T cell receptors that target the NPM1 neoantigen

Abstract

This specification provides T cell receptors (TCRs) or antigen-binding fragments thereof, such as those that recognize or bind to NPM1c neoantigens. In particular, this disclosure relates to TCRs that bind to or recognize specific NPM1c peptides in relation to major histocompatibility complex (MHC) molecules. This disclosure further relates to nucleic acids encoding such TCRs, engineered cells containing such TCRs, methods for isolating such TCRs, and their use in, for example, cell therapy.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 580,503, filed on 5 September 2023, and U.S. Provisional Patent Application No. 63 / 651,653, filed on 24 May 2024, each of which is incorporated herein by reference.

[0002] (Sequence Listing) The sequence list described in the file BSB-0009WO01_SeqList.xml is 383 kilobytes in size, was created on September 3, 2024, and is incorporated herein by reference. (Technical field) This invention relates to a T cell receptor that targets the NPM1 neoantigen. [Background technology]

[0003] Allogeneic hematopoietic stem cell transplantation (HSCT) (HLA-matched or HLA haplotype-matched) can be used to treat diseases or conditions such as hematological malignancies and other non-malignant conditions. Some patients may relapse after alloSCT or are ineligible for alloSCT. Improved treatments are needed to achieve optimal therapeutic outcomes. T cell receptors (TCRs) and engineered T cells containing TCRs are provided to meet such needs. [Overview of the Initiative]

[0004] Anti-NPM1c T cell receptors (anti-AIQDLCLAV TCRs and anti-CLAVEEVSL TCRs) or their antigen-binding fragments that recognize or bind to NPM1c neoantigens (i.e., AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)) are described. In some embodiments, the described anti-NPM1c TCRs bind to or recognize the peptide AIQDLCLAV (SEQ ID NO: 1). In some embodiments, the described anti-NPM1c TCRs bind to or recognize the peptide CLAVEEVSL (SEQ ID NO: 5). In some embodiments, the described anti-NPM1c TCRs bind to or recognize the peptide AIQDLCLAV (SEQ ID NO: 1) in relation to a major histocompatibility complex (MHC) molecule. In some embodiments, the described anti-NPM1c TCRs bind to or recognize the peptide CLAVEEVSL (SEQ ID NO: 5) in relation to a major histocompatibility complex (MHC) molecule. This disclosure further relates to polynucleotides encoding such anti-NPM1c TCRs, purified anti-NPM1c TCRs, purified anti-NPM1c TCR-anti-CD3 fusion proteins, engineered cells containing or expressing such anti-NPM1c TCRs, methods for isolating such anti-NPM1c TCRs, and their use in, for example, T cell therapy, such as engineered autologous T cell therapy.

[0005] This specification provides an anti-NPM1c TCR or its antigen-binding fragment, which comprises an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region. (a) The Vα region or Vγ region contains complementarity-determining region 3 (CDR-3) including sequence number 13, and the Vβ region or Vδ region contains CDR-3 including sequence number 21, (b) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 31, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 39, (c) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 49, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 57, (d) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 67, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 75, (e) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 85, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 93, (f) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 103, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 111, (g) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 121, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 129, (h) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 139, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 147, (i) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 157, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 165, (j) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 175, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 183, (k) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 193, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 201, (l) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO 211, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO 219, (m) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO 229, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO 237, (n) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 247, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 255, (o) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 265, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 273, or (p) A T cell receptor (TCR) or its antigen-binding fragment, wherein the Vα or Vγ region contains CDR-3 containing SEQ ID NO: 283, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO: 291.

[0006] Anti-NPM1c TRCs corresponding to any of (a) to (c) above recognize the AIQDLCLAV (SEQ ID NO: 1) peptide. Anti-NPM1c TRCs corresponding to any of (d) to (p) above recognize the CLAVEEVSL (SEQ ID NO: 5) peptide.

[0007] Furthermore, this specification provides an anti-NPM1c TCR or its antigen-binding fragment, which comprises an α chain containing a Vα region and a β chain containing a Vβ region, or a γ chain containing a Vγ region and a δ chain containing a Vδ region. (a) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 11, CDR-2 containing SEQ ID NO: 12, and CDR-3 containing SEQ ID NO: 13, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 19, CDR-2 containing SEQ ID NO: 20, and CDR-3 containing SEQ ID NO: 21, (b) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 29, CDR-2 containing SEQ ID NO: 30, and CDR-3 containing SEQ ID NO: 31, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 37, CDR-2 containing SEQ ID NO: 38, and CDR-3 containing SEQ ID NO: 39, (c) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 47, CDR-2 containing SEQ ID NO: 48, and CDR-3 containing SEQ ID NO: 49, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 55, CDR-2 containing SEQ ID NO: 56, and CDR-3 containing SEQ ID NO: 57, (d) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 65, CDR-2 containing SEQ ID NO: 66, and CDR-3 containing SEQ ID NO: 67, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 73, CDR-2 containing SEQ ID NO: 74, and CDR-3 containing SEQ ID NO: 75, (e) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 83, CDR-2 containing SEQ ID NO: 84, and CDR-3 containing SEQ ID NO: 85, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 91, CDR-2 containing SEQ ID NO: 92, and CDR-3 containing SEQ ID NO: 93, (f) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 101, CDR-2 containing SEQ ID NO: 102, and CDR-3 containing SEQ ID NO: 103, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 109, CDR-2 containing SEQ ID NO: 110, and CDR-3 containing SEQ ID NO: 111, (g) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 119, CDR-2 containing SEQ ID NO: 120, and CDR-3 containing SEQ ID NO: 121, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 127, CDR-2 containing SEQ ID NO: 128, and CDR-3 containing SEQ ID NO: 129, (h) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 137, CDR-2 containing SEQ ID NO: 138, and CDR-3 containing SEQ ID NO: 139, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 145, CDR-2 containing SEQ ID NO: 146, and CDR-3 containing SEQ ID NO: 147, (i) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 155, CDR-2 containing SEQ ID NO: 156, and CDR-3 containing SEQ ID NO: 157, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 163, CDR-2 containing SEQ ID NO: 164, and CDR-3 containing SEQ ID NO: 165, (j) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 173, CDR-2 containing SEQ ID NO: 174, and CDR-3 containing SEQ ID NO: 175, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 181, CDR-2 containing SEQ ID NO: 182, and CDR-3 containing SEQ ID NO: 183, (k) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 191, CDR-2 containing SEQ ID NO: 192, and CDR-3 containing SEQ ID NO: 193, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 199, CDR-2 containing SEQ ID NO: 200, and CDR-3 containing SEQ ID NO: 201, (l) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 209, CDR-2 containing SEQ ID NO: 210, and CDR-3 containing SEQ ID NO: 211, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 217, CDR-2 containing SEQ ID NO: 218, and CDR-3 containing SEQ ID NO: 219, (m) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 227, CDR-2 containing SEQ ID NO: 228, and CDR-3 containing SEQ ID NO: 229, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 235, CDR-2 containing SEQ ID NO: 236, and CDR-3 containing SEQ ID NO: 237, (n) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 245, CDR-2 containing SEQ ID NO: 246, and CDR-3 containing SEQ ID NO: 247, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 253, CDR-2 containing SEQ ID NO: 254, and CDR-3 containing SEQ ID NO: 255, (o) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 263, CDR-2 containing SEQ ID NO: 264, and CDR-3 containing SEQ ID NO: 265, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 271, CDR-2 containing SEQ ID NO: 272, and CDR-3 containing SEQ ID NO: 273, or (p) A T cell receptor (TCR) or its antigen-binding fragment, wherein the Vα or Vγ region includes CDR-1 containing SEQ ID NO: 281, CDR-2 containing SEQ ID NO: 282, and CDR-3 containing SEQ ID NO: 283, and the Vβ or Vδ region includes CDR-1 containing SEQ ID NO: 289, CDR-2 containing SEQ ID NO: 290, and CDR-3 containing SEQ ID NO: 291.

[0008] Furthermore, this specification provides an anti-NPM1c TCR or its antigen-binding fragment, which comprises an α chain containing a Vα region and a β chain containing a Vβ region, or a γ chain containing a Vγ region and a δ chain containing a Vδ region. (a) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 14, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 22, (b) The Vα region or Vγ region contains a CDR-3 containing a CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 32, and the Vβ region or Vδ region contains a CDR-3 containing a CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 40, (c) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 50, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 58, (d) The Vα region or Vγ region contains a CDR-3 containing a CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 68, and the Vβ region or Vδ region contains a CDR-3 containing a CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 76, (e) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 86, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 94, (f) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 104, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 112, (g) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 122, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 130, (h) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 140, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 148, (i) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 158, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 166, (j) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 176, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 184, (k) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 194, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 202, (l) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 212, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 220, (m) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 230, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 238, (n) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 248, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 256, (o) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 266, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 274, or (p) The Vα region or Vγ region contains a CDR-3 containing a CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 284, and the Vβ region or Vδ region contains a CDR-3 containing a CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 292.

[0009] In some embodiments, (a) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 14, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 22, (b) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 32, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 40, (a) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 14, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 22, (b) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 32, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 40, (c) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 50, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 58, (d) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 68, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 76, (e) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 86, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 94, (f) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 104, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 112, (g) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 122, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 130, (h) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 140, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 148, (i) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 158, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 166, (j) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 176, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 184, (k) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 194, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 202, (l) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 212, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 220, (m) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 230, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 238, (n) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 248, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 256, (o) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 266, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 274, or (p) The Vα region or Vγ region contains CDR-1 and CDR-2, respectively containing CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 284, and the Vβ region or Vδ region contains CDR-1 and CDR-2, respectively containing CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 292.

[0010] Furthermore, an anti-NPM1c TCR or its antigen-binding fragment is provided, which comprises an α chain containing a Vα region and a β chain containing a Vβ region, or a γ chain containing a Vγ region and a δ chain containing a Vδ region. (a) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 14, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 22, (b) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 32, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 40, (c) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 50, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 58, (d) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 68, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 76, (e) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 86, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 94, (f) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 104, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 112, (g) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 122, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 130, (h) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 140, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 148, (i) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 158, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 166, (j) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 176, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 184, (k) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 194, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 202, (l) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 212, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 220, (m) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 230, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 238, (n) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 248, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 256, (o) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 266, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, respectively, which are contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 274, or (p) The Vα region or Vγ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 284, and the Vβ region or Vδ region contains CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 292.

[0011] Furthermore, an anti-NPM1c TCR or its antigen-binding fragment is provided, which comprises an α chain containing a Vα region and a β chain containing a Vβ region, or a γ chain containing a Vγ region and a δ chain containing a Vδ region. (a) The Vα or Vγ region contains sequence number 14 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 22 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 11, CDR-2 containing sequence number 12, and CDR-3 containing sequence number 13, and the Vβ or Vδ region contains CDR-1 containing sequence number 19, CDR-2 containing sequence number 20, and CDR-3 containing sequence number 21, (b) The Vα or Vγ region contains sequence number 32 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 40 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 29, CDR-2 containing sequence number 30, and CDR-3 containing sequence number 31, and the Vβ or Vδ region contains CDR-1 containing sequence number 37, CDR-2 containing sequence number 38, and CDR-3 containing sequence number 38, (c) The Vα or Vγ region contains sequence number 50 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 58 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 47, CDR-2 containing sequence number 48, and CDR-3 containing sequence number 49, and the Vβ or Vδ region contains CDR-1 containing sequence number 55, CDR-2 containing sequence number 56, and CDR-3 containing sequence number 57, (d) The Vα or Vγ region contains sequence number 68 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 76 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 65, CDR-2 containing sequence number 66, and CDR-3 containing sequence number 67, and the Vβ or Vδ region contains CDR-1 containing sequence number 73, CDR-2 containing sequence number 74, and CDR-3 containing sequence number 75, (e) The Vα or Vγ region contains sequence number 86 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 94 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 83, CDR-2 containing sequence number 84, and CDR-3 containing sequence number 85, and the Vβ or Vδ region contains CDR-1 containing sequence number 91, CDR-2 containing sequence number 92, and CDR-3 containing sequence number 93, (f) The Vα or Vγ region contains sequence number 104 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 112 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 101, CDR-2 containing sequence number 102, and CDR-3 containing sequence number 103, and the Vβ or Vδ region contains CDR-1 containing sequence number 109, CDR-2 containing sequence number 110, and CDR-3 containing sequence number 111, (g) The Vα or Vγ region contains sequence number 122 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 130 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 119, CDR-2 containing sequence number 120, and CDR-3 containing sequence number 121, and the Vβ or Vδ region contains CDR-1 containing sequence number 127, CDR-2 containing sequence number 128, and CDR-3 containing sequence number 129, (h) The Vα or Vγ region contains sequence number 140 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 148 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 137, CDR-2 containing sequence number 138, and CDR-3 containing sequence number 139, and the Vβ or Vδ region contains CDR-1 containing sequence number 145, CDR-2 containing sequence number 146, and CDR-3 containing sequence number 147, (i) The Vα or Vγ region contains sequence number 158 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 166 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 155, CDR-2 containing sequence number 156, and CDR-3 containing sequence number 157, and the Vβ or Vδ region contains CDR-1 containing sequence number 163, CDR-2 containing sequence number 164, and CDR-3 containing sequence number 165, (j) The Vα or Vγ region contains sequence number 176 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 184 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 173, CDR-2 containing sequence number 174, and CDR-3 containing sequence number 175, and the Vβ or Vδ region contains CDR-1 containing sequence number 181, CDR-2 containing sequence number 182, and CDR-3 containing sequence number 183, (k) The Vα or Vγ region contains sequence number 194 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 202 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 191, CDR-2 containing sequence number 192, and CDR-3 containing sequence number 193, and the Vβ or Vδ region contains CDR-1 containing sequence number 199, CDR-2 containing sequence number 200, and CDR-3 containing sequence number 201, (l) The Vα or Vγ region contains sequence number 212 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 220 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 209, CDR-2 containing sequence number 210, and CDR-3 containing sequence number 211, and the Vβ or Vδ region contains CDR-1 containing sequence number 217, CDR-2 containing sequence number 218, and CDR-3 containing sequence number 219, (m) The Vα or Vγ region contains sequence number 230 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 238 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 227, CDR-2 containing sequence number 228, and CDR-3 containing sequence number 229, and the Vβ or Vδ region contains CDR-1 containing sequence number 235, CDR-2 containing sequence number 236, and CDR-3 containing sequence number 23, (n) The Vα or Vγ region contains sequence number 248 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 256 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 245, CDR-2 containing sequence number 246, and CDR-3 containing sequence number 247, and the Vβ or Vδ region contains CDR-1 containing sequence number 253, CDR-2 containing sequence number 254, and CDR-3 containing sequence number 255, (o) The Vα or Vγ region contains sequence number 266 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region contains sequence number 274 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region contains CDR-1 containing sequence number 263, CDR-2 containing sequence number 264, and CDR-3 containing sequence number 265, and the Vβ or Vδ region contains CDR-1 containing sequence number 271, CDR-2 containing sequence number 272, and CDR-3 containing sequence number 273, or (p) The Vα or Vγ region includes sequence number 284 or a sequence having at least 90% sequence identity thereto, and the Vβ or Vδ region includes sequence number 292 or a sequence having at least 90% sequence identity thereto, and the Vα or Vγ region includes CDR-1 containing sequence number 281, CDR-2 containing sequence number 282, and CDR-3 containing sequence number 283, and the Vβ or Vδ region includes CDR-1 containing sequence number 289, CDR-2 containing sequence number 290, and CDR-3 containing sequence number 291.

[0012] In some embodiments, (a) The Vα region or Vγ region contains SEQ ID NO: 14, and the Vβ region or Vδ region contains SEQ ID NO: 22, (b) The Vα region or Vγ region contains SEQ ID NO: 32, and the Vβ region or Vδ region contains SEQ ID NO: 40, (c) The Vα region or Vγ region contains SEQ ID NO: 50, and the Vβ region or Vδ region contains SEQ ID NO: 58, (d) The Vα region or Vγ region contains SEQ ID NO: 68, and the Vβ region or Vδ region contains SEQ ID NO: 76, (e) The Vα region or Vγ region contains SEQ ID NO: 86, and the Vβ region or Vδ region contains SEQ ID NO: 94, (f) The Vα region or Vγ region contains SEQ ID NO: 104, and the Vβ region or Vδ region contains SEQ ID NO: 112, (g) The Vα region or Vγ region contains SEQ ID NO: 122, and the Vβ region or Vδ region contains SEQ ID NO: 130, (h) The Vα region or Vγ region contains SEQ ID NO: 140, and the Vβ region or Vδ region contains SEQ ID NO: 148, (i) The Vα region or Vγ region contains sequence number 158, and the Vβ region or Vδ region contains sequence number 166, (j) The Vα region or Vγ region contains SEQ ID NO: 176, and the Vβ region or Vδ region contains SEQ ID NO: 184, (k) The Vα region or Vγ region contains sequence number 194, and the Vβ region or Vδ region contains sequence number 202, (l) The Vα region or Vγ region contains SEQ ID NO: 212, and the Vβ region or Vδ region contains SEQ ID NO: 220, (m) The Vα region or Vγ region contains SEQ ID NO: 230, and the Vβ region or Vδ region contains SEQ ID NO: 238, (n) The Vα region or Vγ region contains SEQ ID NO: 248, and the Vβ region or Vδ region contains SEQ ID NO: 256, (o) The Vα region or Vγ region contains SEQ ID NO: 266, and the Vβ region or Vδ region contains SEQ ID NO: 274, or (p) The TCR or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the Vα region or Vγ region includes SEQ ID NO: 284, and the Vβ region or Vδ region includes SEQ ID NO: 292.

[0013] In some embodiments, the α chain further includes an α-steady (Cα) region and the β chain further includes a β-steady (Cβ) region, or the γ chain further includes a γ-steady (Cγ) region and the δ chain further includes a δ-steady (Cδ) region. In some embodiments, Cα includes SEQ ID NO: 3 and Cβ includes SEQ ID NO: 7.

[0014] In some embodiments, (a) The α chain or γ chain contains SEQ ID NO: 17, and the β chain or δ chain contains SEQ ID NO: 25, (b) The α-chain or γ-chain contains SEQ ID NO: 35, and the β-chain or δ-chain contains SEQ ID NO: 43, (c) The α chain or γ chain contains SEQ ID NO: 53, and the β chain or δ chain contains SEQ ID NO: 61, (d) The α chain or γ chain contains SEQ ID NO: 71, and the β chain or δ chain contains SEQ ID NO: 79, (e) The α-chain or γ-chain contains SEQ ID NO: 89, and the β-chain or δ-chain contains SEQ ID NO: 97, (f) The α chain or γ chain contains SEQ ID NO: 107, and the β chain or δ chain contains SEQ ID NO: 115, (g) The α-chain or γ-chain contains SEQ ID NO: 125, and the β-chain or δ-chain contains SEQ ID NO: 133, (h) The α-chain or γ-chain contains SEQ ID NO: 143, and the β-chain or δ-chain contains SEQ ID NO: 151, (i) The α chain or γ chain contains SEQ ID NO: 161, and the β chain or δ chain contains SEQ ID NO: 169, (j) The α chain or γ chain contains SEQ ID NO: 179, and the β chain or δ chain contains SEQ ID NO: 187, (k) The α or γ chain contains SEQ ID NO: 197, and the β or δ chain contains SEQ ID NO: 205, (l) The α chain or γ chain contains SEQ ID NO: 215, and the β chain or δ chain contains SEQ ID NO: 223, (m) The α-chain or γ-chain contains SEQ ID NO: 233, and the β-chain or δ-chain contains SEQ ID NO: 241, (n) The α chain or γ chain contains SEQ ID NO: 251, and the β chain or δ chain contains SEQ ID NO: 259, (o) The α chain or γ chain contains SEQ ID NO: 269, and the β chain or δ chain contains SEQ ID NO: 277, or (p) The α-chain or γ-chain contains SEQ ID NO: 287, and the β-chain or δ-chain contains SEQ ID NO: 295.

[0015] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment recognizes the NPM1c neoantigen in relation to an MHC molecule. In some embodiments, the MHC molecule is a human leukocyte antigen (HLA)-A molecule. In some embodiments, the HLA-A molecule is a serotype HLA-A * This is from 02:01. In some embodiments, the HLA-A molecule is serotype HLA-A * This is from 02:06. In some embodiments, the HLA-A molecule is serotype HLA-A * This is the 02:03. In some embodiments, the neoantigen of NPM1c is described in SEQ ID NO: 1 or 5.

[0016] Also provided herein are nucleotide sequences (e.g., polynucleotides or nucleic acids) encoding the anti-NPM1c TCR or its antigen-binding fragment, or any of its Vα, Vγ, Vβ, Vδ, α chain, β chain, γ chain, or δ chain. The nucleotide sequences may be provided as pairs of nucleotide sequences, comprising a first nucleotide sequence encoding the Vα region or α chain and a second nucleotide sequence encoding the Vβ region or β chain, or a first nucleotide sequence encoding the Vγ region or γ chain and a second nucleotide sequence encoding the Vδ region or δ chain. The first and second nucleotide sequences may be provided on separate expression cassettes, plasmids, or vectors (e.g., viral vectors), or on a single expression cassette, plasmid, or vector. When provided on a single expression cassette, plasmid, or vector, the first and second nucleotide sequences may be linked by a 2A element (e.g., a P2A element) or an intrasequence ribosome entry sequence (IRES) and expressed from a single promoter.

[0017] In some embodiments, the nucleotide sequence encoding the anti-NPM1c TCR or its antigen-binding fragment comprises a first nucleotide sequence encoding the Vα region and a second nucleotide sequence encoding the Vβ region, or a first nucleotide sequence encoding the Vγ region and a second nucleotide sequence encoding the Vδ region. (a) The first nucleotide sequence codes for SEQ ID NO: 14, and the second nucleotide sequence codes for SEQ ID NO: 22, (b) The first nucleotide sequence codes for SEQ ID NO: 32, and the second nucleotide sequence codes for SEQ ID NO: 40, (c) The first nucleotide sequence codes for SEQ ID NO: 50, and the second nucleotide sequence codes for SEQ ID NO: 58, (d) The first nucleotide sequence codes for SEQ ID NO: 68, and the second nucleotide sequence codes for SEQ ID NO: 76, (e) The first nucleotide sequence codes for SEQ ID NO: 86, and the second nucleotide sequence codes for SEQ ID NO: 94, (f) The first nucleotide sequence codes for sequence number 104, and the second nucleotide sequence codes for sequence number 112, (g) The first nucleotide sequence codes for SEQ ID NO: 122, and the second nucleotide sequence codes for SEQ ID NO: 130, (h) The first nucleotide sequence codes for SEQ ID NO: 140, and the second nucleotide sequence codes for SEQ ID NO: 148, (i) The first nucleotide sequence codes for sequence number 158, and the second nucleotide sequence codes for sequence number 166, (j) The first nucleotide sequence codes for SEQ ID NO: 176, and the second nucleotide sequence codes for SEQ ID NO: 184, (k) The first nucleotide sequence codes for SEQ ID NO: 194 and the second nucleotide sequence codes for SEQ ID NO: 202, (l) The first nucleotide sequence codes for SEQ ID NO: 212, and the second nucleotide sequence codes for SEQ ID NO: 220, (m) The first nucleotide sequence codes for SEQ ID NO: 230, and the second nucleotide sequence codes for SEQ ID NO: 238, (n) The first nucleotide sequence codes for SEQ ID NO: 248, and the second nucleotide sequence codes for SEQ ID NO: 256, (o) The first nucleotide sequence codes for SEQ ID NO: 266 and the second nucleotide sequence codes for SEQ ID NO: 274, or (p) The first nucleotide sequence codes for sequence number 284, and the second nucleotide sequence codes for sequence number 292.

[0018] Anti-NPM1c TRCs corresponding to any of (a) to (c) above recognize the AIQDLCLAV (SEQ ID NO: 1) peptide. Anti-NPM1c TRCs corresponding to any of (d) to (p) above recognize the CLAVEEVSL (SEQ ID NO: 5) peptide.

[0019] In some embodiments, (a) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 15 or SEQ ID NO: 14 and has at least 90% sequence identity with respect to SEQ ID NO: 15, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 23 or SEQ ID NO: 22 and has at least 90% sequence identity with respect to SEQ ID NO: 23, (b) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 33 or SEQ ID NO: 32 and has at least 90% sequence identity with respect to SEQ ID NO: 33, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 41 or SEQ ID NO: 40 and has at least 90% sequence identity with respect to SEQ ID NO: 41, (c) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 51 or SEQ ID NO: 50 and has at least 90% sequence identity with respect to SEQ ID NO: 51, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 59 or SEQ ID NO: 58 and has at least 90% sequence identity with respect to SEQ ID NO: 59, (d) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 69 or SEQ ID NO: 68 and has at least 90% sequence identity with respect to SEQ ID NO: 69, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 77 or SEQ ID NO: 76 and has at least 90% sequence identity with respect to SEQ ID NO: 77, (e) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 87 or SEQ ID NO: 86 and has at least 90% sequence identity with respect to SEQ ID NO: 87, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 95 or SEQ ID NO: 94 and has at least 90% sequence identity with respect to SEQ ID NO: 95, (f) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 105 or SEQ ID NO: 104 and has at least 90% sequence identity with respect to SEQ ID NO: 105, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 113 or SEQ ID NO: 112 and has at least 90% sequence identity with respect to SEQ ID NO: 113, (g) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 123 or SEQ ID NO: 122 and has at least 90% sequence identity with respect to SEQ ID NO: 123, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 131 or SEQ ID NO: 1130 and has at least 90% sequence identity with respect to SEQ ID NO: 131, (h) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 141 or SEQ ID NO: 140 and has at least 90% sequence identity with respect to SEQ ID NO: 141, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 149 or SEQ ID NO: 148 and has at least 90% sequence identity with respect to SEQ ID NO: 149, (i) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 159 or SEQ ID NO: 158 and has at least 90% sequence identity with respect to SEQ ID NO: 159, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 167 or SEQ ID NO: 166 and has at least 90% sequence identity with respect to SEQ ID NO: 167, (j) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 177 or SEQ ID NO: 176 and has at least 90% sequence identity with respect to SEQ ID NO: 177, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 185 or SEQ ID NO: 184 and has at least 90% sequence identity with respect to SEQ ID NO: 185, (k) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 195 or SEQ ID NO: 194 and has at least 90% sequence identity with respect to SEQ ID NO: 195, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 203 or SEQ ID NO: 202 and has at least 90% sequence identity with respect to SEQ ID NO: 203, (l) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 213 or SEQ ID NO: 212 and has at least 90% sequence identity with respect to SEQ ID NO: 213, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 221 or SEQ ID NO: 220 and has at least 90% sequence identity with respect to SEQ ID NO: 221, (m) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 231 or SEQ ID NO: 230 and has at least 90% sequence identity with respect to SEQ ID NO: 231, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 239 or SEQ ID NO: 238 and has at least 90% sequence identity with respect to SEQ ID NO: 239, (n) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 249 or SEQ ID NO: 248 and has at least 90% sequence identity with respect to SEQ ID NO: 249, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 257 or SEQ ID NO: 256 and has at least 90% sequence identity with respect to SEQ ID NO: 257, (o) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 267 or SEQ ID NO: 266 and has at least 90% sequence identity with respect to SEQ ID NO: 267, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 275 or SEQ ID NO: 274 and has at least 90% sequence identity with respect to SEQ ID NO: 275, or (p) The first nucleotide sequence includes a sequence that codes for SEQ ID NO: 285 or SEQ ID NO: 284 and has at least 90% sequence identity with respect to SEQ ID NO: 285, and the second nucleotide sequence includes a sequence that codes for SEQ ID NO: 293 or SEQ ID NO: 292 and has at least 90% sequence identity with respect to SEQ ID NO: 293.

[0020] In some embodiments, one or more nucleic acid sequences encoding Vα, Vγ, Vβ, Vδ, α stationary, γ stationary, β stationary, or δ stationary chains are codon-optimized. In some embodiments, (a) The first nucleotide sequence contains SEQ ID NO: 16 and the second nucleotide sequence contains SEQ ID NO: 24 (b) The first nucleotide sequence contains SEQ ID NO: 34 and the second nucleotide sequence contains SEQ ID NO: 42 (c) The first nucleotide sequence contains SEQ ID NO: 52, and the second nucleotide sequence contains SEQ ID NO: 60, (d) The first nucleotide sequence contains SEQ ID NO: 70, and the second nucleotide sequence contains SEQ ID NO: 78, (e) The first nucleotide sequence contains SEQ ID NO: 88, and the second nucleotide sequence contains SEQ ID NO: 96, (f) The first nucleotide sequence contains SEQ ID NO: 106 and the second nucleotide sequence contains SEQ ID NO: 114 (g) The first nucleotide sequence contains SEQ ID NO: 124, and the second nucleotide sequence contains SEQ ID NO: 132, (h) The first nucleotide sequence contains SEQ ID NO: 142, and the second nucleotide sequence contains SEQ ID NO: 150, (i) The first nucleotide sequence contains SEQ ID NO: 160, and the second nucleotide sequence contains SEQ ID NO: 168, (j) The first nucleotide sequence contains SEQ ID NO: 178 and the second nucleotide sequence contains SEQ ID NO: 186 (k) The first nucleotide sequence contains SEQ ID NO: 196 and the second nucleotide sequence contains SEQ ID NO: 204, (l) The first nucleotide sequence contains SEQ ID NO: 214, and the second nucleotide sequence contains SEQ ID NO: 222, (m) The first nucleotide sequence contains SEQ ID NO: 232, and the second nucleotide sequence contains SEQ ID NO: 240, (n) The first nucleotide sequence contains SEQ ID NO: 250, and the second nucleotide sequence contains SEQ ID NO: 258, (o) The first nucleotide sequence contains SEQ ID NO: 268, and the second nucleotide sequence contains SEQ ID NO: 276, or (p) The first nucleotide sequence includes sequence number 286, and the second nucleotide sequence includes sequence number 294.

[0021] In some embodiments, the nucleotide sequence encoding the anti-NPM1c TCR or its antigen-binding fragment comprises a first nucleotide sequence encoding an α chain and a second nucleotide sequence encoding a β chain, or a first nucleotide sequence encoding a γ chain and a second nucleotide sequence encoding a δ chain. (a) The first nucleotide sequence codes for SEQ ID NO: 17, and the second nucleotide sequence codes for SEQ ID NO: 25, (b) The first nucleotide sequence codes for SEQ ID NO: 35, and the second nucleotide sequence codes for SEQ ID NO: 43, (c) The first nucleotide sequence codes for SEQ ID NO: 53, and the second nucleotide sequence codes for SEQ ID NO: 61, (d) The first nucleotide sequence codes for SEQ ID NO: 71, and the second nucleotide sequence codes for SEQ ID NO: 79, (e) The first nucleotide sequence codes for SEQ ID NO: 89 and the second nucleotide sequence codes for SEQ ID NO: 97 (f) The first nucleotide sequence codes for sequence number 107, and the second nucleotide sequence codes for sequence number 115, (g) The first nucleotide sequence codes for SEQ ID NO: 125, and the second nucleotide sequence codes for SEQ ID NO: 133, (h) The first nucleotide sequence codes for SEQ ID NO: 143, and the second nucleotide sequence codes for SEQ ID NO: 151, (i) The first nucleotide sequence codes for sequence number 161, and the second nucleotide sequence codes for sequence number 169, (j) The first nucleotide sequence codes for SEQ ID NO: 179 and the second nucleotide sequence codes for SEQ ID NO: 187, (k) The first nucleotide sequence codes for SEQ ID NO: 197 and the second nucleotide sequence codes for SEQ ID NO: 205, (l) The first nucleotide sequence codes for SEQ ID NO: 215, and the second nucleotide sequence codes for SEQ ID NO: 223, (m) The first nucleotide sequence codes for SEQ ID NO: 233, and the second nucleotide sequence codes for SEQ ID NO: 241, (n) The first nucleotide sequence codes for SEQ ID NO: 251, and the second nucleotide sequence codes for SEQ ID NO: 259, (o) The first nucleotide sequence codes for SEQ ID NO: 269 and the second nucleotide sequence codes for SEQ ID NO: 277, or (p) The first nucleotide sequence codes for sequence number 287, and the second nucleotide sequence codes for sequence number 295.

[0022] In some embodiments, (a) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 18 or SEQ ID NO: 17 and has at least 90% sequence identity with SEQ ID NO: 18, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 26 or SEQ ID NO: 25 and has at least 90% sequence identity with SEQ ID NO: 26, (b) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 36 or SEQ ID NO: 35 and has at least 90% sequence identity with respect to SEQ ID NO: 36, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 44 or SEQ ID NO: 43 and has at least 90% sequence identity with respect to SEQ ID NO: 44, (c) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 54 or SEQ ID NO: 53 and has at least 90% sequence identity with respect to SEQ ID NO: 54, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 62 or SEQ ID NO: 61 and has at least 90% sequence identity with respect to SEQ ID NO: 62, (d) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 72 or SEQ ID NO: 71 and has at least 90% sequence identity with respect to SEQ ID NO: 72, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 80 or SEQ ID NO: 79 and has at least 90% sequence identity with respect to SEQ ID NO: 80, (e) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 90 or SEQ ID NO: 89 and has at least 90% sequence identity with respect to SEQ ID NO: 90, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 98 or SEQ ID NO: 97 and has at least 90% sequence identity with respect to SEQ ID NO: 98, (f) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 108 or SEQ ID NO: 107 and has at least 90% sequence identity with respect to SEQ ID NO: 108, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 116 or SEQ ID NO: 115 and has at least 90% sequence identity with respect to SEQ ID NO: 116, (g) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 126 or SEQ ID NO: 125 and has at least 90% sequence identity with respect to SEQ ID NO: 126, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 134 or SEQ ID NO: 113 and has at least 90% sequence identity with respect to SEQ ID NO: 134, (h) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 144 or SEQ ID NO: 143 and has at least 90% sequence identity with respect to SEQ ID NO: 144, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 152 or SEQ ID NO: 151 and has at least 90% sequence identity with respect to SEQ ID NO: 152, (i) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 162 or SEQ ID NO: 161 and has at least 90% sequence identity with respect to SEQ ID NO: 162, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 170 or SEQ ID NO: 169 and has at least 90% sequence identity with respect to SEQ ID NO: 170, (j) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 180 or SEQ ID NO: 179 and has at least 90% sequence identity with respect to SEQ ID NO: 180, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 188 or SEQ ID NO: 187 and has at least 90% sequence identity with respect to SEQ ID NO: 188, (k) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 198 or SEQ ID NO: 197 and has at least 90% sequence identity with respect to SEQ ID NO: 198, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 206 or SEQ ID NO: 205 and has at least 90% sequence identity with respect to SEQ ID NO: 206, (l) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 2165 or SEQ ID NO: 215 and has at least 90% sequence identity with SEQ ID NO: 216, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 224 or SEQ ID NO: 223 and has at least 90% sequence identity with SEQ ID NO: 224, (m) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 234 or SEQ ID NO: 233 and has at least 90% sequence identity with respect to SEQ ID NO: 234, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 242 or SEQ ID NO: 241 and has at least 90% sequence identity with respect to SEQ ID NO: 242, (n) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 252 or SEQ ID NO: 251 and has at least 90% sequence identity with respect to SEQ ID NO: 252, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 260 or SEQ ID NO: 259 and has at least 90% sequence identity with respect to SEQ ID NO: 260, (o) The first nucleotide sequence contains a sequence that codes for SEQ ID NO: 270 or SEQ ID NO: 269 and has at least 90% sequence identity with respect to SEQ ID NO: 270, and the second nucleotide sequence contains a sequence that codes for SEQ ID NO: 278 or SEQ ID NO: 277 and has at least 90% sequence identity with respect to SEQ ID NO: 278, or (p) The first nucleotide sequence includes a sequence that codes for SEQ ID NO: 288 or SEQ ID NO: 287 and has at least 90% sequence identity with respect to SEQ ID NO: 288, and the second nucleotide sequence includes a sequence that codes for SEQ ID NO: 296 or SEQ ID NO: 295 and has at least 90% sequence identity with respect to SEQ ID NO: 296.

[0023] In some embodiments, the nucleotide sequences encoding the α-chain and the nucleotide sequences encoding the β-chain reside on a single nucleic acid or vector. In some embodiments, the nucleotide sequences encoding the α-chain and the nucleotide sequences encoding the β-chain reside on a single nucleic acid or vector and are expressed from a single promoter. In some embodiments, the nucleotide sequences encoding the α-chain and the nucleotide sequences encoding the β-chain are separated by a peptide sequence that induces ribosome skipping or autocleavage and reside on a single nucleic acid or vector expressed from a single promoter. The peptide that induces ribosome skipping may, but is not limited to, a 2A peptide. The 2A peptide may, but is not limited to, a P2A peptide. In some embodiments, the P2A peptide includes SEQ ID NO: 301.

[0024] In some embodiments, the nucleotide sequence encodes the amino acid sequence of SEQ ID NOs. 27, 45, 63, 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297.

[0025] In some embodiments, the nucleotide sequence is (a) A sequence that codes for sequence number 28 or sequence number 27 and has at least 90% sequence identity with sequence number 28, (b) A sequence that codes for sequence number 46 or sequence number 45 and has at least 90% sequence identity with sequence number 46, (c) A sequence that codes for sequence number 64 or sequence number 63 and has at least 90% sequence identity with sequence number 64, (d) A sequence that codes for sequence number 82 or sequence number 81 and has at least 90% sequence identity with sequence number 82, (e) A sequence that codes for sequence number 100 or sequence number 99 and has at least 90% sequence identity with sequence number 100, (f) A sequence that codes for sequence number 118 or sequence number 117 and has at least 90% sequence identity with sequence number 118, (g) A sequence that codes for sequence number 136 or sequence number 135 and has at least 90% sequence identity with sequence number 136, (h) A sequence that codes for sequence number 154 or sequence number 153 and has at least 90% sequence identity with sequence number 154, (i) A sequence that codes for sequence number 172 or sequence number 171 and has at least 90% sequence identity with sequence number 172, (j) A sequence that codes for sequence number 190 or sequence number 189 and has at least 90% sequence identity with sequence number 190, (k) A sequence that codes for sequence number 208 or sequence number 207 and has at least 90% sequence identity with sequence number 208, (l) A sequence that codes for sequence number 226 or sequence number 225 and has at least 90% sequence identity with sequence number 226, (m) A sequence that codes for sequence number 244 or sequence number 243 and has at least 90% sequence identity with sequence number 244, (n) A sequence that codes for sequence number 262 or sequence number 261 and has at least 90% sequence identity with sequence number 262, (o) A sequence that codes for sequence number 280 or sequence number 279 and has at least 90% sequence identity with sequence number 280, or (p) A sequence that codes for sequence number 298 or sequence number 297 and has at least 90% sequence identity with sequence number 298.

[0026] Vectors comprising any of the polynucleotides provided herein are also provided herein. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-derived vectors, and examples of retrovirus vectors include, but are not limited to, lentiviruses. In some embodiments, the viral vector is a lentiviral vector. Additional vectors include, but are not limited to, transposon-based systems, minicircles, mRNA, and CRISPR constructs.

[0027] Engineered cells comprising either the anti-NPM1c TCR or its antigen-binding fragment provided herein are also provided herein. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is heterogeneous to the cell. Engineered cells comprising either any of the nucleotide sequences provided herein or any of the vectors provided herein are also provided herein. In some embodiments, the nucleotide sequences encoding the anti-NPM1c TCR or its antigen-binding fragment are heterogeneous to the cell. The cells may be, but are not limited to, T cells, cell line T cells, primary T cells, donor T cells (i.e., T cells derived from a non-patient subject), or autologous T cells (e.g., T cells derived from a subject being treated with the engineered T cells).

[0028] Methods for producing engineered cells are also provided herein, which involve introducing one of the polynucleotides or one of the vectors provided herein into cells to form engineered cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is heterogeneous to the cells. The cells may be, but are not limited to, T cells, cell line T cells, primary T cells, donor T cells (i.e., T cells derived from a non-patient subject), or autologous T cells (i.e., T cells derived from a subject to be treated with the engineered T cells).

[0029] Soluble anti-NPM1c TCRs and anti-NPM1c TCR bispecific molecules are also provided. The anti-NPM1c TCR bispecific molecule comprises a fusion protein containing an antigen-binding domain derived from one of the described anti-NPM1c TCRs and a second antigen-binding domain that specifically binds to a second antigen, the second antigen being not an NPM1c neoantigen. The second antigen may, but is not limited to, a T cell antigen (e.g., CD3), a macrophage antigen, or a natural killer (NK) cell antigen. In some embodiments, the second antigen is an anti-CD3 binding protein. The anti-CD3 binding protein may, but is not limited to, an anti-CD3 scFv.

[0030] In some embodiments, the soluble anti-NPM1c TCR or its antigen-binding fragment is linked to or fused with an immune cell engager. The immune cell engager may be, but is not limited to, a T cell engager (e.g., a CD3-binding domain (e.g., anti-CD3 scFv)), a macrophage engager, or an NK cell engager. In some embodiments, the soluble anti-NPM1c TCR or its antigen-binding fragment is linked to or fused with an Fc peptide, a modified Fc peptide, or a Tc receptor, or a modified Fc receptor.

[0031] Compositions comprising any of the provided anti-NPM1c TCR or its antigen-binding fragment, any of the polynucleotides provided herein, any of the vectors provided herein, or any of the engineered cells provided herein are also provided herein. In some embodiments, the composition also comprises pharmaceutically acceptable excipients.

[0032] Any anti-NPM1c TCR or its antigen-binding fragment provided herein, any polynucleotide provided herein, any vector provided herein, any engineered cell provided herein, or any composition provided herein, NPM1c +Also provided herein are methods of treatment comprising administering to a subject having or diagnosed with cancer. NPM1c in a subject + Also provided herein are any of the anti-NPM1c TCR or antigen-binding fragments thereof, any of the polynucleotides, any of the vectors, any of the engineered cells, or any of the compositions provided herein, for use in the treatment of NPM1c + Also provided herein is use of any of the anti-NPM1c TCR or antigen-binding fragments thereof, any of the polynucleotides, any of the vectors, any of the engineered cells, or any of the compositions provided herein, in the manufacture of a medicament for the treatment of cancer. In some embodiments, the engineered T cells are engineered autologous T cells. In some embodiments, NPM1c + the cancer is AML.

[0033] In some embodiments, NPM1c + Provided herein are methods of treating a subject having or diagnosed with cancer, the method comprising administering to the subject any of the engineered T cells provided herein. In some embodiments, any of the engineered T cells described herein target NPM1c + for use in the treatment of cancer in the subject. In some embodiments, any of the engineered T cells provided herein target NPM1c + can be used in the manufacture of a medicament for the treatment of cancer in a subject. In some embodiments, the engineered T cells are engineered autologous T cells. In some embodiments, NPM1c + the cancer is AML.

[0034] In some embodiments, NPM1c +A method for treating a subject having or diagnosed with cancer is described herein, the method comprising administering to the subject either a soluble anti-NPM1c TCR (or its antigen-binding fragment) or an anti-NPM1c TCR bispecific molecule provided herein. In some embodiments, either the soluble anti-NPM1c TCR (or its antigen-binding fragment) or the anti-NPM1c TCR bispecific molecule described herein is used in NPM1c + It is intended for use in the treatment of cancer. In some embodiments, either the soluble anti-NPM1c TCR (or its antigen-binding fragment) or the anti-NPM1c TCR bispecific molecule provided herein is used in NPM1c + It can be used in the manufacture of pharmaceuticals for the treatment of cancer.

[0035] In some embodiments, the subjects are not eligible for transplantation, which is standard treatment. In some embodiments, the subjects are FLT3 positive. In some embodiments, the subjects have AML or have been diagnosed with it. In some embodiments, the subjects have NPM1c + The subject has cancer and one or more comorbidities or risk factors. In some embodiments, the subject has AML and one or more comorbidities or risk factors. In some embodiments, the subject has NPM1c +The subject has cancer and is ineligible for high-intensity chemotherapy. In some embodiments, the subject has AML and is ineligible for high-intensity chemotherapy. In some embodiments, the subject has relapsed after high-intensity chemotherapy with or without hematopoietic stem cell transplantation (HSCT). In some embodiments, the subject did not respond to high-intensity chemotherapy, did not respond, or failed to achieve complete remission (refractory). In some embodiments, the subject has relapsed after prior hypomethylating therapy. In some embodiments, the subject did not respond to hypomethylating therapy, did not respond, or failed to achieve complete remission (refractory). In some embodiments, the subject has relapsed or did not respond after two prior therapies. Relapse may occur, for example, after complete remission (CR), CR with partial hematological recovery (CRh), or CR with incomplete recovery (CRi). In some embodiments, the subject has relapsed or refractory AML. In some embodiments, the subject has AML and has relapsed after at least one prior therapy. In some embodiments, the subject has AML and has relapsed after at least two prior therapies. In some embodiments, the subject has AML and is refractory to at least one prior treatment. In some embodiments, the subject has AML and is refractory to at least two prior treatments.

[0036] In some embodiments, administration of manipulated cells or compositions induces or enhances cell death associated with malignant hematological disorders, or induces or enhances graft-versus-leukemia (GVL) efficacy in a subject. [Brief explanation of the drawing]

[0037] [Figure 1] This graph shows the activation of engineered T cells expressing TCR D in response to cells pulsed with the CLAVEEVSL (SEQ ID NO: 5) peptide. [Figure 2] This graph shows the target cell death of cells pulsed with the CLAVEEVSL (SEQ ID NO: 5) peptide by engineered T cells expressing TCR D. [Figure 3] This graph shows the target cell death of naturally expressing CLAVEEVSL (SEQ ID NO: 5) peptide by manipulated T cells expressing TCR D. [Figure 4] This graph shows the effectiveness of engineered T cells compared to CAR T cells. [Figure 5] This graph shows the cytotoxicity (top) and EC50 (bottom) of manipulated T cells compared to CAR T cells. [Figure 6] This graph shows the reactivity of TCR D to cysteine- and non-cysteine-treated target peptides. [Figure 7] This graph shows the recognition of the CLAVEEVSL (SEQ ID NO: 5) variant by TCR F. The peptide numbers correspond to the peptides in Table 11. Each peptide was tested at 5 μg / mL, 0.16 μg / mL, 0.015 μg / mL, and 0 μg / mL, respectively. [Figure 8] This graph shows the recognition of the CLAVEEVSL (SEQ ID NO: 5) variant by TCR D. The peptide numbers correspond to the peptides in Table 11. Each peptide was tested at 5 μg / mL, 0.16 μg / mL, 0.015 μg / mL, and 0 μg / mL, respectively. [Modes for carrying out the invention]

[0038] Detailed explanation This specification provides anti-NPM1c TCRs, including recombinant anti-NPM1c TCRs, that bind to or recognize neoantigens associated with NPM1c. Among the embodiments provided are approaches useful in the treatment of such diseases and conditions and / or for targeting cell types such as cancer cells or cells associated with NPM1c expression. In some embodiments, the provided anti-NPM1c TCRs and their antigen-binding fragments specifically bind to or recognize neoantigens having the sequence AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5). In some embodiments, the provided anti-NPM1c TCRs and their antigen-binding fragments specifically bind to or recognize neoantigens having the sequence AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules. In some embodiments, the provided anti-NPM1c TCRs and their antigen-binding fragments HLA-A * 02:01, HLA-A * 02:06, or HLA-A * In relation to 02:03, it specifically binds to or specifically recognizes neoantigens having the sequence:AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5).

[0039] In some embodiments, the described anti-AIQDLCLAV (SEQ ID NO: 1) anti-NPM1c TCR is used on cells (e.g., cancer cells) with HLA subtype A * 02:01, HLA subtype A * 02:06, or HLA subtype A * When presented by 02:03, the AIQDLCLAV (SEQ ID NO: 1) neoantigen specifically binds to the AIQDLCLAV (SEQ ID NO: 1) neoantigen and binds to other HLA subtypes, or HLA subtype A. * 02:01, HLA subtype A * 02:06, or HLA subtype A * It does not specifically bind to other non-target antigens presented by 02:03.

[0040] In some embodiments, the described anti-CLAVEEVSL (SEQ ID NO: 5) anti-NPM1c TCR is used on cells (e.g., cancer cells) with HLA subtype A * 02:01, HLA subtype A * 02:06, or HLA subtype A * When presented by 02:03, the CLAVEEVSL (SEQ ID NO: 5) neoantigen specifically binds to other HLA subtypes, or HLA subtype A. * 02:01, HLA subtype A * 02:06, or HLA subtype A * It does not specifically bind to other non-target antigens presented by 02:03.

[0041] This specification also provides nucleic acid molecules encoding anti-NPM1c TCRs, engineered cells containing anti-NPM1c TCRs, compositions containing anti-NPM1c TCRs or cells, and methods of treatment or use, such as therapeutic applications involving the administration of such anti-NPM1c TCRs, engineered cells, or compositions, as well as uses of such anti-NPM1c TCRs, cells, or compositions. In some embodiments, the provided anti-NPM1c TCRs or engineered cells expressing their antigen-binding fragments exhibit cytotoxic activity against target cells expressing NPM1c neoantigens, such as cancer cells. Methods for identifying anti-NPM1c TCRs that target neoantigens having the sequence AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) are also provided herein.

[0042] Cell therapies (including those involving the administration of cells engineered to express recombinant receptors or anti-NPM1c TCRs specific to the disease or condition of interest, such as recombinant anti-NPM1c TCRs and / or other recombinant antigen receptors), as well as other adoptive immune cell and adoptive T cell therapies, may be effective in treating diseases and disorders. T cells engineered to express the described anti-NPM1c TCRs (or their antigen-binding fragments) recognize and bind to targets within the subject, for example, peptide epitopes of target antibodies, such as AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5), based, for example, the affinity of the antigen-binding domain of the anti-NPM1c TCR to target antibodies.

[0043] In some embodiments, T cells engineered to express a heterologous anti-NPM1c TCR (e.g., anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR) are further modified to knock out the endogenous TCR. Knockout of the endogenous TCR may include knockout of the expression of the endogenous TRAC gene and knockout of the expression of the endogenous TRBC gene. In some embodiments, knockout of the endogenous TCR (endogenous TRAC gene and / or endogenous TRBC gene) can be carried out using CRISPR. Knockout of the endogenous TCR includes, but is not limited to, deletion of all or part of the target gene, insertion of one or more stop codons into the target gene, disruption of the target gene, or disruption of the promoter or splicing region of the target gene. Knockout of the endogenous TCR reduces or eliminates mispairing between the heterologous anti-NPM1c TCR (α and / or β chains) and the endogenous TCR (α and / or β chains).

[0044] In some embodiments, the provided anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR has improved affinity for the NPM1c neoantigen. In some embodiments, engineered T cells expressing either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR have improved activity against cells expressing the NPM-1c neoantigen (e.g., cancer cells). In some embodiments, engineered T cells expressing either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR have improved activity against cells expressing the NPM-1c neoantigen (e.g., cancer cells) with a low effector-to-target (E:T) ratio.

[0045] In some embodiments, for example, in adoptive cell therapy using engineered human T cells expressing the described anti-NPM1c TCR, the therapeutic approach using the described anti-NPM1c TCR targets and kills cancer cells expressing the NPM1c neoantigen, resulting in a graft-versus-leukemia (GVL) effect.

[0046] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for the same extent as each individual publication is incorporated by reference. If any definition described herein contradicts or otherwise does not match any definition described herein in a patent, application, published application, or other publication incorporated herein by reference, the definition described herein shall prevail over the definition incorporated herein by reference.

[0047] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0048] I. T cell receptors that target the NPM1 neoantigen Anti-NPM1c TCRs are provided herein that, in relation to MHC molecules, bind to or recognize peptide epitopes associated with NPM1c, such as peptide neoantigens expressed on the surface of immune cells, cancer cells, and / or cells associated with hematological diseases. In some embodiments, the described anti-NPM1c TCRs bind to or recognize neoantigens AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules. Such anti-NPM1c TCRs and antigen-binding fragments exhibit antigen specificity that binds to or recognizes such peptide epitopes. Also provided herein are (a) a nucleic acid encoding an anti-NPM1c TCR (or its antigen-binding domain), (b) engineered cells expressing an anti-NPM1c TCR (or its antigen-binding domain), (c) a composition comprising an anti-NPM1c TCR, a nucleic acid encoding an anti-NPM1c TCR, or engineered cells expressing an anti-NPM1c TCR, and (d) a therapeutic method comprising administering the described anti-NPM1c TCR (or its antigen-binding domain), nucleic acid, engineered cells, or composition. In some embodiments, the engineered cells expressing the provided anti-NPM1c TCR or its antigen-binding fragment are NPM1c + It exhibits cytotoxic activity against target cells that express the NPM1c neoantigen, such as cancer cells.

[0049] In some embodiments, the described anti-NPM1c TCR recognizes the NPM1c neoantigen expressed by AML cancer cells (e.g., AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)).

[0050] A.NPM1c Nucleophosmin (NPM, also known as nucleolar phosphoprotein B23 or numatrin) is encoded in humans by the NPM1 gene. The NPM1 gene is upregulated and mutates in certain tumor types, such as leukemia, acute myeloid (myelocytic) leukemia (AML), and myelodysplastic syndromes. Mutations in NPM1 often consist of a 4bp insertion or duplication between nucleotides 960 and 961. They result in substitutions at 11 different residues for the last seven amino acids (WQWRKSL), leading to cytoplasmic localization of the protein (NPM1c).

[0051] Examples of NPM1c neoantigens include, but are not limited to, AIQDLCLAV (SEQ ID NO: 1) and CLAVEEVSL (SEQ ID NO: 5). The corresponding peptide sequence in non-mutant NPM1 is AIQDLWQWRKSL (SEQ ID NO: 2). NPM1c neoantigens are class I MHC molecules, such as HLA-A * 02:01, HLA-A * 02:06, or HLA-A * This may be presented in relation to 02:03. The NPM1c neoantigens AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) are typically expressed by leukemia cells (e.g., AML cells) and not typically expressed by non-leukemia cells.

[0052] In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment recognizes or specifically binds to an immunogenic epitope of NPM1c. In some examples, the provided anti-NPM1c TCR or its antigen-binding fragment specifically binds to or recognizes the NPM1c neoantigens AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5), but does not specifically bind to the corresponding normal NPM1 peptide, AIQDLWQWRKSL (SEQ ID NO: 2). In some embodiments, the anti-NPM1c TCR (or its antigen-binding fragment) recognizes or binds to the NPM1c neoantigen (e.g., AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)) in relation to an MHC molecule such as an MHC class I molecule. In some embodiments, the MHC class I molecule is any one or more of its subtypes, e.g., HLA-A * 02:01, * 02:06, or * This is an HLA-A2 molecule containing 02:03.

[0053] In some embodiments, the anti-NPM1c TCR or soluble anti-NPM1c TCR, or its antigen-binding fragment, is isolated or purified. In certain embodiments, either the provided anti-NPM1c TCR or its antigen-binding fragment is recombinant. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is human. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is single-chain. In other embodiments, the anti-NPM1c TCR contains two chains. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is expressed on the surface of a cell (e.g., a T cell, such as a T cell designed to lack endogenous TCR expression). In some embodiments, the soluble anti-NPM1c TCR is secreted from a cell.

[0054] In some embodiments, the provided anti-NPM1c TCR, or its antigen-binding fragment, has one or more specified functional features, such as binding properties including binding to a specific epitope, and / or specific binding affinity, as described herein. In some embodiments, engineered cells, such as engineered T cells, expressing the provided anti-NPM1c TCR (or its antigen-binding fragment), have one or more specified functional features, such as binding properties including binding to a specific epitope, specific binding affinity, activation or stimulation of cellular signaling such as T cell signaling or TCR signaling, cytokine secretion, and / or killing of target cells expressing or presenting an antigen, as described herein.

[0055] Provided are anti-NPM1c TCRs, or antigen-binding fragments thereof, that specifically bind to the NPM1c neoantigen. The TCR is a molecule containing an α chain containing a Vα region and a β chain containing a Vβ region (also known as TCRα and TCRβ, respectively), or a γ chain containing a Vγ region and a δ chain containing a Vδ region (also known as TCRγ and TCRδ, respectively), or their antigen-binding moieties, which can specifically bind to an antigen, such as a peptide antigen or peptide epitope bound to an MHC molecule. In some embodiments, the anti-NPM1c TCR is in the αβ form (e.g., an αβ TCR). In some embodiments, the anti-NPM1c TCR is in the γδ form (e.g., a γδ TCR). Typically, TCRs existing in the αβ or γδ form are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. TCRs can be found on the surface of cells or in a soluble form. Generally, TCRs are found on the surface of T cells, where they are involved in the recognition of antigens, such as peptides bound to MHC molecules.

[0056] In some embodiments, the anti-NPM1c TCRs provided herein may be intact or full-length TCRs, such as TCRs containing full-length α and β chains, or TCRs containing full-length γ and δ chains. In some embodiments, the antigen-binding portion of the TCRs provided herein may be less than a full-length TCR, however, it may bind to a specific peptide bound to an MHC molecule. In some embodiments, the antigen-binding portion or fragment of the TCR may contain only a portion of the structural domains of a full-length or intact TCR, but may still bind to a peptide epitope, such as an MHC-peptide complex to which a full-length TCR binds. In some embodiments, the antigen-binding portion may contain variable domains of the TCR, such as the Vα and Vβ regions or the Vγ and Vδ regions of the TCR, however sufficient to form a binding site for the antigen-binding portion to bind to a specific MHC-peptide complex.

[0057] The variable domain of the TCR contains complementarity-determining regions (CDRs, CDR-1, CDR-2, and CDR-3) that contribute to the antigen recognition and binding ability and specificity of peptides, MHC molecules, and / or MHC-peptide complexes. In some embodiments, one or more CDRs of the TCR form all or substantially all of the antigen-binding sites of a given TCR molecule. The various CDRs within the variable region of the TCR chain are generally separated by a framework region (FR), which generally exhibits less variability between TCRs compared to CDRs. CDR-3 is either the primary CDR responsible for antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and / or interaction with the processed peptide portion of a peptide-MHC complex. In some situations, CDR-1 of the α-chain interacts with the N-terminal portion of a particular antigen peptide. In some situations, CDR-1 of the β-chain interacts with the C-terminal portion of the peptide. In some situations, CDR-2 is either the most strongly contributing CDR responsible for interacting with or recognizing the MHC portion of the MHC-peptide complex, or is the primary CDR.

[0058] In some embodiments, the α and / or β chains of the TCR, or the γ and / or δ chains of the TCR, may also contain one or more of a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. In some embodiments, each chain of the TCR (e.g., α or β) contains one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic tail. In some embodiments, the TCR associates with invariant proteins of the CD3 complex involved in signal transduction, for example, via the cytoplasmic tail. In some embodiments, the structure allows the TCR to associate with other molecules such as CD3 and its subunits. For example, a constant domain-containing TCR with a transmembrane region may immobilize the protein on the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs or ITAMs and are generally involved in the signaling capacity of the TCR complex.

[0059] Various domains or regions of the TCR can be identified. In some embodiments, the exact locus of a domain or region may vary depending on the specific structural or homology modeling or other features used to describe a particular domain. References to amino acids containing specific sequences described as sequence numbers used to describe the domain composition of the TCR are for illustrative purposes only and should not be understood as limiting the scope of the embodiments provided. In some embodiments, a particular domain (e.g., a variable domain or a constant domain) may be longer or shorter than a few amino acids (e.g., one, two, three, or four). In some embodiments, residues of the TCR can be known or identified according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT) numbering system (see Lefranc et al. (2003) Developmental and Comparative Immunology, 27(1); 55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR-1 sequence within the TCR Vα and / or Vβ region may correspond to amino acids located between residue numbers 27 and 38 (including both ends), the CDR-2 sequence within the TCR Vα and / or Vβ region may correspond to amino acids located between residue numbers 56 and 65 (including both ends), and the CDR-3 sequence within the TCR Vα and / or Vβ region may correspond to amino acids located between residue numbers 105 and 117 (including both ends).

[0060] In some embodiments, anti-NPM1c TCRs or their antigen-binding fragments are provided that specifically bind to or recognize the NPM1c neoantigens AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules. In some embodiments, the provided anti-NPM1c TCRs or their antigen-binding fragments do not recognize or specifically bind to the NPM1 peptide AIQDLWQWRKSL (SEQ ID NO: 2). In some embodiments, the provided anti-NPM1c TCRs or their antigen-binding fragments specifically recognize or specifically bind to AIQDLCLAV (SEQ ID NO: 1) and do not specifically recognize or specifically bind to AIQDLWQWRKSL (SEQ ID NO: 2). In some embodiments, the provided anti-NPM1c TCRs or their antigen-binding fragments bind to AIQDLCLAV (SEQ ID NO: 1) with increased affinity compared to binding to AIQDLWQWRKSL (SEQ ID NO: 2). In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment specifically recognizes or binds to CLAVEEVSL (SEQ ID NO: 5) but does not specifically recognize or bind to AIQDLWQWRKSL (SEQ ID NO: 2). In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment binds to CLAVEEVSL (SEQ ID NO: 5) with increased affinity compared to binding to AIQDLWQWRKSL (SEQ ID NO: 2).

[0061] In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment is HLA-A * 02:01, HLA-A * 02:06, or HLA-A * It specifically binds to or recognizes AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) which are complexed with MHC molecules of specific HLA types such as 02:03.

[0062] In some embodiments, the anti-NPM1c TCR provided herein is a full-length TCR. In some embodiments, the anti-NPM1c TCR provided herein is a dimeric TCR (dTCR). In some embodiments, the anti-NPM1c TCR provided herein is a single-chain TCR (scTCR). The anti-NPM1c TCR provided herein may be in a cell-bound or soluble form. In some embodiments, the anti-NPM1c TCR provided herein is in a cell-bound form expressed on the surface of a cell (e.g., a T cell such as a T cell designed to lack the expression of endogenous TCR).

[0063] In some embodiments, the anti-NPM1c TCRs provided herein are scTCRs, which are single-chain amino acids containing α and β chains capable of binding to an MHC-peptide complex. Typically, scTCRs can be produced as described elsewhere. See, for example, International Publication Nos. 96 / 13593, 96 / 18105, 99 / 18129, 04 / 033685, 2006 / 037960, 2011 / 044186; U.S. Patent No. 7,569,664; and Schleuter, C.J. et al. J. Mol. Biol. 256, 859 (1996).

[0064] B. Exemplary Variable Domains This specification provides anti-NPM1c TCRs or antigen-binding fragments thereof that specifically recognize or bind to the neoantigens AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules. Exemplary sequences of AIQDLCLAV (SEQ ID NO: 1)-specific or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs (e.g., CDR, Vα and / or Vβ, or Vγ and / or Vδ, and constant region sequences) are provided.

[0065] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein specifically binds to or recognizes the neoantigen AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) presented on the surface of leukemia cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein specifically binds to or recognizes the neoantigen AIQDLCLAV (SEQ ID NO: 1) presented on the surface of AML cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein specifically binds to or recognizes the neoantigen AIQDLCLAV (SEQ ID NO: 1) presented on the surface of myelodysplastic syndrome cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein specifically binds to or recognizes the neoantigen CLAVEEVSL (SEQ ID NO: 5) presented on the surface of AML cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein specifically binds to or recognizes the neoantigen CLAVEEVSL (SEQ ID NO: 5) presented on the surface of myelodysplastic syndrome cells.

[0066] In some embodiments, the cytotoxic activity of T cells expressing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR is stimulated upon contact between T cells and target cells presenting or expressing the NPM1c(AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)) neoantigen.

[0067] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment contains either the Vα and Vβ regions, or the Vγ and Vδ regions, the described sequence, or a sufficient antigen-binding portion of such a sequence. In some embodiments, the described anti-NPM1c TCR or its antigen-binding fragment contains a sufficient antigen-binding portion containing the Vα and Vβ regions, or the Vγ and Vδ regions, the sequence, or the CDR-3 sequence described herein. In some embodiments, the described anti-NPM1c TCR or its antigen-binding fragment contains a sufficient antigen-binding portion containing the Vα and Vβ regions, or the Vγ and Vδ regions, the sequence, or the CDR-1, CDR-2, and CDR-3 sequences described herein. In addition, some of the provided anti-NPM1c TCRs have sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0068] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein contains a Vα or Vγ region containing a CDR-3 that includes an amino acid sequence described in any of SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, 193, 211, 229, 247, 265, and 283, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the anti-NPM1c TCR or antigen-binding fragments provided herein contain a Vα or Vγ region containing CDR-3 contained within an amino acid sequence described in any of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, and 284, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such a sequence.

[0069] In some embodiments, the Vα or Vγ region contains CDR-1 comprising an amino acid sequence described in any of SEQ ID NOs: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263, and 281, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vα or Vγ region contains CDR-1 contained within an amino acid sequence described in any of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, and 284, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vα or Vγ region contains CDR-2 comprising an amino acid sequence described in any of SEQ ID NOs: 12, 30, 48, 66, 84, 102, 120, 138, 156, 174, 192, 210, 228, 246, 264, and 282, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vα or Vγ region contains CDR-2 contained within an amino acid sequence described in any of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, and 284, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence.

[0070] In some cases, the anti-NPM1c TCR or its antigen-binding fragment provided herein contains a Vβ or Vδ region containing a CDR-3 that includes an amino acid sequence described in any of SEQ ID NOs: 21, 39, 57, 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, and 291, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the anti-NPM1c TCR or antigen-binding fragments provided herein contain a Vβ or Vδ region containing CDR-3 contained within an amino acid sequence described in any of SEQ ID NOs: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, and 292, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such a sequence.

[0071] In some examples, the Vβ or Vδ region contains CDR-1 containing an amino acid sequence described in any of SEQ ID NOs: 19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271, and 289, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vβ or Vδ region contains CDR-1 contained within an amino acid sequence described in any of SEQ ID NOs: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, and 292, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vβ or Vδ region contains CDR-2 comprising an amino acid sequence described in any of SEQ ID NOs: 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272, and 290, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence. In some embodiments, the Vβ or Vδ region contains CDR-2 contained within an amino acid sequence described in any of SEQ ID NOs: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, and 292, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence.

[0072] In some embodiments, the Vα or Vγ region is an amino acid sequence described in any of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, and 284, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with them. The sequence contains a Vβ or Vδ region containing an amino acid sequence described in any of SEQ ID NOs: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, and 292, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with them.

[0073] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein, which has the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, comprises an α-chain having CDR-1 having the amino acid sequence described in SEQ ID NOs: 11, 29, 47, and 65 (or its variants having one or two amino acid modifications), CDR-2 having the amino acid sequence described in SEQ ID NOs: 12, 30, 48, and 66 (or its variants having one or two amino acid modifications), and CDR-3 having the amino acid sequence described in SEQ ID NOs: 13, 31, and 49 (or its variants having one or two amino acid modifications). The β-chain (or δ-chain) may include a γ-chain and a β-chain (or δ-chain) having a CDR-1 having the amino acid sequence described in SEQ ID NOs. 19, 37, and 55 (or its variants having one or two amino acid modifications), a CDR-2 having the amino acid sequence described in SEQ ID NOs. 20, 38, and 56 (or its variants having one or two amino acid modifications), and a CDR-3 having the amino acid sequence described in SEQ ID NOs. Examples of anti-NPM1c TCRs having these CDRs and possessing the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule include TCR A, TCR B, or TCR C (see Table 1).

[0074] In some embodiments, the anti-NPM1c provided herein has the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules. The TCR or its antigen-binding fragment has an α chain (or CDR-1 having the amino acid sequence described in SEQ ID NOs. 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271, or 289 (or its variants having one or two amino acid modifications), SEQ ID NOs. 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272, or 290 (or one or more) The β-chain (or δ-chain) may include a CDR-2 having the amino acid sequence described in SEQ ID NOs. 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, or 291 (or its variant having one or two amino acid modifications), and a CDR-3 having the amino acid sequence described in SEQ ID NOs. Examples of anti-NPM1c TCRs having these CDRs and possessing the ability to specifically bind to CLAVEEVSL (SEQ ID NOs. 5) in relation to MHC molecules include TCR D, TCR E, TCR F, TCR G, TCR H, TCR I, TCR J, TCR K, TCR L, TCR M, TCR N, TCR O, and TCR P (see Table 1).

[0075] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein, which has the ability to specifically bind to the NPM1c neoantigen in relation to an MHC molecule, is CDR-1 having the amino acid sequence described in SEQ ID NOs: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263, or 281 (or its variant having one or two amino acid modifications), SEQ ID NOs: 12, 30, 48, 66, 84, 102, 120, 138, 156, 174, 192, 210, 228, 246, 264 α-chain (or γ-chain) having CDR-2 having the amino acid sequence described in SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, 193, 211, 229, 247, 265, or 283 (or its variant having one or two amino acid modifications), and SEQ ID NOs: CDR-1 having the amino acid sequence described in SEQ ID NOs: 19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271, or 289 (or its variant having one or two amino acid modifications), SEQ ID NOs: 20, 38, 56, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272, or 290 (or its variant having one or two amino acid modifications) A β-chain (or δ-chain) having a CDR-2 having the amino acid sequence described in SEQ ID NO(Variant) (Variant) having no amino acid modification, and a CDR-3 having the amino acid sequence described in SEQ ID NO(21, 39, 57, 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, or 291 (or Variant) (Variant) having one or two amino acid modifications, and which may include any appropriate framework region. For example, such anti-NPM1c TCRs or their antigen-binding fragments are SEQ ID NOs: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263, or 281, which are upstream of the corresponding CDR1 amino acid sequences SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176,Framework region 1 having the entire amino acid sequence described in 194, 212, 230, 248, 266, or 284 (or its variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications), and corresponding CDR1 and CDR2 amidia having the entire amino acid sequence described in SEQ ID NOs. 11 and 12, 29 and 30, 47 and 48, 65 and 66, 83 and 84, 101 and 102, 119 and 120, 137 and 138, 155 and 156, 173 and 174, 191 and 192, 209 and 210, 227 and 228, 245 and 246, 263 and 264, or 281 and 282. Framework region 2 (or its variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284 between the no-acid sequences, and SEQ ID NOs: 12 and 13, 30 and 31, 48 and 49, 66 and 67, 84 and 85, 102 and 103, 120 and 121, 138 and 139, 156 and 157, 174 and 175, 192 and 193, 210 and 211, 228 and Framework region 3 (or its variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284, which lies between the corresponding CDR2 and CDR3 amino acid sequences, 229, 246 and 247, 264 and 264, or 282 and 283, and SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, 193, 211 An α chain comprising: a framework region 4 (or a variant thereof having 1, 2, 3, 4, or 5 amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284 downstream of the corresponding CDR3 amino acid sequence, which is 229, 247, 265, or 283; and SEQ ID NOs: 19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271, or 289,Framework region 1 (or its variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs. 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292 upstream of the corresponding CDR1 amino acid sequence, and SEQ ID NOs. 19 and 20, 37 and 38, 55 and 56, 73 and 74, 91 and 92, 109 and 110, 127 and 128, 145 and 146, 163 and 164, 181 and 182, 199 and 200, 217 Framework region 2 (or its variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs. 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292, which lies between the corresponding CDR1 and CDR2 amino acid sequences, and SEQ ID NOs. 20 and 21, 38 and 39, 56 and 57, 74 and 75, 92 and 93, 110 and 1 Framework region 3 having the entire amino acid sequence described in Sequence IDs 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292, which are between the corresponding CDR2 and CDR3 amino acid sequences, such as 11, 128 and 129, 146 and 147, 164 and 165, 182 and 183, 200 and 201, 218 and 219, 236 and 237, 254 and 255, 272 and 273, or 290 and 291 (or having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications) It may include a β-chain having a variant of the column and a framework region 4 (or a variant of that sequence having 1, 2, 3, 4, or 5 amino acid modifications) having the entire amino acid sequence described in SEQ ID NOs. 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292, which is downstream of the corresponding CDR3 amino acid sequence, which is SEQ ID NOs. 21, 39, 57, 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, or 291.

[0076] In some embodiments, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, may include an α-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence described in SEQ ID NO: 14, 32, or 50, and a β-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence described in SEQ ID NO: 22, 40, or 58. For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence described in SEQ ID NO: 14, 32, or 50, and a β-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence described in SEQ ID NO: 22, 40, or 58. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein may include α-chains and β-chains having amino acid sequences that are 100 percent identical to the amino acid sequences described in (a) SEQ ID NOs: 14 and 22, (b) SEQ ID NOs: 32 and 40, or (c) SEQ ID NOs: 50 and 58.

[0077] In some embodiments, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, may include: (a) an α chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of the Vα domain of any one of TCR A, B, and C (i.e., described in SEQ ID NOs: 14, 32, and 50), provided that it comprises the corresponding CDR1, CDR2, and CRD3 amino acid sequences of the TCRα chain (e.g., SEQ ID NOs: 29, 30, and 31 for TCR B); and (b) a β chain comprising an amino acid sequence having at least 90 percent identity with the amino acids of the Vβ domain of the corresponding TCR (i.e., described in SEQ ID NOs: 22, 40, and 58), provided that it comprises the corresponding CDR1, CDR2, and CRD3 amino acid sequences of the TCRβ chain (e.g., SEQ ID NOs: 37, 38, and 39 for TCR B). For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include (a) an α-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NO: 32 (provided the α-chain includes the amino acid sequences described in SEQ ID NOs: 29, 30, and 31); and (b) a β-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NO: 40 (provided the β-chain includes the amino acid sequences described in SEQ ID NOs: 37, 38, and 39).

[0078] In some embodiments, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, may include an α-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence described in SEQ ID NOs: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284, and a β-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence described in SEQ ID NOs: 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292. For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NOs. 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284, and a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NOs. 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein may include α and β chains having amino acid sequences having 100 percent identity with the amino acid sequences described below: (d) SEQ ID NOs. 68 and 76, (e) SEQ ID NOs. 86 and 94, (f) SEQ ID NOs. 104 and 112, (g) SEQ ID NOs. 122 and 130, (h) SEQ ID NOs. 140 and 148, (i) SEQ ID NOs. 158 and 166, (j) SEQ ID NOs. 176 and 184, (k) SEQ ID NOs. 194 and 202, (l) SEQ ID NOs. 212 and 220, (m) SEQ ID NOs. 230 and 238, (n) SEQ ID NOs. 248 and 256, (o) SEQ ID NOs. 266 and 274, or (p) SEQ ID NOs. 284 and 292.

[0079] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein, which has the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, is (a) an α chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of any one of the Vα domains of TCR D to P (i.e., described in SEQ ID NOs: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284), provided that the corresponding CDR1, CDR2, and CRD3 amino acid sequences of the TCRα chain (e.g., SEQ ID NOs: 65, 66, and 67 for TCR D, SEQ ID NOs: 83, 84, and 85 for TCR E, TCR (b) an α chain comprising (a) the amino acid sequence of the Vβ domain of the corresponding TCR (i.e., described in SEQ ID NOs. 101, 102, and 103 for TCR F), and (b) a β chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of the Vβ domain of the corresponding TCR (i.e., described in SEQ ID NOs. 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292), provided that the β chain comprises the corresponding CDR1, CDR2, and CRD3 amino acid sequences of the TCRβ chain (e.g., SEQ ID NOs. 73, 74, and 75 for TCR D, SEQ ID NOs. 91, 92, and 93 for TCR E, and SEQ ID NOs. 109, 110, and 111 for TCR F). For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include (a) an α-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NO: 68 (provided that the α-chain comprises the amino acid sequences described in SEQ ID NOs: 65, 66, and 67); and (b) a β-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence described in SEQ ID NO: 76 (provided that the β-chain comprises the amino acid sequences described in SEQ ID NOs: 73, 74, and 75).

[0080] In some embodiments, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, may include: (a) an α-chain having the amino acid sequence described in SEQ ID NO: 14, 32, or 50, or the amino acids described in SEQ ID NO: 14 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions); and (b) a corresponding β-chain (see Table 1) having the amino acid sequence described in SEQ ID NO: 22, 40, or H58, or the amino acids described in SEQ ID NO: 22, 40, or 58 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, may include: (a) an α-chain having the amino acid sequence described in SEQ ID NO: 32 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that it includes the CDR amino acid sequence described in SEQ ID NO: 29, 30, and 31; and (b) a β-chain having the amino acid sequence described in SEQ ID NO: 40 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that it includes the CDR amino acid sequence described in SEQ ID NO: 37, 38, or 39.

[0081] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein, having the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, comprises one CDR sequence of any of TCR D-P and (a) the amino acid sequence described in SEQ ID NOs. 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284, or the amino acids described in SEQ ID NOs. 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), the corresponding α chain (see Table 1). (b) a corresponding β-chain (see Table 1) having the amino acid sequence described in SEQ ID NOs. 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292, or the amino acids described in SEQ ID NOs. 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, may include: (a) an α-chain having the amino acid sequence described in SEQ ID NO: 68 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that it includes the CDR amino acid sequence described in SEQ ID NOs: 65, 66, and 67; and (b) a β-chain having the amino acid sequence described in SEQ ID NO: 76 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that it includes the CDR amino acid sequence described in SEQ ID NOs: 73, 73, and 74.

[0082] In some embodiments, the anti-NPM1c TCRs or their antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules, may include α-chains (or γ-chains) and β-chains (or δ-chains) having CDR sequences (α or γ-chains CDR-1, CDR-2 and CDR-3, and δ-chains βCDR-1, CDR-2 and CDR-3) as described in any of the anti-NPM1c TCRs in Table 1. In some embodiments, the anti-NPM1c TCRs or antigen-binding fragments provided herein that have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule may include α-chains (or γ-chains) and β-chains (or δ-chains) having CDR sequences (α or γ-chains CDR-1, CDR-2 and CDR-3, and δ-chains βCDR-1, CDR-2 and CDR-3) described in any of the anti-NPM1c TCRs in Table 1, and one or more of the CDRs may independently be variant CDRs having one or two amino acid modifications from the enumerated CDR sequences in Table 1.

[0083] Exemplary anti-NPM1c TCRs having these CDRs and the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to MHC molecules include TCR A, TCR B, and TCR C. For example, the anti-NPM1c TCRs provided herein that have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to MHC molecules are TCR A, TCR B, and TCR C. The TCR or its antigen-binding fragment may include an α-chain (or γ-chain) having CDR-1 having the amino acid sequence described in SEQ ID NO: 29 (or a variant of SEQ ID NO: 29 having one or two amino acid modifications), CDR-2 having the amino acid sequence described in SEQ ID NO: 30 (or a variant of SEQ ID NO: 30 having one or two amino acid modifications), and CDR-3 having the amino acid sequence described in SEQ ID NO: 31 (or a variant of SEQ ID NO: 31 having one or two amino acid modifications), and a β-chain (or δ-chain) having CDR-1 having the amino acid sequence described in SEQ ID NO: 37 (or a variant of SEQ ID NO: 37 having one or two amino acid modifications), CDR-2 having the amino acid sequence described in SEQ ID NO: 38 (or a variant of SEQ ID NO: 38 having one or two amino acid modifications), and CDR-3 having the amino acid sequence described in SEQ ID NO: 39 (or a variant of SEQ ID NO: 39 having one or two amino acid modifications).

[0084] Examples of anti-NPM1c TCRs having these CDRs and the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules include TCRs D-P. For example, the anti-NPM1c TCRs provided herein that have the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules are shown. The TCR or its antigen-binding fragment may include an α-chain (or γ-chain) having CDR-1 having the amino acid sequence described in SEQ ID NO: 65 (or a variant of SEQ ID NO: 65 having one or two amino acid modifications), CDR-2 having the amino acid sequence described in SEQ ID NO: 66 (or a variant of SEQ ID NO: 66 having one or two amino acid modifications), and CDR-3 having the amino acid sequence described in SEQ ID NO: 67 (or a variant of SEQ ID NO: 67 having one or two amino acid modifications), and a β-chain (or δ-chain) having CDR-1 having the amino acid sequence described in SEQ ID NO: 73 (or a variant of SEQ ID NO: 73 having one or two amino acid modifications), CDR-2 having the amino acid sequence described in SEQ ID NO: 74 (or a variant of SEQ ID NO: 74 having one or two amino acid modifications), and CDR-3 having the amino acid sequence described in SEQ ID NO: 75 (or a variant of SEQ ID NO: 75 having one or two amino acid modifications).

[0085] In some embodiments, the anti-NPM1c TCRs or antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule and have α-chain (or γ-chain) and β-chain (or δ-chain) having CDR sequences (α or γ-chain CDR-1, CDR-2 and CDR-3, and δ-chain βCDR-1, CDR-2 and CDR-3) as described in any of the anti-NPM1c TCRs in Table 1, may include any suitable framework region. In some embodiments, the anti-NPM1c TCRs or antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, and which have an α-chain (or γ-chain) and a β-chain (or δ-chain) having CDR sequences described in any of the anti-NPM1c TCRs in Table 1 (α or γ-chain CDR-1, CDR-2 and CDR-3, and δ-chain βCDR-1, CDR-2 and CDR-3), and which one or more of the CDRs may independently be variant CDRs having one or two amino acid modifications from the enumerated CDR sequences in Table 1, may include any suitable framework regions (i.e., α-chain and β-chain framework regions 1, 2, 3, and 4). The α-chain framework region 1 may contain any amino acid sequence of any of the α-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1 that are downstream of the corresponding CDR-1 sequence, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications. The α-chain framework region 2 may contain any of the α-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1 that are located between the corresponding CDR-1 sequence and the CDR-2 sequence, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications.The α-chain framework region 3 may contain any amino acid sequence of any of the α-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located between the corresponding CDR-2 and CDR-3 sequences, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications. The α-chain framework region 4 may contain any of the α-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located downstream of the corresponding CDR-3 sequence, or a variant of that sequence having 1, 2, 3, 4, or 5 amino acid modifications. The β-chain framework region 1 may contain any of the β-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located downstream of the corresponding CDR-1 sequence, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications. The β-chain framework region 2 may contain any amino acid sequence of any of the β-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located between the corresponding CDR-1 and CDR-2 sequences, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications. The β-chain framework region 3 may contain any of the β-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located between the corresponding CDR-2 and CDR-3 sequences, or a variant of that sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications. The β-chain framework region 4 may contain any of the β-variable (V) region amino acid sequences of any of the anti-NPM1c TCRs described in Table 1, located downstream of the corresponding CDR-3 sequence, or a variant of that sequence having 1, 2, 3, 4, or 5 amino acid modifications. The anti-NPM1c TCR or its antigen-binding fragment may have any of the α-chain framework regions 1, 2, 3, and 4 of the anti-NPM1c TCR described in Table 1, as well as the corresponding β-chain framework regions 1, 2, 3, and 4.For example, a TCR may have α-chain framework regions 1, 2, 3, and 4 of SEQ ID NO: 32 and β-chain framework regions 1, 2, 3, and 4 of SEQ ID NO: 40, or α-chain framework regions 1, 2, 3, and 4 of SEQ ID NO: 68 and β-chain framework regions 1, 2, 3, and 4 of SEQ ID NO: 76.

[0086] In some embodiments, anti-NPM1c TCRs or their antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules, may include an α-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of the α-chain variable (V) region of any of the anti-NPM1c TCRs described in Table 1, and a β-chain comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1. The anti-NPM1c TCR or antigen-binding fragments provided herein may include an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence of the α-chain variable (V) region of any of the anti-NPM1c TCRs described in Table 1, and a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1. In some embodiments, the anti-NPM1c TCR or antigen-binding fragments provided herein may include (a) an α-chain containing an amino acid sequence having 100 percent identity with the amino acid sequence of the α-chain variable (V) region of any of the anti-NPM1c TCRs described in Table 1, and (b) a β-chain containing an amino acid sequence having 100 percent identity with the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1. For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identity with amino acid sequence SEQ ID NO: 32, and a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identity with amino acid sequence SEQ ID NO: 40.As another example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identity with amino acid sequence SEQ ID NO: 68, and a β-chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identity with amino acid sequence SEQ ID NO: 76.

[0087] In some embodiments, anti-NPM1c TCRs or their antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, may include: (a) an α-chain having an amino acid sequence having at least 90 percent identity to the amino acid sequence of the α-chain variable (V) region of any of the anti-NPM1c TCRs described in Table 1 (provided that the α-chain includes CDR-1, CDR-2, and CDR-3 amino acid sequences that are 100% identical to the corresponding α-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1); and (b) a β-chain having an amino acid sequence having at least 90 percent identity to the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1 (provided that the β-chain includes CDR-1, CDR-2, and CDR-3 amino acid sequences that are 100% identical to the corresponding β-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1). The anti-NPM1c TCRs or antigen-binding fragments provided herein are (a) anti-NPM1c as described in Table 1. (b) an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of any of the α-chain variable (V) regions of the TCR (provided that the α-chain includes CDR-1, CDR-2, and CDR-3 amino acid sequences that are 100% identical to the corresponding α-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1), and (b) a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1 (provided that the β-chain includes CDR-1, CDR-2, and CDR-3 amino acid sequences that are 100% identical to the corresponding β-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1).For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of SEQ ID NO: 32 (provided that the α-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NOs: 29, 30, and 31, respectively), and a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of SEQ ID NO: 40 (provided that the β-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NOs: 37, 38, and 39, respectively). As another example, an anti-NPM1c TCR or its antigen-binding fragment provided herein may include an α-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of SEQ ID NO: 68 (provided that the α-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NOs: 65, 66, and 67, respectively), and a β-chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with respect to the amino acid sequence of SEQ ID NO: 76 (provided that the β-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NOs: 73, 74, and 75, respectively).

[0088] In some embodiments, anti-NPM1c TCRs or their antigen-binding fragments provided herein, which have the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to MHC molecules, may include: (a) an α-chain having amino acids in the α-chain variable (V) region of any of the anti-NPM1c TCRs described in Table 1, or having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions); and (b) a β-chain having amino acids in the corresponding β-chain variable (V) region described in Table 1, or having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). The anti-NPM1c TCR or antigen-binding fragment provided herein, which has the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, (a) an α-chain having the amino acid sequence of any of the TCRs described in Table 1 having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (wherein the α-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences which are 100% identical to the corresponding α-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1). (b) a β-chain having the amino acid sequence of the corresponding β-chain variable (V) region described in Table 1, having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), wherein the β-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences which are 100% identical to the corresponding β-chain CDR-1, CDR-2, and CDR-3 sequences described in Table 1.For example, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule, may include an α-chain having the amino acid sequence described in SEQ ID NO: 32 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (where the α-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NO: 29, 30, and 31, respectively), and a β-chain having the amino acid sequence described in SEQ ID NO: 40 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (where the β-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NO: 37, 38, and 39, respectively). As another example, an anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to CLAVEEVSL (SEQ ID NO: 5) in relation to an MHC molecule, may include an α-chain having the amino acid sequence described in SEQ ID NO: 68 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (where the α-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NO: 65, 66, and 67, respectively), and a β-chain having the amino acid sequence described in SEQ ID NO: 76 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions) (where the β-chain includes the CDR-1, CDR-2, and CDR-3 amino acid sequences described in SEQ ID NO: 73, 74, and 75, respectively).

[0089] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein is (a) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 11, 12, and 13, (b) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 29, 30, and 31. (c) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 47, 48, and 49. (d) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 65, 66, and 67. (e) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 83, 84, and 85. (f) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 101, 102, and 103, respectively. (g) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 119, 120, and 121, respectively. (h) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 137, 138, and 139. (i) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 155, 156, and 157, (j) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 173, 174, and 175. (k) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 191, 192, and 193, (l) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 209, 210, and 211. (m) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 227, 228, and 229. (n) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 245, 246, and 247, respectively. (o) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 263, 264, and 265, or (p) Each contains a Vα or Vγ region, containing CDR-1, CDR-2, and CDR-3, respectively, including SEQ ID NOs. 281, 282, and 283.

[0090] Furthermore, some of the TCRs provided have sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0091] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein is (a) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 19, 20, and 21, respectively. (b) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 37, 38, and 39. (c) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 55, 56, and 57. (d) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 73, 74, and 75. (e) CDR-1, CDR-2, and CDR-3, each including sequence numbers 91, 92, and 93, (f) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 109, 110, and 111, respectively. (g) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 127, 128, and 129, respectively. (h) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 145, 146, and 147. (i) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 163, 164, and 165, (j) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 181, 182, and 183. (k) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 199, 200, and 201, respectively. (l) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 217, 218, and 219. (m) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 235, 236, and 237. (n) CDR-1, CDR-2, and CDR-3, each containing sequence numbers 253, 254, and 255, respectively. (o) CDR-1, CDR-2, and CDR-3, respectively, including sequence numbers 271, 272, and 273. (p) Each contains a Vβ or Vδ region, containing CDR-1, CDR-2, and CDR-3, respectively, including SEQ ID NOs. 289, 290, and 291.

[0092] Furthermore, some of the TCRs provided have sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0093] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein comprises a Vα or Vγ region containing CDR-1, CDR-2, and CDR-3, each containing the CDR-1, CDR-2, and CDR-3 amino acid sequences described in Table 1, for example, in each of the columns therein, and a Vβ or Vδ region containing CDR-1, CDR-2, and CDR-3, each containing the CDR-1, CDR-2, and CDR-3 amino acid sequences described in Table 1, for example, in each of the columns therein.

[0094] In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein includes a Vα region or Vγ region containing CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 amino acid sequences within the Vα region or Vγ region amino acid sequences described in Table 1, for example, in each of the columns therein, and a Vβ region or Vδ region containing CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 amino acid sequences within the Vβ region or Vδ region amino acid sequences described in Table 1, for example, in each of the columns therein. In some embodiments, the anti-NPM1c TCR or antigen-binding fragment provided herein includes a Vα region or Vγ region amino acid sequence described in Table 1, for example, in each of the columns therein, and a corresponding Vβ region or Vδ region amino acid sequence. Furthermore, some of the anti-NPM1c TCRs provided contain sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. Exemplary anti-NPM1c TCRs containing such CDRs, or modified versions thereof described elsewhere in this specification, are also described in Table 1, for example, in each of its columns.

[0095] [Table 1]

[0096] In some examples, the anti-NPM1c TCR or its antigen-binding fragment provided herein may be designed to include an α-chain (or γ-chain) (e.g., SEQ ID NOs. 29-31, 65-67, 83-85, or 101-103) containing a set of three CDRs (e.g., CDR-1, CDR-2, and CDR-3) as described in Table 1, and a β-chain (or δ-chain) (e.g., SEQ ID NOs. 37-39, 73-75, 92-92, or 109-111) containing a set of three CDRs (e.g., CDR-1, CDR-2, and CDR-3) as described in Table 1.

[0097] In some embodiments, the Vα region includes CDR-1 containing SEQ ID NO: 29, CDR-2 containing SEQ ID NO: 30, and CDR-3 containing SEQ ID NO: 31, and the Vβ region includes CDR-1 containing SEQ ID NO: 37, CDR-2 containing SEQ ID NO: 38, and CDR-3 containing SEQ ID NO: 39. In some embodiments, the Vα region includes CDR-1 containing SEQ ID NO: 65, CDR-2 containing SEQ ID NO: 66, and CDR-3 containing SEQ ID NO: 67, and the Vβ region includes CDR-1 containing SEQ ID NO: 73, CDR-2 containing SEQ ID NO: 74, and CDR-3 containing SEQ ID NO: 75. In some embodiments, the Vα region includes CDR-1 containing SEQ ID NO: 83, CDR-2 containing SEQ ID NO: 84, and CDR-3 containing SEQ ID NO: 85, and the Vβ region includes CDR-1 containing SEQ ID NO: 92, CDR-2 containing SEQ ID NO: 93, and CDR-3 containing SEQ ID NO: 94. In some embodiments, the Vα region includes CDR-1 containing SEQ ID NO: 101, CDR-2 containing SEQ ID NO: 102, and CDR-3 containing SEQ ID NO: 103, and the Vβ region includes CDR-1 containing SEQ ID NO: 109, CDR-2 containing SEQ ID NO: 110, and CDR-3 containing SEQ ID NO: 111.

[0098] In some embodiments, the Vα region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence of SEQ ID NO: 32, and the Vβ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence of SEQ ID NO: 40. In some embodiments, the Vα region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence of SEQ ID NO: 68, and the Vβ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence of SEQ ID NO: 76. In some embodiments, the Vα region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence of SEQ ID NO: 86, and the Vβ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence of SEQ ID NO: 94. In some embodiments, the Vα region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence of SEQ ID NO: 104, and the Vβ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence of SEQ ID NO: 112.

[0099] In some embodiments, the Vα region includes sequence number 32 or a sequence having at least 90% sequence identity thereto, and the Vβ region includes sequence number 40 or a sequence having at least 90% sequence identity thereto. In some embodiments, the Vα region includes sequence number 68 or a sequence having at least 90% sequence identity thereto, and the Vβ region includes sequence number 76 or a sequence having at least 90% sequence identity thereto. In some embodiments, the Vα region includes sequence number 86 or a sequence having at least 90% sequence identity thereto, and the Vβ region includes sequence number 94 or a sequence having at least 90% sequence identity thereto. In some embodiments, the Vα region includes sequence number 104 or a sequence having at least 90% sequence identity thereto, and the Vβ region includes sequence number 112 or a sequence having at least 90% sequence identity thereto.

[0100] In some embodiments, the Vα region includes sequence number 32 and the Vβ region includes sequence number 40. In some embodiments, the Vα region includes sequence number 68 and the Vβ region includes sequence number 76. In some embodiments, the Vα region includes sequence number 86 and the Vβ region includes sequence number 94. In some embodiments, the Vα region includes sequence number 104 and the Vβ region includes sequence number 112.

[0101] C. Exemplary steady-state domain In some embodiments, the α chain of the anti-NPM1c TCR or its antigen-binding fragment provided herein further contains an α-constant (Cα) region or a portion thereof. In some embodiments, the β chain further contains a β-constant (Cβ) region or a portion thereof. Thus, in some embodiments, the anti-NPM1c TCR (e.g., the anti-AIQDLCLAV or anti-CLAVEEVSL TCR provided herein) or its antigen-binding fragment contains an α chain comprising a Vα region and a Cα domain or a portion thereof, and / or a β chain comprising a Vβ region and a Cβ domain or a portion thereof. In some embodiments, the γ chain of the anti-NPM1c TCR or its antigen-binding fragment provided herein further contains a γ-constant (Cγ) region or a portion thereof. In some embodiments, the δ chain further contains a δ-constant (Cδ) region or a portion thereof. Accordingly, in some embodiments, the anti-NPM1c TCRs provided herein (e.g., the anti-AIQDLCLAV or anti-CLAVEEVSL TCRs provided herein) or their antigen-binding fragments contain a γ chain comprising a Vγ region and a Cγ domain or a portion thereof, and / or a δ chain comprising a Vδ region and a Cδ domain or a portion thereof.

[0102] In some embodiments, the α and β chains, or the γ and δ chains, of the anti-NPM1c TCR provided herein further contain a constant domain. In some embodiments, the Cα and Cβ domains, or the Cγ and Cδ domains, are, individually, mammalian (e.g., human or mouse constant domains). In some embodiments, the constant domains are adjacent to the cell membrane. For example, in some cases, the extracellular portion of the anti-NPM1c TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, where each variable domain contains a CDR.

[0103] In some embodiments, anti-NPM1c TCRs are provided herein that contain human constant domains, such as an α chain containing a human Cα domain and a β chain containing a human Cβ domain, or a γ chain containing a human Cγ domain and a δ chain containing a human Cδ domain. In some embodiments, the provided anti-NPM1c TCRs are fully human. Among the provided anti-NPM1c TCRs are TCRs containing human constant domains, such as fully human TCRs, whose expression and / or activity are not affected, or substantially affected, by the presence of endogenous human TCRs, for example, when expressed in human T cells such as human T cells, such as primary human T cells.

[0104] In some embodiments, each of the Cα and Cβ domains, or each of the Cγ and Cδ domains, is human. In some embodiments, Cα is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, Cβ is encoded by the TRBC1 gene or TRBC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, Cγ is encoded by the TRGC1 gene or TRGC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, Cδ is encoded by the TRDC gene (IMGT nomenclature) or a variant thereof.

[0105] In some embodiments, the Cα domain or its variant has or includes an amino acid sequence described in SEQ ID NO: 3, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO: 3. In some embodiments, the Cα domain has or includes an amino acid sequence described in SEQ ID NO: 3. In some embodiments, the Cβ domain or its variant has or includes an amino acid sequence described in SEQ ID NO: 7, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to SEQ ID NO: 7. In some embodiments, the Cβ domain has or includes an amino acid sequence described in SEQ ID NO: 7. In some embodiments, the anti-NPM1c TCR includes a Cα domain and a Cβ domain, as described in SEQ ID NOs: 3 and 7, respectively.

[0106] In some embodiments, the Cγ domain or its variant has or includes the amino acid sequence described in SEQ ID NO: 312 or 313, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with SEQ ID NO: 312 or 312. In some embodiments, the Cγ domain has or includes the amino acid sequence described in SEQ ID NO: 312. In some embodiments, the Cγ domain has or includes the amino acid sequence described in SEQ ID NO: 313. In some embodiments, the Cδ domain or its variant has or includes an amino acid sequence described in SEQ ID NO: 314, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with SEQ ID NO: 314. In some embodiments, the anti-NPM1c TCR includes a Cγ domain and a Cδ domain described in SEQ ID NO: 312 and 314, respectively. In some embodiments, the anti-NPM1c TCR includes a Cγ domain and a Cδ domain described in SEQ ID NO: 313 and 314, respectively.

[0107] In some embodiments, variants of the Cα domain include at least one substitution of unnatural cysteine, such as any substitution described herein. In some embodiments, variants of the Cβ domain include at least one substitution of unnatural cysteine, such as any substitution described herein.

[0108] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided may be a human / mouse chimeric TCR. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein includes an α-chain and / or a β-chain, or a γ-chain and / or a δ-chain, comprising a mouse constant domain. In some embodiments, the Cα-domain and / or Cβ-domain, or the Cγ-domain and / or Cδ-domain, is a mouse Cα-domain and / or mouse Cβ-domain, or a mouse Cγ-domain and / or mouse Cδ-domain. In some embodiments, the Cα-domain and / or Cβ-domain, or the Cγ-domain and / or Cδ-domain, is any Cα-domain and / or Cβ-domain, or Cγ-domain and / or Cδ-domain described in International Publication Nos. 2015 / 184228, 2015 / 009604, and 2015 / 009606, or includes such Cα-domain and / or Cβ-domain, or Cγ-domain and / or Cδ-domain.

[0109] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment comprises a variant of the α-chain and / or β-chain, or the γ-chain and / or δ-chain. In some embodiments, the variant comprises any amino acid sequence of the anti-NPM1c TCR described herein, having one, two, three, or four or more amino acid substitutions in the constant domain of the α-chain or β-chain. In some embodiments, the anti-NPM1c TCR (or its functional portion) comprising the substituted amino acid sequence advantageously provides one or more of the following compared to the parental TCR comprising the unsubstituted amino acid sequence: reduced mispairing with the endogenous TCR chain, increased expression by host cells, increased recognition of AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5), and increased antitumor activity.

[0110] In some embodiments, the anti-NPM1c TCR may be a heterodimer of α- and β-chains or γ- and δ-chains linked by one or more disulfide bonds. In some embodiments, the constant domain of the anti-NPM1c TCR may contain a short linking sequence in which cysteine ​​residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the anti-NPM1c TCR may have additional cysteine ​​residues in each of the α- and β-chains or γ- and δ-chains so that the TCR contains two disulfide bonds in the constant domain. In some embodiments, each of the constant domain and the variable domain contains a disulfide bond formed by cysteine ​​residues.

[0111] In some embodiments, the anti-NPM1c TCRs provided herein may contain one or more introduced disulfide bonds. In some embodiments, the native disulfide bonds are absent. In some embodiments, one or more native cysteine ​​residues (e.g., in the constant domains of the α and β chains, or the γ and δ chains) that form the native interchain disulfide bonds are substituted with other residues such as serine or alanine. In some embodiments, the introduced disulfide bonds can be formed by mutating non-cysteine ​​residues on the α and β chains, such as in the constant domains of the α and β chains, or the γ and δ chains, to cysteine. Opposing cysteines in the TCR α and β chains or TCR γ and δ chains link the constant domains of the substituted TCR α and β chains or TCR γ and δ chains to each other, providing disulfide bonds that are not present in TCRs containing unsubstituted constant domains with native disulfide bonds, such as unsubstituted native human constant domains or unsubstituted native mouse constant domains. In some embodiments, the presence of non-native cysteine ​​residues in recombinant TCRs (e.g., resulting in one or more non-native disulfide bonds) may be more favorable to the production of the desired recombinant TCR in the cells into which it is introduced than the expression of mismatched TCR pairs containing native TCR chains. In some embodiments, the α or γ chain and β or δ chain of the anti-NPM1c TCR each contain a cysteine ​​amino acid substitution. The cysteine ​​substitution may facilitate the formation of disulfide bonds between the α and β chains or between the γ and δ chains. The formation of disulfide bonds facilitates pairing between α- and β-chains or between γ- and δ-chains, and / or reduces mispairing with endogenous TCR chains. Preferred cysteine ​​substitutions include, but are not limited to, T47C substitution in the α-constant domain chain (at the position relative to SEQ ID NO: 3) and S56C substitution in the β-chain (at the position relative to SEQ ID NO: 7 or 9).

[0112] Exemplary non-natural disulfide bonds of the TCR are described in Published International PCT Patent Applications 2006 / 000830 and 2006 / 037960. In some embodiments, cysteine ​​may be introduced or substituted with residues corresponding to Thr48 in the Cα domain (corresponding to Thr47 in SEQ ID NO: 3) and Ser57 in the Cβ domain (corresponding to Ser56 in SEQ ID NO: 7 or 9), residues corresponding to Thr45 in the Cα domain and Ser77 in the Cβ domain, residues corresponding to Tyr10 in the Cα domain and Ser17 in the Cβ domain, residues corresponding to Thr45 in the Cα domain and Asp59 in the Cβ domain, and / or residues corresponding to Ser15 in the Cα domain and Glu15 in the Cβ domain.

[0113] In some embodiments, any of the provided cysteine ​​mutations can be produced at the corresponding positions of other sequences, such as the human or mouse Cα and / or Cβ domains, or the Cγ and / or Cδ domains described above. The term "corresponding" with respect to protein locations, as in the description in the sequence listing that an amino acid position in the disclosed sequence is a "corresponding" amino acid position, refers to an amino acid position identified when the disclosed sequence is aligned based on structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm. For example, a corresponding residue may be determined by aligning the reference sequence with the Cα sequence described in SEQ ID NO: 3, or the Cβ sequence described in SEQ ID NO: 7 or 9, using a structural alignment method as described herein. By aligning the sequences, corresponding residues can be identified, for example, by using conserved amino acid residues and identical amino acid residues as guides.

[0114] In some embodiments, the anti-AIQDLCLAV TCR or its antigen-binding fragment provided herein includes an α-chain or γ-chain containing at least 90% sequence identity to the amino acid sequence described in any of SEQ ID NOs: 17, 35, or 53, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence, and / or a corresponding β-chain or δ-chain containing the amino acid sequence described in any of SEQ ID NOs: 25, 43, or 63, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence, or a corresponding β-chain or δ-chain containing therein.

[0115] In some embodiments, the anti-CLAVEEVSL TCR or its antigen-binding fragment provided herein is an amino acid sequence described in any of SEQ ID NOs: 71, 89, 107, 125, 143, 161, 179, 197, 215, 233, 251, 269, or 287, or a sequence having at least 90% sequence identity to such a sequence, such as a sequence having at least 90% or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence, or an α-chain or γ-chain containing such a sequence. And / or an amino acid sequence described in any of SEQ ID NOs. 79, 97, 115, 133, 151, 169, 187, 205, 223, 241, 259, 277, or 295, or a sequence having at least 90% sequence identity to such a sequence, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence, or a corresponding β-chain or δ-chain (see Table 2) containing such a sequence.

[0116] Examples of anti-NPM1c TCRs or antigen-binding fragments are those described in Table 2, for example, in each of its columns. In some embodiments, the Vα and Vβ regions, or the Vγ and Vδ regions, contain amino acid sequences corresponding to the sequence numbers described in Table 2, for example, in each of its columns. In some embodiments, the Vα and Vβ regions, or the Vγ and Vδ regions, contain CDR-1, CDR-2, and CDR-3 sequences contained within the Vα and Vβ regions described in Table 2, for example, in each of its columns. In some embodiments, the anti-NPM1c TCR contains a constant α-domain sequence and a constant β-domain sequence, for example, those corresponding to the sequence numbers described in Table 2, for example, in each of its columns. In some embodiments, the anti-NPM1c TCR contains a complete sequence including a variable domain and a constant domain, for example, a sequence corresponding to the sequence number described in Table 2 ("complete α-P2A-β"), for example, in each of its columns. Furthermore, some of the provided anti-NPM1c TCRs contain sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. Exemplary anti-NPM1c TCRs containing such sequences, or modified versions thereof described elsewhere in this specification, are also described in Table 2, for example, in each column therein. In some embodiments, the provided exemplary anti-NPM1c TCRs, when expressed as mature proteins, when fully processed and expressed, include, for example, mature Vα and / or mature Vβ regions, or mature Vγ and / or Vδ mature regions, which do not contain signal sequences (e.g., from cleavage of signal sequences).

[0117] [Table 2]

[0118] In some embodiments, the α-chain or γ-chain includes sequence number 35 or a sequence having at least 90% sequence identity thereto, and the β-chain or δ-chain includes sequence number 43 or a sequence having at least 90% sequence identity thereto. In some embodiments, the α-chain or γ-chain includes sequence number 71 or a sequence having at least 90% sequence identity thereto, and the β-chain or δ-chain includes sequence number 79 or a sequence having at least 90% sequence identity thereto. In some embodiments, the α-chain or γ-chain includes sequence number 89 or a sequence having at least 90% sequence identity thereto, and the β-chain or δ-chain includes sequence number 97 or a sequence having at least 90% sequence identity thereto. In some embodiments, the α-chain or γ-chain includes sequence number 107 or a sequence having at least 90% sequence identity thereto, and the β-chain or δ-chain includes sequence number 115 or a sequence having at least 90% sequence identity thereto.

[0119] In some embodiments, the α chain includes SEQ ID NO: 35 and the β chain includes SEQ ID NO: 43. In some embodiments, the α chain includes SEQ ID NO: 71 and the β chain includes SEQ ID NO: 79. In some embodiments, the α chain includes SEQ ID NO: 89 and the β chain includes SEQ ID NO: 97. In some embodiments, the α chain includes SEQ ID NO: 107 and the β chain includes SEQ ID NO: 115.

[0120] In some embodiments, the anti-AIQDLCLAV TCR includes the amino acid sequence of SEQ ID NOs. 27, 45, or 63, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NOs. 27, 45, or 63. In some embodiments, the anti-CLAVEEVSL TCR includes the amino acid sequence of SEQ ID NOs. 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NOs. 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297.

[0121] In some embodiments, the anti-NPM1c TCR or its antigen-binding moiety provided herein may be a recombinantly produced native protein or a mutant form thereof, in which one or more properties, such as binding properties, are modified. In some embodiments, the nucleic acid is synthetic.

[0122] In some embodiments, any of the described anti-NPM1c TCRs (or nucleic acids encoding anti-NPM1c TCRs) can be modified to increase the affinity of the TCR to a target neoantigen, improve the stability of the TCR, and / or alter the function of the TCR, such as signal transduction. Methods for modifying anti-NPM1c TCRs include, but are not limited to, alanine scanning and peptide sequence optimization. In some embodiments, any of the described anti-NPM1c TCRs may be modified to include a disulfide bond in the constant region of the TCR domain.

[0123] D. Anti-NPM1 TCR single-chain variable fragment Any anti-NPM1 TCR CDR or variable region described herein can be used in the formation of single-chain variable fragments (scFv), bispecific T cell engagers (BiTE), chimeric T cell receptors (CAR), or other proteins containing scFv, BiTE, or CAR.

[0124] "scFv" is a variable heavy chain region (V) of immunoglobulin linked by a short linker peptide. H ) and variable light chain region (V L ), or a fusion protein containing the variable α region (Vα) and variable β region (Vβ) of the TCR. scFv retains the antigen-binding properties of the intact immunoglobulin or TCR from which the variable regions originate. Nucleic acids encoding anti-AIQDLCLAV or CLAVEEVSL scFv are also intended.

[0125] In some embodiments, anti-AIQDLCLAV and anti-CLAVEEVSL scFv are described, comprising the Vα and Vβ regions of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR, where the Vα and Vβ regions are linked via a linker peptide, and the anti-AIQDLCLAV or anti-CLAVEEVSL scFv retains the antigen-binding properties of the anti-AIQDLCLAV or anti-CLAVEEVSL TCR from which the variable region originates. Nucleic acids encoding anti-AIQDLCLAV and anti-CLAVEEVSL scFv are also intended. Anti-AIQDLCLAV or anti-CLAVEEVSL scFv can be formed by linking the C-terminus of the Vα chain to the N-terminus of the Vβ chain, or by linking the C-terminus of Vβ to the N-terminus of the Vα chain. The peptide linker may consist of about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is glycine-rich. The scFv peptide linker is (G4S) x The x is an integer between 2 and 5 (inclusive of both ends), and x can be any integer between 2 and 5. In some embodiments, the linked scFv peptide contains Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (also known as [(Gly)4Ser]3, (G4S)3, or G4S(×3)). In some embodiments, the scFv peptide linker consists of G4S(×3).

[0126] Bispecific T cell engagers (BiTEs) are recombinant molecules containing two flexibly linked antigen-binding domains, such as scFv. In a typical BiTE, one antigen-binding domain is specific to CD3 or other activating antigens on immune cells, while the second antigen-binding domain has affinity for a second antigen, such as a tumor antigen or an antigen on a target cell. BiTEs can be used to target T cells containing the CD3 receptor along with target cells (as described in International Publications 99054440, 2005040220, and 2008119567). BiTEs can transiently bind T cells to target cells and simultaneously activate the cytolytic activity of T cells.

[0127] In some embodiments, anti-AIQDLCLAV and anti-CLAVEEVSL BiTE molecules are described, comprising a first antigen-binding domain (e.g., scFv) specific to an activating antigen on an immune cell, and a second antigen-binding domain comprising an AIQDLCLAV or CLAVEEVSL binding domain or scFv containing a variable region or CDR of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR. In some embodiments, the BiTE comprises an anti-CD3 scFv and an anti-AIQDLCLAV or anti-CLAVEEVSL scFv containing the Vα and Vβ regions of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR. Nucleic acids encoding anti-AIQDLCLAV or anti-CLAVEEVSL BiTE are also intended.

[0128] A “chimeric antigen receptor” or “CAR” is a recombinant protein containing an antigen-binding domain (e.g., an antigen-binding fragment of any of the described anti-NPM1c TCRs) linked to a cell signaling and / or cell activation domain via a transmembrane domain. The cell signaling domain may, but is not limited to, a T cell signaling domain. When used in a CAR, the antigen-binding fragment of the described anti-NPM1c TCR may be provided as an scFv. A CAR may be a first-generation CAR T cell, a second-generation CAR T cell, a third-generation CAR T cell, a fourth-generation CAR T cell, a biantigen receptor CAR T cell, or a CAR T cell with an inducible suicide gene, or a combination thereof. A CAR may have a single signaling and / or cell activation domain, multiple signaling and / or cell activation domains, or one or more signaling and / or cell activation domains and one or more co-stimulatory domains. The signal transduction and / or cell activation domain or co-stimulatory domain may be, but are not limited to, the CD3 zeta domain, CD28 domain, CD137(4-1BB) domain, ICOS domain, CD27 domain, OX40 domain, LFA1 domain, PD-1 domain, CD150 domain, CD244 domain, NKG2D domain, and DAP10 domain. In some embodiments, the CAR has a CD28 transmembrane domain.

[0129] In some embodiments, anti-AIQDLCLAV and anti-CLAVEEVSL CARs are described, comprising an antigen-binding fragment of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR, or an scFv comprising a variable region or CDR, transmembrane domain, and signaling and / or cell activation domain of either of the described TCRs. In some embodiments, the antigen-binding fragment of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR comprises an anti-AIQDLCLAV or anti-CLAVEEVSL scFv, and the anti-AIQDLCLAV or anti-CLAVEEVSL scFv comprises a Vα region and a Vβ region of either the described anti-AIQDLCLAV or anti-CLAVEEVSL TCR. The anti-AIQDLCLAV or anti-CLAVEEVSL CAR may be, but is not limited to, a first-generation CAR T cell, a second-generation CAR T cell, a third-generation CAR T cell, a fourth-generation CAR T cell, a biantigen receptor CAR T cell, or a CAR T cell having an inducible suicide gene, or a combination thereof. Nucleic acids encoding anti-AIQDLCLAV or anti-CLAVEEVSL CAR are also being considered.

[0130] II. Nucleic acids encoding anti-NPM1c TCR Nucleic acids, such as polynucleotides or nucleic acid molecules encoding either the described anti-NPM1c TCR or its antigen-binding fragment, are also provided herein. Nucleic acids may include, for example, those having skeletal modifications, and may include those comprising natural and / or non-natural nucleotides and bases. The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA and RNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides, including a nucleic acid molecule or polynucleotide.

[0131] In some embodiments, the nucleic acid is or contains cDNA. In some embodiments, polynucleotides can be modified for use in the constructs described herein, such as for codon optimization. In some embodiments, the nucleic acid sequence can be designed to contain terminal restriction site sequences for the purpose of cloning into a vector.

[0132] In some embodiments, the anti-NPM1c TCR or its antigen-binding moiety provided herein can be synthesized from knowledge of the amino acid sequence of the TCR.

[0133] A nucleic acid sequence encoding a Vα region, a Vβ region, a Vα region and a Vβ region, an α chain, a β chain, or an α chain and a β chain is described. In some embodiments, the nucleic acid sequence may encode a Vγ region, a Vδ region, a Vγ region and a Vδ region, a γ chain, a δ chain, or a γ chain and a δ chain.

[0134] The nucleotide sequences may be provided as pairs of nucleotide sequences, each comprising a first nucleotide sequence encoding the Vα region or α chain and a second nucleotide sequence encoding the Vβ region or β chain, or a first nucleotide sequence encoding the Vγ region or γ chain and a second nucleotide sequence encoding the Vδ region or δ chain. The first and second nucleotide sequences may be provided on separate expression cassettes, plasmids, or vectors (e.g., viral vectors), or on a single expression cassette, plasmid, or vector.

[0135] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a Vα region or α chain and a Vβ region or β chain (or a Vγ region or γ chain and a Vδ region or δ chain), wherein the nucleic acid sequence encoding the Vα (or Vγ) region or α (or γ) chain is linked to the sequence encoding the Vβ (or Vδ) region or β (or δ) chain via a sequence encoding a peptide sequence that causes ribosome skipping or self-cleavage (e.g., a 2A peptide, such as a P2A peptide, a T2A peptide, an F2A peptide, or an E2A peptide) or an internal ribosome entry site (IRES) sequence. In some embodiments, sequences encoding the Vα and Vβ regions or α and β chains (or the Vγ and Vδ regions or γ and δ chains) are operably linked to a single promoter. In some embodiments, sequences encoding the Vα and Vβ regions or α and β chains (or the Vγ and Vδ regions or γ and δ chains) are each operably linked to a single bidirectional promoter (e.g., a PGK promoter). In some embodiments, the sequence encoding the Vα (or Vγ) region or α (or γ) chain can be 5' to the sequence encoding the Vβ (or Vδ) region or β (or δ) chain. In some embodiments, the sequence encoding the Vβ (or Vδ) region or β (or δ) chain can be 5' to the sequence encoding the Vα (or Vγ) region or α (or γ) chain.

[0136] In some embodiments, the nucleotide sequences encoding the α or γ chain, and / or the β or δ chain, or any domain, region (e.g., Vα and Vβ regions), or portion thereof, are codon-optimized. Typically, codon optimization involves balancing the proportion of selected codons with the published abundance of human transfer RNA to avoid overloading or restricting them. Most amino acids are encoded by two or more codons, and codon usage frequencies vary from organism to organism. Differences in codon usage frequencies between the transfected gene and the host cell can affect protein expression and the immunogenicity of nucleic acid constructs. Generally, for codon optimization, codons are selected to be balanced with human usage frequencies. Typically, amino acid codon duplication is such that different codons encode one amino acid. In some embodiments, when selecting codons for substitution, it may be desirable that the resulting mutations be silent mutations so that the codon changes do not affect the amino acid sequence.

[0137] In some embodiments, the nucleotide sequence encoding the Vα region includes SEQ ID NO: 33 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region includes SEQ ID NO: 41 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region includes SEQ ID NO: 69 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region includes SEQ ID NO: 77 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region includes SEQ ID NO: 87 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region includes SEQ ID NO: 95 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region includes SEQ ID NO: 105 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region includes SEQ ID NO: 113 or a sequence having at least 90% sequence identity thereto.

[0138] In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO: 34, or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO: 42, or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO: 70, or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO: 78, or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO: 88, or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO: 96, or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO: 106, or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO: 114, or a sequence having at least 90% sequence identity thereto.

[0139] In some embodiments, the nucleotide sequence encoding the α-chain or γ-chain includes SEQ ID NO: 36 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the β-chain or δ-chain includes SEQ ID NO: 44 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the α-chain or γ-chain includes SEQ ID NO: 72 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the β-chain or δ-chain includes SEQ ID NO: 80 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the α-chain or γ-chain includes SEQ ID NO: 90 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the β-chain or δ-chain includes SEQ ID NO: 98 or a sequence having at least 90% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the α-chain or γ-chain includes SEQ ID NO: 108 or a sequence having at least 90% sequence identity thereto, and the nucleotide sequence encoding the β-chain or δ-chain includes SEQ ID NO: 116 or a sequence having at least 90% sequence identity thereto. In addition, some of the nucleic acids or polynucleotides provided herein contain sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0140] In some embodiments, the nucleotide sequence encoding the α-chain includes SEQ ID NO: 36, and the nucleotide sequence encoding the β-chain includes SEQ ID NO: 44. In some embodiments, the nucleotide sequence encoding the α-chain includes SEQ ID NO: 72, and the nucleotide sequence encoding the β-chain includes SEQ ID NO: 80. In some embodiments, the nucleotide sequence encoding the α-chain includes SEQ ID NO: 90, and the nucleotide sequence encoding the β-chain includes SEQ ID NO: 98. In some embodiments, the nucleotide sequence encoding the α-chain includes SEQ ID NO: 108, and the nucleotide sequence encoding the β-chain includes SEQ ID NO: 116.

[0141] In some embodiments, the nucleic acid sequence encoding the α chain includes one of sequence numbers 15, 33, or 51, or a degenerate sequence thereof, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with them, and the nucleotide sequence encoding the β chain includes one of the corresponding sequence numbers 23, 41, and 59, or a degenerate sequence thereof, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with them.

[0142] In some embodiments, the nucleic acid sequence encoding the α chain includes one of sequence numbers 69, 87, 105, 123, 141, 159, 177, 195, 213, 231, 249, 267, or 285, or a degenerate sequence thereof, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with them, and the nucleotide sequence encoding the β chain includes one of the corresponding sequence numbers 77, 95, 113, 131, 149, 167, 185, 203, 221, 239, 257, 275, or 293, or a degenerate sequence thereof, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with them.

[0143] In some embodiments, the α- or γ-chain and / or β- or δ-chain of the anti-NPM1c TCR is encoded by a nucleotide sequence containing a signal peptide (also called a leader sequence). Non-limiting examples of such signal peptides are those having or containing the amino acid sequences described in any of SEQ ID NOs: 315-624.

[0144] In some embodiments, the nucleic acids encoding the α-chain or γ-chain and the nucleic acids encoding the β-chain or δ-chain may be linked via a linker, such as those optionally described elsewhere in this specification.

[0145] In some embodiments, the nucleic acid encoding the Vα or Vγ region or the α or γ chain and the nucleic acid encoding the Vβ or Vδ region or the β or δ chain may be linked via a cleavable linker sequence or a peptide that induces ribosome skipping or autocleavage (e.g., a 2A sequence, e.g., T2A, E2A, F2A, or P2A), such as those optionally described elsewhere in this specification. The P2A amino acid sequence may, but is not limited to, the sequence of SEQ ID NO: 301. The nucleic acid sequence encoding the P2A sequence may, but is not limited to, the nucleic acid sequence of SEQ ID NO: 302, a nucleic acid sequence encoding a P2A peptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 301, or a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 302.

[0146] In some embodiments, the nucleic acid encoding the Vα or Vγ region or the α or γ chain and the nucleic acid encoding the Vβ or Vδ region or the β or δ chain may be linked via an IRES sequence.

[0147] In some embodiments, the nucleic acid sequences encoding α and β TCR chains linked via a cleavable linker sequence include the nucleotide sequence of SEQ ID NO: 28, 46, or 64, a nucleotide sequence having at least 90% identity with the nucleic acid sequence of SEQ ID NO: 28, 46, or 64, a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 27, 45, or 63, or a nucleotide sequence encoding a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 27, 45, or 63.

[0148] In some embodiments, the nucleic acid sequences encoding α and β TCR chains linked via a cleavable linker sequence are nucleotide sequences of sequence numbers 82, 100, 118, 136, 154, 172, 190, 208, 226, 244, 262, 280, or 298, having at least 90% identity with the nucleic acid sequences of sequence numbers 82, 100, 118, 136, 154, 172, 190, 208, 226, 244, 262, 280, or 298. The nucleotide sequence comprises an ocidal sequence, a polypeptide having the amino acid sequence of SEQ ID NOs. 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297, or a nucleotide sequence having at least 90% identity with the amino acid sequence of SEQ ID NOs. 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297.

[0149] Vectors or constructs containing such nucleotide sequences are also provided herein. In some embodiments, the vector or construct contains one or more heterologous promoters operably ligated to nucleotides encoding a Vα or Vγ region or an α or γ chain and / or a Vβ or Vδ region or a β or δ chain. In some embodiments, the heterologous promoters are operably ligated to one or more nucleotide sequences.

[0150] In some embodiments, the vector or construct may contain a single promoter that drives the expression of one or more nucleotide sequences. In some embodiments, the promoter is a heterologous promoter. In some embodiments, such a vector or expression construct may be multicistronic (e.g., bicistronic or tricistronic; see, e.g., U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may be operated as a bicistronic unit containing an IRES (intrasequence ribosome entry site), thereby enabling the simultaneous expression of a gene product (e.g., encoding the α or γ chain and / or β or δ chain of the TCR) via a message from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA containing two or three genes (e.g., encoding the α or γ chain and / or β or δ chain of the TCR) separated from each other by sequences encoding self-cleaving peptides (e.g., 2A peptide, e.g., P2A peptide) or protease recognition sites (e.g., furin) in a single open reading frame (ORF). ORFs can encode single polyproteins that are cleaved into individual proteins either during translation (in the case of 2A, e.g., P2A) or post-translation. In some embodiments, peptides such as P2A can result in separation between the end of a 2A sequence and the next downstream peptide by causing ribosomes to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping) (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Examples of 2A cleavage peptides that can induce ribosome skipping include Thosea asigna virus (T2A), porcine rhinitis virus-1 (P2A, e.g., SEQ ID NO: 301), equine rhinitis A virus (E2A), and 2A sequences derived from foot-and-mouth disease virus, such as those described in U.S. Patent Publication No. 2007 / 0116690 (F2A).In some embodiments, the peptide that causes ribosome skipping is a P2A peptide and / or contains the amino acid sequence described in SEQ ID NO: 301.

[0151] In a bisistronic vector, the nucleic acid sequence encoding the Vα or Vγ region or the α or γ chain and the nucleotide sequence encoding the Vβ or Vδ region or the β or δ chain can be in any order and are separated by the nucleotide sequence encoding the peptide sequence that induces ribosome skipping. For example, in some embodiments, the nucleotide sequence includes, in that order, a nucleic acid sequence encoding the β or δ chain, a nucleic acid sequence encoding the peptide sequence that induces ribosome skipping (e.g., the P2A sequence described herein), and a nucleic acid sequence encoding the α or γ chain. In other embodiments, the nucleotide sequence includes, in this order, a nucleic acid sequence encoding the α or γ chain, a nucleic acid sequence encoding the peptide sequence that induces ribosome skipping (e.g., the P2A sequence described herein), and a nucleic acid sequence encoding the β or δ chain.

[0152] In some embodiments, the nucleotide sequences encoding the Vα or Vγ region or α or γ chain of the TCR and / or the Vβ or Vδ region or β or δ chain include one or more nucleic acid sequences corresponding to the sequence numbers described in Table 3 for TCRs A to P. Furthermore, some of the provided nucleotide sequences encoding the TCR contain sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some embodiments, the nucleotide sequences include sequences encoding signal sequences, and the encoded exemplary anti-NPM1c TCR, when fully processed and expressed as a mature protein, includes, for example, a mature Vα region and / or a mature Vβ region, or a mature Vγ region and / or a mature Vδ region, without the signal sequence (e.g., from cleavage of the signal sequence).

[0153] [Table 3]

[0154] Vectors, such as vectors containing any of the nucleic acids described herein, are also provided herein. In some embodiments, one or more nucleic acids encoding one or both strands of the TCR are cloned or assembled into one or more suitable expression vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include plasmids and viruses, and other vectors designed for propagation and expansion, or for expression or both. In some embodiments, the vector is an expression vector.

[0155] III. Methods for Isolating, Evaluating, and Identifying T Cell Receptors In some embodiments, the foregoing provides a method for isolating multiple nucleic acid sequences encoding TCRs specific to AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5). In some embodiments, the AIQDLCLAV (SEQ ID NO: 1)-specific or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs are identified based on the method described herein. In some embodiments, the method also includes isolating nucleic acid sequences, assembling nucleic acid sequences into vectors, evaluating TCR expression and / or activity, and optionally screening and identifying specific TCRs of interest using high-throughput methods.

[0156] The methods described herein also relate to determining the binding activity and functional capacity of candidate TCRs.

[0157] A. TCR donor criteria and methods for isolating NPM1c-specific T cell candidates In some embodiments, whole exome sequencing is used to type a donor for their HLA repertoire and / or haplotype. In some embodiments, the donor subject has cancer. In some embodiments, the donor subject has a type of cancer that is to be treated with engineered T cells. In some embodiments, the donor is a healthy donor. In some embodiments, those subjects having an appropriate HLA type are selected for blood collection. Anti-AIQDLCLAV (SEQ ID NO: 1) reactive T cells from a donor subject can be identified based on their binding to an HLA multimer folded with the neoantigen AIQDLCLAV (SEQ ID NO: 1) peptide. These cells are single-cell sorted, and their TCRs are screened for anti-AIQDLCLAV (SEQ ID NO: 1) reactivity. In some embodiments, the donor is HLA-A * 02:01 + , HLA-A * 02:06 + , or HLA-A * 02:03 + . Anti-CLAVEEVSL (SEQ ID NO: 5) reactive T cells from a donor subject can be identified based on their binding to an HLA multimer folded with the neoantigen CLAVEEVSL (SEQ ID NO: 5) peptide. These cells are single-cell sorted, and their TCRs are screened for anti-CLAVEEVSL (SEQ ID NO: 5) reactivity. In some embodiments, the donor is HLA-A * 02:01 + , HLA-A * 02:06 + , or HLA-A * 02:03 + . Reactive T cells can also be identified by proliferation or expansion, cytokine expression, upregulation of activation / signaling proteins, upregulation or downregulation of other protein metabolites or other cellular components, changes in cell shape or particle size, or other means available in the art.

[0158] B. High-throughput isolation, amplification, and assembly of nucleic acid sequences encoding TCRs In some embodiments, the nucleic acid molecules encoding the TCR can be obtained or identified from various sources. In some embodiments, the TCR can be obtained or identified using high-throughput TCR isolation and screening methods. An example of such a method that may be used is, for example, the method described in International Publication No. 2018 / 102473, which is incorporated herein by reference in its entirety. In some embodiments, the high-throughput TCR isolation and screening method involves amplification of nucleic acids encoding the TCR α chain and / or β chain or the TCR γ chain and / or δ chain from multiple different cells, such as T cells isolated from a donor. Also provided herein are methods relating to isolating or screening multiple different TCRs to obtain TCRs specific to NPM1c neoantigens, such as AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5).

[0159] In some embodiments, the nucleic acid molecule encoding the TCR can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of the encoding nucleic acid in a given cell or a set of cells, or of the encoding nucleic acid isolated therefrom. In some embodiments, the TCR can be obtained from a biological source, such as T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the TCR can be derived from one of various animal species, such as humans, mice, rats, or other mammals. In some embodiments, T cells can be obtained from cells isolated in vivo from normal (or healthy) or diseased subjects, including T cells present in peripheral blood mononuclear cells (PBMCs) or tumor-infiltrating lymphocytes (TILs). In some embodiments, the T cells can be cultured T cell hybridomas or clones. For example, in some embodiments, the α and β chains can be PCR-amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector to generate a vector encoding the TCR. In some embodiments, the α and β chains can be generated synthetically. In some embodiments, the α and β chains are cloned into the same vector.

[0160] As described herein, the methods and materials provided herein may enable users to successfully capture most, but not all, functional TCRs from a selected T cell population. For example, amplification procedures (e.g., nested amplification procedures such as nested PCR) may include using a primer collection designed to amplify all known functional V segments of the two variable strands of a particular mammalian (e.g., human) TCR, or nearly all (>90%, >95%, >96%, >97%, or >98%) of the known V segments (e.g., either the known functional V segments of the α and β variable strands of a particular αβTCR, or either the known functional V segments of the γ and δ variable strands of a particular γδTCR). For humans, the amplification procedure may include a primer collection designed to amplify 45 V segments of all or nearly all (>90%, >95%, >96%, >97%, or >98%) of the α chain currently known to be functional, and 48 V segments of all or nearly all (>90%, >95%, >96%, >97%, or >98%) of the β chain currently known to be functional. Where the α chain TCR V segment is referred to herein, the abbreviation TRAV may be used. Similarly, where the β chain TCR V segment is referred to herein, the abbreviation TRBV may be used. The same applies to the γ chain and δ chain TCR V segments, which may be called TRGV and TRDV, respectively.

[0161] In some embodiments, this document provides methods and materials relating to the cloning of a functional TCR from a single T cell. For example, in some embodiments, this document provides methods and materials for obtaining a nucleic acid encoding a TCR from a single T cell and arranging the nucleic acid to form a nucleic acid vector successfully designed to express a TCR (e.g., a fully intact TCR, such as a fully intact TCR having a variable strand combination present in the single T cell); kits for obtaining a nucleic acid encoding a TCR from a single T cell and arranging the nucleic acid to form a nucleic acid vector successfully designed to express a TCR (e.g., a fully intact TCR, such as a fully intact TCR, having a variable strand combination present in the single T cell); and methods for constructing such kits. A cloned αβ TCR having a variable strand combination as present in the single T cell used to clone the TCR may include a VJα segment combination as present in the single T cell, a VDJβ segment combination as present in the single T cell, a nucleotide sequence of the entire α variable region as present in the single T cell, and a nucleotide sequence of the entire β variable region as present in the single T cell. Similarly, a cloned γδTCR having a variable chain combination as present in a single T cell used to clone the TCR may include a VJγ segment combination as present in a single T cell, a VDJδ segment combination as present in a single T cell, a nucleotide sequence of the entire γ variable region as present in a single T cell, and a nucleotide sequence of the entire δ variable region as present in a single T cell.

[0162] In some embodiments, this document provides a collection of nucleic acid primers designed to amplify the entire coding sequences of both variable regions (e.g., α-variable region and β-variable region, or γ-variable region and δ-variable region) of each expressed V segment (e.g., each expressed αV segment and βV segment, or each expressed γV segment and δV segment) of a functional αβ or γδ TCR in a specific mammalian species (e.g., mouse or human), a method using such a collection of nucleic acid primers to clone a functional TCR from a single T cell, and a kit containing such a collection of nucleic acid primers for cloning a functional TCR from a single T cell.

[0163] In some embodiments, the methods and materials provided herein may enable the cloning of many different TCRs (e.g., hundreds to thousands or more) directly and rapidly (e.g., simultaneously in some cases) from a single T cell, with minimal loss of α / β variable chain combinations (or γ / δ variable chain combinations), even if present. For example, the methods and materials provided herein can be used to directly clone many different αβTCRs (e.g., hundreds to thousands or more different αβTCRs) from a single αβT cell, in a manner that is possible for α / β variable chain combinations of a species (e.g., mouse or human), by losing less than 10 percent of the α variable chain (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) and less than 10 percent of the β variable chain (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent). Similarly, the methods and materials provided herein can be used to clone many different γδTCRs (e.g., hundreds to thousands or more different γδTCRs) directly from a single γδT cell, in a manner that loses less than 10 percent of the γ variable chain (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) and less than 10 percent of the δ variable chain (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) for possible γ / δ variable chain combinations of species (e.g., mouse or human species). In some embodiments, the methods and materials provided herein can be used to (a) obtain a sample of T cells and (b) sort those T cells into isolated locations (e.g., wells), thereby obtaining most, if not all, of the isolated locations (e.g.,(c) each well contains a single T cell, (d) each well contains a single T cell, (e) each well contains a single T cell, (c T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains a single T cell, (c) each T cell contains (f) to produce at least an amplification product containing the nucleic acid sequence of the α variable chain (or γ variable chain) of the TCR of a single T cell at the isolated location, and an amplification product containing the nucleic acid sequence of the β variable chain (or δ variable chain) of the TCR of the same single T cell at the same isolated location, and (f) for each isolated location using the amplification product of the first round of amplification reaction, the primer collection of the second round (e.g., the PCR primer collection of the second round), and a polymerase (e.g., Taq polymerase) as a template. (g) Performing a second round of amplification (e.g., a second round of PCR) of a nested amplification procedure (e.g., a nested PCR procedure) (e.g., simultaneously) to produce at least a first amplification product containing the nucleic acid sequence of the α variable chain (or γ variable chain) of the TCR of a single T cell at the isolated site, and a second amplification product containing the nucleic acid sequence of the β variable chain (or δ variable chain) of the TCR of the same single T cell at the same isolated site, and (g) for each isolated site, the first amplification product and the second amplification product,This may include cloning into an expression vector designed to express a functional TCR having a combination of α / β or γ / δ variable chains (or a part thereof, such as the V segment of the α / β or γ / δ variable chain combination) as present in the single T cell used to generate the amplification product.

[0164] The resulting expression vector can be introduced into cells to cause those cells to express the cloned TCR. The TCRs expressed by such cells, and / or the introduced expression vector, can be screened to identify TCRs with the desired capabilities. For example, cells expressing a cloned TCR that recognizes a specific antigen (e.g., NPM1c neoantigen) can be identified, and these cells, the TCR expression vectors they contain, or the cloned TCR constructs can be used for further analysis or therapeutic applications.

[0165] In some embodiments, when the signaling machinery of a functional TCR is activated, the expression of surface-cloned TCRs and functional TCRs can be evaluated by introducing an expression vector into TCR-negative reporter cells designed to express a measurable marker signal or marker polypeptide. In these cases, functional TCRs can be screened using antibodies designed to nonspecifically activate TCRs (e.g., anti-CD3 antibodies). In some embodiments, cloned TCRs can be screened for antigen specificity. For example, reporter cells expressing cloned TCRs can be screened for recognition of specific antigens (e.g., peptides derived from tumor polypeptides). In some embodiments, primary T cells (e.g., human primary T cells) can be transfected with an expression vector and screened for antigen specificity via a T cell proliferation assay.

[0166] For example, in some cases, the methods and materials provided herein may include a nested amplification procedure (e.g., a nested PCR procedure) comprising a primer collection designed to amplify all known functional V segments of two variable strands of a particular mammalian (e.g., human) TCR (e.g., either of the known functional V segments of the α and β variable strands of a particular αβTCR, or either of the known functional V segments of the γ and δ variable strands of a particular γδTCR).

[0167] In some embodiments, T cells can be stimulated before sorting (e.g., in vitro stimulation), and then RNA expression can be evaluated (e.g., via qPCR) to determine which T cells responded to the stimulation. Any suitable type of stimulation can be used, including, but not limited to, non-specific stimulation, such as stimulation with concanavalin A, phytohemagglutinin-P, phorbol ester + ionomycin, phorbol myristate acetate + calcium ionophore, or antibodies capable of crosslinking the TCR (e.g., anti-CD3 antibody + anti-CD28 antibody, or anti-TCRβ antibody), or antigen-specific stimulation, such as stimulation with one or more specific antigens as described elsewhere (Downward et al., Nature, 346:719-23 (1990); and Dasgupta et al., Proc. Natl. Acad. Sci. USA, 84:1094-8 (1987)). In some embodiments, cytokine expression levels, such as TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, or IL-17 expression levels, can be determined and compared to an unstimulated population. Once single T cells are sorted, methods provided herein can be used to determine which T cells produced specific cytokines in response to stimulation (e.g., in response to a peptide antigen used to stimulate the T cells). In these cases, antigen-specific T cells can be determined without cumbersome methods that expand reactive T cells, which may reduce their ability to effectively clone TCRs, or without destructive methods of paraformaldehyde fixation and intracellular cytokine staining. In such cases, specific TCRs generated from active and antigen-specific T cells can be rapidly identified, in contrast to inactive bystander T cells.

[0168] In some embodiments, cytokine expression levels, such as TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, or IL-17 expression levels, can be determined for a single T cell used to clone a functional TCR, thereby enabling the identification of a specific TCR based on a specific phenotype (e.g., elevated IFN-γ expression) of the T cell that provided the variable chain (or part thereof) of the particular TCR. In such cases, a specific TCR generated from an active rather than inactive T cell can be rapidly identified.

[0169] In some embodiments, TCRs can be obtained using T cells that are unresponsive to stimulation, and the specificity of the cloned TCRs can be tested or screened in cells in which canonical TCR signaling is not suppressed.

[0170] In some embodiments, MHC-peptide complexes (or HLA-peptide complexes) can be used to identify cloned TCRs that recognize such complexes. In these cases, clonal exclusion and / or lack of antigen priming during the immune response may result in TCRs with this specificity that are not present in the activated and / or expanded TCR pool. In such cases, the methods and materials provided herein can be used to clone naive or inactivated TCRs that recognize such complexes, although in some cases only the presence of a single T cell may be required. In some embodiments, a pool of naive T cells can be stained with MHC-peptide tetramers (or HLA-peptide tetramers), and any MHC-peptide (or HLA-peptide)-responsive TCRs in the naive T cells can be used to clone these TCRs using the methods and materials provided herein.

[0171] In some embodiments, the methods provided herein include methods for obtaining a plurality of nucleic acid vectors containing nucleic acids encoding functional T cell receptors. The methods include, or are essentially derived from, (a) obtaining a device comprising a plurality of distinct locations, each of which contains cDNA generated from RNA obtained from a single T cell sorted to a distinct location; (b) performing a nested amplification procedure using each of the cDNAs at the plurality of distinct locations as a template to obtain a first amplification product and a second amplification product for each of the cDNAs at the plurality of distinct locations, wherein the first amplification product comprises a nucleic acid encoding a Vα segment or a Vγ segment, and the second amplification product comprises a nucleic acid encoding a Vβ segment or a Vδ segment; and (c) assembling the first and second amplification products for each of the cDNAs at the plurality of distinct locations into a nucleic acid vector to obtain an assembled nucleic acid vector for each of the cDNAs at the plurality of distinct locations, wherein the assembled nucleic acid vector for each of the cDNAs at the plurality of distinct locations contains a nucleic acid encoding a functional T cell receptor. Multiple nucleic acid vectors may be multiple nucleic acid expression vectors. The device may include a multiwell plate. The multiwell plate may be a 96-well plate, a 384-well plate, or a 1536-well plate. cDNA generated from RNA obtained from a single T cell may include cDNA generated from RNA obtained from a single human T cell. The first amplification product may include a nucleic acid encoding the L sequence of the Vα segment or Vγ segment. The first amplification product may include a nucleic acid encoding the Jα segment or Jγ segment. The first amplification product may include a nucleic acid encoding the 5' portion of the Cα region or Cγ region. The first amplification product may include a nucleic acid encoding the L sequence of the Vα segment or Vγ segment, the Jα segment or Jγ segment, and the 5' portion of the Cα region or Cγ region. The second amplification product may include a nucleic acid encoding the L sequence of the Vβ segment or Vδ segment. The second amplification product may include a nucleic acid encoding the Dβ segment or Dδ segment.The second amplification product may include a nucleic acid encoding a Jβ segment or a Jδ segment. The second amplification product may include a nucleic acid encoding the 5' portion of the Cβ or Cδ region. The second amplification product may include a nucleic acid encoding a Vβ segment or a Vδ segment, a Dβ segment or a Dδ segment, a Jβ segment or a Jδ segment, and an L sequence of the 5' portion of the Cβ or Cδ region. The first amplification product may include an adapter sequence added to the amplified template sequence of cDNA via a second round of amplification in a nested amplification procedure. The second amplification product may include an adapter sequence added to the amplified template sequence of cDNA via a second round of amplification in a nested amplification procedure. The first amplification product may include a first adapter sequence added to the amplified template sequence of cDNA via a second round of amplification in a nested amplification procedure, and the second amplification product may include a second adapter sequence added to the amplified template sequence of cDNA via a second round of amplification in a nested amplification procedure, where the first adapter sequence and the second adapter sequence are different. Each functional T cell receptor in the assembled nucleic acid vector may include a Vα / Vβ combination or a Vγ / Vδ combination, as is present in a single T cell of RNA origin. Each functional T cell receptor in the assembled nucleic acid vector may include (a) a full-length α variable region and a full-length β variable region, or (b) a full-length γ variable region and a full-length δ variable region. Each functional T cell receptor in the assembled nucleic acid vector may include (a) a full-length α variable region and a full-length β variable region, as is present in a single T cell of RNA origin, or (b) a full-length γ variable region and a full-length δ variable region, as is present in a single T cell of RNA origin. Each functional T cell receptor in the assembled nucleic acid vector may include (a) a full-length α constant region and a full-length β constant region, or (b) a full-length γ constant region and a full-length δ constant region. Each assembled nucleic acid vector may contain a nucleic acid sequence encoding a self-cleaved peptide or an internal ribosome entry site (IRES). The method may include a step of sorting a single T cell to a distinct location. The method may include a reverse transcription reaction to obtain cDNA. The assembly step may include seamless cloning.Each assembled nucleic acid vector can be obtained without nucleic acid sequencing. Each assembled nucleic acid vector can be obtained without restriction endonuclease cleavage.

[0172] C. Evaluation of neoantigen-specific T cell receptor expression, activity, and function. Exemplary assays may be used to evaluate the activity, expression, and / or function of the anti-NPM1c TCRs and antigen-binding fragments described herein. The assays described herein should not be construed as limiting and may be used to evaluate the functional capabilities of candidate NPM1c-specific TCRs.

[0173] The functional characterization of anti-NPM1c TCRs can be performed by either a binding assay using fluorescently labeled MHC molecules containing a specific target peptide (tetramer / pentamer / dextramer), or an activation assay involving co-culturing antigen-presenting cells (APCs) displaying the corresponding MHC / peptide complex with anti-NPM1c TCR-expressing cells.

[0174] Cytokine release assays can evaluate the ability of candidate anti-NPM1c TCRs to produce cytokines (e.g., IL-2, TNFα, and / or IFN-γ) or other T cell activation markers (e.g., CD69) after exposure to cells presenting a target antigen. T cells are then subjected to K562 cells loaded with the target AIQDLCLAV(SEQ ID NO: 1) peptide (e.g., AIQDLCLAV(SEQ ID NO: 1)). (neg) / HLA-A * :02:01 (pos) Incubate with ). As a control, load non-target peptides or irrelevant peptide controls into K562 cells. Perform intracellular cytokine staining and FACS analysis following the IL-2 and / or IFN-γ response of T cells.

[0175] A T cell activation / degranulation marker assay can be used to evaluate the ability of candidate anti-NPM1c TCRs to express the surface marker CD107a after exposure to cells presenting a target antigen. CD107a is a marker of T cell degranulation, which is part of the cell death response. T cells are incubated with K562 cells loaded with the target AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptide, or a control peptide. The degranulation response is tracked on T cells by CD107a surface staining and analysis by FACS.

[0176] The killing assay can evaluate the ability of candidate anti-NPM1c TCRs to lyse cells presenting the target antigen. T cells are incubated with a mixture of fluorescently tagged K562 cells differentially loaded with target and control peptides to allow testing of on-target and off-target cytotoxicity within a single test sample. Fluorescent cell counting beads are included as a normalization / counting control. Fluorescently tagged K562 cells are loaded with the target AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptide. As a control, K562 cells labeled with a different fluorescent tag are loaded with a non-target peptide control. The number of gated cells remaining after incubation with T cells—K562 cells loaded with AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptides, and K562 cells loaded with the control peptides—is tracked by FACS.

[0177] The CD34 marker can be used as a surrogate potency measure. See Philip et al. (2014) Blood 124(8):1277-1287. Detection of CD34, a marker of transduction efficiency, may correlate with the functional potency measures described herein.

[0178] In some embodiments, extended and non-extended screening are performed using exactly the same donor for direct comparison of the methods. Extended screening may be performed using specific methods. Methods that generate candidate anti-NPM1c TCRs without an extended screening process may be advantageous in some situations, considering the time sensitivity of screening for anti-NPM1c TCRs with desired specificity. Reduced sample processing may also offer advantages in different situations.

[0179] IV. Manipulated Cells Cells such as cells manipulated to contain or express any of the anti-NPM1c TCRs described herein are also provided herein. Such cells, in which cells expressing an anti-NPM1c TCR constitute at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition, as well as populations of compositions containing and / or enriched with such cells, are also provided herein. In some embodiments, the cells are primary T cells, or T cells or CD8 cells. + Cells or CD4 + These are specific types of cells, such as cellular cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as adoptive cell therapy. Methods of administering cells and compositions to a subject, such as a patient, are also provided herein.

[0180] Accordingly, genetically engineered cells expressing the anti-NPM1c TCR provided herein are also provided herein. The cells are generally eukaryotic cells, such as mammalian cells, and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs and are cells of the immune system, such as cells of the innate or adaptive immune system, such as myeloid cells or lymphoid cells, such as T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or hematopoietic progenitor cells (HPCs). The cells are typically primary cells (e.g., primary T cells), such as those isolated directly from the subject and / or isolated and frozen from the subject. In some embodiments, cells include one or more subsets of T cells or other cell types, such as the whole T cell population, CD4+ cells, CD8+ cells, and their subpopulations, defined, for example, by function, activation state, maturity, differentiation potential, expansion, recirculation potential, localization potential, and / or persistence potential, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, cells may be allogeneic and / or autologous. In some embodiments, methods provided herein include isolating cells from a subject, preparing them as described herein, processing, culturing, and / or manipulating them, and reintroducing them to the same patient before or after cryopreservation.

[0181] T cells (including primary T cells) and / or CD4 included herein + T cells and / or CD8 + T cells (primary CD4 + T cells and / or CD8 + Among the subtypes and subpopulations of cells, there are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, for example, stem cell memory T(T) SCM ), Central Memory T(TCM ), Effector Memory T (T EM These include terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, native and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0182] In some embodiments, the cells are NK cells or memory NK cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0183] In some embodiments, the cells contain one or more nucleic acids introduced through genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterogeneous, i.e., not normally found in the cells being engineered and / or in the organism from which such cells originate, such as those obtained from another organism or cell. In some embodiments, the nucleic acids are non-naturally occurring, such as nucleic acids not found in nature, including those containing chimeric combinations of nucleic acids encoding various domains derived from multiple different cell types.

[0184] In some embodiments, the expression of endogenous TCR chains in manipulated cells is reduced or eliminated. Exemplary methods for reducing or preventing endogenous TCR expression are described; see, for example, U.S. Patent No. 9,273,283, U.S. Patent Application Publication No. 2014 / 0301990.

[0185] In some embodiments, T cells are transfected with nucleic acids encoding anti-AIQDLCLAV or anti-CLAVEEVSL CAR to form anti-AIQDLCLAV or anti-CLAVEEVSL CAR T cells. T cells may include, but are not limited to, primary T cells, cultured T cells, autologous T cells, allogeneic T cells, T cells obtained from bone marrow, T cells obtained from lymph nodes, T cells obtained from the thymus, tumor-infiltrating lymphocytes, T cells obtained from the spleen, T cells from umbilical cord blood, universal allogeneic T cells, universal CAR T cells, CAR T cells, naive T cells, effector T cells, effector memory T cells, CD4+ / CD8+ T cells, helper T cells, CD4+ T cells, CD4+ helper T cells, Th1 T cells, Th2 T cells, Th3 cells, Th9 cells, tH17 cells, Th22 cells, cytotoxic T cells, CD8+ T cells, peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), memory T cells, central memory T cells, regulatory T cells, αβ T cells, γδ T cells, modified T cells, T cells for use in adoptive cell transfer therapy, or TCR-manipulated T cells. CAR T cells may be first-generation CAR T cells, second-generation CAR T cells, third-generation CAR T cells, fourth-generation CAR T cells, biantigen receptor CAR T cells, or CAR T cells possessing an inducible suicide gene, or a combination thereof.

[0186] A. Preparation of cells for genetic manipulation In some embodiments, the preparation of the manipulated cells includes one or more culture and / or preparation steps. Cells for introducing anti-NPM1c TCRs may be isolated from a sample, such as a biological sample, e.g., a sample obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated (donor) is a subject having a disease or condition, or a subject requiring cell therapy, or a subject to which cell therapy is administered (i.e., autologous T cells). In some embodiments, the donor is a healthy donor. In some embodiments, the subject is a human being requiring a specific therapeutic intervention, such as adoptive cell therapy, from which the cells are isolated, processed, and / or manipulated.

[0187] Samples include tissues, bodily fluids, and other samples taken directly from a subject, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples may be samples obtained directly from a biological source or processed samples. Examples of biological samples include, but are not limited to, biopsies, tissue samples, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples (including processed samples derived therefrom).

[0188] In some embodiments, the sample from which cells are derived or isolated is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, PBMCs, leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, gastrointestinal lymphoma tissue, mucosa-associated lymphoma tissue, pancreas, other lymphoma tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, chest, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. Samples may include autologous and allogeneic sources in connection with cell therapy, such as adoptive cell therapy.

[0189] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a heterogeneous source, such as a mouse, rat, non-human primate, or pig.

[0190] B. Vectors and methods for genetic manipulation This specification also provides methods, nucleic acids, compositions, and kits for expressing anti-NPM1c TCR or its antigen-binding fragments, and for generating genetically engineered cells expressing such anti-NPM1c TCR or its antigen-binding fragments. Genetic engineering generally involves introducing nucleic acids encoding anti-NPM1c TCR (or its antigen-binding fragments) into cells, such as by retroviral transduction, nanoparticle delivery, transfection, or transformation.

[0191] In some embodiments, gene transfer is achieved by first stimulating cells, such as by combining them with stimuli that induce responses such as proliferation, survival, and / or activation (measured, for example, by the expression of cytokines or activation markers), and then transducing the activated cells and expanding them to a number sufficient for clinical use during culture.

[0192] Various methods for introducing genetically modified components are well known and may be used in conjunction with the methods and compositions provided herein. Exemplary methods include those provided herein for the transfer of nucleic acids encoding anti-NPM1c TCR or its antigen-binding fragments, including those via viruses, such as retroviral or lentiviral transduction, transposons, electroporation, and nucleofection.

[0193] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles. In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma retroviral vectors.

[0194] In some embodiments, the retroviral vector has a long terminal repeat (LTR) and is derived from, for example, Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen focus forming virus (SFFV). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell source. Retroviruses are typically bispecific, meaning they can infect host cells of several species, including humans. In some embodiments, the expressed nucleic acid substitutes retroviral gag, pol, and / or env sequences. Several exemplary retroviruses are described elsewhere (see, for example, U.S. Patent No. 5,219,740; No. 6,207,453; No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0195] Methods for lentiviral transduction are well known. Exemplary methods are described, for example, in Wang et al. (2012) J.35(9):689-701; Cooper et al. (2003) Blood.101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol.506:97-114; and Cavalieri et al. (2003) Blood.102(2):497-505.

[0196] In some embodiments, recombinant nucleic acids are transferred to T cells via electroporation or nucleofection (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids are transferred to T cells via translocation (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2,e74; and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing nucleic acids provided herein into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-enhanced microparticle bombardment (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0197] Other approaches and vectors for the transfer of nucleic acids encoding anti-NPM1c TCRs, their antigen-binding fragments, or recombinant products provided herein are described elsewhere. See, for example, International Patent Application Publication 2014 / 055668 and U.S. Patent No. 7,446,190.

[0198] In some embodiments, one or more additional nucleic acids can be introduced into cells simultaneously or sequentially with the nucleic acids encoding the anti-NPM1c TCR or its antigen-binding fragment provided herein. In some embodiments, such additional nucleic acids for introduction may improve therapeutic efficacy by, for example, enhancing the viability and / or function of the introduced cells, provide a genetic marker for cell selection and / or evaluation, for example, for assessing survival or localization in vivo, and / or improve safety by making cells sensitive to negative selection in vivo, as described (Lupton SD et al., Mol. and Cell Biol., 11:6 (1991), Riddell et al., Human Gene Therapy 3:319-338 (1992)), international applications PCT / US91 / 08442 and PCT / US94 / 05601, and U.S. Patent No. 6,040,177, describing the use of a bifunctional selectable fusion gene by fusing a dominant positive selectable marker with a negative selectable marker).

[0199] Accordingly, in some embodiments, engineered cells are provided, such as those containing the anti-NPM1c TCR or its antigen-binding fragment, nucleic acid, or vector as described herein. In some embodiments, the cells are produced by transduction of the vector described herein into cells in vitro or ex vivo. In some embodiments, the cells are T cells, such as CD8+ or CD4+ T cells. In some embodiments, the anti-NPM1c TCR is heterogeneous to the cells.

[0200] V. Treatment and prevention methods and use This specification also provides anti-NPM1c TCRs and their antigen-binding fragments provided herein, as well as methods for administering engineered cells expressing anti-NPM1c TCRs or their antigen-binding fragments, and uses such as therapeutic and prophylactic uses. Such methods and uses include, for example, NPM1c + The present invention includes methods and uses of treatment involving the administration of molecules, cells, or compositions containing them to subjects having cancer or a condition. In some embodiments, the molecules, cells, and / or compositions are NPM1c + It is administered in an effective amount to bring about the treatment of cancer or a condition. Uses include the use of anti-NPM1c TCRs and engineered T cells in such methods and treatments, and in the preparation of pharmaceuticals for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering anti-NPM1c TCRs or engineered T cells, or compositions containing them, to a subject who has, has had, or is suspected of having NPM1c+ cancer or a condition. In some embodiments, the method thereby treats NPM1c+ cancer or a condition or condition in the subject.

[0201] The described anti-AIQDLCLAV TCR, BiTE, and CAR, as well as engineered cells expressing anti-AIQDLCLAV TCR, BiTE, and CAR, may be used in the treatment of cancer. Methods for treating cancer in a subject are also described, including administering to a subject any of the cells, such as T cells, expressing any of the described anti-AIQDLCLAV TCR, BiTE, and CAR (engineered anti-AIQDLCLAV T cells). Engineered anti-AIQDLCLAV TCR-expressing T cells may be administered to a subject to induce an immune response, increase T cell infiltration of tumors, reduce or inhibit cancer cell proliferation, reduce or inhibit tumor growth, reduce tumor progression, reduce tumor burden, inhibit or reduce metastasis, reduce or inhibit the development of metastatic cancer, or increase the subject's survival or extend its lifespan.

[0202] The described anti-CLAVEEVSL TCRs, BiTEs, and CARs, as well as engineered cells expressing anti-CLAVEEVSL TCRs, BiTEs, and CARs, may be used in the treatment of cancer. Methods for treating cancer in a subject are also described, including administering to a subject any of the cells, such as T cells, expressing any of the described anti-CLAVEEVSL TCRs, BiTEs, and CARs (engineered anti-CLAVEEVSL T cells). Engineered anti-CLAVEEVSL TCR-expressing T cells may be administered to a subject to induce an immune response, increase T cell infiltration of tumors, reduce or inhibit cancer cell proliferation, reduce or inhibit tumor growth, reduce tumor progression, reduce tumor burden, inhibit or reduce metastasis, reduce or inhibit the development of metastatic cancer, increase the subject's survival or extend its lifespan, or reduce or attenuate an autoimmune response.

[0203] The described anti-NPM1c TCRs and T cells expressing the described anti-NPM1c TCRs may be used to kill or eliminate NPM1c-expressing cancer cells. The killing or elimination of NPM1c-expressing cancer cells in the subject is due to NPM1c + It can be used in the treatment of cancer. In some embodiments, the engineered T cells expressing the anti-NPM1c TCR are engineered autologous T cells (i.e., the T cells originate from the subject being treated).

[0204] Among the diseases treated is cancer. In some embodiments, the disease or condition treated is a humoral tumor. In some embodiments, the disease or condition treated is a hematopoietic tumor. In some embodiments, the disease or condition treated is NPM1c + Leukemia, NPM1c + Acute myeloid leukemia (AML), or NPM1c +The condition is myelodysplastic syndrome (MDS). In some embodiments, the disease state is AML. AML can be diagnosed according to the European LeukemiaNet (ELN) criteria (e.g., Dohner et al., 2022).

[0205] In some embodiments, the described anti-NPM1c TCR may be isolated as a soluble anti-NPM1c TCR and administered to a subject. In some embodiments, any of the described anti-NPM1c TCRs may be isolated and conjugated to a molecule such as a therapeutic molecule or antibody to form an anti-NPM1c TCR-drug conjugate. The conjugated anti-NPM1c TCR may then be administered to a subject. In some embodiments, the therapeutic molecule is a therapeutic molecule that targets AML. Therapeutic molecules that target AML include, but are not limited to, menin-MLL inhibitors. In some embodiments, the therapeutic molecule is a therapeutic molecule anticancer drug. The anticancer drug may be, but is not limited to, Pseudomonas aeruginosa exotoxin, lysine, monomethyl auristatin F, and calitiamycin. In some embodiments, the anti-NPM1c TCR is used as a substitute for the antibody in the antibody-drug conjugate.

[0206] In some embodiments, engineered T cells expressing any of the described anti-NPM1c TCRs are NPM1c + It is administered to a subject for the treatment of cancer, such as AML. In some embodiments, engineered T cells expressing any of the described anti-NPM1c TCRs may be formulated for use as T cell therapy for the treatment of NPM1c+ cancer, such as AML. In some embodiments, the T cell therapy is autologous T cell therapy.

[0207] In some embodiments, either the described anti-NPM1c TCR or engineered cells expressing anti-NPM1c may be used or formulated for use in combination with one or more AML therapies (e.g., menin-MLL inhibitors). One or more AML therapies may be administered to the subject before, concurrently with, or after administration of the anti-NPM1c TCR or engineered cells expressing anti-NPM1c.

[0208] In some embodiments, engineered T cells expressing the described anti-NPM1c TCR and / or the described TCR are used in NPM1c + It is administered to the subject to kill cells. The subject may or may not have received SCT. The subject may or may not be scheduled to receive SCT. The subject may or may not be eligible for SCT.

[0209] In some embodiments, the subject has received one or more prior treatments for cancer. In some embodiments, the subject has relapsed after one or more prior treatments for cancer. In some embodiments, the subject has relapsed after one or more induction therapies. In some embodiments, the subject has relapsed after at least two prior treatments for cancer. In some embodiments, the subject is refractory to one or more prior treatments for cancer. In some embodiments, the subject is refractory to one or more induction therapies. In some embodiments, the subject is refractory to at least two prior treatments for cancer. In some embodiments, the subject has persistent disease after one or more prior treatments for cancer. In some embodiments, the subject has persistent disease after one or more induction therapies for cancer. One or more prior treatments or induction therapies may be, but are not limited to, chemotherapy, azacitidine therapy, FLT3 inhibitor therapy, IDH inhibitor therapy, BCL-2 inhibitor therapy, Hedgehog pathway inhibitor therapy, radiotherapy, aSCT, alloSCT, or menin-MLL inhibitor therapy. In some embodiments, subjects have relapsed, are refractory, or have persistent disease after one or more consolidation therapies. In some embodiments, subjects have relapsed after SCT or alloSCT but have not previously received T-cell therapy.

[0210] In some embodiments, subjects with the NPM1c mutation are FLT3-positive. In some embodiments, subjects with the NPM1c mutation are FLT3-positive, and the manipulated T cells are allogeneic T cells (i.e., T cells derived from an HLA-matched donor). In some embodiments, subjects with the NPM1c mutation are FLT3-positive, the manipulated T cells are allogeneic T cells, and the manipulated T cells are administered in combination with alloSCT.

[0211] In some embodiments, the subject is NPM1c + Patients with cancer who are ineligible for transplantation (e.g., alloSCT with standard treatment). In some embodiments, the subject is NPM1c +The subjects have cancer (e.g., AML) and one or more comorbidities or risk factors. Comorbidities or risk factors include, but are not limited to, advanced age, lung disease, liver disease, renal failure, heart failure, infections, and the risk of infection. In some embodiments, the subjects are 60 years of age or older, 65 years of age or older, or 70 years of age or older.

[0212] In some embodiments, the subject is NPM1c + The subjects have cancer (e.g., AML) and are ineligible for high-intensity chemotherapy. In some embodiments, the subjects have relapsed after high-intensity chemotherapy with or without hematopoietic stem cell transplantation (HSCT). In some embodiments, the subjects did not respond to high-intensity chemotherapy, were unresponsive, or failed to achieve complete remission (refractory). In some embodiments, the subjects are eligible for reduced-intensity chemotherapy. Reduced-intensity chemotherapy can be administered prior to the administration of engineered T cells.

[0213] In some embodiments, subjects are considered eligible for high-intensity treatment but are at moderate or unfavorable risk and have not responded to previous high-intensity chemotherapy or have subsequently relapsed.

[0214] In some embodiments, the subjects had relapsed after prior hypomethylating agent and / or BCL-2 inhibitor therapy. In some embodiments, the subjects were unresponsive to hypomethylating agent therapy, were unresponsive, or failed to achieve complete remission (refractory).

[0215] In some embodiments, subjects have relapsed after stem cell transplantation. In some embodiments, subjects do not respond to stem cell transplantation. In some embodiments, subjects eligible for treatment with T-cell therapy using one of the described anti-NPM1c TCRs have not undergone stem cell transplantation. In some embodiments, subjects eligible for treatment with T-cell therapy using one of the described anti-NPM1c TCRs have not undergone stem cell transplantation within two months of receiving T-cell therapy.

[0216] In some embodiments, NPM1c + Methods for treating subjects having or diagnosed with cancer are described, the methods comprising administering to a subject any of the anti-NPM1c TCR or its antigen-binding fragments provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the engineered cells provided herein, or any of the compositions provided herein, in combination with stem cell transplantation (SCT). In some embodiments, NPM1c + A method for treating a subject having or diagnosed with cancer is described, the method comprising administering to the subject any of the anti-NPM1c TCR or its antigen-binding fragments provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the engineered cells provided herein, or any of the compositions provided herein, before performing SCT on the subject. In some embodiments, any of the anti-NPM1c TCR or its antigen-binding fragments provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the engineered cells provided herein, or any of the compositions provided herein is NPM1c + Anti-NPM1c TCRs are formulated for use in combination with stem cell transplantation for the treatment of subjects with or diagnosed with cancer. Anti-NPM1c TCRs, their antigen-binding fragments, polynucleotides, vectors, engineered cells, or compositions can be formulated for administration to a subject in combination with or prior to an SCT. The SCT may be an autologous SCT (aSCT) or an allogeneic SCT (alloSCT). In some embodiments, anti-NPM1c TCRs are administered as autologous T-cell therapy. + Cancer can be, but is not limited to, AML.

[0217] In some embodiments, NPM1c +Described herein is a method of treating a subject having or diagnosed with cancer, the method comprising administering to the subject any of the anti-NPM1c TCR or antigen-binding fragment thereof provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the engineered cells provided herein, or any of the compositions provided herein. In some embodiments, the method of treatment is not combined with SCT. In some embodiments, any of the anti-NPM1c TCR or antigen-binding fragment thereof provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the engineered cells provided herein, or any of the compositions provided herein is NPM1c + can be formulated for use in the absence of stem cell transplantation for the treatment of a subject having or diagnosed with cancer. In some embodiments, the anti-NPM1c TCR is administered as an autologous T cell therapy. NPM1c + The cancer can be, but is not limited to, AML.

[0218] In some embodiments, the subject has NPM1c + leukemia. In some embodiments, the subject has NPM1c + acute myeloid (myelocytic) leukemia (AML). In some embodiments, the subject has NPM1c with myelodysplastic syndrome + .

[0219] In some embodiments, the subject has not received any prior treatment. In some embodiments, the subject has NPM1c +The subject has leukemia and, as a first therapeutic measure, is administered the described anti-NPM1c-TCR, anti-NPM1c-TCR BiTE, anti-NPM1c-drug conjugate, or engineered T cells expressing anti-NPM1c-TCR. In some embodiments, the subject has received one or more prior therapies to treat the cancer. In some embodiments, the subject has relapsed after one or more prior therapies to treat the cancer. In some embodiments, the subject has relapsed after one or more induction therapies. In some embodiments, the subject is refractory to one or more prior therapies to treat the cancer. In some embodiments, the subject is refractory to one or more induction therapies. In some embodiments, the subject has persistent disease after one or more prior therapies to treat the cancer. In some embodiments, the subject has persistent disease after one or more induction therapies to treat the cancer. One or more prior therapies or induction therapies may be, but are not limited to, chemotherapy, azacitidine therapy, FLT3 inhibitor therapy, IDH inhibitor therapy, BCL-1 inhibitor therapy, Hedgehog pathway inhibitor therapy, radiotherapy, aSCT, or alloSCT. In some embodiments, the subject has relapsed, is refractory, or has persistent disease after one or more consolidation therapies. In some embodiments, the subject has relapsed after SCT or alloSCT but has not previously received T-cell therapy.

[0220] In some embodiments, the subject is NPM1c + Cancer (for example, NPM1c) + The patient has AML (Acute Myelopathy), is undergoing induction therapy, and is in remission.

[0221] In some embodiments, the subject is NPM1c + Cancer (for example, NPM1c) + The patient has been diagnosed with AML and is undergoing induction therapy, and the provided anti-NPM1c TCR, its antigen-binding fragment, polynucleotide, vector, engineered T cells, or compositions thereof are administered to the patient as consolidation therapy.

[0222] In some embodiments, the subject has an NPM1c+ cancer and has an Flt3 ITD mutation. In some embodiments, the subject has NPM1c + cancer with an Flt3 ITD mutation, and the provided anti-NPM1c TCR, antigen-binding fragment thereof, polynucleotide, vector, engineered T cell, or composition thereof is administered to the subject to reduce or eliminate the need for SCT.

[0223] In some embodiments, the subject has NPM1c + cancer and does not have an Flt3 ITD mutation. In some embodiments, the subject has NPM1c + cancer and does not have an Flt3 ITD mutation, and the provided anti-NPM1c TCR, antigen-binding fragment thereof, polynucleotide, vector, engineered T cell, or composition thereof is administered to the subject to reduce the risk or recurrence.

[0224] In some embodiments, a subject diagnosed with or suspected of having leukemia undergoes genetic testing for the NPM1 gene. NPM1 genetic testing can be performed on a sample obtained from bone marrow or peripheral blood. NPM1 genetic testing may be any test known in the art for detecting the presence of an NPM1c mutation, or for detecting a type A or type D NPM1 mutation. In some embodiments, NPM1 genetic testing comprises PCR testing or next-generation sequencing (NGS). If genetic testing indicates that the subject has NPM1c+ cancer, the subject can be treated with engineered T cells expressing any of the described anti-NPM1c TCRs.

[0225] In some embodiments, subjects who test positive for NPM1c+ cancer are screened to assess their eligibility or suitability for engineered T-cell therapy. Screening may include, but is not limited to, NPM1 testing, organ function testing (e.g., liver and kidney function), the subject's performance status, and the presence of adverse events from previous treatments. In some embodiments, screening is performed up to approximately 45 days before the expected administration of engineered T-cell therapy.

[0226] Leukocyte apheresis is performed on the subject to collect leukocytes. In some embodiments, CD8 + T cells are obtained from leukocyte apheresis products via positive selection. In some embodiments, CD8 + T cells are activated using an activator. Approximately 24 hours after activation, the T cells are manipulated to knock out the endogenous TCR. Knockout of the endogenous TCR can be performed using CRISPR. The nucleic acid sequences encoding any of the described anti-NPM1c TCRs are CD8 + The T cells are inserted. Insertion of the nucleic acid sequence encoding the anti-NPM1c TCR can be carried out using any method available in the art for inserting a transgene into T cells. Such methods include, but are not limited to, lentiviral vector transduction. The engineered T cells are then grown to obtain a sufficient number for therapy. Leukocyte apheresis can be performed approximately 21 to 45 days before the expected administration of engineered T cell therapy to the subject.

[0227] Prior to T-cell therapy, subjects are treated to induce lymphocyte depletion to enable the engraftment of the engineered T cells (e.g., TCR-T cells) to be injected. Lymphocyte depletion can be performed using any method available in the field for lymphocyte depletion. Preferred lymphocyte depletion regimens include, but are not limited to, cyclophosphamide treatment and fludarabine treatment. Lymphocyte depletion can be performed approximately 1 to 7 days before the expected administration of engineered T-cell therapy to the subject.

[0228] For T-cell therapy, engineered T cells are infused at a predetermined dose. The dose is not limited, but is approximately 0.3 × 10⁻⁶. 6 cells ~ approx. 1 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 1 x 10 7 Cells, or approximately 3 × 10 6 cells ~ approx. 1 x 10 7 The dose can be in units of cells. In some embodiments, the dose is about 0.3 × 10⁻⁶. 6 cells, approximately 0.4 x 10 6 cells, approximately 0.5 x 10 6 cells, approximately 0.6 x 10 6 cells, approximately 0.7 x 10 6 cells, approximately 0.8 x 10 6 cells, approximately 0.9 x 10 6 cells, approximately 1 x 10 6 cells, approximately 2 x 10 6 cells, approximately 3 x 10 6 cells, approximately 4 x 10 6 cells, approximately 5 x 10 6 cells, approximately 6 x 10 6 cells, approximately 7 x 10 6 cells, approximately 8 x 10 6 cells, approximately 9 x 10 6 Cells, or approximately 1 × 10⁻⁶ 7 It is a cell.

[0229] The terms “to treat” and “treatment” mean methods or processes taken to reduce, improve or alleviate one or more symptoms or pathological outcomes of a disease, disorder, or condition in a subject. Treatment may be preventive in that it prevents or partially prevents the disease, or the symptoms or conditions of the disease. Prevention includes providing prevention with respect to the onset or recurrence of the disease in a subject who is susceptible to the disease but has not yet been diagnosed with the disease. Prevention also includes providing prevention with respect to the onset or recurrence of the symptoms or pathological outcomes of the disease in a subject who is susceptible to the symptoms or pathological outcomes of the disease but has not yet been diagnosed with the symptoms or pathological outcomes of the disease. Treatment may also be preventive in that it delays the onset of the disease or the symptoms or conditions of the disease. Delay in the onset of the disease or the symptoms or pathological outcomes of the disease indicates postponement, interference, slowing, delay, stabilization, suppression, and / or deferral of the onset of the disease or the symptoms or pathological outcomes of the disease. The delay may be of varying lengths, depending on the disease history and / or the individual being treated. Treatment may include inhibiting a disease, disorder, or condition, e.g., preventing its progression, and reducing a disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treatment may also mean an extended survival period compared to the survival period expected without treatment. Treatment may be therapeutic in relation to the partial or complete cure of a disease, condition, symptom, or adverse effects resulting from a disease, disorder, or condition. The term treatment includes (a) prevention of the onset of a disease in a person who is susceptible to the disease but has not yet been diagnosed with it, (b) inhibition of the disease, i.e., cessation of its onset, and (c) reduction of the disease, i.e., relief or improvement of the disease and / or its symptoms or condition. Treatment may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those who require treatment (those who require it) may include those who have been previously diagnosed with a disease, disorder, or condition, or those who have been identified as being at risk of developing a disease, disorder, or condition.Treating a disease, disorder, or condition may include improving at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology remains unaffected.

[0230] The “effective dose” of a drug, such as a pharmaceutical preparation, TCR, cell, or composition, means the amount / quantity and duration of administration that is effective in order to achieve a desired outcome, such as a therapeutic or prophylactic result.

[0231] The “therapeutic effective dose” of a drug, such as a pharmaceutical preparation, TCR, or cells, refers to the amount effective in the dose and duration necessary to achieve the desired therapeutic outcome, such as the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. The therapeutic effective dose may vary depending on factors such as the disease condition of the subject, age, sex, and weight, as well as the population of cells being administered. In some embodiments, the methods provided involve administering the TCR, cells, and / or composition in an effective dose, e.g., a therapeutic effective dose.

[0232] As used herein, “subject” means a mammal (e.g., human or other animal), and is typically human.

[0233] VI.Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or jargon used herein are intended to have the same meaning as those generally understood by those skilled in the art to which the claimed subject matter pertains. In some embodiments, terms having a generally understood meaning are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those generally understood in the art.

[0234] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the T cell receptor, its antigen-binding fragment, and other peptides such as linkers, may contain amino acid residues, including native and / or non-native amino acid residues. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications to their native or native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as those resulting from site-directed mutagenesis, or accidental, such as those resulting from mutations in the host producing the protein or errors resulting from PCR amplification.

[0235] "Isolated" nucleic acids refer to nucleic acid molecules separated from components in their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells that contain nucleic acid molecules, but the nucleic acid molecules are located outside or at chromosomal locations different from their natural chromosomal locations.

[0236] "An isolated nucleic acid molecule encoding a TCR" refers to a single nucleic acid molecule (e.g., a single vector) that encodes a TCR, such as a functional α / β TCR or a functional γ / δ TCR.

[0237] "An isolated nucleic acid molecule encoding the antigen-binding fragment of a TCR" refers to a single nucleic acid molecule (e.g., a single vector) that encodes the antigen-binding fragment of a TCR.

[0238] "Isolated nucleic acid molecules encoding a TCR" refers to two or more distinct nucleic acid molecules (e.g., two or more vectors) that together encode a TCR, such as a functional α / β TCR or a functional γ / δ TCR. Each of these two or more nucleic acid molecules may be located at a different position within the host cell.

[0239] "Isolated nucleic acid molecules encoding the antigen-binding fragment of the TCR" refers to two or more nucleic acid molecules (e.g., two or more vectors) that together encode the antigen-binding fragment of the TCR. Each of these two or more nucleic acid molecules may be located at a different position within the host cell.

[0240] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, although they may not be completely identical to the parent cells in terms of nucleic acid content. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0241] As used herein, "percent (%) amino acid sequence identity" and "percent identity" are defined, when used in relation to an amino acid sequence (reference polypeptide sequence), as the percentage of amino acid residues in a candidate sequence (e.g., a target T cell receptor or fragment) that are identical to the amino acid residues in the reference polypeptide sequence after the sequences have been aligned, gaps introduced as necessary to achieve maximum percent sequence identity, and no conservative substitutions have been considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, for example. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0242] Amino acid substitutions may include replacing one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into the target TCR or its antigen-binding fragment, and the product may be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved cytolytic activity.

[0243] Amino acids can generally be classified according to the following common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0244] In some embodiments, a conservative substitution may involve the exchange of one member of these classes for another member of the same class. In some embodiments, a non-conservative amino acid substitution may involve the exchange of one member of these classes for another class.

[0245] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are operably ligated. Such vectors are referred herein as “expression vectors.”

[0246] As used herein, the singular “one” (“a,” “an,” and “the”) also includes multiple references unless otherwise indicated. For example, “a” or “an” means “at least one” or “one or more.” The embodiments and variations described herein are understood to include “consisting of” and / or “essentially consisting of.”

[0247] Throughout this disclosure, various aspects of the claimed subject matter are presented in scope form. It should be understood that the scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, the scope should be considered to encompass all specifically disclosed possible sub-scopes and the individual numerical values ​​within those scopes. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range, and any other stated values ​​or intermediate values ​​within that stated range, are included within the claimed subject matter. These smaller upper and lower limits may be independently included within the smaller range and are also included within the claimed subject matter, subject to any specifically excluded limits within the stated range. If the stated range includes one or both of the limit values, the range excluding either one or both of those limit values ​​is also included within the claimed subject matter. This applies regardless of the width of the range.

[0248] As used herein, the terms “about” and “approximately” indicate a reasonable deviation from the numerical value and from values ​​known to those skilled in the art when used to modify a quantity specified by a number or range. In some embodiments, the term “about” means within a typical acceptable range in the art. In some embodiments, the term “about” means within one or two standard deviations from the mean. In some embodiments, the term “about” means ±10%. In some embodiments, the term “about” means ±5%. When the term “about” precedes a series of numbers or a range, it is understood that “about” may modify each of the numbers in that series or range.

[0249] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. This may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0250] As used herein, the statement that a cell or cell population is “positive” for a particular marker means the detectable presence of a particular marker, typically a surface marker, on or within the cell. When referring to a surface marker, the term means the presence of surface expression, which can be detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the stain is detectable by flow cytometry at a level substantially higher than the stain detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that for cells known to be positive for the marker, and / or at a level substantially higher than that for cells known to be negative for the marker.

[0251] As used herein, the statement that a cell or population of cells is “negative” for a particular marker means the absence of substantial detectable presence of the particular marker, typically a surface marker, on or within the cell. When referring to a surface marker, the term means the absence of surface expression that can be detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the stain is not detectable by flow cytometry at a level substantially higher than the stain detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cells known to be positive for the marker, and / or at a level substantially the same as that for cells known to be negative for the marker.

[0252] When referring to a binding region (e.g., a TCR binding region), the term "specifically binds" to the target refers to a binding reaction in which the binding region binds to the target with higher affinity, higher avidity, and / or longer duration than when binding to structurally different targets. When assayed under the same affinity assay conditions, a binding region may have at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more affinity to a particular target compared to an unrelated target. [Examples]

[0253] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. CDR3β

[0254] Example 1. Exemplary identified TCR. Table 4 lists the sequence identifiers (SEQ ID NOs) for the amino acid (aa) or nucleotide (nt) sequences of isolated, evaluated, and sequenced AIQDLCLAV (SEQ ID NO: 1)-specific TCRs and CLAVEEVSL (SEQ ID NO: 5)-specific TCRs (i.e., anti-AIQDLCLAV TCRs and anti-CLAVEEVSL TCRs). The table also lists the sequence identifiers (SEQ ID NOs) corresponding to exemplary full lengths, including amino acid sequences containing the α-chain and β-chain sequences of each TCR, isolated by a sequence encoding a constant domain, a ribosome skipped P2A sequence (the P2A linker described in SEQ ID NO: 301, encoded by the nucleotide described in SEQ ID NO: 302) (named "α-P2A-β").

[0255] [Table 4]

[0256] Example 2. Characteristics of an exemplary AIQDLCLAV (SEQ ID NO: 1)-specific TCR. As described herein, AIQDLCLAV (SEQ ID NO: 1) and / or CLAVEEVSL (SEQ ID NO: 5) are expressed on cancer cells (e.g., AML cells) but not on non-cancerous cells. For this reason, T cells targeting AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) are less likely to cause GVHD.

[0257] Example 3. Exemplary binding specificity of NPM1-specific TCRs The binding specificity of AIQDLCLAV (SEQ ID NO: 1) and CLAVEEVSL (SEQ ID NO: 5) specific TCRs was determined using tetramers complexed with AIQDLCLAV (SEQ ID NO: 1), CLAVEEVSL (SEQ ID NO: 5), or a control peptide.

[0258] HEK293 suspension cells were engineered to express human CD3γ, CD3δ, CD3ε, CD3ζ (poly-CD3), and human CD8α / β. CD3 and CD8 were cloned from pooled PBMCs derived from two healthy blood donors and stably transduced into HEK293. The HEK293-CD3-CD8 suspension cells were transiently transfected with plasmid DNA containing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR and the fluorescent protein mCherry as a control for transfection efficiency.

[0259] Twenty-four hours after transfection, cells were stained with an amine-reactive survival dye (violet 510 Ghost dye), anti-CD3, and immunogenic AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptide tetramer or a control peptide tetramer. Cells were collected and analyzed by flow cytometry.

[0260] The dual fluorescence positivity of CD3 and the specific binding of HLA tetramers complexed with AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptides indicates a functionally reactive TCR.

[0261] Example 4. In vitro evaluation of the reactivity of manipulated T cells to AIQDLCLAV (SEQ ID NO: 1). The isolated and identified TCRs, as described in Examples 1-3 above, were recombinantly expressed in Jurkat cell lines manipulated to stably express CD8, and EC 50 Further characteristics and evaluations of the functions, including decision-making, were identified and evaluated. Cytokine secretion, T cell activation, and binding specificity can also be determined for each TCR.

[0262] For exemplary AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs, Jurkat T cells expressing anti-AIQDLCLAV TCRs or anti-CLAVEEVSL TCRs were co-cultured with K562 cells pulsed with AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptide or a control peptide. The same number of Jurkat T cells and K562 cells loaded with an increased concentration (e.g., 0.1–31.6 ng / mL) of either the AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptide or the control peptide were co-cultured for 16 hours. Jurkat CD69 expression was determined by FACS.

[0263] A. Evaluation of the early activation marker CD69 Jurkat T cells expressing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR were co-cultured with APCs loaded with AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptides, and the expression of CD69, a marker of T cell activation and function, was evaluated.

[0264] Jurkat T cells expressing various anti-AIQDLCLAV TCRs or anti-CLAVEEVSL TCRs are prepared as described. Jurkat T cells transduced with anti-AIQDLCLAV TCRs or anti-CLAVEEVSL TCRs are subjected to a 1:1 E / T ratio while increasing the concentration of AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptides. *02:01 Incubated overnight with LCL. EC 50 The value is CD69 + The calculations were based on XLFit (ID Business Solutions) on a plot of cell percentage (y-axis) versus peptide concentration (logarithmic x-axis).

[0265] For exemplary TCRs, anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR-expressing T cells were co-cultured in two pairs with peptide-loaded K562 cells in a 1:1 effector-to-target ratio. After 16–24 hours of culture, cells were washed, stained with Ghost Dye, anti-CD3, and anti-CD69, and evaluated by flow cytometry. Data were analyzed by XLfit. The estimated EC values ​​for each of the five experiments were... 50 This was determined by evaluating the ratio of CD69-positive Jurkat cells to total viable cells.

[0266] Evaluation of BT cell receptor specificity The specificity of candidate anti-AIQDLCLAV or anti-CLAVEEVSL TCRs to the AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) peptides will also be evaluated, relative to the corresponding wild-type NPM1 peptide presented by restriction HLA, or potentially other HLA molecules, or other HLA molecules that present other peptides.

[0267] 2. Alloreactivity HLA-A loaded with unrelated peptides (such as HPV peptides) * 02:01 Alloreactivity was tested using a tetramer.

[0268] Example 5. Target cell death by primary T cells transduced via AIQDLCLAV (SEQ ID NO: 1)-specific TCR. To determine the potential for inducing graft-versus-host leukemia (GvL) effects, we evaluated the anti-leukemia target cell death activity of primary T cells transduced with anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR.

[0269] A. Expression of AIQDLCLAV (SEQ ID NO: 1)-specific or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs in primary cells First Generation Human CD8 + T cells were enriched from PBMCs by negative selection. CD8 + The endogenous TCR of T cells was knocked out using CRISPR. Subsequently, CD8 + T cells were transduced with a lentiviral vector (pLVG.M containing the MNDU3 promoter) encoding either an anti-AIQDLCLAV TCR or an anti-CLAVEEVSL TCR and an RQR8 tag. The percentage of transduced T cells was evaluated on day 5 post-transduction by flow cytometry using an anti-CD34 antibody to assess RQR8 expression.

[0270] B. Target cell death The target cell death specificity of anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR was evaluated. HLA-A * 02:01 + / AIQDLCLAV + (Sequence ID 1) OCI-AML2 cells or HLA-A * 02:01 + / CLAVEEVSL + (SEQ ID NO: 5) OCI-AML2 cells were used as APCs. OCI-AML2 cells were stained with Violet Proliferation Dye 450 (VPD450). VPD450 labels target cells by binding the dye to intracellular proteins, indicating T cell-mediated target cell death. Apoptosis of labeled cells results in the loss of their detection in the live cell gate in flow cytometry. Primary human T cells expressing anti-AIQDLCLAV TCR or CLAVEEVSL TCR were started with an E:T ratio of 10:1 and terminated with a 0:1 E:T ratio (transduced CD34). + Cells (positive cells) were started with serial dilutions and co-cultured with LCL for 18 hours. Cells were stained with 7-AAD (to assess cell viability), acquired, and evaluated by flow cytometry.

[0271] Example 6. Target cell death activity The target cell death activity of exemplary TCRs was further evaluated using different effector:target (E:T) ratios.

[0272] OCI-AML3 cells were genotyped based on AIQDLCLAV (SEQ ID NO: 1) and / or CLAVEEVSL (SEQ ID NO: 5) expression. To distinguish between cell populations, OCI-AML3 cells displaying the AIQDLCLAV (SEQ ID NO: 1) and / or CLAVEEVSL (SEQ ID NO: 5) peptides were labeled with a high concentration of VPD450 (0.5 μM), while OCI-AML2 cells displaying the corresponding wild-type NPM1 peptide were labeled with a low concentration (0.025 μM) of VPD450. OCI-AML2 and OCI-AML3 cells were stained with VPD450 at 37°C for 15 minutes. OCI-AML2 and OCI-AML3 cells containing the target (AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)) and control peptides were mixed in a 1:1 ratio and incubated while increasing the number of transduced primary T cells expressing AIQDLCLAV (SEQ ID NO: 1)-specific or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs. Furthermore, the target OCI-AML2 or OCI-AML3 cells were stained and individually incubated with TCR-T cells in a 1:1 ratio. Subsequently, the E:T ratio EC50 death curves could be determined.

[0273] OCI-AML2 and OCI-AML3 cells were co-cultured for 16 hours while increasing the number of primary T cells (E:T ratio 1:16 to 10:1). The cell mixture was stained with LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific), anti-CD8, and anti-CD19, and two different target cell populations were evaluated by VPD450 staining levels. Cells were acquired and analyzed by flow cytometry to evaluate CD8- cells (to exclude effector cells) and VPD450 high vs. VPD450 low populations.

[0274] After incubation with non-transduced T cells or transduced T cells with anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR, the number of viable OCI-AML2 and OCI-AML3 cells presenting either the target or control peptide was determined. As the effector-to-target (E:T) ratio increased, simultaneous loss of viable cells presenting the target peptide (high VPD450 staining) and retention of viable cells presenting the control peptide (low VPD450 staining) was observed, indicating selective death of target cells presenting AIQDLCLAV (SEQ ID NO: 1) and / or CLAVEEVSL (SEQ ID NO: 5) by T cells genetically engineered to express AIQDLCLAV (SEQ ID NO: 1)-specific or CLAVEEVSL (SEQ ID NO: 5)-specific TCRs.

[0275] Example 7. In silico prediction of HLA binding of AIQDLCLAV (SEQ ID NO: 1) peptide. The binding of AIQDLCLAV, IQDLCLAV, and AIQDLCLA to HLA was predicted using the ANN 4.0 option on the IEDB (see below). Predicted IC for AIQDLCLAV binding to HLA-A alleles. 50 The values ​​are shown in Tables 6a-d. Based on the predicted binding of AIQDLCLAV, IQDLCLAV, and AIQDLCLA, HLA-AHLA-A * 02:06, HLA-A * 02:03, HLA-A * Subjects with 02:01 are expected to be suitable candidates.

[0276] [Table 5]

[0277] [Table 6-1]

[0278] [Table 6-2]

[0279] [Table 6-3]

[0280] [Table 6-4]

[0281] Example 8. TCR-induced signaling in manipulated T cells TCR D, HLA * The ability of the A02:01-presented target peptide (CLAVEEVSL, SEQ ID NO: 5) to induce T cell signaling was evaluated. Briefly, Jurkat cells were manipulated to express α-TCR D. K562 HLA * A02:01-expressing cells were peptide pulsed with a target peptide, and then co-cultured to evaluate TCR signaling. CD69 expression on Jurkat cells was evaluated as a surrogate marker for T cell activation induced by the target peptide.

[0282] Jurkat and K562 cell lines were cultured in RPMI and IMDM, respectively. Peptides were reconstituted and diluted in PBS prior to peptide pulses.

[0283] CD8 + Jurkat cells were transduced with a lentivirus using TCR D, and purified by FACS-based sorting on TCR-positive cells (Jurkat-TCR D cells). K562 was generated by pan HLA class I knockout, and HLA * The virus was transduced using A02:01 and purified by FACS sorting prior to the reporter assay.

[0284] For the reporter assay, engineered K562 cells were peptide pulsed with the target peptide at a titration concentration for 1 hour and then co-cultured. Jurkat-TCR D cells were co-cultured with peptide-pulsed K562 cells in a 96-well plate at a 1:1 E:T ratio. After 16-18 hours, the co-cultured cells were stained with fluorescently tagged antibodies for CD3 (a Jurkat-specific marker), CD34 (a marker for Jurkat cells transduced with the target TCR), and CD69 (to evaluate Jurkat-TCR D activation after co-culture). Cells were also stained using live / dead staining and analyzed using NovacyteQuanteon. EC of peptide-induced Jurkat activation 50 This was determined based on the percentage of Jurkat CD69-positive cells.

[0285] Peptide-mediated Jurkat-TCR D activation, as measured by CD69 expression, is dose-responsive, and EC 50 The concentration was 45.54 ng / mL (Figure 1). The results demonstrate that TCR D can induce TCR signaling and, when manipulated in primary cells, has the potential to induce CLAVEEVSL-specific cytotoxic signaling.

[0286] Example 9. TCR-mediated toxicity to target cells. Anti-CLAVEEVSL TCR, HLA expressing NPM1c mutation * A02:01 The cytotoxicity against AML cell lines (OCI-AML3) was evaluated. In short, HLA * A02:01 First CD8 from a negative healthy donor + T cells were manipulated to knock out the endogenous TCRα and β constant chain domains. Subsequently, CD8 + TCR was transduced into the cells. OCI-AML2 cells (HLA * A02:01 + ) is peptide pulsed with the target peptide, and then the manipulated primary CD8 + TCR cytotoxicity was evaluated by co-culturing with T cells.

[0287] Frozen PBMCs were stimulated with a fever-inducing CD3 antibody, and activated CD8 T cells were purified. Subsequently, the cells were transduced using CRISPR to delete the endogenous TCR, and TCR D, TCR E, or TCR F were transduced using a lentivirus. Primary cells were grown for 10 days in CTS medium containing a serum replacement optimizer supplemented with 50 U IL-2 and 10 ng / mL IL-7. After 10 days, the transduced T cells were purified using CD34 (transduction marker) bead selection. + TCR-T cell operation was confirmed by FACS analysis using CD3, CD8 (primary T cell-specific marker), CD34, and NPM1-specific tetramers, as well as viability staining.

[0288] For cytotoxic assays, OCI-AML2 cells were peptide pulsed with CLAVEEVSL (SEQ ID NO: 5) peptide at titration concentrations for 1 hour and then co-cultured.

[0289] Manipulated CD8 +TCR-T cells were co-cultured in 96-well plates with control or peptide-pulsed OCI-AML2 cells at a 1:1 E:T ratio, or with OCI-AML3 cells or OCI-AML2 cells (negative control cells) at various E:T ratios. For the TCR-T negative control condition, HPV-specific TCR-T cells were prepared as described above and co-cultured as described above. No cytotoxicity was observed in cells that did not express the target peptide or cells that were not pulsed with the target peptide (data not shown). After 16-18 hours, the co-cultured cells were stained with fluorescently tagged antibodies or cell dyes for CD8 (primary T cell-specific marker), VPD450 (to identify OCI-AML2 or OCI-AML3 cells), and live / dead staining. Data are presented as percentages of pulsed (containing the target peptide) OCI-AML2 cells versus unpulsed (not containing the target peptide) OCI-AML2 cells (Figure 2), or percentages of OCI-AML3 (expressing the target peptide) versus unpulsed OCI-AML3 cells (Figure 3). The results shown for TCR D indicate that TCR was effective in killing cells pulsed with the target peptide or cells that naturally express the target peptide.

[0290] Co-culture assays were performed as described above using multiple TCRs and donors. The previously published Leiden (US Patent No. 11352389) TCR was included as a control. Data are presented as the percentage of peptide-pulsed OCI-AML2 cells (sensitive to immunogenic death) compared to OCI-AML2 cells without the target peptide. EC for peptide-induced TCR cytotoxicity. 50 This was determined based on the number of target cells in peptide-pulsed OCI-AML2 cells.

[0291] Example 10.E:T's EC 50 decision Co-culture assays were performed as described above using TCR D, TCR E, and TCR F-modified T cells collected from three healthy donors, as well as three different donor cells known to express NPM1c. Leiden's previously published TCR (US Patent No. 11352389) was included as a control. Cytotoxicity was determined by the E:T ratio of the modified T cells. 50 This was determined based on the number of OCI-AML3 target cells. The data in Table 7 show that engineered T cells expressing TCR D, TCR E, or TCR F had an E:T ratio of EC, respectively. 50 We demonstrated potent cytotoxic activity against cell lines expressing NPM1c CLAVEEVSL (SEQ ID NO: 5) with a value less than 1. Furthermore, the observed cytotoxicity was not significantly different from that of publicly published engineered T cells. The demonstrated killing of OCI-AML3 cells by T cells engineered with TCR D, TCR E, and TCR F indicates mutation-specific cytotoxicity against cells expressing NPM1c mutations presented in patient AML blast cells.

[0292] [Table 7]

[0293] Example 11. Selection of CLAVEEVSL-reactive CD8+ T cell clones based on tetramers. T cell isolation was performed by stimulating T cells with activated dendritic cells. Dendritic cells were generated by stimulating purified CD14 monocytes derived from PBMCs with 10 ng / mL of LPS, IL-4, INF-γ, and GM-CSF for 4 days. The stimulated dendritic cells were pulsed with 10 ng / mL of CLAVEEVL (SEQ ID NO: 5) peptide on day 5. CD8 T cells isolated from the same donor were maintained in XVIVO-15 medium containing 5% human serum from day 1 to day 4. +The cells were co-cultured with pulsed dendritic cells. On day 5, the cells were maintained in XVIVO-15 medium supplemented with 30 ng / mL of IL-21, and then supplemented with 5 ng / mL of IL-7 and IL-15 on days 8, 10, and 12. On day 16, CD8 + T cells were stained with two 1:40 CLAVEEVSL tetramers stained for allophycocyanin (APC) and R-phycoerythrin (PE) for 1 hour at room temperature, and then FACS screening was performed for tetramer-positive T cells. After cloning and sequencing of the positive T cells, the TCRs identified by tetramer double-positive screening were further evaluated.

[0294] CD8 + Selection based on tetramers of T cell clones was performed for the AIQDCLAV peptide (SEQ ID NO: 1), as described above for the CLAVEEVSL peptide (SEQ ID NO: 5) (data not shown).

[0295] Stable transduced Jurkat cells are given CD3, CD34 and CLAVEEVSL or C * TCR / peptide specificity was confirmed by staining with LAVEEVSL tetramer (1:40).

[0296] As demonstrated by FACS plots, we were able to isolate TCRs specific to NPM1c CLAVEEVSL and AIQDCLAV neoantigen peptide tetramers. Furthermore, the isolated anti-CLAVEEVSL TCRs were found to be specific to non-cysteine ​​CLAVEEVSL and cysteine ​​C. * We were able to demonstrate that it can bind to both sides of the LAVEEVSL tetramer. *Binding to LAVEEVSL (SEQ ID NO: 5) has been reported to be important for the death of mutNPM1c target cells (van der Lee DI et al., "Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia," J Clin Invest 2019 129(2):774-785). Manipulated cells expressing TCR D showed 34.40% and 39.8% CLAVEEVSL tetramer binding (by FACS analysis) and 83.85% and 83.24% C binding in separate experiments. * The LAVEEVSL tetramer bond was shown.

[0297] Further activation, tetramer bonding, and EC50 data are shown in Tables 8 and 9.

[0298] [Table 8] * Primary CD8+ T cells co-cultured with OCI-AML3 ** Primary CD8+ T cells co-cultured with OCI-AML2 and pulsed with CLAV peptide.

[0299] [Table 9]

[0300] Example 12. Modified TCR. TCR D was modified to introduce additional disulfide bonds, reducing the likelihood of its α- or β-chain forming a chimeric TCR with the endogenous primary CD8 TCR, thereby reducing potential toxicity caused by chimeric TCRs that do not target the NPM1c neoepitope. TCR D was modified to introduce cysteine ​​at position 186 of the α-chain (T186C substitution, relative to SEQ ID NO: 71) and position 191 of the β-chain (S191C, relative to SEQ ID NO: 79). See Table 10.

[0301] Jurkat cells were transduced with either unmodified TCR D or disulfide-modified TCR D, as previously described. Disulfide-modified TCR D binding showed increased binding to the CLAVEEVSL (95.48%) tetramer compared to TCR D (88.45%), as determined by FACS analysis. Therefore, disulfide binding improves potency, likely by increasing the proportion of anti-NPM1c TCR α / β pairing in the manipulated T cells. The same improvement in potency is expected when similar cysteine ​​substitutions are made in other described TCRs.

[0302] Furthermore, TCR D was also manipulated with α and β constant regions substituted with Ig domains in the γ and δ constant regions. Jurkat cells were transduced with either unmodified TCR D or domain-exchanged TCR D, as previously described. Tetramer binding was not observed in negative control domain-exchanged TCR D.

[0303] [Table 10]

[0304] Example 13. Manipulated T cells are superior to CAR T cells. As described, modifications were made to generate an anti-NPM1c chimeric T cell receptor containing anti-AIQDCLAV scFv (Xie G et al. "CAR-T cells targeting a nucleophosmin neoepitope exhibit potent specific activity in mouse models of acute myeloid leukaemia" Nat Biomed Eng. 2021). The modifications used a P2A element instead of a T2A element and an RQR8 tag instead of an EGFR tag. These modifications are known in the art to not affect the targeting of the 5′CAR-T construct.

[0305] Manipulated T cells and CAR-T cells, CD8+ T cells were isolated, activated with TransACT, and proliferated using G-REX to produce the cells. Transduction, CRISPR editing, and cytokine replacement were performed as previously described.

[0306] The reporter assay was performed as described above to evaluate anti-AIQDCLAV CAR-T cells and engineered TCR BT cells. Target cells were pulsed with the AIQDCLAV (SEQ ID NO: 1) target peptide, as described above for the CLAVEEVSL (SEQ ID NO: 5) peptide. Furthermore, engineered TCR DT cells were evaluated in parallel with cells pulsed with the CLAVEEVSL (SEQ ID NO: 5) target peptide. In the pulsed peptide reporter assay using K562 cells as antigen-presenting cells, HLA * Using A02.01, K562 HLA * A24:02 cells were used as a negative control.

[0307] Engineered Jurkat cells transduced with chimeric T cell receptor, TCR B, or TCR D were exogenously loaded with a titrated concentration of the target peptide into target cells A. * 02:01 K562 or A * The responsiveness to K562 was analyzed at 24:02. Cells were co-cultured in a 1:1 E:T ratio for 16-18 hours. Jurkat cell activation values ​​were measured by flow cytometry using CD69. + This was evaluated as a percentage of cells (Figure 4). Manipulated TCR DT cells showed robust TCR signaling induction. Manipulated anti-AIQDCLAV (TCR B) T cells also induced robust TCR signaling, as determined by CD69 expression. Manipulated T cells of both TCR B and TCR D showed substantially stronger signaling than CAR-T cells. As expected, K562 HLA * No signal transduction was observed in A24:02 cells.

[0308] TCR B and TCR D are HLA-A *Jurkat CD8-positive cells co-cultured with K562 cells pulsed with NPM1 expressing 02:01 exhibit superior CD69 reporter signaling compared to publicly reported (Xie et al. 2021) anti-AIQDCLAV CAR-T cells.

[0309] Example 14. Comparison of CAR-T versus TCR-T in a cytotoxic assay. Primary CD8 cells derived from healthy donors that have been genetically edited and transduced using either TCR D (natural type) or disulfide-modified TCR D (DB). + Cells, or anti-AIQDCLAV CAR-T cells, as previously described, possess HLA-A with NPM1c. * 02:01 The recognition of immunogenic AML cell lines (OCI-AML3) was tested. Cells were co-cultured for 16-18 hours at different E:T ratios. The mortality rate was assessed by flow cytometry, and EC was measured using GraphPad Prism. 50 This was generated (Figure 5).

[0310] Both T cells manipulated with TCR D and disulfide-modified TCR D exhibit substantially higher mortality and lower EC compared to CAR-T cells. 50 The results showed that, since the endogenous TCR was deleted in the manipulated T cells, no difference in cell death was observed between T cells manipulated with TCR D and T cells manipulated with disulfide-added TCR D. TCR knockout (KO) T cells were used as a negative control.

[0311] Example 15. TCR D targets the neoantigen (CLAVEEVSL) with or without post-translational modification. The killing assay was performed as previously described with minor modifications. The cytotoxic assay was performed for strains lacking post-translational modification or where the first cysteine ​​in the target peptide was cysteinylated (C *The assay was performed using OCI-AML2 cells pulsed with either the CLAVEEVSL (SEQ ID NO: 5) peptide (represented as LAVEEVSL). Pulsed peptide assays were performed using 10 ng / mL of peptide at E:T ratios of 0.5:1 and 1:1. Wild-type NPM1 was used as the negative peptide control, and anti-HPV TCR-modified T cells were used as the negative T cell control.

[0312] C * The cytotoxicity of LAVEEVSL is NPM1c + It has been shown to be important for T-cell therapy for cancer (van der Lee et al. 2019). Previously published anti-CLAVEEVSL TCRs are CLAVEEVSL or C * Upon binding to LAVEEVSL, differential activation, as measured by INFγ, was observed. Clonal reactivity was more potent against non-cysteine ​​target peptides. In contrast, T cells engineered with TCR D showed high cytotoxicity against both cysteine ​​and non-cysteine ​​target peptides (Figures 7-8), suggesting a potential cytotoxic advantage over previously published anti-CLAVEEVSL TCRs (US Patent No. 11352389).

[0313] Example 16. Identification of amino acids of CLAVEEVSL (SEQ ID NO: 5) recognized by anti-CLAVEEVSL TCR. Alanine scanning was performed on the CLAVEEVSL (SEQ ID NO: 5) peptide. The peptides used are listed in Table 11.

[0314] The reporter assay was performed as previously described, with minor modifications. Briefly, target K562 cells were pulsed for 1 hour with the listed peptides at various peptide concentrations. CD8 + Jurkat cells were engineered to express TCR D and TCR F. The reporter assay was performed using a 1:1 E:T ratio. The control condition included either a wild-type negative control peptide or an immunogenic positive control peptide (CLAVEEVSL, SEQ ID NO: 5).

[0315] [Table 11]

[0316] The CLAVEEVSL (SEQ ID NO: 5) residues important for recognition by anti-CLAVEEVLS TCRs were evaluated by the reduction or loss of CD69 signaling when amino acids were substituted with alanine. The amino acids at positions 2, 5, 6, and 7 (relative to SEQ ID NO: 5) were found to be important for recognition by both TCRs (Figures 7 and 8). Surprisingly, residue 4 was important only for recognition by TCR D, and residue 9 was important only for recognition by TCR F.

[0317] The present invention is not intended to limit its scope to any particular disclosed embodiment, provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described herein will become apparent from the description and teachings herein. Such modifications may be carried out without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the disclosure.

[0318] [Table 12-1]

[0319] [Table 12-2]

[0320] [Table 12-3]

[0321] [Table 12-4]

[0322] Table 12-5

[0323] Table 12-6

[0324] Table 12-7

[0325] Table 12-8

[0326] Table 12-9

[0327] Table 12-10

[0328] Table 12-11

[0329] Table 12-12

[0330] Table 12-13

[0331] Table 12-14

[0332] Table 12-15

[0333] Table 12-16

[0334] Table 12-17

[0335] Table 12-18

[0336] Table 12-19

[0337] Table 12-20

[0338] Table 12-21

[0339] Table 12-22

[0340] Table 12-23

[0341] Table 12-24

[0342] Table 12-25

[0343] Table 12-26

[0344] Table 12-27

[0345] Table 12-28

[0346] Table 12-29

[0347] Table 12-30

[0348] Table 12-31

[0349] Table 12-32

[0350] Table 12-33

[0351] Table 12-34

[0352] Table 12-35

[0353] Table 12-36

[0354] Table 12-37

[0355] Table 12-38

[0356] Table 12-39

[0357] Table 12-40

[0358] Table 12-41

[0359] Table 12-42

[0360] Table 12-43

[0361] Table 12-44

[0362] Table 12-45

[0363] Table 12-46

[0364] Table 12-47

[0365] Table 12-48

[0366] Table 12-49

[0367] Table 12-50

Claims

1. A T cell receptor (TCR) or its antigen-binding fragment, An α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or It comprises a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region, (a) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 67, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 75, (b) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 85, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 93, (c) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 103, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 111, (d) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 31, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 39, (e) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 49, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 57, (f) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 121, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 129, (g) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 139, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 147, (h) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 157, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 165, (i) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 175, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 183, (j) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 193, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 201, (k) The Vα region or Vγ region includes CDR-3 containing SEQ ID NO: 211, and the Vβ region or Vδ region includes CDR-3 containing SEQ ID NO: 219, (l) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 229, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 237, (m) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 247, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 255, (n) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 265, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 273, (o) The Vα region or Vγ region contains CDR-3 containing SEQ ID NO: 283, and the Vβ region or Vδ region contains CDR-3 containing SEQ ID NO: 291, or (p) A TCR or antigen-binding fragment thereof, wherein the Vα region or Vγ region includes a complementarity-determining region 3 (CDR-3) containing SEQ ID NO: 13, and the Vβ region or Vδ region includes a CDR-3 containing SEQ ID NO:

21.

2. (a) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 65 and CDR-2 containing SEQ ID NO: 66, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 73 and CDR-2 containing SEQ ID NO: 74, (b) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 83 and CDR-2 containing SEQ ID NO: 84, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 91 and CDR-2 containing SEQ ID NO: 92, (c) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 101 and CDR-2 containing SEQ ID NO: 102, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 109 and CDR-2 containing SEQ ID NO: 110, (d) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 29 and CDR-2 containing SEQ ID NO: 30, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 37 and CDR-2 containing SEQ ID NO: 38, (e) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 47 and CDR-2 containing SEQ ID NO: 48, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 55 and CDR-2 containing SEQ ID NO: 56, (f) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 119 and CDR-2 containing SEQ ID NO: 120, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 127 and CDR-2 containing SEQ ID NO: 128, (g) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 137 and CDR-2 containing SEQ ID NO: 138, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 145 and CDR-2 containing SEQ ID NO: 146, (h) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 155 and CDR-2 containing SEQ ID NO: 156, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 163 and CDR-2 containing SEQ ID NO: 164, (i) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 173 and CDR-2 containing SEQ ID NO: 174, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 181 and CDR-2 containing SEQ ID NO: 182, (j) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 191 and CDR-2 containing SEQ ID NO: 192, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 199 and CDR-2 containing SEQ ID NO: 200, (k) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 209 and CDR-2 containing SEQ ID NO: 210, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 217 and CDR-2 containing SEQ ID NO: 218, (l) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 227 and CDR-2 containing SEQ ID NO: 228, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 235 and CDR-2 containing SEQ ID NO: 236, (m) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 245 and CDR-2 containing SEQ ID NO: 246, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 253 and CDR-2 containing SEQ ID NO: 254, (n) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 263 and CDR-2 containing SEQ ID NO: 264, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 271 and CDR-2 containing SEQ ID NO: 272, (o) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 281 and CDR-2 containing SEQ ID NO: 282, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 289 and CDR-2 containing SEQ ID NO: 290, or (p) The TCR or antigen-binding fragment according to claim 1, wherein the Vα region or Vγ region comprises a complementarity-determining region 1 (CDR-1) containing SEQ ID NO: 11 and a complementarity-determining region 2 (CDR-2) containing SEQ ID NO: 12, and the Vβ region or Vδ region comprises CDR-1 containing SEQ ID NO: 19 and CDR-2 containing SEQ ID NO:

20.

3. A T cell receptor (TCR) or its antigen-binding fragment, An α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or It comprises a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region, (a) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 65, CDR-2 containing SEQ ID NO: 66, and CDR-3 containing SEQ ID NO: 67, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 73, CDR-2 containing SEQ ID NO: 74, and CDR-3 containing SEQ ID NO: 75, (b) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 83, CDR-2 containing SEQ ID NO: 84, and CDR-3 containing SEQ ID NO: 85, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 91, CDR-2 containing SEQ ID NO: 92, and CDR-3 containing SEQ ID NO: 93, (c) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 101, CDR-2 containing SEQ ID NO: 102, and CDR-3 containing SEQ ID NO: 103, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 109, CDR-2 containing SEQ ID NO: 110, and CDR-3 containing SEQ ID NO: 111, (d) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 29, CDR-2 containing SEQ ID NO: 30, and CDR-3 containing SEQ ID NO: 31, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 37, CDR-2 containing SEQ ID NO: 38, and CDR-3 containing SEQ ID NO: 38, (e) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 47, CDR-2 containing SEQ ID NO: 48, and CDR-3 containing SEQ ID NO: 49, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 55, CDR-2 containing SEQ ID NO: 56, and CDR-3 containing SEQ ID NO: 57, (f) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 119, CDR-2 containing SEQ ID NO: 120, and CDR-3 containing SEQ ID NO: 121, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 127, CDR-2 containing SEQ ID NO: 128, and CDR-3 containing SEQ ID NO: 129, (g) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 137, CDR-2 containing SEQ ID NO: 138, and CDR-3 containing SEQ ID NO: 139, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 145, CDR-2 containing SEQ ID NO: 146, and CDR-3 containing SEQ ID NO: 147, (h) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 155, CDR-2 containing SEQ ID NO: 156, and CDR-3 containing SEQ ID NO: 157, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 163, CDR-2 containing SEQ ID NO: 164, and CDR-3 containing SEQ ID NO: 165, (i) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 173, CDR-2 containing SEQ ID NO: 174, and CDR-3 containing SEQ ID NO: 175, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 181, CDR-2 containing SEQ ID NO: 182, and CDR-3 containing SEQ ID NO: 183, (j) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 191, CDR-2 containing SEQ ID NO: 192, and CDR-3 containing SEQ ID NO: 193, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 199, CDR-2 containing SEQ ID NO: 200, and CDR-3 containing SEQ ID NO: 201, (k) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 209, CDR-2 containing SEQ ID NO: 210, and CDR-3 containing SEQ ID NO: 211, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 217, CDR-2 containing SEQ ID NO: 218, and CDR-3 containing SEQ ID NO: 219, (l) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 227, CDR-2 containing SEQ ID NO: 228, and CDR-3 containing SEQ ID NO: 229, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 235, CDR-2 containing SEQ ID NO: 236, and CDR-3 containing SEQ ID NO: 237, (m) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 245, CDR-2 containing SEQ ID NO: 246, and CDR-3 containing SEQ ID NO: 247, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 253, CDR-2 containing SEQ ID NO: 254, and CDR-3 containing SEQ ID NO: 255, (n) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 263, CDR-2 containing SEQ ID NO: 264, and CDR-3 containing SEQ ID NO: 265, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 271, CDR-2 containing SEQ ID NO: 272, and CDR-3 containing SEQ ID NO: 273, (o) The Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 281, CDR-2 containing SEQ ID NO: 282, and CDR-3 containing SEQ ID NO: 283, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 289, CDR-2 containing SEQ ID NO: 290, and CDR-3 containing SEQ ID NO: 291, or (p) A TCR or antigen-binding fragment thereof, wherein the Vα region or Vγ region includes CDR-1 containing SEQ ID NO: 11, CDR-2 containing SEQ ID NO: 12, and CDR-3 containing SEQ ID NO: 13, and the Vβ region or Vδ region includes CDR-1 containing SEQ ID NO: 19, CDR-2 containing SEQ ID NO: 20, and CDR-3 containing SEQ ID NO:

21.

4. A T cell receptor (TCR) or its antigen-binding fragment, An α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or It comprises a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region, (a) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 68, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 76, (b) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 86, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 94, (c) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 104, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 112, (d) The Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 32, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 40; (e) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 50, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 58, (f) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 122, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 130, (g) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 140, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 148, (h) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 158, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 166, (i) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 176, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 184, (j) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 194, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 202, (k) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 212, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 220, (l) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 230, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 238, (m) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 248, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 256, (n) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 266, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 274, (o) The Vα region or Vγ region contains CDR-3 such that it is contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 284, and the Vβ region or Vδ region contains CDR-3 such that it is contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO: 292, (p) A TCR or antigen-binding fragment thereof, wherein the Vα region or Vγ region contains CDR-3 containing CDR-3 contained within the Vα region sequence or Vγ region sequence of SEQ ID NO: 14, and the Vβ region or Vδ region contains CDR-3 containing CDR-3 contained within the Vβ region sequence or Vδ region sequence of SEQ ID NO:

22.

5. (a) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 68, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 76, (b) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 86, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 94, (c) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 104, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 112, (d) The Vα region or Vγ region contains CDR-1 and CDR-2, each containing CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 32, and the Vβ region or Vδ region contains CDR-1 and CDR-2, each containing CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 40, (e) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 50, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 58, (f) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 122, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 130, (g) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 140, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 148, (h) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 158, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 166, (i) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 176, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 184, (j) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 194, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 202, (k) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 212, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 220, (l) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 230, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 238, (m) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 248, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 256, (n) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 266, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 274, (o) The Vα region or Vγ region includes CDR-1 and CDR-2 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 284, and the Vβ region or Vδ region includes CDR-1 and CDR-2 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 292, or (p) The TCR or antigen-binding fragment according to claim 4, wherein the Vα region or Vγ region comprises CDR-1 and CDR-2, each comprising CDR-1 and CDR-2 contained in the Vα region sequence or Vγ region sequence of Sequence ID No. 14, and the Vβ region or Vδ region comprises CDR-1 and CDR-2, each comprising CDR-1 and CDR-2, each comprising CDR-1 and CDR-2 contained in the Vβ region sequence or Vδ region sequence of Sequence ID No.

22.

6. A T cell receptor (TCR) or its antigen-binding fragment, An α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or It comprises a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region, (a) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 68, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 76, (b) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 86, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 94, (c) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 104, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 112, (d) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 32, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 40, (e) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 50, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 58, (f) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 122, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 130, (g) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 140, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 148, (h) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 158, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 166, (i) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 176, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 184, (j) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 194, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 202, (k) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 212, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 220, (l) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 230, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 238, (m) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 248, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 256, (n) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 266, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 274, (o) The Vα region or Vγ region includes CDR-1, CDR-2, and CDR-3 contained within the Vα region sequence or Vγ region sequence of Sequence ID No. 284, and the Vβ region or Vδ region includes CDR-1, CDR-2, and CDR-3 contained within the Vβ region sequence or Vδ region sequence of Sequence ID No. 292, (p) A TCR or antigen-binding fragment thereof, wherein the Vα region or Vγ region comprises CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained in the Vα region sequence or Vγ region sequence of Sequence ID No. 14, and the Vβ region or Vδ region comprises CDR-1, CDR-2, and CDR-3, each containing CDR-1, CDR-2, and CDR-3 contained in the Vβ region sequence or Vδ region sequence of Sequence ID No.

22.

7. A T cell receptor (TCR) or its antigen-binding fragment, An α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, or It comprises a γ chain containing a variable γ (Vγ) region and a δ chain containing a variable δ (Vδ) region, (a) The Vα region or Vγ region contains sequence number 68 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 76 or a sequence having at least 90% sequence identity thereto, (b) The Vα region or Vγ region contains sequence number 86 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 94 or a sequence having at least 90% sequence identity thereto, (c) The Vα region or Vγ region contains sequence number 104 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 112 or a sequence having at least 90% sequence identity thereto, (d) The Vα region or Vγ region contains sequence number 32 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 40 or a sequence having at least 90% sequence identity thereto, (e) The Vα region or Vγ region contains sequence number 50 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 58 or a sequence having at least 90% sequence identity thereto, (f) The Vα region or Vγ region contains sequence number 122 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 130 or a sequence having at least 90% sequence identity thereto, (g) The Vα region or Vγ region contains sequence number 140 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 148 or a sequence having at least 90% sequence identity thereto, (h) The Vα region or Vγ region contains sequence number 158 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 166 or a sequence having at least 90% sequence identity thereto, (i) The Vα region or Vγ region contains sequence number 176 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 184 or a sequence having at least 90% sequence identity thereto, (j) The Vα region or Vγ region contains sequence number 194 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 202 or a sequence having at least 90% sequence identity thereto, (k) The Vα region or Vγ region contains sequence number 212 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 220 or a sequence having at least 90% sequence identity thereto, (l) The Vα region or Vγ region contains sequence number 230 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 238 or a sequence having at least 90% sequence identity thereto, (m) The Vα region or Vγ region contains sequence number 248 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 256 or a sequence having at least 90% sequence identity thereto, (n) The Vα region or Vγ region contains sequence number 266 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 274 or a sequence having at least 90% sequence identity thereto, (o) The Vα region or Vγ region contains sequence number 284 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains sequence number 292 or a sequence having at least 90% sequence identity thereto, (p) A TCR or antigen-binding fragment thereof, wherein the Vα region or Vγ region contains SEQ ID NO: 14 or a sequence having at least 90% sequence identity thereto, and the Vβ region or Vδ region contains SEQ ID NO: 22 or a sequence having at least 90% sequence identity thereto.

8. (a) The Vα region or Vγ region includes SEQ ID NO: 68, and the Vβ region or Vδ region includes SEQ ID NO: 76, (b) The Vα region or Vγ region includes SEQ ID NO: 86, and the Vβ region or Vδ region includes SEQ ID NO: 94, (c) The Vα region or Vγ region includes sequence number 104, and the Vβ region or Vδ region includes sequence number 112, (d) The Vα region or Vγ region includes SEQ ID NO: 32, and the Vβ region or Vδ region includes SEQ ID NO: 40, (e) The Vα region or Vγ region includes SEQ ID NO: 50, and the Vβ region or Vδ region includes SEQ ID NO: 58, (f) The Vα region or Vγ region includes SEQ ID NO: 122, and the Vβ region or Vδ region includes SEQ ID NO: 130, (g) The Vα region or Vγ region includes SEQ ID NO: 140, and the Vβ region or Vδ region includes SEQ ID NO: 148, (h) The Vα region or Vγ region includes sequence number 158, and the Vβ region or Vδ region includes sequence number 166, (i) The Vα region or Vγ region includes sequence number 176, and the Vβ region or Vδ region includes sequence number 184, (j) The Vα region or Vγ region includes SEQ ID NO: 194, and the Vβ region or Vδ region includes SEQ ID NO: 202, (k) The Vα region or Vγ region includes SEQ ID NO: 212, and the Vβ region or Vδ region includes SEQ ID NO: 220, (l) The Vα region or Vγ region includes SEQ ID NO: 230, and the Vβ region or Vδ region includes SEQ ID NO: 238, (m) The Vα region or Vγ region includes sequence number 248, and the Vβ region or Vδ region includes sequence number 256, (n) The Vα region or Vγ region includes sequence number 266, and the Vβ region or Vδ region includes sequence number 274, (o) The Vα region or Vγ region includes SEQ ID NO: 284, and the Vβ region or Vδ region includes SEQ ID NO: 292, or (p) The TCR or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the Vα region or Vγ region includes SEQ ID NO: 14, and the Vβ region or Vδ region includes SEQ ID NO:

22.

9. The α chain further includes an α-steady (Cα) region, and the β chain further includes a β-steady (Cβ) region, or The TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the γ chain further comprises a γ constant (Cγ) region, and the δ chain further comprises a δ constant (Cδ) region.

10. The TCR or antigen-binding fragment thereof according to claim 9, wherein the Cα comprises SEQ ID NO: 3 and the Cβ comprises SEQ ID NO:

7.

11. (a) The α-chain or γ-chain contains SEQ ID NO: 71, and the β-chain or δ-chain contains SEQ ID NO: 79, (b) The α-chain or γ-chain contains SEQ ID NO: 89, and the β-chain or δ-chain contains SEQ ID NO: 97, (c) The α-chain or γ-chain contains SEQ ID NO: 107, and the β-chain or δ-chain contains SEQ ID NO: 115, (d) The α-chain or γ-chain contains SEQ ID NO: 35, and the β-chain or δ-chain contains SEQ ID NO: 43, (e) The α-chain or γ-chain contains SEQ ID NO: 53, and the β-chain or δ-chain contains SEQ ID NO: 61, (f) The α-chain or γ-chain contains SEQ ID NO: 125, and the β-chain or δ-chain contains SEQ ID NO: 133, (g) The α-chain or γ-chain contains SEQ ID NO: 143, and the β-chain or δ-chain contains SEQ ID NO: 151, (h) The α chain or γ chain contains SEQ ID NO: 161, and the β chain or δ chain contains SEQ ID NO: 169, (i) The α-chain or γ-chain contains SEQ ID NO: 179, and the β-chain or δ-chain contains SEQ ID NO: 187, (j) The α-chain or γ-chain contains SEQ ID NO: 197, and the β-chain or δ-chain contains SEQ ID NO: 205, (k) The α-chain or γ-chain contains SEQ ID NO: 215, and the β-chain or δ-chain contains SEQ ID NO: 223, (l) The α-chain or γ-chain contains SEQ ID NO: 233, and the β-chain or δ-chain contains SEQ ID NO: 241, (m) The α-chain or γ-chain contains SEQ ID NO: 251, and the β-chain or δ-chain contains SEQ ID NO: 259, (n) The α-chain or γ-chain contains SEQ ID NO: 269, and the β-chain or δ-chain contains SEQ ID NO: 277, (o) The α-chain or γ-chain contains SEQ ID NO: 287, and the β-chain or δ-chain contains SEQ ID NO: 295, or (p) The TCR or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the α chain or γ chain comprises SEQ ID NO: 17, and the β chain or δ chain comprises SEQ ID NO:

25.

12. The TCR or antigen-binding fragment according to any one of claims 1 to 11, wherein the TCR or antigen-binding fragment therein recognizes the NPM1c neoantigen in relation to an MHC molecule.

13. The TCR or antigen-binding fragment thereof according to claim 12, wherein the MHC molecule is a human leukocyte antigen (HLA)-A molecule.

14. The aforementioned HLA-A molecule is serotype HLA-A ＊ 02:01, HLA-A ＊ 02:06, or HLA-A ＊ A TCR or antigen-binding fragment thereof according to claim 13, which is in the 02:03 ratio.

15. The TCR or antigen-binding fragment thereof according to any one of claims 12 to 14, wherein the NPM1c neoantigen comprises CLAVEEVSL (SEQ ID NO: 5) or AIQDLCLAV (SEQ ID NO: 1).

16. The TCR or its antigen-binding fragment, (a) Recognizes CLAVEEVSL (SEQ ID NO: 5) with higher affinity than AIQDLWQWRKSL (SEQ ID NO: 2), or (b) The TCR or antigen-binding fragment thereof according to 15, which recognizes AIQDLCLAV (SEQ ID NO: 1) with higher affinity than AIQDLWQWRKSL (SEQ ID NO: 2).

17. A nucleic acid sequence encoding a TCR or its antigen-binding fragment, or its α-chain, β-chain, γ-chain, or δ-chain, according to any one of claims 1 to 16.

18. The nucleic acid sequence includes a first nucleotide sequence encoding the Vα region and a second nucleotide sequence encoding the Vβ region, or a first nucleotide sequence encoding the Vγ region and a second nucleotide sequence encoding the Vδ region. (a) The first nucleotide sequence includes sequence number 69 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 77 or a sequence having at least 90% sequence identity thereto, (b) The first nucleotide sequence includes sequence number 87 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 95 or a sequence having at least 90% sequence identity thereto, (c) The first nucleotide sequence includes sequence number 105 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 113 or a sequence having at least 90% sequence identity thereto, (d) The first nucleotide sequence includes sequence number 33 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 41 or a sequence having at least 90% sequence identity thereto, (e) The first nucleotide sequence includes sequence number 51 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 59 or a sequence having at least 90% sequence identity thereto, (f) The first nucleotide sequence includes sequence number 123 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 131 or a sequence having at least 90% sequence identity thereto, (g) The first nucleotide sequence includes sequence number 141 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 149 or a sequence having at least 90% sequence identity thereto, (h) The first nucleotide sequence includes sequence number 159 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 167 or a sequence having at least 90% sequence identity thereto, (i) The first nucleotide sequence includes sequence number 177 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 185 or a sequence having at least 90% sequence identity thereto, (j) The first nucleotide sequence includes sequence number 195 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 203 or a sequence having at least 90% sequence identity thereto, (k) The first nucleotide sequence includes sequence number 213 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 221 or a sequence having at least 90% sequence identity thereto, (l) The first nucleotide sequence includes sequence number 231 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 239 or a sequence having at least 90% sequence identity thereto, (m) The first nucleotide sequence includes sequence number 249 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 257 or a sequence having at least 90% sequence identity thereto, (n) The first nucleotide sequence includes sequence number 267 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 275 or a sequence having at least 90% sequence identity thereto, (o) The first nucleotide sequence includes sequence number 285 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 293 or a sequence having at least 90% sequence identity thereto, (p) The first nucleotide sequence includes sequence number 15 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 23 or a sequence having at least 90% sequence identity thereto, (aa) The first nucleotide sequence includes sequence number 70 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 78 or a sequence having at least 90% sequence identity thereto, (bb) The first nucleotide sequence includes sequence number 88 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 96 or a sequence having at least 90% sequence identity thereto, (cc) The first nucleotide sequence includes sequence number 106 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 114 or a sequence having at least 90% sequence identity thereto, (dd) The first nucleotide sequence includes sequence number 34 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 42 or a sequence having at least 90% sequence identity thereto, (ee) The first nucleotide sequence includes sequence number 52 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 60 or a sequence having at least 90% sequence identity thereto, (ff) The first nucleotide sequence includes sequence number 124 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 132 or a sequence having at least 90% sequence identity thereto, (gg) The first nucleotide sequence includes sequence number 142 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 150 or a sequence having at least 90% sequence identity thereto, (hh) The first nucleotide sequence includes sequence number 160 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 168 or a sequence having at least 90% sequence identity thereto, (ii) The first nucleotide sequence includes sequence number 178 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 186 or a sequence having at least 90% sequence identity thereto, (jj) The first nucleotide sequence includes sequence number 196 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 204 or a sequence having at least 90% sequence identity thereto, (kk) The first nucleotide sequence includes sequence number 214 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 222 or a sequence having at least 90% sequence identity thereto, (ll) The first nucleotide sequence includes sequence number 232 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 240 or a sequence having at least 90% sequence identity thereto, (mm) The first nucleotide sequence includes sequence number 250 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 258 or a sequence having at least 90% sequence identity thereto, (nn) The first nucleotide sequence includes sequence number 268 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 276 or a sequence having at least 90% sequence identity thereto, (oo) The first nucleotide sequence includes sequence number 286, or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 294, or a sequence having at least 90% sequence identity thereto, (pp) The nucleic acid sequence according to claim 17, wherein the first nucleotide sequence includes sequence number 16 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 24 or a sequence having at least 90% sequence identity thereto.

19. (a) The first nucleotide sequence includes sequence number 72 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 80 or a sequence having at least 90% sequence identity thereto, (b) The first nucleotide sequence includes sequence number 90 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 98 or a sequence having at least 90% sequence identity thereto, (c) The first nucleotide sequence includes sequence number 108 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 116 or a sequence having at least 90% sequence identity thereto, (d) The first nucleotide sequence includes sequence number 36 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 44 or a sequence having at least 90% sequence identity thereto, (e) The first nucleotide sequence includes sequence number 54 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 62 or a sequence having at least 90% sequence identity thereto, (f) The first nucleotide sequence includes sequence number 126 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 134 or a sequence having at least 90% sequence identity thereto, (g) The first nucleotide sequence includes sequence number 144 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 152 or a sequence having at least 90% sequence identity thereto, (h) The first nucleotide sequence includes sequence number 162 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 170 or a sequence having at least 90% sequence identity thereto, (i) The first nucleotide sequence includes sequence number 180 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 188 or a sequence having at least 90% sequence identity thereto, (j) The first nucleotide sequence includes sequence number 198 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 206 or a sequence having at least 90% sequence identity thereto, (k) The first nucleotide sequence includes sequence number 216 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 224 or a sequence having at least 90% sequence identity thereto, (l) The first nucleotide sequence includes sequence number 234 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 242 or a sequence having at least 90% sequence identity thereto, (m) The first nucleotide sequence includes sequence number 252 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 260 or a sequence having at least 90% sequence identity thereto, (n) The first nucleotide sequence includes sequence number 270 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 278 or a sequence having at least 90% sequence identity thereto, (o) The first nucleotide sequence includes sequence number 288 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 296 or a sequence having at least 90% sequence identity thereto, (p) The nucleic acid sequence according to claim 17 or 18, wherein the first nucleotide sequence includes sequence number 18 or a sequence having at least 90% sequence identity thereto, and the second nucleotide sequence includes sequence number 26 or a sequence having at least 90% sequence identity thereto.

20. The nucleic acid sequence according to any one of claims 17 to 19, wherein the first nucleotide and the second nucleotide are present on a single expression vector and expressed from a single promoter.

21. The nucleic acid sequence according to any one of claims 17 to 20, wherein the first nucleotide sequence and the second nucleotide sequence are separated by a peptide sequence that causes ribosome skipping.

22. The nucleic acid sequence according to claim 21, wherein the peptide that causes ribosome skipping is a P2A peptide.

23. The nucleic acid sequence according to claim 22, wherein the P2A peptide comprises SEQ ID NO:

301.

24. The nucleic acid sequence according to claim 23, wherein the sequence encoding the P2A peptide is described in sequence number 302.

25. The nucleic acid sequence according to any one of claims 17 to 24, wherein the nucleic acid sequence encodes the amino acid sequence of sequence numbers 81, 99, 117, 45, 63, 135, 153, 171, 189, 207, 225, 243, 261, 279, 297, or 27.

26. The nucleic acid sequence according to any one of claims 20 to 25, wherein the single expression vector comprises the nucleic acid sequence of SEQ ID NOs: 82, 100, 118, 46, 64, 136, 154, 172, 190, 208, 226, 244, 262, 280, 298, or 28.

27. A vector comprising the nucleic acid sequence described in any one of claims 17 to 26.

28. The vector according to claim 27, wherein the vector is a viral vector.

29. The vector according to claim 28, wherein the viral vector is a lentiviral vector.

30. A manipulated cell comprising a TCR or its antigen-binding fragment according to any one of claims 1 to 16.

31. An engineered cell comprising a TCR or its antigen-binding fragment, wherein the TCR or its antigen-binding fragment is encoded by a nucleic acid sequence according to any one of claims 17 to 26.

32. The manipulated cell according to claim 30 or 31, wherein the TCR or its antigen-binding fragment is heterogeneous to the cell.

33. The manipulated cells according to any one of claims 30 to 32, wherein the manipulated cells are derived from a cell line.

34. The manipulated cells according to any one of claims 30 to 32, wherein the manipulated cells are derived from primary cells obtained from the subject.

35. The manipulated cell according to any one of claims 30 to 34, wherein the manipulated cell is a T cell, and optionally the T cell is a primary T cell, a natural killer T cell, or a cytotoxic T cell.

36. The manipulated cells according to any one of claims 30 to 35, wherein the expression of one or more endogenous TCR chains of the T cells is reduced or eliminated, and optionally, the expression of the endogenous TRAC gene and the endogenous TRBC gene of the T cells is knocked out.

37. The manipulated cells according to claim 35, wherein the manipulated cells have cytotoxic activity against cells expressing CLAVEEVSL (SEQ ID NO: 5) or AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule.

38. The manipulated cell according to claim 36, wherein the MHC molecule is a human leukocyte antigen (HLA)-A molecule.

39. The aforementioned HLA-A molecule is serotype HLA-A ＊ 02:01, HLA-A ＊ 02:06, or HLA-A ＊ The manipulated cell according to claim 37, which is 02:

03.

40. The manipulated cells according to claim 39, wherein the manipulated cells exhibit increased cytotoxic activity against cells expressing CLAVEEVSL (SEQ ID NO: 5) or AIQDLCLAV (SEQ ID NO: 1) compared to the cytotoxic activity against cells expressing AIQDLWQWRKSL (SEQ ID NO: 2).

41. A method for producing engineered cells, comprising introducing a nucleic acid sequence according to any one of claims 17 to 26 or a vector according to any one of claims 27 to 29 into cells in vitro or ex vivo, wherein the cells are optionally T cells, and optionally the T cells are primary T cells, natural killer T cells, or cytotoxic T cells.

42. The method according to claim 41, further comprising knocking out the expression of one or more endogenous TCR genes in the T cells.

43. A composition comprising a TCR or antigen-binding fragment thereof according to any one of claims 1 to 16, a nucleic acid sequence according to any one of claims 17 to 26, or a vector according to any one of claims 27 to 29.

44. A composition comprising the manipulated cells described in any one of claims 30 to 40.

45. The composition according to claim 43 or 44, further comprising a pharmaceutically acceptable excipient.

46. A method for identifying T cell receptors (TCRs) that target NPM1c neoantigen, the method comprising identifying a functional TCR that recognizes the NPM1c neoantigen from among a plurality of functional TCRs, wherein the plurality of functional TCRs are encoded by a plurality of functional TCR coding nucleic acid vectors generated by a high-throughput nucleic acid amplification and assembly method using nucleic acids obtained from a single T cell among a plurality of T cells, and the plurality of T cells are derived from a donor subject.

47. A method for identifying T cell receptors (TCRs) that target NPM1c neoantigen, wherein the method is: (i) Generating multiple functional TCR coding nucleic acid vectors by a high-throughput nucleic acid amplification and assembly method using nucleic acids obtained from a single T cell among multiple T cells, wherein the T cell is derived from a donor subject. (ii) A method comprising identifying a functional TCR that recognizes the NPM1c neoantigen from among a plurality of functional TCRs encoded by the plurality of functional TCR coding nucleic acid vectors.

48. The method according to claim 46 or 47, wherein the donor subject is a healthy human donor.

49. The method according to any one of claims 46 to 48, wherein the identified functional TCR recognizes the peptide CLAVEEVSL (SEQ ID NO: 5) or AIQDLCLAV (SEQ ID NO: 1) in relation to an MHC molecule.

50. The method according to claim 49, wherein the MHC molecule is a human leukocyte antigen (HLA)-A molecule.

51. Said HLA-A molecule is serotype HLA-A ＊ 02:01, HLA-A ＊ 02:06, or HLA-A ＊ 02:03, the method according to claim 50.

52. The method according to any one of claims 46 to 51, wherein the T cells derived from the donor subject are cultured under conditions for cell expansion of the T cells before the generation of the plurality of functional TCR coding nucleic acid vectors.

53. The method according to any one of claims 46 to 51, wherein the T cells derived from the donor subject are not cultured under conditions for cell expansion of the T cells prior to the generation of the plurality of functional TCR coding nucleic acid vectors.

54. The high-throughput nucleic acid amplification and assembly method described above is (1) Amplifying a first amplification product and a second amplification product from complementary DNA (cDNA) generated from RNA obtained from a single T cell among the plurality of T cells selected to each of a plurality of distinct locations of the device, The amplification is such that the first amplification product includes a nucleotide sequence encoding the fully variable-length α (Vα) region or the fully variable-length γ (Vγ) region of the TCR, and the second amplification product includes a nucleotide sequence encoding the fully variable-length β (Vβ) region or the fully variable-length δ (Vδ) region of the TCR. (2) Assembling the first amplification product and the second amplification product from each of the plurality of separate locations into a nucleic acid vector to obtain an assembled nucleic acid vector containing a nucleotide sequence encoding a functional TCR for each of the plurality of separate locations, The method according to any one of claims 46 to 53, wherein the functional TCR comprises (i) a full-length Vα region and a full-length Vβ region derived from the single T cell, or (ii) a full-length Vγ region and a full-length Vδ region derived from the single T cell.

55. A manipulated cell comprising a TCR identified by the method of any one of claims 1 to 16 or a nucleic acid sequence according to any one of claims 17 to 26.

56. The manipulated cells according to claim 55, wherein the manipulated cells exhibit increased cytotoxic activity against cells expressing CLAVEEVLS (SEQ ID NO: 5) or AIQDLCLAV (SEQ ID NO: 1) compared to the cytotoxic activity against cells expressing AIQDLWQWRKSL (SEQ ID NO: 2).

57. A composition comprising the manipulated cells described in claim 55 or 56.

58. The composition according to claim 52, further comprising a pharmaceutically acceptable excipient.

59. A treatment method comprising administering to a subject having a disease or condition the composition described in any one of claims 44, 45, 57, or 58, the engineered T cells described in any one of claims 30 to 40, or the engineered T cells expressing the TCR or its antigen-binding fragment described in any one of claims 1 to 16, or the nucleic acid sequence described in any one of claims 17 to 26.

60. A treatment method, wherein the method is applied to a subject having a disease or condition. (a) The TCR or antigen-binding fragment according to any one of claims 1 to 16, (b) A cell expressing the TCR or antigen-binding fragment described in any one of claims 1 to 16, (c) A protein comprising a TCR or antigen-binding fragment as described in any one of claims 1 to 16, or (d) A therapeutic method comprising administering cells expressing the TCR or antigen-binding fragment described in any one of claims 1 to 16.

61. The method according to claim 59 or 60, wherein the protein comprising the TCR or antigen-binding fragment according to any one of claims 1 to 16 comprises a bispecific T cell engager or a chimeric T cell receptor.

62. The aforementioned target is NPM1c ＋ Leukemia, NPM1c ＋ Acute myeloid leukemia (AML), or NPM1c ＋ The method according to any one of claims 59 to 61, wherein the person has or has been diagnosed with myelodysplastic syndrome (MDS).

63. The method according to any one of claims 59 to 61, wherein the subject has a humoral tumor, a hematopoietic tumor, or chronic myeloid leukemia, or has been diagnosed with a humoral tumor, a hematopoietic tumor, or chronic myeloid leukemia.

64. The method according to any one of claims 61 to 63, wherein the treatment induces or enhances cell death of cells associated with the malignant hematological disorder, or induces or enhances the graft-to-leukemia effect (GVL) in the subject.

65. The method according to claim 59 or 90, wherein the disease or condition is a non-malignant disorder or an autoimmune disorder.

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