Treatment of hematopoietic malignancies

Abstract

Novel nucleic acid sequences, vectors, modified cells, peptides and pharmaceutical compositions are provided that are useful for treating human subjects with ΔNPM1-positive hematopoietic tumors. Corresponding methods and uses are also provided.

Description

[Technical Field]

[0001] Novel nucleic acid sequences, vectors, modified cells, peptides and pharmaceutical compositions useful for treating human subjects with ΔNPM1-positive hematopoietic malignancies are provided. Corresponding methods and uses are also provided. [Background technology]

[0002] background Hematopoietic tumors are cancers that affect the blood and lymphatic system. Cancers can begin in blood-forming tissues (e.g., bone marrow) or in cells of the immune system. Examples of hematopoietic tumors include myeloid tumors, such as acute myeloid leukemia (AML).

[0003] Acute myeloid leukemia (AML) is a malignant disease of the bone marrow characterized by the accumulation of differentiated myeloid progenitor cells. Currently, standard treatment involves induction chemotherapy followed by intensive consolidation chemotherapy or high-dose therapy and autologous or allogeneic hematopoietic stem cell transplantation (alloSCT), resulting in a 5-year survival rate of 40-45% in patients aged ≤65 years and only 10% in patients aged >65 years. 1-2 Although alloSCT is associated with a low relapse rate, 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. Relapse occurs within 3 years after chemotherapy in the majority of patients, and there is an urgent need for novel targeted therapies with high efficacy and no or limited toxicity to treat and improve survival in patients with AML. 2 .

[0004] Molecular characterization of AML has accelerated in recent decades. Whole genome and exome sequencing indicates that AML has a low mutational burden, with an average of 13 coding region mutations per patient. For cancer types with a high mutational burden, such as melanoma and lung cancer, a small proportion of somatic mutations have been shown to encode neoantigens. 3Neoantigens are peptides resulting from tumor-specific DNA mutations that can be recognized by specific T cells when presented on tumor cells under the influence of HLA. The formation of these antigens is a stochastic process, with each additional mutation increasing the chance of generating a neoantigen. Due to the low mutational burden in AML, the number of neoantigens is presumed to be limited. 3 .

[0005] Neoantigens can serve as in vivo cancer rejection antigens following treatment with checkpoint inhibitors or adoptive transfer of in vitro expanded tumor-infiltrating lymphocytes (TILs) 3-4 Checkpoint inhibitors are antibodies that block T cell inhibitory signals mediated by CTLA-4 (ipilimumab) or PD-1 (pembrolizumab and nivolumab), thereby promoting the immune system's targeting of neoantigens. Checkpoint inhibitors and TIL treatments have proven successful in tumors with high mutational burden but are ineffective in tumors with low mutational burden. However, although the overall mutational burden in AML is low, somatic aberrations often occur in a limited number of driver genes that are recurrently mutated in multiple patients. 5 Consequently, neoantigens arising from recurrent mutations in AML are suitable for the development of targeted immunotherapies. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for new immunotherapies to treat hematopoietic malignancies, including myeloid malignancies such as AML. [Means for solving the problem]

[0007] Summary of the Invention The present inventors have identified a mutant form of nucleophosmin (ΔNPM1 or NPM1 mut ) recognized that the formation of this mutant protein is restricted to malignant hematopoietic cells and therefore represents an ideal target for immunotherapy of hematopoietic malignancies such as myeloid tumors (particularly AML).

[0008] Nucleophosmin (NPM1) is a driver gene that is frequently mutated in approximately 30% of patients with AML. 5 Mutant NPM1 has also been observed in other types of hematopoietic malignancies (e.g., other myeloid tumors), but is much less common in tumors other than AML. mut Patients with ΔNPM1 carry a characteristic four-base pair (4-bp) frameshift insertion in exon 12 of the gene. The resulting ΔNPM1 protein is four amino acids (AA) longer than its wild-type counterpart, and its C-terminal 11 AA are translated into an alternative reading frame (CLAVEEVSLRK). As a result, the ΔNPM1 protein translocates from the nucleolus, where it functions as a nucleocytoplasmic shuttle protein, to the cytoplasm. 6 The ΔNPM1 protein is thus localized intracellularly, however, HLA-restricted ΔNPM1-derived peptides are accessible at the cell surface to T cell receptors and can therefore be recognized by T cells.

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

[0010] Advantageously, T cells expressing TCRs specific for peptides selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29) can be used as an effective immunotherapy for the treatment of ΔNPM1-positive AML. A TCR gene transfer approach using these peptide-specific TCRs may therefore provide a novel treatment modality for patients with ΔNPM1-positive AML.

[0011] The present inventors have demonstrated for the first time that CLAVEEVSL binds to HLA-A * The peptide was shown to be presented on the surface of primary AML cells isolated from patients with 02:01-positive AML. Advantageously, the peptide therefore binds to HLA-A * The modified cells described herein can be used as therapeutic agents (e.g., vaccines) to treat or prevent ΔNPM1-positive AML in 02:01-positive human patients, or as target antigens for the treatment of patients with such cells (e.g., peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TILs) bearing T cell receptors that specifically recognize CLAVEEVSL).

[0012] HLA-A * To investigate whether T cells bearing T cell receptors (TCRs) specific for CLAVEEVSL presented under the influence of 02:01 are present in the T cell repertoire from healthy individuals, HLA-A * The 02:01 tetramer was generated for CLAVEEVSL and its 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 and HLA-A. * The most reactive clone (1A2) was sequenced for T cell receptors and showed recognition of CD8 + and CD4 + T cells, which express HLA-A in a co-receptor-independent manner. *Specific recognition and lysis of 02:01-positive primary AML cells by ΔNPM1 was demonstrated.

[0013] The inventors have therefore identified a TCR that specifically binds to the neoantigen CLAVEEVSL.

[0014] Advantageously, T cells expressing TCR specific for CLAVEEVSL are therefore HLA-A positive for ΔNPM1-positive AML. * Therefore, a TCR gene transfer approach using a CLAVEEVSL-specific TCR can be used as an effective immunotherapy in the treatment of HLA-A positive patients with ΔNPM1-positive AML. * This may lead to novel treatment modalities for 02:01 positive patients.

[0015] Furthermore, the peptide CLAVEEVSL (and in particular its cysteinylated form, i.e., C * LAVEEVSL) and HLA-A * The peptides themselves can therefore also be useful, for example, in isolated form or when formulated as pharmaceutical compositions.

[0016] By studying the HLA class I ligandome of primary AML, we also identified additional 9-mer and 11-mer peptides encoded by alternative reading frames of ΔNPM1 (AVEEVSLRK and CLAVEEVSLRK, respectively). * 03:01 and HLA-A * The binding of each of AVEEVSLRK and CLAVEEVSLRK to 11:01 was confirmed by monomer folding to produce tetramers (as detailed herein for the CLAVEEVSL peptide). * 03:01 or HLA-A *These peptides can be used as therapeutic agents (e.g., vaccines) to treat or prevent ΔNPM1-positive AML in 11:01-positive human patients. Alternatively, each of these peptides can be used as a target antigen for the treatment of patients who have the modified cells described herein (e.g., peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TILs) bearing T cell receptors that specifically recognize AVEEVSLRK or CLAVEEVSLRK, respectively).

[0017] Monomer folding for tetramer production also involves HLA-A * AVEEVSLRK using 01:01 has been well documented (as detailed here for the CLAVEEVSL peptide). * Its ability to bind to 01:01 has therefore been confirmed. AVEEVSLRK also binds to HLA-A * 03:01 and HLA-A * HLA-A lacking 11:01 * It has also been identified in the HLA class I ligandome from a 01:01-positive AML subject (AML4443) (see Figure 2). Thus, the AVEEVSLRK peptide also has a similar affinity to HLA-A * The peptides can be used as therapeutic agents (e.g., vaccines) to treat or prevent ΔNPM1-positive AML in 01:01-positive human patients. Alternatively, the peptides can be used as target antigens for the treatment of patients with the modified cells described herein (e.g., peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TILs) bearing T cell receptors that specifically recognize AVEEVSLRK).

[0018] HLA-A * 03:01 or HLA-A * To investigate whether T cells bearing T cell receptors (TCRs) specific for AVEEVSLRK presented under the influence of 11:01 are present in the T cell repertoire from healthy individuals, we performed HLA-A * 03:01Tetramers and HLA-A *11:01 tetramers were generated for AVEEVSLRK, 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, one of which was HLA-A * It was shown that the T cell clone specifically binds to AVEEVSLRK under the influence of 03:01 (reactive clone (3B3); Figure 37). Furthermore, one T cell clone was found to be HLA-A * These clones were shown to have specific binding to AVEEVSLRK under the influence of 11:01 (reactive clone (6F11); Figure 37). The reactivity of each of these clones (6F11 and 3B3) was also tested (Figures 38 and 39), where cytokine release was demonstrated by each clone when present with the ΔNPM1 peptide under the influence of the appropriate HLA-A alone.

[0019] We therefore propose that HLA-A * 03:01 (TCR from clone 3B3) or HLA-A * Two TCRs were identified that specifically bound to the neoantigen AVEEVSLRK under the influence of 11:01 (TCR from clone 6F11).

[0020] Advantageously, T cells expressing TCR specific for AVEEVSLRK are therefore HLA-A positive for ΔNPM1-positive AML. * 03:01, HLA-A * 11:01 or HLA-A * A TCR gene transfer approach using AVEEVSLRK-specific TCRs can therefore be used as an effective immunotherapy in the treatment of HLA-A 01:01-positive patients with ΔNPM1-positive AML. * 03:01, HLA-A * 11:01 or HLA-A * This may lead to novel treatment modalities for 01:01 positive patients.

[0021] Furthermore, the peptide AVEEVSLRK binds to HLA-A * 03:01, HLA-A * 11:01 or HLA-A *It can be used as a therapeutic agent (e.g., a vaccine) for the treatment or prevention of ΔNPM1-positive AML in 01:01-positive patients. The peptide itself is therefore also useful, for example, in isolated form or when formulated as a pharmaceutical composition.

[0022] The inventors have demonstrated that the peptide CLAVEEVSLRK (SEQ ID NO: 27) binds to HLA-A * 03:01 or HLA-A * 11:01. Therefore, specific binding to any of these peptides can occur under the influence of the appropriate HLA (i.e., specific binding to a peptide can only occur when presented by the appropriate HLA, as described above).

[0023] HLA-A * To investigate whether T cells bearing T cell receptors (TCRs) specific for CLAVEEVSLRK presented under the influence of 03:01 are present in the T cell repertoire from healthy individuals, HLA-A * The 03:01 tetramer was generated for CLAVEEVSLRK and its cysteinylation 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, one of which was HLA-A. * Under the influence of 03:01 C * It was identified as having specific binding to LAVEEVSLRK (reactive clone (1F2); Figure 37).

[0024] We therefore propose that HLA-A * Neoantigen C at 03:01 * A TCR (TCR from clone 1F2) that specifically binds to LAVEEVSLRK was identified.

[0025] Advantageously, CLAVEEVSLRK (and in particular the cysteinylated form, i.e. C * T cells expressing TCR specific for ΔNPM1-positive AML are therefore HLA-A positive. * 03:01 or HLA-A *It can be used as an effective immunotherapy in the treatment of 11:01-positive patients. * Therefore, a TCR gene transfer approach using a (LAVEEVSLRK)-specific TCR is suitable for patients with HLA-A AML who have ΔNPM1-positive AML. * 03:01 and HLA-A * This may lead to novel treatment modalities for 11:01-positive patients.

[0026] Furthermore, the peptide CLAVEEVSLRK (and in particular its cysteinylated form, i.e., C * LAVEEVSLRK) to HLA-A * 03:01 or HLA-A * It can be used as a therapeutic agent (e.g., a vaccine) to treat or prevent ΔNPM1-positive AML in 11:01-positive patients. The peptide itself is therefore also useful, for example, in isolated form or when formulated as a pharmaceutical composition.

[0027] The present invention has specific application to the treatment of patients with ΔNPM1-positive AML. However, ΔNPM1 is also present in a subset of patients with other forms of hematopoietic malignancies, particularly myeloid malignancies. The present invention is therefore equally applicable to patients with ΔNPM1-positive hematopoietic malignancies, such as, but not limited to, myeloid malignancies (e.g., AML).

[0028] Thus, in one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) a polypeptide comprising a 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 a 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). An isolated nucleic acid sequence encoding

[0029] The nucleic acid sequence can encode both (a) and (b), where (a) and (b) together specifically bind to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29).

[0030] The encoded polypeptide may specifically bind to CLAVEEVSL (SEQ ID NO: 1). The peptide may be in a cysteinylated form. The encoded polypeptide may therefore be C * It can specifically bind only to LAVEEVSL (cysteinylated form).

[0031] Alternatively, the encoded polypeptide may specifically bind to AVEEVSLRK (SEQ ID NO:26).

[0032] Alternatively, the encoded polypeptide may specifically bind to CLAVEEVSLRK (SEQ ID NO: 27). The peptide may be in a cysteinylated form. The encoded polypeptide may therefore be C * It can specifically bind only to LAVEEVSLRK (cysteinylated form).

[0033] CDR3 of (a) may have an amino acid sequence having at least 90% sequence identity to CAVTGARLMF (SEQ ID NO: 2). Optionally, CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0034] CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity to CASSPGGLSNEQF (SEQ ID NO: 5). Optionally, CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0035] The CDR3 of (a) may be within a TCR alpha chain variable region 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).

[0036] (a) may further comprise a TCR α chain constant region. In other words, the polypeptide of (a) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to the selected peptide.

[0037] The TCR alpha chain variable region may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8. Optionally, the TCR alpha chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a genetically modified 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 (b) may be within a TCR β chain variable region 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] (b) may further comprise a TCR β chain constant region. In other words, the polypeptide of (b) may comprise a full-length TCR α chain variable region and a full-length TCR α chain constant region that specifically binds to the selected peptide.

[0040] The TCR β chain variable region of (b) may have an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. Optionally, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneracy of the genetic code).

[0041] The CDR3 of (a) may be within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 8, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 2. Optionally, (a) comprises a TCR alpha chain constant region.

[0042] In any of the embodiments described herein, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO:14 and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO:15.

[0043] The CDR3 of (b) may be within a TCR β chain variable region having at least 90% sequence identity to SEQ ID NO: 11, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 5. Optionally, (b) comprises a TCR β chain constant region.

[0044] 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.

[0045] The selected peptide CLAVEEVSL (SEQ ID NO: 1) can be cysteinylated.

[0046] The selected peptide CLAVEEVSLRK (SEQ ID NO: 27) can be cysteinylated.

[0047] The nucleic acid sequence may encode a T cell receptor.

[0048] For the avoidance of doubt, we have determined that the peptide CLAVEEVSL (SEQ ID NO: 1) binds to HLA-A *Presented by 02:01 (i.e., HLA-A * Furthermore, the present inventors have identified that the peptides AVEEVSLRK and CLAVEEVSLRK each bind to HLA-A. * 03:01 or HLA-A * Presented by 11:01, AVEEVSLRK also has HLA-A * 01:01. Therefore, specific binding to any of these peptides can occur with the appropriate HLA (i.e., specific binding to the peptide can only occur when presented by the appropriate HLA, as described above).

[0049] The nucleic acid sequence of the present invention can be a non-naturally occurring nucleic acid sequence (e.g., the entire sequence can not occur in nature in its entirety).For example, the nucleic acid sequence of the present invention can be operably linked to a promoter, where the promoter is not naturally associated with the equivalent human nucleic acid sequence in nature (e.g., a human TCR sequence or a fragment thereof); that is, it is not the entire promoter naturally associated with the nucleic acid in its natural environment.In this situation, such a promoter can be considered an exogenous promoter.Examples of suitable promoters are described elsewhere.

[0050] In a further aspect, the present invention provides a vector comprising a nucleic acid sequence of the present invention.

[0051] The vector can 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, as described above, is operably linked.

[0052] In a further aspect, the present invention provides modified cells transfected or transduced with a nucleic acid sequence of the invention or a vector of the invention.

[0053] The transfected or transduced nucleic acid sequence of the invention or the vector of the invention may be operably linked to a promoter, as described above.

[0054] Modified cells include CD8 T cells, CD4 T cells, NK cells, NKT cells, gamma-delta T cells, hematopoietic stem cells, and progenitor cells. cell, It may be selected from the group consisting of a T cell line or an NK-92 cell line.

[0055] The modified cells can be human cells.

[0056] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (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) An isolated peptide is provided, comprising an amino acid sequence selected from:

[0057] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0058] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0059] The peptide may have 20 or fewer amino acids.

[0060] The peptide is (i) SEQ ID NO: 1 (wherein the cysteine ​​amino acid may or may not be cysteinylated); (ii) SEQ ID NO: 26; (iii) 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 The sequence may consist of a sequence selected from:

[0061] In certain embodiments, the peptide may consist of the sequence of SEQ ID NO: 1, wherein the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0062] In certain embodiments, the peptide may consist of the sequence of SEQ ID NO: 27, wherein the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0063] In a further aspect, the present invention provides an isolated nucleic acid sequence encoding a peptide of the present invention.

[0064] In a further aspect, the present invention provides a vector comprising a nucleic acid sequence of the present invention. R .

[0065] In a further aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid sequence of the invention, a vector of the invention, a modified cell of the invention or an isolated peptide of the invention and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0066] A pharmaceutical composition can be formulated as a vaccine when the composition comprises an isolated peptide of the present invention (or a nucleic acid or vector encoding the isolated peptide). Suitable vaccine formulations for peptides and nucleic acids are well known in the art.

[0067] In a further aspect, the present invention provides a method for treating or preventing a ΔNPM1-positive hematopoietic tumor in a human subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0068] A method for treating or preventing ΔNPM1-positive hematopoietic tumors in a human subject may include administering to the subject a therapeutically effective amount of a peptide described herein (or a nucleic acid (e.g., RNA or DNA) or vector encoding the peptide).

[0069] For example, (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) An isolated peptide comprising or consisting of an amino acid sequence selected from may be administered as an immunotherapy (eg, as a vaccine).

[0070] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0071] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0072] The hematopoietic malignancy can be a bone marrow malignancy.

[0073] The bone marrow tumor can be acute myeloid leukemia.

[0074] The method may induce or enhance a cell-mediated immune response in a subject.

[0075] For the avoidance of doubt, the inventors have confirmed that the peptide CLAVEEVSL (SEQ ID NO: 1) binds to HLA-A * Furthermore, the present inventors have identified that the peptides AVEEVSLRK and CLAVEEVSLRK are HLA-A 02:01-restrictive. *03:01 and HLA-A * 11:01, and AVEEVSLRK is also HLA-A * 01:01. CLAVEEVSL and / or CLAVEEVSLRK may be particularly in a cysteinylated form.

[0076] Therefore, HLA-A * Methods for treating or preventing ΔNPM1-positive hematopoietic tumors in 02:01-positive human subjects may preferentially use a nucleic acid sequence encoding a polypeptide that specifically binds to CLAVEEVSL, a vector encoding such a nucleic acid sequence, modified cells containing such a nucleic acid sequence or vector, or a pharmaceutical composition comprising a nucleic acid encoding CLAVEEVSL, a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence CLAVEEVSL (all of which are described in more detail elsewhere herein). CLAVEEVSL may particularly be in a cysteinylated form.

[0077] Similarly, HLA-A in human subjects * 03:01 or HLA-A * Methods for treating or preventing ΔNPM1-positive hematopoietic tumors that are 11:01 positive may preferentially use a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK or CLAVEEVSLRK, a vector encoding such a nucleic acid sequence, modified cells containing such a nucleic acid sequence or vector, or a pharmaceutical composition comprising a nucleic acid encoding AVEEVSLRK or CLAVEEVSLRK, a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK or CLAVEEVSLRK (all of which are described in more detail elsewhere herein).

[0078] Furthermore, HLA-A in human subjects *Methods for treating or preventing ΔNPM1-positive hematopoietic tumors that are 01:01 positive may preferentially use a nucleic acid sequence encoding a polypeptide that specifically binds to AVEEVSLRK, a vector encoding such a nucleic acid sequence, modified cells containing such a nucleic acid sequence or vector, or pharmaceutical compositions containing a nucleic acid encoding AVEEVSLRK, a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence AVEEVSLRK (all of which are described in more detail elsewhere herein).

[0079] CLAVEEVSLRK may be in particular a cysteinylated form.

[0080] In a further aspect, the present invention provides a pharmaceutical composition of the invention for use in the treatment or prevention of a ΔNPM1-positive hematopoietic tumor in a human subject.

[0081] Pharmaceutical compositions of the invention for use in treating or preventing ΔNPM1-positive hematopoietic tumors in human subjects may contain a therapeutically effective amount of a peptide described herein (or a nucleic acid (e.g., RNA or DNA) or vector encoding the peptide).

[0082] By way of example, the pharmaceutical composition may comprise: (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) The peptide may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0083] The pharmaceutical composition may be used as an immunotherapy (eg, as a vaccine).

[0084] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0085] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0086] The hematopoietic malignancy can be a bone marrow malignancy.

[0087] The bone marrow tumor can be acute myeloid leukemia.

[0088] The pharmaceutical composition may be for use in eliciting or enhancing a cell-mediated immune response in a subject.

[0089] As mentioned above, the present inventors have demonstrated that the peptide CLAVEEVSL (SEQ ID NO: 1) binds to HLA-A * Furthermore, the present inventors have identified that the peptides AVEEVSLRK and CLAVEEVSLRK are HLA-A 02:01-restrictive. * 03:01 and HLA-A * Presented by 11:01, AVEEVSLRK also has HLA-A * It was identified as being presented by 01:01.

[0090] CLAVEEVSL and / or CLAVEEVSLRK may be particularly in a cysteinylated form.

[0091] Therefore, a pharmaceutical composition comprising a nucleic acid sequence encoding a polypeptide that specifically binds to CLAVEEVSL, 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, a vector encoding such a nucleic acid sequence, or a protein or peptide comprising the sequence CLAVEEVSL (all of which are described in more detail elsewhere herein) can be used to specifically bind to HLA-A *It may be preferentially used in the treatment or prevention of ΔNPM1-positive hematopoietic tumors in 02:01-positive human subjects. CLAVEEVSL may be particularly in a cysteinylated form.

[0092] Similarly, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to AVEEVSLRK or CLAVEEVSLRK, vectors encoding such nucleic acid sequences, modified cells containing such nucleic acid sequences or vectors, or nucleic acids encoding AVEEVSLRK or CLAVEEVSLRK, vectors encoding such nucleic acid sequences, or proteins or peptides comprising the sequence AVEEVSLRK or CLAVEEVSLRK (all of which are described in more detail elsewhere herein) may be used to identify HLA-A * 03:01 or HLA-A * It may be preferentially used in the treatment or prevention of ΔNPM1-positive hematopoietic tumors in human subjects who are 11:01 positive.

[0093] Additionally, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to AVEEVSLRK, vectors encoding such nucleic acid sequences, modified cells containing such nucleic acid sequences or vectors, or nucleic acids encoding AVEEVSLRK, vectors encoding such nucleic acid sequences, or proteins or peptides comprising the sequence AVEEVSLRK (all of which are described in more detail elsewhere herein) can be used to identify HLA-A * It may be preferentially used in the treatment or prevention of ΔNPM1-positive hematopoietic tumors in human subjects who are 01:01 positive.

[0094] CLAVEEVSLRK may be in particular a cysteinylated form.

[0095] In a further aspect, the present invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of a ΔNPM1-positive hematopoietic tumor in a human subject.

[0096] A pharmaceutical composition can include a therapeutically effective amount of a peptide described herein (or a nucleic acid (eg, RNA or DNA) or vector encoding the peptide).

[0097] By way of example, the pharmaceutical composition may comprise: (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) The peptide may comprise an isolated peptide comprising or consisting of an amino acid sequence selected from the group consisting of:

[0098] The pharmaceutical composition may be used as an immunotherapy (eg, as a vaccine).

[0099] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0100] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0101] The hematopoietic malignancy can be a bone marrow malignancy.

[0102] The bone marrow tumor can be acute myeloid leukemia.

[0103] As described elsewhere herein, the inventors have demonstrated that the peptide CLAVEEVSL (SEQ ID NO: 1) binds to HLA-A * Furthermore, the present inventors have identified that the peptides AVEEVSLRK and CLAVEEVSLRK are HLA-A 02:01-restrictive. * 03:01 and HLA-A *Presented by 11:01, AVEEVSLRK also has HLA-A * 01:01. CLAVEEVSL and / or CLAVEEVSLRK may be particularly in a cysteinylated form.

[0104] Thus, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to CLAVEEVSL, vectors encoding such nucleic acid sequences, modified cells containing such nucleic acid sequences or vectors, or isolated peptides comprising the sequence CLAVEEVSL (all of which are described in more detail elsewhere herein) can be used to identify HLA-A * It may be preferentially used for the manufacture of a medicament for the treatment or prevention of ΔNPM1-positive hematopoietic tumors in 02:01-positive human subjects. CLAVEEVSL may particularly be in a cysteinylated form.

[0105] Similarly, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to AVEEVSLRK or CLAVEEVSLRK, vectors encoding such nucleic acid sequences, modified cells containing such nucleic acid sequences or vectors, or isolated peptides comprising the sequence AVEEVSLRK or CLAVEEVSLRK (all of which are described in more detail elsewhere herein) can be used to bind to HLA-A * 03:01 or HLA-A * It may be preferentially used for the manufacture of a medicament for the treatment or prevention of ΔNPM1-positive hematopoietic tumors in human subjects who are 11:01-positive.

[0106] Additionally, pharmaceutical compositions comprising nucleic acid sequences encoding polypeptides that specifically bind to AVEEVSLRK, vectors encoding such nucleic acid sequences, modified cells containing such nucleic acid sequences or vectors, or isolated peptides comprising the sequence AVEEVSLRK (all of which are described in more detail elsewhere herein) can be used to inhibit HLA-A * It may be preferentially used for the manufacture of a medicament for the treatment or prevention of ΔNPM1-positive hematopoietic tumors in human subjects who are 01:01-positive.

[0107] CLAVEEVSLRK may be in particular a cysteinylated form.

[0108] In a further aspect, the present invention provides a method of producing a T cell receptor comprising contacting a cell with a nucleic acid sequence of the present invention under conditions such that the nucleic acid sequence is taken up by and expressed in the cell to produce 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.

[0109] The method can be ex vivo.

[0110] As noted above, specific binding to any of CLAVEEVSL, AVEEVSLRK and CLAVEEVSLRK can occur in the context of the appropriate HLA (e.g., specific binding to a peptide can only occur when it is presented by the appropriate HLA, as described above).

[0111] In a further aspect, the present invention provides the use of a peptide as a biomarker for ΔNPM1-positive hematopoietic tumors in a human subject, wherein the peptide is (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 selected from.

[0112] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0113] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0114] In a further aspect, the present invention provides a method for diagnosing a ΔNPM1-positive hematopoietic tumor in a human subject, comprising: determining the presence of a peptide in a sample isolated from a subject, wherein the peptide is selected from (i) CLAVEEVSL (SEQ ID NO: 1), wherein the cysteine ​​amino acid may or may not be 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); wherein the presence of the peptide in the sample identifies the subject as having a ΔNPM1-positive hematopoietic tumor, and the absence of the peptide identifies the subject as not having a ΔNPM1-positive hematopoietic tumor. A method is provided.

[0115] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0116] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0117] In a further aspect, the present invention provides a method of treating or preventing a ΔNPM1-positive hematopoietic tumor in a human subject, comprising: (i) determining the presence of a peptide in a sample isolated from a subject, wherein the peptide is selected from CLAVEEVSL (SEQ ID NO: 1) (wherein the cysteine ​​amino acid may or may not be cysteinylated), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27) (wherein the cysteine ​​amino acid may or may not be cysteinylated); VEEVSLRK (SEQ ID NO: 28); and AVEEVSLR (SEQ ID NO: 29); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention. The present invention provides a method for detecting a temperature difference between a plurality of electrodes.

[0118] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0119] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0120] The above descriptions regarding each of the peptides and their HLA-restricted nature (and particularly in the context of suitable pharmaceutical compositions for use in methods for treating or preventing ΔNPM1-positive hematopoietic tumors in subjects with particular HLA status) apply equally here.

[0121] In a further aspect, the present invention provides a pharmaceutical composition of the invention for use in treating or preventing a ΔNPM1-positive hematopoietic tumor in a human subject, wherein the subject has been identified as having a ΔNPM1-positive hematopoietic tumor by the presence of a peptide in a sample isolated from said subject, wherein 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.

[0122] In this embodiment, the subject who has been identified as having a ΔNPM1-positive hematopoietic tumor has already been diagnosed as having a ΔNPM1-positive hematopoietic tumor prior to treatment by the presence of a peptide in a sample isolated from the subject, wherein 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.

[0123] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 1 is cysteinylated.

[0124] In certain embodiments, the cysteine ​​amino acid of SEQ ID NO: 27 is cysteinylated.

[0125] The above descriptions regarding each of the peptides and their HLA-restricted nature (and in particular in suitable pharmaceutical compositions for use in the treatment or prevention of ΔNPM1-positive hematopoietic tumors in subjects with particular HLA status) apply equally here.

[0126] Throughout the specification and claims of this application, the terms "comprise" and "comprises" and grammatical variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, ingredients, integers or steps.

[0127] Throughout the specification and claims of this application, the singular includes the plural unless the context requires otherwise. In particular, when the indefinite article is used, the description should be construed as contemplating both the plural and the singular unless the context requires otherwise.

[0128] Any property, integer, feature, compound, compound moiety or group described in connection with a particular aspect, embodiment or example of the invention should be construed as being applicable to any other aspect, embodiment or example described herein, unless inconsistent.

[0129] The patent, scientific, and technical literature cited herein demonstrates the knowledge available to those skilled in the art at the time of filing. The entire disclosures of issued patents, published and pending patent applications, and other publications cited herein are hereby incorporated by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference. In the case of any conflict, the present specification will control.

[0130] Various aspects of the invention are described in further detail below.

[0131] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be further described with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0132] [Figure 1] Figure 1 provides validation of C*LAVEEVSL as a peptide eluted from HLA-A*02:01-positive AML with ΔNPM1. Mass spectra are shown for the eluted peptide from HLA-A*02:01-positive AML10197 with ΔNPM1 (top) and the synthetic peptide C*LAVEEVSL after cysteinylation of the first residue (bottom). The data show a perfect match of the mass spectra between both peptides.

[0133] [Figure 2]Figure 2 provides validation of ΔNPM1 peptides eluted from primary AML. Tandem mass spectra are shown for eluted peptides from AML (top) and synthetic peptides (bottom). C* = cysteinylation of Cys residues. (A) Tandem mass spectra for eluted peptides from AML10197 (top) and AML3361 (center) and the synthetic peptide C*LAVEEVSL, and for eluted peptides from AML3361 and the synthetic peptide AVEEVSLRK (bottom). (B) Tandem mass spectra for eluted peptides from AML9448 (top), AML5444 (center), and AML5518 (bottom) and the synthetic peptide AVEEVSLRK. (C) Tandem mass spectra for eluted peptides from AML6498 (top) and AML4443 (center) and the synthetic peptide AVEEVSLRK, and for eluted peptides from AML9448 and the synthetic peptide C*LAVEEVSLRK (bottom). (D) Tandem mass spectra for the eluted peptide from AML6498 and the synthetic peptide C*LAVEEVSLRK (top), the eluted peptide from AML3361 and the synthetic peptide VEEVSLRK (middle), and the eluted peptide from AML5518 and the synthetic peptide AVEEVSLR (bottom).

[0134] [Figure 3]Figure 3 shows that CD8 cells for ΔNPM1 were single-cell isolated from PBMCs from HLA-A*02:01-positive healthy individuals using a mix of ΔNPM1-CLA and ΔNPM1-C*LA pMHC tetramers. A. Expanding T cell clones were tested for staining with pMHC tetramers. T cell clones 1A2 (top) and 4A8 (bottom) were both positive for ΔNPM1-CLA tetramers; only clone 1A2 stained with ΔNPM1-C*LA. B. Tetramer-positive T-cell clones 1A2 (top) and 4A8 (bottom) were tested by IFN-γ ELISA for reactivity against HLA-A*02:01-positive T2 cells exogenously loaded with titrated concentrations of the noncysteinylated ΔNPM1 peptide CLAVEEVSL (circles), the cysteinylated ΔNPM1 peptide C*LAVEEVSL (squares), or the irrelevant HLA-A*02:01-restricted CMV peptide NLVPMVATV (triangles). Only clone 1A2 showed recognition of both cysteinylated and noncysteinylated ΔNPM1 peptides. No reactivity was observed against the irrelevant NLVPMVATV peptide. The mean IFN-γ release (ng / ml) from duplicate wells is shown. C. Clones 1A2 (top) and 4A8 (middle) were tested by IFN-γ ELISA for reactivity against five HLA-A*02:01-positive primary AML. The panel included three AMLs with ΔNPM1 and two AML1s with wtNPM. T cell clone 1A2 reacted to various degrees with all three AMLs with ΔNPM1, while clone 4A8 recognized only two of the three AMLs. Both T cell clones failed to recognize AML1 with wtNPM. An HLA-A*02:01-specific alloreactive T cell clone (Allo-A2 clone; bottom) was included as a positive control. The mean release of IFN-γ (ng / ml) from duplicate wells is shown.

[0135] [Figure 4]Figure 4 shows specificity for ΔNPM1 after TCR gene transfer. The ΔNPM1-specific TCR α and β chain genes of clone 1A2 were cloned into a modified MP71-TCR-flex retroviral vector for TCR gene transfer. CD8 and CD4 cells isolated from HLA-A*02:01-positive healthy individuals were retrovirally transduced with a TCR for ΔNPM1 and, as a control, a TCR for the HLA-A*02:01-restricted CMV peptide NLVPMVATV. Six days after transduction, TCR-transduced T cells were purified using an APC-conjugated antibody against mouse TCR-Cβ and magnetic anti-APC beads. A. Seven days after transduction, TCR-transduced T cells were analyzed by flow cytometry using CD8 or CD4 and antibodies against the pMHC tetramer for CLAVEEVSL (ΔNPM1-CLA; left) or NLVPMVATV (CMV-NLV; right). CD8 (CD8ФNPM1) and CD4 (CD4ФNPM1) cells transduced with a TCR for ΔNPM1 stained with ΔNPM1-CLA tetramers but not with CMV-NLV tetramers. In contrast, CD8 (CD8ФCMV) and CD4 (CD4ФCMV) T cells transduced with a CMV-specific TCR showed binding to CMV-NLV tetramers but not to ΔNPM1-CLA tetramers. Results are shown for donor 1, but similar results were obtained for donor 2. B. TCR-transduced T cells were analyzed for reactivity to their target peptides by IFN-γ ELISA. TCR-transduced CD8 and CD4 cells were co-incubated with T2 cells exogenously loaded with titrated concentrations of the ΔNPM1 peptide CLAVEEVSL (circles) or the CMV-derived peptide NLVPMVATV (squares). CD8ФNPM1 (upper left) and CD4ФNPM1 (lower left) show half-maximal recognition of T2 cells loaded with 30-100 nM CLAVEEVSL (dotted line), but not with NLVPMVATV. Conversely, CD8ФCMV (upper right) and CD4ФCMV (lower right) were reactive with NLVPMVATV-loaded T2 cells, but not with CLAVEEVSL.The mean release of IFN-γ (ng / ml) from duplicate wells is shown for donor 1; results were similar for donor 2. C. TCR-transduced T cells (CD8 ΦNPM1 and CD4 ΦNPM1 are shown as black bars; CD8 ΦCMV and CD4 ΦCMV are shown as gray bars) were tested by IFN-γ ELISA for recognition of the HLA-A*02:01-positive AML cell lines ΔNPM1 (OCI-AML3) or wtNPM1 (OCI-AML2) in the absence or presence of blocking antibodies against HLA class I (W6 / 32) or HLA class II (PdV5.1). Recognition of OCI-AML3 by CD8 and CD4 cells transduced with the TCR for ΔNPM1 is mediated by HLA class I. The mean release of IFN-γ (ng / ml) from duplicate wells is shown for donor 2.

[0136] [Figure 5] Figure 5 shows the recognition of ΔNPM1 by primary AML after TCR gene transfer. TCR-transduced CD8 and CD4 cells were tested by IFN-γ ELISA for reactivity against a panel of 13 HLA-A*02:01-positive primary AML, including nine ΔNPM1 samples and four wtNPM1 samples. CD8 (CD8 ΦNPM1; upper panel; black bars) and CD4 (CD4 ΦNPM1; lower panel; black bars) cells transduced with the TCR for ΔNPM1 reacted with all nine AMLs with ΔNPM1 but not with AML1s with wtNPM1, whereas none of the 13 AML samples were recognized by CD8 (CD8 ΦCMV; upper panel; dark gray bars) or CD4 (CD4 ΦCMV; lower panel; dark gray bars) cells after transfer of a CMV-specific TCR. TCR-transduced CD8 and CD4 cells also did not recognize HLA-A*02:01-negative AML with ΔNPM1 (data not shown). The allo-A2 clone (light gray bar) is included as a positive control. The mean release of IFN-γ (ng / ml) in duplicate wells is shown for donor 1.

[0137] [Figure 6]Figure 6 shows TCR-transduced T cells tested with monocyte-derived mature DCs by IFN-γ ELISA. T cells from donor 2 transduced with TCRs for ΔNPM1 (CD8 ΦNPM1 and CD4 ΦNPM1; black bars) or CMV (CD8 ΦCMV and CD4 ΦCMV; medium gray bars) were tested for reactivity against autologous monocyte-derived mature DCs and ΔNPM1-positive AML that were HLA-A*02:01 positive (AML8861) or negative (AML587). For autologous mature DCs and AML8861, allo-A2 clones (light gray bars) were included as a positive control, and AML587 was transduced with HLA-B*07:02-binding SMCY peptide and tested for recognition by SMCY-specific CD8 T cell clones as a positive control (dark gray bars). The mean IFN-γ release (ng / ml) from duplicate wells is shown.

[0138] [Figure 7] Figure 7 shows the lysis of primary AML with ΔNPM1 after TCR gene transfer in a 51Cr release assay. TCR-transduced CD8 and CD4 cells were tested for cytolytic capacity in a 9-hour 51Cr release assay of a panel of six HLA-A*02:01-positive primary AMLs, including four ΔNPM1 samples and two wtNPM1 samples. CD8 (CD8ФNPM1; closed circles) and CD4 (CD4ФNPM1; closed squares) cells transduced with the TCR for ΔNPM1 showed specific lysis of all four AML with ΔNPM1, but not AML with wtNPM1, whereas none of the six AML samples were specifically lysed by CD8 (CD8ФCMV; gray circles) or CD4 (CD4ФCMV; lower panel; gray squares) cells after transfer of a CMV-specific TCR. The allo-A2 clone (gray triangle) is included as a positive control. The average percentage of specific lysis in triplicate wells is shown for donor 2 at an E:T ratio of 30:1, although results were similar for donor 1.

[0139] [Figure 8]Figure 8 shows the immunogenic peptide amino acid sequence of SEQ ID NO: 1. It should be noted that the cysteine ​​amino acid in SEQ ID NO: 1 may or may not be cysteinylated.

[0140] [Figure 9] FIG. 9 shows the amino acid sequence of CDR3 (TCR alpha chain) (SEQ ID NO: 2).

[0141] [Figure 10] FIG. 10 shows the non-optimized nucleic acid sequence encoding CDR3 (TCR alpha chain) (SEQ ID NO: 3).

[0142] [Figure 11] FIG. 11 shows the optimized nucleic acid sequence encoding CDR3 (TCR alpha chain) (SEQ ID NO: 4).

[0143] [Figure 12] FIG. 12 shows the amino acid sequence of CDR3 (TCR β chain) (SEQ ID NO: 5).

[0144] [Figure 13] FIG. 13 shows the non-optimized nucleic acid sequence encoding CDR3 (TCR β chain) (SEQ ID NO: 6).

[0145] [Figure 14] FIG. 14 shows the optimized nucleic acid sequence encoding CDR3 (TCR β chain) (SEQ ID NO: 7).

[0146] [Figure 15] FIG. 15 shows the amino acid sequence of the α chain variable region (SEQ ID NO: 8) (CDR3 underlined).

[0147] [Figure 16] FIG. 16 shows the non-optimized nucleic acid sequence encoding the α chain variable region (SEQ ID NO: 9) (CDR3 underlined).

[0148] [Figure 17]FIG. 17 shows the optimized nucleic acid sequence encoding the α chain variable region (SEQ ID NO: 10) (CDR3 underlined).

[0149] [Figure 18] FIG. 18 shows the amino acid sequence of the β chain variable region (SEQ ID NO: 11) (CDR3 underlined).

[0150] [Figure 19] FIG. 19 shows the non-optimized nucleic acid sequence encoding the β chain variable region (SEQ ID NO: 12) (CDR3 underlined).

[0151] [Figure 20] FIG. 20 shows the optimized nucleic acid sequence encoding the β chain variable region (SEQ ID NO: 13) (CDR3 underlined).

[0152] [Figure 21] FIG. 21 shows the amino acid sequence of CDR1 (TCR alpha chain) (SEQ ID NO: 14).

[0153] [Figure 22] FIG. 22 shows the amino acid sequence of CDR2 (TCR alpha chain) (SEQ ID NO: 15).

[0154] [Figure 23] FIG. 23 shows the amino acid sequence of CDR1 (TCR β chain) (SEQ ID NO: 16).

[0155] [Figure 24] FIG. 24 shows the amino acid sequence of CDR2 (TCR β chain) (SEQ ID NO: 17).

[0156] [Figure 25] FIG. 25 shows the non-optimized nucleic acid sequence encoding CDR1 (TCR alpha chain) (SEQ ID NO: 18).

[0157] [Figure 26] FIG. 26 shows the optimized nucleic acid sequence encoding CDR1 (TCR alpha chain) (SEQ ID NO: 19).

[0158] [Figure 27] FIG. 27 shows the non-optimized nucleic acid sequence encoding CDR2 (TCR alpha chain) (SEQ ID NO: 20).

[0159] [Figure 28] FIG. 28 shows the optimized nucleic acid sequence encoding CDR2 (TCR alpha chain) (SEQ ID NO: 21).

[0160] [Figure 29] FIG. 29 shows the non-optimized nucleic acid sequence encoding CDR1 (TCR β chain) (SEQ ID NO: 22).

[0161] [Figure 30] FIG. 30 shows the optimized nucleic acid sequence encoding CDR1 (TCR β chain) (SEQ ID NO: 23).

[0162] [Figure 31] FIG. 31 shows the non-optimized nucleic acid sequence encoding CDR2 (TCR β chain) (SEQ ID NO: 24).

[0163] [Figure 32] FIG. 32 shows the optimized nucleic acid sequence encoding CDR2 (TCR β chain) (SEQ ID NO: 25).

[0164] [Figure 33] FIG. 33 shows the immunogenic peptide amino acid sequence of SEQ ID NO:26.

[0165] [Figure 34] Figure 34 shows the immunogenic peptide amino acid sequence of SEQ ID NO: 27. It should be noted that the cysteine ​​amino acid in SEQ ID NO: 27 may or may not be cysteinylated.

[0166] [Figure 35] FIG. 35 shows the immunogenic peptide amino acid sequence of SEQ ID NO:28.

[0167] [Figure 36] FIG. 36 shows the immunogenic peptide amino acid sequence of SEQ ID NO:29.

[0168] Figures 37 to 39 show A * 03;01 and A * T cells against ΔNPM1 peptides at 11:01 are shown. ΔNPM1 peptides AVEEVSLRK (AVE), CLAVEEVSLRK (CLA) and C * LAVEEVSLRK(C * PE-conjugated HLA-A with LA; first residue cysteinylation * 03:01 Mixture of tetramers or single HLA-A with AVEEVSLRK * 11:01 tetramer was used to target specific T cells to HLA-A * 03:01 and / or HLA-A * 11:01 were isolated from healthy individuals. * LAVEEVSLRK and HLA-A with CLAVEEVSLRK as control * 03:01 Tetramers were UV-exchanged (UV-C * LA and UV-CLA).

[0169] [Figure 37] Figure 37 shows T cell clone binding to PE-conjugated HLA-A*03:01 UV-C*LA (clone 1F2; left), A*03:01 AVE (clone 3B3; center), and A*11:01 AVE (clone 6F11; right) tetramers.

[0170] [Figure 38]Figure 38 shows the reactivity of T cell clone 3B3 (left) to T2 cells transduced with HLA-A*03:01 exogenously pulsed with titrated concentrations of the ΔNPM1 peptide AVEEVSLRK (triangles), CLAVEEVSLRK (circles), or C*LAVEEVSLRK (squares). GM-CSF release (ng / ml) at various peptide concentrations (nM) is shown. T cell clone 6F11 (right) shows the reactivity of T2 cells transduced with HLA-A*11:01 exogenously pulsed with titrated concentrations of the ΔNPM1 peptide AVEEVSLRK (triangles). IFN-γ release (ng / ml) at various peptide concentrations (nM) is shown.

[0171] [Figure 39] Figure 39 shows T cell clones 3B3 (left) and 6F11 (right) tested for reactivity against K562 cells, K562 transduced with HLA-A*03:01 or A*11:01, K562 transduced with HLA-A*03:01 or A*11:01, and K562 transduced with full-length wild-type or ΔNPM1-encoding genes, and OCI-AML2 and OCI-AML3 cell lines transduced with A*03:01 or A*11:01, which endogenously express wild-type and ΔNPM1, respectively. GM-CSF (clone 3B3) or IFN-γ (clone 6F11) release is shown in ng / ml. DETAILED DESCRIPTION OF THE INVENTION

[0172] Detailed Description The immunogenicity of peptides derived from mutant NPM1 (ΔNPM1) has been previously tested. 14 Therefore, in silico screening of the entire amino acid sequence of ΔNPM1 was performed to identify which peptides bind to HLA-A. * HLA-A, including CLAVEEVSL, was used to predict whether a patient was likely to be presented by 02:01. * Peptides predicted to bind 02:01 were synthetically produced. CD8 isolated from healthy individuals and ΔNPM1 AML patients +T cells were stimulated with the peptides and T cell responses were measured. Only two of the tested peptides (AIQDLCLAV and AIQDLCVAV) were found to elicit an immune response in vitro. These peptides were therefore considered to be the most prominent epitopes of ΔNPM1 and were therefore used for further studies.

[0173] According to the present invention, the inventors have identified five distinct 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.

[0174] The present inventors have also demonstrated that, with all possible peptides within ΔNPM1, the CLAVEEVSL peptide binds to HLA-A * We have surprisingly shown that 02:01 is presented on the surface of primary ΔNPM1 AML cells isolated from cancer patients, and further that the peptide is found in a cysteinylated form on the surface of isolated primary AML cells.

[0175] The present inventors have also found that the ΔNPM1-derived peptides AVEEVSLRK and CLAVEEVSLRK each bind to HLA-A * 03:01 and HLA-A * Presented by 11:01, AVEEVSLRK also has HLA-A * I also surprisingly discovered that it is presented by 01:01.

[0176] The present inventors have investigated the effects of HLA-A * We isolated and cloned T cell receptors reactive with CLAVEEVSL from the T cell repertoire of 02:01-positive individuals. These T cell receptors can be used to genetically engineer peripheral blood lymphocytes, and the genetically modified lymphocytes express HLA-A ΔNPM1. * These TCRs were shown to efficiently kill 02:01-positive AML. *It can be advantageously used as an effective immunotherapy in the treatment of patients with 02:01-positive ΔNPM1-positive AML.

[0177] The present inventors also investigated the effect of HLA-A * We also isolated a T cell clone reactive with CLAVEEVSLRK (specifically, a cysteinylated variant of this peptide) from the T cell repertoire of a 03:01-positive individual. The TCR from this clone was associated with HLA-A * It can be advantageously used as an effective immunotherapy in the treatment of patients with 03:01-positive ΔNPM1-positive AML.

[0178] The present inventors also performed a randomized controlled trial of the HLA-A * 03:01 and HLA-A * Two T cell clones reactive with AVEEVSLRK were also isolated from the T cell repertoire of an 11:01-positive individual. The TCRs from these clones were expressed by HLA-A receptors with ΔNPM1-positive AML. * 03:01 and HLA-A * It can be advantageously used as an effective immunotherapy in the treatment of 11:01 positive patients.

[0179] Nucleic acid sequences encoding TCR polypeptide components The present invention provides nucleic acid sequences encoding T cell receptor components that 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). The nucleic acid sequence may form part of a larger nucleic acid sequence encoding larger components of the T cell receptor (e.g., a TCR alpha chain variable region, a TCR beta chain variable region, a TCR alpha chain, a TCR beta chain, etc.). The nucleic acid sequence may also form part of a larger nucleic acid sequence encoding a functional T cell receptor (i.e., encoding a functional TCR alpha chain and a functional TCR beta chain, optionally separated by a linker sequence that allows coordinated expression of the two proteins or polypeptides from the same vector. Further details in this regard are provided below.

[0180] The nucleic acid sequence may encode only a small component of the T cell receptor, such as the CDR3 domain of a TCR α chain polypeptide or the CDR3 domain of a TCR β chain polypeptide. The nucleic acid sequence may therefore be considered a "component" that provides the essential component of peptide specificity. The nucleic acid sequence of the present invention may be incorporated into another nucleic acid sequence (e.g., a vector) encoding other elements of the TCR variable chain, such that when the nucleic acid sequence of the present invention is incorporated, a novel 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 produced. The nucleic acid sequence of the present invention may therefore be useful as the essential component of TCR specificity for a selected ΔNPM1 peptide and thus may be used to generate nucleic acid sequences encoding TCR variable regions with the necessary antigen binding activity and specificity for targeting ΔNPM1-positive AML.

[0181] T cell receptors (TCRs) are molecules found on the surface of T cells (T lymphocytes) that are responsible for recognizing peptides bound to (presented by) major histocompatibility complex (MHC) molecules on target cells. The present invention provides a method for identifying specific peptides in the context of the appropriate serotype of MHC, e.g., HLA-A. * CLAVEEVSL under the influence of 02:01; or HLA-A, respectively * 03:01 and HLA-A * 11:01 under the influence of either AVEEVSLRK or CLAVEEVSLRK or HLA-A * This relates to a nucleic acid sequence encoding a TCR that interacts with AVEEVSLRK under the influence of 01:01.

[0182] HLA-A * 02:01 is a common human leukocyte antigen serotype worldwide within the HLA-A serogroup. * The peptide presented by the TCR against 02:01 is expressed as "HLA-A * 02:01 It is described as "limited".

[0183] HLA-A * 03:01, HLA-A * 11:01 and HLA-A * 01:01 is also a common human leukocyte antigen serotype within the HLA-A serogroup. * The peptide presented by the TCR against 03:01 is expressed as "HLA-A * 03:01 is described as "limited." Similarly, HLA-A * The peptide presented by the TCR against 11:01 is expressed as "HLA-A * 11:01 is described as "HLA-A limited." * The peptide presented by the TCR against 01:01 is expressed as "HLA-A * 01:01 It is described as "limited".

[0184] TCRs consist of two distinct 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 TCRs are associated with an HLA (e.g., appropriately HLA-A * 02:01, HLA-A * 03:01 or HLA-A * When T cells bind to the peptide under the influence of IgG1 (11:01), they are activated by signal transduction.

[0185] The alpha and beta chains of the TCR are highly variable in sequence. Each chain consists of two extracellular domains, a variable region (V) and a constant region (C). The constant region is proximal to the T cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / HLA-A complex.

[0186] The variable region of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Thus, the TCR alpha chain contains CDR1, CDR2 and CDR3, and the TCR beta chain also contains (different) CDR1, CDR2 and CDR3. In each of the alpha and beta chains, it is CDR3 that is primarily responsible for recognizing peptides presented by HLA-A.

[0187] In one aspect, the present invention provides an isolated nucleic acid sequence encoding (a) a polypeptide comprising a CDR3 of a TCR alpha 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 a CDR3 of a TCR beta 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).

[0188] In certain embodiments, the encoded polypeptide specifically binds to CLAVEEVSL (SEQ ID NO: 1). CLAVEEVSL can be in a cysteinylated form. The encoded polypeptide can specifically bind only to the cysteinylated form.

[0189] In certain embodiments, the encoded polypeptide specifically binds to AVEEVSLRK (SEQ ID NO: 26).

[0190] In certain embodiments, the encoded polypeptide specifically binds to CLAVEEVSLRK (SEQ ID NO: 27). CLAVEEVSLRK can be in a cysteinylated form. The encoded polypeptide can specifically bind only to the cysteinylated form.

[0191] The nucleic acid sequence can encode (a), (b), or (a) and (b). The nucleic acid sequence therefore encodes at least one polypeptide comprising a CDR3 of a T cell receptor polypeptide, wherein the CDR3 specifically binds to one of the following peptides: CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28), or AVEEVSLR (SEQ ID NO: 29).

[0192] The nucleic acid sequence can comprise an alpha chain CDR3 and a beta chain CDR3, where the alpha chain CDR3 and the beta chain CDR3 together specifically bind to a selected peptide.

[0193] The nucleic acid sequence therefore encodes the "CDR3 of a TCR alpha chain polypeptide" (also referred to herein as alpha chain CDR3 or alpha chain CDR3) and / or the "CDR3 of a TCR beta chain polypeptide" (also referred to herein as beta chain CDR3 or beta chain CDR3).

[0194] The alpha chain CDR3 can be that of SEQ ID NO: 2 or one of the variants below. Similarly, the beta chain CDR3 can be that of SEQ ID NO: 5 or one of the variants 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: 1.

[0195] The permutations described below for (a) can be combined with those described for (b), e.g., to form a suitable nucleic acid sequence encoding a functional T cell receptor (i.e., encoding a functional TCR α chain and a TCR β chain, optionally separated by a linker sequence that allows coordinate expression of the two proteins or polypeptides from the same vector).

[0196] Polypeptide (a) - component of the TCR alpha chain In certain embodiments, the CDR3 of (a) has the amino acid sequence of SEQ ID NO:2 or can be a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:2. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:2 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0197] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 2 that does not specifically bind to SEQ ID NO: 1. Non-functional variants generally 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: 2. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0198] In some embodiments, 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: 2 while maintaining the ability to specifically bind to the peptide of SEQ ID NO: 2. In other words, functional CDR3s having one amino acid substitution compared to the sequence of SEQ ID NO: 2 are also encompassed. As mentioned above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 2).

[0199] In the example where the CDR3 of (a) has the amino acid sequence of SEQ ID NO: 2, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 4 is a codon-optimized version of the nucleic acid sequence of CDR3 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 3). Thus, the polypeptide of (a) can be encoded by the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0200] In certain embodiments, the polypeptide of (a) comprises a CDR3 within a TCR alpha chain variable region 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 TCR alpha chain variable region that comprises a particular CDR3, wherein the TCR alpha chain variable region (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 1. As will be clear to those skilled in the art, the term "TCR alpha chain variable region" refers to the variable (V) region (extracellular domain) of the TCR alpha chain, and thus includes the three hypervariable regions (CDR1, CDR2, and particular CDR3) and intervening sequences, but does not include the constant (C) regions of the alpha chain that do not form part of the variable chain.

[0201] The encoded TCR alpha chain variable region may include, in addition to a specific CDR3, a CDR1 having the amino acid sequence of SEQ ID NO: 14, or a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the N-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: 14. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 14 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0202] 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 generally include 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.

[0203] In one embodiment, CDR1 (e.g., within the alpha chain variable region) of (a) is SEQ ID NO: 1 Nope The CDR1 may have an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 14 while maintaining the ability to specifically bind to the N-terminus of a peptide. In other words, a functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 14 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as the percentage of identity relative to the entire length of the reference sequence (e.g., SEQ ID NO: 14).

[0204] In the example where CDR1 (e.g., in the alpha chain variable region) of (a) has the amino acid sequence of SEQ ID NO: 14, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 19 is a codon-optimized version of the nucleic acid sequence of CDR1 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 18). Thus, the polypeptide of (a) can be encoded by the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0205] The encoded TCR α chain variable region may comprise, in addition to a specific CDR3 (and optionally the specific CDR1), the amino acid sequence of SEQ ID NO: 15 or a functional variant thereof (i.e., wherein the variant is 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" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 15 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0206] Non-functional variants include HLA-A * and amino acid sequence variants of SEQ ID NO: 15 that do not specifically bind to 02:01. Non-functional variants generally 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: 15. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0207] In some embodiments, the CDR2 (e.g., within the alpha chain variable region) of (a) is * The CDR2 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 encompassed. As mentioned above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 15).

[0208] In the example where CDR2 (e.g., in the alpha chain variable region) of (a) has the amino acid sequence of SEQ ID NO: 15, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 21 is a codon-optimized version of the nucleic acid sequence of CDR2 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 20). Thus, the polypeptide of (a) can be encoded by the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0209] The polypeptide of (a) may therefore comprise a TCR alpha chain variable region comprising the CDRs detailed above (either sequence-specific or variants thereof), with appropriate intervening sequences between the CDRs.

[0210] The TCR alpha chain variable region of (a) may have the amino acid sequence of SEQ ID NO: 8 or a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 8 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0211] 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 generally 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.

[0212] In certain embodiments, the TCR alpha 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 to 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 alpha chain variable regions having one or more amino acid substitutions relative to the sequence of SEQ ID NO:8 are also encompassed. As noted above, the amino acid substitutions may be conservative amino acid substitutions. The sequence variability relative to SEQ ID NO:8 may all be in regions that do not form the CDRs of the TCR alpha chain variable region (i.e., a 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 relative to SEQ ID NO:8). In other words, the sequence of the CDRs of SEQ ID NO: 8 is maintained while varying the remainder of the sequence as appropriate within the "at least 75% identity" parameters specified above. Similarly, percent identity can be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 8).

[0213] By way of example, the polypeptide of (a) may comprise a CDR3 within a TCR alpha chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein CDR3 has the amino acid sequence of SEQ ID NO: 2. In this example, the TCR alpha chain variable region CDR1 may have the amino acid sequence of SEQ ID NO: 14, and the TCR alpha chain variable region CDR2 may have the amino acid sequence of SEQ ID NO: 15.

[0214] In the example where the TCR alpha chain variable region of (a) has the amino acid sequence of SEQ ID NO:8, the TCR alpha chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO:10 is a codon-optimized version of the nucleic acid sequence of the TCR alpha chain variable region of clone 1A2 (the non-optimized sequence is SEQ ID NO:9). Thus, the polypeptide of (a) can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0215] For the avoidance of doubt, the polypeptide of (a) may comprise a TCR alpha chain variable region (as specified above) and a TCR alpha chain constant region. An example of a suitable constant region is encoded by GenScript's MP71-TCR-flex retroviral vector used herein. However, the present invention is not limited to this specific constant region and encompasses any suitable TCR alpha chain constant region. The constant region may be murine, human, or humanized. Methods for identifying or producing suitable constant regions are well known and well within the routine capabilities of those skilled in the art.

[0216] By way of example only, constant regions can be encoded by or derived from vectors such as lentivirus, retrovirus, or plasmid vectors, into which mouse or human constant regions have been pre-cloned, as well as adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, or herpesvirus vectors. Recently, minicircles have also been reported for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors, published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek in Leukemia 2016). Furthermore, naked (synthetic) DNA / RNA can also be used for TCR transduction. For example, the pMSGV retroviral vector, which carries pre-cloned TCR-Ca and Cb genes as described by LV Coren et al., BioTechniques 2015, can also be used to provide appropriate constant regions.

[0217] Polypeptide (b) - a component of the TCR beta chain In certain embodiments, the CDR3 of (b) has the amino acid sequence of SEQ ID NO: 5 or can be a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 5. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 5 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0218] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 5 that does not specifically bind to SEQ ID NO: 1. Non-functional variants generally 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: 5. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0219] In some embodiments, 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 maintaining the ability to specifically bind to the peptide of SEQ ID NO: 5. In other words, functional CDR3 having one amino acid substitution compared to the sequence of SEQ ID NO: 5 is also encompassed. As mentioned above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 5).

[0220] In the example where the CDR3 of (b) has the amino acid sequence of SEQ ID NO:5, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO:7 is a codon-optimized version of the nucleic acid sequence of CDR3 of clone 1A2 (the non-optimized sequence is SEQ ID NO:6). Thus, the polypeptide of (b) can be encoded by the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0221] In one embodiment, the polypeptide of (b) comprises a CDR3 (e.g., the CDR3 of SEQ ID NO: 5, or a variant thereof, as defined above) within a TCR β chain variable region that specifically binds to the peptide of SEQ ID NO: 1. In other words, the polypeptide of (b) comprises a TCR β chain variable region that comprises a particular CDR3, wherein the TCR β chain variable region (and the CDR3 therein) specifically binds to the peptide of SEQ ID NO: 1. As will be clear to those skilled in the art, the term "TCR β chain variable region" refers to the variable (V) region (extracellular domain) of the TCR β chain, and thus includes the three hypervariable regions (CDR1, CDR2, and particular CDR3) and intervening sequences, but does not include the constant (C) regions of the β chain that do not form part of the variable chain.

[0222] The encoded TCR β chain variable region may include, in addition to a specific CDR3, a CDR1 having the amino acids of SEQ ID NO: 16 or a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the C-terminus of a 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 includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 16 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0223] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 16 that does not specifically bind to the C-terminus of the peptide of SEQ ID NO: 1. Non-functional variants generally include 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: 16. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0224] In some embodiments, the CDR1 (e.g., in the β-chain variable region) of (b) may have an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 16 while maintaining the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO: 1. In other words, functional CDR1 having one amino acid substitution compared to the sequence of SEQ ID NO: 16 is also encompassed. As described above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 16).

[0225] In an example where CDR1 (e.g., in the β-chain variable region) of (b) has the amino acid sequence of SEQ ID NO: 16, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 23 is a codon-optimized version of the nucleic acid sequence of CDR1 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 22). Thus, the polypeptide of (b) can be encoded by the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a genetically modified sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0226] The encoded TCR β chain variable region may comprise, in addition to a specific CDR3 (and optionally the specific CDR1), the amino acid sequence of SEQ ID NO: 17 or a functional variant thereof (i.e., wherein the variant is 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" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 17 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0227] Non-functional variants include HLA-A *and amino acid sequence variants of SEQ ID NO: 17 that do not specifically bind to 02:01. Non-functional variants generally include 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: 17. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0228] In some embodiments, the CDR2 (e.g., within the β chain variable region) of (b) is * The CDR2 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 encompassed. As mentioned above, the amino acid substitution may be a conservative amino acid substitution. Similarly, the percent identity may be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 17).

[0229] In an example where CDR2 (e.g., in the β-chain variable region) of (b) has the amino acid sequence of SEQ ID NO: 17, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 25 is a codon-optimized version of the nucleic acid sequence of CDR2 of clone 1A2 (the non-optimized sequence is SEQ ID NO: 24). Thus, the polypeptide of (b) can be encoded by the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0230] The polypeptide of (b) may therefore comprise a TCR β chain variable region comprising the CDRs detailed above (either sequence-specific or variants thereof), with appropriate intervening sequences between the CDRs.

[0231] The TCR β chain variable region of (b) may have the amino acid sequence of SEQ ID NO: 11 or a functional variant thereof (i.e., where the variant retains the ability to specifically bind to the peptide of SEQ ID NO: 1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 11. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 11 or non-critical amino acid substitutions, deletions, or insertions in non-critical regions of the protein.

[0232] 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 generally 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: 11. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0233] In certain embodiments, the TCR β chain variable region of (b) may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to 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 that have one or more amino acid substitutions relative to the sequence of SEQ ID NO: 11 are also encompassed. As noted above, the amino acid substitutions may be conservative amino acid substitutions. All of the sequence variability relative to SEQ ID NO: 11 may be in regions that do not form the CDRs of the TCR β chain variable region (i.e., a 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 relative to SEQ ID NO: 11). In other words, the sequence of the CDRs of SEQ ID NO: 11 is maintained while varying the remainder of the sequence as appropriate within the "at least 75% identity" parameters specified above. Similarly, percent identity can be calculated as a percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 11).

[0234] For example, (b) may comprise a CDR3 within the TCR β chain variable region having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 11, wherein 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.

[0235] In the example where the TCR β chain variable region of (b) has the amino acid sequence of SEQ ID NO: 11, the TCR β chain variable region can be encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy). It should be noted that SEQ ID NO: 13 is a codon-optimized version of the nucleic acid sequence of the TCR β chain variable region of clone 1A2 (the non-optimized sequence is SEQ ID NO: 12). Thus, the polypeptide of (b) can be encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, or a genetically modified version thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic code degeneracy).

[0236] For the avoidance of doubt, (b) may include a TCR β chain variable region (as specified above) and a TCR β chain constant region. An example of a suitable constant region is that encoded by GenScript's MP71-TCR-flex retroviral vector used herein. However, the present invention is not limited to this specific constant region and encompasses any suitable TCR β chain constant region. The constant region may be murine, human, or humanized. Methods for identifying or producing suitable constant regions are well known and well within the routine capabilities of those skilled in the art.

[0237] By way of example only, constant regions can be encoded by or derived from vectors such as lentivirus, retrovirus, or plasmid vectors into which mouse or human constant regions have been previously cloned, as well as adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, or herpesvirus vectors. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek in Leukemia 2016). Furthermore, naked (synthetic) DNA / RNA can also be used for TCR transduction. For example, the MP71-TCR-flex retroviral vector with pre-cloned TCR-Ca and Cb genes used by the present inventors or the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes described in LV Coren et al., BioTechniques 2015 may also be used to provide appropriate constant regions.

[0238] In examples where the nucleic acid molecule of the present invention is encoded by both (a) and (b), the polypeptide of (a) can be linked to the polypeptide of (b) via a linker, such as a linker that allows for the translation of two proteins or polypeptides from the same vector. For example, a linker containing the porcine teschovirus-1 2A (P2A) sequence can be used, such as a 2A sequence from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), or Thosea asigna virus (T2A) or a 2A-like sequence published by AL Szymczak et al., Nature Biotechnology 22, 589-594 (2004). The 2A and 2A-like sequences are linkers that are cleavable once the nucleic acid molecule is transcribed and translated. Another example of a linker is an internal ribosome entry site (IRES), which allows for the translation of two proteins or polypeptides from the same transcript. Any other suitable linker can also be used. Identifying a suitable linker is well within the routine capabilities of one of ordinary skill in the art. As a further example, a nucleic acid sequence encoding (a) and a nucleic acid sequence encoding (b) can be cloned into a vector with dual internal promoters (see, e.g., S Jones et al., Human Gene Ther 2009).

[0239] Additional suitable polypeptide domains can also be encoded by the nucleic acid sequences of the present invention.By way of example only, the nucleic acid sequence can comprise a membrane targeting sequence, which provides for the transport of the encoded polypeptide to the cell surface membrane of modified cells.Other suitable additional domains are well known and are described, for example, in WO2016 / 071758.

[0240] In some embodiments, the nucleic acid sequence of the present invention can encode a soluble TCR. For example, the nucleic acid sequence can encode (a) and (b), where (a) and (b) comprise the variable regions of the TCR alpha and beta chains, respectively, and optionally an immunomodulator molecule such as a CD3 agonist (e.g., anti-CD3 scFv). The CD3 antigen is presented on mature human T cells, thymocytes, and a subset of natural killer cells. It associates with the TCR and is involved in TCR signaling. Antibodies specific for the human CD3 antigen are well known. One such antibody is the murine monoclonal antibody OKT3, the first monoclonal antibody approved by the FDA. Other CD3-specific antibodies have also been reported (see, e.g., WO 2004 / 106380; U.S. Patent Application Publication No. 2004 / 0202657; U.S. Patent No. 6,750,325). Immune mobilizing anti-cancer mTCR (ImmTAC; Immunocore Limited, Milton Park, Abington, Oxon, United Kingdom) 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 can be combined with a radioisotope or a toxic drug. Suitable radioisotopes and / or toxic drugs are well known in the art and can be readily identified by those skilled in the art.

[0241] In one embodiment, the nucleic acid sequences of the invention can encode a chimeric single-chain TCR in which a polypeptide (a) (e.g., a TCR alpha chain variable region) is linked to a polypeptide (b) (e.g., a TCR beta chain variable region) and a constant region fused to, for example, a CD3 zeta signaling domain. In this example, the linker is non-cleavable. In another embodiment, the nucleic acid sequences of the invention can encode a chimeric two-chain TCR in which a polypeptide (a) (e.g., a TCR alpha chain variable region) and a polypeptide (b) (e.g., a TCR beta chain variable region) are each linked to a CD3 zeta signaling domain. Methods for producing such single-chain and two-chain TCRs are well known in the art; see, for example, RA Willemsen et al., Gene Therapy 2000.

[0242] The present invention also provides isolated nucleic acid sequences (and corresponding vectors) encoding the peptides of the present invention. All general descriptions regarding nucleic acid sequences and vectors apply equally. Those skilled in the art will readily identify appropriate nucleic acid sequences and vectors based on the peptide sequences provided herein.

[0243] Vectors and modified cells In one embodiment, the present invention provides a vector comprising the nucleic acid sequence described herein. Any suitable vector can be used. By way of example only, the vector can be a plasmid or a viral vector, such as a retroviral or lentiviral vector. Adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, herpes virus, minicircle vectors, and naked (synthetic) DNA / RNA can also be used (for details of minicircle vectors, see, for example, "Non-viral Sleeping Beauty Transposition from Minicircle Vectors" published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek in Leukemia 2016).

[0244] Optionally, the vector comprises a nucleic acid sequence operably linked to a promoter.

[0245] As used herein, the term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it is operably linked. A vector can be capable of autonomous replication or can integrate into host DNA. A vector may contain restriction enzyme sites for insertion of recombinant DNA and may contain one or more selectable markers or suicide genes. A vector can be a nucleic acid sequence in the form of a plasmid, bacteriophage, or cosmid. Preferably, the vector is suitable for expression in a cell (i.e., the vector is an "expression vector"). Preferably, the vector is a vector that encodes a CD8 + T cells or CD4 + The vector is suitable for expression in human T cells, such as T cells. In some 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 adenovirus, vaccinia virus, canarypox virus, herpes virus, minicircle vector, and synthetic DNA or synthetic RNA.

[0246] Preferably, the (expression) vector is capable of replicating in the host cell and being stably transmitted to progeny.

[0247] As used herein, "operably linked" refers to coding sequence sequences that are in a functional relationship with each other, e.g., linked in such a way as to direct expression of the coding sequence, in conjunction with one or a combination of the following regulatory elements:

[0248] A vector may contain a regulatory sequence. As used herein, "regulatory sequence" refers to a DNA or RNA element that can control gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA boxes, internal ribosome entry sites (IRES), transcription factor attachment sites, transcription terminators, polyadenylation sites, etc. If desired, a vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression as well as tissue-specific regulatory and / or inducible sequences.

[0249] A vector may contain a promoter. As used herein, a "promoter" refers to a nucleotide sequence in DNA to which RNA polymerase binds and initiates transcription. A promoter may be inducible or constitutively expressed. Alternatively, a promoter may be under the control of a repressor or stimulatory protein. A promoter may not be naturally found in a host cell (e.g., it may be an exogenous promoter). Those skilled in the art will recognize appropriate promoters to use for expression of a target protein, where the selected promoter will depend on the host cell.

[0250] The vector may contain a transcription terminator. As used herein, "transcription terminator" refers to a DNA element that stops the function of RNA polymerase, which is responsible for translating DNA into RNA. A preferred transcription terminator is characterized by a series of T residues preceded by a GC-rich bisecting symmetric region.

[0251] The vector may contain a translational regulatory element. As used herein, "translational regulatory element" refers to a DNA or RNA element that controls the translation of mRNA. A preferred translational regulatory element is a ribosome binding site. Preferably, the translational regulatory element is derived from a system homologous to a promoter, such as a promoter and its associated ribosome binding site. Preferred ribosome binding sites are known and depend on the selected host cell.

[0252] A vector may contain a restriction enzyme recognition site. As used herein, a "restriction enzyme recognition site" refers to a DNA motif recognized by a restriction enzyme.

[0253] 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 a phenotype on the cell that allows for the selection of cells expressing the selectable marker gene. Generally, this can be a protein that confers a novel advantageous property on the host cell (e.g., antibiotic resistance) or a protein that is expressed on the cell surface and is therefore available for antibody binding. Suitable selectable markers are well known in the art.

[0254] Optionally, the vector may also contain a suicide gene. As used herein, the term "suicide gene" refers to a protein that induces the death of modified cells upon treatment with a specific drug. For example, suicide can be induced in cells modified with the herpes simplex virus thymidine kinase gene by treatment with nucleoside analogs, including ganciclovir, in cells modified with human CD20 by treatment with anti-CD20 monoclonal antibodies, and in cells modified with inducible caspase 9 (iCasp9) 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.

[0255] Preferably, the vector contains the genetic elements necessary for the expression of a polypeptide described herein by a host cell. Elements necessary for transcription and translation in a host cell include a promoter, a translation region for the protein of interest, and a transcription terminator.

[0256] Those skilled in the art will be well aware of the molecular techniques available for preparing (expression) vectors and how they can be transduced or transfected into suitable host cells (thereby producing the modified cells of the invention). The (expression) vectors of the invention can be introduced into cells by conventional techniques such as transformation, transfection or transduction. "Transformation," "transfection," and "transduction" generally refer to methods for introducing heterologous (exogenous) nucleic acid sequences into host cells and therefore include methods such as electroporation, microinjection, biolistic delivery, transduction with retroviral, lentiviral, or adeno-associated viral vectors, lipofection, superfection, etc. The specific method used will generally depend 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, NY; Ausubel et al. (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al. (1988) Mol. Microbiol. 2, 637-646. Further conventional methods suitable for preparing expression vectors and introducing them into suitable host cells are described in detail, for example, in WO2016 / 071758.

[0257] It is understood that in some embodiments, the host cell is contacted with the vector (e.g., a viral vector) in vitro, ex vivo, and in some embodiments, the host cell is contacted with the vector (e.g., a viral vector) in vivo.

[0258] The term "host cell" refers to any cell into which a nucleic acid sequence or vector of the invention can be introduced (e.g., transduced). Once a nucleic acid molecule or vector has been introduced into a cell, it may be referred to herein as a "modified cell." Once a nucleic acid molecule or vector has been introduced into a host cell, the resulting modified cell must be capable of expressing the encoded polypeptide (and, e.g., correctly localizing the encoded polypeptide for its intended function, e.g., transporting an encoded TCR to the cell surface).

[0259] The term "modified cell" refers to a cell that has been genetically altered (e.g., transformed or transfected). The term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because some modifications may occur in progeny due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell but still be within the scope of the term as used herein.

[0260] Host cells (and modified cells thereof) are generally eukaryotic cells and particularly human cells (e.g., CD8 + T cells or CD4 + The host cells (and modified cells thereof) are autologous cells (e.g., CD8 T cells, T cells, or mixtures thereof), meaning cells derived from the same individual to whom they are subsequently administered. + T cells or CD4 + The host cells (and modified cells thereof) may be isolated T cells from the subject to be treated. Preferably, the host cells (and modified cells thereof) may be isolated from a blood sample, for example by leukapheresis.

[0261] The host cells (and modified cells thereof) can be any cells that can confer anti-tumor immunity after TCR gene transfer. Non-limiting examples of suitable cells include autologous or allogeneic natural killer (NK) cells, NKT cells, gamma-delta T cells, hematopoietic stem cells or other progenitor cells. Pelvic floorand any other autologous or allogeneic cells or cell lines (e.g., NK-92 or T cell lines) that can confer anti-tumor immunity after TCR gene transfer.

[0262] Advantageously, the modified cells are capable of expressing a polypeptide encoded by a nucleic acid sequence or vector (e.g., a TCR or TCR component portion) of the invention such that the modified cells specifically target ΔNPM1 malignant cells and thus provide an immunotherapy that can be used to treat or prevent ΔNPM1-bearing hematopoietic tumors, further details of which use are provided below.

[0263] Immunogenic peptides The inventors identified five peptides present in the HLA class I ligandome of ΔNPM1-positive primary AML patients: 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).

[0264] For clarity, unless otherwise specified, a general reference herein to "SEQ ID NO: 1" encompasses both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSL.

[0265] As an isolated peptide, the present invention particularly provides an isolated peptide comprising the amino acid sequence CLAVEEVSL (SEQ ID NO: 1), including when the amino acid cysteine ​​in CLAVEEVSL (SEQ ID NO: 1) is cysteinylated.

[0266] In certain embodiments, the amino acid cysteine ​​in CLAVEEVSL is cysteinylated.

[0267] For clarity, unless otherwise specified, a general reference herein to "SEQ ID NO:27" encompasses both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSLRK.

[0268] As an isolated peptide, the present invention particularly provides an isolated peptide comprising the amino acid sequence CLAVEEVSLRK (SEQ ID NO: 27), including when the amino acid cysteine ​​in CLAVEEVSLRK (SEQ ID NO: 27) is cysteinylated.

[0269] In certain embodiments, the amino acid cysteine ​​in CLAVEEVSLRK is cysteinylated.

[0270] The present invention therefore provides isolated peptides comprising an amino acid sequence selected from (i) CLAVEEVSL (SEQ ID NO: 1) (wherein the amino acid cysteine ​​may or may not be cysteinylated); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27) (wherein the amino acid cysteine ​​may or may not be cysteinylated); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29).

[0271] In certain embodiments, the amino acid cysteine ​​in CLAVEEVSL or CLAVEEVSLRK is cysteinylated.

[0272] As used herein, an "isolated peptide" refers to a peptide that is not in its natural environment. The peptide may therefore be of synthetic origin (or may be naturally occurring but isolated from its natural environment).

[0273] The isolated peptide may be relatively short (i.e., 20 or fewer amino acids; for example, not more than 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids). The peptide may consist solely of the amino acid sequence of SEQ ID NO: 1 (wherein the amino acid cysteine ​​may or may not be cysteinylated), SEQ ID NO: 26, SEQ ID NO: 27 (wherein the amino acid cysteine ​​may or may not be cysteinylated), SEQ ID NO: 28, or SEQ ID NO: 29.

[0274] In certain embodiments, the amino acid cysteine ​​in CLAVEEVSL or CLAVEEVSLRK is cysteinylated.

[0275] The isolated peptide can be administered to human subjects to treat or prevent ΔNPM1 positive hematopoietic tumors.For example, the isolated peptide can be administered to subjects to induce or enhance their immune response.The peptide can therefore be administered to subjects to induce T cell activation (for example, in vivo T cell activation) in subjects, where activated T cells are specific for the peptide (and therefore specifically target ΔNPM1 positive malignant cells).

[0276] The isolated peptides may be administered as peptide vaccines for the treatment or prevention of ΔNPM1-positive AML.The isolated peptides may be administered to induce or enhance the activation of T cells specific for ΔNPM1-positive malignant cells.

[0277] The present 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 to the T cell repertoire of ΔNPM1-positive subjects in vivo. Binding of these peptides to T cells is demonstrated herein. These peptides therefore represent bona fide immunogenic ΔNPM1-specific antigens that can be further explored for the development of personalized vaccines, which may be particularly useful as adjuvants to other therapies (e.g., the ACTs described herein). These immunogenic peptides can therefore be used as immunotherapies in the form of peptide, RNA, DNA, dendritic cell-based therapies, and adoptive TCR transgenic T cell-based therapies (for a suitable review, see ref. 23).

[0278] The inventors also show that (iv) VEEVSLRK (SEQ ID NO: 28) and (v) AVEEVSLR (SEQ ID NO: 29) are HLA-binding peptides presented on the surface of primary AML.

[0279] Isolated peptides comprising an amino acid sequence selected from (i) CLAVEEVSL (SEQ ID NO: 1) (wherein the amino acid cysteine ​​may or may not be cysteinylated); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27) (wherein the amino acid cysteine ​​may or may not be cysteinylated); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29) may therefore be useful as immunotherapies. For example, such isolated peptides may be used as immunotherapy for subjects who have, are at risk of developing, or are suspected of having ΔNPM1-positive AML. Nucleic acid sequences and vectors encoding these peptides may also be useful for this purpose.

[0280] The particular peptide for administration may be selected based on the subject's HLA-A status. As explained elsewhere herein, peptides comprising the sequence of SEQ ID NO: 1 may be administered to the subject based on HLA-A status. * 02:01 positive subjects, whereas peptides comprising the sequence of SEQ ID NO: 26 or SEQ ID NO: 27 may be particularly suitable for administration to subjects who are HLA-A positive. * 03:01 or HLA-A * It may be particularly suitable for administration to subjects who are 11:01 positive.

[0281] The isolated peptides of the present invention may also be provided in compositions comprising more than one of the above peptides. By way of example, the isolated peptides may include (a) an isolated peptide comprising the amino acid sequence CLAVEEVSL (SEQ ID NO: 1), wherein the amino acid cysteine ​​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 amino acid cysteine ​​in CLAVEEVSL (SEQ ID NO: 1) is cysteinylated. Not yet1) as a composition comprising a mixture of isolated peptides containing SEQ ID NO: 1. This composition can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), where the activated T cells have a TCR that is specific for one (or both) of the cysteinylated and / or non-cysteinylated forms of the peptide of SEQ ID NO: 1 and therefore bind to HLA-A. * It may be particularly useful for treating or preventing ΔNPM1-positive hematopoietic tumors in subjects who are 02:01 positive.

[0282] As another example, a peptide composition can be provided that comprises a mixture of (a) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 26 and (b) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27. This composition is compatible with HLA-A * 03:01 or HLA-A * Both of these peptides may be particularly useful in the treatment or prevention of ΔNPM1-positive hematopoietic malignancies in subjects who are 11:01 positive, as they are both presented by either of these HLA-A serotypes.

[0283] Similar to the peptide CLAVEEVSL, the peptide of SEQ ID NO: 27 can also be used for vaccination in its cysteinylated and non-cysteinylated forms. Thus, as another example, isolated peptides include: (a) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27, in which the amino acid cysteine ​​is cysteinylated; and (b) an isolated peptide comprising the amino acid sequence of SEQ ID NO: 27, in which the amino acid cysteine ​​is cysteinylated. Not yet 27)。 The composition can be used to induce T cell activation (e.g., in vivo T cell activation in a subject), where the activated T cells have a TCR that is specific for one (or both) of the cysteinylated and / or non-cysteinylated forms of the peptide of SEQ ID NO: 27 and therefore bind to HLA-A * 03:01 or HLA-A * It may be particularly useful for treating or preventing ΔNPM1-positive hematopoietic tumors in subjects who are 11:01 positive.

[0284] Pharmaceutical Composition The nucleic acid sequences, vectors, modified cells, isolated proteins, or peptides described herein can be provided as part of a pharmaceutical composition. Advantageously, such compositions can be administered to a human subject with a ΔNPM1-positive hematopoietic tumor for the treatment or prevention of the ΔNPM1-positive hematopoietic tumor (e.g., by eliciting or enhancing a ΔNPM1-target-specific immune response). Particularly suitable compositions can be selected based on the HLA-A serotype of the human subject, as detailed elsewhere herein.

[0285] Pharmaceutical compositions may include the nucleic acid sequences, vectors, modified cells or isolated proteins or peptides described herein together with pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers.

[0286] The compositions may contain, always in pharmaceutically acceptable concentrations, salts, buffering agents, preservatives, compatible carriers, adjuvants and supplemental immune enhancing agents such as cytokines and other therapeutic agents or compounds as desired.

[0287] As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., a substance that may be administered to an individual without causing any undesired biological effects in conjunction with the selected nucleic acid sequence, vector, modified cell or isolated peptide or interacting in a deleterious way with any of the other components of the pharmaceutical composition in which it is included.

[0288] Additives are natural or synthetic substances formulated with an active ingredient (e.g., a nucleic acid sequence, vector, modified cell, or isolated peptide provided herein) to bulk the formulation or enhance the therapeutic effect of the active ingredient in the final dosage form, e.g., to promote drug absorption or solubility. Additives may also be useful during the manufacturing process to aid in handling of the associated active ingredient, such as by promoting powder flowability or non-stick properties, as well as to aid in in vitro stability, such as preventing degradation during the expected storage period. Pharmaceutically acceptable additives are well known in the art. Suitable additives can therefore be readily identified by those skilled in the art. By way of example, suitable pharmaceutically acceptable additives include water, saline, aqueous dextrose, glycerol, ethanol, etc.

[0289] Adjuvants are pharmacological and / or immunological agents that modify the effects of other agents in the formulation. Pharmaceutically acceptable adjuvants are well known in the art. Suitable adjuvants can therefore be easily identified by those skilled in the art.

[0290] A diluent is an agent that dilutes. Pharmaceutically acceptable diluents are well known in the art. Suitable diluents can therefore be easily identified by those skilled in the art.

[0291] The carrier is non-toxic to recipients at the dosage and concentration used, and is compatible with other ingredients in the formulation.The term "carrier" refers to a natural or synthetic, organic or inorganic component with which the active ingredient is combined to facilitate administration.Pharmaceutically acceptable carriers are well known in the art.Suitable carriers can therefore be easily identified by those skilled in the art.

[0292] Target treatment The compositions of the present invention can be advantageously used to treat or prevent ΔNPM1-positive hematopoietic tumors in human subjects. Suitable compositions can be selected based on the HLA-A serotype of the human subject, as detailed elsewhere herein.

[0293] In one embodiment, the methods of treating or preventing ΔNPM1-positive hematopoietic tumors described herein result in the induction or enhancement of an immune response (e.g., a cell-mediated response) in a subject (e.g., a targeted immune response against malignant cells that present HLA-A-restricted peptides).

[0294] The term "inducing or enhancing an immune response" refers to an increase in a subject's immune response (e.g., a cell-mediated immune response, such as a T cell-mediated immune response) during or after treatment compared to the immune response before treatment. An "induced or enhanced" immune response therefore encompasses any measurable increase in an immune response that directly or indirectly targets the ΔNPM1-positive hematopoietic tumor being treated.

[0295] The compositions of the present invention can be used for the treatment or prevention of ΔNPM1-positive hematopoietic tumors, particularly ΔNPM1-positive myeloid tumors, more particularly ΔNPM1-positive AML, in human subjects.

[0296] Those skilled in the art will be well aware of hematopoietic tumors that may be ΔNPM1 positive and therefore treatable by the present invention. Similarly, those skilled in the art will be well aware of myeloid tumors that may be ΔNPM1 positive and therefore treatable by the present invention.

[0297] As used herein, the terms "treatment" and "treating" are intended to include interventions performed with the intent of preventing or altering the pathological progression of a condition, disorder, or symptom (i.e., in this case, a hematopoietic tumor). Accordingly, "treatment" refers to both therapeutic treatments and prophylactic or preventative measures whose purpose is to prevent or slow (reduce) the target condition, disorder, or symptom. Thus, "treatment" refers to a reduction, slowing, or inhibition of the amount or concentration of malignant cells by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, as measured in a sample obtained from a subject, compared to the amount or concentration of malignant cells before treatment. Methods for measuring the amount or concentration of malignant cells include, for example, qRT-PCR and quantification of ΔNPM1-positive hematopoietic tumor-specific biomarkers, such as CLAVEEVSL, AVEEVSLRK, CLAVEEVSLRK, VEEVSLRK, and / or AVEEVSLR, in a sample obtained from a subject.

[0298] As used herein, the term "subject" refers to an individual, e.g., a human, having or at risk of having a described condition, disorder, or symptom. A subject may be a patient, i.e., a subject in need of treatment according to the present invention. A subject may be undergoing treatment for the condition, disorder, or symptom. Alternatively, a subject may not have been treated prior to treatment according to the present invention.

[0299] The compositions described herein can be administered to a subject by any conventional route, including injection or slow infusion over time, for example, by infusion or intramuscular, intravascular, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, or transdermal administration.

[0300] The compositions described herein can be in any form suitable for the above-described administration modes. For example, compositions containing modified cells can be in any form suitable for infusion. By way of 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 or local delivery. Identifying appropriate dosages for the compositions of the present invention is well within the routine capabilities of one of ordinary skill in the art.

[0301] Advantageously, the compositions of the invention can be formulated for use with T cell receptor (TCR) gene transfer, an approach that is rapid, reliable, and capable of generating large numbers of T cells with specificity 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)), regardless of the patient's pre-existing immune repertoire. Through the use of TCR gene transfer, modified autologous cells suitable for infusion can be generated within a few days.

[0302] Advantageously, the compositions of the present invention can 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) can be formulated as a pharmaceutical composition suitable for use as a peptide vaccine). Suitable peptide vaccine formulations are well known in the art.

[0303] Preferably, the pharmaceutical composition of the present invention is a vaccine, preferably a peptide-based vaccine. Such peptide-based vaccines can be used for the prevention or treatment of ΔNPM1-positive hematopoietic tumors, such as AML.

[0304] The pharmaceutical composition is preferably for administration to a subject, preferably a human or animal subject, and is therefore suitably formulated. Preferably, administration is parenteral, such as intravenous, subcutaneous, intramuscular, intradermal, intradermal and / or intratumoral administration, i.e., injection.

[0305] Preferably, the pharmaceutical composition comprises or consists of an amount of peptide constituting a pharmaceutical dosage unit. Pharmaceutical dosage unit herein refers to the amount of active ingredient (i.e., the total amount of peptide in a peptide-based vaccine) applied to a subject at a given time. The pharmaceutical dosage unit may be applied to a subject in one volume, i.e., a single shot, or in two, three, four, five, or more separate volumes or shots, preferably applied to different locations on the body, for example, the right and left limbs. It is understood that the compositions of the separate volumes of a pharmaceutical dosage may differ, i.e., may contain different species or compositions of active ingredient and / or adjuvant.

[0306] A single injection volume or shot (i.e., a volume applied to a single location at one time), including the total pharmaceutical dose or portions thereof in the case of multiple shots administered substantially simultaneously, can be 100 μl to 2 mL or 100 μl to 1 mL. A single injection volume can be 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 3 mL, or any value therebetween.

[0307] Preferably, the pharmaceutical dosage unit or total amount of peptide administered to a subject at any one time, whether as a single or multiple injections at any one time, is in the range of 0.1 μg to 20 mg, for example about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, The pharmaceutical dosage units may contain an amount of peptide of 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 any value therebetween. Preferred ranges for pharmaceutical dosage units 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 to 4 mg.

[0308] The compositions described herein are intended to be administered in an effective amount. An "effective amount" is an amount that, alone or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used depends, for example, on the therapeutic (or non-therapeutic) purpose, the route of administration, and the condition of the patient / subject. For example, the appropriate dosage of the compositions of the present invention for a given patient / subject can be determined by the attending physician (or the person administering the composition) taking into account various factors known to modify the action of the compositions of the present invention, such as the severity and type of hematopoietic tumor, body weight, sex, dietary habits, time and route of administration, other drug treatments, and other relevant clinical factors. The dosage and schedule can vary depending on the specific condition, disorder, or symptom, and the overall condition of the patient / subject. The effective dosage can be determined by in vitro or in vivo methods.

[0309] The compositions of the present invention are advantageously presented in unit dosage form.

[0310] Methods for producing TCRs In one aspect, the present invention provides a method for producing a T cell receptor that specifically binds to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28) and AVEEVSLR (SEQ ID NO: 29), comprising contacting a nucleic acid sequence (or vector) of the present invention with a host cell under conditions wherein the nucleic acid sequence (or vector) is taken up by the cell, expressed, and produces the T cell receptor.

[0311] The method can be carried out on host cells ex vivo or in vitro. Alternatively, the method can be carried out in vivo, wherein the nucleic acid sequence (or vector) is administered to a subject and contacted with host cells in vivo under the conditions that the nucleic acid sequence is incorporated into the host cell, expressed, and produces T cell receptor. In some embodiments, the method is not a method for treating human or animal body.

[0312] Suitable in vivo, in vitro and ex vivo methods for contacting a nucleic acid sequence (or vector) with a host cell under conditions whereby the nucleic acid sequence (or vector) is taken up by the cell and expressed are well known, as described elsewhere herein.

[0313] general definition 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 a polynucleotide sequence. A 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), and analogs of DNA or RNA produced by the use of nucleotide analogs.

[0314] As used herein, an "isolated nucleic acid sequence" refers to a nucleic acid sequence that is not in its natural environment when associated with its naturally associated sequences, which are also in their natural environment. In other words, an isolated nucleic acid sequence is not a naturally occurring nucleotide sequence, where "natively occurring nucleotide sequence" refers to an entire nucleotide sequence in its natural environment and when operably associated with the entire promoter with which it is naturally associated, the promoter also in its natural environment.

[0315] As used herein, "specifically binds to CLAVEEVSL" refers to selective binding to the CLAVEEVSL peptide. Under certain conditions, e.g., in an immunoassay described herein, a polypeptide that "specifically binds to CLAVEEVSL" selectively binds to this peptide and does not bind to a significant amount to other peptides. Thus, the polypeptide may bind to CLAVEEVSL with at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold greater affinity than it binds to a control antigenic peptide. Selective binding can also be determined indirectly in the context of modified cells expressing a nucleic acid or vector of the invention. For example, in an assay such as that described herein, the modified cells may selectively bind to the HLA-A * Specifically reactive to cells presenting CLAVE-EVSL under the influence of 02:01 (e.g., primary ΔNPM1 HLA-A * 02:01-positive AML cells or any HLA-A carrying the ΔNPM1 gene * 02:01 positive cell line). Therefore, modified cells are HLA-A * HLA-A was significantly increased under the influence of 02:01 compared to its reactivity against a control cell line that does not present CLAVEEVSL. * It may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold more to cells displaying CLAVEEVSL in the context of 02:01.

[0316] Selective binding is HLA-A * In other words, in certain embodiments, a polypeptide that specifically binds to CLAVEEVSL may be in the context of CLAVEEVSL according to HLA-A 02:01. *When presented (i.e., bound) by 02:01 or HLA-A * This can only happen if the structural configuration is equivalent to that presented by 02:01.

[0317] Unless otherwise specified, a polypeptide that "specifically binds to CLAVEEVSL" may bind to (i) the cysteinylated form of CLAVEEVSL, (ii) the non-cysteinylated form of CLAVEEVSL, or (iii) both the cysteinylated and non-cysteinylated forms of CLAVEEVSL. Similarly, unless otherwise specified, a general reference to "SEQ ID NO: 1" or "CLAVEEVSL" encompasses both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSL.

[0318] As used herein, "specifically binds to AVEEVSLRK" refers to selective binding to the AVEEVSLRK peptide. Under certain conditions, e.g., in an immunoassay described herein, a polypeptide that "specifically binds to AVEEVSLRK" selectively binds to this peptide and does not bind to a significant amount to other peptides. Thus, the polypeptide may bind to AVEEVSLRK with at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold greater affinity than it binds to a control antigenic peptide. Selective binding can also be determined indirectly by the influence of modified cells expressing a nucleic acid or vector of the invention. For example, in an assay such as that described herein, the modified cells may bind to an HLA-A * 03:01, HLA-A * 11:01 or HLA-A * Specifically reactive to cells presenting AVEEVSLRK under the influence of 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 with ΔNPM1 gene transfer * 03:01, HLA-A * 11:01 or HLA-A *01:01 positive cell line). Therefore, modified cells are HLA-A * 03:01, HLA-A * 11:01 or HLA-A * HLA-A when compared with its reactivity to a control cell line that does not present AVEEVSLRK in the 01:01 context * 03:01, HLA-A * 11:01 or HLA-A * It may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold more to cells displaying AVEEVSLRK under the influence of 01:01.

[0319] Selective binding is HLA-A * 03:01, HLA-A * 11:01 or HLA-A * In other words, in one embodiment, a polypeptide that specifically binds to AVEEVSLRK may be under the influence of AVEEVSLRK only by HLA-A 01:01. * 03:01, HLA-A * When presented (i.e., bound) by 11:01 or HLA-A * 01:01 or HLA-A * 03:01, HLA-A * 11:01 or HLA-A * This can only happen if the structural configuration is equivalent to that presented by 01:01.

[0320] Similarly, as used herein, "RK that specifically binds to CLAVEEVSL" refers to selective binding to the CLAVEEVSLRK peptide. Under certain conditions, e.g., in an immunoassay described herein, a "RK that specifically binds to CLAVEEVSLRK" polypeptide selectively binds to this peptide and does not bind to any appreciable amount to other peptides. Thus, the polypeptide may bind to CLAVEEVSLRK with at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold greater affinity than it binds to a control antigenic peptide. Selective binding can also be determined indirectly by the influence of modified cells expressing a nucleic acid or vector of the invention. For example, in an assay such as that described herein, the modified cells may bind to an HLA-A * 03:01 or HLA-A * Specifically reactive to cells presenting CLAVEEVSLRK in the context of 11:01 (e.g., primary ΔNPM1 HLA-A * 03:01 or HLA-A * 11:01-positive AML cells or any HLA-A with ΔNPM1 gene transfer * 03:01 or HLA-A * 11:01 positive cell line). Therefore, modified cells are HLA-A * 03:01 or HLA-A * HLA-A when compared with its reactivity to a control cell line that does not present CLAVEEVSLRK in the context of 11:01 * 03:01 or HLA-A * It can bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold more to cells displaying CLAVEEVSLRK in the context of 11:01.

[0321] Selective binding is HLA-A * 03:01 or HLA-A * In other words, in one embodiment, the RK polypeptide that specifically binds to CLAVEEVSL is a polypeptide that specifically binds to HLA-A. * When presented (i.e., bound) by 03:01 or HLA-A *11:01 or HLA-A * 03:01 or HLA-A * This can only happen if the structural configuration is equivalent to that presented by 11:01.

[0322] Unless otherwise specified, a polypeptide that "specifically binds to CLAVEEVSLRK" may bind to (i) the cysteinylated form of CLAVEEVSLRK, (ii) the non-cysteinylated form of CLAVEEVSLRK, or (iii) both the cysteinylated and non-cysteinylated forms of CLAVEEVSLRK. Similarly, unless otherwise specified, a general reference to "SEQ ID NO:27" or "CLAVEEVSLRK" encompasses both the cysteinylated and non-cysteinylated forms of the peptide CLAVEEVSLRK.

[0323] As used herein, "specifically binds to VEEVSLRK" refers to selective binding to the VEEVSLRK peptide. Under certain conditions, e.g., in an immunoassay described herein, a polypeptide that "specifically binds to VEEVSLRK" selectively binds to this peptide and does not bind to significant amounts to other peptides. Thus, the polypeptide may bind to VEEVSLRK with at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold greater affinity than it binds to a control antigenic peptide. Selective binding can also be determined indirectly in the context of modified cells expressing a nucleic acid or vector of the invention. For example, in an assay such as that described herein, the modified cells are specifically reactive to cells that present VEEVSLRK in the context of an appropriate HLA-A (e.g., primary ΔNPM1-positive AML cells or any appropriate HLA-A-positive cell line into which the ΔNPM1 gene has been introduced). Therefore, the modified cells may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold more to cells presenting VEEVSLRK under the influence of the appropriate HLA-A when compared to their reactivity to a control cell line that does not present VEEVSLRK under the influence of the appropriate HLA-A.

[0324] Selective binding can be in the context of VEEVSLRK only by the appropriate HLA-A. In other words, in certain embodiments, a polypeptide that specifically binds to VEEVSLRK can do so only when presented (i.e., bound) by the appropriate HLA-A or when in a structural form equivalent to when presented by the appropriate HLA-A.

[0325] As used herein, "specifically binds to AVEEVSLR" refers to selective binding to the AVEEVSLR peptide. Under certain conditions, e.g., in an immunoassay described herein, a polypeptide that "specifically binds to AVEEVSLR" selectively binds to this peptide and does not bind to significant amounts to other peptides. Thus, the polypeptide may bind to AVEEVSLR with at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold greater affinity than it binds to a control antigenic peptide. Selective binding can also be determined indirectly in the context of modified cells expressing a nucleic acid or vector of the invention. For example, in an assay such as that described herein, the modified cells are specifically reactive to cells that present AVEEVSLR in the context of an appropriate HLA-A (e.g., primary ΔNPM1-positive AML cells or any appropriate HLA-A-positive cell line into which the ΔNPM1 gene has been introduced). Therefore, the modified cells may bind at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold more to cells presenting AVEEVSLR in the appropriate HLA-A context when compared to their reactivity to a control cell line that does not present AVEEVSLR in the appropriate HLA-A context.

[0326] Selective binding can be under the influence of VEEVSLRK only by the appropriate HLA-A. In other words, in certain embodiments, a polypeptide that specifically binds to VEEVSLRK can do so only when presented (i.e., bound) by the appropriate HLA-A or when in a structural form equivalent to when presented by the appropriate HLA-A.

[0327] A "non-essential" (or "non-critical") amino acid residue refers to a residue that can be modified from a wild-type sequence (e.g., a sequence identified by a SEQ ID NO: herein) without abolishing, or more preferably without substantially altering, biological activity, whereas an "essential" (or "critical") amino acid residue is one that can be modified in this way. For example, conserved amino acid residues are predicted to be particularly unamenable to modification, with the exception that amino acid residues within the hydrophobic core of a domain can generally be replaced with other residues of approximately equivalent hydrophobicity without significantly altering activity.

[0328] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential (or non-critical) amino acid residues in a protein are preferably replaced with other amino acid residues from the same side chain family. Alternatively, in other embodiments, mutations can be introduced randomly and the resulting mutants screened for activity to identify mutants that retain activity.

[0329] Calculations of sequence homology or identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0330] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be inserted into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the position shared by the sequences, taking into account the number of gaps and the length of each gap that needed to be introduced for optimal alignment of the two sequences.

[0331] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, 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 of the GCG software package (available at http: / / www.gcg.com) using a BLOSUM 62 matrix or a PAM250 matrix, 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 another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, 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 which should be used if the practitioner is unsure which parameters to apply in determining whether a molecule falls within the sequence identity or homology limits of the invention) is the BLOSUM 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0332] Alternatively, percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers et al. ((1989) CABIOS 4:11-17) as 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.

[0333] The nucleic acid and protein sequences described herein can be used as "query sequences" to conduct searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ((1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, 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 utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.<http: / / www.ncbi.nlm.nih.gov> reference.

[0334] The polypeptides and nucleic acid molecules described herein can have an amino acid sequence or a nucleic acid sequence that is fully or substantially identical to a sequence identified by a SEQ ID NO. The terms "sufficiently identical" or "substantially identical" are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains) to a second amino acid or nucleotide sequence, such that the first and second amino acid or nucleotide sequences share a common structural domain or a common functional activity. For example, multiple amino acid or nucleotide sequences that share a common structural domain with at least about 60% or 65% identity, approximately 75% identity, or even 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity are defined herein as being fully or substantially identical.

[0335] Unless otherwise defined, 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 pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of most of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein will be useful in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined below are more fully described with reference to the specification as a whole. Also, as used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly requires otherwise. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the circumstances used by those skilled in the art.

[0336] Aspects of the present invention are illustrated by the following non-limiting examples. [Example]

[0337] material and method Test Design The objectives of this study were (1) to identify HLA class I ligands from ΔNPM1 in primary AML, (2) to isolate CD8 cells bearing TCRs specific for HLA class I ligands from ΔNPM1, and (3) to identify TCRs for HLA class I ligands from ΔNPM1 that can mediate specific recognition and lysis of primary AML after gene transfer. HLA class I ligandome data were generated from 12 primary AML using tandem mass spectrometry, and peptides matching the alternative reading frames of ΔNPM1 were searched for. HLA-A * 02:01 pMHC tetramers were generated for each identified peptide and analyzed using six HLA-A * 02:01 positive AML patient and 6 healthy HLA-A * The TCR from one highly reactive T cell clone was sequenced and cloned into the retroviral MP71-TCR-flex vector. This vector was then used to isolate specific CD8+ cells from two healthy individuals. T cell clones isolated from healthy individuals were screened for pMHC tetramer staining, and tetramer-positive T cell clones were tested for reactivity against peptide-loaded T2 cells and primary AML with ΔNPM1 or wtNPM1 by IFN-γ ELISA. * CD8 and CD4 cells from 02:01-positive individuals were used to transduce the TCR, and TCR-transduced T cells were assessed by flow cytometry. TCR-transduced T cells were identified as HLA-A by IFN-γ ELISA. * 02:01 was tested for recognition of positive AML cell lines and primary AML with ΔNPM1 or wtNPM1, and specific lysis was observed. 51 Cr release assay.

[0338] Sample collection and cell culture After approval by the Institutional Review Board of the Leiden University Medical Center with informed consent in accordance with the Declaration of Helsinki, peripheral blood and bone marrow samples were collected from patients with AML and healthy individuals. Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll-Isopaque separation and cryopreserved. HLA-A *Buffy coats for PBMC isolation from healthy individuals with type 02:01 were ordered from Sanquin (Amsterdam, 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, United States), 5% human serum, 1.5% L-glutamine (Lonza), 1% penicillin / streptomycin (Lonza), and 100 IU / ml IL-2 (Novartis, Basel, Switzerland). AML cell lines expressing wild-type NPM1 (OCI-AML2) and ΔNPM1 (OCI-AML3) were 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 PBMCs using a magnetic cell separator (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) with CliniMACS CD14 beads (Miltenyi Biotec). Isolated monocytes were cultured for 7 days in medium containing 100 ng / mL GM-CSF (Novartis, Basel, Switzerland) and 500 IU / mL IL-4 (Schering-Plough, Kenilworth, NJ) to produce immature dendritic cells (DCs).During the last 2 days, maturation was induced by adding 100 ng / mL GM-CSF, 10 ng / mL TNF-α (Cellgenix, Freiburg, Germany), 10 ng / mL IL-1β (Cellgenix), 10 ng / mL IL-6 (Cellgenix), 1 μg / mL prostaglandin E2 (Sigma-Aldrich, St. Louis, MO), and 500 IU / mL IFN-γ (Boehringer-Ingelheim, Ingelheim, Germany).

[0339] 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 ethylenediaminetetraacetic acid, and 0.5% Zwittergent 3-12 (pH 8.0), and Complete protease inhibitor (Sigma-Aldrich, St. Louis, Missouri, United States) was added. After 2 hours of incubation with end-over-end in the lysis buffer at 4°C, the preparations were centrifuged for 10 minutes at 1000 g at 4°C. The supernatants were transferred to new tubes and incubated for 35 minutes at 13,000 g at 4°C. The supernatant was precleared with Protein A Sepharose CL-4B beads (GE Healthcare Life Sciences, Chicago, Illinois, United States) and then loaded onto 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 lysis buffer and 5–10 column volumes of 1 M, 120 mM, and NaCl-free 10 mM Tris-HCl (pH 8.0) buffer, bound HLA class I-peptide complexes were eluted and dissociated from the column with 3–4 column volumes of 10% acetic acid. Peptides were separated from HLA class I molecules through a 10 kDa membrane (Macrosep Advance Centrifugal Devices With Supor Membrane, Pall Corporation, Port Washington, New York, United States). The filtrate was lyophilized.

[0340] The eluted peptide pool was fractionated by strong cation exchange chromatography (SCX) using a homemade SCX column (320 μm i.d., 15 cm, polysulfoethyl A 3 μm, Poly LC) run at 4 μl / min. A gradient was run at 100% solvent A (100 / 0.1 water / trifluoroacetic acid v / v) for 10 min, after which a linear gradient was started to reach 100% solvent B (65 / 35 / 0.1 250 mM KCl / acetonitrile / trifluoroacetic acid v / v / v) over 15 min, followed by 100% solvent C (65 / 35 / 0.1 500 mM KCl / acetonitrile / trifluoroacetic acid v / v / v) over an additional 15 min. The gradient was maintained at 100% solvent C for 5 min, then switched back to 100% solvent A. Twenty 4-μl fractions were collected in vials prefilled with 20 μl 95 / 3 / 0.1 water / acetonitrile / FA v / v / v. Peptide fractions were lyophilized, dissolved in 95 / 3 / 0.1 water / acetonitrile / formic acid v / v / v, and analyzed by data-dependent tandem mass spectrometry (MS) on either an LTQ-FT Ultra equipped with a nanoflow liquid chromatography 1100 HPLC system (Agilent Technologies, Santa Clara, California, United States) or a Q-Exactive equipped with an easy-nLC1000, as previously described (38). Peptides were trapped on a 1.5 cm column (100 μm i.d.; ReproSil-Pur C18-AQ, 3 μm, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) at 6–10 μl / min and eluted on a 20 cm column (50 μm i.d.; ReproSil-Pur C18-AQ, 3 μm) at 150 μl / min. The column was developed with a 120 min gradient of 0–40% acetonitrile in 0.1% formic acid. The bottom of the column was withdrawn onto a tip (approximately 5 μm i.d.), from which the eluent was sprayed into the mass spectrometer. Full scan MS spectra were acquired on an FT-ICR at a resolution of 25,000 and a target value of 3,000,000.The two most intense ions were isolated for accurate mass measurement using a selected ion monitoring scan in the FT-ICR at a resolution of 50,000 and a target accumulation of 50,000. The selected ions were then fragmented in a linear ion trap using collision-induced dissociation with a target of 10,000. The Q-Exactive mass spectrometer was operated in top10 mode. The parameters were an intensity threshold of 17,000, an AGC target of 70,000 with a 20 ms (full scan) AGC target of 3,000,000 / max fill time for MS / MS, and a resolution of 17,500 with an AGC target of 100,000 / max fill time for 60 ms. The Apex trigger was set to 1-10 s, and the charge tolerance was 2-6. Proteome Discoverer version 2.1 (Thermo Fisher Scientific) was used for peptide and protein identification. The mascot node for identification was used with mascot version 2.2.04, along with the UniProt Homo Sapiens database (UP000005640; January 2015; 67,911 entries). Cysteine ​​methionine oxidation and cysteinylation were set as variable modifications. Peptide assignment was performed with a precursor tolerance of 10 ppm and MS / MS fragmentation tolerance of 20 mmu for Q-Exactive data and 2 ppm and 0.5 Da for LTQ-FT-ultra data, respectively. The identity of ΔNPM1-derived peptides was determined by their synthetic counterparts.

[0341] Peptide synthesis and pMHC tetramer production Peptides were synthesized using standard Fmoc chemistry and dissolved in dimethyl sulfoxide. Cysteinylation of the peptides was performed by treating 1 mM peptide with 2 mM 1,4-dithiothreitol in 50 mM ammonium bicarbonate for 15 min at 50°C, followed by the addition of 10 mM free cysteine ​​and 15 mM H2O2 for 30 min at RT. pMHC tetramers were produced as previously outlined (7, 8). Recombinant HLA-A *Monomers consisting of the 02:01 heavy chain and human β2-microglobulin were purified by gel filtration HPLC and biotinylation. After folding with the appropriate peptide, pMHC tetramers were generated by the addition of PE-conjugated streptavidin (Invitrogen, Thermo Fisher Scientific). UV-exchanged pMHC tetramers were then transferred to biotinylated HLA-A containing UV-sensitive peptides. * 02:01 monomers were generated by exposure to 366 nm UV light in the presence of cysteinylated ΔNPM1 peptide. After 1 h of peptide exchange, the monomers were incubated for 1 h at 4°C, 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.

[0342] Antibodies and FACS analysis T cells were stained with FITC-conjugated antibodies against CD3, CD4, CD8 (BD Biosciences, San Jose, California, United States), and TCR-Vβ5.1 (Beckman Coulter, Brea, California, United States), APC-conjugated antibodies against CD3, CD4, CD8, and mouse 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 analysis was performed with FlowJo software (Flowjo, LLC, Ashland, Oregon, United States).

[0343] T cell isolation and culture pMHC tetramer-positive CD8 T cells were isolated from PBMCs from patients with AML and healthy individuals as previously described (9). *PBMCs from 02:01-positive AML patients and healthy individuals were stained with ΔNPM1 peptide-containing PE-conjugated pMHC tetramers for 1 hour at 4°C, followed by MACS isolation using anti-PE MicroBeads (Miltenyi Biotec). Isolated cells were stained with CD8-Alexa Fluor 700 (Invitrogen), CD4-FITC, CD14-FITC, and CD19-FITC (BD Biosciences) antibodies, and pMHC tetramer-positive CD8 T cells were single-cell sorted on 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, 5000 irradiated allogeneic EBV-LCLs, and 0.8 μg / ml PHA (Oxoid Microbiology Products, Thermo Fisher Scientific) in 100 μl TCM per well in 96-well U-bottom culture plates (Costar, Sigma-Aldrich). Expanding T cell clones were restimulated with irradiated feeder cells and PHA every 10–14 days.

[0344] 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 (2,000 cells) in 40 μl TCM per well in a 384-well flat-bottom plate (Greiner Bio One, Kremsmuenster, Austria). After overnight co-incubation, culture supernatants were collected and IFN-γ release was measured. In peptide recognition assays, T2 cells (15,000 cells) were incubated with titrated peptide concentrations for 30 min at 37°C, washed twice, and then co-incubated with T cells. In blocking assays, target cells (10,000 cells) were pre-incubated for 60 min at RT with saturating concentrations of antibody-blocking HLA class I (W6 / 32) or HLA class II (PdV5.2) antibodies, followed by addition of T cells. T cell-mediated cytotoxicity was measured. 51Chromium release assay was performed. Primary AML cells were treated with 100 μCi Na2 51 They were labeled with CrO4 for 1 hour at 37°C, washed, and co-incubated with T cells at various E:T ratios in 100 μl TCM per well of a 96-well U-bottom culture plate (Costar). 51 Cr release was measured in separate plates containing 100 μl TCM or 100 μl TCM with 1% Triton-X100 (Sigma-Aldrich) per well, respectively. After 9 h of co-incubation, 25 μl of culture supernatant was harvested and transferred to a 96-well LumaPlate (PerkinElmer, Waltham, Massachusetts, United States). The release rate in counts per minute (cpm) was 1.0 μg / well. 51 Cr release, 2450 Microbeta 2 Measurement was performed using a plate counter (PerkinElmer).

[0345] TCR cloning and production of retroviral supernatants TCR α and β chain usage of clone 1A2 was determined as previously described (9) with minor modifications. 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, California, United States). During first-strand cDNA synthesis, SMARTScribe reverse transcriptase adds a 3′ non-templated polycytosine tail, which allows annealing of the TSO and second-strand cDNA synthesis. TCR amplification was performed by PCR using Phusion Flash (Thermo Fisher Scientific), anchor-specific primers, and nested primers for annealing to the TCR-Cα or Cβ region. The TCR sequences for clone 1A2 were 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) using GenScript (Piscataway, New Jersey, United States). In MP71-TCR-flex, the murine TCR-Cα and -Cβ regions contained an additional cysteine ​​residue linked to the porcine teschovirus-derived P2A sequence to facilitate TCR selective pairing and expression. The constructs were transfected into packaging cells Φ-NX-A (ATCC, Manassas, Virginia, United States), and retroviral supernatants were harvested 48 and 72 hours posttransfection and frozen at -80°C. CMV-derived HLA-A *MP71-TCR-flex, encoding a TCR for the 02:01 restricted peptide NLVPMVATV, was kindly provided by Prof. Dr. TN Schumacher (Division of Immunology, Netherlands Cancer Institute, Amsterdam, the Netherlands).

[0346] TCR gene transfer Two HLA-As * PBMCs from 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 a CD8 T cell isolation kit (Miltenyi Biotec). CD8 and CD4 cells were stimulated in 24-well flat-bottom culture plates (Costar) with irradiated autologous feeders and 0.8 μg / ml PHA. Two days after stimulation, T cells were transferred to 24-well flat-bottom suspension culture plates (Greiner Bio-One) for retroviral transduction as previously described (9, 12). Prior to the addition of T cells, plates were coated with 30 mg / mL retronectin (Takara, Clontech) and blocked with 2% human serum albumin (Sanquin). Retroviral supernatant was added, and plates were centrifuged at 3,000 g for 20 minutes at 4°C. T cells were added to the plates together with the viral supernatant at 300,000 cells per well. After overnight incubation, T cells were transferred to 24-well flat-bottom culture plates. Six days 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 and EBV-LCL and 0.8 μg / ml PHA. Prior to analysis, restimulated TCR-transduced T cells were enriched by MACS using anti-mouse TCR-Cβ-APC and anti-APC MicroBeads as described above.

[0347] result Presence of ΔNPM1 in the HLA class I ligandome of primary AML To investigate whether ΔNPM1 peptides are processed and presented by 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 classification of the 12 AML samples and their NPM1 mutation status. A 4-bp frameshift insertion in exon 12 of the NPM1 gene is a recurrent mutation occurring in 30% of primary AML cases. PCR fragment analysis demonstrated the presence of ΔNPM1 in 8 of the 12 primary AML cases. All patients had a normal karyotype by cytogenetics, except for one AML patient carrying a known chromosomal rearrangement, inv(16)(p13q22). Blast percentages measured in peripheral blood or bone marrow samples ranged from 55 to 98%.

[0348] [Table 1] a ΔNPM1 patients carried a 4-bp insertion in exon 12 of the NPM1 gene that causes a frameshift at the C-terminus of the protein. b Blast percentages measured in peripheral blood and bone marrow samples from patients with AML. * Anti-HLA-A * 02:01 AML sample on which peptide elution was performed using antibody BB7.2.

[0349] A recurrent 4-bp insertion in exon 12 results in a ΔNPM1 protein that is 4 AA longer at the C-terminus than its wild-type counterpart (11 AA, CLAVEEVSLRK) translated in an alternative reading frame. From this alternative ΔNPM1 protein, a protein region extending from the 10 N-terminal residues in the normal reading frame to the 11 C-terminal AA in the alternative reading frame (MTDQEAIQDLCLAVEEVSLRK) was searched for matching peptides in the HLA class I ligandome analyzed from 12 primary AMLs. This confirmed the presence of two 8-mer peptides (VEEVSLRK and AVEEVSLR), two 9-mer peptides (CLAVEEVSL and AVEEVSLRK), and one 11-mer peptide (CLAVEEVSLRK) in AML with ΔNPM1 but not in AML with wild-type NPM (Table 1). All five ligands eluted from seven primary AMLs were validated by mass spectrometry with synthetic peptides (Figures 1 and 2). Tandem mass spectral validation of CLAVEEVSL and CLAVEEVSLRK with synthetic peptides was performed by in vitro cysteinylation of the first residue. Prediction of HLA class I binding affinity by NetMHCpan 3.0 revealed that the epitope CLAVEEVSL binds to HLA-A. * 02:01, whereas epitopes AVEEVSLRK and CLAVEEVSLRK bind to HLA-A * 03:01 and HLA-A * HLA-A for 11:01 and possibly AVEEVSLRK * The possibility of binding to 01:01 was suggested (Table 2). * Binding to 02:01 and HLA-A of AVEEVSLRK and CLAVEEVSLRK * 03:01 and A * Binding to 11:01 was confirmed by monomeric refolding. * Since 02:01 is expressed in 50% of the Caucasian population, we hypothesized that HLA-A with 2 of 3 ΔNPM1 *We focused on CLAVEEVSL, which was detected in 02:01-positive AML (AML10197 and AML3361).

[0350] [Table 2] a Lower limit of predicted binding affinity (nM) for all other HLA class I alleles expressed by the primary AML sample from which the peptide was eluted. NA Not applicable.

[0351] T cell recognition of ΔNPM1 on primary AML HLA-A * To test whether the 02:01-restricted epitope CLAVEEVSL is a neoantigen that can be targeted by immunotherapy, we tested specific T cells from patients with AML. PE-conjugated pMHC tetramers were conjugated to CLAVEEVSL (ΔNPM1-CLA) and its cysteinylated variant C. * LAVEEVSL(ΔNPM1-C * These tetramer mixtures were then administered to six patients with ΔNPM1 AML who were in remission after chemotherapy. * We used this method to isolate specific T cells from PBMCs of 02:01-positive patients. T cells that bound one or both pMHC tetramers were enriched with magnetic anti-PE beads, and single-tetramer-positive CD8+ cells were isolated by flow cytometry (Table 3).

[0352] [Table 3] a Days since diagnosis. b PBMC, peripheral blood mononuclear cells. HLA-A bearing ΔNPM1 AML was used for T cell isolation. * 02:01 PBMC counts from positive patients are shown. cPBMCs were incubated with anti-CD8-ALX700 and CLAVEEVSL and its cysteinylated variant C * Staining was performed with a mixture of PE-conjugated pMHC tetramers against LAVEEVSL. The numbers of sorted tetramer-positive CD8 cells are shown. d ΔNPM1-CLA or ΔNPM1-C * Number of T cell clones positive for LA tetramer.

[0353] A total of 41 tetramer-positive CD8 cells were collected from 42 of the 4 patients. * 10 6 Five T cell clones from three patients were isolated from PBMCs and clonally expanded in them. However, none of the five T cell clones expressed either the ΔNPM1-CLA tetramer or the ΔNPM1-C * The HLA-A tetramer staining was not observed, indicating that T cells against CLAVEEVSL were absent or at a frequency below the detection threshold. * Specific T cells were searched for in multiple PBMCs from 02:01-positive healthy individuals (Table 4). Tetramer-positive CD8 cells, with numbers ranging from 8 to 55, were found in total from 460 to 1970 × 10 cells from each healthy individual. 6 From these cells, 31 clonally expanded T cells from 5 individuals and 13 T cell clones from 4 individuals were positive for the ΔNPM1-CLA tetramer. Of these 13 clones, 3 T cell clones were positive for the ΔNPM1-CLA tetramer. * Staining with LA tetramer was possible (Table and Fig. 3A).

[0354] [Table 4] a PBMC, peripheral blood mononuclear cells. HLA-A was used for T cell isolation. * PBMC numbers from 02:01 positive healthy individuals are shown. b PBMCs were treated with anti-CD8-ALX700 and CLAVEEVSL and its cysteinylated variant C* Staining was performed with a mixture of PE-conjugated pMHC tetramers against LAVEEVSL. The numbers of sorted tetramer-positive CD8 cells are shown. c Number of expanding T cell clones positive for ΔNPM1-CLA tetramer. d ΔNPM1-C * Number of expanding T cell clones positive for LA tetramer. nd not implemented.

[0355] To determine which of the 13 tetramer-positive CD8 clones were reactive to their target peptide, the clones were cloned into CLAVEEVSL, a cysteinylated variant of C. * LAVEEVSL or unrelated HLA-A * HLA-A binding to the exogenously bound 02:01-restricted CMV peptide NLVPMVATV * We tested the recognition of 02:01-positive T2 cells. Among 13 ΔNPM1-CLA tetramer-positive clones, two T cell clones (1A2 and 4A8) showed specific reactivity to CLAVEEVSL-loaded T2 cells but not to the control peptide NLVPMVATV (Figure 3B). Clone 1A2 also showed specific reactivity to CLAVEEVSL-loaded T2 cells. * 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 consistent with that of CLAVEEVSL as well as C. * To test the antitumor potential of clones 1A2 and 4A8, T cell reactivity was assessed against five HLA-A antigens, including three samples with ΔNPM1 and two samples with wtNPM1. *The reactivity of T cell clone 1A2 to AML with all three ΔNPM1 variants was measured in a panel of 02:01-positive primary AML. T cell clone 1A2 showed varying degrees of reactivity to AML with all three ΔNPM1 variants, whereas clone 4A8 showed relatively low reactivity to samples with two of the three ΔNPM1 variants (Figure 3C). The strong T cell reactivity of clone 1A2 to AML is explained by its ability to recognize cysteinylated ΔNPM1 as well as non-cysteinylated ΔNPM1 eluted from the cell surface of primary AML. HLA-A with wild-type NPM1 * 02:01-positive AML (Fig. 3C) or HLA-A with ΔNPM1 * No T cell reactivity was observed against 02:01-negative AML (data not shown). These data suggest that T cells with TCRs specific for ΔNPM1 are present in the T cell repertoire of healthy individuals and that these T cells specifically target HLA-A receptors on primary AML with ΔNPM1. * Figure 1 shows that the antibodies recognized CLAVEEVSL as an endogenous neoantigen presented by 02:01.

[0356] TCR gene transfer to target ΔNPM1 in primary AML ΔNPM1 is a recurrent 4-bp insertion that occurs in 30% of primary AML and is associated with HLA-A * Because 02:01 is expressed in 50% of the Caucasian population, we considered CLAVEEVSL an ideal target for TCR gene transfer. Primary AML was most strongly recognized by clone 1A2, from which mRNA was isolated and cDNA was generated for sequences of the variable regions of the TCR α and β chains against ΔNPM1. Codon-optimized gene sequences for TRAV12-2 and TRBV5-1 expressed by T cell clone 1A2 were synthesized and cloned into a modified MP71-TCR-flex retroviral vector. To promote preferential binding and expression of the TCR α and β chains, the variable regions of the TCR were cloned in frame with the murine constant region linked to the P2A sequence. The TCR against ΔNPM1 and, as a control, HLA-A were used. * 02:01 TCR against the restricted CMV peptide NLVPMVATV was induced by healthy HLA-A *CD8 and CD4 cells isolated from PBMCs from 02:01-positive individuals (donors 1 and 2) were transduced with the TCR. Six days after transduction, TCR-transduced CD8 and CD4 cells were purified using an APC-conjugated antibody against mouse TCR-Cβ and magnetic anti-APC beads. Flow cytometry analysis demonstrated specific binding of the ΔNPM1-CLA tetramer to CD8 cells transduced with the TCR for ΔNPM1 (CD8ФNPM1) (Figure 4A). In contrast, CMV-NLV tetramers did not bind to CD8 cells transduced with the TCR for ΔNPM1, whereas CD8 cells transduced with a CMV-specific TCR (CD8ФCMV) could be stained with the CMV-NLV tetramer but not with the ΔNPM1-CLA tetramer. For TCR-transduced CD4 cells (CD4ФNPM1 and CD4ФCMV), the results were similar to those for CD8 cells, indicating that binding of ΔNPM1-CLA tetramers to TCR-transduced T cells occurred independently of the CD8 coreceptor. TCR-transduced CD8 and CD4 cells could also be stained with antibodies against mouse TCR-Cβ, and CD8 and CD4 cells transduced with the TCR for ΔNPM1 also showed specific binding to antibodies against human TCR-Vβ5.1 (data not shown).

[0357] We then analyzed the functionality of TCR-transduced CD8 and CD4 cells for ΔNPM1 and HLA-A loaded with CLAVEEVSL but not by co-incubation with the CMV peptide NLVPMVATV. * We demonstrated that co-incubation with 02:01-positive T2 cells specifically released IFN-γ (Figure 4B). Specific release of IFN-γ was also observed upon co-incubation with the AML cell line OCI-AML3, which harbors ΔNPM1, but not with the AML cell line OCI-AML2, which harbors wtNPM1 (Figure 4C).

[0358] TCR-transduced CD8 and CD4 cells were then transduced with 13 HLA-A antigens, including 9 samples with ΔNPM1 and 4 samples with wtNPM1. *The reactivity of 02:01-positive primary AMLs was tested. After transduction with the TCR for ΔNPM1, both CD8 and CD4 cells showed recognition of all nine ΔNPM1-bearing primary AMLs, but specific recognition of AML1 bearing wtNPM was not observed (Figure 5). CD8 and CD4 cells transduced with the TCR for ΔNPM1 were also found to recognize HLA-A binding with ΔNPM1. * The transduced T cells also lacked reactivity to 02:01-negative AML and mature DCs bearing wtNPM1 (Figure 6). Next, we transduced the T cells to monocyte-derived mature DCs from donors 1 and 2, as well as 40 HLA-A antigens as non-malignant cell types with strong antigen processing and presentation capabilities. * We tested for reactivity against a panel of 02:01-positive third-party EBV-LCL. CD8 and CD4 cells transduced with the TCR for ΔNPM1 failed to recognize mature DCs or EBV-LCL, indicating that selective translation of the wtNPM1 gene did not occur and that this gene did not produce peptides mimicking CLAVEEVSL. In summary, the data demonstrate that gene transfer into CD8 and CD4 cells enabled the TCR for ΔNPM1 to recognize HLA-A receptors on primary AML with ΔNPM1. * This results in the specific recognition of CLAVEEVSL as an endogenous neoantigen presented by 02:01.

[0359] Finally, we tested the cytolytic potential of TCR-transduced CD8 and CD4 cells against primary AML. TCR-transduced T cells were incubated for 9 h. 51 Six HLA-A samples, including four with ΔNPM1 and two with wtNPM1 in the chromium release assay * The CLAVEVSL antibody was tested on a panel of 02:01-positive primary AML. Both CD8 and CD4 cells transduced with TCR for ΔNPM1 showed specific lysis of AML with ΔNPM1, but not AML with wtNPM1 (Figure 7). In conclusion, the results demonstrate that CLAVEEVSL is a therapeutic neoantigen expressed in primary AML with ΔNPM1 that can be efficiently targeted by TCR gene transfer in a coreceptor-independent manner.

[0360] Consideration We have demonstrated that HLA-A, encoded by ΔNPM1 (CLAVEEVSL), in the HLA class I ligandome of primary AML. * We identified a 02:01-restricted 9-mer peptide. T cell clones bearing TCRs specific for this peptide were isolated from healthy individuals. The TCR from one of these clones specifically recognized and lysed primary AML bearing ΔNPM1 upon retroviral transduction into CD8 and CD4 cells, demonstrating that CLAVEEVSL is a therapeutic neoantigen on AML that can be targeted by TCR gene transfer in a coreceptor-independent manner.

[0361] Using HLA class I ligandome data from 12 primary AMLs, we identified C as a peptide endogenously processed and presented in AML with ΔNPM1. * Although no match was found between the eluted peptide and the synthetic peptide in the absence of cysteinylation, the data provide strong evidence that the non-cysteinylated CLAVEEVSL peptide is also presented in AML. Clone 1A2, selected for TCR cloning and gene transfer, was a synthetic C * This clone also stained with both pMHC tetramers, but binding of the tetramers to the cysteinylated peptides was weak. Experiments testing serine-substituted synthetic peptides confirmed that the first residue of the epitope was not essential for the reactivity of clone 1A2. However, results were different for clone 4A8. Clone 4A8 recognized CLAVEEVSL but not C. *Only pMHC tetramers bearing noncysteinylated peptides, not LAVEEVSL, could bind to T cell clones. The importance of the first residue of the peptide for T cell recognition was confirmed by the lack of reactivity of clone 4A8 with a synthetic peptide in which the first residue was replaced with serine. Notably, despite its inability to recognize cysteinylated peptides, clone 4A8 showed reactivity with two AMLs with ΔNPM1. Based on this reactivity pattern of clones 1A2 and 4A8, we suggested that cysteinylated and noncysteinylated ΔNPM1 peptide variants are expressed on the cell surface, but the expression level of the epitope may differ between AMLs. The reason for the absence of CLAVEEVSL in the HLA class I ligandome is unclear but may be explained by low surface expression or poor quality of the mass spectra of the eluted peptides.

[0362] Interestingly, patients carrying ΔNPM1 in the absence of a concomitant internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD) have improved survival after chemotherapy, thus often avoiding the need for allogeneic stem cell transplantation. 1, 2, 5, 13 In vivo immune responses to ΔNPM1-derived peptides highlight this favorable prognosis, particularly because ΔNPM1 protein translocates from the nucleolus to the cytoplasm where it is susceptible to proteasomal degradation and subsequent processing by the HLA class I antigen presentation pathway. 6 Greiner et al. 14 In vitro, nine HLA-A antigens derived from ΔNPM1, including CLAVEEVSL, were identified in healthy volunteers and patients with AML. * They searched for T cell responses to the 02:01-restricted peptide. After co-culture of CD8 cells with peptide-loaded PBMCs, T cell responses to two of the nine peptides were demonstrated in both healthy volunteers and patients, but no immune responses to CLAVEEVSL were measured. They subsequently screened 25 patients with ΔNPM1 AML and confirmed that patients with immune responses to these two ΔNPM1-derived peptides had significantly higher overall survival than patients without immune responses. 15However, the number of patients screened in this study was small, and FLT3 mutation status was not determined. Recent data suggest that patients with FLT3-ITD, especially those with a high allele ratio of mutant to wild-type FLT3 gene expression, have a worse prognosis than patients without FLT3-ITD, regardless of NPM1 mutation status. 13 This observation reinforces that in vivo induction of an immune response against ΔNPM1 supports a favorable prognosis, but also supports the importance of intrinsic factors for favorable AML tumor growth and the relevance of ΔNPM1 TCR gene therapy for treating AML with an unfavorable prognosis.

[0363] AML arises from a single founder clone carrying hundreds of somatic mutations with only a few driver mutations. Subclones can emerge from the founder clone through the accumulation of additional mutations that confer a survival advantage to the cells. As a result, the majority of mutations in subclones are shared by the founder clone, while a minority are clone-specific. This heterogeneous composition of AML increases the chance of clonal evolution in persistent or recurrent disease after induction or consolidation therapy. Targeting neoantigens resulting from shared mutations is an attractive immunotherapeutic strategy for eradicating founder clones as well as subclones. Neoantigens resulting from passenger mutations can be easily lost as a result of tumor immunoediting by T cells, resulting in tumor immune escape. Immune escape is unlikely when neoantigens generated by driver mutations are targeted because they are essential for malignant transformation and are presented to all tumor cells. 16 To date, only a few neoantigens resulting from driver mutations have been identified, among which mutant KRAS is expressed in 45% of pancreatic cancers and 13% of colorectal cancers. 17 Although immune escape caused by driver mutations and neoantigens is unlikely, Tran et al. *This study demonstrates that loss of 08:02 confers escape of mutant KRAS from TCR gene therapy. Furthermore, ΔNPM1, as a clonal driver mutation arising early in leukemogenesis, remains an attractive target for immunotherapy. ΔNPM1 is also an ideal target based on its high mutation frequency of 30% in primary AML. 6 The characteristic 4-bp frameshift insertion occurs at a limited number of positions (859, 860, and 861) in the translated sequence, and although the exact 4-bp sequence can vary, the majority of mutations encode the same 11 amino acid alternative reading frame (CLAVEEVSLRK). * We identified a TCR that targets the first nine residues of this alternative reading frame in 02:01. This TCR may be used in future gene therapy for the treatment of patients with AML associated with ΔNPM1. Clinical trials will assess the efficacy and potential toxicity of ΔNPM1 TCR gene therapy and its association with HLA-A. * This will show whether immune evasion due to loss of 02:01 is an important obstacle to long-term remission of AML.

[0364] The TCR isolated from clone 1A2 transduced into CD8 and CD4 cells and expressed HLA-A ΔNPM1. *These results demonstrate that CD4 TCRs mediate the specific recognition and lysis of 02:01-positive AML cells, suggesting that the TCR can redirect immune reactivity to AML in a coreceptor-independent manner. The important role of CD4 cells in antitumor immunity has become more evident in recent decades. Traditionally, CD4 cells have been known to provide support for CD8 cells, resulting in improved tumor elimination and the induction of immunological memory. However, increasing evidence suggests that CD4 cells can mediate tumor rejection even in the absence of CD8 cells. Patients with hematological malignancies who received CD8-depleted allogeneic bone marrow transplants or donor lymphocyte infusions developed graft-versus-leukemia responses similar to those of patients who received unmodified stem cell transplants or donor lymphocytes, while the incidence and severity of graft-versus-host disease were reduced. In autologous settings, adoptive transfer of CD4 cells against HLA class II-restricted tumor-associated antigens or neoantigens resulted in tumor regression in patients with metastatic melanoma and cholangiocarcinoma, respectively. 18 However, because tumors often do not express HLA class II, administration of a mixture of CD8 and CD4 cells expressing HLA class II-unrelated antigen-receptors is preferable and may result in superior antitumor immunity. Indeed, Turtle et al. 19、20 demonstrated that administration of a fixed ratio of CD8 and CD4 cells expressing the same CD19-specific chimeric antigen receptor resulted in complete remission in a significant number of patients with relapsed or refractory B-cell non-Hodgkin's lymphoma and B-cell acute lymphoblastic leukemia. Similarly, as previously shown in mice, adoptive transfer of CD8 and CD4 cells expressing the same TCR directed against an HLA class I-restricted epitope, such as CLAVEEVSL, can lead to potent antitumor immunity.

[0365] Recently, ΔNPM1 has been described as a reliable marker for measuring minimal residual disease in patients with AML. 21Persistence of ΔNPM1 transcripts, detected by quantitative RT-PCR in the peripheral blood of patients after chemotherapy, correlated with disease recurrence 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 mutant DNA methyltransferase 3 alpha (DNMT3A). 1、2、5、13 A key finding for the authors was that the presence of ΔNPM1 transcripts after the second chemotherapy cycle in patients with a favorable molecular signature at diagnosis (absence of FLT3-ITD or mutated DNMT3A) characterized a group of patients with a relatively poor prognosis, whereas the absence of ΔNPM1 transcripts after the second chemotherapy cycle in patients with an unfavorable molecular profile (FLT3-ITD, mutated DNMT3A, or both) was prominent in patients with a relatively good prognosis. 21 Therefore, using ΔNPM1 as a marker of disease status will enable the selection of patients eligible for alloSCT and the optimal timing and selection of patients for ΔNPM1 TCR gene therapy to treat persistent or recurrent disease after chemotherapy. Ultimately, if clinical trials demonstrate that AML can be effectively treated by ΔNPM1 TCR gene transfer with low treatment-related mortality, it may replace alloSCT as the standard of care for patients with poor-prognosis ΔNPM1 AML based on adverse molecular abnormalities at diagnosis or detectable persistent or recurrent disease after chemotherapy, thereby improving overall survival for patients with AML.

[0366] References 1. Doehner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015 Sep 17;373(12):1136-52. 2. Doehner H, Estey EH, Amadori S, Appelbaum FR, Buechner T, Burnett AK et al; European LeukemiaNet. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010 Jan 21;115(3):453-74. 3. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015 Apr 3;348(6230):69-74. 4. Tran E, Robbins PF, Rosenberg SA. 'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol. 2017 Feb 15;18(3):255-262. 5. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med. 2016 Jun 9;374(23):2209-21. 6. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005 Jan 20;352(3):254-66. 7. Burrows SR, Kienzle N, Winterhalter A, Bharadwaj M, Altman JD, Brooks A. Peptide-MHC class I tetrameric complexes display exquisite ligand specificity. J Immunol. 2000 Dec 1;165(11):6229-34. 8. Rodenko B, Toebes M, Hadrup SR, van Esch WJ, Molenaar AM, Schumacher TN, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc. 2006;1(3):1120-32. 9. Jahn L, Hombrink P, Hagedoorn RS, Kester MG, van der Steen DM, Rodriguez T et al. TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1. Blood.2017 Jan 4. 10. Lefranc MP, Giudicelli V, Ginestoux C, Bodmer J, Mueller W, Bontrop R et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 1999 Jan 1;27(1):209-12. 11. Linnemann C, Heemskerk B, Kvistborg P, Kluin RJ, Bolotin DA, Chen X et al. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 Nov;19(11):1534-41. 12. Heemskerk MH, Hoogeboom M, de Paus RA, Kester MG, van der Hoorn MA, Goulmy E et al. Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region. Blood. 2003 Nov 15;102(10):3530-40. 13. Versluis J, In 't Hout FE, Devillier R, van Putten WL, Manz MG, Vekemans MC et al. Comparative value of post-remission treatment in cytogenetically normal AML subclassified by NPM1 and FLT3-ITD allelic ratio. Leukemia. 2017 Jan;31(1):26-33. 14. Greiner J, Ono Y, Hofmann S, Schmitt A, Mehring E, Goetz M et al. Mutated regions of nucleophosmin 1 elicit both CD4(+) and CD8(+) T-cell responses in patients with acute myeloid leukemia. Blood. 2012 Aug 9;120(6):1282-9. 15. Greiner J, Schneider V, Schmitt M, Goetz M, Doehner K, Wiesneth M et al. Immune responses against the mutated region of cytoplasmatic NPM1 might contribute to the favorable clinical outcome of AML patients with NPM1 mutations (NPM1mut). Blood. 2013 Aug 8;122(6):1087-8. 16. Blankenstein T, Leisegang M, Uckert W, Schreiber H. Targeting cancer-specific mutations by T cell receptor gene therapy. Curr Opin Immunol. 2015 Apr;33:112-9. 17. Tran E, Robbins PF, Lu YC, Prickett TD, Gartner JJ, Jia L et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N Engl J Med. 2016 Dec 8;375(23):2255-2262. 18. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 2014 May 9;344(6184):641-5. 19. Turtle CJ, Hanafi LA, Berger C, Hudecek M, Pender B, Robinson E et al. Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells. Sci Transl Med. 2016 Sep 7;8(355):355ra116. 20. Turtle CJ, Hanafi LA, Berger C, Gooley TA, Cherian S, Hudecek M et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest. 2016 Jun 1;126(6):2123-38. 21. Ivey A, Hills RK, Simpson MA, Jovanovic JV, Gilkes A, Grech A et al; UK National Cancer Research Institute AML Working Group. Assessment of Minimal Residual Disease in Standard-Risk AML. N Engl J Med. 2016 Feb 4;374(5):422-33. 22. H. D. Meiring, E. van der Heeft, G. J. ten Hove, A. P. J. M. de Jong, Nanoscale LC MS(n): technical design and applications to peptide and protein analysis. J Sep Sci 25, 557-568 (2002). 23. Michal Bassani-Sternberg and George Coukos: Mass spectrometry-based antigen discovery for cancer immunotherapy. Current opinion in Immunology (2016) 41:9-17. Furthermore, the present invention encompasses the following aspects. 1. (a) a polypeptide comprising a CDR3 of a TCR alpha 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 a 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). An isolated nucleic acid sequence encoding 2. The isolated nucleic acid sequence of paragraph 1, wherein the nucleic acid sequence encodes both (a) and (b), and wherein (a) and (b) together specifically bind to a peptide selected from CLAVEEVSL (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 26), CLAVEEVSLRK (SEQ ID NO: 27), VEEVSLRK (SEQ ID NO: 28), and AVEEVSLR (SEQ ID NO: 29). 3. The isolated nucleic acid sequence of paragraph 1 or 2, wherein the encoded polypeptide specifically binds to CLAVEEVSL (SEQ ID NO: 1). 4. The isolated nucleic acid sequence of paragraph 3, wherein the CDR3 of (a) has an amino acid sequence having at least 90% sequence identity to CAVTGARLMF (SEQ ID NO: 2). 5. The isolated nucleic acid sequence of paragraph 4, wherein the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 6. The isolated nucleic acid sequence according to any one of items 3 to 5, wherein the CDR3 of (b) has an amino acid sequence having at least 90% sequence identity with CASSPGGLSNEQF (SEQ ID NO: 5). 7. The isolated nucleic acid sequence of paragraph 6, wherein the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. 8. The isolated nucleic acid sequence of any of items 3 to 7, wherein the CDR3 of (a) is within a TCR α chain variable region that specifically binds to SEQ ID NO: 1, and optionally (a) further comprises a TCR α chain constant region. 9. The isolated nucleic acid sequence of paragraph 8, wherein the TCR alpha chain variable region has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8. 10. The isolated nucleic acid sequence of paragraph 9, wherein the TCR alpha chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 11. The isolated nucleic acid sequence of any of items 3 to 10, wherein the CDR3 of (b) is within a TCR β chain variable region that specifically binds to SEQ ID NO: 1, and optionally (b) further comprises a TCR β chain constant region. 12. The isolated nucleic acid sequence of paragraph 11, wherein the TCR β chain variable region has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11. 13. The isolated nucleic acid sequence of paragraph 12, wherein the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. 14. The isolated nucleic acid sequence of any of items 3 to 13, wherein the CDR3 of (a) is within a TCR alpha chain variable region having at least 90% sequence identity to SEQ ID NO: 8, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 2, and optionally (a) comprises a TCR alpha chain constant region. 15. The isolated nucleic acid sequence of paragraph 14, wherein the TCR alpha chain variable region CDR1 has the amino acid sequence of SEQ ID NO: 14 and the TCR alpha chain variable region CDR2 has the amino acid sequence of SEQ ID NO: 15. 16. The isolated nucleic acid sequence of any of items 3 to 15, wherein the CDR3 of (b) is within a TCR β chain variable region having at least 90% sequence identity to SEQ ID NO: 11, wherein the CDR3 has the amino acid sequence of SEQ ID NO: 5, and optionally (b) comprises a TCR β chain constant region. 17. The isolated nucleic acid sequence of paragraph 16, wherein the TCR β chain variable region CDR1 has the amino acid sequence of SEQ ID NO: 16 and the TCR β chain variable region CDR2 has the amino acid sequence of SEQ ID NO: 17. 18. The isolated nucleic acid sequence of any of paragraphs 1 to 17, wherein the peptide CLAVEEVSL (SEQ ID NO: 1) is cysteinylated. 19. The isolated nucleic acid sequence of any of paragraphs 1 to 18, wherein the nucleic acid sequence encodes a T cell receptor. 20. A vector comprising the nucleic acid sequence according to any one of items 1 to 19. 21. The vector of paragraph 20, wherein the vector is a plasmid or a viral vector, and optionally the vector is selected from the group consisting of retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canarypox virus, herpes virus, minicircle vector, and synthetic DNA or RNA. 22. A modified cell transfected or transduced with a nucleic acid sequence according to any one of paragraphs 1 to 19 or a vector according to paragraph 20 or 21. 23. The modified cells of paragraph 22, wherein the modified cells are selected from the group consisting of CD8 T cells, CD4 T cells, NK cells, NKT cells, gamma-delta T cells, hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines. 24. The modified cell of paragraph 22 or 23, wherein the modified cell is a human cell. 25. (i) CLAVEEVSL (SEQ ID NO: 1) (wherein the amino acid cysteine ​​is cysteinylated); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29) An isolated peptide comprising an amino acid sequence selected from: 26. The peptide according to item 25, wherein the amino acid cysteine ​​of CLAVEEVSLRK (sequence number 27) is cysteinylated. 27. The peptide of paragraph 25 or 26, wherein the peptide has 20 or fewer amino acids. 28. Peptides (i) SEQ ID NO: 1 (wherein the amino acid cysteine ​​is cysteinylated); (ii) SEQ ID NO: 26; (iii) SEQ ID NO: 27; (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29) 28. The peptide according to Item 27, consisting of a sequence selected from: 29. An isolated nucleic acid sequence encoding the peptide according to any one of items 25 to 28. 30. A vector comprising the nucleic acid sequence described in item 29. 31. A pharmaceutical composition comprising the nucleic acid sequence of any one of items 1 to 19 or the vector of item 29, item 20, item 21, or item 30, the modified cell of any one of items 22 to 24, or the isolated peptide of any one of items 25 to 28, and a pharmaceutically acceptable additive, adjuvant, diluent, and / or carrier. 32. The pharmaceutical composition according to Item 31, wherein the composition comprises the isolated peptide according to any one of Items 25 to 28, the nucleic acid according to Item 29, or the vector according to Item 30, and is formulated as a vaccine. 33. A method for treating or preventing ΔNPM1-positive hematopoietic tumors in a human subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in paragraph 31 or 32. 34. The method according to paragraph 33, wherein the hematopoietic tumor is a bone marrow tumor. 35. The method of paragraph 34, wherein the myeloid tumor is acute myeloid leukemia. 36. The method of any of paragraphs 33 to 35, wherein the method induces or enhances a cell-mediated immune response in the subject. 37. The pharmaceutical composition according to paragraph 31 or 32, for use in treating or preventing ΔNPM1-positive hematopoietic tumors in a human subject. 38. A pharmaceutical composition for use according to item 37, wherein the hematopoietic tumor is a bone marrow tumor. 39. A pharmaceutical composition for use according to item 37 or 38, wherein the myeloid tumor is acute myeloid leukemia. 40. The pharmaceutical composition for use according to any one of items 37 to 39, wherein the pharmaceutical composition is for use in inducing or enhancing a cell-mediated immune response in a subject. 41. Use of the pharmaceutical composition according to paragraph 31 or 32 in the manufacture of a medicament for treating or preventing a ΔNPM1-positive hematopoietic tumor in a human subject. 42. The use described in paragraph 41, wherein the hematopoietic tumor is a bone marrow tumor. 43. The use described in paragraph 42, wherein the myeloid tumor is acute myeloid leukemia. 44. A method for producing a T cell receptor, comprising contacting a cell with the nucleic acid sequence of any one of paragraphs 1 to 19 under conditions in which the nucleic acid sequence is taken up by the cell and expressed, so as to produce 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, or SEQ ID NO:29. 45. The method of paragraph 44, wherein the method is ex vivo. 46. ​​Use of a peptide as a biomarker for ΔNPM1-positive hematopoietic tumors in a human subject, wherein the peptide is: (i) CLAVEEVSL (SEQ ID NO: 1); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29) The use is selected from: 47. A method for diagnosing a ΔNPM1-positive hematopoietic tumor 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); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29); wherein the presence of the peptide in the sample identifies the subject as having a ΔNPM1-positive hematopoietic tumor, and the absence of the peptide identifies the subject as not having a ΔNPM1-positive hematopoietic tumor. method. 48. A method for treating or preventing a ΔNPM1-positive hematopoietic tumor 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); and (ii) administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 31 or 32; A method comprising: 49. The pharmaceutical composition according to paragraph 31 or 32, for use in treating or preventing a ΔNPM1-positive hematopoietic tumor in a human subject, wherein the subject has been identified as having a ΔNPM1-positive hematopoietic tumor by the presence of a peptide in a sample isolated from the subject, wherein the peptide is (i) CLAVEEVSL (SEQ ID NO: 1); (ii) AVEEVSLRK (SEQ ID NO: 26); (iii) CLAVEEVSLRK (SEQ ID NO: 27); (iv) VEEVSLRK (SEQ ID NO: 28); and (v) AVEEVSLR (SEQ ID NO: 29) The pharmaceutical composition is selected from the group consisting of:

Claims

1. A TCR that binds to a peptide having the amino acid sequence CLAVEEVSL (SEQ ID NO: 1) (a) a polypeptide comprising an alpha chain variable region having a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 2; and (b) a polypeptide comprising a β-chain variable region having a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 5; An isolated nucleic acid encoding the polypeptide (a), wherein a nucleic acid sequence encoding the polypeptide (b) and a nucleic acid sequence encoding the polypeptide (a) are operably linked to a promoter, separated by a linker sequence.

2. (i) the polypeptide of (a) further comprises a TCR alpha chain constant region; and / or (ii) the polypeptide of (b) further comprises a TCR β chain constant region; The isolated nucleic acid of claim 1. (i) the TCR alpha chain has the amino acid sequence of SEQ ID NO: 8; and (ii) the TCR β chain has the amino acid sequence of SEQ ID NO: 11; 3. The isolated nucleic acid of claim 1 or 2.

4. A vector comprising the nucleic acid according to any one of claims 1 to 3.

5. A modified cell transfected or transduced with a nucleic acid according to any one of claims 1 to 3 or a vector according to claim 4.

6. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 3, a vector according to claim 4, or a modified cell according to claim 5, and a pharmaceutically acceptable additive, adjuvant, diluent and / or carrier.

7. 7. The pharmaceutical composition of claim 6 for use in the treatment or prevention of ΔNPM1-positive hematopoietic tumors in a human subject.

8. 10. Use of the pharmaceutical composition of claim 6 in the manufacture of a medicament for the treatment or prevention of ΔNPM1-positive hematopoietic tumors in a human subject.

9. A method for producing a TCR ex vivo, comprising contacting a cell with a nucleic acid according to any one of claims 1 to 3 under conditions in which the nucleic acid sequence is taken up by the cell and expressed to produce a TCR that binds to the peptide of SEQ ID NO: 1.

Images