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 having ΔNPM1-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 the treatment of human subjects having ΔNPM1-positive hematological malignancies. Corresponding methods and uses are also provided.

Background Art

[0002] Hematological malignancies (hematopoietic tumors) are cancers that affect the blood and lymphatic systems. This cancer may originate in hematopoietic tissues (e.g., bone marrow) or in cells of the immune system. Examples of hematological malignancies include myeloid malignancies such as acute myeloid leukemia (AML).

[0003] Acute myeloid leukemia is a malignant disease of the bone marrow characterized by the accumulation of myeloid progenitor cells with arrested differentiation. Currently, standard therapy consists of induction chemotherapy followed by high-dose therapy combined with intensive consolidation chemotherapy or autologous or allogeneic hematopoietic stem cell transplantation (alloSCT), which provides a 5-year survival rate of 40-45% in patients under 65 years old, but only a survival rate of 10% in patients over 65 years old. 1~2 AlloSCT is associated with a low relapse rate, but this benefit is limited by high toxicity. Therefore, treatment with alloSCT is limited to patients with good performance status but 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 an urgent need for new targeted therapies that are highly effective and have no or limited toxicity to treat patients with AML and improve their survival. 2

[0004] The molecular characterization of AML has been accelerating over the last few decades. Whole-genome and exome sequencing has revealed that AML has a low mutation burden, with an average of 13 coding mutations per patient. For cancer types with a high mutation burden, such as melanoma and lung cancer, it has been shown that only a small fraction of somatic mutations encode neoantigens. 3 Neoantigens are peptides that arise from tumor-specific DNA mutations and can be recognized by specific T cells when presented on tumor cells in the context of HLA. The formation of these antigens is a stochastic process where each additional mutation increases the chance of generating a neoantigen. Given the low mutation burden in AML, the number of neoantigens is expected to be limited. 3

[0005] Neoantigens can function as in vivo cancer rejection antigens after therapy with checkpoint inhibitors or adoptive transfer of tumor-infiltrating lymphocytes (TIL) expanded in vitro. 3~4 Checkpoint inhibitors are antibodies that block inhibitory signals on T cells mediated by CTLA-4 or PD-1 (ipilimumab for CTLA-4, pembrolizumab and nivolumab for PD-1), thereby stimulating the immune system to target neoantigens. Checkpoint inhibitors and TIL therapy have proven successful for tumors with a high mutation burden but are not effective for tumors with a low mutation burden. However, although the overall mutation burden in AML is low, somatic abnormalities often occur in a limited number of driver genes that are recurrently mutated in multiple patients. 5 As a result, neoantigens arising from recurrent mutations in AML are relevant to the development of targeted immunotherapies.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0007] There is a need for a novel immunotherapy for treating hematological malignancies including myeloid malignancies such as AML.

Means for Solving the Problems

[0008] The inventors recognized that since the formation of a mutant form of nucleophosmin (ΔNPM1 or NPM1 mut ) is limited to malignant hematopoietic cells, this mutant protein is an ideal target for immunotherapy of hematological malignancies such as myeloid malignancies (especially AML).

[0009] Nucleophosmin (NPM1) is a driver gene that frequently mutates in about 30% of patients with AML.5 Mutant NPM1 is also observed in other types of hematological malignancies (such as other myeloid malignancies), but the frequency in tumors other than AML is much lower. Patients with mutant NPM1 (ΔNPM1 or NPM1 mut ) carry a characteristic 4-base pair (4bp) frameshift insertion in exon 12 of the gene. The resulting ΔNPM1 protein is 4 amino acids (AA) longer than the wild-type counterpart, and the 11 AAs at its C-terminus are translated in an alternative reading frame (CLAVEEVSLRK (SEQ ID NO: 27)). As a result, the ΔNPM1 protein translocates from the nucleolus and functions there as a nucleocytoplasmic shuttle protein to the cytoplasm. 6 Thus, the ΔNPM1 protein is localized inside the cell. However, HLA-restricted ΔNPM1-derived peptides are accessible to the T cell receptor on the cell surface and can therefore be recognized by T cells.

[0010] By studying the HLA class I ligandome of primary AML, the inventors have identified five peptides encoded by the alternative reading frame of ΔNPM1 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 (such as vaccines) for treating or preventing ΔNPM1-positive AML 23 . Alternatively, they can be used as target antigens for the treatment of such patients by modified cells (such as peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL)) described herein having a T cell receptor that specifically recognizes one of the above-specified peptides.

[0011] Advantageously, T cells expressing a TCR specific for 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) can be used as an effective immunotherapy in the treatment of ΔNPM1-positive AML. Therefore, a TCR gene transfer approach using a TCR specific for these peptides can provide a novel treatment modality for patients with ΔNPM1-positive AML.

[0012] The inventors have shown for the first time that CLAVEEVSL (SEQ ID NO: 1) is presented on the surface of primary AML cells isolated from HLA-A * 02:01-positive patients with AML. Therefore, advantageously, the above peptides can be used as therapeutic agents (e.g., vaccines) for treating or preventing ΔNPM1-positive AML in HLA-A * 02:01-positive human patients. Alternatively, the peptide can be used as a target antigen for treating such patients by the modified cells (e.g., peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TILs)) described herein that have a T cell receptor that specifically recognizes CLAVEEVSL (SEQ ID NO: 1).

[0013] HLA-A * To examine whether T cells having a T cell receptor (TCR) specific for CLAVEEVSL (SEQ ID NO: 1) presented in the context of HLA-A * 02:01 are present in the T cell repertoire of healthy individuals, an HLA-A * 02:01 tetramer was generated for CLAVEEVSL (SEQ ID NO: 1), and its cysteinylated (cysteinylated) variants and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy individuals. Several tetramer-positive T cell clones were tested, and only two showed specific binding to CLAVEEVSL (SEQ ID NO: 1) and recognition of HLA-A +and CD4 + were introduced into T cells, which demonstrated specific recognition and lysis of HLA-A with ΔNPM1 in a coreceptor (coreceptor) - independent manner * of primary (primary) AML positive for 02:01.

[0014] Therefore, the inventors identified a TCR that specifically binds to the neoantigen CLAVEEVSL (SEQ ID NO: 1).

[0015] Therefore, advantageously, T cells expressing a TCR specific for CLAVEEVSL (SEQ ID NO: 1) can be used as an effective immunotherapy in the treatment of HLA-A * positive patients with ΔNPM1-positive AML. Therefore, the TCR gene transfer approach using the CLAVEEVSL (SEQ ID NO: 1)-specific TCR can provide a novel treatment modality for HLA-A * positive patients with ΔNPM1-positive AML.

[0016] Furthermore, the peptide CLAVEEVSL (SEQ ID NO: 1) (specifically, its cysteinylated form, i.e., C * LAVEEVSL) can be used as a therapeutic agent (e.g., a vaccine) for treating or preventing ΔNPM1-positive AML in HLA-A * positive patients. Therefore, this peptide itself also has utility when formulated, for example, in an isolated form or as a pharmaceutical composition.

[0017] By studying the HLA class I ligandome of primary AML, the inventors also identified distinct nonameric and undecameric peptides encoded by the alternative reading frame of ΔNPM1 (AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27), respectively). Each of AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) of HLA-A * 03:01 and HLA-A *The binding to 11:01 was confirmed by monomer folding for tetramer formation (described in detail herein for the CLAVEEVSL (SEQ ID NO: 1) peptide). Thus, each of the AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) peptides can be used as a therapeutic agent (e.g., a vaccine) for treating or preventing ΔNPM1-positive AML in HLA-A * 03:01 or HLA-A * positive human patients. Alternatively, each of these peptides can be used as a target antigen for treating such patients by the modified cells (e.g., peripheral blood lymphocytes or tumor infiltrating lymphocytes (TILs)) described herein having T cell receptors that specifically recognize AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27), respectively.

[0018] Monomer folding for tetramer formation was also successfully demonstrated for AVEEVSLRK (SEQ ID NO: 26) using HLA-A * 01:01 (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 was confirmed. AVEEVSLRK (SEQ ID NO: 26) was also identified in the HLA class I ligandome from an HLA-A * 03:01 and HLA-A * 11:01-deficient HLA-A * 01:01-positive AML subject (AML4443) (see Figure 2). Thus, the AVEEVSLRK (SEQ ID NO: 26) peptide was also found to be associated with HLA-A *01:01 It can be used as a therapeutic agent (e.g., vaccine) for treating or preventing ΔNPM1-positive AML in positive human patients. Alternatively, this peptide can be used as a target antigen for treating such patients as described herein by modified cells (e.g., peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL)) having a T cell receptor that specifically recognizes AVEEVSLRK (SEQ ID NO: 26).

[0019] HLA-A * 03:01 or HLA-A * 11:01 To examine whether T cells having a T cell receptor (TCR) specific for AVEEVSLRK (SEQ ID NO: 26) presented in relation to HLA-A * 03:01 tetramer and HLA-A * 11:01 tetramers were generated for AVEEVSLRK (SEQ ID NO: 26), and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy individuals. Several tetramer-positive T cell clones were tested, and two were identified as having specific binding to AVEEVSLRK (SEQ ID NO: 26) in relation to HLA-A * 03:01 (reactive clone (3B3) and reactive clone (31.3.F1); FIGS. 37 and 40). Furthermore, two T cell clones were identified as having specific binding to AVEEVSLRK (SEQ ID NO: 26) in relation to HLA-A * 11:01 (reactive clone (6F11) and reactive clone (26.2.D6); FIGS. 37 and 40). The reactivity of each of these clones (6F11, 26.2.D6, 31.3.F1, and 3B3) was also tested (FIGS. 38, 39, and 40), and cytokine release was demonstrated by each clone only when presented with the ΔNPM1 peptide in relation to the appropriate HLA-A.

[0020] Therefore, the inventors have found that four TCRs that specifically bind to the neoantigen AVEEVSLRK (SEQ ID NO: 26) in relation to HLA-A * 03:01 or HLA-A * 11:01* For 03:01, the TCR derived from clone 3B3 and the TCR derived from clone 31.3.F1, HLA-A * For 11:01, the TCR derived from clone 6F11 and the TCR derived from clone 26.2.D6 were identified.

[0021] The T cell receptors of clones 26.2.D6 and 6F11 were sequenced and introduced into CD8 + T cells. These TCR-T cells showed specific binding to the PE-labeled pHLA-A * 11:01-AVEEVSLRK tetramer. In IFN-γ ELISA, the TCR-T cells specifically reacted against HLA-A * 11:01-positive AML.

[0022] The T cell receptor of clone 31.3.F1 was also sequenced and introduced into CD8 + T cells. These TCR-T cells showed specific binding to the PE-labeled pHLA-A * 03:01-AVEEVSLRK tetramer. In IFN-γ ELISA, the TCR-T cells specifically reacted against HLA-A * 03:01-positive AML.

[0023] Therefore, the inventors identified and sequenced three TCRs that specifically bind to AVEEVSLRK (SEQ ID NO: 26). Therefore, advantageously, T cells expressing a TCR specific for AVEEVSLRK (SEQ ID NO: 26) can be used as an effective immunotherapy in the treatment of HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01-positive patients. Therefore, a TCR gene transfer approach using an AVEEVSLRK (SEQ ID NO: 26)-specific TCR can be used for HLA-A * 03:01, HLA-A * 11:01 or HLA-A *01:01 It can bring about a new treatment modality for positive patients. The inventors have found that the TCRs derived from clone 6F11 and 26.2.D6 strongly bind to AVEEVSLRK (SEQ ID NO: 26), and also bind, although to a lesser extent, to 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. Thus, TCRs having "specific binding to AVEEVSLRK (SEQ ID NO: 26)" as described herein include those that bind to AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27), but do not include TCRs that bind only to CLAVEEVSLRK (SEQ ID NO: 27) (the latter TCRs are described as being specific only to CLAVEEVSLRK (SEQ ID NO: 27)).

[0024] Furthermore, the peptide AVEEVSLRK (SEQ ID NO: 26) can be used as a therapeutic agent (e.g., a vaccine) for treating or preventing ΔNPM1-positive AML in HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01 positive patients. Therefore, this peptide itself also has utility when formulated, for example, in isolated form or as a pharmaceutical composition.

[0025] The inventors have shown 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 context of an appropriate HLA (i.e., specific binding to this peptide may occur only when the peptide is presented by an appropriate HLA as described above).

[0026] HLA-A * To examine whether T cells having a T cell receptor (TCR) specific for CLAVEEVSLRK (SEQ ID NO: 27) presented in the context of HLA-A 03:01 are present in the T cell repertoire of healthy individuals, HLA-A * 03:01 tetramers were generated for CLAVEEVSLRK (SEQ ID NO: 27), and their cysteinylated variants and tetramer-positive CD8 T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy individuals. Several tetramer-positive T cell clones were tested, and one was found to have specific binding to C * LAVEEVSLRK in the context of HLA-A 03:01 (reactive clone (1F2); Figure 37). * Identified as having specific binding to C

[0027] Therefore, the inventors have identified a TCR (TCR derived from clone 1F2) that specifically binds to the neoantigen C * LAVEEVSLRK in the context of HLA-A 03:01. * Identified as having specific binding to C

[0028] Therefore, advantageously, T cells expressing a TCR specific for CLAVEEVSLRK (SEQ ID NO: 27) (specifically the cysteinylated form of SEQ ID NO: 27, i.e., C * LAVEEVSLRK) can be used as an effective immunotherapy in the treatment of HLA-A 03:01 or HLA-A * 11:01 positive patients with ΔNPM1-positive AML. Therefore, a TCR gene transfer approach using a CLAVEEVSLRK (SEQ ID NO: 27) (specifically C * LAVEEVSLRK)-specific TCR can provide a novel treatment modality for HLA-A 03:01 and HLA-A * 11:01 positive patients with ΔNPM1-positive AML. * 03:01 and HLA-A * 11:01 positive patients.

[0029] Furthermore, the peptide CLAVEEVSLRK (specifically its cysteinylated form, i.e., C *LAVEEVSLRK) is useful for HLA-A * 03:01 or HLA-A * 11:01 positive patients as a therapeutic agent (e.g., vaccine) for treating or preventing ΔNPM1-positive AML. Therefore, this peptide itself is also useful when formulated, for example, in isolated form or as a pharmaceutical composition.

[0030] The present invention has a specific use in the treatment of 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 equally applicable to patients with ΔNPM1-positive hematological malignancies such as, but not limited to, myeloid malignancies (e.g., AML).

[0031] Accordingly, in one aspect, the present invention provides (a) a polypeptide comprising the CDR3 of a TCR α-chain polypeptide 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), and / or (b) a polypeptide comprising the CDR3 of a TCR β-chain polypeptide 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) and provides an isolated nucleic acid sequence encoding the same.

[0032] The nucleic acid sequence may encode both (a) and (b), and both (a) and (b) 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).

[0033] The encoded polypeptide may specifically bind to CLAVEEVSL (SEQ ID NO: 1). This peptide may be in a cysteinylated form. Therefore, the encoded polypeptide may specifically bind only to C * LAVEEVSL (SEQ ID NO: 1 in cysteinylated form).

[0034] Alternatively, the encoded polypeptide may specifically bind to AVEEVSLRK (SEQ ID NO: 26). Alternatively, the encoded polypeptide may specifically bind to CLAVEEVSLRK (SEQ ID NO: 27). This peptide may be in a cysteinylated form. Therefore, the encoded polypeptide may specifically bind only to C * LAVEEVSLRK (SEQ ID NO: 27 in cysteinylated form).

[0035] In one example, the isolated nucleic acid sequence may include one or more features of the TCR of clone 1A2 described herein.

[0036] For example, the CDR3 of (a) may have an amino acid sequence having at least 90% sequence identity with CAVTGARLMF (SEQ ID NO: 2). Optionally, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0037] (b)'s CDR3 may have an amino acid sequence having at least 90% sequence identity with CASSPGGLSNEQF (SEQ ID NO: 5). Optionally, (b)'s CDR3 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0038] The CDR3 of (a) may be within the variable region of the TCR α-chain that specifically binds to the selected peptide (i.e., SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29).

[0039] (a) may further include the TCR α-chain constant region. In other words, the polypeptide of (a) may include the full-length variable region of the TCR α-chain and the full-length constant region of the TCR α-chain that specifically binds to the selected peptide.

[0040] The variable region of the TCR α-chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8. Optionally, the variable region of the TCR α-chain 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0041] (b) The CDR3 may be within the variable region of the TCR β-chain that specifically binds to the selected peptide (i.e., SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29).

[0042] (b) may further include the TCR β-chain constant region. In other words, the polypeptide of (b) may include the full-length variable region of the TCR β-chain and the full-length constant region of the TCR β-chain that specifically binds to the selected peptide.

[0043] (b) The variable region of the TCR β-chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. Optionally, the variable region of the TCR β-chain 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0044] (a)'s CDR3 may be within the TCR α-chain variable region having at least 90% sequence identity with SEQ ID NO: 8, and this CDR3 has the amino acid sequence of SEQ ID NO: 2. Optionally, (a) includes the TCR α-chain constant region.

[0045] In any of the embodiments described herein, the TCR α-chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 14, and the TCR α-chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 15.

[0046] (b)'s CDR3 may be within the TCR β-chain variable region having at least 90% sequence identity with SEQ ID NO: 11, and this CDR3 has the amino acid sequence of SEQ ID NO: 5. Optionally, (b) includes the TCR β-chain constant region.

[0047] 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. In one example, the isolated nucleic acid sequence may include one or more features of the TCR of clone 26.2.D6 described herein.

[0048] For example, (a)'s CDR3 may have an amino acid sequence having at least 90% sequence identity with CAESKGQNFVF (SEQ ID NO: 35). Optionally, (a)'s CDR3 is encoded by the nucleic acid sequence of SEQ ID NO: 36 or its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0049] (b)'s CDR3 may have an amino acid sequence having at least 90% sequence identity with CASTTWGTGGHEQYF (SEQ ID NO: 43). Optionally, (b)'s CDR3 is encoded by the nucleic acid sequence of SEQ ID NO: 44 or its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0050] The CDR3 of (a) may be within the variable region of the TCR α chain that specifically binds to the selected peptide (e.g., SEQ ID NO: 26).

[0051] (a) may further include the TCR α chain constant region. In other words, the polypeptide of (a) may include the full-length variable region of the TCR α chain and the full-length constant region of the TCR α chain that specifically binds to the selected peptide.

[0052] The variable region of the TCR α chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37. Optionally, the variable region of the TCR α chain of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 38 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0053] (b) The CDR3 may be within the variable region of the TCR β chain that specifically binds to the selected peptide (e.g., SEQ ID NO: 26).

[0054] (b) may further include the TCR β chain constant region. In other words, the polypeptide of (b) may include the full-length variable region of the TCR β chain and the full-length constant region of the TCR β chain that specifically binds to the selected peptide.

[0055] (b) The variable region of the TCR β chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45. Optionally, the variable region of the TCR β chain of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 46 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0056] (a) The CDR3 may be within the variable region of the TCR α chain having at least 90% sequence identity with SEQ ID NO: 37, and this CDR3 has the amino acid sequence of SEQ ID NO: 35. Optionally, (a) includes the TCR α chain constant region.

[0057] In any of the embodiments described herein, 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. The CDR3 of (b) may be within the TCR β-chain variable region having at least 90% sequence identity with SEQ ID NO: 45, and this CDR3 has the amino acid sequence of SEQ ID NO: 43. Optionally, (b) includes a TCR β-chain constant region.

[0058] 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.

[0059] In one example, the isolated nucleic acid sequence may include one or more features of the TCR of clone 6F11 described herein.

[0060] For example, the CDR3 of (a) may have an amino acid sequence having at least 90% sequence identity with CAVSPAGNQFYF (SEQ ID NO: 51). Optionally, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 52 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0061] The CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity with CASSLGSNQPQHF (SEQ ID NO: 59). Optionally, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 60 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0062] The CDR3 of (a) may be within the TCR α-chain variable region that specifically binds to a selected peptide (e.g., SEQ ID NO: 26).

[0063] (a) may further include a TCR α-chain constant region. In other words, the polypeptide of (a) may include a full-length TCR α-chain variable region and a full-length TCR α-chain constant region that specifically binds to the selected peptide.

[0064] The TCR α-chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 53. Optionally, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0065] The CDR3 of (b) may be within the TCR β-chain variable region that specifically binds to the selected peptide (e.g., SEQ ID NO: 26).

[0066] (b) may further include a TCR β-chain constant region. In other words, the polypeptide of (b) may include a full-length TCR β-chain variable region and a full-length TCR β-chain constant region that specifically binds to the selected peptide.

[0067] 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. Optionally, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0068] The CDR3 of (a) may be within the TCR α-chain variable region having at least 90% sequence identity with SEQ ID NO: 53, and this CDR3 has the amino acid sequence of SEQ ID NO: 51. Optionally, (a) includes a TCR α-chain constant region. In any of the embodiments described herein, 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.

[0069] (b)'s CDR3 may be within the TCR β-chain variable region having at least 90% sequence identity with SEQ ID NO: 61, and this CDR3 has the amino acid sequence of SEQ ID NO: 59. Optionally, (b) includes the TCR β-chain constant region.

[0070] 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.

[0071] In one example, the isolated nucleic acid sequence may include one or more features of the TCR of clone 31.3.F1 described herein.

[0072] For example, (a)'s CDR3 may have an amino acid sequence having at least 90% sequence identity with CALSGGGQNFVF (SEQ ID NO: 67). Optionally, (a)'s CDR3 is encoded by the nucleic acid sequence of SEQ ID NO: 68 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). (b)'s CDR3 may have an amino acid sequence having at least 90% sequence identity with CASSQGSGFRHF (SEQ ID NO: 75). Optionally, (b)'s CDR3 is encoded by the nucleic acid sequence of SEQ ID NO: 76 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0073] (a)'s CDR3 may be within the TCR α-chain variable region that specifically binds to a selected peptide (e.g., SEQ ID NO: 26).

[0074] (a) may further include the TCR α-chain constant region. In other words, the polypeptide of (a) may include the full-length TCR α-chain variable region and the full-length TCR α-chain constant region that specifically binds to the selected peptide.

[0075] The variable region of the TCR α chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 69. Optionally, the variable region of the TCR α chain of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 70 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0076] (b)'s CDR3 may be within the variable region of the TCR β chain that specifically binds to the selected peptide (e.g., SEQ ID NO: 26).

[0077] (b) may further include a TCR β chain constant region. In other words, the polypeptide of (b) may include a full-length variable region of the TCR β chain and a full-length constant region of the TCR β chain that specifically binds to the selected peptide.

[0078] (b)'s variable region of the TCR β chain may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 77. Optionally, the variable region of the TCR β chain of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 78 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0079] (a)'s CDR3 may be within the variable region of the TCR α chain having at least 90% sequence identity with SEQ ID NO: 69, and this CDR3 has the amino acid sequence of SEQ ID NO: 67. Optionally, (a) includes a TCR α chain constant region.

[0080] In any of the embodiments described herein, the CDR1 of the variable region of the TCR α chain may have the amino acid sequence of SEQ ID NO: 71, and the CDR2 of the variable region of the TCR α chain may have the amino acid sequence of SEQ ID NO: 73.

[0081] (b)'s CDR3 may be within the variable region of the TCR β chain having at least 90% sequence identity with SEQ ID NO: 77, and this CDR3 has the amino acid sequence of SEQ ID NO: 75. Optionally, (b) includes a TCR β chain constant region.

[0082] 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. The selected peptide CLAVEEVSL (SEQ ID NO: 1) may be cysteinylated.

[0083] The selected peptide CLAVEEVSLRK (SEQ ID NO: 27) may be cysteinylated. The nucleic acid sequence may encode a T cell receptor.

[0084] To avoid misunderstanding, the inventors have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) is presented by HLA-A * 02:01 (i.e., HLA-A * 02:01 restricted). Furthermore, 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 one of these peptides may occur in the context of the appropriate HLA (i.e., specific binding to the peptide may occur only when the peptide is presented by the appropriate HLA as described above).

[0085] The nucleic acid sequences of the present invention may be non-naturally occurring nucleic acid sequences (for example, the nucleic acid sequences of the present invention may be such that the entire sequence does not naturally occur as a whole). For example, the nucleic acid sequences of the present invention may be operably linked to a promoter, which is not naturally associated with a native equivalent human nucleic acid sequence (for example, a human TCR sequence or a fragment thereof), that is, the promoter is not the entire promoter that is naturally associated with the nucleic acid in its natural environment. In this context, such a promoter may be considered an exogenous promoter. Examples of suitable promoters are described elsewhere.

[0086] In a further aspect, the present invention provides a vector comprising the nucleic acid sequence of the present invention. The vector may be a plasmid or a viral vector. Optionally, the vector is selected from the group consisting of 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.

[0087] 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.

[0088] The transfected or transduced nucleic acid sequence of the present invention or the vector of the present invention may be operably linked to a promoter, as described above.

[0089] The modified cell may be 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. The modified cell may be a human cell.

[0090] In a further aspect, the present invention provides (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) and provides an isolated peptide comprising an amino acid sequence selected from the group consisting of.

[0091] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0092] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0093] The peptide may have 20 or fewer amino acids.

[0094] The peptide (i) may be SEQ ID NO: 1, wherein the cysteine amino acid may or may not be cysteinylated, (ii) SEQ ID NO: 26, (iii) may be SEQ ID NO: 27, wherein the cysteine amino acid may or may not be cysteinylated, (iv) SEQ ID NO: 28, and (v) SEQ ID NO: 29 and may consist of a sequence selected from the group consisting of.

[0095] In a specific embodiment, the peptide may consist of the sequence of SEQ ID NO: 1, and the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0096] In a specific embodiment, the peptide may consist of the sequence of SEQ ID NO: 27, and the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0097] In a further aspect, the present invention provides an isolated nucleic acid sequence encoding the peptide of the present invention.

[0098] In a further aspect, the present invention provides a vector comprising the nucleic acid sequence of the present invention.

[0099] In a further aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid sequence of the present invention, the vector of the present invention, the modified cell of the present invention, or the isolated peptide of the present invention, and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0100] When the pharmaceutical composition contains the isolated peptide according to the present invention (or a nucleic acid or vector encoding this isolated peptide), it may be formulated as a vaccine. Suitable vaccine formulations for peptides and nucleic acids are well known in the art.

[0101] In a further aspect, the present invention provides a method for treating or preventing ΔNPM1-positive hematological malignancies in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0102] The method for treating or preventing ΔNPM1-positive hematological malignancies in a human subject may comprise administering to the subject a therapeutically effective amount of the peptide described herein (or a nucleic acid (e.g., RNA or DNA) or vector encoding the peptide).

[0103] By way of example, (i) CLAVEEVSL (SEQ ID NO: 1), where the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), in which the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) An isolated peptide comprising or consisting of an amino acid sequence selected from the above may be administered as an immunotherapy (e.g., as a vaccine).

[0104] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0105] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated. The hematological malignancy may be a myeloid malignancy.

[0106] The myeloid malignancy may be acute myeloid leukemia.

[0107] The method may induce or enhance a cell-mediated immune response in a subject. To avoid misunderstanding, the inventors have identified that the peptide CLAVEEVSL (SEQ ID NO: 1) is restricted by HLA-A * 02:01. Furthermore, the inventors have identified 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 that 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 cysteinylated form.

[0108] Therefore, HLA-A *02:01 A method for treating or preventing ΔNPM1-positive hematological malignancies in a positive human subject may preferably 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 cysteinylated form.

[0109] Similarly, HLA-A * 03:01 or HLA-A * 11:01 A method for treating or preventing ΔNPM1-positive hematological malignancies in a human subject positive for HLA-A may preferably 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).

[0110] Furthermore, HLA-A *For treating or preventing ΔNPM1-positive hematological malignancies in a human subject who is positive for 01:01, a method may preferably 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 in this specification).

[0111] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteinylated form.

[0112] In a further aspect, the present invention provides a pharmaceutical composition of the present invention for use in the treatment or prevention of ΔNPM1-positive hematological malignancies in a human subject.

[0113] The pharmaceutical composition of the present invention for use in the treatment or prevention of ΔNPM1-positive hematological malignancies in a human subject may comprise a therapeutically effective amount of a peptide (or a nucleic acid (e.g., RNA or DNA) or vector encoding such peptide) described herein.

[0114] By way of example, the pharmaceutical composition may (i) CLAVEEVSL (SEQ ID NO: 1), where the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), where the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) and may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from these.

[0115] The pharmaceutical composition may be for use as an immunotherapy (for example, as a vaccine).

[0116] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0117] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0118] The hematological malignancy may be a myeloid malignancy.

[0119] The myeloid malignancy may be acute myeloid leukemia. The pharmaceutical composition may be for use in inducing or enhancing a cell-mediated immune response in a subject.

[0120] As described above, the inventors have identified that the peptide CLAVEEVSL (SEQ ID NO: 1) is restricted by HLA-A * 02:01. Furthermore, the inventors have identified 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 that AVEEVSLRK (SEQ ID NO: 26) is also presented by HLA-A * 01:01.

[0121] CLAVEEVSL (SEQ ID NO: 1) and / or CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteinylated form.

[0122] Therefore, 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 containing 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 containing the sequence CLAVEEVSL (SEQ ID NO: 1) (all of which are described in more detail elsewhere in this specification) in a pharmaceutical composition may be preferentially used when treating or preventing ΔNPM1-positive hematological malignancies in HLA-A * 02:01-positive human subjects. CLAVEEVSL (SEQ ID NO: 1) may specifically be in a cysteinylated form.

[0123] Similarly, 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 containing 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 containing the sequence AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) (all of which are described in more detail elsewhere in this specification) in a pharmaceutical composition may be preferentially used when treating or preventing ΔNPM1-positive hematological malignancies in human subjects that are positive for HLA-A * 03:01 or HLA-A * 11:01.

[0124] Furthermore, 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 containing 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 containing the sequence AVEEVSLRK (SEQ ID NO: 26) (all of which are described in more detail elsewhere in this specification), a pharmaceutical composition may be preferentially used when treating or preventing ΔNPM1-positive hematological malignancies in a human subject who is positive for HLA-A * 01:01.

[0125] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteinylated form.

[0126] In a further aspect, the present invention provides the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in a human subject.

[0127] The pharmaceutical composition may comprise a therapeutically effective amount of a peptide described herein (or a nucleic acid (e.g., RNA or DNA) or vector encoding such peptide).

[0128] By way of example, the pharmaceutical composition may (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) and may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from these.

[0129] The pharmaceutical composition may be for use as an immunotherapy (for example, as a vaccine).

[0130] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0131] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0132] The hematological malignancy may be a myeloid malignancy.

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

[0134] As mentioned elsewhere herein, the inventors have identified that the peptide CLAVEEVSL (SEQ ID NO: 1) is restricted by HLA-A * 02:01. Furthermore, the inventors have identified 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 that 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 cysteinylated form.

[0135] Therefore, 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 an isolated peptide comprising the sequence CLAVEEVSL (SEQ ID NO: 1) (all of which are described in more detail elsewhere herein) is restricted by HLA-A *It may be preferentially used in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in a positive human subject. CLAVEEVSL (SEQ ID NO: 1) may specifically be in a cysteinylated form.

[0136] Similarly, 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 containing such a nucleic acid sequence or vector, or an isolated peptide containing the sequence AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) (all of which are described in more detail elsewhere in this specification) in a pharmaceutical composition may be used preferentially in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in a human subject who is positive for HLA-A * 03:01 or HLA-A * 11:01. Furthermore, 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 containing such a nucleic acid sequence or vector, or an isolated peptide containing the sequence AVEEVSLRK (SEQ ID NO: 26) (all of which are described in more detail elsewhere in this specification) in a pharmaceutical composition may be used preferentially in the manufacture of a medicament for treating or preventing ΔNPM1-positive hematological malignancies in a human subject who is positive for HLA-A * 01:01.

[0137] CLAVEEVSLRK (SEQ ID NO: 27) may specifically be in a cysteinylated form.

[0138] In a further aspect, the invention provides a method of generating a T cell receptor, the method comprising contacting a nucleic acid sequence of the invention with a cell under conditions such that the nucleic acid sequence is incorporated and expressed by the cell to generate a T cell receptor that specifically binds to a peptide selected from SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

[0139] The method may be an ex vivo method.

[0140] As described above, specific binding to any one of CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), and CLAVEEVSLRK (SEQ ID NO: 27) may occur in the context of an appropriate HLA (e.g., specific binding to the peptide may occur only when the peptide is presented by an appropriate HLA, as described above).

[0141] In a further aspect, use of a peptide as a biomarker for ΔNPM1-positive hematological malignancies in a human subject, the peptide being (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) is provided.

[0142] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0143] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated. In a further aspect, the present invention is a method for diagnosing a ΔNPM1-positive hematological malignancy in a human subject, comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is selected from (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated; (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated; (iv) VEEVSLRK (SEQ ID NO: 28); and AVEEVSLR (SEQ ID NO: 29); wherein the presence of the peptide in the sample identifies the subject as having a ΔNPM1-positive hematological malignancy, and the absence of the peptide in the sample identifies the subject as not having a ΔNPM1-positive hematological malignancy.

[0144] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0145] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0146] In a further aspect, the present invention is a method for treating or preventing a ΔNPM1-positive hematological malignancy in a human subject, comprising: (i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is 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), wherein the cysteine amino acid may or may not be cysteinylated; and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention. A method is provided that includes these steps.

[0147] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0148] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0149] The previously provided description regarding each of the peptides and their HLA-restricted properties (specifically, regarding suitable pharmaceutical compositions for use in methods of treating or preventing ΔNPM1-positive hematological malignancies in a subject having a specific HLA status) applies equally in this regard.

[0150] In a further aspect, the present invention is a pharmaceutical composition of the present invention for use in the treatment or prevention of ΔNPM1-positive hematological malignancies in a human subject, wherein the subject is identified as having a ΔNPM1-positive hematological malignancy by the presence of a peptide in a sample isolated from the subject, and the peptide is (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine residue may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine residue may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) and provides a pharmaceutical composition of the present invention selected from the above.

[0151] In this aspect, a subject identified as having a ΔNPM1-positive hematological malignancy is a subject already diagnosed as having a ΔNPM1-positive hematological malignancy prior to treatment due to the presence of a peptide in a sample isolated from the subject, and the peptide is (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine residue may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine residue may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) is selected from.

[0152] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 1 is cysteinylated.

[0153] In a specific embodiment, the cysteine amino acid of SEQ ID NO: 27 is cysteinylated.

[0154] The previous descriptions provided for each of the peptides and their HLA-restricted properties (specifically, regarding pharmaceutical compositions suitable for use in treating or preventing ΔNPM1-positive hematological malignancies in a subject having a specific HLA status) apply equally in this aspect.

[0155] Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variations mean "including but not limited to" and are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0156] Throughout the description and claims of this specification, the singular form includes the plural unless the context requires otherwise. In particular, when an indefinite article is used, the specification should be understood as intending both the singular and the plural unless the context requires otherwise.

[0157] Features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are to be understood as applicable to any other aspect, embodiment or example described herein, unless inconsistent therewith.

[0158] The patents, scientific and technical literature referred to herein establish the knowledge available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications cited herein are hereby incorporated by reference into this specification to the same extent as if each were specifically and individually indicated to be incorporated by reference. In case of any conflict, the present disclosure shall prevail.

[0159] Various aspects of the invention are described in more detail below.

Brief Description of the Drawings

[0160] Embodiments of the invention are described in detail below with reference to the drawings.

[0161]

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Mode for Carrying Out the Invention

[0162] The immunogenicity of peptides derived from the mutant NPM1 (ΔNPM1) has been previously studied 14 and in silico screening of the entire amino acid sequence of ΔNPM1 was used to predict which peptides could be presented by HLA-A * 02:01. Peptides predicted to have HLA-A * 02:01 binding, including CLAVEEVSL (SEQ ID NO: 1), were synthetically generated. CD8 + T cells isolated from healthy individuals and ΔNPM1 AML patients were stimulated with the above peptides and the T cell response was measured. Only two of the peptides tested (AIQDLCLAV (SEQ ID NO: 32) and AIQDLCVAV (SEQ ID NO: 33)) were found to induce an immune response in vitro. Therefore, these peptides were considered to be the most critical epitopes of ΔNPM1 and were thus used in further studies.

[0163] The inventors have now identified five different peptides (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)) present in the HLA class I ligandome of ΔNPM1-positive primary AML.

[0164] The inventors have surprisingly shown that among all possible peptides within ΔNPM1, the CLAVEEVSL (SEQ ID NO: 1) peptide is presented on the surface of primary ΔNPM1 AML cells isolated from HLA-A * 02:01 cancer patients, and furthermore that the peptide is found in cysteinylated form on the surface of isolated primary AML cells.

[0165] The inventors have surprisingly shown 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 HLA-A* It was also found in the present study that it can also be presented by 01:01.

[0166] The inventors isolated and cloned T cell receptors reactive with CLAVEEVSL (SEQ ID NO: 1) from the T cell repertoire of healthy HLA-A * 02:01-positive individuals. The inventors demonstrated that these T cell receptors can be used for genetic manipulation of peripheral blood lymphocytes, and that genetically modified lymphocytes effectively kill HLA-A * 02:01-positive AML with ΔNPM1. Advantageously, these TCRs can be used as an effective immunotherapy in the treatment of HLA-A * 02:01-positive patients with ΔNPM1-positive AML.

[0167] The inventors also isolated T cell clones reactive with CLAVEEVSLRK (SEQ ID NO: 27) (specifically, cysteinylated variants of this peptide) from the T cell repertoire of healthy HLA-A * 03:01-positive individuals. Advantageously, TCRs derived from this clone can be used as an effective immunotherapy in the treatment of HLA-A * 03:01-positive patients with ΔNPM1-positive AML.

[0168] The inventors 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 was isolated from an HLA-A * 11:01-negative (but 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 derived from these clones can be used as an effective immunotherapy in the treatment of HLA-A *03:01 and HLA-A * It can be used as an effective immunotherapy in the treatment of patients positive for 11:01.

[0169] Nucleic acid sequence encoding a TCR polypeptide component 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 may form part of a larger nucleic acid sequence encoding a larger component part of the T cell receptor (e.g., TCR α-chain variable region, TCR β-chain variable region, TCR α-chain, TCR β-chain, etc.). This nucleic acid sequence may 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 that may be separated by a linker sequence that allows for co-expression of two proteins or polypeptides by the same vector). Further details regarding this are provided below.

[0170] When the nucleic acid sequence may encode only a small component of the T cell receptor, for example, the CDR3 domain of the TCR α-chain polypeptide, or only the CDR3 domain of the TCR β-chain polypeptide. Therefore, this nucleic acid sequence may be regarded as a "building block" that provides an essential component of peptide specificity. When the nucleic acid sequence of the present invention is incorporated, a new nucleic acid sequence encoding 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) is generated. Thus, the nucleic acid sequence of the present invention may be incorporated into a separate nucleic acid sequence (e.g., a vector) encoding other elements of the TCR variable chain. Therefore, the nucleic acid sequence of the present invention has utility as an essential component of TCR specificity for the selected ΔNPM1 peptide and can thus be used to generate a nucleic acid sequence encoding a TCR variable region having the antigen-binding activity and specificity necessary to target ΔNPM1-positive AML.

[0171] The T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is involved in recognizing peptides (presented by this molecule) that bind to major histocompatibility complex (MHC) molecules on target cells. The present invention is directed to a nucleic acid sequence encoding a TCR that interacts with AVEEVSLRK (SEQ ID NO: 26) with respect to a specific peptide, for example HLA-A * with respect to 02:01 for CLAVEEVSL (SEQ ID NO: 1); or, respectively, HLA-A * 03:01 and HLA-A * one of AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) with respect to one of 11:01, or HLA-A * 01:01.

[0172] HLA-A * 02:01 is a human leukocyte antigen serotype that is inclusively common within the HLA-A serotype group. HLA-A* The peptide presented to the TCR by 02:01 is described as being "HLA-A * 02:01 restricted".

[0173] 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. The peptide presented to the TCR by HLA-A * 03:01 is described as being "HLA-A * 03:01 restricted". Similarly, the peptide presented to the TCR by HLA-A * 11:01 is described as being "HLA-A * 11:01 restricted". Similarly, the peptide presented to the TCR by HLA-A * 01:01 is described as being "HLA-A * 01:01 restricted".

[0174] The TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively). When the TCR engages a peptide associated with HLA (e.g., HLA-A * 02:01, HLA-A * 03:01 or HLA-A * 11:01), the T cell is activated by signal transduction. The α and β chains of the TCR have highly variable sequences. 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 and is followed by a transmembrane region and a short cytoplasmic tail (cytoplasmic side terminus), while the variable region binds to the peptide / HLA-A complex.

[0175] The variable region of each lock has three hypervariable regions (high-frequency variable regions), also called complementarity-determining regions (CDRs). Thus, the TCR α chain contains CDR1, CDR2, and CDR3, and the TCR β chain also contains (different) CDR1, CDR2, and CDR3. In each of the α and β chains, CDR3 is mainly involved in recognizing the peptide presented by HLA-A.

[0176] In one aspect, the present invention provides an isolated nucleic acid sequence encoding a polypeptide comprising the CDR3 of a TCR α-chain polypeptide 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), and / or a polypeptide comprising the CDR3 of a TCR β-chain polypeptide 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).

[0177] In a specific embodiment, the encoded polypeptide specifically binds to CLAVEEVSL (SEQ ID NO: 1). CLAVEEVSL (SEQ ID NO: 1) may be in a cysteinylated form. The encoded polypeptide may specifically bind only to the cysteinylated form.

[0178] In a specific embodiment, the encoded polypeptide specifically binds to AVEEVSLRK (SEQ ID NO: 26).

[0179] In a specific embodiment, the encoded polypeptide specifically binds to CLAVEEVSLRK (SEQ ID NO: 27). CLAVEEVSLRK (SEQ ID NO: 27) may be in a cysteinylated form. The encoded polypeptide may specifically bind only to the cysteinylated form.

[0180] The nucleic acid sequence may encode (a), (b), or both (a) and (b). Therefore, the nucleic acid sequence encodes at least one polypeptide comprising the CDR3 of a T cell receptor polypeptide, and 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).

[0181] The nucleic acid sequence may include an α-chain CDR3 and a β-chain CDR3, and both the α-chain CDR3 and the β-chain CDR3 specifically bind to a selected peptide.

[0182] Therefore, the nucleic acid sequence encodes the "CDR3 of the TCR α-chain polypeptide" (also referred to herein as the alpha-chain CDR3 or α-chain CDR3) and / or the "CDR3 of the TCR β-chain polypeptide" (also referred to herein as the beta-chain CDR3 or β-chain CDR3).

[0183] The α-chain CDR3 may be the one of SEQ ID NO: 2 or one of the variants described below. Similarly, the β-chain CDR3 may be the one of SEQ ID NO: 5 or one of the variants described below. It should be noted that these specific CDRs have been found by the inventors to specifically bind to the peptide of SEQ ID NO: 1.

[0184] Alternatively, the α-chain CDR3 may be the one of SEQ ID NO: 35 or one of the variants described below. Similarly, the β-chain CDR3 may be the one of SEQ ID NO: 43 or one of the variants described below. It should be noted that these specific CDRs have been found by the inventors to specifically bind to the peptide of SEQ ID NO: 26.

[0185] Alternatively, the α-chain CDR3 may be the one of SEQ ID NO: 51 or one of the variants described below. Similarly, the β-chain CDR3 may be the one of SEQ ID NO: 59 or one of the variants described below. It should be noted that these specific CDR3s have been found by the inventors to specifically bind to the peptide of SEQ ID NO: 26.

[0186] Alternatively, the α-chain CDR3 may be the one of SEQ ID NO: 67 or one of the variants described below. Similarly, the β-chain CDR3 may be the one of SEQ ID NO: 75 or one of the variants described below. It should be noted that these specific CDR3s have been found by the inventors to specifically bind to the peptide of SEQ ID NO: 26.

[0187] (For example, in order to form an appropriate nucleic acid sequence encoding a functional T cell receptor (i.e., encoding a functional TCR α-chain and a functional TCR β-chain which may be separated by a linker sequence allowing co-expression of two proteins or polypeptides by the same vector),) any of the replacement examples described below for (a) may be combined with the replacement examples described below for (b).

[0188] Polypeptide (a) - a component of the TCR α-chain (common to the TCR of clone 1A2 described herein) 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., this 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" includes homologs. Functional variants typically 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.

[0189] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 2 that does not specifically bind to SEQ ID NO: 1. The non-functional variant typically contains non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 2, or premature truncation (shortening), or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0190] 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: 2 while retaining the ability to specifically bind to the peptide of SEQ ID NO: 1. In other words, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 2 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 2).

[0191] In an 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 4 is the codon-optimized version of the nucleic acid sequence for the CDR3 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 3). Accordingly, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0192] In one embodiment, the polypeptide of (a) comprises a CDR3 within the variable region of the TCR α-chain that specifically binds to the peptide of SEQ ID NO: 1 (e.g., the CDR3 of SEQ ID NO: 2 as defined above or a variant thereof). In other words, the polypeptide of (a) may comprise a variable region of the TCR α-chain that contains the specified CDR3, and this variable region of the TCR α-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 1. As will be apparent to those skilled in the art, the phrase "variable region of the TCR α-chain" refers to the variable (V) region (extracellular domain) of the TCR α-chain, and thus includes three hypervariable regions (CDR1, CDR2, and the specific CDR3), as well as intervening sequences, but does not include the constant (C) region of the α-chain, which does not form part of the variable chain. In addition to the specified CDR3, the encoded variable region of the TCR α-chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 14, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 1). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 14. The term "variant" also encompasses homologs. Functional variants typically 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.

[0193] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 14. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0194] In one embodiment, the CDR1 of (a), (e.g., within the α-chain variable region), may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 14 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 14). In an example where the CDR1 of (a), (e.g., within 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 19 is the codon-optimized version of the nucleic acid sequence for the CDR1 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 18). Thus, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0195] The encoded TCR α-chain variable region, in addition to the specified CDR3 (and optionally the specified CDR1 as above), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 15, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 02:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 15. The term "variant" includes homologs. Functional variants typically 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.

[0196] Non-functional variants are HLA-A *It is a variant of the amino acid sequence of SEQ ID NO: 15 that does not specifically bind to 02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 15, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0197] In one embodiment, the CDR2 of (a) (e.g., within the α-chain variable region) is HLA-A * It may have an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 15 while retaining the ability to bind to 02:01. In other words, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 15 are also included. As mentioned above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 15).

[0198] 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: 15, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or its genetically degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 21 is the codon-optimized version of the nucleic acid sequence for the CDR2 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 20). Thus, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or its genetically degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0199] Therefore, the polypeptide of (a) may include a TCR α-chain variable region containing the CDRs (specifically those referred to by SEQ ID NO: or their variants) mentioned in detail above, with appropriate intervening sequences between those CDRs.

[0200] (a) The TCR α-chain variable region may have the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 8. The term "variant" also includes homologs. Functional variants typically 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.

[0201] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 8. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0202] 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 included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 8 may be in regions of the TCR α-chain variable region that do not form CDRs (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 25% or less sequence variability compared to SEQ ID NO: 8). In other words, the sequence of the CDRs of SEQ ID NO: 8 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 8).

[0203] By way of 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 with the amino acid sequence of SEQ ID NO: 8, and this CDR3 has the amino acid sequence of SEQ ID NO: 2. In this example, the TCR α-chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 14, and the TCR α-chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 15.

[0204] 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 may be encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 10 is the codon-optimized version of the nucleic acid sequence for the TCR α-chain variable region of clone 1A2 (the non-optimized sequence is SEQ ID NO: 9). Thus, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0205] To avoid misunderstanding, the polypeptide of (a) may include the TCR α-chain variable region and the TCR α-chain constant region (specified above). Examples of suitable constant regions are encoded by the MP71-TCR-flex retroviral vector by Genscript used herein. However, the present invention is not limited to this particular constant region and encompasses any suitable TCR α-chain constant region. This constant region may be of murine origin, of human origin, or humanized. Methods for identifying or generating suitable constant regions are well known to those skilled in the art and are well within the routine capabilities of those skilled in the art. By way of example only, the constant region may be encoded by or derived from a vector, such as a lentiviral vector, retroviral vector or plasmid vector, in which the constant region of a mouse or human has been pre-cloned, and further an adenoviral vector, adeno-associated viral vector, vaccinia viral vector, canarypox viral vector or herpes viral vector. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors, published in Leukemia 2016 by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. By way of example, a pMSGV retroviral vector having pre-cloned TCR-Ca and Cb genes, described by LV Coren et al. in BioTechniques 2015, may be used to provide an appropriate constant region.

[0206] Polypeptide (b) - a component of the TCR β-chain (common with the TCR of clone 1A2 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 5. The term "variant" includes homologs. Functional variants typically 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.

[0207] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 5 that does not specifically bind to SEQ ID NO: 1. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 5, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0208] In one embodiment, the CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity with 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 5 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 5).

[0209] 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 7 is the codon-optimized version of the nucleic acid sequence for the CDR3 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 6). Thus, 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., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0210] In one embodiment, the polypeptide of (b) comprises a CDR3 within the variable region of the TCR β-chain that specifically binds to the peptide of SEQ ID NO: 1 (e.g., the CDR3 of SEQ ID NO: 5 as defined above or a variant thereof). In other words, the polypeptide of (b) comprises a variable region of the TCR β-chain that contains the specified CDR3, and the variable region of the TCR β-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 1. As will be apparent to those skilled in the art, the phrase "variable region of the TCR β-chain" refers to the variable (V) region (extracellular domain) of the TCR β-chain, and thus includes the three hypervariable regions (CDR1, CDR2, and the specified CDR3) as well as intervening sequences, but does not include the constant (C) region of the β-chain, which does not form part of the variable chain.

[0211] In addition to the specified CDR3, the encoded variable region of the TCR β-chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 16, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 1). 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 typically 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.

[0212] A non-functional variant is a variant of the amino acid sequence of SEQ ID NO: 16 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 1. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 16, or premature truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0213] In one embodiment, the CDR1 of (b) (e.g., within the β-chain variable region) may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 16 are also included. As mentioned above, this amino acid substitution may be a conservative amino acid substitution. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 16). In an example where the CDR1 of (b) (e.g., within the β-chain variable region) 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 a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 23 is the codon-optimized version of the nucleic acid sequence for the CDR1 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 22). Thus, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0214] The encoded TCR β-chain variable region, in addition to the specified CDR3 (and optionally the specified CDR1 as above), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 17, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 02:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 17. The term "variant" includes homologs. Functional variants typically 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.

[0215] Non-functional variants are HLA-A *It is an amino acid sequence variant of SEQ ID NO: 17 that does not specifically bind to 02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 17, or early truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0216] In one embodiment, the CDR2 of (b) (e.g., within the β-chain variable region) is HLA-A * It 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 02:01. In other words, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 17 are also included. As mentioned above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 17). In an example where the CDR2 of (b) (e.g., within the β-chain variable region) 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 a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 25 is the codon-optimized version of the nucleic acid sequence for the CDR2 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 24). Thus, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25 or a genetically degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0217] Therefore, the polypeptide of (b) may include a TCR β-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NO: or variants thereof) mentioned in detail above, with appropriate intervening sequences between those CDRs.

[0218] (b) The TCR β-chain variable region may have the amino acid sequence of SEQ ID NO: 11, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 11. The term "variant" includes homologs. Functional variants typically 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.

[0219] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 11, or premature truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0220] In one embodiment, the TCR β-chain variable region of (b) has 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: 11 while retaining the ability to specifically bind to the peptide of SEQ ID NO: 1. In other words, functional TCR β-chain variable regions having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 11 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 11 may be in regions of the TCR β-chain variable region that do not form CDRs (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 25% or less sequence variability compared to SEQ ID NO: 11). In other words, the sequence of the CDRs of SEQ ID NO: 11 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 11).

[0221] By way of 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, and this CDR3 has the amino acid sequence of SEQ ID NO: 5. In this example, 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.

[0222] 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 may 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 that encode the same protein as a result of the degeneracy of the genetic code). Note that SEQ ID NO: 13 is the codon-optimized version of the nucleic acid sequence for the TCR β-chain variable region of clone 1A2 (the non-optimized sequence is SEQ ID NO: 12). Thus, the polypeptide of (b) may 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 that encode the same protein as a result of the degeneracy of the genetic code).

[0223] To avoid misunderstanding, (b) may include the TCR β-chain variable region and the TCR β-chain constant region (identified above). Examples of suitable constant regions are encoded by the Genscript MP71-TCR-flex retroviral vector used herein. However, the present invention is not limited to this particular constant region and encompasses any suitable TCR β-chain constant region. This constant region may be of murine origin, of human origin, or humanized. Methods for identifying or generating suitable constant regions are well known to those skilled in the art and are well within the routine capabilities of those skilled in the art.

[0224] As a mere example, the constant region may be encoded by, or be derived from, a vector, such as a lentiviral vector, a retroviral vector or a plasmid vector, in which the constant region of a mouse or a human has been pre-cloned, and further an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector or a herpes viral vector. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors, published in Leukemia 2016 by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. As an example, the MP71-TCR-flex retroviral vector having pre-cloned TCR-Ca and Cb genes used by the inventors, or the pMSGV retroviral vector having pre-cloned TCR-Ca and Cb genes described by LV Coren et al., in BioTechniques 2015, may be used to provide an appropriate constant region.

[0225] Alternative embodiments of polypeptide (a) - components of the TCR α chain (common to the TCR of clone 26.2.D6 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 35. The term "variant" includes homologs. 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.

[0226] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 35 that does not specifically bind to SEQ ID NO: 26. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 35, or early truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0227] In one embodiment, the CDR3 of (a) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 35 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 35). In an 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 (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0228] In one embodiment, the polypeptide of (a) comprises a CDR3 within the variable region of the TCR α-chain that specifically binds to the peptide of SEQ ID NO: 26 (e.g., the CDR3 of SEQ ID NO: 35 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a variable region of the TCR α-chain that contains the specified CDR3, and the variable region of the TCR α-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0229] The TCR α-chain variable region to be encoded may, in addition to the specified CDR3, include a CDR1 having the amino acid sequence of SEQ ID NO: 39, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 39. The term "variant" also includes homologs. Functional variants typically 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.

[0230] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 39. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0231] In one embodiment, the CDR1 of (a) (e.g., within the α-chain variable region) may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 39 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage identity can be calculated as the percentage identity to the full length of the reference sequence (e.g., SEQ ID NO: 39). 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, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 40 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0232] The encoded TCR α-chain variable region, in addition to the specified CDR3 (and optionally the above-specified CDR1), has a CDR2 having the amino acid sequence of SEQ ID NO: 41, or a functional variant thereof (i.e., this variant * also retains the ability to specifically bind to HLA-A 11:01). 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 typically 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.

[0233] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions or premature truncations or substitutions, insertions or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 41. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0234] In one embodiment, the CDR2 of (a) (e.g., within the α-chain variable region) may have an amino acid sequence having 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, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 41 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage identity can be calculated as the percentage identity to the full length of the reference sequence (e.g., SEQ ID NO: 41).

[0235] 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, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 42 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0236] Therefore, the polypeptide of (a) may include a TCR α-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NOs, or variants thereof) referred to in detail above, with appropriate intervening sequences between those CDRs.

[0237] The TCR α-chain variable region of (a) may have the amino acid sequence of SEQ ID NO: 37, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 37. The term "variant" also includes homologs. Functional variants typically 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.

[0238] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 37. Methods for identifying functional and non-functional variants are well known to those of skill in the art.

[0239] 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, functional TCR α-chain variable regions having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 37 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 37 may be in regions of the TCR α-chain variable region that do not form CDRs (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 25% or less sequence variability compared to SEQ ID NO: 37). In other words, the sequence of the CDRs of SEQ ID NO: 37 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 37).

[0240] As an example, the polypeptide of (a) may include 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 with the amino acid sequence of SEQ ID NO: 37, and this 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.

[0241] In the example where the variable region of the TCR α-chain of (a) has the amino acid sequence of SEQ ID NO: 37, the variable region of the TCR α-chain may be encoded by the nucleic acid sequence of SEQ ID NO: 38 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0242] To avoid misunderstanding, the polypeptide of (a) may include the variable region of the TCR α-chain (identified above) and the constant region of the TCR α-chain. Examples of suitable constant regions are described in detail elsewhere in this specification and are equally applicable to this embodiment.

[0243] Alternative embodiments of polypeptide polypeptide (b) - components of the TCR β-chain (shared with the TCR of clone 26.2.D6 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 43. The term "variant" includes homologs. 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.

[0244] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 43, or premature truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0245] In one embodiment, the CDR3 of (b) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 43 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 43).

[0246] In an 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 its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0247] In one embodiment, the polypeptide of (b) comprises a CDR3 (e.g., the CDR3 of SEQ ID NO: 43 or a variant thereof as defined above) within the variable region of the TCR β-chain that specifically binds to the peptide of SEQ ID NO: 26. In other words, the polypeptide of (b) comprises a variable region of the TCR β-chain that contains the specified CDR3, and the variable region of the TCR β-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0248] In addition to the specified CDR3, the encoded variable region of the TCR β-chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 47, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 47. The term "variant" also includes homologs. Functional variants typically 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.

[0249] The 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 47, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art. In one embodiment, the CDR1 of (b) (e.g., within the β-chain variable region) may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 47 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 47).

[0250] 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, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 48 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0251] The encoded TCR β-chain variable region, in addition to the specified CDR3 (and optionally the above-specified CDR1), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 49, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 11:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 49. The term "variant" also includes homologs. Functional variants typically 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. The non-functional variant is HLA-A *It is a variant of the amino acid sequence of SEQ ID NO: 49 that does not specifically bind to 11:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 49, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0252] In one embodiment, the CDR2 of (b) (e.g., within the β-chain variable region) is HLA-A * It 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 11:01. In other words, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 49 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 49).

[0253] In the example where the CDR2 of (b) (e.g., within the β-chain variable region) has the amino acid sequence of SEQ ID NO: 49, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 50 or its genetically degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0254] Therefore, the polypeptide of (b) may include a TCR β-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NO) mentioned in detail above, or variants thereof, with appropriate intervening sequences between those CDRs. (b) The TCR β-chain variable region may have the amino acid sequence of SEQ ID NO: 45, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 45. The term "variant" also encompasses homologs. A functional variant typically contains 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.

[0255] 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 typically contains non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 45. Methods for identifying functional and non-functional variants are well known to those of skill in the art.

[0256] In one embodiment, the TCR β-chain variable region of (b) 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, functional TCR β-chain variable regions having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 45 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 45 may be in regions of the TCR β-chain variable region that do not form CDRs (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 25% or less sequence variability as compared to SEQ ID NO: 45). In other words, the sequence of the CDRs of SEQ ID NO: 45 may be retained while the remainder of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 45).

[0257] By way of 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: 45, and this 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.

[0258] In the example where the variable region of the TCR β-chain in (b) has the amino acid sequence of SEQ ID NO: 45, the variable region of the TCR β-chain may be encoded by the nucleic acid sequence of SEQ ID NO: 46 or its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0259] To avoid misunderstanding, (b) may include the variable region of the TCR β-chain (identified above) and the constant region of the TCR β-chain. Examples of suitable constant regions are provided elsewhere in this specification and are equally applicable to this embodiment.

[0260] Alternative embodiments of polypeptide (a) - Components of the TCR α-chain (shared with the TCR of clone 6F11 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 51. The term "variant" includes homologs. 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.

[0261] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 51, or premature truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0262] In one embodiment, the CDR3 of (a) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 51 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 51). In an 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 (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0263] In one embodiment, the polypeptide of (a) comprises a CDR3 within the variable region of the TCR α-chain that specifically binds to the peptide of SEQ ID NO: 26 (e.g., the CDR3 of SEQ ID NO: 51 or a variant thereof as defined above). In other words, the polypeptide of (a) may comprise a variable region of the TCR α-chain that contains the specified CDR3, and the variable region of the TCR α-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0264] The encoded variable region of the TCR α-chain may, in addition to the specified CDR3, include a CDR1 having the amino acid sequence of SEQ ID NO: 55, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 55. The term "variant" includes homologs. Functional variants typically 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.

[0265] The 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 55, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0266] 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 55 are also included. As mentioned above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 55).

[0267] 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, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 56 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0268] The encoded TCR α-chain variable region, in addition to the specified CDR3 (and optionally the specified CDR1 as described above), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 57, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 11:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 57. The term "variant" also includes homologs. Functional variants typically 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.

[0269] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 57 that does not specifically bind to HLA-A * 11:01. The non-functional variant typically contains non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 57, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0270] In one embodiment, the CDR2 of (a) (e.g., within the α-chain variable region) may have an amino acid sequence having 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, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 57 are also included. As mentioned above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 57).

[0271] 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, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 58 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0272] Therefore, the polypeptide of (a) may include a TCR α-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NO) mentioned in detail above, or variants thereof, with appropriate intervening sequences between those CDRs.

[0273] (a) The TCR α-chain variable region may have the amino acid sequence of SEQ ID NO: 53, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 53. The term "variant" includes homologs. Functional variants typically 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. 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 53. Methods for identifying functional and non-functional variants are well known to those of skill in the art.

[0274] 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: 53 while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, functional TCR α-chain variable regions having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 53 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 53 may be in regions of the TCR α-chain variable region that do not form CDRs (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 25% or less sequence variability as compared to SEQ ID NO: 53). In other words, the sequence of the CDRs of SEQ ID NO: 53 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 53).

[0275] By way of 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 with the amino acid sequence of SEQ ID NO: 53, and this 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.

[0276] 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 may be encoded by the nucleic acid sequence of SEQ ID NO: 54 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). To avoid misunderstanding, the polypeptide of (a) may include the TCR α-chain variable region (identified above) and the TCR α-chain constant region. Examples of suitable constant regions are described in detail elsewhere in this specification and are equally applicable to this embodiment.

[0277] Alternative embodiments of polypeptide polypeptide (b) - Components of the TCR β-chain (shared with the TCR of clone 6F11 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 59. The term "variant" includes homologs. Functional variants typically 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.

[0278] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 59, or premature truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0279] In one embodiment, the CDR3 of (b) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 59 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 59).

[0280] In an 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 its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0281] In one embodiment, the polypeptide of (b) comprises a CDR3 within the variable region of the TCR β chain that specifically binds to the peptide of SEQ ID NO: 26 (e.g., the CDR3 of SEQ ID NO: 59 or a variant thereof as defined above). In other words, the polypeptide of (b) comprises a variable region of the TCR β chain that contains the specified CDR3, and the variable region of the TCR β chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0282] In addition to the specified CDR3, the encoded variable region of the TCR β chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 63, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 63. The term "variant" includes homologs. Functional variants typically 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.

[0283] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 63, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0284] In one embodiment, the CDR1 of (b) (e.g., within the β-chain variable region) may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 63 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 63).

[0285] In an example where the CDR1 of (b) (e.g., within the β-chain variable region) has the amino acid sequence of SEQ ID NO: 63, the CDR1 may 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 as a result of the degeneracy of the genetic code).

[0286] The encoded TCR β-chain variable region, in addition to the specified CDR3 (and optionally the specified CDR1 as above), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 65, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 11:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 65. The term "variant" includes homologs. Functional variants typically 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.

[0287] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 65 that does not specifically bind to HLA-A * 11:01. The non-functional variant typically contains non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 65, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0288] In one embodiment, the CDR2 of (b) (e.g., within the β-chain variable region) may have an amino acid sequence having 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, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 65 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage identity can be calculated as the percentage identity to the full length of the reference sequence (e.g., SEQ ID NO: 65).

[0289] In an example where the CDR2 of (b) (e.g., within the β-chain variable region) has the amino acid sequence of SEQ ID NO: 65, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 66 or its genetically degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0290] Therefore, the polypeptide of (b) may include a TCR β-chain variable region containing the CDRs (specifically those referred to by SEQ ID NO: or variants thereof) detailed above, with appropriate intervening sequences between those CDRs. (b) The TCR β-chain variable region may have the amino acid sequence of SEQ ID NO: 61, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 61. The term "variant" also includes homologs. Functional variants typically 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.

[0291] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 61. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0292] In one embodiment, the TCR β-chain variable region of (b) has 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: 61 while retaining the ability to specifically bind to the peptide of SEQ ID NO: 26. In other words, functional TCR β-chain variable regions having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 61 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 61 may be in regions of the TCR β-chain variable region that do not form CDRs (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 25% or less sequence variability as compared to SEQ ID NO: 61). In other words, the sequence of the CDRs of SEQ ID NO: 61 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 61).

[0293] By way of 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: 61, and this 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.

[0294] In the example where the variable region of the TCR β-chain in (b) has the amino acid sequence of SEQ ID NO: 61, the variable region of the TCR β-chain may be encoded by the nucleic acid sequence of SEQ ID NO: 62 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0295] To avoid misunderstanding, (b) may include the variable region of the TCR β-chain (identified above) and the constant region of the TCR β-chain. Examples of suitable constant regions are provided elsewhere in this specification and are equally applicable to this embodiment.

[0296] Alternative embodiments of polypeptide (a) - components of the TCR α-chain (shared with the TCR of clone 31.3.F1 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 67. The term "variant" includes homologs. 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. 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 67. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0297] In one embodiment, the CDR3 of (a) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 67 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 67).

[0298] In an 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 its genetically degenerate sequence (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0299] In one embodiment, the polypeptide of (a) includes a CDR3 within the variable region of the TCR α chain that specifically binds to the peptide of SEQ ID NO: 26 (e.g., the CDR3 of SEQ ID NO: 67 or a variant thereof as defined above). In other words, the polypeptide of (a) may include a variable region of the TCR α chain that includes the specified CDR3, and the variable region of the TCR α chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0300] In addition to the specified CDR3, the encoded variable region of the TCR α chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 71, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 71. The term "variant" also includes homologs. Functional variants typically 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.

[0301] The 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. The non-functional variant typically contains non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 71, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0302] In one embodiment, the CDR1 of (a) (e.g., within the α-chain variable region) may have an amino acid sequence having 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, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 71 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 71).

[0303] In an example where the CDR1 of (a) (e.g., within the α-chain variable region) has the amino acid sequence of SEQ ID NO: 71, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 72 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0304] The encoded TCR α-chain variable region, in addition to the specified CDR3 (and optionally the above-specified CDR1), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 73, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 03:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 73. The term "variant" includes homologs. Functional variants typically 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.

[0305] The non-functional variant is an amino acid sequence variant of SEQ ID NO: 73 that does not specifically bind to HLA-A * 03:01. The non-functional variant typically contains non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 73, or early truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art. In one embodiment, the CDR2 of (a) (e.g., within the α-chain variable region) may have an amino acid sequence having 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, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 73 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 73). In an example where the CDR2 of (a) (e.g., within the α-chain variable region) has the amino acid sequence of SEQ ID NO: 73, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 74 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code). Therefore, the polypeptide of (a) may include a TCR α-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NO: or variants thereof) detailed above, with appropriate intervening sequences between those CDRs.

[0306] (a) The TCR α-chain variable region may have the amino acid sequence of SEQ ID NO: 69, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 69. The term "variant" also includes homologs. Functional variants typically 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.

[0307] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 69. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0308] 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, functional TCR α-chain variable regions having one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 69 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 69 may be in regions of the TCR α-chain variable region that do not form CDRs (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 25% or less sequence variability as compared to SEQ ID NO: 69). In other words, the sequence of the CDR of SEQ ID NO: 53 may be retained while the rest of the sequence may vary within the "at least 75% identity" parameter specified above as needed. Suitably, the percentage identity can be calculated as the percentage identity to the full length of the reference sequence (e.g., SEQ ID NO: 69).

[0309] As an example, the polypeptide of (a) may include a 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: 69, and this 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.

[0310] In the example where the variable region of the TCR α-chain in (a) has the amino acid sequence of SEQ ID NO: 69, the variable region of the TCR α-chain may be encoded by the nucleic acid sequence of SEQ ID NO: 70 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0311] To avoid misunderstanding, the polypeptide in (a) may include the variable region of the TCR α-chain (specified above) and the constant region of the TCR α-chain. Examples of suitable constant regions are described in detail elsewhere in this specification and are equally applicable to this embodiment.

[0312] Alternative embodiments of polypeptide polypeptide (b) - components of the TCR β-chain (shared with the TCR of clone 31.3.F1 described herein) 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., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 75. The term "variant" also encompasses homologs. 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.

[0313] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 75. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0314] In one embodiment, the CDR3 of (b) may have an amino acid sequence having 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, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 75 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 75).

[0315] In an 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 (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0316] In one embodiment, the polypeptide of (b) comprises a CDR3 (e.g., the CDR3 of SEQ ID NO: 75 or a variant thereof as defined above) within the variable region of the TCR β-chain that specifically binds to the peptide of SEQ ID NO: 26. In other words, the polypeptide of (b) comprises a variable region of the TCR β-chain that contains the specified CDR3, and the variable region of the TCR β-chain (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 26.

[0317] In addition to the specified CDR3, the encoded variable region of the TCR β-chain may include a CDR1 having the amino acid sequence of SEQ ID NO: 79, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 79. The term "variant" also includes homologs. Functional variants typically 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.

[0318] The 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 79, or early truncations or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art. In one embodiment, the CDR1 of (b) (e.g., within the β-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: 79 while retaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 26. In other words, functional CDR1s having one amino acid substitution compared to the sequence of SEQ ID NO: 79 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 79).

[0319] In an example where the CDR1 of (b) (e.g., within the β-chain variable region) has the amino acid sequence of SEQ ID NO: 79, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO: 80 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). The encoded TCR β-chain variable region, in addition to the specified CDR3 (and optionally the specified CDR1 above), may also include a CDR2 having the amino acid sequence of SEQ ID NO: 81, or a functional variant thereof (i.e., this variant retains the ability to specifically bind to HLA-A * 03:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 81. The term "variant" includes homologs. Functional variants typically 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.

[0320] The non-functional variant is HLA-A *It is a variant of the amino acid sequence of SEQ ID NO: 81 that does not specifically bind to 03:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 81, or early truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0321] In one embodiment, the CDR2 of (b) (e.g., within the β-chain variable region) is HLA-A * It may have an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 81 while retaining the ability to bind to 03:01. In other words, functional CDR2s having one amino acid substitution compared to the sequence of SEQ ID NO: 81 are also included. As described above, this amino acid substitution may be a conservative amino acid substitution. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 81).

[0322] In an example where the CDR2 of (b) (e.g., within the β-chain variable region) has the amino acid sequence of SEQ ID NO: 81, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO: 82 or its genetically degenerate sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0323] Therefore, the polypeptide of (b) may include a TCR β-chain variable region that includes the CDRs (specifically those referred to by SEQ ID NO: or variants thereof) mentioned in detail above, with appropriate intervening sequences between those CDRs.

[0324] (b)'s TCR β-chain variable region may have the amino acid sequence of SEQ ID NO: 77 or may be a functional variant thereof (i.e., this variant retains the ability to specifically bind to the peptide of SEQ ID NO: 26). Such a functional variant may be a naturally occurring, synthetic, or synthetically improved functional variant of SEQ ID NO: 77. The term "variant" includes homologs. Functional variants typically 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.

[0325] 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. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions of the amino acid sequence of SEQ ID NO: 77. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0326] In one embodiment, the TCR β-chain variable region of (b) has 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, functional TCR β-chain variable regions having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 77 are also included. As described above, such amino acid substitutions may be conservative amino acid substitutions. All variations in the sequence compared to SEQ ID NO: 77 may be in regions of the TCR β-chain variable region that do not form CDRs (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 25% or less sequence variability compared to SEQ ID NO: 77). In other words, the sequence of the CDRs of SEQ ID NO: 77 may be retained while the rest of the sequence varies within the "at least 75% identity" parameter specified above as needed. Suitably, the percent identity can be calculated as the percent identity to the full length of the reference sequence (e.g., SEQ ID NO: 77).

[0327] By way of 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: 77, and this 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. In an example where the TCR β-chain variable region of (b) has the amino acid sequence of SEQ ID NO: 77, the TCR β-chain variable region may be encoded by the nucleic acid sequence of SEQ ID NO: 78 or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0328] To avoid misunderstanding, (b) may include the TCR β-chain variable region and the TCR β-chain constant region (identified above). Examples of suitable constant regions are provided elsewhere in this specification and are equally applicable to this embodiment.

[0329] In an example where the nucleic acid molecule of the present invention encodes both (a) and (b), the polypeptide of (a) may be linked to the polypeptide of (b) via a linker, for example, a linker that enables the expression of two proteins or polypeptides by the same vector. As an example, a linker containing a porcine teschovirus-1 2A (P2A) sequence, such as a 2A sequence derived from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A) or Thosea asigna virus (T2A), or a 2A-like sequence, as published by A. L. Szymczak et al., Nature Biotechnology 22, 589-594 (2004), may be used. 2A sequences and 2A-like sequences are linkers that are cleavable when the nucleic acid molecule is transcribed and translated. Another example of a linker is an internal ribosomal entry site (IRES) that enables the translation of two proteins or polypeptides by the same transcript. Any other suitable linker may also be used. Identification of suitable linkers is well within the routine capabilities of those skilled in the art. As a further example, the nucleic acid sequence encoding (a) and the nucleic acid sequence encoding (b) may be cloned into a vector having a dual internal promoter (see, for example, S Jones et al., Human Gene Ther 2009).

[0330] Additional suitable polypeptide domains may also be encoded by the nucleic acid sequences of the present invention. By way of mere example, the nucleic acid sequences may include membrane targeting sequences that provide for the transport of the encoded polypeptide to the cell surface membrane of the modified cell. Other suitable additional domains are well known and are described, for example, in WO 2016 / 071758 pamphlet.

[0331] In one embodiment, the nucleic acid sequence of the present invention may encode a soluble TCR. For example, the nucleic acid sequence may encode (a) and (b), which are, respectively, the variable regions of the TCR α and β chains, and optionally, an immune modulator molecule, such as a CD3 agonist (e.g., anti-CD3 scFv). The CD3 antigen is present on mature human T cells, thymocytes, and a subset of natural killer cells. It is associated with the TCR and is involved in TCR signaling. Antibodies specific for the human CD3 antigen are well known. One such antibody is the mouse monoclonal antibody OKT3, which was the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see, for example, WO 2004 / 106380 pamphlet, US 2004 / 0202657 A1, US 6,750,325 B2). Immune mobilising mTCR Against Cancer (ImmTAC; Immunocore Limited, Milton Partk, Abington, Oxon, UK) is a bifunctional protein that combines 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 may be combined with a radioisotope or a toxic drug. Suitable radioisotopes and / or toxic drugs are well known in the art and can be readily identified by those skilled in the art.

[0332] In one embodiment, the nucleic acid sequence of the invention may encode a chimeric single-chain TCR in which the polypeptide of (a) (e.g., the variable region of the TCR α-chain) is linked to a constant region fused to the polypeptide of (b) (e.g., the variable region of the TCR β-chain) and, for example, the CD3ζ signaling domain. In this example, the linker is not cleavable. In an alternative embodiment, the nucleic acid sequence of the invention may encode a chimeric double-chain TCR in which the polypeptide of (a) (e.g., the variable region of the TCR α-chain) and the polypeptide of (b) (e.g., the variable region of the TCR β-chain) are each linked to the CD3ζ 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.

[0333] The invention also provides an isolated nucleic acid sequence encoding a peptide (and corresponding vector) of the invention. All of the general descriptions herein regarding nucleic acid sequences and vectors apply equally. One of ordinary skill in the art will readily identify appropriate nucleic acid sequences and vectors based on the peptide sequences provided herein.

[0334] Vectors and Modified Cells In one aspect, the invention provides a vector comprising the nucleic acid sequence described herein. Any suitable vector can be used. By way of mere example, the vector may be a plasmid vector or a viral vector, such as a retroviral vector or a lentiviral vector. Adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, canarypox viral vectors, herpes viral vectors, minicircle vectors, and naked (synthetic) DNA / RNA may also be used (for details regarding minicircle vectors, see, for example, Non-viral Sleeping Beauty transposition from minicircle vectors, published in Leukemia 2016 by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek). Optionally, the vector comprises a nucleic acid sequence operably linked to a promoter.

[0335] As used herein, the term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid sequence operably linked thereto. The vector can be capable of autonomous replication or can be integrated into the host DNA. The vector may contain restriction enzyme sites for the insertion of recombinant DNA and may contain one or more selectable markers or suicide genes. The vector may be a nucleic acid sequence in the form of a plasmid, bacteriophage or cosmid. Preferably, the vector is suitable for expression in cells (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 certain embodiments, 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 the group consisting of an adenoviral vector, a vaccinia viral vector, a canarypox viral vector, a herpes viral vector, a minicircle vector and synthetic DNA or synthetic RNA.

[0336] Preferably, the (expression) vector can grow in the host cell and be stably transmitted to future generations.

[0337] "Operably linked" as used herein refers to the sole or combined presence of the following control elements with a coding sequence that are in a functional relationship with each other, e.g., linked so as to direct the expression of the coding sequence.

[0338] The vector may contain a regulatory sequence. As used herein, "regulatory sequence" refers to a DNA element or an RNA element capable of controlling 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, and the like. Optionally, the vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. The regulatory sequences include those that direct constitutive expression, as well as tissue-specific regulatory sequences and / or inducible sequences.

[0339] The vector may contain a promoter. As used herein, "promoter" refers to a nucleotide sequence in DNA to which RNA polymerase binds to initiate transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or a stimulatory protein. The promoter may be a promoter not naturally found in the host cell (e.g., it may be an exogenous promoter). Those skilled in the art are well aware of suitable promoters for use in expressing a target protein, and the promoter selected depends on the host cell.

[0340] The vector may contain a transcription terminator. As used herein, "transcription terminator" refers to a DNA element that terminates the function of RNA polymerase responsible for transcribing DNA into RNA. Preferred transcription terminators are characterized by a stretch of T residues preceded by a GC rich dyad symmetrical region.

[0341] The vector may contain a translation control element. As used herein, a "translation control element" refers to a DNA element or an RNA element that controls the translation of mRNA. A preferred translation control element is a ribosome binding site. Preferably, the translation control element is homologous to a promoter, for example, derived from a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known and depend on the host cell to be selected.

[0342] The vector may contain a restriction enzyme recognition site. As used herein, a "restriction enzyme recognition site" refers to a motif on DNA that is recognized by a restriction enzyme. The vector may contain a selectable marker. As used herein, a "selectable marker" refers to a protein that, when expressed in a host cell, confers on the cell a phenotype that allows the selection of cells expressing the selectable marker gene. Generally, this may be a protein that confers a new beneficial property (e.g., antibiotic resistance) on the host cell, or a protein that is expressed on the cell surface and thus available for antibody binding. Suitable selectable markers are well known in the art.

[0343] Optionally, the vector may also contain a suicide gene. As used herein, "suicide gene" refers to a protein that induces the death of modified cells when treated with a specific drug. By way of example, the suicide of cells modified by the herpes simplex virus thymidine kinase gene can be induced by treatment with specific nucleoside analogs including ganciclovir, the suicide of cells modified by human CD20 can be induced by treatment with anti-CD20 monoclonal antibodies, and the suicide of cells modified by inducible caspase 9 (iCasp9) can be induced by treatment with AP1903 (reviewed by BS Jones, LS Lamb, F Goldman, A Di Stasi; Improving the safety of cell therapy products by suicide gene transfer, Front Pharmacol. (2014) 5:254). Suitable suicide genes are well known in the art.

[0344] Preferably, the vector contains the genetic elements necessary for the expression of the polypeptides described herein by the host cell. Elements necessary for transcription and translation in the host cell include a promoter, the coding region of the protein of interest, and a transcription terminator.

[0345] Those skilled in the art are well aware of the molecular techniques available for preparing (expression) vectors and how the (expression) vectors may be transduced or transfected into a suitable host cell (thereby generating the modified cells of the present invention). The (expression) vectors of the present 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 thus include methods such as electroporation, microinjection, gene gun delivery, transduction by retroviral vectors, lentiviral vectors or adeno-associated vectors, lipofection, superfection, etc. The specific method used typically depends on both the type of vector and the cell. Suitable methods for introducing nucleic acid sequences and vectors into host cells such as 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, New York; Ausubel et al. (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al. (1988) Mol. Microbiol. 2, 637-646. Further conventional methods suitable for preparing expression vectors and introducing them into suitable host cells are described in detail, for example, in WO 2016 / 071758 pamphlet.

[0346] It is understood that in some embodiments, the host cell is contacted with the vector (e.g., a viral vector) in vitro or ex vivo, and in some embodiments, the host cell is contacted with the vector (e.g., a viral vector) in vivo. The term "host cell" includes any cell into which a nucleic acid sequence or vector of the cells of the invention may be introduced (e.g., transduced). When a nucleic acid molecule or vector is introduced into a cell, that cell may be referred to herein as a "modified cell." When a nucleic acid molecule or vector is introduced into a host cell, the resulting modified cell should be able to express the encoded polypeptide (and, for example, accurately localize the encoded polypeptide for its intended function, e.g., transport the encoded TCR to the cell surface).

[0347] The term "modified cell" refers to a cell that has been genetically modified (e.g., transformed or transfected). This term refers to a particular target cell and also to the progeny or potential progeny of such a cell. Because certain modifications can occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term as used herein.

[0348] Host cells (and thus modified cells) are typically eukaryotic cells, particularly human cells (e.g., CD8 + T cells or CD4 + T cells such as T cells, or mixtures thereof). Host cells (and thus modified cells) may be autologous cells (e.g., autologous CD8 + T cells or CD4 + T cells such as T cells, or mixtures thereof), where such autologous cells refer to cells derived from the same individual to be administered later. In other words, host cells (and thus modified cells) may be isolated T cells from the subject to be treated. Suitably, host cells (and thus modified cells) may be isolated from a blood sample, for example, by leukapheresis (leukocyte removal).

[0349] The host cell (and thus the modified cell) may be any cell capable of conferring anti-tumor immunity after TCR gene transfer. Non-limiting examples of suitable cells include autologous or allogeneic natural killer (NK) cells, NKT cells, γ-δ T cells, hematopoietic stem cells or other progenitor cells, and any other autologous or allogeneic cell or cell line capable of conferring anti-tumor immunity after TCR gene transfer (e.g., NK-92 or a T cell line).

[0350] Advantageously, the modified cell is capable of expressing a polypeptide encoded by the nucleic acid sequence or vector of the present invention (e.g., a TCR or a TCR component part), such that the modified cell provides an immunotherapy that specifically targets ΔNPM1 malignant cells and can thus be used to treat or prevent hematological malignancies having ΔNPM1. Further details regarding this use are provided below.

[0351] Immunogenic peptide The inventors have identified five peptides present in the HLA class I ligandome 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).

[0352] To avoid misunderstanding, unless otherwise specified, general references herein to "SEQ ID NO: 1" include both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSL (SEQ ID NO: 1).

[0353] With respect to the 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 cysteinylated.

[0354] In a specific embodiment, the cysteine amino acid in CLAVEEVSL (SEQ ID NO: 1) is cysteinylated.

[0355] To avoid misunderstanding, unless otherwise specified, general references herein to "SEQ ID NO: 27" include both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSLRK (SEQ ID NO: 27).

[0356] With respect to 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 cysteinylated. In a specific embodiment, the cysteine amino acid in CLAVEEVSLRK (SEQ ID NO: 27) is cysteinylated.

[0357] Therefore, the present invention provides an isolated peptide comprising an amino acid sequence selected from (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated; (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated; (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29).

[0358] In a specific embodiment, the cysteine amino acid in CLAVEEVSL (SEQ ID NO: 1) or CLAVEEVSLRK (SEQ ID NO: 27) is cysteinylated.

[0359] As used herein, "isolated peptide" refers to a peptide that is not in its natural environment. Therefore, this peptide may be of synthetic origin (alternatively, it may be of natural origin but isolated from its natural environment).

[0360] The isolated peptide may be relatively short (i.e., 20 or fewer amino acids; for example, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer amino acids). The peptide may consist of only the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 26, which may or may not be cysteinylated at the cysteine amino acid, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29, which may or may not be cysteinylated at the cysteine amino acid. In a specific embodiment, the cysteine amino acid in CLAVEEVSL (SEQ ID NO: 1) or CLAVEEVSLRK (SEQ ID NO: 27) is cysteinylated.

[0361] The isolated peptide may be administered to a human subject for treating or preventing ΔNPM1-positive hematologic malignancies. For example, the isolated peptide may be administered to the subject to induce or enhance the subject's immune response. Therefore, the peptide may be administered to the subject to induce T cell activation (e.g., in vivo T cell activation) in the subject, and the activated T cells are specific for this peptide (and thus specifically target ΔNPM1-positive malignant cells).

[0362] The isolated peptide may be administered as a peptide vaccine for treating or preventing ΔNPM1-positive AML. The isolated peptide may be administered to induce or enhance the activation of T cells specific for ΔNPM1-positive malignant cells. The inventors have shown 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 are presented in vivo to the T cell repertoire of ΔNPM1-positive subjects. The binding of these peptides to T cells has been demonstrated herein. Therefore, these peptides represent true immunogenic ΔNPM1-specific antigens that may be further utilized in the development of personalized vaccines, which may be particularly useful as adjuvants 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 (see reference 23 for a suitable review).

[0363] The inventors have also shown that (iv) VEEVSLRK (SEQ ID NO: 28) and (v) AVEEVSLR (SEQ ID NO: 29) are also HLA-binding peptides presented on the surface of primary AML.

[0364] Therefore, an isolated peptide comprising an amino acid sequence selected from (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine amino acid may or may not be cysteinylated, (ii) AVEEVSLRK (SEQ ID NO: 26), (iii) CLAVEEVSLRK (SEQ ID NO: 27), wherein the cysteine amino acid may or may not be cysteinylated, (iv) VEEVSLRK (SEQ ID NO: 28), and (v) AVEEVSLR (SEQ ID NO: 29) may be useful as an immunotherapy. For example, such an isolated peptide may be used as an immunotherapy for a subject having, at risk of developing, or suspected of having ΔNPM1-positive AML. Nucleic acid sequences and vectors encoding these peptides may also be useful for this purpose.

[0365] Specific peptides for administration may be selected based on the HLA-A status of the subject. As described elsewhere herein, peptides comprising the sequence of SEQ ID NO: 1 may be particularly suitable for administration to subjects positive for HLA-A * 02:01, whereas peptides comprising the sequence of SEQ ID NO: 26 or SEQ ID NO: 27 may be particularly suitable for administration to subjects positive for HLA-A * 03:01 or HLA-A * 11:01.

[0366] The isolated peptides of the invention may also be provided in a composition comprising a plurality of the peptides discussed above. By way of example, the isolated peptide may 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 cysteinylated, 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 not cysteinylated. This composition can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), and the activated T cells have a TCR specific for one (or both) of the cysteinylated and / or non-cysteinylated forms of the peptide of SEQ ID NO: 1, so this composition may be particularly useful for treating or preventing ΔNPM1-positive hematologic malignancies in subjects positive for HLA-A * 02:01. As an alternative, a peptide composition may be provided comprising 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. This composition is HLA-A * 03:01 or HLA-A *It may be particularly useful for treating or preventing ΔNPM1-positive hematological malignancies in subjects who are positive for 11:01, because both of these peptides are presented by any one of these HLA-A serotypes.

[0367] Similar to the peptide CLAVEEVSL (SEQ ID NO: 1), the peptide of SEQ ID NO: 27 can also be used for vaccination in its cysteinylated and non-cysteinylated forms. Therefore, as an alternative, the isolated peptide may 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 cysteinylated, and (b) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27, wherein the cysteine amino acid is not cysteinylated. This composition can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), and the activated T cells have a TCR specific for one (or both) of the cysteinylated and / or non-cysteinylated forms of the peptide of SEQ ID NO: 27. Therefore, this composition is HLA-A * 03:01 or HLA-A * It may be particularly useful for treating or preventing ΔNPM1-positive hematological malignancies in subjects who are positive for 11:01.

[0368] Pharmaceutical composition The nucleic acid sequences, vectors, modified cells, isolated proteins or peptides described herein may be provided as part of a pharmaceutical composition. Advantageously, such a composition may be administered to a human subject having a ΔNPM1-positive hematological malignancy for treating or preventing a ΔNPM1-positive hematological malignancy (e.g., by inducing or enhancing a ΔNPM1-target specific immune response). Particularly suitable compositions may be selected based on the HLA-A serotype of the human subject, as described in detail elsewhere herein.

[0369] The pharmaceutical composition may contain the nucleic acid sequences, vectors, modified cells or isolated proteins or peptides described herein, together with pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers.

[0370] The composition may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, co-immunopotentiators such as adjuvants and cytokines, and optionally other therapeutic agents or compounds. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance does not cause any undesirable biological effects or interact in a harmful manner with any of the other components of the pharmaceutical composition containing the selected nucleic acid sequence, vector, modified cell or isolated peptide, and may be administered to an individual together with the selected nucleic acid sequence, vector, modified cell or isolated peptide.

[0371] Excipients are natural or synthetic substances that are formulated with the active ingredient (e.g., the nucleic acid sequences, vectors, modified cells or isolated peptides provided herein), for the purpose of bulking up the formulation or imparting a therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption or solubility. Excipients can also be useful in the manufacturing process, in addition to helping to prevent degradation over the expected shelf life and thus assisting in the handling of the active substances involved, for example by promoting powder flowability or non-stick properties. Pharmaceutically acceptable excipients are well known in the art. Therefore, suitable excipients can be readily identified by those skilled in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0372] An adjuvant is a pharmacological and / or immunological agent that modifies the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. Therefore, suitable adjuvants can be readily identified by those skilled in the art.

[0373] A diluent is a drug to be diluted. Pharmaceutically acceptable diluents are well known in the art. Therefore, suitable diluents can be readily identified by those skilled in the art.

[0374] A carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. The term "carrier" refers to a natural or synthetic, organic or inorganic component with which the active ingredient is combined to facilitate its application. Pharmaceutically acceptable carriers are well known in the art. Therefore, suitable carriers can be readily identified by those skilled in the art.

[0375] Treatment of a subject The compositions of the present invention may advantageously be used to treat or prevent ΔNPM1-positive hematological malignancies in a human subject. Suitable compositions may be selected based on the HLA-A serotype of the human subject, as discussed in detail elsewhere herein.

[0376] In one embodiment, the methods for treating or preventing ΔNPM1-positive hematological malignancies described herein result in an induced or enhanced immune response (e.g., a cell-mediated response) (e.g., a targeted immune response against malignant cells presenting HLA-A restricted peptides) in a subject.

[0377] The phrase "induced or enhanced immune response" refers to an increase in the immune response (e.g., a cell-mediated immune response such as a T cell-mediated immune response) of a subject during or after treatment compared to the immune response prior to treatment. Therefore, an "induced or enhanced" immune response encompasses any measurable increase in an immune response that is directly or indirectly targeted to the ΔNPM1-positive hematological malignancy being treated.

[0378] The compositions of the present invention may be used to treat or prevent ΔNPM1-positive hematological malignancies in a human subject, particularly ΔNPM1-positive myeloid malignancies, and more specifically ΔNPM1-positive AML.

[0379] One of ordinary skill in the art will fully recognize hematological malignancies that may be ΔNPM1 positive and thus may be treated in accordance with the present invention. Similarly, one of ordinary skill in the art will fully recognize myeloid malignancies that may be ΔNPM1 positive and thus may be treated in accordance with the present invention.

[0380] As used herein, the terms "treat", "treating", and "treatment" are construed to include an intervention that is intended to prevent the onset of a condition, disorder, or symptom (i.e., in this case, a hematological malignancy) or to alter its pathology. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow (mitigate) the targeted condition, disorder or symptom. Therefore, "treatment" includes a decrease, slowdown or inhibition of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the amount or concentration of malignant cells measured in a sample obtained from a subject, for example, as compared to the amount or concentration of malignant cells prior to treatment. Methods for measuring the amount or concentration of malignant cells include, for example, qRT-PCR and quantification of ΔNPM1 positive hematological malignancy-specific biomarkers in a sample obtained from a subject, 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).

[0381] As used herein, the term "subject" refers to an individual, such as a human, who has or is at risk of having a specified condition, disorder or symptom. The subject may be a patient, i.e., a subject in need of treatment according to the present invention. The subject may have been treated for a condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment according to the present invention.

[0382] The compositions described herein can be administered to a subject by any conventional route including injection or by gradual infusion over time. Administration can be, for example, by injection or by intramuscular, intravenous, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, or transdermal administration.

[0383] The compositions described herein can be in any form suitable for the above-described modes of administration. For example, a composition containing modified cells can be in any form suitable for injection. As a further example, forms suitable for parenteral injection (including subcutaneous, intramuscular, intravenous 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 can be by direct injection into the target area, or by regional delivery, or by local delivery. Determination of an appropriate dosage of the compositions of the present invention is well within the routine capabilities of those skilled in the art.

[0384] Advantageously, the compositions of the present invention are a rapid and reliable approach, regardless of the patient's existing immune repertoire, that can generate large numbers of T cells specific for a ΔNPM1 - specific peptide (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)) and may be formulated for use in T - cell receptor (TCR) gene transfer. When using TCR gene transfer, modified autologous cells suitable for injection may be generated within a few days.

[0385] Advantageously, the compositions of the invention may be formulated for use as a vaccine (e.g., 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)), and may be formulated as a pharmaceutical composition suitable for use as a peptide vaccine. Suitable peptide vaccine formulations are well known in the art.

[0386] Preferably, the pharmaceutical composition of the invention is a vaccine, preferably a peptide-based vaccine. Such peptide-based vaccines may be used for the prevention or treatment of ΔNPM1-positive hematological malignancies such as AML.

[0387] The pharmaceutical composition is preferably for administration to a subject, preferably a human or animal subject, and is thus 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, intracutaneous, and / or intratumoral administration, i.e., by injection.

[0388] Preferably, the pharmaceutical composition comprises or consists of an amount of peptide that constitutes a pharmaceutical dosage unit. A pharmaceutical dosage unit is defined herein as the amount of active ingredient (i.e., the total amount of peptide in the peptide-based vaccine) administered to a subject at a given point in time. The pharmaceutical dosage unit may be administered to the subject in a single volume, i.e., a single shot, or preferably in 2, 3, 4, 5 or more separate volumes or shots administered at different locations on the body, such as the right and left limbs. It should be understood that separate volumes of the pharmaceutical dosage may have different compositions, i.e., may contain different types or compositions of active ingredient and / or adjuvant.

[0389] A single injection volume or shot (i.e., the volume administered at one location at a given time point) containing the total pharmaceutical dosage, or a portion thereof if multiple shots are administered at substantially the same time point, may be 100 μL to 2 mL, or 100 μL to 1 mL. The single injection volume may 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 in between.

[0390] Preferably, the total amount of the pharmaceutical dosage unit or peptide administered to the subject at a given time point, either as a single injection or multiple injections at a given time point, is 0.1 μg to 20 mg, such as 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, 400 μg, 450 μg, 500 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 15 mg or about 20 mg, or an amount of peptide of any value in between. Preferred ranges for the pharmaceutical dosage unit are 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 - 4 mg.

[0391] The compositions described herein are for administration in an effective amount. An "effective amount" is an amount that produces the desired (therapeutic or non-therapeutic) response, either alone or in conjunction with further dosages. The effective amount used will depend, for example, on the purpose of the treatment (or non-treatment), the route of administration, and the condition of the patient / subject. For example, the appropriate dosage of the composition of the present invention for a given patient / subject will be determined by the attending physician (or the person administering the composition) taking into account various factors known to modify the action of the composition of the present invention, such as the severity and type of hematological malignancy, body weight, gender, diet, time and route of administration, other medicaments, and other relevant clinical factors. The dosage and schedule may vary according to the particular condition, disorder or symptom and the overall condition of the patient / subject. The effective dosage may be determined by any method, either in vitro or in vivo.

[0392] The composition of the present invention is preferably presented in unit dosage form.

[0393] Method for generating TCR In one aspect, the present invention provides a method for generating 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 host cell with a nucleic acid sequence (or vector) of the present invention under conditions such that the nucleic acid sequence (or vector) is incorporated and expressed by the cell to generate a T cell receptor.

[0394] The method may be carried out ex vivo or in vitro with respect to the host cell. Alternatively, the method may be carried out in vivo, and the nucleic acid sequence (or vector) is administered to a subject and contacted in vivo with the host cell under conditions such that the nucleic acid sequence (or vector) is incorporated and expressed by the host cell to generate a T cell receptor. In one embodiment, the method is not a method of treating the human or animal body.

[0395] Suitable in vivo, in vitro, and ex vivo methods for contacting a nucleic acid sequence (or vector) with a host cell under conditions where the nucleic acid sequence (or vector) is incorporated and expressed by the cell are well known, as described elsewhere herein.

[0396] General Definitions As used herein, the terms "nucleic acid sequence", "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence. The nucleotide sequence may be of genomic, synthetic, or recombinant origin and may be double-stranded or single-stranded (representing the sense or antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g., mRNA), as well as analogs of DNA or RNA generated, for example, by the use of nucleotide analogs. In one example, the nucleotide sequence lacks introns. In other words, the nucleotide sequence is a nucleic acid sequence without introns. For example, the nucleotide sequence may be a DNA sequence that does not contain an intron sequence. As used herein, an "isolated nucleic acid sequence" refers to a nucleic acid sequence that is not in its natural environment, and the nucleic acid sequence, when in its natural environment, is linked to its naturally associated sequences that are also in the natural environment. In other words, an isolated nucleic acid sequence is not a native nucleotide sequence, where the term "native nucleotide sequence" means the entire nucleotide sequence in its natural environment, and if it is operably linked to all promoters to which it is naturally associated, those promoters are also in their natural environment.

[0397] As used herein, "specifically binds to CLAVEEVSL (SEQ ID NO: 1)" refers to the selective binding of the CLAVEEVSL (SEQ ID NO: 1) peptide. Under certain conditions, for example, in the immunoassays described herein, a polypeptide that "specifically binds to CLAVEEVSL (SEQ ID NO: 1)" selectively binds to this peptide and does not bind in significant amounts to other peptides that do not contain this sequence. Thus, the polypeptide may bind to CLAVEEVSL (SEQ ID NO: 1) with an affinity that is at least 10, 20, 30, 40, 50, or 100 times higher than it binds to a control antigen peptide. Selective binding may also be determined indirectly in the context of modified cells expressing the nucleic acids or vectors of the invention. For example, in an assay such as the assays discussed herein, the modified cells are specifically reactive against cells presenting CLAVEEVSL (SEQ ID NO: 1) associated with HLA-A * 02:01 (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 is introduced). Thus, the modified cells bind to cells presenting CLAVEEVSL (SEQ ID NO: 1) associated with HLA-A * 02:01 with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times higher compared to their reactivity against a control cell line that does not present CLAVEEVSL (SEQ ID NO: 1) associated with HLA-A * 02:01.

[0398] Selective binding may also be in the context of presentation of CLAVEEVSL (SEQ ID NO: 1) by HLA-A * 02:01. In other words, in certain embodiments, a polypeptide that "specifically binds to CLAVEEVSL (SEQ ID NO: 1)" binds as such only when it is presented by (i.e., bound by) HLA-A * 02:01, or only when it is in a conformational formation equivalent to when it is presented by HLA-A * 02:01. * 02:01.

[0399] Unless stated to the contrary, a polypeptide that "specifically binds to CLAVEEVSL (SEQ ID NO: 1)" may bind to either (i) the cysteinylated form of CLAVEEVSL (SEQ ID NO: 1), (ii) the non-cysteinylated form of CLAVEEVSL (SEQ ID NO: 1), or (iii) both the cysteinylated form and the non-cysteinylated form of CLAVEEVSL (SEQ ID NO: 1). Similarly, unless otherwise specified, general references herein to "SEQ ID NO: 1" or "CLAVEEVSL" (SEQ ID NO: 1) include both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSL (SEQ ID NO: 1). As used herein, "specifically binds to AVEEVSLRK (SEQ ID NO: 26)" refers to the selective binding of the AVEEVSLRK (SEQ ID NO: 26) peptide. Under certain conditions, e.g., in the immunoassays described herein, a polypeptide that "specifically binds to AVEEVSLRK (SEQ ID NO: 26)" selectively binds to this peptide and does not bind significantly to other peptides that do not contain this sequence. Thus, the polypeptide may bind to AVEEVSLRK (SEQ ID NO: 26) with an affinity that is at least 10, 20, 30, 40, 50, or 100 times higher than it binds to a control antigen peptide. Selective binding may also be determined indirectly in the context of modified cells expressing the nucleic acid or vector of the invention. For example, in an assay such as the assays discussed herein, the modified cells are 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 into which the ΔNPM1 gene is introduced * 03:01, HLA-A * 11:01 or HLA-A *01:01 It is specifically reactive against cells presenting AVEEVSLRK (SEQ ID NO: 26) associated with the positive cell line). Thus, the modified cells are HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01 and may bind with at least 10, 20, 30, 40, 50, or 100 times higher reactivity compared to its reactivity against control cell lines that do not present AVEEVSLRK (SEQ ID NO: 26) associated with HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01.

[0400] Specific binding may be only in the context of AVEEVSLRK (SEQ ID NO: 26) presentation 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)" binds as such only when it is presented (i.e., bound) by HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01, or only when it is in a conformational state equivalent to when it is presented by HLA-A * 03:01, HLA-A * 11:01 or HLA-A * 01:01.

[0401] Similarly, as used herein, "specifically binds to CLAVEEVSLRK (SEQ ID NO: 27)" refers to the selective binding of the CLAVEEVSLRK peptide. Under certain conditions, for example, in the immunoassays described herein, a polypeptide that "specifically binds to CLAVEEVSLRK (SEQ ID NO: 27)" selectively binds to this peptide and does not bind significantly to other peptides that do not contain this sequence. 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 higher than binding to a control antigen peptide. Selective binding may also be determined indirectly in the context of modified cells expressing the nucleic acid or vector of the invention. For example, in an assay such as the assays discussed herein, a modified cell is specifically reactive with cells presenting CLAVEEVSLRK (SEQ ID NO: 27) associated with 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 cell may bind with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times higher compared to its reactivity with a control cell line that does not present CLAVEEVSLRK (SEQ ID NO: 27) associated with HLA-A * 03:01 or HLA-A * 11:01. The modified cell binds to cells presenting CLAVEEVSLRK (SEQ ID NO: 27) associated with HLA-A * 03:01 or HLA-A * 11:01 and not presenting CLAVEEVSLRK (SEQ ID NO: 27) associated with HLA-A

[0402] Selective binding is HLA-A * 03:01 or HLA-A *It may be only in the context of presentation by CLAVEEVSLRK (SEQ ID NO: 27) at 11:01. In other words, in certain embodiments, a polypeptide that "specifically binds to CLAVEEVSLRK (SEQ ID NO: 27)" is such that it binds only when it is presented by HLA-A * 03:01 or HLA-A * presented by 11:01 (i.e., bound by these), or when it is in a conformational state equivalent to when it is presented by HLA-A * 03:01 or HLA-A * presented by 11:01. It may bind in this way only when it is in a conformational state equivalent to when it is presented by HLA-A 11:01.

[0403] Unless stated to the contrary, a polypeptide that "specifically binds to CLAVEEVSLRK (SEQ ID NO: 27)" may bind to either (i) a cysteinylated form of CLAVEEVSLRK, (ii) a non-cysteinylated form of CLAVEEVSLRK (SEQ ID NO: 27), or (iii) both a cysteinylated form of CLAVEEVSLRK and a non-cysteinylated form of CLAVEEVSLRK (SEQ ID NO: 27). Similarly, unless otherwise indicated, general references herein to "SEQ ID NO: 27" or "CLAVEEVSLRK" include both cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSLRK (SEQ ID NO: 27).

[0404] As used herein, "specifically binds to VEEVSLRK (SEQ ID NO: 28)" refers to the selective binding of the VEEVSLRK (SEQ ID NO: 28) peptide. Under certain conditions, for example, in the immunoassays described herein, a polypeptide that "specifically binds to VEEVSLRK (SEQ ID NO: 28)" selectively binds to this peptide and does not bind in significant amounts to other peptides that do not contain this sequence. Thus, the polypeptide may bind to VEEVSLRK (SEQ ID NO: 28) with an affinity that is at least 10, 20, 30, 40, 50, or 100 times higher than binding to a control antigen peptide. Selective binding may also be determined indirectly in the context of modified cells expressing the nucleic acid or vector of the invention. For example, in an assay such as the assays discussed herein, a modified cell is specifically reactive with a cell presenting VEEVSLRK (SEQ ID NO: 28) associated with a suitable HLA-A (e.g., primary ΔNPM1-positive AML cells, or any suitable HLA-A-positive cell line into which the ΔNPM1 gene is introduced). Thus, the modified cell may bind with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times higher compared to its reactivity with a control cell line that does not present VEEVSLRK associated with a suitable HLA-A to a cell presenting VEEVSLRK (SEQ ID NO: 28) associated with a suitable HLA-A.

[0405] Selective binding may be only in the context of VEEVSLRK (SEQ ID NO: 28) presentation by a suitable HLA-A. In other words, in certain embodiments, a polypeptide that "specifically binds to VEEVSLRK (SEQ ID NO: 28)" may bind as such only when it is presented (i.e., bound by) by a suitable HLA-A, or only when it is in a conformational state equivalent to when it is presented by a suitable HLA-A.

[0406] As used herein, "specifically binds to AVEEVSLR (SEQ ID NO: 29)" refers to the selective binding of the AVEEVSLR (SEQ ID NO: 29) peptide. Under certain conditions, for example, in the immunoassays described herein, a polypeptide that "specifically binds to AVEEVSLR (SEQ ID NO: 29)" selectively binds to this peptide and does not bind significantly to other peptides that do not contain this sequence. Thus, the polypeptide may bind to AVEEVSLR (SEQ ID NO: 29) with an affinity that is at least 10, 20, 30, 40, 50, or 100 times higher than binding to a control antigen peptide. Selective binding may also be determined indirectly in the context of modified cells expressing the nucleic acid or vector of the invention. For example, in an assay such as the assays discussed herein, a modified cell is specifically reactive with a cell presenting AVEEVSLR (SEQ ID NO: 29) associated with a suitable HLA-A (e.g., primary ΔNPM1-positive AML cells, or any suitable HLA-A-positive cell line into which the ΔNPM1 gene is introduced). Thus, the modified cell may bind with a reactivity that is at least 10, 20, 30, 40, 50, or 100 times higher compared to its reactivity with a cell presenting AVEEVSLR (SEQ ID NO: 29) associated with a suitable HLA-A and a control cell line that does not present AVEEVSLR (SEQ ID NO: 29) associated with a suitable HLA-A.

[0407] Selective binding may be only in the context of presentation of AVEEVSLR (SEQ ID NO: 29) by a suitable HLA-A. In other words, in certain embodiments, a polypeptide that "specifically binds to AVEEVSLR (SEQ ID NO: 29)" may bind as such only when it is presented (i.e., bound by) by a suitable HLA-A, or only when it is in a conformational state equivalent to when it is presented by a suitable HLA-A.

[0408] "Non-essential" (or "non-critical") amino acid residues can be changed from the wild-type sequence (e.g., the sequence specified by the SEQ ID NOs herein) without loss of biological activity, or more preferably without substantial change, whereas "essential" (or "critical") amino acid residues result in such a change. For example, conserved amino acid residues are particularly predicted to be intolerant to change, with the exception that generally amino acid residues within the hydrophobic core of a domain can be substituted by other residues having substantially equivalent hydrophobicity without significantly altering activity.

[0409] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, non-essential (or non-critical) amino acid residues in a protein are preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly and the resulting mutants screened for activity to identify mutants that retain activity.

[0410] Calculation of sequence homology or identity between sequences (these terms are used interchangeably herein) is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment purposes, and non-homologous sequences can be disregarded for comparison purposes) for optimal 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%, 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. Then, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0411] The comparison of arrays and determination of percent identity between two arrays can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453 algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either the BLOSUM 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the parameters to be used when the physician is unclear as to which parameters should be applied to determine whether a molecule is within the sequence identity or homology limits of the invention) is the BLOSUM62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0412] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the Meyers et al. (1989) CABIOS 4:11-17 algorithm incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0413] The nucleic acid and protein sequences described herein can be used, for example, as "query sequences" to perform searches against public databases 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 carried out using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be carried out using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>. The polypeptides and nucleic acid molecules described herein can have amino acid sequences or nucleic acid sequences that are fully or substantially identical to the sequences identified by SEQ ID NO. The terms "fully identical" or "substantially identical" are used herein to mean that the first and second amino acid sequences or nucleotide sequences have a common structural domain or common functional activity, such that the first amino acid sequence or nucleotide sequence contains a sufficient or minimal number of identical or equivalent (e.g., having similar side chains) amino acid residues or nucleotides relative to the second amino acid sequence or nucleotide sequence. For example, amino acid sequences or nucleotide sequences containing a common structural domain having at least about 60% or 65% identity, perhaps 75% identity, perhaps 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as being fully or substantially identical.

[0414] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, New York (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in this invention. Any methods and materials similar or equivalent to those described herein can be used in the practice of the invention, but the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully explained by reference to the entire specification. Also, as used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in a 5' to 3' orientation, and amino acid sequences are written left to right in an amino to carboxy orientation. It is to be understood that the invention is not limited to the particular methodologies, protocols, and reagents described, as these may vary depending on the context in which one of ordinary skill in the art uses them.

[0415] Aspects of the invention are demonstrated by the following non-limiting examples.

Examples

[0416] Example 1 Materials and Methods Study Design 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 of HLA class I ligands from ΔNPM1 that could mediate specific recognition and lysis of primary AML after gene transfer. HLA class I ligandome data were generated for 12 primary AMLs using tandem mass spectrometry, and peptides matching the alternative reading frame of ΔNPM1 were searched for. For one identified peptide, HLA-A * 02:01 pMHC tetramers were generated and used to isolate specific CD8 cells from 6 HLA-A * 02:01-positive AML patients and 6 healthy HLA-A * 02:01-positive individuals. T cell clones isolated from healthy individuals were screened for pMHC tetramer staining, and tetramer-positive T cell clones were tested by IFN-γ ELISA for reactivity against peptide-loaded T2 cells and primary AML with ΔNPM1 or wtNPM1. TCRs were sequenced from one strongly reactive T cell clone and cloned into the retroviral MP71-TCR-flex vector. This vector was used to transduce the above TCRs into CD8 and CD4 cells from 2 healthy HLA-A * 02:01-positive individuals, and the purity of the TCR-transduced T cells was evaluated by flow cytometry. TCR-transduced T cells were tested by IFN-γ ELISA for recognition of HLA-A * 02:01-positive AML cell lines and primary AML with ΔNPM1 or wtNPM1, and specific lysis was 51 measured by 51Cr release assay.

[0417] Sample collection and cell culture Peripheral blood and bone marrow samples were obtained from AML patients and healthy individuals with informed consent according to the Helsinki Declaration and after approval by the Institutional Review Board of the Leiden University Medical Center. Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll-Isopaque separation and cryopreserved. HLA-A *For the isolation of PBMC from healthy individuals of the 02:01 type, buffy coats were ordered from Sanquin (Amsterdam, the Netherlands). T cells were cultured in T cell medium (TCM) consisting 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, USA), 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 AML cell lines expressing ΔNPM1 (OCI-AML3) were ordered from DSMZ (Braunschweig, Germany) and cultured in Minimum 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. Monocytes were isolated from PBMC by magnetic-activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) using CliniMACS CD14 beads (Miltenyi Biotec). The isolated monocytes were cultured for 7 days to immature dendritic cells (DC) in a medium containing 100 ng / mL of GM-CSF (Novartis, Basel, Switzerland) and 500 IU / mL of IL-4 (Schering-Plough, Kenilworth, New Jersey).For the last two days, 100 ng / mL of GM-CSF, 10 ng / mL of TNF-α (Cellgenix, Freiburg, Germany), 10 ng / mL of IL-1β (Cellgenix), 10 ng / mL of IL-6 (Cellgenix), 1 μg / mL of prostaglandin E2 (Sigma-Aldrich, St. Louis, Missouri), and 500 IU / mL of IFN-γ (Boehringer-Ingelheim, Ingelheim, Germany) were added to induce maturation.

[0418] HLA class I ligandome of primary AML 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 inhibitor (Sigma-Aldrich, St. Louis, Missouri, USA). After incubating for 2 hours with tumbling of the cells in the lysis buffer 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-cleared with Protein A Sepharose CL-4B beads (GE Healthcare Life Sciences, Chicago, Illinois, USA), and applied to an immunoaffinity column using dimethyl pimelimidate (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 5-10 column volumes with 10 mM Tris-HCl (pH 8.0) buffer containing 120 mM NaCl, 1 M NaCl, and no NaCl, 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 passage through a 10 kDa membrane (Macrosep Advance Centrifugal Devices With Supor Membrane, Pall Corporation, Port Washington, New York, USA). The filtrate was lyophilized.

[0419] The eluted peptide pool was fractionated by strong cation exchange chromatography (SCX) using a homemade SCX column (inner diameter 320 μm, 15 cm, polysulfoethyl A 3 μm, Poly LC) that was run at 4 μl / min. The gradient was run for 10 minutes with 100% solvent A (100 / 0.1 water / trifluoroacetic acid v / v), after which a linear gradient was started and reached 100% solvent B (65 / 35 / 0.1 250 mM KCl / acetonitrile / trifluoroacetic acid v / v / v) over 15 minutes, followed by reaching 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 and then switched back to 100% solvent A. Twenty 4-μl fractions were collected into vials pre-filled with 20 μl of 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 subsequently analyzed by data-dependent tandem mass spectrometry (MS) using either an LTQ-FTUltra equipped with a nanoflow liquid chromatography 1100 HPLC system (Agilent Technologies, Santa Clara, California, USA) as previously described (38), or a Q-Exactive equipped with an easy-nLC1000. Peptides were trapped on a 1.5-cm column (inner diameter 100 μm; ReproSil-Pur C18-AQ, 3 μm, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) at 6–10 μl / min and eluted onto a 20-cm column (inner diameter 50 μm; ReproSil-Pur C18-AQ, 3 μm) at 150 nl / min. The column was developed with a 120-minute gradient of 0–40% acetonitrile in 0.1% formic acid. The end of the column was pulled to a tip (inner diameter of approximately 5 μm), from which the eluent was sprayed into the mass spectrometer. Full-scan MS spectra were acquired in FT-ICR at a resolution of 25,000 with a target value of 3,000,000.The two most intense ions were isolated for accurate mass measurement by selected ion monitoring scans in an FT-ICR with a resolution of 50,000 at a target accumulation value of 50,000. The selected ions were then fragmented in a linear ion trap using collision-induced dissociation at a target value of 10,000. The Q-Exactive mass spectrometer was operated in top 10 mode. The parameters were a resolution of 70,000 at an AGC target value of 3,000,000 / 20 ms maximum fill time (full scan) for MS / MS at an intensity threshold of 17,000 and a resolution of 17,500 at an AGC target value of 100,000 / 60 ms maximum fill time. The apex trigger was set to 1 - 10 seconds and the allowed charge was 2 - 6. Proteome Discoverer version 2.1 (Thermo Fisher Scientific) was used with mascot version 2.2.04 having the UniProt Homo Sapiens database (UP000005640; January 2015; 67911 entries) to identify peptides and proteins using the mascot node for identification. Methionine oxidation and cysteine cysteinylation were set as variable modifications. Peptide assignments were made with a precursor tolerance of 10 ppm and an MS / MS fragment tolerance of 20 mmu for Q-Exactive data and 2 ppm and 0.5 Da for LTQ-FTUltra data. Identification of ΔNPM1-derived peptides was confirmed by their synthetic counterparts.

[0420] Peptide synthesis and pMHC tetramer generation Peptides were synthesized by standard Fmoc chemistry and dissolved in dimethyl sulfoxide. 1 mM peptides were treated with 2 mM 1,4-dithiothreitol in 50 mM ammonium bicarbonate at 50 °C for 15 min, followed by addition of 10 mM free cysteine and 15 mM H 2 O 2 to cysteinylate the peptides by adding at room temperature for 30 min. pMHC tetramers were generated as previously outlined (7, 8). Briefly, recombinant HLA-A *02:01 The monomer consisting of a heavy chain and human β2-microglobulin was purified by gel filtration HPLC and biotinylated. After folding with an appropriate peptide, pMHC tetramers were generated by adding PE-conjugated streptavidin (Invitrogen, Thermo Fisher Scientific). UV-exchange pMHC tetramers contain a biotinylated HLA-A with a UV-sensitive peptide * 02:01 Monomers were generated by exposing them to UV light at 366 nm in the presence of cysteinylated ΔNPM1 peptide. One hour after peptide exchange, the monomers were incubated at 4 °C for 1 h, followed by centrifugation at 4000 g for 10 min at 15 °C. Tetramers were generated by adding streptavidin-conjugated PE to the supernatant. pMHC tetramers were stored at 4 °C.

[0421] Antibodies and FACS analysis T cells were stained with FITC-conjugated antibodies against CD3, CD4, CD8 (BD Biosciences, San Jose, CA, USA), FITC-conjugated antibody against TCR-Vβ5.1 (Beckman Coulter, Brea, CA, USA), APC-conjugated antibodies against CD3, CD4, CD8 and murine TCR-Cβ, and PE-conjugated pMHC tetramers and antibodies against CD3, CD4 and CD8 (BD Biosciences). Cells were measured on a BD FACSCalibur II (BD Biosciences) using BD CellQuest Pro software (BD Biosciences), and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

[0422] Isolation and culture of T cells pMHC tetramer-positive CD8 T cells were isolated from PBMCs from AML patients and healthy individuals as previously described (9). Briefly, HLA- *PBMCs derived from positive AML patients and healthy individuals were stained with a PE-conjugated pMHC tetramer containing the ΔNPM1 peptide at 4 °C for 1 hour and subsequently isolated by MACS using anti-PE microbeads (Miltenyi Biotec). The 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 sorted as single cells by a BD FACSAria III cell sorter (BD Biosciences) using BD FACSDiva v6 software (BD Biosciences). Single T cells were stimulated with 50,000 irradiated allogeneic PBMCs, 5,000 irradiated allogeneic EBV-LCLs, and 0.8 μg / ml PHA (Oxoid Microbiology Products, Thermo Fisher Scientific) in 100 μl of TCM per well in a 96-well U-bottom culture plate (Costar, Sigma-Aldrich). Expanding T cell clones were restimulated with irradiated feeder cells and PHA every 10 - 14 days.

[0423] T cell reactivity assay 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 of TCM per well in a 384-well flat-bottom plate (Greiner Bio One (Greiner Bio One), Kremsmuenster, Austria) overnight. After overnight co-incubation, the culture supernatant was collected and IFN-γ release was measured. In the peptide recognition assay, T2 cells (15,000 cells) were pre-incubated with titrated peptide concentrations at 37 °C for 30 minutes, washed twice, and then co-incubated with T cells. In the blocking assay, target cells (10,000 cells) were pre-incubated with antibodies that block (block) HLA class I (W6 / 32) or HLA class II (PdV5.2) at saturation concentration for 60 minutes at room temperature, and then T cells were added. T cell-mediated cytotoxicity was 51 measured by a chromium release assay. Primary AML cells were labeled with 100 μCi of Na 2 51 CrO 4 for 1 hour at 37 °C, washed, and co-incubated with T cells at various E:T ratios in 100 μl of TCM per well in a 96-well U-bottom culture plate (Costar). Spontaneous and maximum 51 Cr release was measured in separate plates containing 100 μl of TCM or 100 μl of TCM containing 1% Triton-X100 (Sigma-Aldrich) per well, respectively. After 9 hours of co-incubation, 25 μl of the culture supernatant was collected and transferred to 96-well LumaPlates (PerkinElmer, Waltham, Massachusetts, USA). 51 Cr release in counts per minute (cpm) was measured with a 2450 Microbeta 2 plate counter (PerkinElmer).

[0424] TCR cloning and production of retroviral supernatant The use of the α and β chains of the TCR of clone 1A2 was determined as previously described (9) with minor modifications. Briefly, T cells were lysed and mRNA was isolated using the Dynabeads mRNA DIRECT kit (Invitrogen). TCR-specific cDNA was generated using two TCR-Cβ-specific primers, a TCR-Cα-specific primer, a SA.rt anchor template-switching oligonucleotide (TSO), and SMARTScribe reverse transcriptase (Takara, Clontech, Mountain View, CA, USA). During first-strand cDNA synthesis, SMARTScribe reverse transcriptase adds a 3’ non-templated polycytosine tail, which enables annealing of the TSO and second-strand cDNA synthesis. TCR amplification was performed by PCR using Phusion Flash (Thermo Fisher Scientific), an anchor-specific primer, and nested primers that anneal to the TCR-Cα or -Cβ regions. The TCR sequence of clone 1A2 was identified as TRAV12-2 and TRBV5-1 by Sanger sequencing (Macrogen, Amsterdam, 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, NJ, USA). In MP71-TCR-flex, the murine TCR-Cα and -Cβ regions are linked by a porcine teschovirus-derived P2A sequence and contain additional cysteine residues to facilitate preferential pairing and expression of the TCR. This construct was transfected into packaging cells φ-NX-A (ATCC, Manassas, VA, USA), and retroviral supernatants were harvested 48 and 72 hours after transfection and frozen at -80°C. CMV-derived HLA-A* MP71-TCR-flex, which encodes a TCR specific for the restricted peptide NLVPMVATV, was kindly provided by Dr. T. N. Schumacher, professor, Division of Immunology, Netherlands Cancer Institute, Amsterdam, the Netherlands.

[0425] TCR gene transfer Two HLA-A * PBMCs from two HLA-A 02:01-positive healthy individuals (donors 1 and 2) were thawed, and CD4 and CD8 cells were isolated by MACS using anti-CD4 MicroBeads (Miltenyi Biotec), followed by the CD8 T Cell Isolation Kit (Miltenyi Biotec). CD8 and CD4 cells were stimulated with irradiated autologous feeders and 0.8 μg / ml PHA in 24-well flat-bottom culture plates (Costar). Two days after stimulation, the T cells were transferred to 24-well flat-bottom suspension culture plates (Greiner Bio-One) and transduced with retrovirus as previously described (9, 12). Before adding the T cells, the plates were coated with 30 mg / mL of retronectin (Takara, Clontech) and blocked with 2% human serum albumin (Sanquin). The retroviral supernatant was added, and the plates were centrifuged at 3000 g for 20 minutes at 4°C. T cells were added to the plates containing the viral supernatant at 300,000 cells / well. After incubation overnight, the T cells were transferred to 24-well flat-bottom culture plates. On day 6 after transduction, TCR-transduced T cells were stained with an APC-conjugated antibody against mouse TCR-Cβ for 15 minutes at 4°C, followed by MACS isolation using anti-APC MicroBeads (Miltenyi Biotec). TCR-transduced T cells were restimulated every 10 - 14 days with irradiated allogeneic PBMCs, EBV-LCLs, and 0.8 μg / ml PHA. Before analysis, the restimulated TCR-transduced T cells were concentrated by MACS using anti-mouse TCR-Cβ-APC and anti-APC MicroBeads as described above.

[0426] Result Presence of ΔNPM1 in the HLA class I ligandome of primary AML To examine whether ΔNPM1 peptides are processed and presented in HLA class I, we immunoprecipitated HLA class I surface molecules from 12 primary AML samples, eluted peptides from the binding groove, and analyzed the peptidome by mass spectrometry. Table I shows the HLA class I typing of 12 AML samples and their mutation status for NPM1. A 4-bp frameshift insertion in exon 12 of the NPM1 gene is a recurrent mutation that occurs in 30% of primary AML. In 8 out of 12 primary AMLs, the presence of ΔNPM1 was demonstrated by PCR fragment analysis. All patients had a cytogenetically normal karyotype, except for one AML with the known chromosomal rearrangement inv(16)(p13q22). The blast percentage measured in peripheral blood or bone marrow samples ranged from 55% to 98%.

[0427] [Table 1] a ΔNPM1 patients carry a 4-bp insertion in exon 12 of the NPM1 gene that causes a frameshift at the C-terminus of the protein. b Blast percentage measured in peripheral blood and bone marrow samples from AML patients. * Anti-HLA-A * AML samples in which peptide elution was performed using the 02:01 antibody BB7.2

[0428] The repetitive 4bp insertion in exon 12 results in a ΔNPM1 protein that is 4AA longer than its wild-type counterpart (CLAVEEVSLRK (SEQ ID NO: 27)) with 11AA at the C-terminus that is translated in the alternative reading frame. From this alternative ΔNPM1 protein, a protein region (MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 34)) spanning 10 N-terminal residues in the normal reading frame followed by 11 C-terminal AAs in the alternative reading frame was searched for matching peptides in the HLA class I ligandome analyzed from 12 primary AMLs. This revealed the presence of two octameric peptides (VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29)), two nonameric peptides (CLAVEEVSL (SEQ ID NO: 1) and AVEEVSLRK (SEQ ID NO: 26)) and one undecameric peptide (CLAVEEVSLRK (SEQ ID NO: 27)) in AML with ΔNPM1, but not in AML with wtNPM1 (Table 1). All five ligands eluted from seven primary AMLs were verified by mass spectrometry using synthetic peptides (Figures 1 and 2). Verification of the tandem mass spectra of CLAVEEVSL (SEQ ID NO: 1) and CLAVEEVSLRK (SEQ ID NO: 27) using synthetic peptides was performed after in vitro cysteinylation of the first residue. Prediction of HLA class I binding affinity by NetMHCpan3.0 suggested binding of the epitope CLAVEEVSL (SEQ ID NO: 1) to HLA-A * 02:01, while the epitopes AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) were most likely to bind to HLA-A * 03:01 and HLA-A * 11:01, and for AVEEVSLRK (SEQ ID NO: 26) probably also to HLA-A * 01:01 (Table 2). Binding of CLAVEEVSL (SEQ ID NO: 1) to HLA-A * 02:01, and of AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) to HLA-A * 03:01 and A* The binding to 11:01 was confirmed by monomer refolding. HLA-A * 02:01 is expressed in 50% of the Caucasian population. Therefore, the inventors focused their research on CLAVEEVSL (SEQ ID NO: 1) detected in two of the three HLA-A * 02:01-positive AMLs (AML10197 and AML3361) with ΔNPM1.

[0429]

Table 2

[0430] T cell recognition of ΔNPM1 on primary AML HLA-A * To investigate whether the HLA-A * 02:01-restricted epitope CLAVEEVSL (SEQ ID NO: 1) is a neoantigen that can be targeted by immunotherapy, the inventors searched for specific T cells in patients with AML. CLAVEEVSL (SEQ ID NO: 1) (ΔNPM1-CLA) and its cysteinylated variant C * LAVEEVSL (cysteinylated form of SEQ ID NO: 1) (ΔNPM1-C * LA) were used to generate PE-conjugated pMHC tetramers. A mixture of these tetramers was used to isolate specific T cells from PBMCs from six HLA-A

[0431]

Table 3

[0432] A total of 41 tetramer-positive CD8 cells were isolated from 42×10 6 PBMC from 4 patients, and 5 T cells from 3 of these patients were clonally expanded. However, none of the 5 T cell clones could be stained with either the ΔNPM1-CLA tetramer or the ΔNPM1-C * LA tetramer, indicating the absence or frequency below the detection threshold of T cells specific for CLAVEEVSL (SEQ ID NO: 1). Therefore, following the same strategy, specific T cells in a large number of PBMC from 6 HLA-A * 02:01 positive healthy individuals were searched for (Table 4). Various numbers of 8 - 55 tetramer-positive CD8 cells were isolated from 460 - 1970×10 6 Total PBMC from each healthy individual. Of these cells, 31 T cells from 5 individuals were clonally expanded, and 13 T cell clones from 4 individuals were positive for the ΔNPM1-CLA tetramer. Among these 13 clones, 3 T cell clones could also be stained with the ΔNPM1-C * LA tetramer (Table 4 and Figure 3A).

[0433]

Table 4

[0434] To determine whether 13 tetramer-positive CD8 clones were reactive against their target peptides, CLAVEEVSL (SEQ ID NO: 1), cysteinylated variant C * LAVEEVSL (cysteinylated form of SEQ ID NO: 1) or an irrelevant HLA-A * 02:01 restricted CMV peptide NLVPMVATV (SEQ ID NO: 31) was exogenously loaded onto HLA-A * 02:01 positive T2 cells and the clones were tested for recognition. Of the 13 ΔNPM1-CLA tetramer-positive clones, two T cell clones (1A2 and 4A8) showed specific reactivity against T2 cells loaded with CLAVEEVSL (SEQ ID NO: 1), but not against the control peptide NLVPMVATV (SEQ ID NO: 31) (Figure 3B). Clone 1A2 also showed recognition of T2 cells loaded with C * LAVEEVSL (cysteinylated form of SEQ ID NO: 1). These results are consistent with the tetramer data and indicate that cysteinylation of the first residue abolishes T cell recognition by clone 4A8. However, peptide recognition by clone 1A2 was independent of CLAVEEVSL (SEQ ID NO: 1) and C * LAVEEVSL (cysteinylated form of SEQ ID NO: 1). These results are consistent with the tetramer data and indicate that cysteinylation of the first residue abolishes T cell recognition by clone 4A8. However, peptide recognition by clone 1A2 was independent of CLAVEEVSL (SEQ ID NO: 1) and C * LAVEEVSL (cysteinylated form of SEQ ID NO: 1). These results are consistent with the tetramer data and indicate that cysteinylation of the first residue abolishes T cell recognition by clone 4A8. However, peptide recognition by clone 1A2 was independent of CLAVEEVSL (SEQ ID NO: 1) and C *It is independent of cysteinylation, as shown by its specific reactivity against LAVEEVSL (cysteineylated form of SEQ ID NO: 1). To examine the anti-tumor ability of clones 1A2 and 4A8, five HLA-A samples including three samples with ΔNPM1 and two samples with wtNPM1 * T cell reactivity was measured against a panel of HLA-A 02:01-positive primary AML. T cell clone 1A2 showed reactivity to varying degrees against all three AMLs with ΔNPM1, while clone 4A8 showed relatively low reactivity against two of the three samples with ΔNPM1 (Figure 3C). The strong T cell reactivity of clone 1A2 against AML may be explained by its ability to recognize cysteinylated ΔNPM1 and non-cysteinylated ΔNPM1 eluted from the cell surface of primary AML. HLA-A with wtNPM1 * No T cell reactivity was observed against HLA-A 02:01-positive AML (Figure 3C) or HLA-A with ΔNPM1 * 02:01-negative AML (data not shown). These data indicate that T cells with a TCR specific for ΔNPM1 are present 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.

[0435] TCR gene transfer targeting ΔNPM1 on primary AML ΔNPM1 is a recurrent 4bp insertion that occurs in 30% of primary AML, HLA-A *Since 02:01 is expressed in 50% of the white population, the inventors consider CLAVEEVSL (SEQ ID NO: 1) to be an ideal target for TCR gene transfer. Since primary AML was most strongly recognized by clone 1A2, mRNA was isolated from this clone and cDNA was prepared to sequence the variable regions of the TCR α and β chains for ΔNPM1. The codon-optimized gene sequences of TRAV12-2 and TRBV5-1 expressed by T cell clone 1A2 were synthesized and cloned into the modified MP71-TCR-flex retroviral vector. To facilitate the 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 linked by the P2A sequence. The TCR for ΔNPM1 and HLA-A as a control * The TCR for the 02:01-restricted CMV peptide NLVPMVATV (SEQ ID NO: 31) was introduced into CD8 and CD4 cells isolated from PBMCs from healthy HLA-A * 02:01-positive individuals (donors 1 and 2). On day 6 after transduction, TCR-transduced CD8 and CD4 cells were purified using an APC-binding antibody against mouse TCR-Cβ and magnetic anti-APC beads. Flow cytometry analysis demonstrated specific binding of the ΔNPM1-CLA tetramer to CD8 cells (CD8φNPM1) transduced with the TCR for ΔNPM1 (Figure 4A). In contrast, the CMV-NLV tetramer did not bind to CD8 cells transduced with the TCR for ΔNPM1, whereas CD8 cells (CD8φCMV) transduced with the CMV-specific TCR could be stained with the CMV-NLV tetramer but not with the ΔNPM1-CLA tetramer. For TCR-transduced CD4 cells (CD4φNPM1 and CD4φCMV), the results were the same as for CD8 cells, indicating that binding of the ΔNPM1-CLA tetramer to TCR-transduced T cells occurs independently of the CD8 coreceptor. CD8 and CD4 cells transduced with the TCR could also be stained with an antibody against mouse TCR-Cβ, and CD8 and CD4 cells transduced with the TCR for ΔNPM1 also showed specific binding to an antibody against human TCR-Vβ5.1 (data not shown).

[0436] Next, the inventors analyzed the functionality of CD8 and CD4 cells transduced with the TCR for ΔNPM1 and co-incubated them with HLA-A * 02:01-positive T2 cells and found specific release of IFN-γ during co-incubation with CLAVEEVSL (SEQ ID NO: 1), but no release during co-incubation with the CMV peptide NLVPMVATV (SEQ ID NO: 31) (Figure 4B). Specific release of IFN-γ was also observed during co-incubation with the AML cell line OCI-AML3 having ΔNPM1, but not with stimulation by the AML cell line OCI-AML2 having wtNPM1 (Figure 4C). Subsequently, TCR-transduced CD8 and CD4 cells were tested for reactivity against a panel of 13 HLA-A * 02:01-positive primary AML samples including 9 samples having ΔNPM1 and 4 samples having wtNPM1. Upon transduction with the TCR for ΔNPM1, both CD8 and CD4 cells showed recognition of all 9 primary AMLs having ΔNPM1, whereas specific recognition of AMLs having wtNPM1 was not observed (Figure 5). CD8 and CD4 cells transduced with the TCR for ΔNPM1 lacked reactivity against HLA-A * 02:01-negative AMLs and mature DCs having wtNPM1 as well (Figure 6). Next, the transduced T cells were incubated with monocyte-derived mature DCs from donors 1 and 2 and 40 HLA-A *02:01 Reactivity against a panel of positive third-party EBV-LCLs was tested. CD8 and CD4 cells transduced with TCRs for ΔNPM1 were unable to recognize mature DCs or EBV-LCLs, indicating that alternative translation of the wtNPM1 gene does not occur and that this gene does not produce peptides similar to CLAVEEVSL (SEQ ID NO: 1). In summary, this data shows that TCRs for ΔNPM1 upon gene transfer into CD8 and CD4 cells result in specific recognition of CLAVEEVSL (SEQ ID NO: 1) as an endogenous neoantigen presented by HLA-A * 02:01. Finally, the inventors tested the cytolytic ability of TCR-transduced CD8 and CD4 cells against primary AML. TCR-transduced T cells were tested in a 9-hour 51 chromium release assay against a panel of six HLA-A * 02:01 positive primary AMLs, including four samples with ΔNPM1 and two samples with wtNPM1. Both CD8 and CD4 cells transduced with TCRs for ΔNPM1 showed specific lysis of AMLs with ΔNPM1, but not of AMLs with wtNPM1 (Figure 7). In conclusion, these results indicate that CLAVEEVSL (SEQ ID NO: 1) is a therapeutic neoantigen expressed on primary AMLs with ΔNPM1 that can be efficiently targeted by TCR gene transfer in a coreceptor-independent manner.

[0437] Discussion The inventors identified HLA-A encoded by ΔNPM1 in the HLA class I ligandome of primary AML *02:01 Identified the 9-mer peptide (CLAVEEVSL (SEQ ID NO: 1)). T cell clones with TCRs specific for this peptide were isolated from healthy individuals, and it was shown that the TCR from one of the clones mediates specific recognition and lysis of primary AML with ΔNPM1 upon 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.

[0438] Using HLA class I ligandome data from 12 primary AMLs, the inventors identified C * LAVEEVSL (the cysteinylated form of SEQ ID NO: 1) as a peptide that is endogenously processed and presented on AML with ΔNPM1. No match was seen between the eluted and synthetic peptides in the absence of cysteinylation, but the data provide strong evidence that the non-cysteinylated CLAVEEVSL (SEQ ID NO: 1) peptide is also presented on AML. Clone 1A2 was selected for TCR cloning and gene transfer, and this clone 1A2 was shown to recognize both synthetic C * LAVEEVSL (the cysteinylated form of SEQ ID NO: 1) and the CLAVEEVSL (SEQ ID NO: 1) peptide. This clone could also be stained with both pMHC tetramers, but the binding to the tetramer with the cysteinylated peptide was weaker. Experiments testing synthetic peptides with serine substitutions confirmed that the first residue of this epitope is not essential for the reactivity of clone 1A2. However, the results were different for clone 4A8. Clone 4A8 recognized CLAVEEVSL (SEQ ID NO: 1), but C *It did not recognize LAVEEVSL (the cysteinylated form of SEQ ID NO: 1), and only the non-cysteinylated pMHC tetramer with the peptide was able to bind to this T cell clone. The importance of the first residue of the peptide for T cell recognition was confirmed by the lack of reactivity of clone 4A8 to the synthetic peptide in which the first residue was substituted with serine. Surprisingly, despite the inability to recognize the cysteinylated peptide, clone 4A8 showed reactivity to two of the three AMLs with ΔNPM1. Based on the reactivity patterns of clones 1A2 and 4A8, the inventors suggest that cysteinylated and non-cysteinylated ΔNPM1 peptide variants are presented on the cell surface, but the expression levels of the epitopes may vary among AMLs. The reason for the absence of CLAVEEVSL (SEQ ID NO: 1) in the HLA class I ligandome is unknown, but it may be explained by low surface expression or low quality of the mass spectrum of the eluted peptide.

[0439] Interestingly, patients bearing ΔNPM1 without a concurrent intragenic tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD) show improved survival after chemotherapy, often eliminating the need for allogeneic stem cell transplantation. 1、2、5、13 In particular, the ΔNPM1 protein translocates from the nucleolus to the cytoplasm, where the ΔNPM1 protein is susceptible to subsequent processing by proteasomal degradation and the HLA class I antigen presentation pathway, 6 and an in vivo immune response against peptides derived from ΔNPM1 may underlie this favorable prognosis. Greiner et al. 14 showed that, in healthy volunteers and patients with AML, nine HLA-A derived from ΔNPM1 containing CLAVEEVSL (SEQ ID NO: 1) *The T cell responses to the defined peptides were explored in vitro. After co-culturing CD8 cells with peptide-loaded PBMCs, T cell responses to two of the nine peptides were revealed in both healthy volunteers and patients, but the immune response to CLAVEEVSL (SEQ ID NO: 1) was not measured. They then screened 25 patients with ΔNPM1 AML and revealed that the overall survival (duration) of patients with an immune response to these two ΔNPM1-derived peptides was significantly higher than that of patients without an immune response. 15 However, the number of patients screened in this study was small and the FLT3 mutation status was not determined. Recent data indicate that patients with FLT3-ITD, especially those with a high allelic ratio of mutant FLT3 gene expression to wild-type FLT3 gene, have a poor prognosis compared to patients without FLT3-ITD, regardless of the NPM1 mutation status. 13 This observation may negate the induction of an in vivo immune response to ΔNPM1 underlying the good prognosis, but emphasizes the importance of endogenous factors in AML tumor growth and the relevance of ΔNPM1 TCR gene therapy for treating AML with poor prognosis.

[0440] AML arises from a single founding clone carrying numerous somatic mutations with only a few driver mutations (driver gene mutations). Subclones can emerge from the founding clone through the accumulation of additional mutations that confer a survival advantage to the cells. As a result, most of the mutations in subclones are shared with the founding clone, and a minority are clone-specific. This heterogeneous composition of AML increases the opportunity for clonal evolution in persistent or recurrent disease after induction or consolidation therapy. Targeting neoantigens arising from shared mutations is an attractive immunotherapy strategy for eradicating the founding clone and subclones. Neoantigens arising from passenger mutations (passenger gene mutations) can be readily lost as a result of tumor immune editing by T cells, leading to tumor immune evasion. When neoantigens produced by driver mutations are targeted, the likelihood of immune escape is low. This is because they are essential for malignant transformation and are present in all tumor cells. 16 So far, only a few neoantigens arising from driver mutations have been identified, among which the mutant KRAS gives rise to antigens expressed in 45% of pancreatic cancers and 13% of colorectal cancers. 17 Although immune escape is less likely for neoantigens arising from driver mutations, Tran et al. demonstrated the escape from TCR gene therapy of mutant KRAS in patients with metastatic colorectal cancer through the loss of HLA-C * 08:02. Still, ΔNPM1 as a clonal driver mutation occurring early in leukemogenesis remains an attractive target for immunotherapy. ΔNPM1 is also an ideal target based on its high mutation frequency in 30% of primary AML 6 . The characteristic 4bp frameshift insertion occurs at a limited number of positions (859, 860, and 861) in the coding sequence, and although the exact 4bp sequence can vary, most of the mutations encode the same 11-amino acid alternative reading frame (CLAVEEVSLRK (SEQ ID NO: 27)). We have *A TCR targeting the first 9 residues of this alternative reading frame at 02:01 was identified. This TCR can be used for future gene therapy to treat AML patients with ΔNPM1. Clinical studies will show whether there is any significance in the immune escape due to the loss of HLA-A * 02:01 in preventing long-term remission of AML.

[0441] The TCR isolated from clone 1A2 was shown to mediate specific recognition and lysis of HLA-A * 02:01-positive AML cells, demonstrating that this TCR can redirect immunoreactivity in an HLA co-receptor-independent manner to AML. The critical role of CD4 cells in anti-tumor immunity has become clearer over the past few decades. Conventionally, CD4 cells are known to assist CD8 cells, leading to improved tumor clearance and induction of 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 transplantation or donor lymphocyte infusion developed a graft-versus-leukemia response similar to that of patients who received non-modified stem cell transplantation or donor lymphocytes, but the incidence and severity of graft-versus-host disease were reduced. In the autologous setting, adoptive transfer of CD4 cells directed against HLA class II-restricted tumor-associated antigens or neoantigens resulted in tumor shrinkage 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 is preferred and may result in superior anti-tumor immunity. Indeed, Turtle et al. 19、20It has been demonstrated that administration of defined ratios of CD8 and CD4 cells expressing the same CD19-specific chimeric antigen receptor results in complete remission in a significant number of patients with relapsed or refractory B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. Similarly, adoptive transfer of CD8 and CD4 cells expressing the same TCR directed against an HLA class I-restricted epitope such as CLAVEEVSL (SEQ ID NO: 1) may result in potent antitumor immunity, as previously demonstrated in mice.

[0442] In recent years, ΔNPM1 has been described as a reliable marker for measuring minimal residual disease in patients with AML. 21 In the peripheral blood of patients after chemotherapy, the persistence of ΔNPM1 transcripts detected by quantitative RT-PCR was associated with disease relapse within 3 years of follow-up. The prognostic value of ΔNPM1 was shown to be independent of other risk factors such as the presence of FLT3-ITD or mutated DNA methyltransferase 3α (DNMT3A). 1、2、5、13 Crucially, the authors reported that the presence of ΔNPM1 transcripts after the second chemotherapy cycle in patients with a favorable molecular signature (without FLT3-ITD or mutated DNMT3A) at diagnosis characterized a group of patients with relatively poor outcomes, whereas the absence of ΔNPM1 transcripts after the second chemotherapy cycle in patients with an unfavorable molecular profile (FLT3-ITD, mutated DNMT3A, or both) distinguished patients with relatively good prognoses. 21Therefore, ΔNPM1 as a marker of disease state can be used to select patients eligible for alloSCT, enabling optimal timing and patient selection for ΔNPM1 TCR gene therapy for treating persistent or relapsed disease after chemotherapy. Ultimately, if clinical studies show that AML can be effectively treated by ΔNPM1 TCR gene transfer with a low treatment-related mortality rate, it may replace alloSCT as the standard therapy for patients with ΔNPM1 AML having a poor prognosis based on deleterious molecular abnormalities at diagnosis or detectable persistent or relapsed disease after chemotherapy, thereby potentially improving the overall survival of AML patients.

[0443] Example 2 Healthy individuals were screened according to the same strategy as described for T cell clone 1A2 recognizing CLAVEEVSL (SEQ ID NO: 1) at HLA-A * 03:01 or for T cell clones recognizing AVEEVSLRK (SEQ ID NO: 26) or CLAVEEVSLRK (SEQ ID NO: 27) at HLA-A * 11:01. PE-labeled pHLA-tetramers were generated for AVEEVSLRK (SEQ ID NO: 26) at HLA-A * 02:01, for CLAVEEVSLRK (SEQ ID NO: 27) at HLA-A * 03:01, for AVEEVSLRK (SEQ ID NO: 26) at HLA-A * 03:01, for CLAVEEVSLRK (SEQ ID NO: 27) at HLA-A * 11:01, and for AVEEVSLRK (SEQ ID NO: 26) and CLAVEEVSLRK (SEQ ID NO: 27) at HLA-A * 11:01. Peripheral blood mononuclear cells from healthy individuals were incubated with a mixture of the 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 transduced with untransduced or HLA-A * 03:01 or HLA-A *The reactivity against either OCI-AML2 (wild-type NPM1) or OCI-AML3 (mutant NPM1) AML cell lines transduced with 11:01 was analyzed by IFN-γ ELISA. HLA-A * 03:01 or HLA-A * T cell clones selectively recognizing OCI-AML3 transduced with 11:01 were analyzed by flow cytometry for pHLA-tetramer staining and re-analyzed by IFN-γ ELISA for reactivity against T2 cells pulsed with titrated concentrations of the peptide of interest and transduced with the same panel of AML cell lines and related HLA-restricted alleles.

[0444] HLA-A * 03:01 and / or HLA-A * A total of 42 healthy individuals positive for 11:01 were screened. HLA-A * 03:01 or HLA-A * OCI-AML3 transduced with 11:01 and HLA-A * 03:01 or HLA-A * Three pHLA tetramer-positive T cell clones capable of specifically reacting against peptide-pulsed T2 cells transduced with 11:01 were isolated. T cell clone 31.3.F1 is specific for AVEEVSLRK (SEQ ID NO: 26) in HLA-A * 03:01 (Figure 40A), and T cell clones 6F11 and 26.2.D6 are specific for AVEEVSLRK (SEQ ID NO: 26) in HLA-A * 11:01 (Figure 40B). All three T cell clones were able to react against primary AML with mutant NPM1 expressing the related HLA-restricted alleles, but did not react against AML cases with wild-type NPM1. T cell clones 31.3.F1 and 6F11 were isolated from individuals positive for the related HLA-A * 03:01 and HLA-A * 11:01 restricted alleles, respectively, whereas T cell clone 26.2.D6 was isolated from an HLA-A * 11:01 negative individual.

[0445] Similar to T cell clone 1A2, the 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. The human TCR α and β variable regions were sequenced and cloned into MP71-flex containing pre-cloned mouse TCR α and β constant genes. CD8 + cells were isolated from healthy individuals positive for HLA-A * 03:01 or HLA-A * 11:01 and stimulated with PHA, IL-2, and autologous irradiated peripheral blood mononuclear cells as feeder cells. On day 2, CD8+ cells were transduced with the TCR for mutant NPM1. As a control, CD8+ cells were transduced with the HLA-A * 03:01-restricted TCR for EBNA3A or the HLA-A * 11:01-restricted TCR for EBNA3B. On day 10, TCR-transduced T cells (TCR-T) were stained with an APC-labeled antibody against mouse TCR-Cβ and isolated 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 for reactivity against OCI-AML2 (wild-type NPM1) and OCI-AML3 (mutant NPM1) cell lines transfected with HLA-A * 03:01 or HLA-A * 11:01 by IFN-γ ELISA. 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 against OCI-AML3 transfected with HLA-A * 03:01 (Figure 41A). The TCRs of clones 6F11 and 26.2.D6 showed specific binding to the pHLA-A * 11:01-AVEEVSLRK tetramer and reactivity against HLA-A *It showed specific reactivity against OCI-AML3 transfected with 11:01 (Figs. 41B and 41C).

[0446] For dNPM1-A11 TCR-T cells, T cell clones 26.2.D6 and 6F11 and the corresponding TCR-T cells were compared for their in vitro reactivity against primary human AML samples and in vivo reactivity against the AML cell line OCI-AML3 in NSG mice. The T cell clone and TCR of clone 26.2.D6 (isolated from an HLA-A * 11:01-negative donor) are particularly advantageous. This was most clearly demonstrated in NSG mice in which 26.2.D6 TCR-T cells induced an anti-tumor effect against OCI-AML3 similar to that of the HLA-A * 02:01-restricted TCR-T cells, and this anti-tumor effect was superior to that observed for 6F11 TCR-T cells.

[0447] For dNPM1-A3 TCR-T cells, the T cell clone 31.3.F1 is also particularly advantageous due to its specificity for AVEEVSLRK at A * 03:01. Clone 31.3.F1 clearly reacts against primary human AML samples in vitro.

[0448] Additional sequences SEQ ID NOs: 35 to 50 relate to the novel dNPM1-A * 11:01 TCR (clone 26.2.D6) (TCR alpha chain - TRAV5 * 01, TRAJ26 * 01; TCR beta chain - TRBV19 * 03, TRBJ2-7 * 01).

[0449] SEQ ID NO: 35 - Amino acid sequence of CDR3 (TCR alpha chain, clone 26.2.D6): CAESKGQNFVF

[0450] SEQ ID NO: 36 - Nucleic acid sequence encoding CDR3 (TCR alpha chain, clone 26.2.D6): TGTGCAGAGAGTAAAGGTCAGAATTTTGTCTTT

[0451] Amino acid sequence of the variable region of the α-chain, clone 26.2.D6, SEQ ID NO: 37: GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESKGQNFVFGPGTRLSVLP

[0452] Nucleic acid sequence encoding the variable region of the α-chain, clone 26.2.D6, SEQ ID NO: 38: GGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAGTAAAGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCC

[0453] Amino acid sequence of CDR1 (TCR α-chain, clone 26.2.D6), SEQ ID NO: 39: DSSSTY

[0454] Nucleic acid sequence encoding CDR1 (TCR α-chain, clone 26.2.D6), SEQ ID NO: 40: GACAGCTCCTCCACCTAC Amino acid sequence of CDR2 (TCR α-chain, clone 26.2.D6), SEQ ID NO: 41: IFSNMDM

[0455] Nucleic acid sequence encoding CDR2 (TCR α-chain, clone 26.2.D6), SEQ ID NO: 42: ATTTTTTCAAATATGGACATG

[0456] Amino acid sequence of SEQ ID NO: 43 - CDR3 (TCR β chain, clone 26.2.D6): CASTTWGTGGHEQYF

[0457] Nucleic acid sequence encoding SEQ ID NO: 44 - CDR3 (TCR β chain, clone 26.2.D6): TGTGCCAGTACTACATGGGGGACAGGGGGCCACGAGCAGTACTTC Amino acid sequence of SEQ ID NO: 45 - Variable region of β chain, clone 26.2.D6: DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSHIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASTTWGTGGHEQYFGPGTRLTVT

[0458] Nucleic acid sequence encoding SEQ ID NO: 46 - Variable region of β chain, clone 26.2.D6: GATGGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACTACTCACACATAGTAAATGACTTTCAGAAAGGAGATATAGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCTCACTGTGACATCGGCCCAAAAGAACCCGACAGCTTTCTATCTCTGTGCCAGTACTACATGGGGGACAGGGGGCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA

[0459] Amino acid sequence of SEQ ID NO: 47 - CDR1 (TCR β chain, clone 26.2.D6): LNHDA

[0460] Nucleic acid sequence encoding SEQ ID NO:48 - CDR1 (TCR β chain, clone 26.2.D6): TTGAACCACGATGCC

[0461] Amino acid sequence of SEQ ID NO:49 - CDR2 (TCR β chain, clone 26.2.D6): SHIVND

[0462] Nucleic acid sequence encoding SEQ ID NO:50 - CDR2 (TCR β chain, clone 26.2.D6): TCACACATAGTAAATGAC

[0463] SEQ ID NOs:51 - 66 are 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).

[0464] Amino acid sequence of SEQ ID NO:51 - CDR3 (TCR α chain, clone 6F11): CAVSPAGNQFYF

[0465] Nucleic acid sequence encoding SEQ ID NO:52 - CDR3 (TCR α chain, clone 6F11): TGTGCTGTGAGTCCCGCCGGTAACCAGTTCTATTTT

[0466] Amino acid sequence of SEQ ID NO:53 - variable region of α chain, clone 6F11: AQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVKGINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVSPAGNQFYFGTGTSLTVIP

[0467] Nucleic acid sequence encoding SEQ ID NO:54 - variable region of α chain, clone 6F11: GCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGAGTCCCGCCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAG

[0468] SEQ ID NO: 55 - Amino acid sequence of CDR1 (TCR α chain, clone 6F11): SSVSVY

[0469] SEQ ID NO: 56 - Nucleic acid sequence encoding CDR1 (TCR α chain, clone 6F11): TCGTCTGTTTCAGTGTAT

[0470] SEQ ID NO: 57 - Amino acid sequence of CDR2 (TCR α chain, clone 6F11): YLSGSTLV

[0471] SEQ ID NO: 58 - Nucleic acid sequence encoding CDR2 (TCR α chain, clone 6F11): TATTTATCAGGATCCACCCTGGTT

[0472] SEQ ID NO: 59 - Amino acid sequence of CDR3 (TCR β chain, clone 6F11): CASSLGSNQPQHF

[0473] SEQ ID NO: 60 - Nucleic acid sequence encoding CDR3 (TCR β chain, clone 6F11): TGTGCCAGCAGCCTGGGTAGCAATCAGCCCCAGCATTTT

[0474] SEQ ID NO: 61 - Amino acid sequence of the variable region of the β chain, clone 6F11: GAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLGSNQPQHFGDGTRLSIL

[0475] Nucleotide sequence encoding the β-chain variable region, clone 6F11, SEQ ID NO: 62: GGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCCCAACAAGACAAATCAGGGCTGCCCAATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATCCAGCGCACAGAGCAGCGGGACTCGGCCATGTATCGCTGTGCCAGCAGCCTGGGTAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTA

[0476] Amino acid sequence of CDR1 (TCR β-chain, clone 6F11), SEQ ID NO: 63: SGHVS Nucleotide sequence encoding CDR1 (TCR β-chain, clone 6F11), SEQ ID NO: 64: TCGGGTCATGTATCC

[0477] Amino acid sequence of CDR2 (TCR β-chain, clone 6F11), SEQ ID NO: 65: FNYEAQ

[0478] Nucleotide sequence encoding CDR2 (TCR β-chain, clone 6F11), SEQ ID NO: 66: TTCAATTATGAAGCCCAA

[0479] SEQ ID NOS: 67 to 82 are 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 * Regarding 01).

[0480] Amino acid sequence of SEQ ID NO: 67 - CDR3 (TCR α chain, clone 31.3.F1): CALSGGGQNFVF

[0481] Nucleic acid sequence encoding SEQ ID NO: 68 - CDR3 (TCR α chain, clone 31.3.F1): TGTGCCCTCTCCGGCGGGGGTCAGAATTTTGTCTTT Amino acid sequence of SEQ ID NO: 69 - variable region of α chain, clone 31.3.F1: QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCALSGGGQNFVFGPGTRLSVLP

[0482] Nucleic acid sequence encoding SEQ ID NO: 70 - variable region of α chain, clone 31.3.F1: CAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCCTCTCCGGCGGGGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCC

[0483] Amino acid sequence of SEQ ID NO: 71 - CDR1 (TCR α chain, clone 31.3.F1): DRGSQS

[0484] Nucleic acid sequence encoding SEQ ID NO:72 - CDR1 (TCR α chain, clone 31.3.F1): GACCGAGGTTCCCAGTCC

[0485] Amino acid sequence of SEQ ID NO:73 - CDR2 (TCR α chain, clone 31.3.F1): IYSNGD Nucleic acid sequence encoding SEQ ID NO:74 - CDR2 (TCR α chain, clone 31.3.F1): ATATACTCCAATGGTGAC

[0486] Amino acid sequence of SEQ ID NO:75 - CDR3 (TCR β chain, clone 31.3.F1): CASSQGSGFRHF

[0487] Nucleic acid sequence encoding SEQ ID NO:76 - CDR3 (TCR β chain, clone 31.3.F1): TGTGCCAGCAGCCAAGGATCAGGTTTTCGGCATTTT

[0488] Amino acid sequence of SEQ ID NO:77 - variable region of β chain, clone 31.3.F1: EAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELE...

Claims

1. (a) A polypeptide comprising CDR3, a TCR α-chain polypeptide that specifically binds to peptide AVEEVSLRK (SEQ ID NO: 26), (b) A polypeptide containing CDR3 of a TCR β chain polypeptide that specifically binds to peptide AVEEVSLRK (SEQ ID NO: 26) An isolated nucleic acid sequence encoding, wherein (a) and (b) both specifically bind to the peptide AVEEVSLRK (SEQ ID NO: 26), (i) The CDR3 in (a) has the amino acid sequence of SEQ ID NO: 35, and the CDR3 is located within the TCR α chain variable region that specifically binds to SEQ ID NO:

26. (ii)(b) The CDR3 has the amino acid sequence of SEQ ID NO: 43, and the CDR3 is located within the TCR β chain variable region that specifically binds to SEQ ID NO:

26. Isolated nucleic acid sequence.

2. (i) The CDR3 in (a) is located within a TCR α chain variable region having at least 90% sequence identity with SEQ ID NO: 37, (a) may also include a TCR α chain constant region, and furthermore, 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. (ii) The CDR3 in (b) is located within a TCR β-chain variable region having at least 90% sequence identity with SEQ ID NO: 45, and (b) may also include a TCR β-chain constant region. Furthermore, 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. The isolated nucleic acid sequence according to claim 1.

3. The isolated nucleic acid sequence according to claim 1, wherein the nucleic acid sequence encodes a T cell receptor.

4. A vector comprising the nucleic acid sequence described in any one of claims 1 to 3.

5. The vector according to claim 4, wherein the vector is a plasmid or a viral vector, and the vector may be selected from the group consisting of retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, vaccinia viruses, canary poxviruses, herpesviruses, minicircle vectors, and synthetic DNA or RNA.

6. Modified cells transfected or transfected with a nucleic acid sequence according to any one of claims 1 to 3 or a vector containing a nucleic acid sequence according to any one of claims 1 to 3.

7. The modified cells according to claim 6, wherein the modified cells are 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 the modified cells may be human cells.

8. (a) an isolated nucleic acid sequence encoding a polypeptide comprising CDR3 of a TCR α chain polypeptide that specifically binds to peptide AVEEVSLRK (SEQ ID NO: 26), (b) Isolated nucleic acid sequences encoding a polypeptide containing CDR3, a TCR β-chain polypeptide that specifically binds to peptide AVEEVSLRK (SEQ ID NO: 26), and A pharmaceutical composition comprising (a) and (b), wherein both are specifically bound to the peptide AVEEVSLRK (SEQ ID NO: 26), (i) The CDR3 in (a) has the amino acid sequence of SEQ ID NO: 35, and the CDR3 is located within the TCR α chain variable region that specifically binds to SEQ ID NO:

26. (ii)(b) The CDR3 has the amino acid sequence of SEQ ID NO: 43, and the CDR3 is located within the TCR β chain variable region that specifically binds to SEQ ID NO:

26. Pharmaceutical composition.

9. A pharmaceutical composition for treating or preventing ΔNPM1-positive hematological malignancies, (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, (b) pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers A pharmaceutical composition containing the following:

10. Use of the pharmaceutical composition according to claim 9 in the manufacture of a pharmaceutical for treating or preventing ΔNPM1-positive hematological malignancies in human subjects.

11. The use according to claim 10, wherein the hematological malignancy is a myeloid malignancy, and the myeloid malignancy may be acute myeloid leukemia.

12. The use of the pharmaceutical composition according to claim 10, wherein the pharmaceutical composition induces or enhances a cell-mediated immune response in the subject.

13. The pharmaceutical composition according to claim 9 for use in the treatment or prevention of ΔNPM1-positive hematological malignancies in human subjects.

14. The pharmaceutical composition for use according to claim 13, wherein the hematological malignancy is a myeloid malignancy, and the myeloid malignancy may be acute myeloid leukemia.

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

16. A method for generating a T cell receptor, comprising contacting a nucleic acid sequence according to any one of claims 1 to 3 with a cell under conditions in which the nucleic acid sequence is incorporated and expressed by the cell, in order to generate a T cell receptor that specifically binds to the peptide of SEQ ID NO:

26.

17. The method according to claim 16, which is an exovivo method.

18. Use of the pharmaceutical composition according to claim 8 in the manufacture of a pharmaceutical for treating or preventing ΔNPM1-positive hematological malignancies in human subjects.

19. The pharmaceutical composition according to claim 8 for use in the treatment or prevention of ΔNPM1-positive hematological malignancies in human subjects.

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