVSL (SEQ ID NO: 1) (circles) or the CMV-derived peptide NLVPMVATV (SEQ ID NO: 31) (squares). (top left) and (Lower left) shows half-high recognition (dashed line) of T2 cells loaded with CLAVEEVSL (SEQ ID NO: 1) at concentrations of 30-100 nM, but no recognition of T2 cells loaded with NLVPMVATV. (upper right) and (Bottom right) Responded to T2 cells loaded with NLVPMVATV (SEQ ID NO:31) but not to T2 cells loaded with CLAVEEVSL (SEQ ID NO:1). For donor 1, the average release of IFN-γ in duplicate wells (ng / ml) is shown, but the results for donor 2 were similar. C. TCR-transduced T cells were tested by IFN-γ ELISA in the presence or absence of blocking antibodies against HLA class I (W6 / 32) or HLA class II (PdV5.1) (black bars indicate and Gray bars indicate and ) Recognition of HLA-A*02:01 positive AML cell lines with ΔNPM1 (OCI-AML3) or wtNPM1 (OCI-AML2). Recognition of OCI-AML3 by CD8 and CD4 cells transduced with TCR for ΔNPM1 is mediated by HLA class I. Mean IFN-γ release (ng / ml) from donor 2 in duplicate wells is shown.

[0224] Figure 5 Figure 3 shows recognition of primary AML by ΔNPM1 after TCR gene transfer. TCR-transduced CD8 and CD4 cells were tested for reactivity against a panel of 13 HLA-A*02:01-positive primary AMLs, including 9 samples with ΔNPM1 and 4 samples with wtNPM1, by IFN-γ ELISA. upper panel; black bars) and CD4 ( Lower panel; black bars) cells responded to all 9 AMLs with ΔNPM1 but not to AMLs with wtNPM1, whereas none of the 13 AML samples were activated by CD8( upper panel; dark grey bars) or CD4 ( Lower panel; dark grey bars) Cell-specific recognition. TCR-transduced CD8 and CD4 cells also failed to recognize HLA-A*02:01-negative AML carrying ΔNPM1 (data not shown). The allo-A2 clone (light grey bars) was included as a positive control. The average IFN-γ release (ng / ml) of donor 1 in duplicate wells is shown.

[0225] Figure 6 TCR-transduced T cells tested on monocyte-derived mature DCs by IFN-γ ELISA are shown. T cells from donor 2 were tested and these T cells were transduced with ΔNPM1 ( and Black bar) or CMV( and Figure 2. TCR transduction of autologous monocyte-derived mature DCs and ΔNPM1-positive AMLs that were HLA-A*02:01 positive (AML8861) or negative (AML587) was tested for reactivity to autologous monocyte-derived mature DCs and HLA-A*02:01 positive (AML8861) or negative (AML587). For autologous mature DCs and AML8861, an allo-A2 clone (light gray bar) was included as a positive control, while AML587 was loaded with an HLA-B*07:02-binding SMCY peptide and recognition by a SMCY-specific CD8 T cell clone was tested as a positive control (dark gray bar). The average release of IFN-γ (ng / ml) in duplicate wells is shown.

[0226] Figure 7 Shows 51 Lysis of primary AML with ΔNPM1 after TCR gene transfer in a Cr release assay. 51 The cytolytic capacity of TCR-transduced CD8 and CD4 cells was tested in a panel of 6 HLA-A*02:01-positive primary AML (including 4 samples with ΔNPM1 and 2 samples with wtNPM1) using a Cr release assay. black circles) and CD4 ( Black squares) cells showed specific lysis of all four AMLs with ΔNPM1 but not AMLs with wtNPM1, whereas none of the six AML samples were lysed by CD8( gray circle) or CD4( Lower panel; grey squares) Cell-specific lysis. The allo-A2 clone (grey triangles) was included as a positive control. For donor 2, the average percentage of specific lysis in triplicate wells is shown at an E:T ratio of 30:1, but the results for donor 1 were similar.

[0227] Figure 8 Shown is the immunogenic peptide amino acid sequence of SEQ ID NO: 1. Of note, the cysteine ​​amino acid of SEQ ID NO: 1 may or may not be cystylated.

[0228] Fig. 9 The amino acid sequence of CDR3 (TCR alpha chain) is shown (SEQ ID NO: 2).

[0229] Fig.10 A non-optimized nucleic acid sequence encoding CDR3 (TCR alpha chain) is shown (SEQ ID NO: 3).

[0230] Fig.11The optimized nucleic acid sequence encoding CDR3 (TCR alpha chain) is shown (SEQ ID NO: 4).

[0231] Fig.12 The amino acid sequence of CDR3 (TCR β chain) is shown (SEQ ID NO: 5).

[0232] Fig.13 A non-optimized nucleic acid sequence encoding CDR3 (TCR β chain) is shown (SEQ ID NO: 6).

[0233] Fig.14 The optimized nucleic acid sequence encoding CDR3 (TCR β chain) is shown (SEQ ID NO: 7).

[0234] Fig.15 The amino acid sequence of the alpha chain variable region (SEQ ID NO: 8) is shown (CDR3, underlined).

[0235] Fig.16 The non-optimized nucleic acid sequence encoding the alpha chain variable region is shown (SEQ ID NO:9) (CDR3 is underlined).

[0236] Fig.17 The optimized nucleic acid sequence encoding the alpha chain variable region is shown (SEQ ID NO: 10) (CDR3 is underlined).

[0237] Fig.18 The amino acid sequence of the beta chain variable region is shown (SEQ ID NO: 11) (CDR3 is underlined).

[0238] Fig.19 The non-optimized nucleic acid sequence encoding the beta chain variable region is shown (SEQ ID NO: 12) (CDR3 is underlined).

[0239] Fig. 20 The optimized nucleic acid sequence encoding the beta chain variable region is shown (SEQ ID NO: 13) (CDR3 is underlined).

[0240] Fig.21 The amino acid sequence of CDR1 (TCR alpha chain) is shown (SEQ ID NO: 14).

[0241] Fig. 22 The amino acid sequence of CDR2 (TCR alpha chain) is shown (SEQ ID NO: 15).

[0242] Fig.23 The amino acid sequence of CDR1 (TCR β chain) is shown (SEQ ID NO: 16).

[0243] Fig.24 The amino acid sequence of CDR2 (TCR β chain) is shown (SEQ ID NO: 17).

[0244] Fig.25 The non-optimized nucleic acid sequence encoding CDR1 (TCR alpha chain) is shown (SEQ ID NO: 18).

[0245] Fig.26 The optimized nucleic acid sequence encoding CDR1 (TCR alpha chain) is shown (SEQ ID NO: 19).

[0246] Fig. 27 The non-optimized nucleic acid sequence encoding CDR2 (TCR alpha chain) is shown (SEQ ID NO: 20).

[0247] Fig.28 The optimized nucleic acid sequence encoding CDR2 (TCR alpha chain) is shown (SEQ ID NO: 21).

[0248] Fig.29 The non-optimized nucleic acid sequence encoding CDR1 (TCR β chain) is shown (SEQ ID NO: 22).

[0249] Fig.30 The optimized nucleic acid sequence encoding CDR1 (TCR β chain) is shown (SEQ ID NO: 23).

[0250] Fig.31 The non-optimized nucleic acid sequence encoding CDR2 (TCR β chain) is shown (SEQ ID NO: 24).

[0251] Fig.32 The optimized nucleic acid sequence encoding CDR2 (TCR β chain) is shown (SEQ ID NO: 25).

[0252] Fig.33 The immunogenic peptide amino acid sequence of SEQ ID NO: 26 is shown.

[0253] Fig.34 The immunogenic peptide amino acid sequence of SEQ ID NO: 27 is shown. Of note, the cysteine ​​amino acid of SEQ ID NO: 27 may or may not be cystylated.

[0254] Fig.35 The immunogenic peptide amino acid sequence of SEQ ID NO: 28 is shown.

[0255] Fig.36 The immunogenic peptide amino acid sequence of SEQ ID NO: 29 is shown.

[0256] Figure 37 to Figure 39 T cells for ΔNPM1 peptide in A*03:01 and A*11:01 are shown. A mixture of PE-coupled HLA-A*03:01 tetramers with ΔNPM1 peptides AVEEVSLRK (SEQ ID NO:26) (AVE), CLAVEEVSLRK (SEQ ID NO:27) (CLA) and C*LAVEEVSLRK (cystylated form of SEQ ID NO:27) (C*LA; first residue cystylated) or a single HLA-A*11:01 tetramer with AVEEVSLRK (SEQ ID NO:26) was used to isolate specific T cells from HLA-A*03:01 and / or HLA-A*11:01 positive healthy individuals, respectively. HLA-A*03:01 tetramers with C*LAVEEVSLRK (SEQ ID NO: 27 in cysteine ​​form) and CLAVEEVSLRK (SEQ ID NO: 27) as a control were generated by UV exchange (UV-C*LA and UV-CLA, respectively).

[0257] Fig.37 T cell clones binding to PE-conjugated HLA-A*03:01UV-C*LA (clone 1F2; left), A*03:01AVE (clone 3B3; center), and A*11:01AVE (clone 6F11; right) tetramers are shown.

[0258] Fig.38 The responsiveness of T cell clone 3B3 (left) was tested against T2 cells transduced with HLA-A*03:01, which were exogenously pulsed with titrated concentrations of the ΔNPM1 peptide AVEEVSLRK (SEQ ID NO:26) (triangles), CLAVEEVSLRK (SEQ ID NO:27) (circles), or C*LAVEEVSLRK (cystylated form of SEQ ID NO:27) (squares). Indicated is the release of GM-CSF (ng / ml) at different peptide concentrations (nM). The responsiveness of T cell clone 6F11 (right) was tested against T2 cells transduced with HLA-A*11:01, which were exogenously pulsed with titrated concentrations of the ΔNPM1 peptide AVEEVSLRK (SEQ ID NO:26) (triangles). Indicated is the release of IFN-γ (ng / ml) at different peptide concentrations (nM).

[0259] Fig.39T cell clones 3B3 (left) and 6F11 (right) were tested for their reactivity against K562 cells, K562 transduced with HLA-A*03:01 or A*11:01, K562 transduced with HLA-A*03:01 or A*11:01 and a gene encoding full-length wild-type or ΔNPM1, and OCI-AML2 and OCI-AML3 cell lines endogenously expressing wild-type and ΔNPM1, respectively, transduced with A*03:01 or A*11:01. The amount of GM-CSF (clone 3B3) or IFN-γ (clone 6F11) released (in ng / ml) is indicated.

[0260] Fig.40 T cell clones for mutant NPM1 in HLA-A*03:01 or HLA-A*11:01 are shown. Fig.40 (A) shows the activity of T cell clone 31.3.F1 isolated from a HLA-A*03:01-positive healthy individual. The T cell clone showed specific binding to PE-labeled pHLA-A*03:01-AVEEVSLRK (SEQ ID NO: 26) tetramers, but not to PE-labeled pHLA-A*03:01-CLAVEEVSLRK (SEQ ID NO: 27) tetramers (upper figure). Both pHLA tetramers did not bind to the control T cell clones (lower two histograms). In IFN-γ ELISA, the T cell clone (black bar) specifically reacted to OCI-AML3 (mutant NPM1; dNPM1) transduced with HLA-A*03:01 and T2 cells transduced with HLA-A*03:01 pulsed with titrated concentrations of AVEEVSLRK (SEQ ID NO:26) peptide, but not to OCI-AML3, OCI-AML3 transduced with HLA-A*11:01, OCI-AML2 (wild-type NPM1; wtNPM1), and OCI-AML2 transduced with HLA-A*03:01 or HLA-A*11:01 (middle panel). The T cell clone also reacted to HLA-A*03:01-positive primary AML with mutant NPM1, but not to HLA-A*03:01-positive AML with wild-type NPM1 (lower panel). Indicated are the average amounts of IFN-γ released in the culture supernatants of duplicate wells. Fig.40(B) shows the activity of T cell clones 6F11 and 26.2.D6, which were isolated from HLA-A*11:01-positive and HLA-A*11:01-negative healthy individuals, respectively. The T cell clones showed specific binding to PE-labeled pHLA-A*11:01-AVEEVSLRK (SEQ ID NO: 26) tetramers, and to a lower extent to PE-labeled pHLA-A*11:01-CLAVEEVSLRK (SEQ ID NO: 27) (upper figure). Both pHLA tetramers did not bind to the control T cell clones (lower two histograms). In the IFN-γ ELISA, T cell clones 6F11 (grey shaded bars) and 26.2.D6 (black bars) specifically reacted against OCI-AML3 (mutant NPM1; dNPM1) transduced with HLA-A*11:01 and T2 cells transduced with HLA-A*11:01 pulsed with titrated concentrations of AVEEVSLRK (SEQ ID NO:26) peptide, but did not react to OCI-AML3, OCI-AML3 transduced with HLA-A*03:01, OCI-AML2 (wild-type NPM1; wtNPM1), and OCI-AML2 transduced with HLA-A*03:01 or HLA-A*11:01 (middle panel). This T cell clone also reacted to HLA-A*11:01-positive primary AML with mutant NPM1, but not to HLA-A*11:01-positive AML with wild-type NPM1 (lower panel). Indicated are the average release amounts of IFN-γ in the culture supernatants of duplicate wells.

[0261] Fig.41 The activity of TCR-T cells against mutant NPM1 in HLA-A*03:01 or HLA-A*11:01 is shown. Fig.41(A) HLA-A*03:01 restricted TCR involving mutant NPM1 for clone 31.3.F1, which has been sequenced, cloned and introduced into CD8+ cells from an HLA-A*03:01 positive healthy individual (donor 10231). As a control, an HLA-A*03:01 restricted TCR for EBNA3A was introduced. TCR-T cells were isolated, restimulated, and tested for binding to pHLA-tetramers and reactivity to AML by IFN-γ ELISA. TCR-T cells showed specific binding to PE-labeled pHLA*03:01-AVEEVSLRK tetramers (top). pHLA-tetramers did not bind to EBNA3A TCR-T cells (bottom histogram). In the IFN-γ ELISA, TCR-T cells for mutant NPM1 in HLA-A*03:01 (black bars) specifically reacted to OCI-AML3 (mutant NPM1; dNPM1) transduced with HLA-A*03:01 and EBV-B cells from HLA-A*03:01-positive individuals pulsed with a mixture of dNPM1 and EBNA3A peptides, but not to unpulsed EBV-B cells, OCI-AML3, OCI-AML3 transduced with HLA-A*11:01, OCI-AML2 (wild-type NPM1; wtNPM1), and OCI-AML2 transduced with HLA-A*03:01 or HLA-A*11:01 (middle graph). Indicated is the average amount of IFN-γ released in the culture supernatant of duplicate wells. Fig.41(B) HLA-A*11:01 restricted T cell receptor (TCR) involving mutant NPM1 used for cloning 6F11, which has been sequenced, cloned and introduced into CD8+ cells from an HLA-A*11:01 positive healthy individual (donor 10095). As a control, an HLA-A*11:01 restricted TCR for EBNA3B was introduced. TCR-T cells were isolated, restimulated, and tested for pHLA-tetramer binding and reactivity to AML by IFN-γ ELISA. The TCR-T cells showed specific binding to PE-labeled pHLA*11:01-AVEEVSLRK tetramers and to a lesser extent to PE-labeled pHLA-A*11:01-CLAVEEVSLRK tetramers (top). Neither pHLA tetramer binds to EBNA3B TCR-T cells (bottom two histograms). In the IFN-γ ELISA, TCR-T cells for dNPM1 in HLA-A*11:01 (black bars) specifically reacted against OCI-AML3 (mutant NPM1; dNPM1) transduced with HLA-A*11:01 and EBV-B cells from HLA-A*11:01-positive individuals pulsed with a mixture of dNPM1 and BNA3B peptides, but not against unpulsed EBV-B cells, OCI-AML3, OCI-AML3 transduced with HLA-A*03:01, OCI-AML2 (wild-type NPM1; wtNPM1), and OCI-AML2 transduced with HLA-A*11:01 or HLA-A*03:01 (middle graph). Indicated is the average amount of IFN-γ released in the culture supernatant of duplicate wells. Fig.41(C) HLA-A*11:01 restricted T cell receptor (TCR) involving mutant NPM1 for clone 26.2.D6, which has been sequenced, cloned and introduced into CD8+ cells from an HLA-A*11:01 positive healthy individual (donor 10231). As a control, an HLA-A*11:01 restricted TCR for EBNA3B was introduced. TCR-T cells were isolated, restimulated and tested for pHLA-tetramer binding and reactivity to AML by IFN-γ ELISA. The TCR-T cells showed specific binding to PE-labeled pHLA*11:01-AVEEVSLRK tetramers and to a lesser extent to PE-labeled pHLA*11:01-CLAVEEVSLRK tetramers (top). Neither pHLA tetramer binds to EBNA3B TCR-T cells (bottom two histograms). In the IFN-γ ELISA, TCR-T cells for dNPM1 in HLA-A*11:01 (black bars) specifically reacted to OCI-AML3 (mutant NPM1; dNPM1) transduced with HLA-A*11:01 and EBV-B cells from HLA-A*11:01-positive individuals pulsed with a mixture of dNPM1 and EBNA3B peptides, but not to unpulsed EBV-B cells, OCI-AML3, OCI-AML3 transduced with HLA-A*03:01, OCI-AML2 (wild-type NPM1; wtNPM1), and OCI-AML2 transduced with HLA-A*11:01 or HLA-A*03:01 (middle graph). Indicated are the average IFN-γ release in the culture supernatant of duplicate wells.

[0262] Fig.42 The reactivity of T cell clones for mutant NPM1 in HLA-A*11:01 against healthy hematopoietic cell types is shown. T cell clones 26.2.D6 and 6F11 specific for mutant NPM1 in HLA-A*11:01 were tested by IFN-γ ELISA against PHA-T cells, PBMCs and monocyte-derived dendritic cells (moDCs) from HLA-A*11:01-positive healthy donor PBL3499. Both T cell clones failed to recognize healthy hematopoietic cell types, whereas all cell types were recognized after exogenous pulsing with mutant NPM1-derived AVEEVSLRK peptide.

[0263] Fig.43TCR-T cells for mutant NPM1 in HLA-A*11:01 were shown to be reactive against AML in vitro. CD8 cells from HLA-A*11:01-positive healthy individuals were transduced with HLA-A*11:01-restricted mutant NPM1-specific (dNPM1-A11) 26.2D6 or 6F11 TCRs and tested for reactivity against OCI-AML2 (wild-type NPM1) transduced with HLA-A*11:01, OCI-AML3 (mutant NPM1) transduced with HLA-A*11:01, as well as patient AML case 10535 (mutant NPM1; HLA-A*11:01) and AML case JHZ8388 (wild-type NPM1; HLA-A*11:01) by IFN-γ ELISA. dNPM1-A11TCR-T cells were generated with wild-type endogenous human TCRα and β genes or using both genes knocked out by CRISPR / Cas9 (TCRαβKO).

[0264] Fig.44 TCR-T cells for mutant NPM1 in HLA-A*11:01 were shown to be reactive against AML in vivo. Immunodeficient NSG-hIL7 / hIL15 mice were implanted with 1x10^6 OCI-AML3 (mutant NPM1) cells transduced with HLA-A*11:01. The OCI-AML3 cell line was also transduced with luciferase to track tumor growth in vivo. On day 11 after injection, when tumor cells were detectable, mice received an infusion of 3x10^6 CD8 cells transduced with HLA-A*11:01 restricted mutant NPM1 specific (dNPM1-A11) 6F11 or 26.2D6 TCRs. Mice were treated with CD8 cells transduced with HLA-A*02:01 restricted mutant NPM1 specific 1A2 TCR (as a positive control) and with CD8 cells transduced with HLA-A*11:01 restricted EBNA3B specific TCR (as a negative control). Mice were sacrificed on day 7 after TCR-T cell infusion. DETAILED DESCRIPTION

[0265] The immunogenicity of peptides derived from mutant NPM1 (ΔNPM1) has been previously studied 14 , in which computer screening of the entire amino acid sequence of ΔNPM1 was used to predict which peptides might be presented by HLA-A*02:01. Peptides predicted to bind to HLA-A*02:01 were synthetically generated and included CLAVEEVSL (SEQ ID NO: 1). CD8 +T cells were isolated from healthy individuals and cells from ΔNPM1 AML patients stimulated with peptides, and T cell responses were measured. Only two of the tested peptides (AIQDLCLAV (SEQ ID NO: 32) and AIQDLCVAV (SEQ ID NO: 33)) were found to be able to elicit an immune response in vitro. Therefore, these peptides were considered to be the most important epitopes of ΔNPM1 and were therefore used for further studies.

[0266] The inventors have now identified five different peptides present in the HLA class I ligand set of ΔNPM1-positive primary AML (i.e., CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0267] Now, the inventors have also unexpectedly demonstrated that, among all possible peptides within ΔNPM1, the CLAVEEVSL (SEQ ID NO: 1) peptide is present on the surface of primary ΔNPM1 AML cells isolated from HLA-A*02:01 cancer patients, and that this peptide is present on the surface of isolated primary AML cells in a cysteine-ylated form.

[0268] Now, the inventors have also surprisingly found that the ΔNPM1 derived peptides AVEEVSLRK (SEQ ID NO:26) and CLAVEEVSLRK (SEQ ID NO:27) are presented by HLA-A*03:01 and HLA-A*11:01, respectively, and that AVEEVSLRK is also presented by HLA-A*01:01.

[0269] The inventors isolated and cloned T cell receptors reactive with CLAVEEVSL (SEQ ID NO: 1) from the T cell pool of healthy HLA-A*02:01-positive individuals. They demonstrated that these T cell receptors can be used for genetic engineering of peripheral blood lymphocytes, and that genetically engineered lymphocytes can effectively kill HLA-A*02:01-positive AML with ΔNPM1. Advantageously, these TCRs can be used as effective immunotherapy for treating HLA-A*02:01-positive patients with ΔNPM1-positive AML.

[0270] The inventors also isolated a T cell clone reactive with CLAVEEVSLRK (SEQ ID NO: 27) (specifically, a cysteine ​​variant of the peptide) from the T cell repertoire of a healthy HLA-A*03:01-positive individual. Advantageously, the TCR from this clone can be used as an effective immunotherapy for treating HLA-A*03:01-positive patients with ΔNPM1-positive AML.

[0271] The inventors have also isolated four T cell clones reactive with AVEEVSLRK (SEQ ID NO: 26) from the T cell repertoire of healthy HLA-A*03:01 or HLA-A*11:01 positive individuals. In particular, T cell clone 26.2.D6 has been isolated from an HLA-A*11:01 negative (but not A*03:01 positive) individual. 6F11 and 31.3.F1 were isolated from HLA-A*11:01+ and A*03:01+ individuals, respectively. Advantageously, TCRs from these clones can be used as effective immunotherapy for treating HLA-A*03:01 and HLA-A*11:01 positive patients with ΔNPM1 positive AML.

[0272] Nucleic acid sequences encoding TCR polypeptide components

[0273] The present invention provides a nucleic acid sequence encoding a T cell receptor component that specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29). The nucleic acid sequence can form part of a larger nucleic acid sequence encoding a larger component of a T cell receptor (e.g., a TCR α chain variable region, a TCR β chain variable region, a TCR α chain, a TCR β chain, etc.). The nucleic acid sequence can also form part of a larger nucleic acid sequence encoding a functional T cell receptor (i.e., encoding a functional TCR α chain and a functional TCR β chain, which are optionally separated by a linker sequence that enables coordinated expression of two proteins or polypeptides by the same vector). More details on this are provided below.

[0274] The nucleic acid sequence may encode only a small component of a T cell receptor, such as a CDR3 domain of a TCR α chain polypeptide, or a CDR3 domain of a TCR β chain polypeptide. Therefore, the nucleic acid sequence may be regarded as a "building block" that provides the necessary components for peptide specificity. The nucleic acid sequence of the present invention may be incorporated into a unique nucleic acid sequence (e.g., a vector) encoding other elements of a TCR variable chain, such that when the nucleic acid sequence of the present invention is incorporated, a new nucleic acid sequence is generated that encodes a TCR α chain variable region and / or a TCR β chain variable region that specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29). Therefore, the nucleic acid sequences of the present invention can be used as essential components of TCR specificity for the selected ΔNPM1 peptide and can thus be used to generate nucleic acid sequences encoding TCR variable regions having the desired antigen binding activity and having specificity for targeting ΔNPM1-positive AML.

[0275] The T cell receptor (TCR) is a molecule present on the surface of T cells (T lymphocytes) that is responsible for recognizing peptides bound to (presented by) major histocompatibility complex (MHC) molecules on target cells. The present invention relates to nucleic acid sequences encoding TCRs that interact with specific peptides in the context of an appropriate MHC serotype, such as CLAVEEVSL (SEQ ID NO: 1) in the context of HLA-A*02:01; or AVEEVSLRK (SEQID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) in the context of HLA-A*03:01 and HLA-A*11:01, or AVEEVSLRK (SEQID NO: 26) in the context of HLA-A*01:01, respectively.

[0276] HLA-A*02:01 is a globally common human leukocyte antigen serotype in the HLA-A serotype group. Peptides presented to the TCR by HLA-A*02:01 are described as "HLA-A*02:01 restricted".

[0277] HLA-A*03:01, HLA-A*11:01, and HLA-A*01:01 are also common human leukocyte antigen serotypes within the HLA-A serotype group. Peptides presented to the TCR by HLA-A*03:01 are described as "HLA-A*03:01 restricted". Similarly, peptides presented to the TCR by HLA-A*11:01 are described as "HLA-A*11:01 restricted". Similarly, peptides presented to the TCR by HLA-A*01:01 are described as "HLA-A*01:01 restricted".

[0278] The TCR consists of two different polypeptide chains. In humans, 95% of TCRs are composed of an alpha chain and a beta chain (encoded by TRA and TRB, respectively). When the TCR binds to a peptide in the context of an HLA (e.g., in the context of HLA-A*02:01, HLA-A*03:01, or HLA-A*11:01, as appropriate), the T cell is activated through signal transduction. The sequences of the alpha and beta chains of the TCR are highly variable. Each chain consists of two extracellular domains, a variable region (V) and a constant region (C). The constant region is close to the T cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / HLA-A complex.

[0279] The variable region of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Thus, the TCR alpha chain contains CDR1, CDR2 and CDR3, while the TCR beta chain also contains (different) CDR1, CDR2 and CDR3. In each alpha and beta chain, CDR3 is primarily responsible for recognizing peptides presented by HLA-A.

[0280] In one aspect, the present invention provides an isolated nucleic acid sequence encoding: (a) a polypeptide comprising the CDR3 of a TCR alpha chain polypeptide, which TCR alpha chain polypeptide specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29); and / or (b) a polypeptide comprising the CDR3 of a TCR beta chain polypeptide, which TCR beta chain polypeptide specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0281] In a specific embodiment, the encoded polypeptide specifically binds to CLAVEEVSL (SEQ ID NO: 1). CLAVEEVSL (SEQ ID NO: 1) may be a cysteine-form. The encoded polypeptide may specifically bind only to the cysteine-form.

[0282] In a specific embodiment, the encoded polypeptide specifically binds AVEEVSLRK (SEQ ID NO: 26).

[0283] In one embodiment, the encoded polypeptide specifically binds to CLAVEEVSLRK (SEQ ID NO: 27). CLAVEEVSLRK (SEQ ID NO: 27) may be a cysteine-ylated form. The encoded polypeptide may specifically bind only to the cysteine-ylated form.

[0284] The nucleic acid sequence may encode (a), (b) or (a) and (b). Thus, the nucleic acid sequence encodes at least one polypeptide comprising a CDR3 of a T cell receptor polypeptide, wherein the CDR3 specifically binds to one of the following peptides: CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) or AVEEVSLR (SEQ ID NO: 29).

[0285] The nucleic acid sequence may include an α chain CDR3 and a β chain CDR3, wherein the α chain CDR3 and the β chain CDR3 together specifically bind to a selected peptide.

[0286] Therefore, the nucleic acid sequence encodes "CDR3 of TCR α chain polypeptide" (also referred to herein as α chain CDR3 or α chain CDR3) and / or "CDR3 of TCR β chain polypeptide" (also referred to herein as β chain CDR3 or β chain CDR3).

[0287] The α chain CDR3 may be the α chain CDR3 of SEQ ID NO: 2 or one of the variants described below. Similarly, the β chain CDR3 may be the β chain CDR3 of SEQ ID NO: 5 or one of the variants described below. Notably, the inventors have discovered that these particular CDR3s can specifically bind to the peptide of SEQ ID NO: 1.

[0288] Alternatively, the α chain CDR3 may be the α chain CDR3 of SEQ ID NO: 35 or one of the variants described below. Similarly, the β chain CDR3 may be the β chain CDR3 of SEQ ID NO: 43 or one of the variants described below. Notably, the inventors have found that these particular CDR3s can specifically bind to the peptide of SEQ ID NO: 26.

[0289] Alternatively, the α chain CDR3 may be the α chain CDR3 of SEQ ID NO: 51 or one of the variants described below. Similarly, the β chain CDR3 may be the β chain CDR3 of SEQ ID NO: 59 or one of the variants described below. Notably, the inventors have found that these particular CDR3s can specifically bind to the peptide of SEQ ID NO: 26.

[0290] Alternatively, the α chain CDR3 may be the α chain CDR3 of SEQ ID NO: 67 or one of the variants described below. Similarly, the β chain CDR3 may be the β chain CDR3 of SEQ ID NO: 75 or one of the variants described below. Notably, the inventors have discovered that these particular CDR3s can specifically bind to the peptide of SEQ ID NO: 26.

[0291] Any arrangement described below for (a) may be combined with the arrangement described below for (b) (e.g., to form an appropriate nucleic acid sequence encoding a functional T cell receptor (i.e., encoding a functional TCR α chain and TCR β chain, which are optionally separated by a linker sequence to enable coordinated expression of both proteins or polypeptides by the same vector).

[0292] Polypeptide (a) - component of TCR alpha chain (same as TCR of clone 1A2 described herein)

[0293] In one embodiment, the CDR3 of (a) may have the amino acid sequence of SEQ ID NO:2, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:2. The term "variant" also encompasses homologues. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:2, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0294] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 2 that does not specifically bind to SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 2, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0295] In one embodiment, the CDR3 of (a) may have an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 1. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 2 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 2).

[0296] In the example where the CDR3 of (a) has the amino acid sequence of SEQ ID NO: 2, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO: 4 is a codon-optimized version of the CDR3 nucleic acid sequence of clone 1A2 (the unoptimized sequence is SEQ ID NO: 3). Therefore, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0297] In one embodiment, the polypeptide of (a) comprises a CDR3 within a TCR α chain variable region that specifically binds to a peptide of SEQ ID NO: 1 (e.g., a CDR3 of SEQ ID NO: 2 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a TCR α chain variable region containing a specified CDR3, wherein the TCR α chain variable region (and CDR3 therein) specifically binds to a peptide of SEQ ID NO: 1. It will be clear to those skilled in the art that the phrase "TCR α chain variable region" refers to the variable (V) region (extracellular domain) of the TCR α chain, and therefore includes three hypervariable regions (CDR1, CDR2, and specified CDR3), as well as intervening sequences, but does not include the constant (C) region of the α chain, which does not constitute part of the variable chain.

[0298] The encoded TCR α chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 14, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 1), in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 14. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 14, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0299] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 14 that does not specifically bind to the N-terminus of the peptide of SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 14, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0300] In one embodiment, the CDR1 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 14, while retaining the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 1. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 14 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 14).

[0301] In the example where the CDR1 of (a) (e.g., in the α chain variable region) has the amino acid sequence of SEQ ID NO: 14, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO: 19 is a codon-optimized version of the CDR1 nucleic acid sequence of clone 1A2 (the unoptimized sequence is SEQ ID NO: 18). Therefore, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0302] The encoded TCR α chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 15 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*02:01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 15. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 15, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0303] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 15 that does not specifically bind to HLA-A*02:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 15, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0304] In one embodiment, the CDR2 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 15, while retaining the ability to bind to HLA-A*02:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 15 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 15).

[0305] In the example where the CDR2 of (a) (e.g., in the α chain variable region) has the amino acid sequence of SEQ ID NO: 15, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NQ: 20 or SEQ ID NO: 21 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO: 21 is a codon-optimized version of the CDR2 nucleic acid sequence of clone 1A2 (the unoptimized sequence is SEQ ID NQ: 20). Therefore, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NQ: 20 or SEQ ID NO: 21 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0306] Thus, the polypeptide of (a) may comprise a TCR alpha chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), and appropriate intervening sequences between the CDRs.

[0307] The TCR α chain variable region of (a) may have the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 8, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0308] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 8 that does not specifically bind to SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 8, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0309] In one embodiment, the TCR α chain variable region of (a) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 8, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 1. In other words, functional TCR α chain variable regions having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 8 are also encompassed. As described above, the amino acid substitutions may be conservative amino acid substitutions. Compared to SEQ ID NO: 8, the variability of the sequence may all be located within the TCR α chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 2, SEQ ID NO: 14 and / or SEQ ID NO: 15, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 8). In other words, the CDR sequences of SEQ ID NO: 8 may be retained while the rest of the sequence may be altered, as appropriate, within the parameters of "at least 75% identity" specified above. Suitably, the percent identity may be calculated as the percent identity over the entire length of the reference sequence (e.g. SEQ ID NO: 8).

[0310] As an example, the polypeptide of (a) may include CDR3 within a TCR α chain variable region that has at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity with the amino acid sequence of SEQ ID NO: 8, wherein CDR3 has the amino acid sequence of SEQ ID NO: 2. In this example, TCR α chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 14, and TCR α chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 15.

[0311] In the example where the TCR α chain variable region of (a) has the amino acid sequence of SEQ ID NO: 8, the TCR α chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NQ: 10 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is worth noting that SEQ ID NQ: 10 is a codon-optimized version of the nucleic acid sequence of the TCR α chain variable region of clone 1A2 (the unoptimized sequence is SEQ ID NO: 9). Therefore, the polypeptide of (a) can be encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0312] For the avoidance of doubt, the polypeptide of (a) may comprise a TCR alpha chain variable region (as described above) and a TCR alpha chain constant region. An example of a suitable constant region is encoded by GenScript in the MP71-TCR-flex retroviral vector used herein. However, the present invention is not limited to this specific constant region, but encompasses any suitable TCR alpha chain constant region. The constant region may be mouse, human or humanized. Methods for identifying or generating suitable constant regions are well known to those skilled in the art and are fully within their conventional capabilities.

[0313] By way of example only, the constant region may be encoded by or derived from a vector, such as a lentivirus, retrovirus or plasmid vector, and may also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpes virus vector, in which the mouse or human constant region is pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). In addition, naked (synthetic) DNA / RNA may also be used to introduce TCR. As an example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes described in LV Coren et al., BioTechniques 2015 may be used to provide appropriate constant regions.

[0314] Polypeptide (b) - component of TCR β chain (same as TCR of clone 1A2 described herein)

[0315] In one embodiment, the CDR3 of (b) may have the amino acid sequence of SEQ ID NO:5, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:5. The term "variant" also encompasses homologues. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:5, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0316] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 5 that does not specifically bind to SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 5, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0317] In one embodiment, the CDR3 of (b) may have an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5, while retaining the ability to specifically bind to the peptide of SEQ ID NO:1. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO:5 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO:5).

[0318] In the example where the CDR3 of (b) has the amino acid sequence of SEQ ID NO:5, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO:7 is a codon-optimized version of the CDR3 nucleic acid sequence of clone 1A2 (the unoptimized sequence is SEQ ID NO:6). Therefore, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0319] In one embodiment, the polypeptide of (b) comprises a CDR3 within a TCR β chain variable region that specifically binds to a peptide of SEQ ID NO: 1 (e.g., a CDR3 of SEQ ID NO: 5 or a variant thereof as defined above). In other words, the polypeptide of (b) comprises a TCR β chain variable region containing a specified CDR3, wherein the TCR β chain variable region (and CDR3 therein) specifically binds to a peptide of SEQ ID NO: 1. It will be clear to those skilled in the art that the phrase "TCR β chain variable region" refers to the variable (V) region (extracellular domain) of the TCR β chain, and therefore includes three hypervariable regions (CDR1, CDR2, and specified CDR3) and intervening sequences, but does not include the constant (C) region of the β chain, which does not constitute part of the variable chain.

[0320] The encoded TCR β chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 16, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 1), in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 16. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 16, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0321] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 16 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 16, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0322] In one embodiment, the CDR1 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 16, while retaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 1. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 16 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 16).

[0323] In the example where the CDR1 of (b) (e.g., in the variable region of the beta chain) has the amino acid sequence of SEQ ID NO: 16, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO: 23 is a codon-optimized version of the CDR1 nucleic acid sequence of clone 1A2 (the unoptimized sequence is SEQ ID NO: 22). Therefore, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0324] The encoded TCR β chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 17, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*02:01), in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 17. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 17, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0325] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 17 that does not specifically bind to HLA-A*02:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 17, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0326] In one embodiment, the CDR2 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17, while retaining the ability to bind to HLA-A*02:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 17 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 17).

[0327] In the example where the CDR2 of (b) (e.g., in the variable region of the beta chain) has the amino acid sequence of SEQ ID NO: 17, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is noteworthy that SEQ ID NO: 25 is a codon-optimized version of the nucleic acid sequence of the CDR2 of clone 1A2 (the unoptimized sequence is SEQ ID NO: 24). Therefore, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0328] Thus, the polypeptide of (b) may comprise a TCR beta chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID or variants thereof), with appropriate intervening sequences between the CDRs.

[0329] The TCR β chain variable region of (b) may have the amino acid sequence of SEQ ID NO: 11, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 11. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 11, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0330] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 11 that does not specifically bind to SEQ ID NO: 1. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 11, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0331] In one embodiment, the TCR β chain variable region of (b) may have an amino acid sequence with at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 11, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 1. In other words, a functional TCR β chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 11 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. The sequence variability compared to SEQ ID NO: 11 may all be located in the TCR β chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 5, SEQ ID NO: 16 and / or SEQ ID NO: 17, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 11). In other words, the CDR sequences of SEQ ID NO: 11 can be retained while the rest of the sequence can be altered, optionally within the "at least 75% identity" parameter specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (eg SEQ ID NO: 11).

[0332] As an example, (b) may include CDR3 within a TCR β chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity with the amino acid sequence of SEQ ID NO: 11, wherein CDR3 has the amino acid sequence of SEQ ID NO: 5. In this example, TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 16, and TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 17.

[0333] In the example where the TCR β chain variable region of (b) has the amino acid sequence of SEQ ID NO: 11, the TCR β chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). It is worth noting that SEQ ID NO: 13 is a codon-optimized version of the nucleic acid sequence of the TCR β chain variable region of clone 1A2 (the unoptimized sequence is SEQ ID NO: 12). Therefore, the polypeptide of (b) can be encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or its genetically degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0334] For the avoidance of doubt, (b) may include TCR β chain variable regions (as described above) and TCR β chain constant regions. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector used in this article by GenScript. However, the present invention is not limited to this specific constant region, and encompasses any suitable TCR β chain constant region. The constant region may be mouse-derived, human-derived or humanized. Methods for identifying or generating suitable constant regions are well known to those skilled in the art and are fully within their conventional capabilities.

[0335] By way of example only, the constant region may be encoded by or derived from a vector, such as a lentivirus, a retrovirus or a plasmid vector, and may also be an adenovirus, adeno-associated virus, vaccinia virus, canary pox virus or herpes virus vector, in which the mouse or human constant region is pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). In addition, naked (synthetic) DNA / RNA may also be used to introduce TCR. As an example, the MP71-TCR-flex retroviral vector with pre-cloned TCR-Ca and Cb genes used by the inventors or the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes described in LV Coren et al., BioTechniques 2015 may be used to provide appropriate constant regions.

[0336] Alternative embodiment of polypeptide (a) - components of the TCR alpha chain (same as the TCR of clone 26.2.D6 described herein)

[0337] In one embodiment, the CDR3 of (a) may have the amino acid sequence of SEQ ID NO:35, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:35. The term "variant" also encompasses homologues. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:35, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0338] A non-functional variant is an amino acid sequence variant of SEQ ID NO:35 that does not specifically bind to SEQ ID NO:26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO:35, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0339] In one embodiment, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 35, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 35 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 35).

[0340] In the example where the CDR3 of (a) has the amino acid sequence of SEQ ID NO: 35, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 36 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0341] In one embodiment, the polypeptide of (a) comprises a CDR3 within a TCR α chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 35 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a TCR α chain variable region comprising a specified CDR3, wherein the TCR α chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0342] The encoded TCR α chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 39, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 39. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 39, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0343] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 39 that does not specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 39, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0344] In one embodiment, the CDR1 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 39, while retaining the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 39 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 39).

[0345] In the example where the CDR1 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:39, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:40 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0346] The encoded TCR α chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 41, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*11: 01), in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 41. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 41, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0347] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 41 that does not specifically bind to HLA-A*11:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 41, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0348] In one embodiment, the CDR2 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 41, while retaining the ability to bind to HLA-A*11:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 41 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 41).

[0349] In the example where the CDR2 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:41, CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO:42 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0350] Thus, the polypeptide of (a) may comprise a TCR alpha chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), with appropriate intervening sequences between the CDRs.

[0351] The TCR α chain variable region of (a) may have an amino acid sequence of SEQ ID NO: 37, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 37. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 37, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0352] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 37 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 37, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0353] In one embodiment, the TCR α chain variable region of (a) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 37, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR α chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 37 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 37, the variability of the sequence may all be located within the TCR α chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 35, SEQ ID NO: 39 and / or SEQ ID NO: 41, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 37). In other words, the CDR sequences of SEQ ID NO: 37 can be retained while the rest of the sequence can be altered, optionally within the "at least 75% identity" parameters specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 37).

[0354] As an example, the polypeptide of (a) may comprise CDR3 within a TCR α chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 37, wherein CDR3 has the amino acid sequence of SEQ ID NO: 35. In this example, the TCR α chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 39, and the TCR α chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 41.

[0355] In the example where the TCR α chain variable region of (a) has the amino acid sequence of SEQ ID NO: 37, the TCR α chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 38 or its genetic degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0356] For the avoidance of doubt, the polypeptide of (a) may comprise a TCR alpha chain variable region (as described above) and a TCR alpha chain constant region. Examples of suitable constant regions are described in detail elsewhere herein and are equally applicable here.

[0357] Alternative embodiment of polypeptide (b) - component of TCR beta chain (same as TCR of clone 26.2.D6 described herein)

[0358] In one embodiment, the CDR3 of (b) may have the amino acid sequence of SEQ ID NO:43, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:43. The term "variant" also encompasses homologues. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:43, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0359] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 43 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 43, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0360] In one embodiment, the CDR3 of (b) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 43, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 43 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 43).

[0361] In the example where the CDR3 of (b) has the amino acid sequence of SEQ ID NO:43, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO:44 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0362] In one embodiment, the polypeptide of (b) comprises a CDR3 within a TCR β chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 43 or a variant thereof as defined above). In other words, the polypeptide of (b) comprises a TCR β chain variable region that contains a specified CDR3, wherein the TCR β chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0363] The encoded TCR β chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 47, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 47. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 47, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0364] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 47 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 47, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0365] In one embodiment, the CDR1 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 47, while retaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 47 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 47).

[0366] In the example where the CDR1 of (b) (e.g., within the β chain variable region) has the amino acid sequence of SEQ ID NO:47, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:48 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0367] The encoded TCR β chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 49 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*11: 01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 49. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 49, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0368] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 49 that does not specifically bind to HLA-A*11:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 49, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0369] In one embodiment, the CDR2 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 49, while retaining the ability to bind to HLA-A*11:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 49 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 49).

[0370] In the example where the CDR2 of (b) (e.g., within the variable region of the β chain) has the amino acid sequence of SEQ ID NO:49, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:50 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0371] Thus, the polypeptide of (b) may comprise a TCR β chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID or variants thereof), with appropriate intervening sequences between the CDRs.

[0372] The TCR β chain variable region of (b) may have an amino acid sequence of SEQ ID NO: 45 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 45. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 45, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0373] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 45 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 45, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0374] In one embodiment, the TCR β chain variable region of (b) may have an amino acid sequence that has at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 45, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR β chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 45 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 45, the variability of the sequence may be all within the TCR β chain variable region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 43, SEQ ID NO: 47 and / or SEQ ID NO: 49, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 45). In other words, the CDR sequences of SEQ ID NO: 45 can be retained while the rest of the sequence can be altered, optionally within the "at least 75% identity" parameters specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (eg SEQ ID NO: 45).

[0375] As an example, (b) may include a CDR3 within a TCR β chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 45, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 43. In this example, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 47, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 49.

[0376] In the example of (b) where the TCR β chain variable region has the amino acid sequence of SEQ ID NO:45, the TCR β chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO:46 or its genetic degenerate sequence (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0377] For the avoidance of doubt, (b) may comprise a TCR beta chain variable region (as described above) and a TCR beta chain constant region. Examples of suitable constant regions are provided elsewhere herein and are equally applicable here.

[0378] Alternative embodiment of polypeptide (a) - components of the TCR alpha chain (same as the TCR of clone 6F11 described herein)

[0379] In one embodiment, the CDR3 of (a) may have the amino acid sequence of SEQ ID NO:51, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:51. The term "variant" also encompasses homologues. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:51, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0380] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 51 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 51, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0381] In one embodiment, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 51, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 51 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 51).

[0382] In the example where the CDR3 of (a) has the amino acid sequence of SEQ ID NO:51, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO:52 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0383] In one embodiment, the polypeptide of (a) comprises a CDR3 within a TCR α chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 51 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a TCR α chain variable region comprising a specified CDR3, wherein the TCR α chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0384] The encoded TCR α chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 55, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 55. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 55, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0385] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 55 that does not specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 55, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0386] In one embodiment, the CDR1 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 55, while retaining the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 55 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 55).

[0387] In the example where the CDR1 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:55, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:56 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0388] The encoded TCR α chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 57 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*11: 01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 57. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 57, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0389] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 57 that does not specifically bind to HLA-A*11:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 57, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0390] In one embodiment, the CDR2 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 57, while retaining the ability to bind to HLA-A*11: 01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 57 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 57).

[0391] In the example where the CDR2 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:57, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:58 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0392] Thus, the polypeptide of (a) may comprise a TCR alpha chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), with appropriate intervening sequences between the CDRs.

[0393] The TCR α chain variable region of (a) may have an amino acid sequence of SEQ ID NO: 53 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 53. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 53, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0394] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 53 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 53, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0395] In one embodiment, the TCR α chain variable region of (a) may have an amino acid sequence that has at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 53, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR α chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 53 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 53, the variability of the sequence may all be located within the TCR α chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 51, SEQ ID NO: 55 and / or SEQ ID NO: 57, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 53). In other words, the CDR sequences of SEQ ID NO: 53 can be retained while the rest of the sequence can be altered, optionally within the "at least 75% identity" parameter specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (eg SEQ ID NO: 53).

[0396] As an example, the polypeptide of (a) may include CDR3 within a TCR α chain variable region that has at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity with the amino acid sequence of SEQ ID NO: 53, wherein CDR3 has the amino acid sequence of SEQ ID NO: 51. In this example, the TCR α chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 55, and the TCR α chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 57.

[0397] In the example where the TCR α chain variable region of (a) has the amino acid sequence of SEQ ID NO: 53, the TCR α chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 54 or its genetic degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0398] For the avoidance of doubt, the polypeptide of (a) may comprise a TCR alpha chain variable region (as described above) and a TCR alpha chain constant region. Examples of suitable constant regions are described in detail elsewhere herein and are equally applicable here.

[0399] Alternative embodiment of polypeptide (b) - components of the TCR beta chain (same as the TCR of clone 6F11 described herein)

[0400] In one embodiment, the CDR3 of (b) may have the amino acid sequence of SEQ ID NO:59, or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:59. The term "variant" also encompasses homologues. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:59, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0401] A non-functional variant is an amino acid sequence variant of SEQ ID NO:59 that does not specifically bind to SEQ ID NO:26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO:59, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0402] In one embodiment, the CDR3 of (b) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 59 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 59).

[0403] In the example where the CDR3 of (b) has the amino acid sequence of SEQ ID NO: 59, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 60 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0404] In one embodiment, the polypeptide of (b) comprises a CDR3 within a TCR β chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 59 or a variant thereof as defined above). In other words, the polypeptide of (b) comprises a TCR β chain variable region that contains a specified CDR3, wherein the TCR β chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0405] The encoded TCR β chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 63 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 63. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 63, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0406] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 63 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 63, or a substitution, insertion or deletion of a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0407] In one embodiment, the CDR1 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 63, while retaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 63 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 63).

[0408] In the example where the CDR1 of (b) (e.g., within the β chain variable region) has the amino acid sequence of SEQ ID NO:63, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:64 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0409] The encoded TCR β chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 65 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*11: 01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 65. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 65, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0410] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 65 that does not specifically bind to HLA-A*11:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 65, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0411] In one embodiment, the CDR2 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 65, while retaining the ability to bind to HLA-A*11:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 65 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 65).

[0412] In the example where the CDR2 of (b) (e.g., within the variable region of the β chain) has the amino acid sequence of SEQ ID NO:65, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:66 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0413] Thus, the polypeptide of (b) may comprise a TCR β chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), with appropriate intervening sequences between the CDRs.

[0414] The TCR β chain variable region of (b) may have an amino acid sequence of SEQ ID NO: 61 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 61. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 61, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0415] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 61 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 61, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0416] In one embodiment, the TCR β chain variable region of (b) may have an amino acid sequence that has at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 61, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR β chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 61 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 61, the variability of the sequence may all be located within the TCR β chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 59, SEQ ID NO: 63 and / or SEQ ID NO: 65, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 61). In other words, the CDR sequences of SEQ ID NO: 61 can be retained while the rest of the sequence can be altered, optionally within the parameters of "at least 75% identity" specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (eg SEQ ID NO: 61).

[0417] As an example, (b) may include a CDR3 within a TCR β chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity with the amino acid sequence of SEQ ID NO: 61, wherein CDR3 has the amino acid sequence of SEQ ID NO: 59. In this example, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 63, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 65.

[0418] In the example of (b) where the TCR β chain variable region has the amino acid sequence of SEQ ID NO:61, the TCR β chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO:62 or its genetic degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0419] For the avoidance of doubt, (b) may comprise a TCR beta chain variable region (as described above) and a TCR beta chain constant region. Examples of suitable constant regions are provided elsewhere herein and are equally applicable here.

[0420] Alternative embodiment of polypeptide (a) - components of the TCR alpha chain (same as the TCR of clone 31.3.F1 described herein)

[0421] In one embodiment, the CDR3 of (a) may have the amino acid sequence of SEQ ID NO:67 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:67. The term "variant" also encompasses homologues. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:67, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0422] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 67 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 67, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0423] In one embodiment, the CDR3 of (a) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 67 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 67).

[0424] In the example where the CDR3 of (a) has the amino acid sequence of SEQ ID NO: 67, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 68 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0425] In one embodiment, the polypeptide of (a) comprises a CDR3 within a TCR α chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 67 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a TCR α chain variable region comprising a specified CDR3, wherein the TCR α chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0426] The encoded TCR α chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 71 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 71. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 71, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0427] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 71 that does not specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 71, or a substitution, insertion or deletion in a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0428] In one embodiment, the CDR1 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 71, while retaining the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 71 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 71).

[0429] In the example where the CDR1 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:71, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:72 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0430] The encoded TCR α chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 73 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*03: 01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 73. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 73, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0431] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 73 that does not specifically bind to HLA-A*03:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 73, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0432] In one embodiment, the CDR2 of (a) (e.g., within the α chain variable region) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 73, while retaining the ability to bind to HLA-A*03:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 73 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 73).

[0433] In the example where the CDR2 of (a) (e.g., within the α chain variable region) has the amino acid sequence of SEQ ID NO:73, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:74 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0434] Thus, the polypeptide of (a) may comprise a TCR alpha chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), with appropriate intervening sequences between the CDRs.

[0435] The TCR α chain variable region of (a) may have an amino acid sequence of SEQ ID NO: 69 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 69. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 69, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0436] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 69 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 69, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0437] In one embodiment, the TCR α chain variable region of (a) may have an amino acid sequence that has at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 69, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR α chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 69 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 69, the sequence variability may all be located within the TCR α chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 67, SEQ ID NO: 71 and / or SEQ ID NO: 73, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 69). In other words, the CDR sequences of SEQ ID NO: 53 can be retained while the rest of the sequence can be altered, optionally within the parameters of "at least 75% identity" specified above. Suitably, the percentage identity can be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 69).

[0438] As an example, the polypeptide of (a) may comprise a CDR3 within a TCR α chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 69, wherein CDR3 has the amino acid sequence of SEQ ID NO: 67. In this example, the TCR α chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 71, and the TCR α chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 73.

[0439] In the example where the TCR α chain variable region of (a) has the amino acid sequence of SEQ ID NO:69, the TCR α chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO:70 or its genetic degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0440] For the avoidance of doubt, the polypeptide of (a) may comprise a TCR alpha chain variable region (as described above) and a TCR alpha chain constant region. Examples of suitable constant regions are described in detail elsewhere herein and are equally applicable here.

[0441] Alternative embodiment of polypeptide (b) - components of the TCR beta chain (same as the TCR described herein for clone 31.3.F1)

[0442] In one embodiment, the CDR3 of (b) may have the amino acid sequence of SEQ ID NO:75 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:75. The term "variant" also encompasses homologues. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:75, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0443] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 75 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 75, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0444] In one embodiment, the CDR3 of (b) may have an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 75, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 75 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 75).

[0445] In the example where the CDR3 of (b) has the amino acid sequence of SEQ ID NO: 75, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 76 or a genetically degenerate sequence thereof (ie, other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0446] In one embodiment, the polypeptide of (b) comprises a CDR3 within a TCR β chain variable region that specifically binds to a peptide of SEQ ID NO: 26 (e.g., a CDR3 of SEQ ID NO: 75 or a variant thereof as defined above). In other words, the polypeptide of (b) comprises a TCR β chain variable region that contains a specified CDR3, wherein the TCR β chain variable region (and the CDR3 therein) specifically binds to a peptide of SEQ ID NO: 26.

[0447] The encoded TCR β chain variable region may also include a CDR1 having an amino acid sequence of SEQ ID NO: 79 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26) in addition to the specified CDR3. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 79. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 79, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0448] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 79 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 79, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0449] In one embodiment, the CDR1 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 79, while retaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 79 is also encompassed. As previously described, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 79).

[0450] In the example where the CDR1 of (b) (e.g., within the variable region of the β chain) has the amino acid sequence of SEQ ID NO: 79, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 80 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0451] The encoded TCR β chain variable region may also include a CDR2 having an amino acid sequence of SEQ ID NO: 81 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to HLA-A*03:01) in addition to the specified CDR3 (and optionally the CDR1 specified above). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 81. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 81, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0452] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 81 that does not specifically bind to HLA-A*03:01. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 81, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0453] In one embodiment, the CDR2 of (b) (e.g., within the variable region of the beta chain) may have an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 81, while retaining the ability to bind to HLA-A*03:01. In other words, a functional CDR2 having one amino acid substitution compared to the sequence of SEQ ID NO: 81 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Suitably, the percentage identity may be calculated as the percentage of identity over the entire length of the reference sequence (e.g., SEQ ID NO: 81).

[0454] In the example where the CDR2 of (b) (e.g., within the variable region of the β chain) has the amino acid sequence of SEQ ID NO:81, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:82 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code).

[0455] Thus, the polypeptide of (b) may comprise a TCR β chain variable region comprising the CDRs mentioned in detail above (particularly by SEQ ID, or variants thereof), with appropriate intervening sequences between the CDRs.

[0456] The TCR β chain variable region of (b) may have an amino acid sequence of SEQ ID NO: 77 or a functional variant thereof (i.e., wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 77. The term "variant" also encompasses homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 77, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0457] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 77 that does not specifically bind to SEQ ID NO: 26. A non-functional variant generally contains a non-conservative substitution, deletion, insertion or premature truncation of the amino acid sequence of SEQ ID NO: 77, or a substitution, insertion or deletion within a key amino acid or key region. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0458] In one embodiment, the TCR β chain variable region of (b) may have an amino acid sequence that has at least 75%, at least 80%, at least 85% or at least 90% (or 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%) sequence identity with the amino acid sequence of SEQ ID NO: 77, while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, a functional TCR β chain variable region having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 77 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Compared to SEQ ID NO: 77, the variability of the sequence may all be located within the TCR β chain variable region region that does not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 75, SEQ ID NO: 79 and / or SEQ ID NO: 81, and still have a sequence variability of 25% (or less) compared to SEQ ID NO: 77). In other words, the CDR sequences of SEQ ID NO: 77 can be retained while the rest of the sequence can be altered, optionally within the "at least 75% identity" parameter specified above. Suitably, the percent identity can be calculated as the percentage of identity over the entire length of the reference sequence (eg, SEQ ID NO: 77).

[0459] As an example, (b) may include a CDR3 within a TCR β chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 77, wherein CDR3 has the amino acid sequence of SEQ ID NO: 75. In this example, the TCR β chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 79, and the TCR β chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 80.

[0460] In the example of (b) where the TCR β chain variable region has the amino acid sequence of SEQ ID NO: 77, the TCR β chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 78 or its genetic degenerate sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0461] For the avoidance of doubt, (b) may comprise a TCR beta chain variable region (as described above) and a TCR beta chain constant region. Examples of suitable constant regions are provided elsewhere herein and are equally applicable here.

[0462] In the example where the nucleic acid molecule of the present invention encodes both (a) and (b), the polypeptide of (a) can be joined to the polypeptide of (b) by a linker, such as a linker that enables two proteins or polypeptides to be expressed by the same vector. For example, a linker comprising the porcine Teschovirus-1 2A (P2A) sequence can be used, such as a 2A sequence or 2A-like sequence from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A) or Thosea asigna virus (T2A) published in AL Szymczak et al., Nature Biotechnology 22, 589-594 (2004). 2A and 2A-like sequences are linkers that can be cut after transcription and translation of nucleic acid molecules. Another example of a linker is an internal ribosome entry site (IRES), which enables the same transcript to translate two proteins or polypeptides. Any other suitable linker can also be used. The determination of a suitable linker is entirely within the routine capabilities of those skilled in the art. As a further example, a nucleic acid sequence encoding (a) and a nucleic acid sequence encoding (b) can be cloned into a vector with a double internal promoter (see, for example, S Jones et al., Human Gene Ther 2009).

[0463] Other suitable polypeptide domains can also be encoded by the nucleic acid sequence of the present invention. For example only, the nucleic acid sequence can include a membrane targeting sequence that provides for transporting the encoded polypeptide to the cell surface membrane of the modified cell. Other suitable additional domains are also well known and described, for example, in WO2016 / 071758.

[0464] In one embodiment, the nucleic acid sequence of the present invention can encode a soluble TCR. For example, the nucleic acid sequence can encode (a) and (b), wherein (a) and (b) respectively comprise the variable regions of the TCR α chain and β chain, and optional immunomodulatory molecules, such as CD3 agonists (e.g., anti-CD3 scFv). The CD3 antigen is present on a subset of mature human T cells, thymocytes, and natural killer cells. It is associated with TCR and participates in the signal transduction of TCR. Antibodies specific to human CD3 antigens are well known. One such antibody is the mouse monoclonal antibody OKT3, which is the first monoclonal antibody approved by the FDA. Other antibodies specific to CD3 have also been reported (e.g., see WO2004 / 106380; U.S. Patent Application Publication No. 2004 / 0202657; U.S. Patent No. 6,750,325). Immunomobilization mTCR anticancer drugs (ImmTAC; Immunocore Limited, Milton Partk, Abington, Oxon, United Kingdom) are bifunctional proteins that combine affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism of action (i.e., anti-CD3 scFv). In another example, the soluble TCR of the present invention can be combined with a radioisotope or a toxic drug. Suitable radioisotopes and / or toxic drugs are well known in the art and can be easily identified by a person of ordinary skill in the art.

[0465] In one embodiment, the nucleic acid sequence of the invention can encode a chimeric single-chain TCR, wherein the polypeptide of (a) (e.g., TCR α chain variable region) is linked to the polypeptide of (b) (e.g., TCR β chain variable region) and a constant region, which is, for example, fused to a CD3 ζ signaling domain. In this example, the linker is not cleavable. In an alternative embodiment, the nucleic acid sequence of the invention can encode a chimeric double-chain TCR, wherein the polypeptide of (a) (e.g., TCR α chain variable region) and the polypeptide of (b) (e.g., TCR β chain variable region) are each linked to a CD3 ζ signaling domain. Methods for preparing such single-chain TCRs and double-chain TCRs are well known in the art; see, for example, RA Willemsen et al, Gene Therapy 2000.

[0466] The present invention also provides the nucleic acid sequence (and corresponding vector) of the separation of encoding peptide of the present invention.All general statements related to nucleic acid sequence and vector herein are equally applicable.Those skilled in the art can easily identify suitable nucleic acid sequence and vector based on the peptide sequence provided herein.

[0467] Vectors and modified cells

[0468] In one aspect, the present invention provides a vector comprising a nucleic acid sequence as described herein. Any suitable vector can be used. By way of example only, the vector can be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector. Adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, herpes virus, minicircle vectors and naked (synthetic) DNA / RNA can also be used (for details on minicircle vectors, see, for example, R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek, published in Leukemia 2016, Non-viral Sleeping Beauty Transposition from Minicircle Vectors).

[0469] Optionally, the vector comprises a nucleic acid sequence operably linked to a promoter.

[0470] The term "vector" as used herein refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it is operably linked. A vector can replicate autonomously or can be integrated into a host DNA. A vector can include restriction enzyme sites for inserting recombinant DNA and can include one or more selectable markers or suicide genes. A vector can be a nucleic acid sequence in the form of a plasmid, phage or cosmid. Preferably, the vector is suitable for expression in a cell (i.e., the vector is an "expression vector"). Preferably, the vector is suitable for expression in human T cells, such as CD8 + T cells or CD4 + T cells. In some aspects, the vector is a viral vector, such as a retroviral vector, a lentiviral vector, or an adeno-associated vector. Optionally, the vector is selected from adenovirus, vaccinia virus, canarypox virus, herpes virus, minicircle vector, and synthetic DNA or synthetic RNA.

[0471] Preferably, the (expression) vector is capable of propagating in the host cell and being stably passed on to progeny.

[0472] As used herein, "operably linked" means that one or a combination of the following control elements and a coding sequence are in a functional relationship with each other, such as being in a linked relationship to direct the expression of the coding sequence.

[0473] The vector may comprise a regulatory sequence. "Regulatory sequence" as used herein refers to a DNA or RNA element that can control gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA boxes, internal ribosome entry sites (IRES), attachment sites for transcription factors, transcription terminators, polyadenylation sites, etc. Optionally, the vector comprises one or more regulatory sequences that are operably connected to the nucleic acid sequence to be expressed. Regulatory sequences include sequences that instruct constitutive expression, and tissue-specific regulatory sequences and / or inducing sequences.

[0474] The vector may include a promoter. As used herein, "promoter" refers to a nucleotide sequence in DNA that binds to RNA polymerase to initiate transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter may be controlled by a repressor or stimulatory protein. The promoter may be a non-natural promoter in the host cell (e.g., it may be an exogenous promoter). Those skilled in the art are familiar with promoters suitable for expressing a target protein, wherein the selected promoter will depend on the host cell.

[0475] The vector may contain a transcription terminator. As used herein, a "transcription terminator" refers to a DNA element that terminates the function of an RNA polymerase responsible for transcribing DNA into RNA. A preferred transcription terminator is characterized in that a series of T residues is preceded by a GC-rich twofold symmetric region.

[0476] The vector may comprise a translation control element. As used herein, a "translation control element" refers to a DNA or RNA element that controls the translation of mRNA. A preferred translation control element is a ribosome binding site. Preferably, the translation control element is from a system homologous to the promoter, such as a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known and depend on the selected host cell.

[0477] The vector may contain a restriction enzyme recognition site. As used herein, "restriction enzyme recognition site" refers to a motif on DNA that is recognized by a restriction enzyme.

[0478] The vector may contain a selectable marker. As used herein, a "selectable marker" refers to a protein that confers a cell phenotype when expressed in a host cell, thereby allowing selection of cells expressing the selectable marker gene. In general, this can be a protein that confers new beneficial properties (e.g., antibiotic resistance) to the host cell, or a protein that is expressed on the cell surface and is therefore available for antibody binding. Suitable selectable markers are well known in the art.

[0479] Alternatively, the vector may also include a suicide gene. As used herein, "suicide gene" refers to a protein that can induce the death of modified cells after treatment with a specific drug. For example, after treatment with a specific nucleoside analog (including ganciclovir), cells modified by the herpes simplex virus thymidine kinase gene can be induced to commit suicide, after treatment with an anti-CD20 monoclonal antibody, cells modified by human CD20 can be induced to commit suicide, and after treatment with AP1903, cells modified by inducible caspase 9 (iCasp9) can be induced to commit suicide (BS Jones, LS Lamb, F Goldman, A Di Stasi review; Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol. (2014) 5: 254). Appropriate suicide genes are well known in the art.

[0480] Preferably, the vector comprises those genetic elements necessary for the host cell to express the polypeptide described herein. Elements required for transcription and translation in the host cell include a promoter, a coding region of the target protein, and a transcription terminator.

[0481] Those skilled in the art will be fully aware of the molecular techniques that can be used to prepare (expression) vectors and how to transduce or transfect (expression) vectors into appropriate host cells (thereby generating modified cells of the invention). The (expression) vectors of the invention can be introduced into cells by conventional techniques such as transformation, transfection or transduction. "Transformation", "transfection" and "transduction" generally refer to techniques for introducing foreign (exogenous) nucleic acid sequences into host cells, and therefore include methods such as electroporation, microinjection, gene gun delivery, transduction using retrovirus, lentivirus or adeno-associated vectors, lipofection, supertransfection, etc. The specific method commonly used depends on the type of vector and cell. Suitable methods for introducing nucleic acid sequences and vectors into host cells (e.g., human cells) are well known in the art; see, for example, Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637-646). For example, further conventional methods suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in WO2016 / 071758.

[0482] It will be appreciated that in some embodiments, the host cell is contacted with a vector (eg, a viral vector) in vitro, ex vivo, and in some embodiments, the host cell is contacted with a vector (eg, a viral vector) in vivo.

[0483] The term "host cell" includes any cell into which a nucleic acid sequence or vector of a cell of the invention can be introduced (e.g., transduced). Once a nucleic acid molecule or vector has been introduced into a cell, the cell may be referred to herein as a "modified cell." Once a nucleic acid molecule or vector has been introduced into a host cell, the resulting modified cell should be able to express the encoded polypeptide (and, for example, correctly localize the encoded polypeptide to achieve its intended function, such as transporting the encoded TCR to the cell surface).

[0484] The term "modified cell" refers to a cell that has been genetically altered (e.g., transformed or transfected). The term refers to a specific subject cell and the progeny or potential progeny of such a cell. Due to mutation or environmental influences, certain modifications may occur in the progeny, so such progeny may not actually be the same as the parent cell, but are still included within the scope of the term as used herein.

[0485] The host cell (and thus the cell modified) is typically a eukaryotic cell, and in particular a human cell (e.g., a T cell, such as a CD8 + T cells or CD4 + T cells, or mixtures thereof). The host cell (and thus the modified cell) may be an autologous cell (e.g., an autologous T cell, such as CD8 + T cells or CD4 + T cells, or mixtures thereof), which refers to cells from the same individual to which they are subsequently administered. In other words, the host cells (and thus modified cells) may be T cells isolated from the subject to be treated. Suitably, the host cells (and thus modified cells) may be isolated from a blood sample, for example by leukapheresis.

[0486] Host cells (and thus modified cells) can be any cells that can confer anti-tumor immunity after TCR gene transfer. Non-limiting examples of suitable cells include autologous or allogeneic natural killer (NK) cells, NKT cells, γ-δT cells, hematopoietic stem cells or other progenitor cells, and any other autologous or allogeneic cells or cell lines (e.g., NK-92 or T cell lines) that can confer anti-tumor immunity after TCR gene transfer.

[0487] Advantageously, the modified cells are capable of expressing a polypeptide encoded by a nucleic acid sequence or vector (e.g., a TCR or TCR component) of the invention, such that the modified cells provide immunotherapy specifically targeting ΔNPM1 malignant cells and can therefore be used to treat or prevent hematological malignancies with ΔNPM1. More details on this use are given below.

[0488] Immunogenic peptides

[0489] The present inventors have identified five peptides present in the HLA class I ligand panel of ΔNPM1-positive primary AML patients, namely CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0490] For the avoidance of doubt, unless expressly stated otherwise, general references herein to "SEQ ID NO: 1" encompass both cystylated and non-cystylated forms of the peptide CLAVEEVSL (SEQ ID NO: 1).

[0491] In the context of an isolated peptide, the present invention specifically provides an isolated peptide comprising the amino acid sequence CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) may or may not be cystylated.

[0492] In a specific embodiment, the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) is cystylated.

[0493] For the avoidance of doubt, unless expressly stated otherwise, general references herein to "SEQ ID NO: 27" encompass both cystylated and non-cystylated forms of the peptide CLAVEEVSLRK (SEQ ID NO: 27).

[0494] In the context of an isolated peptide, the present invention specifically provides an isolated peptide comprising the amino acid sequence CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​amino acid in CLAVEEVSLRK (SEQ ID NO: 27) may or may not be cystylated.

[0495] In a specific embodiment, the cysteine ​​amino acid in CLAVEEVSLRK (SEQ ID NO: 27) is cystylated.

[0496] Accordingly, the present invention provides an isolated peptide comprising an amino acid sequence selected from the group consisting of: (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cysteine-ylated; (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​amino acid may or may not be cysteine-ylated; (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29).

[0497] In a specific embodiment, the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) or CLAVEEVSLRK (SEQ ID NO: 27) is cystylated.

[0498] As used herein, "isolated peptide" refers to a peptide that is not present in its natural environment. Thus, the peptide may be of synthetic origin (or alternatively of natural origin, but separated from its natural environment).

[0499] The isolated peptide may be relatively short (i.e., no more than 20 amino acids; e.g., no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids). The peptide may consist solely of the amino acid sequence of SEQ ID NO: 1 (wherein the cysteine ​​amino acid may or may not be cystylated), SEQ ID NO: 26, SEQ ID NO: 27 (wherein the cysteine ​​amino acid may or may not be cystylated), SEQ ID NO: 28, or SEQ ID NO: 29.

[0500] In a specific embodiment, the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) or CLAVEEVSLRK (SEQ ID NO: 27) is cystylated.

[0501] The isolated peptides can be administered to human subjects to treat or prevent ΔNPM1-positive hematological malignancies. For example, the isolated peptides can be administered to subjects to induce or enhance their immune response. Thus, the peptides can be administered to subjects to induce T cell activation in the subject (e.g., in vivo T cell activation), wherein the activated T cells are specific for the peptide (and therefore will specifically target ΔNPM1-positive malignant cells).

[0502] The isolated peptides can be administered as peptide vaccines for the treatment or prevention of ΔNPM1-positive AML. The isolated peptides can be administered to induce or enhance the activation of T cells specific for ΔNPM1-positive malignant cells.

[0503] The inventors have demonstrated that (i) CLAVEEVSL (SEQ ID NO: 1), (ii) AVEEVSLRK (SEQ ID NO: 26) and (iii) CLAVEEVSLRK (SEQ ID NO: 27) are ΔNPM1 peptides that bind to T cells. These peptides were presented to the T cell repertoire of ΔNPM1 positive subjects in vivo. Binding of these peptides to T cells has been demonstrated herein. Therefore, these peptides represent true immunogenic ΔNPM1-specific antigens, which can be further used to develop personalized vaccines, which may be particularly useful as adjuncts to other therapies (e.g., ACT as described herein). Therefore, these immunogenic peptides can be used as immunotherapies in the form of peptides, RNA, DNA, dendritic cell-based therapies, and adoptive TCR transgenic T cell-based therapies (for a suitable review, see reference 23).

[0504] The inventors have also demonstrated that (iv) VEEVSLRK (SEQ ID NO: 28) and (v) AVEEVSLR (SEQ ID NO: 29) are HLA binding peptides presented on the surface of primary AML.

[0505] Thus, isolated peptides comprising an amino acid sequence selected from the group consisting of (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be cysteine; (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine ​​amino acid may or may not be cysteine; (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29). For example, such isolated peptides may be used as immunotherapies for subjects having, at risk of, or suspected of having ΔNPM1-positive AML. Nucleic acid sequences and vectors encoding these peptides may also be used for this purpose.

[0506] The specific peptides for administration may be selected based on the HLA-A status of the subject. As explained elsewhere herein, a peptide comprising the sequence of SEQ ID NO: 1 may be particularly suitable for administration to subjects who are positive for HLA-A*02:01, while a peptide comprising the sequence of SEQ ID NO: 26 or SEQ ID NO: 27 may be particularly suitable for administration to subjects who are positive for HLA-A*03:01 or HLA-A*11:01.

[0507] The isolated peptides of the present invention can also be provided in the form of a composition comprising more than one of the above peptides. For example, the isolated peptides can be provided (and / or administered) as a composition comprising a mixture of: (a) an isolated peptide comprising the amino acid sequence CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) is cysteine-terminated; and (b) an isolated peptide comprising the amino acid sequence CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid in CLAVEEVSL (SEQ ID NO: 1) is cysteine-terminated. no Cysteinylated. This composition may be particularly useful for treating or preventing ΔNPM1-positive hematological malignancies in subjects who are positive for HLA-A*02:01 because it can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), wherein the activated T cells have a TCR specific for one (or both) of the cysteine ​​and / or non-cysteine ​​forms of the peptide of SEQ ID NO: 1.

[0508] As an alternative example, a peptide composition may be provided that comprises a mixture of: (a) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 26 and (b) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27. Such a composition may be particularly useful for treating or preventing ΔNPM1-positive hematological malignancies in subjects who are positive for HLA-A*03:01 or HLA-A*11:01, as these peptides are presented by either of these HLA-A serotypes.

[0509] Similar to the peptide CLAVEEVSL (SEQ ID NO: 1), the peptide of SEQ ID NO: 27 can also be used for vaccination in its cysteine ​​form as well as in its non-cysteine ​​form. Thus, as an alternative example, the isolated peptide can be provided (and / or administered) as a composition comprising a mixture of: (a) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27, wherein the cysteine ​​amino acid is cysteine; and (b) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27, wherein the cysteine ​​amino acid is cysteine; no This composition may be particularly useful for treating or preventing ΔNPM1-positive hematological malignancies in subjects who are positive for HLA-A*03:01 or HLA-A*11:01 because it can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), wherein the activated T cells have a TCR specific for one (or both) of the cystylated and / or non-cystylated forms of the peptide of SEQ ID NO:27.

[0510] Pharmaceutical composition

[0511] The nucleic acid sequences, vectors, modified cells, isolated proteins or peptides described herein can be provided as part of a pharmaceutical composition. Advantageously, such compositions can be administered to human subjects with ΔNPM1-positive hematological malignancies to treat or prevent ΔNPM1-positive hematological malignancies (e.g., by inducing or enhancing ΔNPM1-target-specific immune responses). Particularly suitable compositions can be selected based on the HLA-A serotype of the human subject, as described in detail elsewhere herein.

[0512] A pharmaceutical composition may comprise a nucleic acid sequence, vector, modified cell, or isolated protein or peptide described herein and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0513] The compositions may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplemental immunopotentiators (such as adjuvants and cytokines), and optionally other therapeutic agents or compounds.

[0514] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual together with a selected nucleic acid sequence, vector, modified cell or isolated peptide without causing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition containing it.

[0515] Excipients are natural or synthetic substances formulated with active ingredients (e.g., nucleic acid sequences, vectors, modified cells, or isolated peptides provided herein), including those whose purpose is to increase the bulk of the formulation or enhance the therapeutic effect of the active ingredient in the final dosage form, such as promoting the absorption or solubility of the drug. Excipients are also useful during the manufacturing process, where they aid in handling the associated active substances, such as by promoting powder flowability or non-stickiness, and also aid in in vitro stability, such as preventing denaturation during the expected shelf life.

[0516] Pharmaceutically acceptable excipients are well known in the art. Therefore, those of ordinary skill in the art can easily determine suitable excipients. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous glucose solutions, glycerol, ethanol, etc.

[0517] Adjuvants are pharmacological and / or immunological agents that can alter the effects of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. Therefore, one of ordinary skill in the art can easily determine a suitable adjuvant.

[0518] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. Therefore, a person of ordinary skill in the art can easily determine a suitable diluent.

[0519] The carrier is nontoxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients in the formulation. The term "carrier" refers to an organic or inorganic ingredient, natural or synthetic, that is combined with the active ingredient to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Therefore, one of ordinary skill in the art can easily determine a suitable carrier.

[0520] Treatment of subjects

[0521] The compositions of the invention may be advantageously used to treat or prevent ΔNPM1 -positive hematological malignancies in human subjects. Appropriate compositions may be selected based on the HLA-A serotype of the human subject, as discussed in detail elsewhere herein.

[0522] In one embodiment, the methods of treating or preventing a ΔNPM1 -positive hematological malignancy described herein produce an induced or enhanced immune response (eg, a cell-mediated response) in a subject (eg, a targeted immune response against malignant cells presenting an HLA-A restricted peptide).

[0523] The phrase "induced or enhanced immune response" means that the subject has an increased immune response (e.g., a cell-mediated immune response, such as a T cell-mediated immune response) during or after treatment compared to the immune response before treatment. Thus, an "induced or enhanced" immune response encompasses any measurable increase in an immune response that directly or indirectly targets the ΔNPM1-positive hematological malignancy being treated.

[0524] The compositions of the present invention can be used to treat or prevent ΔNPM1-positive hematological malignancies, particularly ΔNPM1-positive myeloid malignancies, and more particularly ΔNPM1-positive AML in human subjects.

[0525] Those skilled in the art will be fully aware of hematological malignancies that may be positive for ΔNPM1 and thus may be treated in accordance with the present invention. Similarly, those skilled in the art will be fully aware of myeloid malignancies that may be positive for ΔNPM1 and thus may be treated in accordance with the present invention.

[0526] As used herein, the terms "treat", "treating" and "treatment" include interventions performed to prevent the pathological development or changes of a condition, disorder or symptom (i.e., in this case, a hematological malignancy). Thus, "treat" refers to therapeutic treatment as well as prophylactic or preventive measures, wherein the purpose is to prevent or mitigate (alleviate) the target condition, disorder or symptom. Thus, "treat" includes reducing, mitigating or inhibiting the number or concentration of malignant cells, for example, as measured in a sample obtained from a subject, compared to the number or concentration of malignant cells before treatment, reducing, mitigating or inhibiting by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Methods for measuring the number or concentration of malignant cells include, for example, qRT-PCR, and quantifying ΔNPM1-positive hematological malignancy-specific biomarkers, such as CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28), and / or AVEEVSLR (SEQ ID NO: 29) in a sample obtained from a subject.

[0527] The term "subject" as used herein refers to an individual, such as a human, who suffers from or is at risk of suffering from a specified condition, disorder or symptom. The subject can be a patient, i.e., a subject in need of treatment according to the present invention. The subject may have already received treatment for the condition, disorder or symptom. Alternatively, the subject has not received treatment before being treated according to the present invention.

[0528] The compositions described herein can be administered to a subject by any conventional route, including injection or gradual infusion over time. Administration can be, for example, by infusion or by intramuscular, intravascular, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, transdermal administration.

[0529] The compositions described herein may be in any form suitable for the above-described modes of administration. For example, the composition comprising the modified cells may be in any form suitable for infusion. As a further example, forms suitable for parenteral injection (including subcutaneous, intramuscular, intravascular or infusion) include sterile solutions, suspensions or emulsions; forms suitable for topical administration include ointments or creams; and forms suitable for rectal administration include suppositories. Alternatively, the route of administration may be direct injection into the target area, or by regional delivery or by local delivery. It is well within the routine capabilities of those skilled in the art to determine the appropriate dosage of the compositions of the present invention.

[0530] Advantageously, the compositions of the invention can be formulated for T cell receptor (TCR) gene transfer, which is rapid, reliable and capable of generating large numbers of T cells specific for ΔNPM1-specific peptides (e.g., CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) or AVEEVSLR (SEQ ID NO: 29)), regardless of the patient's prior immune repertoire. Using TCR gene transfer, modified autologous cells suitable for infusion can be generated within days.

[0531] Advantageously, the compositions of the invention can be formulated for use as vaccines, for example, a composition comprising one or more peptides selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) or AVEEVSLR (SEQ ID NO: 29) can be formulated as a pharmaceutical composition suitable for use as a peptide vaccine. Suitable peptide vaccine formulations are well known in the art.

[0532] Preferably, the pharmaceutical composition of the present invention is a vaccine, preferably a peptide-based vaccine. Such a peptide-based vaccine can be used to prevent or treat ΔNPM1-positive hematological malignancies such as AML.

[0533] The pharmaceutical composition is preferably intended for and is therefore formulated to be suitable for administration to a subject, preferably a human or animal subject. Preferably, the administration is parenteral, such as intravenous, subcutaneous, intramuscular, intradermal, intrathecal and / or intratumoral administration, ie by injection.

[0534] Preferably, the pharmaceutical composition comprises or is composed of peptides constituting the amount of a pharmaceutical dosage unit. A pharmaceutical dosage unit is defined herein as the amount of active ingredient applied to a subject at a given time point (i.e., the total amount of peptides in a peptide-based vaccine). A pharmaceutical dosage unit can be applied to a subject in a single volume (i.e., a single injection), or can be applied to a subject in 2, 3, 4, 5 or more separate volumes or injections, which are preferably applied to different locations of the body, such as in the right and left limbs. It should be understood herein that the separate volumes of a pharmaceutical dosage may be different in composition, i.e., may contain active ingredients and / or adjuvants of different types or compositions.

[0535] The single injection volume or injection amount (i.e., the volume applied to one location at a specific time point) can be 100 to 2 mL or 100 to 1 mL, which includes the total drug dose, or if multiple injections are applied at substantially the same time point, includes a portion of the total drug dose. The single injection volume can be 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 3 mL, or any value therebetween.

[0536] Preferably, the total amount of the pharmaceutical dosage unit or peptide applied to a subject at a given time point in the form of a single injection or multiple injections at specific time points comprises an amount of peptide in the range of 0.1 μg to 20 mg, for example, about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg. The preferred range of the pharmaceutical dosage unit is 0.1 μg to 20 mg, 1 μg to 10 mg, 10 μg to 5 mg, 0.5 mg to 2 mg, 0.5 mg to 10 mg or 1 mg to 5 mg or 2 to 4 mg.

[0537] The compositions described herein are used to be administered in an effective amount. An "effective amount" refers to the amount that produces the desired (therapeutic or non-therapeutic) response alone or together with other dosages. For example, the effective amount to be used will depend on the treatment (or non-therapeutic) purpose, the route of administration, and the condition of the patient / subject. For example, for a given patient / subject, the appropriate dosage of the compositions of the present invention will be determined by the attending physician (or the person administering the compositions), while considering various factors known to change the effect of the compositions of the present invention, such as the severity and type of hematological malignancies, body weight, sex, diet, time and route of administration, other drugs, and other relevant clinical factors. Dosage and schedule can vary according to the specific condition, illness or symptom, and overall condition of the patient / subject. The effective dose can be determined by in vitro or in vivo methods.

[0538] The compositions of the invention are advantageously presented in unit dosage form.

[0539] Methods for generating TCRs

[0540] In one aspect, the present invention provides a method for producing a T cell receptor that specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29), the method comprising contacting a nucleic acid sequence (or vector) of the present invention with a host cell under conditions where the nucleic acid sequence (or vector) is incorporated into the cell and expressed by the cell, to produce a T cell receptor.

[0541] The method can be performed in vitro or on a host cell. Alternatively, the method can be performed in vivo, wherein the nucleic acid sequence (or vector) is administered to a subject and contacted with a host cell in vivo, under which conditions the nucleic acid sequence is incorporated into the host cell and expressed to produce a T cell receptor. In one embodiment, the method is not a method for treating a human or animal body.

[0542] As described elsewhere herein, suitable in vivo, in vitro and ex vivo methods for contacting a nucleic acid sequence (or vector) with a host cell under conditions whereby the nucleic acid sequence (or vector) is incorporated into the cell and expressed are well known.

[0543] General Definition

[0544] As used herein, "nucleic acid sequence", "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to refer to an oligonucleotide sequence or a polynucleotide sequence. The nucleotide sequence can be of genomic, synthetic or recombinant origin, and can be double-stranded or single-stranded (representing a sense strand or an antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g., mRNA) and DNA or RNA analogs produced, for example, by using nucleotide analogs. In an example, the nucleotide sequence lacks introns. In other words, it is an intron-free nucleic acid sequence. For example, the nucleotide sequence can be a DNA sequence that does not contain an intron sequence.

[0545] As used herein, an "isolated nucleic acid sequence" refers to a nucleic acid sequence that is not in its native environment, where, when in its native environment, it is linked to naturally associated sequences that are also in its native environment. In other words, an isolated nucleic acid sequence is not a native nucleotide sequence, where "native nucleotide sequence" refers to an intact nucleotide sequence in its native environment, and when operably linked to an intact promoter with which it is naturally associated, the promoter is also in its native environment.

[0546] As used herein, "specifically binds to CLAVEEVSL (SEQ ID NO: 1)" refers to selective binding to the CLAVEEVSL (SEQ ID NO: 1) peptide. Under certain conditions, such as in the immunoassays described herein, a polypeptide that "specifically binds to CLAVEEVSL" (SEQ ID NO: 1) will selectively bind to the peptide and will not bind to other peptides that do not include the sequence in significant amounts. Thus, the binding affinity of the polypeptide to CLAVEEVSL (SEQ ID NO: 1) may be at least 10, 20, 30, 40, 50, or 100 times greater than its binding affinity to a control antigenic peptide. Selective binding may also be determined indirectly in the context of modified cells expressing a nucleic acid or vector of the invention. In an assay (e.g., an assay discussed herein), the modified cells are specifically reactive to cells (e.g., primary ΔNPM1 HLA-A*02:01-positive AML cells, or any HLA-A*02:01-positive cell line into which the ΔNPM1 gene has been introduced) that present CLAVEEVSL (SEQ ID NO: 1) in an HLA-A*02:01 background. Thus, the modified cells can bind to cells that present CLAVEEVSL (SEQ ID NO: 1) in an HLA-A*02:01 background with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times greater than their reactivity to a control cell line that does not present CLAVEEVSL (SEQ ID NO: 1) in an HLA-A*02:01 background.

[0547] Selective binding may occur in the context of HLA-A*02:01 presenting CLAVEEVSL (SEQ ID NO: 1). In other words, in certain embodiments, a polypeptide that "specifically binds to CLAVEEVSL" (SEQ ID NO: 1) will only bind when it is presented by HLA-A*02:01 (i.e., bound by HLA-A*02:01), or will only bind when it is presented by HLA-A*02:01 in an equivalent structural formation.

[0548] Unless otherwise specified, a polypeptide that "specifically binds to CLAVEEVSL" (SEQ ID NO: 1) can bind to (i) a cysteine ​​form of CLAVEEVSL (SEQ ID NO: 1), (ii) a non-cysteine ​​form of CLAVEEVSL (SEQ ID NO: 1), or (iii) a cysteine ​​form of CLAVEEVSL (SEQ ID NO: 1) and a non-cysteine ​​form of CLAVEEVSL (SEQ ID NO: 1). Likewise, unless otherwise expressly specified, general references herein to "SEQ ID NO: 1" or "CLAVEEVSL" (SEQ ID NO: 1) encompass both cysteine ​​and non-cysteine ​​forms of the peptide CLAVEEVSL (SEQ ID NO: 1).

[0549] As used herein, "specifically binds to AVEEVSLRK" (SEQ ID NO: 26) refers to selective binding to the AVEEVSLRK (SEQ ID NO: 26) peptide. Under certain conditions, such as in the immunoassays described herein, a polypeptide that "specifically binds to AVEEVSLRK" (SEQ ID NO: 26) will selectively bind to the peptide and will not bind to other peptides that do not include the sequence in significant amounts. Thus, the binding affinity of the polypeptide to AVEEVSLRK (SEQ ID NO: 26) may be at least 10, 20, 30, 40, 50, or 100 times greater than its binding affinity to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell expressing a nucleic acid or vector of the invention. In an assay (e.g., the assay discussed herein), the modified cells are specifically reactive to cells presenting AVEEVSLRK (SEQ ID NO:26) in the context of HLA-A*03:01, HLA-A*11:01, or HLA-A*01:01 (e.g., primary ΔNPM1 HLA-A*03:01, HLA-A*11:01, or HLA-A*01:01-positive AML cells, or any HLA-A*03:01, HLA-A*11:01, or HLA-A*01:01-positive cell line into which the ΔNPM1 gene has been introduced). Thus, the modified cells can bind to cells presenting AVEEVSLRK (SEQ ID NO:26) in the context of HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01 with a reactivity that is at least 10, 20, 30, 40, 50 or 100 times higher than their reactivity to a control cell line that does not present AVEEVSLRK (SEQ ID NO:26) in the context of HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01.

[0550] Selective binding may occur only in the context of AVEEVSLRK (SEQ ID NO: 26) presented by HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01. In other words, in certain embodiments, a polypeptide that "specifically binds to AVEEVSLRK" (SEQ ID NO: 26) will only bind when it is presented (i.e., it is bound) by HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01, or will only bind when it is presented by HLA-A*03:01, HLA-A*11:01 or HLA-A*01:01 in an equivalent structural formation.

[0551] Similarly, "specifically binds to CLAVEEVSLRK" (SEQ ID NO: 27), as used herein, refers to selective binding to the CLAVEEVSLRK peptide. Under certain conditions, such as in the immunoassays described herein, a polypeptide that "specifically binds to CLAVEEVSLRK" (SEQ ID NO: 27) will selectively bind to that peptide and will not bind to other peptides that do not include that sequence in significant amounts. Thus, the polypeptide may bind to CLAVEEVSLRK (SEQ ID NO: 27) with an affinity that is at least 10, 20, 30, 40, 50, or 100 times greater than its affinity for a control antigenic peptide. Selective binding may also be determined indirectly in the context of modified cells expressing a nucleic acid or vector of the invention. In an assay (e.g., an assay discussed herein), the modified cells are specifically reactive to cells presenting CLAVEEVSLRK (SEQ ID NO:27) in the context of HLA-A*03:01 or HLA-A*11:01 (e.g., primary ΔNPM1 HLA-A*03:01 or HLA-A*11:01-positive AML cells, or any HLA-A*03:01 or HLA-A*11:01-positive cell line into which the ΔNPM1 gene has been introduced). Thus, the modified cells can bind to cells presenting CLAVEEVSLRK (SEQ ID NO:27) in the context of HLA-A*03:01 or HLA-A*11:01 with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times higher than their reactivity to a control cell line that does not present CLAVEEVSLRK (SEQ ID NO:27) in the context of HLA-A*03:01 or HLA-A*11:01.

[0552] Selective binding may occur only in the context of CLAVEEVSLRK (SEQ ID NO: 27) presented by HLA-A*03:01 or HLA-A*11:01. In other words, in certain embodiments, a polypeptide that "specifically binds to CLAVEEVSLRK" (SEQ ID NO: 27) will only bind when presented (i.e., bound) by HLA-A*03:01 or HLA-A*11:01, or will only bind when presented by HLA-A*03:01 or HLA-A*11:01 and in an equivalent structural formation.

[0553] Unless otherwise indicated, a polypeptide that "specifically binds to CLAVEEVSLRK" (SEQ ID NO: 27) can bind to (i) a cysteine ​​form of CLAVEEVSLRK, (ii) a non-cysteine ​​form of CLAVEEVSLRK (SEQ ID NO: 27), or (iii) a cysteine ​​form of CLAVEEVSLRK and a non-cysteine ​​form of CLAVEEVSLRK (SEQ ID NO: 27). Likewise, unless otherwise expressly indicated, general references herein to "SEQ ID NO: 27" or "CLAVEEVSLRK" encompass both cysteine ​​and non-cysteine ​​forms of the peptide CLAVEEVSLRK (SEQ ID NO: 27).

[0554] As used herein, "specifically binds to VEEVSLRK" (SEQ ID NO: 28) refers to selective binding to the VEEVSLRK (SEQ ID NO: 28) peptide. Under certain conditions, such as in the immunoassays described herein, a polypeptide that "specifically binds to VEEVSLRK" (SEQ ID NO: 28) will selectively bind to the peptide and will not bind to other peptides that do not include the sequence in significant amounts. Thus, the binding affinity of the polypeptide to VEEVSLRK (SEQ ID NO: 28) is at least 10, 20, 30, 40, 50, or 100 times greater than its binding affinity to a control antigenic peptide. Selective binding can also be determined indirectly in the context of a modified cell expressing a nucleic acid or vector of the invention. In an assay (e.g., an assay discussed herein), the modified cell is specifically reactive to cells presenting VEEVSLRK (SEQ ID NO: 28) in the appropriate HLA-A context (e.g., primary ΔNPM1-positive AML cells, or any appropriate HLA-A-positive cell line into which the ΔNPM1 gene has been introduced). Thus, the modified cells can bind to cells presenting VEEVSLRK (SEQ ID NO:28) in any appropriate HAL-A background with at least 10, 20, 30, 40, 50 or 100 times greater reactivity than to a control cell line that does not present VEEVSLRK in the appropriate HLA-A background.

[0555] Selective binding may occur only in the context of presentation of VEEVSLRK (SEQ ID NO: 28) by an appropriate HLA-A. In other words, in certain embodiments, a polypeptide that "specifically binds to VEEVSLRK" (SEQ ID NO: 28) will only bind when it is presented (i.e., bound) by an appropriate HLA-A, or will only bind when presented by an appropriate HLA-A in an equivalent structural formation.

[0556] As used herein, "specifically binds to AVEEVSLR" (SEQ ID NO: 29) refers to selective binding to the AVEEVSLR (SEQ ID NO: 29) peptide. Under certain conditions, such as in the immunoassays described herein, a polypeptide that "specifically binds to AVEEVSLR" (SEQ ID NO: 29) will selectively bind to the peptide and will not bind to other peptides that do not include the sequence in significant amounts. Thus, the binding affinity of the polypeptide to AVEEVSLR (SEQ ID NO: 29) is at least 10, 20, 30, 40, 50, or 100 times greater than its binding affinity to a control antigenic peptide. Selective binding can also be determined indirectly in the context of a modified cell expressing a nucleic acid or vector of the invention. In an assay (e.g., an assay discussed herein), the modified cell is specifically reactive to cells presenting AVEEVSLR (SEQ ID NO: 29) in the appropriate HLA-A context (e.g., primary ΔNPM1-positive AML cells, or any appropriate HLA-A-positive cell line into which the ΔNPM1 gene has been introduced). Thus, the modified cells can bind to cells presenting AVEEVSLR (SEQ ID NO:29) in any appropriate HLA-A background, and their reactivity can be at least 10, 20, 30, 40, 50 or 100 times higher than their reactivity to a control cell line that does not present AVEEVSLR (SEQ ID NO:29) in the appropriate HLA-A background.

[0557] Selective binding may occur only in the context of presentation of AVEEVSLR (SEQ ID NO: 29) by an appropriate HLA-A. In other words, in certain embodiments, a polypeptide that "specifically binds to AVEEVSLR" (SEQ ID NO: 29) will only bind when presented (i.e., bound) by an appropriate HLA-A, or will only bind when presented by an appropriate HLA-A in an equivalent structural formation.

[0558] A "non-essential" (or "non-critical") amino acid residue is a residue that can be altered from a wild-type sequence (e.g., a sequence identified herein by a SEQ ID NO) without eliminating or, more preferably, substantially altering biological activity, whereas an "essential" (or "critical") amino acid residue can result in such a change. For example, amino acid residues that are predicted to be conserved are particularly difficult to alter, but amino acid residues within the hydrophobic core of a domain can often be replaced by other residues of roughly comparable hydrophobicity without significantly altering activity.

[0559] "Conservative amino acid substitution" is where an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential (or non-critical) amino acid residues in proteins are preferentially replaced by another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly, and the activity of the resulting mutants can be screened to identify mutants that retain activity.

[0560] Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0561] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, it is necessary to align the sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (amino acid or nucleic acid "identity" as used herein is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences.

[0562] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the comparison of sequences and determination of percent identity between two sequences is performed using the Needleman et al. (1970) J. Mol. Biol. 48: 444-453 algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a BLOSLIM 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the parameter set that should be used when the practitioner is unsure what parameters should be applied to determine whether a molecule is within the sequence identity or homology limits of the invention) is the BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0563] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0564] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search public databases, for example to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402) can be utilized. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See<http: / / www.ncbi.nlm.nih.gov> .

[0565] The polypeptides and nucleic acid molecules described herein may have an amino acid sequence or nucleic acid sequence that is sufficiently or substantially identical to a sequence identified by a SEQ ID NO. The terms "sufficiently identical" or "substantially identical" as used herein refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains) to a second amino acid or nucleotide sequence, such that the first and second amino acid or nucleotide sequences have a common structural domain or a common functional activity. For example, amino acid or nucleotide sequences containing a common structural domain having at least about 60% or 65% identity, possibly 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as being sufficiently or substantially identical.

[0566] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art described in the present invention. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1 94); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary of many terms used in the present invention for those skilled in the art. Although any method and material similar or equivalent to the methods and materials described herein can be used to practice the present invention, preferred methods and materials are described herein. Therefore, the terms defined immediately below will be more fully described by reference to the entire specification. In addition, as used herein, the singular terms "a", "an" and "the" include plural references unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written from left to right in the direction of 5' to 3'; amino acid sequences are written from left to right in the direction of amino to carboxyl. It is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context in which they are used by one skilled in the art.

[0567] Aspects of the invention are demonstrated by the following non-limiting examples.

[0568] Example

[0569] Example 1

[0570] Materials and methods

[0571] Study Design

[0572] The objectives of this study were to: (1) identify HLA class I ligands from ΔNPM1 on primary AML, (2) isolate CD8 cells with TCRs specific for HLA class I ligands from ΔNPM1, and (3) identify TCRs for HLA class I ligands from ΔNPM1 that are able to mediate specific recognition and lysis of primary AML after gene transfer. HLA class I ligandome data for 12 primary AMLs were generated using tandem mass spectrometry and peptides matching alternative reading frames of ΔNPM1 were searched. HLA-A*02:01 pMHC tetramers were generated against one of the identified peptides and used to isolate specific CD8 cells from six HLA-A*02:01-positive AML patients and six healthy HLA-A*02:01-positive individuals. T cell clones isolated from healthy individuals were screened by pMHC tetramer staining, and tetramer-positive T cell clones were tested for reactivity to peptide-loaded T2 cells and primary AML with ΔNPM1 or wtNPM1 by IFN-γ ELISA. From one highly reactive T cell clone, the TCR was sequenced and cloned into the retroviral MP71-TCR-flex vector. This vector was used to introduce the TCR into CD8 and CD4 cells from two healthy HLA-A*02:01-positive individuals, and the purity of the TCR-transduced T cells was assessed by flow cytometry. The recognition of the TCR-transduced T cells to HLA-A*02:01-positive AML cell lines and primary AML with ΔNPM1 or wtNPM1 was tested by IFN-γ ELISA, and the purity of the TCR-transduced T cells was assessed by flow cytometry. 51 The Cr release assay measures specific cleavage.

[0573] Sample collection and cell culture

[0574] Peripheral blood and bone marrow samples were collected from patients with AML and healthy individuals after obtaining approval from the Leiden University Medical Center Institutional Review Board and obtaining informed consent according to the Declaration of Helsinki. Peripheral blood and bone marrow mononuclear cells were separated by Ficoll-Isopaque separation and cryopreserved. In order to separate PBMCs from HLA-A*02:01-type healthy individuals, buffy coats were ordered from Sanquin (Amsterdam, the Netherlands). T cells were cultured in T cell medium (TCM), which consisted of Iscove's modified Dulbecco's medium (IMDM; Lonza, Basel, Switzerland) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, United States), 5% human serum, 1.5% L-glutamine (Lonza), 1% penicillin / streptomycin (Lonza), and 100 IU / ml IL-2 (Novartis, Basel, Switzerland). AML cell lines expressing wild-type NPM1 (OCI-AML2) and ΔNPM1 (OCI-AML3) were purchased from DSMZ (Braunschweig, Germany) and cultured in minimal essential medium alpha (MEMα; Gibco) containing 20% ​​FBS and 1% penicillin / streptomycin. Primary AML and T2 cells were cultured in IMDM containing 10% FBS, 1.5% L-glutamine and 1% penicillin / streptomycin. Magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) and CliniMACS CD14 beads (Miltenyi Biotec) were used to separate monocytes from PBMCs. In a culture medium containing 100 ng / mL GM-CSF (Novartis, Basel, Switzerland) and 500 IU / mL IL-4 (Schering-Plough, Kenilworth, NJ), isolated monocytes were cultured for 7 days to immature dendritic cells (DC).On the last two days, 100 ng / mL GM-CSF, 10 ng / mL TNF-α (Cellgenix, Freiburg, Germany), 10 ng / mL IL-1β (Cellgenix), 10 ng / mL IL-6 (Cellgenix), 1 μg / mL prostaglandin E2 (Sigma-Aldrich, St. Louis, MO), and 500 IU / mL IFN-γ (Boehringer-Ingelheim, Ingelheim, Germany) were added to induce maturation.

[0575] HLA Class I Ligand Panel in Primary AML

[0576] Cell pellets from 12 primary AML samples were lysed in 50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetate, and 0.5% Zwittergent 3-12 (pH 8.0), supplemented with complete protease inhibitors (Sigma-Aldrich, St. Louis, Missouri, United States). After tumbling incubation of cells in lysis buffer for 2 hours at 4°C, the preparation was centrifuged at 1000 g for 10 minutes at 4°C. The supernatant was transferred to a new tube and centrifuged at 13,000 g for 35 minutes at 4°C. The supernatant was pre-clarified with protein A Sepharose CL-4B beads (GE Healthcare Life Sciences, Chicago, Illinois, United States) and then processed through an immunoaffinity column with dimethyl pimelidate (DMP)-immobilized W6 / 32 antibody (3 mg / ml resin) on protein A Sepharose CL-4B beads at a flow rate of 1 ml / min. After washing with lysis buffer and 10 mM Tris-HCl (pH 8.0) buffer (containing 1 M, 120 mM, and without NaCl) for 5-10 column volumes, the bound HLA class I-peptide complex was eluted from the column and dissociated with 3-4 column volumes of 10% acetic acid. The peptides were separated from the HLA class I molecules by passing through a 10 kDa membrane (Macrosep Advance centrifugal device with Supor membrane, Pall Corporation, Port Washington, New York, United States). The filtrate was freeze-dried.

[0577] The eluted peptide pool was fractionated by strong cation exchange chromatography (SCX) using a homemade SCX column (320 μm inner diameter, 15 cm, polysulfoethyl A 3 μm, poly LC) running at 4 μl / min. The gradient was run at 100% solvent A (100 / 0.1 water / trifluoroacetic acid v / v) for 10 minutes, after which a linear gradient was started to reach 100% solvent B (65 / 35 / 0.1 250 mM KCl / / acetonitrile / trifluoroacetic acid v / v / v) over 15 minutes, and then to 100% solvent C (65 / 35 / 0.1 500 mM KCl / acetonitrile / trifluoroacetic acid v / v / v) over the next 15 minutes. The gradient was maintained at 100% solvent C for 5 minutes before switching to 100% solvent A. 20 4 μl fractions were collected into vials pre-filled with 20 μl 95 / 3 / 0.1 water / acetonitrile / FA v / v / v. The peptide fractions were lyophilized, dissolved in 95 / 3 / 0.1 water / acetonitrile / formic acid v / v / v, and analyzed by data-dependent tandem mass spectrometry (MS) on a LTQ-FTUltra (Agilent Technologies, Santa Clara, California, United States) equipped with a nanoflow HPLC 1100 HPLC system as described previously (38) or on a Q-Exactive equipped with an Easy-nLC 1000. Peptides were captured at 6-10 μl / min on a 1.5 cm column (100-μm id; ReproSil-Pur C18-AQ, 3 μm, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) and eluted at 150 nl / min onto a 20 cm column (50-μm id; ReproSil-Pur C18-AQ, 3 μm). The column was developed with a 120 min gradient from 0 to 40% acetonitrile in 0.1% formic acid. The end of the column was directed to a tip (~5 μm inner diameter) from which the eluent was sprayed into the mass spectrometer. Full scan MS spectra were acquired in the FT-ICR with a resolution of 25,000 and a target value of 3,000,000. The two most intense ions were isolated by a selected ion monitoring scan in the FT-ICR (resolution of 50,000 and target accumulation of 50,000) for accurate mass measurement. The selected ions were then fragmented in a linear ion trap using collision induced dissociation with a target value of 10,000. The Q-Exactive mass spectrometer was operated in the front 10 mode. The parameters were: resolution 70,000, AGC target 3,000,000 / max fill time 20 ms (full scan); and resolution 17,500, AGC target 100,000 / max fill time 60 ms, MS / MS intensity threshold 17,000.The Apex trigger was set to 1 to 10 s, and the number of charges allowed was 2-6. Peptide and protein identification was performed using Proteome Discoverer version 2.1 (Thermo Fisher Scientific), using the mascot node for identification, using mascot version 2.2.04 and the UniProt Homo Sapiens database (UP000005640; January 2015; 67911 entries). Methionine oxidation and cysteine ​​cysteinization were set as variable modifications. For Q-Exactive data, the precursor tolerance for peptide assignment was 10 ppm and the MS / MS fragment tolerance was 20 mmu, while for LTQ-FTUItra data, it was 2 ppm and 0.5 Da. The identity of the ΔNPM1-derived peptide was confirmed by its synthetic counterpart.

[0578] Peptide synthesis and pMHC tetramer production

[0579] Peptides were synthesized by standard Fmoc chemistry and dissolved in dimethyl sulfoxide. Cysteinylation of the peptide was performed by treating 1 mM peptide with 2 mM 1,4-dithiothreitol in 50 mM ammonium bicarbonate at 50°C for 15 min, followed by the addition of 10 mM free cysteine ​​and 15 mM H2O2 for 30 min at RT. pMHC tetramers were produced as previously described (7, 8). Briefly, monomers composed of recombinant HLA-A*02:01 heavy chain and human β2-microglobulin were purified by gel filtration HPLC and biotinylated. After folding with the appropriate peptide, pMHC tetramers were generated by the addition of PE-coupled streptavidin (Invitrogen, Thermo Fisher Scientific). Biotinylated HLA-A*02:01 monomers containing UV-sensitive peptides were exposed to 366 nm UV light in the presence of cysteine-labeled ΔNPM1 peptides to generate UV-exchanged pMHC tetramers. After 1 hour of peptide exchange, monomers were incubated at 4°C for 1 hour and then centrifuged at 4000 g for 10 minutes at 15°C. Tetramers were generated by adding streptavidin-conjugated PE to the supernatant. pMHC tetramers were stored at 4°C.

[0580] Antibodies and FACS analysis

[0581] T cells were stained with FITC-coupled antibodies to CD3, CD4, CD8 (BD Biosciences, San Jose, California, United States) and TCR-Vβ5.1 (Beckman Coulter, Brea, California, United States), APC-coupled antibodies to CD3, CD4, CD8 and mouse TCR-Cβ, and PE-coupled pMHC tetramers and antibodies to CD3, CD4 and CD8 (BD Biosciences). Cells were measured on a BD FACSCalibur II (BD Biosciences) using BD CellQuest Pro software (BD BiosClences) and analyzed using FlowJo software (Flowjo, LLC, Ashland, Oregon, United States).

[0582] T cell isolation and culture

[0583] pMHC tetramer-positive CD8 T cells were isolated from PBMCs from patients with AML and healthy individuals as described previously (9). Briefly, PBMCs from HLA-*02:01-positive AML patients and healthy individuals were stained with PE-conjugated pMHC tetramers containing ΔNPM1 peptide for 1 h at 4°C and then subjected to MACS separation using anti-PE microbeads (Miltenyi Biotec). Isolated cells were stained with CD8-Alexa Fluor 700 (Invitrogen), CD4-FITC, CD14-FITC, and CD19-FITC (BD Biosciences) antibodies, and pMHC tetramer-positive CD8 T cells were single-cell sorted using BD FACSDiva v6 software (BD Biosciences) using a BD FACSAria III cell sorter (BD Biosciences). Single T cells were stimulated with 50,000 irradiated allogeneic PBMCs, 5000 irradiated allogeneic EBV-LCLs and 0.8 μg / ml PHA (Oxoid Microbiology Products, Thermo Fisher Scientific) in 100 μl TCM per well in 96-well U-bottom plates (Costar, Sigma-Aldrich). Growing T cell clones were restimulated with irradiated feeder cells and PHA every 10-14 days.

[0584] T cell reactivity assay

[0585] T cell recognition was measured by IFN-γ ELISA (Sanquin). Stimulator cells (30,000 cells) were co-incubated with T cells (2000 cells) in 40 μl TCM per well in 384-well flat-bottom plates (Greiner Bio One, Kremsmünster, Austria). After overnight co-incubation, culture supernatants were harvested to measure IFN-γ release. In peptide recognition assays, T2 cells (15,000 cells) were pre-incubated with titrated peptide concentrations for 30 min at 37°C and washed twice before co-incubation with T cells. In blocking assays, target cells (10,000 cells) were pre-incubated for 60 min at RT with saturating concentrations of antibodies blocking HLA class I (W6 / 32) or HLA class II (PdV5.2) before the addition of T cells. 51 T cell-mediated cytotoxicity was measured in a chromium release assay. The cells were incubated at 37°C with 100 μCi Na2 51 Primary AML cells were labeled with CrO4 for 1 hour, washed and co-incubated with T cells at different E:T ratios in 100 μl TCM per well in 96-well U-bottom plates (Costar). Spontaneous and maximal cell death were measured in separate plates containing either 100 μl TCM or 100 μl TCM with 1% Triton-X100 (Sigma-Aldrich) per well. 51 Cr release. After 9 hours of co-incubation, 25 μl of culture supernatant was harvested and transferred to 96-well LumaPlates (PerkinElmer, Waltham, Massachusetts, United States). 2 The number of cells per minute was measured on a plate counter (PerkinElmer). 51 Cr release counts (cpm).

[0586] TCR cloning and production of retroviral supernatant

[0587] The use of the TCR α and β chains of clone 1A2 was determined as previously described (9) with slight modifications. In brief, T cells were lysed and mRNA was isolated by Dynabeads mRNA DIRECT kit (Invitrogen). TCR-specific cDNA was generated using two TCR-Cβ specific primers, one TCR-Cα specific primer, a SA.rt anchor template-switching oligonucleotide (TSO) and SMARTScribe reverse transcriptase (Takara, Clontech, Mountain View, California, United States). During the first-strand cDNA synthesis, SMARTScribe reverse transcriptase adds 3' non-templated polycytosine ends, which allows TSO annealing and second-strand cDNA synthesis. TCR amplification was performed by PCR using Phusion Flash (Thermo Fisher Scientific), anchor-specific primers and nested primers annealed to the TCR-Cα or -Cβ region. The TCR sequences of clone 1A2 were determined to be TRAV12-2 and TRBV5-1 by Sanger sequencing (Macrogen, Amsterdam, the Netherlands) and the ImMunoGeneTics (IMGT) database (10). Codon-optimized TRAV12-2 and TRBV5-1 sequences were synthesized and cloned into the MP71-TCR-flex retroviral vector (11) by GenScript (Piscataway, New Jersey, United States). In MP71-TCR-flex, the murine TCR-Cα and -Cβ regions contain additional cysteine ​​residues to promote preferential pairing and expression of the TCR, connected by a porcine teschovirus-derived P2A sequence. The constructs were transfected into packaging cells φ-NX-A (ATCC, Manassas, Virginia, United States), and the retroviral supernatants were harvested 48 and 72 hours after transfection and frozen at -80°C. MP71-TCR-flex encoding the TCR for the CMV-derived HLA-A*02:01-restricted peptide NLVPMVATV was kindly provided by Prof. Dr. TN Schumacher (Division of Immunology, Netherlands Cancer Institute, Amsterdam, the Netherlands).

[0588] TCR gene transfer

[0589] PBMC from two HLA-A*02:01 positive healthy individuals (donor 1 and donor 2) were thawed and passed through MACS using anti-CD4 microbeads (Miltenyi Biotec), followed by separation of CD4 and CD8 cells using a CD8T cell separation kit (Miltenyi Biotec). In a 24-well flat-bottomed culture plate (Costar), CD8 and CD4 cells were stimulated with irradiated autologous feeders and 0.8 μg / ml PHA. After two days of stimulation, T cells were transferred to a 24-well flat-bottomed suspension culture plate (Greiner Bio-One) and retroviral transduction (9, 12) was performed as described previously. Before adding T cells, the plate was coated with 30 mg / mL retronectin (Takara, Clontech) and closed with 2% human serum albumin (Sanquin). Retroviral supernatant was added and the plate was centrifuged for 20 minutes at 4°C with 3000 g. T cells were added to plates containing viral supernatants at 300,000 cells per well. After overnight incubation, T cells were transferred to 24-well flat-bottom culture plates. On the 6th day after transduction, TCR-transduced T cells were stained with APC-coupled antibodies against mouse TCR-Cβ at 4°C for 15 minutes and then separated by MACS using anti-APC microbeads (Miltenyi Biotec). TCR-transduced T cells were restimulated with irradiated allogeneic PBMCs and EBV-LCL and 0.8μg / ml PHA every 10-14 days. Prior to analysis, restimulated TCR-transduced T cells were enriched by MACS using anti-mouse TCR-Cβ-APC and anti-APC microbeads as described above.

[0590] result

[0591] ΔNPM1 is present in the HLA class I ligand panel of primary AML

[0592] To investigate whether the ΔNPM1 peptide is processed and presented in HLA class I, the inventors immunoprecipitated HLA class I surface molecules from 12 primary AML samples, eluted peptides from the binding groove and analyzed the peptide group by mass spectrometry. Table I shows the HLA class I typing of the 12 AML samples and their mutation status of NPM1. The 4bp frameshift insertion in exon 12 of the NPM1 gene is a recurrent mutation occurring in 30% of primary AML. In 8 of the 12 primary AMLs, the presence of ΔNPM1 was confirmed by PCR fragment analysis. The cytogenetic karyotype of all patients was normal, except for one AML carrying a known chromosomal rearrangement inv(16)(p13q22). The percentage of primitive cells (blasts) measured in peripheral blood or bone marrow samples ranged from 55-98%.

[0593] Table 1. Presence of ΔNPM1 in the HLA class I-ligand panel of primary AML

[0594]

[0595]

[0596] a ΔNPM1 patients have a 4-bp insertion in exon 12 of the NPM1 gene, resulting in a frameshift at the C-terminus of the protein.

[0597] b The percentage of blasts measured in peripheral blood and bone marrow samples from patients with AML.

[0598] * AML samples were subjected to peptide elution using the anti-HLA-A*02:01 antibody BB7.2.

[0599] Recurrent 4-bp insertions in exon 12 generate a ΔNPM1 protein that is 4 AA longer than its wild-type counterpart, with 11 AA at the C-terminus (CLAVEEVSLRK (SEQ ID NO: 27)) translated in an alternative reading frame. From this alternative ΔNPM1 protein, a protein region spanning 10 N-terminal residues in the normal reading frame followed by 11 C-terminal AAs in the alternative reading frame ((MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO:34)) was searched for peptides matching the HLA class I ligand set analyzed from 12 primary AMLs. The results showed that two 8-mer peptides (VEEVSLRK (SEQ ID NO:28) and AVEEVSLR (SEQ ID NO:29)), two 9-mer peptides (CLAVEEVSL (SEQ ID NO:1) and AVEEVSLRK (SEQ ID NO:26)) and one 11-mer peptide (CLAVEEVSLRK (SEQ ID NO:27)) were present in AMLs with ΔNPM1, but not in AMLs with wtNPM1 (Table 1). All five ligands eluted from seven primary AMLs were validated by mass spectrometry using synthetic peptides. Figure 1and Figure 2). After in vitro cysteine ​​ligation of the first residue, tandem mass spectrometry validation of CLAVEEVSL (SEQ ID NO: 1) and CLAVEEVSLRK (SEQ ID NO: 27) was performed with synthetic peptides. The prediction of HLA class I binding affinity by NetMHCpan 3.0 indicated that the epitope CLAVEEVSL (SEQ ID NO: 1) binds to HLA-A*02:01, while the epitopes AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) are most likely to bind to HLA-A*03:01 as well as HLA-A*11:01, and for AVEEVSLRK (SEQ ID NO: 26), it is possible to bind to HLA-A*01:01 (Table 2). The binding of CLAVEEVSL (SEQ ID NO: 1) to HLA-A*02:01 and the binding of AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) to HLA-A*03:01 and A*11:01 were confirmed by monomer refolding. Since HLA-A*02:01 is expressed in 50% of the Caucasian population, the inventors focused on CLAVEEVSL (SEQ ID NO: 1), and CLAVEEVSL (SEQ ID NO: 1) was detected in 2 of the 3 HLA-A*02:01-positive AMLs with ΔNPM1 (AML10197 and AML3361).

[0600] Table 2: Binding affinities of peptides predicted by NetMHCpan3.0 for HLA class I alleles

[0601]

[0602] a Lower limit of predicted binding affinity (nM) for all other HLA class I alleles expressed by the primary AML sample from which the peptide was eluted.

[0603] NA Not applicable.

[0604] T cell recognition of ΔNPM1 on primary AML

[0605] To examine whether the HLA-A*02:01 restricted epitope CLAVEEVSL (SEQ ID NO: 1) is a new antigen that can be targeted by immunotherapy, the inventors searched for specific T cells in patients with AML. PE-coupled pMHC tetramers were prepared for CLAVEEVSL (SEQ ID NO: 1) (ΔNPM1-CLA) and its cysteine ​​variant C*LAVEEVSL (cysteine ​​form of SEQ ID NO: 1) (ΔNPM1-C*LA), and a mixture of these tetramers was used to isolate specific T cells from PBMCs of 6 HLA-A*02:01-positive patients with ΔNPM1 AML who were in remission after chemotherapy. T cells bound to one or two pMHC tetramers were enriched by magnetic anti-PE beads, and single tetramer-positive CD8 cells were isolated by flow cytometry (Table 3).

[0606] Table 3. Isolation of T cells from patients with HLA-A*02:01 positive ΔNPM1 AML.

[0607]

[0608]

[0609] a Days since diagnosis.

[0610] b PBMC, peripheral blood mononuclear cells. Indicated are the numbers of PBMCs from HLA-A*02:01-positive patients with ΔNPM1 AML used for T cell isolation.

[0611] c PBMCs were stained with anti-CD8-ALX700 and a mixture of PE-coupled pMHC tetramers for CLAVEEVSL (SEQ ID NO: 1) and its cysteine ​​variant C*LAVEEVSL (cysteine ​​version of SEQ ID NO: 1). Indicated are the numbers of sorted tetramer-positive CD8 cells.

[0612] d Number of T cell clones positive for ΔNPM1-CLA or ΔNPM1-C*LA tetramers.

[0613] From 42*10 from 4 patients 6A total of 41 tetramer-positive CD8 cells were isolated from PBMCs, of which 5 T cells from 3 patients were cloned and expanded. However, none of the 5 T cell clones could be stained with ΔNPM1-CLA tetramers or ΔNPM1-C*LA tetramers, indicating that there were no T cells for CLAVEEVSL (SEQ ID NO: 1) or the frequency was below the detection threshold. Therefore, the same strategy was followed to search for specific T cells in a large number of PBMCs from 6 HLA-A*02:01-positive healthy individuals (Table 4). From 460-1970x10 6 From these cells, 31 T cells from 5 individuals were clonally expanded and 13 T cell clones from 4 individuals were positive for ΔNPM1-CLA tetramer. Among these 13 clones, 3 T cell clones could also be stained with ΔNPM1-CLA tetramer (Table 4 and Table 5 ). Figure 3 A).

[0614] Table 4. Isolation of T cells for ΔNPM1 from healthy individuals

[0615]

[0616]

[0617] a PBMC, peripheral blood mononuclear cells. Indicated are the numbers of PBMCs from HLA-A*02:01-positive healthy individuals used for T cell isolation.

[0618] b PBMCs were stained with anti-CD8-ALX700 and a mixture of PE-coupled pMHC tetramers for CLAVEEVSL (SEQ ID NO: 1) and its cysteine ​​variant C*LAVEEVSL (cysteine ​​version of SEQ ID NO: 1). Indicated are the numbers of sorted tetramer-positive CD8 cells.

[0619] c Number of growing T cell clones positive for ΔNPM1-CLA tetramer.

[0620] d Number of growing T cell clones positive for ΔNPM1-C*LA tetramer.

[0621] ndNot tested.

[0622] To determine whether the 13 tetramer-positive CD8 clones were reactive to their target peptides, the clones were tested for recognition of HLA-A*02:01-positive T2 cells loaded with CLAVEEVSL (SEQ ID NO: 1), the cysteine ​​variant C*LAVEEVSL (cysteine ​​form of SEQ ID NO: 1), or the irrelevant HLA-A*02:01-restricted CMV peptide NLVPMVATV (SEQ ID NO: 31). Of the 13 ΔNPM1-CLA tetramer-positive clones, two T cell clones (1A2 and 4A8) showed specific reactivity to T2 cells loaded with CLAVEEVSL (SEQ ID NO: 1), but not to the control peptide NLVPMVATV (SEQ ID NO: 31) ( Figure 3 B). Clone 1A2 also showed recognition of T2 cells loaded with C*LAVEEVSL (cystylated SEQ ID NO: 1). These results are consistent with the tetramer data, indicating that cystylation of the first residue abolished T cell recognition of clone 4A8. However, peptide recognition by clone 1A2 was independent of cystylation, as indicated by its specific reactivity to CLAVEEVSL (SEQ ID NO: 1) as well as C*LAVEEVSL (cystylated form of SEQ ID NO: 1).

[0623] To investigate the antitumor potential of clones 1A2 and 4A8, T cell reactivity was measured against a panel of 5 HLA-A*02:01-positive primary AMLs, including 3 samples with ΔNPM1 and 2 samples with wtNPM1. T cell clone 1A2 showed varying degrees of reactivity against all 3 AMLs with ΔNPM1, whereas clone 4A8 showed relatively low reactivity against 2 of the 3 samples with ΔNPM1 ( Figure 3 C). The strong T cell reactivity of clone 1A2 against AML can be explained by its ability to recognize cystylated ΔNPM1 eluted from the surface of primary AML cells as well as uncystylated ΔNPM1. No T cell reactivity was observed against HLA-A*02:01-positive AML ( Figure 3 C) or T cell reactivity of HLA-A*02:01-negative AML with ΔNPM1 (data not shown). These data indicate that T cells with TCRs specific for ΔNPM1 exist in the T cell repertoire of healthy individuals and that these T cells can specifically recognize CLAVEEVSL (SEQ ID NO: 1) as an endogenous neoantigen presented by HLA-A*02:01 on primary AML with ΔNPM1.

[0624] TCR gene transfer to target ΔNPM1 on primary AML

[0625] Since ΔNPM1 is a recurrent 4-bp insertion that occurs in 30% of primary AML and HLA-A*02:01 is expressed in 50% of the Caucasian population, the inventors believed that CLAVEEVSL (SEQ ID NO: 1) is an ideal target for TCR gene transfer. Since primary AML is most strongly recognized by clone 1A2, mRNA was isolated from this clone and cDNA was generated to sequence the variable regions of the TCR α and β chains of ΔNPM1. Codon-optimized gene sequences for TRAV12-2 and TRBV5-1 as expressed by T cell clone 1A2 were synthesized and cloned into a modified MP71-TCR-flex retroviral vector. To promote preferential binding and expression of the TCR α and β chains, the variable regions of the TCR were cloned in frame with the mouse constant regions connected by the P2A sequence. TCR for ΔNPM1 and TCR for HLA-A*02:01 restricted CMV peptide NLVPMVATV (SEQ ID NO:31) as a control were introduced into CD8 and CD4 cells isolated from PBMCs from healthy HLA-A*02:01 positive individuals (donor 1 and donor 2). On day 6 after transduction, TCR-transduced CD8 and CD4 cells were purified using APC-coupled antibodies against mouse TCR-Cβ and magnetic anti-APC beads. Flow cytometric analysis showed that ΔNPM1-CLA tetramers were activated by using TCR for ΔNPM1. Specific binding of transduced CD8 cells ( Figure 4 A) In contrast, CMV-NLV tetramers did not bind CD8 cells transduced with the TCR for ΔNPM1, but rather with the CMV-specific TCR Transduced CD8 cells can be stained with CMV-NLV tetramers, but not with ΔNPM1-CLA tetramers. For TCR-transduced CD4 cells ( and ), the results were similar to those of CD8 cells, indicating that the binding of ΔNPM1-CLA tetramers to TCR-transduced T cells is independent of the CD8 co-receptor. TCR-transduced CD8 and CD4 cells can also be stained with antibodies against mouse TCR-Cβ, and CD8 and CD4 cells transduced with TCR for ΔNPM1 also showed specific binding with antibodies against human TCR-Vβ5.1 (data not shown).

[0626] Subsequently, the inventors analyzed the functionality of CD8 and CD4 cells transduced with the TCR for ΔNPM1 and demonstrated that IFN-γ was specifically released when co-incubated with HLA-A*02:01-positive T2 cells loaded with CLAVEEVSL (SEQ ID NO: 1), but not when co-incubated with the CMV peptide NLVPMVATV (SEQ ID NO: 31). Figure 4 B). Specific release of IFN-γ was also observed upon co-incubation with the AML cell line OCI-AML3 harboring ΔNPM1, but not upon stimulation with the AML cell line OCI-AML2 harboring wtNPM1 ( Figure 4 C).

[0627] The TCR-transduced CD8 and CD4 cells were then tested for reactivity against a panel of 13 HLA-A*02:01-positive primary AMLs, including 9 samples with ΔNPM1 and 4 samples with wtNPM1. After transduction with the TCR for ΔNPM1, both CD8 and CD4 cells showed recognition of all 9 primary AMLs with ΔNPM1, whereas no specific recognition of AMLs with wtNPM1 was observed ( Figure 5 CD8 and CD4 cells transduced with TCR for ΔNPM1 also lacked reactivity to HLA-A*02:01-negative AML with ΔNPM1 as well as mature DC with wtNPM1 ( Figure 6 ). Next, the transduced T cells were tested for reactivity against monocyte-derived mature DCs from donors 1 and 2, as well as a panel of 40 HLA-A*02:01-positive third-party EBV-LCLs (non-malignant cell types with strong antigen processing and presentation capabilities). CD8 and CD4 cells transduced with the TCR for ΔNPM1 were unable to recognize mature DCs or EBV-LCLs, indicating that alternative translation of the wtNPM1 gene did not occur and that the gene did not produce a peptide similar to CLAVEEVSL (SEQ ID NO: 1). In summary, the data indicate that the TCR for ΔNPM1, when gene transferred to CD8 and CD4 cells, results in specific recognition of CLAVEEVSL (SEQ ID NO: 1) as an endogenous neoantigen presented by HLA-A*02:01 on primary AML with ΔNPM1.

[0628] Finally, the inventors tested the cytolytic capacity of TCR-transduced CD8 and CD4 cells against primary AML. 51TCR-transduced T cells were tested in a chromium release assay against a panel of 6 HLA-A*02:01-positive primary AMLs (including 4 samples with ΔNPM1 and 2 samples with wtNPM1). Both CD8 and CD4 cells transduced with the TCR for ΔNPM1 showed specific lysis of AML with ΔNPM1, but not AML with wtNPM1 ( Figure 7 ). In summary, the results indicate that CLAVEEVSL (SEQ ID NO: 1) is a therapeutic neoantigen expressed on primary AML with ΔNPM1 and can be effectively targeted by TCR gene transfer in a co-receptor-independent manner.

[0629] discuss

[0630] The inventors have identified an HLA-A*02:01 restricted 9-mer peptide (CLAVEEVSL (SEQ ID NO: 1)) encoded by ΔNPM1 in the HLA class I ligand panel of primary AML. T cell clones with TCRs specific for this peptide were isolated from healthy individuals, and TCRs from one of the clones have been shown to mediate specific recognition and lysis of primary AML with ΔNPM1 after retroviral introduction into CD8 and CD4 cells, indicating that CLAVEEVSL (SEQ ID NO: 1) is a therapeutic neoantigen on AML that can be targeted by TCR gene transfer in a co-receptor-independent manner.

[0631] Using HLA class I ligand data from 12 primary AMLs, the inventors identified C*LAVEEVSL (a cystylated form of SEQ ID NO: 1) as a peptide that is endogenously processed and presented on AML with ΔNPM1. Although no matches were found between the eluted peptide and the synthetic peptide in the absence of cystylation, the data provided strong evidence that the non-cystylated CLAVEEVSL (SEQ ID NO: 1) peptide is also presented on AML. Clone 1A2, which was selected for TCR cloning and gene transfer, has been shown to recognize synthetic C*LAVEEVSL (a cystylated form of SEQ ID NO: 1) and CLAVEEVSL (SEQ ID NO: 1) peptides. This clone can also be stained by two pMHC tetramers, although the binding to the tetramers with the cystylated peptide is weaker. Testing experiments with synthetic peptides with serine substitutions confirmed that the first residue of the epitope is not essential for the reactivity of clone 1A2. However, for clone 4A8, the results were different. Clone 4A8 recognized CLAVEEVSL (SEQ ID NO: 1) but not C*LAVEEVSL (a cysteine ​​form of SEQ ID NO: 1), and only pMHC tetramers with the non-cysteine ​​peptide could bind to the T cell clone. The lack of reactivity of clone 4A8 to a synthetic peptide in which the first residue was substituted with serine confirms the importance of the first residue of the peptide for T cell recognition. Surprisingly, although clone 4A8 failed to recognize the cysteine ​​peptide, it showed reactivity to 2 of the 3 AMLs with ΔNPM1. Based on the reactivity patterns of clones 1A2 and 4A8, the inventors believe that both cysteine ​​and non-cysteine ​​ΔNPM1 peptide variants are presented on the cell surface, but the expression level of the epitope may vary between AMLs. The reason for the absence of CLAVEEVSL (SEQ ID NO: 1) in the HLA class I ligand panel is unclear, but may be explained by low surface expression or poor mass spectrometry quality of the eluted peptide.

[0632] Interestingly, patients carrying ΔNPM1 and lacking the accompanying internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD) show improved survival after chemotherapy, thus generally eliminating the need for allogeneic stem cell transplantation. 1,2,5,13 In vivo immune responses against peptides derived from ΔNPM1 may underlie this favorable prognosis, particularly because the ΔNPM1 protein translocates from the nucleolus to the cytoplasm, where it is susceptible to proteasomal degradation and subsequent processing by the HLA class I antigen presentation pathway. 6 Greiner et al. 14T cell responses to nine HLA-A*02:01-restricted peptides derived from ΔNPM1, including CLAVEEVSL (SEQ ID NO: 1), were searched in vitro in healthy volunteers and AML patients. After co-culturing CD8 cells with peptide-loaded PBMCs, T cell responses to two of the nine peptides were shown in both healthy volunteers and patients, but no immune response to CLAVEEVSL (SEQ ID NO: 1) was measured. They then screened 25 patients with ΔNPM1 AML and found that patients who had immune responses to the two ΔNPM1-derived peptides had significantly better overall survival than those who did not. 15 However, the number of patients screened in this study was small, and FLT3 mutation status had not been determined. Recent data have shown that patients with FLT3-ITD, especially those with a high allele ratio of mutant to wild-type FLT3 gene expression, have a worse prognosis compared with patients without FLT3-ITD, regardless of NPM1 mutation status. 13 This observation may refute that in vivo induction of an immune response against ΔNPM1 underlies a favorable prognosis but emphasizes the importance of intrinsic factors for AML tumor growth and the relevance of ΔNPM1 TCR gene therapy for AML to a poor prognosis.

[0633] AML originates from a single founding clone that contains hundreds of somatic mutations, of which only a few are driver mutations. Subclones can emerge from the founding clone by accumulating additional mutations that confer a survival advantage to the cells. Therefore, most mutations in a subclone are shared with the founding clone, while a few mutations are clone-specific. This heterogeneous composition of AML increases the chances of clonal evolution in persistent or recurrent disease after induction or consolidation therapy. Targeting neoantigens generated by shared mutations is an attractive immunotherapeutic strategy that can eradicate the founding clone as well as subclones. Neoantigens generated by passenger mutations are easily lost by immune editing of tumors by T cells, leading to tumor immune escape. Immune escape is less likely to occur when targeting neoantigens generated by driver mutations because these neoantigens are essential for malignant transformation and are present in all tumor cells. 16 So far, only a few neoantigens caused by driver mutations have been discovered, with mutant KRAS resulting in one antigen expressed in 45% of pancreatic cancers and 13% of colorectal cancers. 17Although neoantigens caused by driver mutations have a lower potential for immune evasion, Tran et al. demonstrated that patients with metastatic colorectal cancer could escape TCR gene therapy for mutant KRAS through loss of HLA-C*08:02. Nevertheless, ΔNPM1 remains an attractive target for immunotherapy as a clonal driver mutation that occurs early in leukemogenesis. Based on its high mutation frequency in 30% of primary AML, ΔNPM1 is also an ideal target. 6 Characteristic 4 bp frameshift insertions occur at a limited number of positions in the coding sequence (859, 860, and 861) and although the exact 4-bp sequence may differ, most mutations encode the same 11 amino acid alternative reading frame (CLAVEEVSLRK (SEQ ID NO: 27)). The inventors have identified a TCR that targets the first 9 residues of this alternative reading frame in HLA-A*02:01. This TCR can be used for future gene therapy to treat patients with AML with ΔNPM1. Clinical studies will show the efficacy and potential toxicity of ΔNPM1 TCR gene therapy, and whether immune escape caused by HLA-A*02:01 loss will have a significant impact on preventing long-term remission of AML.

[0634] It has been shown that the TCR isolated from clone 1A2 is able to mediate specific recognition and lysis of HLA-A*02:01-positive AML cells with ΔNPM1 after transfer to CD8 and CD4 cells, indicating that the TCR is able to redirect immune responses to AML in a co-receptor-independent manner. Over the past few decades, the critical role of CD4 cells in antitumor immunity has become more evident. Traditionally, it has been known that CD4 cells can provide help to CD8 cells, leading to improved tumor clearance and inducing immune memory. However, increasing evidence suggests that CD4 cells can also mediate tumor rejection in the absence of CD8 cells. Patients with hematological malignancies who received CD8-depleted allogeneic bone marrow transplants or donor lymphocyte infusions developed graft-versus-leukemia reactions that were similar to those in patients who received unmodified stem cell transplants or donor lymphocytes, while the incidence and severity of graft-versus-host disease were reduced. In an autologous setting, adoptive transfer of CD4 cells directed against HLA class II-restricted tumor-associated antigens or neoantigens resulted in tumor regression in patients with metastatic melanoma and cholangiocarcinoma, respectively. 18 However, since tumors often do not express HLA class II, administration of a mixture of CD8 and CD4 cells expressing HLA class II-independent antigen-receptors may be more preferable and may result in better antitumor immunity. In fact, Turtle et al. 19,20It was demonstrated that administration of a specific ratio of CD8 and CD4 cells expressing the same CD19-specific chimeric antigen receptor resulted in complete remission in a large number of patients with relapsed or refractory B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. Similarly, as previously demonstrated in mice, adoptive transfer of CD8 and CD4 cells expressing the same TCR against an HLA class I restricted epitope such as CLAVEEVSL (SEQ ID NO: 1) resulted in potent anti-tumor immunity.

[0635] Recently, ΔNPM1 was described as a reliable marker for measuring minimal residual disease in patients with AML. 21 The persistence of ΔNPM1 transcripts detected by quantitative RT-PCR in the peripheral blood of patients after chemotherapy was associated with disease recurrence within 3 years of follow-up. The prognostic value of ΔNPM1 has been shown to be independent of other risk factors such as FLT3-ITD or mutant DNA methyltransferase 3α (DNMT3A). 1,2,5,13 Importantly, the authors report that among patients with a favorable molecular profile at diagnosis (no FLT3-ITD or mutated DNMT3A), the presence of ΔNPM1 transcripts after the second chemotherapy cycle marked a group of patients with a relatively poor prognosis, whereas the absence of ΔNPM1 transcripts after the second chemotherapy cycle distinguished patients with a relatively good prognosis among patients with an unfavorable molecular profile (FLT3-ITD, mutated DNMT3A, or both). 21 Therefore, ΔNPM1 as a marker of disease status could be used to select patients eligible for alloSCT and allow for the optimal timing and selection of patients for ΔNPM1 TCR gene therapy to treat persistent or recurrent disease after chemotherapy. Ultimately, when clinical studies demonstrate that AML can be effectively treated with ΔNPM1 TCR gene transfer with a low treatment-related mortality, it could replace alloSCT as the standard of care for ΔNPM1 AML patients with a poor prognosis based on adverse molecular abnormalities at diagnosis or detectable persistent or recurrent disease after chemotherapy, thereby improving overall survival of AML patients.

[0636] Example 2

[0637] Following the same strategy as described for T cell clone 1A2 recognizing CLAVEEVSL (SEQ ID NO: 1) in HLA-A*02:01, T cell clones recognizing AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) in HLA-A*03:01 or HLA-A*11:01 were screened from healthy individuals. PE-labeled pHLA-tetramers were generated for AVEEVSLRK (SEQ ID NO: 26) in HLA-A*03:01, CLAVEEVSLRK (SEQ ID NO: 27) in HLA-A*03:01, AVEEVSLRK (SEQ ID NO: 26) in HLA-A*11:01, and CLAVEEVSLRK (SEQ ID NO: 27) in HLA-A*11:01. Peripheral blood mononuclear cells from healthy individuals were incubated with a mixture of four PE-labeled pHLA-tetramers, and pHLA-tetramer-positive T cells were isolated using magnetic anti-PE beads. In the next step, single pHLA-tetramer-positive CD8+ T cells were isolated by flow cytometry, expanded, and analyzed by IFN-γ ELISA for reactivity against the AML cell lines OCI-AML2 (wild-type NPM1) and OCI-AML3 (mutant NPM1), which were either non-transduced or transduced with HLA-A*03:01 or HLA-A*11:01. T cell clones that selectively recognized OCI-AML3 transduced with HLA-A*03:01 or HLA-A*11:01 were analyzed by flow cytometry for pHLA-tetramer staining and reanalyzed by IFN-γ ELISA for reactivity against the same panel of AML cell lines and T2 cells transduced with the relevant HLA-restricted alleles and pulsed with titrated concentrations of the peptide of interest.

[0638] A total of 42 healthy individuals positive for HLA-A*03:01 and / or HLA-A*11:01 were screened. Three pHLA-tetramer positive T cell clones were isolated that were able to specifically react to OCI-AML3 transduced with HLA-A*03:01 or HLA-A*11:01 and peptide-pulsed T2 cells transduced with HLA-A*03:01 or HLA-A*11:01. T cell clone 31.3.F1 was specific for AVEEVSLRK (SEQ ID NO:26) in HLA-A*03:01 ( Fig.40 A), and T cell clones 6F11 and 26.2.D6 are specific for AVEEVSLRK (SEQ ID NO: 26) in HLA-A*11:01 ( Fig.40B). All three T cell clones were able to react against primary AML with mutant NPM1 expressing relevant HLA-restricted alleles, but not against AML cases with wild-type NPM1. T cell clones 31.3.F1 and 6F11 were isolated from individuals positive for relevant HLA-A*03:01 and HLA-A*11:01-restricted alleles, respectively, while T cell clone 26.2.D6 was isolated from an HLA-A*11:01-negative individual.

[0639] Similar to T cell clone 1A2, TCRs of T cell clones 6F11, 31.3.F1, and 26.2.D6 were sequenced, cloned, and tested for reactivity against AML by IFN-γ ELISA. Human TCR α and β variable regions were tested and cloned in MP71-flex, which contains precloned murine TCR α and β constant genes. CD8+ cells were isolated from HLA-A*03:01 or HLA-A*11:01 positive healthy individuals and stimulated with PHA, IL-2, and autologous irradiated peripheral blood mononuclear cells as feeder cells. On day 2, CD8+ cells were transduced with TCR for mutant NPM1. As controls, CD8+ cells were transduced with HLA-A*03:01 restricted TCR for EBNA3A or HLA-A*11:01 restricted TCR for EBNA3B. On day 10, TCR-transduced T cells (TCR-T) cells were stained with APC-labeled antibodies against murine TCR-Cβ and separated by magnetic anti-APC beads. On day 11, TCR-T cells were restimulated with PHA, IL-2 and irradiated allogeneic peripheral blood mononuclear cells as feeder cells. Two weeks after restimulation, TCR-T cells were analyzed by flow cytometry for pHLA-tetramer staining and by IFN-γ ELISA for their reactivity to OCI-AML2 (wild-type NPM1) and OCI-AML3 (mutant NPM1) cell lines transduced with HLA-A*03:01 or HLA-A*11:01. The TCR of clone 31.3.F1 showed specific binding to the pHLA-A*03:01-AVEEVSLRK (SEQ ID NO:26) tetramer and specific reactivity to OCI-AML3 transduced with HLA-A*03:01 ( Fig.41 A). TCRs of clones 6F11 and 26.2.D6 showed specific binding to pHLA-A*11:01-AVEEVSLRK tetramer and specific reactivity to OCI-AML3 transduced with HLA-A*11:01 ( Fig.41 B and Fig.41 C).

[0640] In the context of dNPM1-A11 TCR-T cells, the in vitro reactivity of T cell clones 26.2.D6 and 6F11 and the corresponding TCR-T cells against primary human AML samples and in vivo reactivity against the AML cell line OCI-AML3 in NSG mice were compared. T cell clones and the TCR of clone 26.2.D6 (isolated from an HLA-A*11:01 negative donor) were particularly favorable. This was most clearly demonstrated in NSG mice, where 26.2.D6 TCR-T cells induced an antitumor effect against OCI-AML3 that was similar to that of NPM1-specific HLA-A*02:01 restricted TCR-T cells and superior to that observed from 6F11 TCR-T cells.

[0641] In the context of dNPM1-A3 TCR-T cells, T cell clone 31.3.F1 was also particularly advantageous due to its specificity for AVEEVSLRK in A*03:01. Clone 31.3.F1 also showed significant reactivity against primary human AML samples in vitro.

[0642] Other sequences

[0643] SEQ ID NOs: 35 to 50 relate to a novel dNPM1-A*11:01 TCR (clone 26.2.D6) (TCR α chain - TRAV5*01, TRAJ26*01; TCR β chain - TRBV19*03, TRBJ2-7*01):

[0644] SEQ ID NO:35—Amino acid sequence of CDR3 (TCR alpha chain, clone 26.2.D6):

[0645] CAESKGQNFVF

[0646] SEQ ID NO:36—Nucleic acid sequence encoding CDR3 (TCR alpha chain, clone 26.2.D6):

[0647] TGTGCAGAGAGTAAAGGTCAGAATTTTGTCTTT

[0648] SEQ ID NO:37—Amino acid sequence of alpha chain variable region, clone 26.2.D6:

[0649] GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESKGQNFVFGPGTRLSVLP

[0650] SEQ ID NO:38—Nucleic acid sequence encoding the α chain variable region, clone 26.2.D6

[0651] GGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACA TGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAGTAAAGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCC

[0652] SEQ ID NO:39—Amino acid sequence of CDR1 (TCR alpha chain, clone 26.2.D6): DSSSTY

[0653] SEQ ID NO:40—Nucleic acid sequence encoding CDR1 (TCR alpha chain, clone 26.2.D6):

[0654] GACAGCTCCTCCACCTAC

[0655] SEQ ID NO:41 - Amino acid sequence of CDR2 (TCR alpha chain, clone 26.2.D6): IFSNMDM

[0656] SEQ ID NO:42—Nucleic acid sequence encoding CDR2 (TCR alpha chain, clone 26.2.D6):

[0657] ATTTTTTCAAATATGGACATG

[0658] SEQ ID NO:43 - Amino acid sequence of CDR3 (TCR β chain, clone 26.2.D6):

[0659] CASTTWGTGGHEQYF

[0660] SEQ ID NO:44—Nucleic acid sequence encoding CDR3 (TCR β chain, clone 26.2.D6):

[0661] TGTGCCAGTACTACATGGGGGACAGGGGGCCACGAGCAGTACTTC

[0662] SEQ ID NO:45—Amino acid sequence of the variable region of the beta chain, clone 26.2.D6:

[0663] DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSHIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASTTWGTGGHEQYFGPGTRLTVT

[0664] SEQ ID NO:46—Nucleic acid sequence encoding the variable region of the beta chain, clone 26.2.D6:

[0665] GATGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACTACTCACACATAGTAAATGACTTTCAGAAA GGAGATATAGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCTCACTGTGACATCGGCCCAAAAGAACCCGACAGCTTTCTATCTCTGTGCCAGTACTACATGGGGGACAGGGGGCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA

[0666] SEQ ID NO:47—Amino acid sequence of CDR1 (TCRβ chain, clone 26.2.D6): LNHDA

[0667] SEQ ID NO:48—Nucleic acid sequence encoding CDR1 (TCRβ chain, clone 26.2.D6):

[0668] TTGAACCACGATGCC

[0669] SEQ ID NO:49—Amino acid sequence of CDR2 (TCRβ chain, clone 26.2.D6): SHIVND

[0670] SEQ ID NO:50—Nucleic acid sequence encoding CDR2 (TCRβ chain, clone 26.2.D6):

[0671] TCACACATAGTAAATGAC

[0672] SEQ ID NOs: 51 to 66 relate to a novel dNPM1-A*11:01 TCR (clone 6F11) (TCR α chain - TRAV8-6*02, TRAJ49*01; TCR β chain - TRBV7-6*01, TRBJ1-5*01):

[0673] SEQ ID NO:51 - Amino acid sequence of CDR3 (TCR alpha chain, clone 6F11): CAVSPAGNQFYF

[0674] SEQ ID NO:52—Nucleic acid sequence encoding CDR3 (TCR alpha chain, clone 6F11):

[0675] TGTGCTGTGAGTCCCGCCGGTAACCAGTTCTATTTT

[0676] SEQ ID NO:53—Amino acid sequence of alpha chain variable region, clone 6F11:

[0677] AQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVKGINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVSPAGNQFYFGTGTSLTVIP

[0678] SEQ ID NO:54—Nucleic acid sequence encoding the α chain variable region, clone 6F11:

[0679] GCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTG GTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGAGTCCCGCCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAG

[0680] SEQ ID NO:55—Amino acid sequence of CDR1 (TCR alpha chain, clone 6F11): SSVSVY

[0681] SEQ ID NO:56—Nucleic acid sequence encoding CDR1 (TCR alpha chain, clone 6F11):

[0682] TCGTCTGTTTCAGTGTAT

[0683] SEQ ID NO:57—Amino acid sequence of CDR2 (TCR alpha chain, clone 6F11): YLSGSTLV

[0684] SEQ ID NO:58—Nucleic acid sequence encoding CDR2 (TCR alpha chain, clone 6F11):

[0685] TATTTATCAGGATCCACCCTGGTT

[0686] SEQ ID NO:59—Amino acid sequence of CDR3 (TCRβ chain, clone 6F11):

[0687] CASSLGSNQPQHF

[0688] SEQ ID NO:60—Nucleic acid sequence encoding CDR3 (TCRβ chain, clone 6F11):

[0689] TGTGCCAGCAGCCTGGGTAGCAATCAGCCCCAGCATTTT

[0690] SEQ ID NO:61—Amino acid sequence of the variable region of the beta chain, clone 6F11:

[0691] GAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLGSNQPQHFGDGTRLSIL

[0692] SEQ ID NO:62—Nucleic acid sequence encoding the variable region of the beta chain, clone 6F11:

[0693] GGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCCCAACAAGACA AATCAGGGCTGCCCAATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATCCAGCGCACAGAGCAGCGGGACTCGGCCATGTATCGCTGTGCCAGCAGCCTGGGTAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTA

[0694] SEQ ID NO:63—Amino acid sequence of CDR1 (TCRβ chain, clone 6F11): SGHVS SEQ ID NO:64—Nucleic acid sequence encoding CDR1 (TCRβ chain, clone 6F11):

[0695] TCGGGTCATGTATCC

[0696] SEQ ID NO:65—Amino acid sequence of CDR2 (TCRβ chain, clone 6F11): FNYEAQ SEQ ID NO:66—Nucleic acid sequence encoding CDR2 (TCRβ chain, clone 6F11):

[0697] TTCAATTATGAAGCCCAA

[0698] SEQ ID NOs: 67 to 82 relate to a novel dNPM1-A*03:01 TCR (clone 31.3.F1) (TCR α chain - TRAV12-2*02, TRAJ26*01; TCR β chain - TRBV14*01, TRBJ1-5*01):

[0699] SEQ ID NO:67—Amino acid sequence of CDR3 (TCR alpha chain, clone 31.3.F1):

[0700] CALSGGGQNFVF

[0701] SEQ ID NO:68—Nucleic acid sequence encoding CDR3 (TCR alpha chain, clone 31.3.F1):

[0702] TGTGCCCTCTCCGGCGGGGGTCAGAATTTTGTCTTT

[0703] SEQ ID NO:69—Amino acid sequence of alpha chain variable region, clone 31.3.F1:

[0704] QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCALSGGGQNFVFGPGTRLSVLP

[0705] SEQ ID NO:70—Nucleic acid sequence encoding the α chain variable region, clone 31.3.F1:

[0706] CAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGAC AAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCCTCTCCGGCGGGGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCC

[0707] SEQ ID NO:71 - Amino acid sequence of CDR1 (TCR alpha chain, clone 31.3.F1): DRGSQS

[0708] SEQ ID NO:72—Nucleic acid sequence encoding CDR1 (TCR alpha chain, clone 31.3.F1):

[0709] GACCGAGGTTCCCAGTCC

[0710] SEQ ID NO:73—Amino acid sequence of CDR2 (TCR alpha chain, clone 31.3.F1): IYSNGD

[0711] SEQ ID NO:74—Nucleic acid sequence encoding CDR2 (TCR alpha chain, clone 31.3.F1):

[0712] ATATACTCCAATGGTGAC

[0713] SEQ ID NO:75—Amino acid sequence of CDR3 (TCRβ chain, clone 31.3.F1):

[0714] CASSQGSGFRHF

[0715] SEQ ID NO:76—Nucleic acid sequence encoding CDR3 (TCR β chain, clone 31.3.F1):

[0716] TGTGCCAGCAGCCAAGGATCAGGTTTTCGGCATTTT

[0717] SEQ ID NO:77—Amino acid sequence of the variable region of the beta chain, clone 31.3.F1:

[0718] EAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSQGSGFRHFGDGTRLSIL

[0719] SEQ ID NO:78—Nucleic acid sequence encoding the variable region of the beta chain, clone 31.3.F1:

[0720] GAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGAT GAGTCCGGTATGCCCAACAATCGATTCTTAGCTGAAAGGACTGGAGGGACGTATTCTACTCTGAAGGTGCAGCCTGCAGAACTGGAGGATTCTGGAGTTTATTTCTGTGCCAGCAGCCAAGGATCAGGTTTTCGGCATTTTGGTGATGGGACTCGACTCTCCATCCTA

[0721] SEQ ID NO:79—Amino acid sequence of CDR1 (TCRβ chain, clone 31.3.F1): SGHDN

[0722] SEQ ID NO:80—Nucleic acid sequence encoding CDR1 (TCRβ chain, clone 31.3.F1):

[0723] TCTGGACATGATAAT

[0724] SEQ ID NO:81 - Amino acid sequence of CDR2 (TCRβ chain, clone 31.3.F1): FVKESK

[0725] SEQ ID NO:82—Nucleic acid sequence encoding CDR2 (TCRβ chain, clone 31.3.F1):

[0726] TTTGTGAAAGAGTCTAAA

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[0751] The present invention may be described with reference to the following paragraphs:

[0752] Paragraph 1. An isolated nucleic acid sequence encoding:

[0753] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to a peptide selected from the group consisting of CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29); and / or

[0754] (b) a polypeptide comprising the CDR3 of a TCR β chain polypeptide, which TCR β chain polypeptide specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0755] Paragraph 2. The isolated nucleic acid sequence of paragraph 1, wherein the nucleic acid sequence encodes (a) and (b), wherein ...

Claims

1. An isolated nucleic acid sequence comprising a promoter operably linked to a nucleic acid sequence encoding: (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide AVEEVSLRK (SEQ ID NO: 26); and (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to the peptide AVEEVSLRK (SEQ ID NO: 26), wherein (a) and (b) together specifically bind to the peptide AVEEVSLRK (SEQ ID NO: 26), and wherein: (i) The CDR3 of (a) has an amino acid sequence of SEQ ID NO: 35, wherein the CDR3 is located within a TCR alpha chain variable region that specifically binds to SEQ ID NO: 26; and (ii) The CDR3 of (b) has the amino acid sequence of SEQ ID NO:43, wherein the CDR3 is located within the TCR β chain variable region that specifically binds to SEQ ID NO:

26.

2. The isolated nucleic acid sequence of claim 1, wherein: (i) the CDR3 of (a) is located within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 37; and optionally wherein (a) comprises a TCR alpha chain constant region, and further optionally wherein the TCR alpha chain variable region CDR1 has the amino acid sequence of SEQ ID NO: 39 and the TCR alpha chain variable region CDR2 has the amino acid sequence of SEQ ID NO: 41; and (ii) The CDR3 of (b) is located within a TCR β chain variable region having at least 90% sequence identity with SEQ ID NO:45; and optionally wherein (b) comprises a TCR β chain constant region, and further optionally wherein the TCR β chain variable region CDR1 has the amino acid sequence of SEQ ID NO:47 and the TCR β chain variable region CDR2 has the amino acid sequence of SEQ ID NO:

49.

3. The isolated nucleic acid sequence of claim 1 or 2, wherein the nucleic acid sequence encodes a T cell receptor. A vector comprising the nucleic acid sequence according to any one of claims 1 to 3.

5. The vector of claim 4, wherein the vector is a plasmid or a viral vector, optionally wherein the vector is selected from retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canarypox virus, herpes virus, minicircle vector and synthetic DNA or RNA.

6. A modified cell transfected or transduced with the nucleic acid sequence of any one of claims 1 to 3 or a vector comprising the nucleic acid sequence of any one of claims 1 to 3.

7. The modified cell of claim 6, wherein the modified cell is selected from the group consisting of CD8 T cells, CD4 T cells, NK cells, NKT cells, γ-δ T cells, hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines, and optionally wherein the modified cell is a human cell.

8. A pharmaceutical composition for treating or preventing ΔNPM1-positive hematological malignancies, comprising: (a) a nucleic acid sequence according to any one of claims 1 to 3, a vector comprising the nucleic acid sequence according to any one of claims 1 to 3, or a modified cell comprising the nucleic acid sequence according to any one of claims 1 to 3, and (b) pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers.

9. A method for treating or preventing a ΔNPM1-positive hematological malignancy in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the hematological malignancy is a myeloid malignancy, and optionally wherein the myeloid malignancy is acute myeloid leukemia.

11. The method of claim 9 or 10, wherein the method induces or enhances a cell-mediated immune response in a subject.

12. The pharmaceutical composition of claim 8, for use in treating or preventing ΔNPM1-positive hematological malignancies in human subjects.

13. The pharmaceutical composition for use of claim 12, wherein the hematological malignancy is a myeloid malignancy, and optionally wherein the myeloid malignancy is acute myeloid leukemia.

14. The pharmaceutical composition for use according to claim 12 or 13, wherein the pharmaceutical composition induces or enhances a cell-mediated immune response in a subject.

15. A method for producing a T cell receptor, comprising contacting the nucleic acid sequence of any one of claims 1 to 3 with a cell, the contact being carried out under conditions where the nucleic acid sequence is incorporated into the cell and expressed in the cell, to produce a T cell receptor that specifically binds to the peptide of SEQ ID NO:

26.

16. The method of claim 15, wherein the method is an ex vivo method.

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