T cell receptors targeting npm1 neoantigens

Abstract

Provided herein are T cell receptors (TCRs) or antigen-binding fragments thereof, such as those that recognize or bind NPM1c neoantigens. In particular, the present disclosure relates to TCRs that bind or recognize specific NPM1c peptides in the context of major histocompatibility complex (MHC) molecules. The present disclosure also relates to nucleic acids encoding such TCRs, engineered cells comprising such TCRs, methods of isolating such TCRs, and uses thereof, e.g., in cell therapy.

Description

Cross-references to related applications

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 580,503, filed September 5, 2023, and U.S. Provisional Application No. 63 / 651,653, filed May 24, 2024, each of which is incorporated herein by reference.

[0002] sequence list The sequence list, written in the file BSB-0009WO01_SeqList.xml, is 383 kilobytes in size, was created on September 3, 2024, and is incorporated by reference. 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 hematologic malignancies and other non-malignant conditions. Some subjects may relapse after alloSCT or be unsuitable for alloSCT. Improved treatment methods are needed to achieve optimal outcomes. T-cell receptors (TCRs) and engineered T cells containing TCRs have been developed to meet these needs. Summary of the Invention

[0004] Anti-NPM1c T-cell receptors (anti-AIQDLCLAV TCR and anti-CLAVEEVSL TCR) or antigen-binding fragments thereof that recognize or bind to NPM1c neoantigens (i.e., AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5)). In some embodiments, the described anti-NPM1c TCR binds to or recognizes the peptide AIQDLCLAV (SEQ ID NO:1). In some embodiments, the described anti-NPM1c TCR binds to or recognizes the peptide CLAVEEVSL (SEQ ID NO:5). In some embodiments, the described anti-NPM1c TCR binds to or recognizes the peptide AIQDLCLAV (SEQ ID NO:1) in the context of major histocompatibility complex (MHC) molecules. In some embodiments, the described anti-NPM1c TCR binds to or recognizes the peptide CLAVEEVSL (SEQ ID NO:5) in the context of major histocompatibility complex (MHC) molecules. This disclosure also 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, for example, in T-cell therapy (e.g., engineered autologous T-cell therapy).

[0005] This article provides an anti-NPM1c TCR or its antigen-binding fragment, wherein the anti-NPM1c TCR or its antigen-binding fragment 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; wherein: (a) The Vα or Vγ region contains a complementary determination region 3 (CDR-3) containing SEQ ID NO:13, and the Vβ or Vδ region contains a CDR-3 containing SEQ ID NO:21; (b) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:31, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:39; (c) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:49, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:57; (d) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:67, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:75; (e) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:85, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:93; (f) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:103, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:111; (g) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:121, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:129; (h) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:139, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:147; (i) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:157, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:165; (j) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:175, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:183; (k) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:193, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:201; (l) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:211, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:219; (m) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:229, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:237; (n) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:247, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:255; (o) The Vα or Vγ region contains CDR-3 containing SEQ ID NO:265, and the Vβ or Vδ region contains CDR-3 containing SEQ ID NO:273; or (p) 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] The anti-NPM1c TRC recognition corresponding to any of (a)-(c) as described above is the AIQDLCLAV (SEQ ID NO: 1) peptide. The anti-NPM1c TRC recognition corresponding to any of (d)-(p) as described above is the CLAVEEVSL (SEQ ID NO: 5) peptide.

[0007] This article also provides an anti-NPM1c TCR or its antigen-binding fragment, wherein the anti-NPM1c TCR or its antigen-binding fragment 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; wherein: (a) The Vα 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 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 or Vγ region contains 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 or Vδ region contains 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 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 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 or Vγ region contains 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 or Vδ region contains 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 or Vγ region contains 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 or Vδ region contains 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 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 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 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 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 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 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 or Vγ region contains 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 or Vδ region contains 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 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 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 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 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 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 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 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 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 or Vγ region contains 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 or Vδ region contains 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 or Vγ region comprises 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 or Vδ region comprises CDR-1 containing SEQ ID NO:271, CDR-2 containing SEQ ID NO:272, and CDR-3 containing SEQ ID NO:273; or (p) 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] This article also provides an anti-NPM1c TCR or its antigen-binding fragment, wherein the anti-NPM1c TCR or its antigen-binding fragment 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; wherein: (a) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:14, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:22; (b) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:32, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:40; (c) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:50, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:58. (d) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:68, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:76. (e) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:86, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:94. (f) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:104, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:112. (g) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:122, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:130. (h) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:140, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:148. (i) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:158, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:166. (j) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:176, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:184. (k) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:194, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:202. (l) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:212, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:220. (m) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:230, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:238. (n) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:248, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:256. (o) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:266, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:274; or (p) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:284, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:292.

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

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

[0011] Also provided is an anti-NPM1c TCR or its antigen-binding fragment, said anti-NPM1c TCR or its antigen-binding fragment comprising: 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, wherein: (a) The Vα or Vγ region contains SEQ ID NO:14 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:22 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:11, CDR-2 containing SEQ ID NO:12 and CDR-3 containing SEQ ID NO:13, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:32 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:40 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:29, CDR-2 containing SEQ ID NO:30 and CDR-3 containing SEQ ID NO:31, and wherein the Vβ or Vδ region contains CDR-1 containing SEQ ID NO:37, CDR-2 containing SEQ ID NO:38 and CDR-3 containing SEQ ID NO:38; (c) The Vα or Vγ region contains SEQ ID NO:50 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:58 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:47, CDR-2 containing SEQ ID NO:48 and CDR-3 containing SEQ ID NO:49, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:68 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:76 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:65, CDR-2 containing SEQ ID NO:66 and CDR-3 containing SEQ ID NO:67, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:86 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:94 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:83, CDR-2 containing SEQ ID NO:84 and CDR-3 containing SEQ ID NO:85, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:104 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:112 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:101, CDR-2 containing SEQ ID NO:102 and CDR-3 containing SEQ ID NO:103, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:122 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:130 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:119, CDR-2 containing SEQ ID NO:120 and CDR-3 containing SEQ ID NO:121, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:140 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:148 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:137, CDR-2 containing SEQ ID NO:138 and CDR-3 containing SEQ ID NO:139, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:158 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:166 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:155, CDR-2 containing SEQ ID NO:156 and CDR-3 containing SEQ ID NO:157, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:176 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:184 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:173, CDR-2 containing SEQ ID NO:174 and CDR-3 containing SEQ ID NO:175, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:194 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:202 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:191, CDR-2 containing SEQ ID NO:192 and CDR-3 containing SEQ ID NO:193, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:212 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:220 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:209, CDR-2 containing SEQ ID NO:210 and CDR-3 containing SEQ ID NO:211, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:230 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:238 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:227, CDR-2 containing SEQ ID NO:228 and CDR-3 containing SEQ ID NO:229, and wherein the Vβ or Vδ region contains CDR-1 containing SEQ ID NO:235, CDR-2 containing SEQ ID NO:236 and CDR-3 containing SEQ ID NO:23; (n) The Vα or Vγ region contains SEQ ID NO:248 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:256 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:245, CDR-2 containing SEQ ID NO:246 and CDR-3 containing SEQ ID NO:247, and wherein the Vβ or Vδ region contains 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 or Vγ region contains SEQ ID NO:266 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:274 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:263, CDR-2 containing SEQ ID NO:264, and CDR-3 containing SEQ ID NO:265, and wherein the Vβ or Vδ region contains CDR-1 containing SEQ ID NO:271, CDR-2 containing SEQ ID NO:272, and CDR-3 containing SEQ ID NO:273; or (p) The Vα or Vγ region contains SEQ ID NO:284 or a sequence having at least 90% sequence identity with it, and the Vβ or Vδ region contains SEQ ID NO:292 or a sequence having at least 90% sequence identity with it, wherein the Vα or Vγ region contains CDR-1 containing SEQ ID NO:281, CDR-2 containing SEQ ID NO:282 and CDR-3 containing SEQ ID NO:283, and wherein the Vβ or Vδ region contains CDR-1 containing SEQ ID NO:289, CDR-2 containing SEQ ID NO:290 and CDR-3 containing SEQ ID NO:291.

[0012] In some implementation schemes, (a) The Vα or Vγ region contains SEQ ID NO:14, and the Vβ or Vδ region contains SEQ ID NO:22; (b) The Vα or Vγ region contains SEQ ID NO:32, and the Vβ or Vδ region contains SEQ ID NO:40; (c) The Vα or Vγ region contains SEQ ID NO:50, and the Vβ or Vδ region contains SEQ ID NO:58; (d) The Vα or Vγ region contains SEQ ID NO:68, and the Vβ or Vδ region contains SEQ ID NO:76; (e) The Vα or Vγ region contains SEQ ID NO:86, and the Vβ or Vδ region contains SEQ ID NO:94; (f) The Vα or Vγ region contains SEQ ID NO:104, and the Vβ or Vδ region contains SEQ ID NO:112; (g) The Vα or Vγ region contains SEQ ID NO:122, and the Vβ or Vδ region contains SEQ ID NO:130; (h) The Vα or Vγ region contains SEQ ID NO:140, and the Vβ or Vδ region contains SEQ ID NO:148; (i) The Vα or Vγ region contains SEQ ID NO:158, and the Vβ or Vδ region contains SEQ ID NO:166; (j) The Vα or Vγ region contains SEQ ID NO:176, and the Vβ or Vδ region contains SEQ ID NO:184; (k) The Vα or Vγ region contains SEQ ID NO:194, and the Vβ or Vδ region contains SEQ ID NO:202; (l) The Vα or Vγ region contains SEQ ID NO:212, and the Vβ or Vδ region contains SEQ ID NO:220; (m) The Vα or Vγ region contains SEQ ID NO:230, and the Vβ or Vδ region contains SEQ ID NO:238; (n) The Vα or Vγ region contains SEQ ID NO:248, and the Vβ or Vδ region contains SEQ ID NO:256; (o) The Vα or Vγ region contains SEQ ID NO:266, and the Vβ or Vδ region contains SEQ ID NO:274; or (p) The Vα or Vγ region contains SEQ ID NO:284, and the Vβ or Vδ region contains SEQ ID NO:292.

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

[0014] In some implementation schemes, (a) The α or γ chain comprises SEQ ID NO:17, and the β or δ chain comprises SEQ ID NO:25; (b) The α or γ chain comprises SEQ ID NO:35, and the β or δ chain comprises SEQ ID NO:43; (c) The α or γ chain comprises SEQ ID NO:53, and the β or δ chain comprises SEQ ID NO:61; (d) The α or γ chain contains SEQ ID NO:71, and the β or δ chain contains SEQ ID NO:79; (e) The α or γ chain contains SEQ ID NO:89, and the β or δ chain contains SEQ ID NO:97; (f) The α or γ chain contains SEQ ID NO:107, and the β or δ chain contains SEQ ID NO:115; (g) The α or γ chain comprises SEQ ID NO:125, and the β or δ chain comprises SEQ ID NO:133; (h) The α or γ chain comprises SEQ ID NO:143, and the β or δ chain comprises SEQ ID NO:151; (i) The α or γ chain contains SEQ ID NO:161, and the β or δ chain contains SEQ ID NO:169; (j) The α or γ chain contains SEQ ID NO:179, and the β or δ chain contains SEQ ID NO:187; (k) The α or γ chain comprises SEQ ID NO:197, and the β or δ chain comprises SEQ ID NO:205; (l) The α or γ chain comprises SEQ ID NO:215, and the β or δ chain comprises SEQ ID NO:223; (m) The α or γ chain contains SEQ ID NO:233, and the β or δ chain contains SEQ ID NO:241; (n) The α or γ chain contains SEQ ID NO:251, and the β or δ chain contains SEQ ID NO:259; (o) The α or γ chain comprises SEQ ID NO:269, and the β or δ chain comprises SEQ ID NO:277; or (p) The α or γ chain contains SEQ ID NO:287, and the β 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 the context of MHC molecules. In some embodiments, the MHC molecule is a human leukocyte antigen (HLA)-A molecule. In some embodiments, the HLA-A molecule is a serum-type HLA-A molecule. 02:01. In some implementations, the HLA-A molecule is serum HLA-A. 02:06. In some implementations, the HLA-A molecule is serum HLA-A. 02:03. In some embodiments, the NPM1c neoantigen is shown in SEQ ID NO:1 or 5.

[0016] Also provided are nucleotide sequences (e.g., polynucleotides or nucleic acids) encoding any of the anti-NPM1c TCRs described herein, or their antigen-binding fragments, or their Vα, Vγ, Vβ, Vδ, α chain, β chain, γ chain, or δ chain. The nucleotide sequences may be provided as a pair 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. If 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 internal ribosome entry sequence (IRES) and expressed by 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; wherein: (a) The first nucleotide sequence encodes SEQ ID NO:14 and the second nucleotide sequence encodes SEQ ID NO:22; (b) The first nucleotide sequence encodes SEQ ID NO:32 and the second nucleotide sequence encodes SEQ ID NO:40; (c) The first nucleotide sequence encodes SEQ ID NO:50 and the second nucleotide sequence encodes SEQ ID NO:58; (d) The first nucleotide sequence encodes SEQ ID NO:68 and the second nucleotide sequence encodes SEQ ID NO:76; (e) The first nucleotide sequence encodes SEQ ID NO:86 and the second nucleotide sequence encodes SEQ ID NO:94; (f) The first nucleotide sequence encodes SEQ ID NO:104 and the second nucleotide sequence encodes SEQ ID NO:112; (g) The first nucleotide sequence encodes SEQ ID NO:122 and the second nucleotide sequence encodes SEQ ID NO:130; (h) The first nucleotide sequence encodes SEQ ID NO:140 and the second nucleotide sequence encodes SEQ ID NO:148; (i) The first nucleotide sequence encodes SEQ ID NO:158 and the second nucleotide sequence encodes SEQ ID NO:166; (j) The first nucleotide sequence encodes SEQ ID NO:176 and the second nucleotide sequence encodes SEQ ID NO:184; (k) The first nucleotide sequence encodes SEQ ID NO:194 and the second nucleotide sequence encodes SEQ ID NO:202; (l) The first nucleotide sequence encodes SEQ ID NO:212 and the second nucleotide sequence encodes SEQ ID NO:220; (m) The first nucleotide sequence encodes SEQ ID NO:230 and the second nucleotide sequence encodes SEQ ID NO:238; (n) The first nucleotide sequence encodes SEQ ID NO:248 and the second nucleotide sequence encodes SEQ ID NO:256; (o) The first nucleotide sequence encodes SEQ ID NO:266 and the second nucleotide sequence encodes SEQ ID NO:274; or (p) The first nucleotide sequence encodes SEQ ID NO:284 and the second nucleotide sequence encodes SEQ ID NO:292.

[0018] The anti-NPM1c TRC recognition corresponding to any of (a)-(c) as described above is the AIQDLCLAV (SEQ ID NO: 1) peptide. The anti-NPM1c TRC recognition corresponding to any of (d)-(p) as described above is the CLAVEEVSL (SEQ ID NO: 5) peptide.

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

[0020] In some implementations, one or more nucleic acid sequences encoding Vα, Vγ, Vβ, Vδ, α constant strand, γ constant strand, β constant strand, or δ constant strand are codon-optimized. In some implementations, (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 contains SEQ ID NO:286, and the second nucleotide sequence contains SEQ ID NO:294.

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

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

[0023] In some embodiments, the nucleotide sequences encoding the α-chain and the nucleotide sequences encoding the β-chain are present on a single nucleic acid or vector. In some embodiments, the nucleotide sequences encoding the α-chain and the β-chain are present on a single nucleic acid or vector and expressed by a single promoter. In some embodiments, the nucleotide sequences encoding the α-chain and the β-chain are present on a single nucleic acid or vector, separated by a peptide sequence that induces ribosome jumping or self-cleavage, and expressed by a single promoter. The peptide that induces ribosome jumping can be, but is not limited to, a 2A peptide. The 2A peptide can be, but is not limited to, a P2A peptide. In some embodiments, the P2A peptide comprises SEQ ID NO:301.

[0024] In some implementations, the nucleotide sequence encodes an amino acid sequence of SEQ ID NO: 27, 45, 63, 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297.

[0025] In some implementations, the nucleotide sequence includes: (a) A sequence that is SEQ ID NO:28 or SEQ ID NO:27 and has at least 90% sequence identity with SEQ ID NO:28; (b) A sequence that is SEQ ID NO:46 or encoded as SEQ ID NO:45 and has at least 90% sequence identity with SEQ ID NO:46: (c) A sequence that is SEQ ID NO:64 or SEQ ID NO:63 and has at least 90% sequence identity with SEQ ID NO 64; (d) A sequence that is SEQ ID NO:82 or encoded as SEQ ID NO:81 and has at least 90% sequence identity with SEQ ID NO:82; (e) A sequence that is SEQ ID NO:100 or encoded as SEQ ID NO:99 and has at least 90% sequence identity with SEQ ID NO:100; (f) A sequence that is SEQ ID NO:118 or encoded as SEQ ID NO:117 and has at least 90% sequence identity with SEQ ID NO:118; (g) A sequence that is SEQ ID NO:136 or encoded as SEQ ID NO:135 and has at least 90% sequence identity with SEQ ID NO:136; (h) A sequence that encodes SEQ ID NO:154 or SEQ ID NO:153 and has at least 90% sequence identity with SEQ ID NO:154; (i) A sequence that is SEQ ID NO:172 or encoded as SEQ ID NO:171 and has at least 90% sequence identity with SEQ ID NO:172; (j) A sequence that is SEQ ID NO:190 or encoded as SEQ ID NO:189 and has at least 90% sequence identity with SEQ ID NO:190; (k) A sequence that is SEQ ID NO:208 or SEQ ID NO:207 and has at least 90% sequence identity with SEQ ID NO:208; (l) A sequence that is SEQ ID NO:226 or encoded as SEQ ID NO:225 and has at least 90% sequence identity with SEQ ID NO:226; (m) A sequence that is SEQ ID NO:244 or encoded as SEQ ID NO:243 and has at least 90% sequence identity with SEQ ID NO:244; (n) A sequence that is SEQ ID NO:262 or encoded as SEQ ID NO:261 and has at least 90% sequence identity with SEQ ID NO:262; (o) A sequence encoding SEQ ID NO:280 or SEQ ID NO:279 that has at least 90% sequence identity with SEQ ID NO:280; or (p) A sequence that is SEQ ID NO:298 or encoded as SEQ ID NO:297 and has at least 90% sequence identity with SEQ ID NO:298.

[0026] This document also provides vectors containing any of the polynucleotides provided herein. In some embodiments, the vector is a viral vector. Viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, and adeno-derived vectors, with retroviral vectors including, but 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, small loops, mRNA, and CRISPR constructs.

[0027] This document also provides engineered cells comprising any of the anti-NPM1c TCRs or their antigen-binding fragments provided herein. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is heterologous to the cell. This document also provides engineered cells comprising any of the nucleotide sequences provided herein or any of the vectors provided herein. In some embodiments, the nucleotide sequence encoding the anti-NPM1c TCR or its antigen-binding fragment is heterologous to the cell. The cell may be, but is not limited to, T cells, cell line T cells, primary T cells, donor T cells (i.e., T cells derived from a subject who is not a patient), or autologous T cells (e.g., T cells derived from a subject to be treated with engineered T cells).

[0028] This document also provides methods for generating engineered cells, which include introducing any of the polynucleotides provided herein or any of the vectors provided herein into cells to form engineered cells. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment is heterologous 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 subject who is not a patient), or autologous T cells (i.e., T cells derived from a subject to be treated with engineered T cells).

[0029] Soluble anti-NPM1c TCR and anti-NPM1c TCR bispecific molecules are also provided. The anti-NPM1c TCR bispecific molecules comprise fusion proteins containing an antigen-binding domain from any of the described anti-NPM1c TCRs and a second antigen-binding domain that specifically binds to a second antigen, wherein the second antigen is not a neo-NPM1c antigen. The second antigen may be, 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 be, 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 or fused to an immune cell adaptor. The immune cell adaptor may be, but is not limited to, a T cell adaptor (e.g., a CD3-binding domain (e.g., anti-CD3 scFv)), a macrophage adaptor, or an NK cell adaptor. In some embodiments, the soluble anti-NPM1c TCR or its antigen-binding fragment is linked or fused to an Fc peptide, a modified Fc peptide, or a Tc receptor or a modified Fc receptor.

[0031] This document also provides compositions comprising any of the provided anti-NPM1c TCRs or their antigen-binding fragments, any of the provided polynucleotides, any of the provided vectors, or any of the provided engineered cells. In some embodiments, the composition further comprises pharmaceutically acceptable excipients.

[0032] This article also provides treatment options, including those for patients or those diagnosed with NPM1c. + Cancer subjects are administered any of the anti-NPM1c TCRs or their 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. This document also provides information on the use of NPM1c in treating subjects. + This document describes the use of any of the anti-NPM1c TCRs or their 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 the manufacture of NPM1c TCRs for treating subjects. The document also provides for the use of any of the anti-NPM1c TCRs or their 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 the manufacture of NPM1c TCRs for treating subjects. + Use in cancer drugs. In some implementations, the engineered T cells are engineered autologous T cells. In some implementations, NPM1c + Cancer is AML.

[0033] In some implementations, treatment for patients with or diagnosed with NPM1c is described. + Methods for treating cancer subjects, including administering any of the engineered T cells described herein to the subject. In some embodiments, any of the engineered T cells described herein are used to treat the subject with NPM1c. + Cancer. In some implementations, any of the engineered T cells described herein can be used to manufacture NPM1c for treating subjects. + In cancer drugs, in some implementations, the engineered T cells are engineered autologous T cells. In some implementations, NPM1c... + Cancer is AML.

[0034] In some implementations, treatment for patients with or diagnosed with NPM1c is described. + Methods for treating cancer subjects, including administering to a subject either a soluble anti-NPM1c TCR (or its antigen-binding fragment) or an anti-NPM1c TCR bispecific molecule as described 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 to treat a subject with NPM1c. + Cancer. In some embodiments, either the soluble anti-NPM1c TCR (or its antigen-binding fragment) or the anti-NPM1c TCR bispecific molecule provided herein can be used to manufacture NPM1c for treating subjects. + In cancer medications.

[0035] In some implementations, the subject is not a suitable candidate for standard care transplantation. In some implementations, the subject is FLT3 positive. In some implementations, the subject has or has been diagnosed with AML. In some implementations, the subject has NPM1c. + Cancer and one or more comorbidities or risk factors. In some implementations, the subject has AML and one or more comorbidities or risk factors. In some implementations, the subject has NPM1c. + Cancer and unsuitable for high-intensity chemotherapy. In some embodiments, the subject has AML and is unsuitable 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 has no response to high-intensity chemotherapy, has not responded, or has failed to achieve complete remission (refractory to high-intensity chemotherapy). In some embodiments, the subject has relapsed after prior hypomethylating agent therapy. In some embodiments, the subject has no response to hypomethylating agent therapy, has not responded, or has failed to achieve complete remission (refractory to hypomethylating agent therapy). In some embodiments, the subject has relapsed or has not responded to at least two prior therapies. Relapse can occur, for example, after complete remission (CR), CR with partial hematologic 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 therapy. In some implementations, the subject has AML and is refractory to at least two prior therapies.

[0036] In some implementations, the administration of engineered cells or compositions induces or enhances cell death associated with malignant blood disorders in subjects, or induces or enhances graft-versus-leukemia (GVL) effects. Attached Figure Description

[0037] Figure 1 A graph illustrating the activation of engineered T cells expressing TCR D in response to pulses of the CLAVEEVSL (SEQ ID NO:5) peptide.

[0038] Figure 2 A graph illustrating the killing of target cells by engineered T cells expressing TCR D by pulsed cells with the CLAVEEVSL (SEQ ID NO:5) peptide.

[0039] Figure 3 A graph illustrating the killing of target cells by engineered T cells expressing TCR D against cells naturally expressing the CLAVEEVSL (SEQ ID NO:5) peptide.

[0040] Figure 4 A chart illustrating the efficacy of engineered T cells compared to CAR T cells.

[0041] Figure 5 Examples of cytotoxicity (top) and EC50 of engineered T cells compared to CAR T cells. 50 The chart at the bottom.

[0042] Figure 6 A graph illustrating the reactivity of TCR D to cysteylated and non-cysteylated target peptides.

[0043] Figure 7 A graph illustrating the TCR F identification of the CLAVEEVSL (SEQ ID NO:5) variant is shown. The peptide numbers correspond to the peptides in Table 11. Each peptide was tested sequentially at concentrations of 5 μg / mL, 0.16 μg / mL, 0.015 μg / mL, and 0 μg / mL.

[0044] Figure 8 A graph illustrating the TCR D identification of the CLAVEEVSL (SEQ ID NO:5) variant is shown. The peptide numbers correspond to the peptides in Table 11. Each peptide was tested sequentially at concentrations of 5 μg / mL, 0.16 μg / mL, 0.015 μg / mL, and 0 μg / mL. Detailed Implementation

[0045] This document provides anti-NPM1c TCRs, including recombinant anti-NPM1c TCRs, that bind to or recognize neoantigens associated with NPM1c. The provided embodiments are methods for treating 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 TCR and its antigen-binding fragment 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 TCR and its antigen-binding fragment specifically bind to or recognize neoantigens having the sequence AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules. In some embodiments, the provided anti-NPM1c TCR and its antigen-binding fragment are HLA-A 02:01, HLA-A 02:06 or HLA-A In the context of 02:03, it specifically binds to or recognizes new antigens with the sequence AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5).

[0046] In some embodiments, the described anti-AIQDLCLAV (SEQ ID NO:1) anti-NPM1c TCR targets HLA subtype A on cells (e.g., cancer cells). 02:01, HLA subtype A 02:06 or HLA subtype A 02:03 Upon presentation, it specifically binds to the AIQDLCLAV (SEQ ID NO:1) neoantigen, in conjunction with HLA subtype A 02:01, HLA subtype A 02:06 or HLA subtype A 02:03 Other HLA subtypes or other non-target antigens presented do not specifically bind to the AIQDLCLAV (SEQ ID NO:1) neoantigen.

[0047] In some embodiments, the described anti-CLAVEEVSL (SEQ ID NO: 5) anti-NPM1c TCR targets HLA subtype A on cells (e.g., cancer cells). 02:01, HLA subtype A 02:06 or HLA subtype A 02:03 Upon presentation, it specifically binds to the CLAVEEVSL (SEQ ID NO: 5) neoantigen, in conjunction with HLA subtype A. 02:01, HLA subtype A 02:06 or HLA subtype A 02:03 Other HLA subtypes or other non-target antigens presented do not specifically bind to the CLAVEEVSL (SEQ ID NO:5) neoantigen.

[0048] This document also provides nucleic acid molecules encoding anti-NPM1c TCRs, engineered cells containing anti-NPM1c TCRs, compositions containing anti-NPM1c TCRs or cells, and therapeutic methods or uses (such as therapeutic uses) relating to 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 aspects, engineered cells expressing the provided anti-NPM1c TCRs or their antigen-binding fragments exhibit cytotoxic activity against target cells (such as cancer cells) expressing NPM1c neoantigens. This document also provides methods for identifying anti-NPM1c TCRs targeting neoantigens having the sequence AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5).

[0049] Cell therapies (including those involving the administration of cells engineered to express recombinant receptors or anti-NPM1c TCRs (such as recombinant anti-NPM1c TCRs and / or other recombinant antigen receptors) specific to a disease or condition of interest) and other adoptive immune cell and adoptive T cell therapies can be effective in treating diseases and disorders. T cells engineered to express the described anti-NPM1c TCR (or its antigen-binding fragment) recognize and bind to targets within the subject (e.g., target antigens, such as peptide epitopes of AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5)) based on the affinity of the antigen-binding domain of the anti-NPM1c TCR for its target antigen.

[0050] In some embodiments, T cells engineered to express heterologous anti-NPM1c TCRs (e.g., anti-AIQDLCLAV TCRs or anti-CLAVEEVSL TCRs) are further modified to knock out endogenous TCRs. Knockout of endogenous TCRs may include knockout of expression of endogenous TRAC genes and endogenous TRBC genes. In some embodiments, CRISPR may be used to knock out endogenous TCRs (endogenous TRAC genes and / or endogenous TRBC genes). Knockout of endogenous TCRs includes, but is not limited to, deleting the entire target gene or a port of the target gene, inserting one or more stop codons into the target gene, disrupting the target gene, or disrupting the promoter or splice region of the target gene. Knockout of endogenous TCRs reduces or eliminates mismatches between heterologous anti-NPM1c TCRs (α and / or β chains) and endogenous TCRs (α and / or β chains).

[0051] In some respects, the provided anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR exhibits improved affinity for the NPM1c neoantigen. In some respects, engineered T cells expressing either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR exhibit improved activity against cells expressing the NPM-1c neoantigen (e.g., cancer cells). In some respects, engineered T cells expressing either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR exhibit improved activity against cells expressing the NPM-1c neoantigen at a low effector to target (E:T) ratio (e.g., cancer cells).

[0052] In some respects, the use of the described anti-NPM1c TCR treatments, such as in adoptive cell therapy using engineered human T cells expressing the described anti-NPM1c TCR, enables the targeting and killing of cancer cells expressing the NPM1c neoantigen, thereby producing a graft-versus-leukemia (GVL) effect.

[0053] All publications (including patent documents, scientific papers, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated separately by reference. If any definition set forth herein is contrary to or otherwise inconsistent with the definitions shown in patents, applications, published applications, and other publications incorporated herein by reference, the definitions shown herein shall prevail over those incorporated herein by reference.

[0054] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0055] I. T-cell receptors targeting the NPM1 neoantigen This document provides anti-NPM1c TCRs, such as those that bind to or recognize NPM1c-associated peptide epitopes (such as peptide neoantigens expressed on the surface of immune cells, cancer cells, and / or cells associated with blood disorders) in the context of MHC molecules. 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 the context of MHC molecules. Such anti-NPM1c TCRs and antigen-binding fragments exhibit antigen specificity for binding to or recognizing such peptide epitopes. Also provided are (a) nucleic acids encoding anti-NPM1c TCRs (or their antigen-binding domains), (b) engineered cells expressing anti-NPM1c TCRs (or their antigen-binding domains), (c) compositions comprising anti-NPM1c TCRs, nucleic acids encoding anti-NPM1c TCRs, or engineered cells expressing anti-NPM1c TCRs, and (d) treatment methods comprising administering the described anti-NPM1c TCRs (or their antigen-binding domains), nucleic acids, engineered cells, or compositions. In some respects, engineered cells expressing the provided anti-NPM1c TCR or its antigen-binding fragment are effective against target cells expressing NPM1c neoantigens (such as NPM1c TCR). + Cancer cells exhibit cytotoxic activity.

[0056] In some implementations, the described anti-NPM1c TCR recognizes neoantigens of NPM1c expressed by AML cancer cells (e.g., AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5)).

[0057] A.NPM1c Nucleophosphorus proteins (NPMs, also known as nucleolar phosphoprotein B23 or numatrin) are encoded by the NPM1 gene in humans. The NPM1 gene is upregulated and mutated in certain tumor types, including leukemia, acute myeloid leukemia (AML), and myelodysplastic syndromes. Mutations in NPM1 typically consist of a 4 bp insertion or duplication between nucleotides 960 and 961. These result in the substitution of the last seven amino acids (WQWRKSL) with 11 different residues, leading to the cytoplasmic localization of the protein (NPM1c).

[0058] 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-mutated NPM1 is AIQDLWQWRKSL (SEQ ID NO:2). NPM1c neoantigens can be found in class I MHC molecules (e.g., HLA-A). 02:01, HLA-A 02:06 or HLA-A Presented in the context of 02:03). The NPM1c neoantigen AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) is typically expressed by leukemia cells (e.g., AML cells) and is not typically expressed by non-leukemia cells.

[0059] In some aspects, the provided anti-NPM1c TCR or its antigen-binding fragment recognizes or specifically binds to the immunogenic epitope of NPM1c. In some examples, the provided anti-NPM1c TCR or its antigen-binding fragment specifically binds to or recognizes the NPM1c neoantigen 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 aspects, 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 the context of MHC molecules such as MHC class I molecules. In some aspects, the MHC class I molecule is an HLA-A2 molecule, including any one or more of its subtypes, such as HLA-A 02:01 02:06 or 02:03.

[0060] In some embodiments, anti-NPM1c TCRs or soluble anti-NPM1c TCRs or their antigen-binding fragments are isolated or purified. In certain embodiments, any of the provided anti-NPM1c TCRs or their antigen-binding fragments are recombinant. In some aspects, the anti-NPM1c TCRs or their antigen-binding fragments are human. In some aspects, the anti-NPM1c TCRs or their antigen-binding fragments are single-stranded. In other embodiments, the anti-NPM1c TCRs contain two strands. In some embodiments, the anti-NPM1c TCRs or their antigen-binding fragments are expressed on the surface of cells (e.g., T cells, such as T cells engineered to lack endogenous TCR expression). In some embodiments, the soluble anti-NPM1c TCRs are secreted by the cells.

[0061] In some aspects, the provided anti-NPM1c TCR or its antigen-binding fragment has one or more specific functional characteristics, such as binding properties, including, for example, binding to a specific epitope and / or a specific binding affinity, as described herein. In some aspects, engineered cells (such as engineered T cells) expressing the provided anti-NPM1c TCR (or its antigen-binding fragment) have one or more specific functional characteristics, such as binding properties, including, for example, binding to a specific epitope, a specific binding affinity, activation or stimulation of cell signaling (such as T cell signaling or TCR signaling), secretion of cytokines, and / or killing of target cells expressing or presenting antigens, as described herein.

[0062] This provides anti-NPM1c TCRs or their antigen-binding fragments that specifically bind to the NPM1c neoantigen. A TCR is an α-chain containing a Vα region and a β-chain containing a Vβ region (also referred to as TCRα and TCRβ, respectively), or a γ-chain containing a Vγ region and a δ-chain containing a Vδ region (also referred to as TCRα and TCRβ, respectively). TCRs (and TCRδ) molecules or their antigen-binding portions are capable of specifically binding to antigens, such as peptide antigens or peptide epitopes that bind to MHC molecules. In some embodiments, anti-NPM1c TCRs are in the αβ form (e.g., αβ TCRs). In some embodiments, anti-NPM1c TCRs are in the γδ form (e.g., γδ TCRs). Generally, TCRs present in αβ or γδ forms are structurally similar, but T cells expressing them may have different anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells, where they are generally responsible for recognizing antigens, such as peptides that bind to MHC molecules.

[0063] In some embodiments, the anti-NPM1c TCR provided herein may be a full-length or complete TCR, such as a TCR containing a full-length α chain and a full-length β chain, or a TCR containing a full-length γ chain and a full-length δ chain. In some embodiments, the antigen-binding moiety of the TCR provided herein may be smaller than that of the full-length TCR, provided that it binds to a specific peptide bound to an MHC molecule. In some embodiments, the antigen-binding moiety or fragment of the TCR may contain only a portion of the domains of a full-length or complete TCR, but may still be capable of binding to peptide epitopes bound to a full-length TCR, such as MHC-peptide complexes. In some embodiments, the antigen-binding moiety contains variable domains of the TCR, such as the Vα and Vβ regions or the Vγ and Vδ regions of the TCR provided herein, provided that the antigen-binding moiety is sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0064] The variable domains of a TCR contain 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 domain of the TCR chain are typically separated by frame regions (FRs), which generally exhibit less variability between TCRs compared to the CDRs. CDR-3 is the dominant CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on the variable domain of a given TCR for antigen recognition and / or for interaction with the processed peptide moiety of the peptide-MHC complex. In some contexts, CDR-1 of the α-chain interacts with the N-terminal moiety of certain antigenic peptides. In some contexts, CDR-1 of the β-chain interacts with the C-terminal moiety of the peptide. In some contexts, CDR-2 contributes most strongly to the dominant CDR responsible for interaction with or recognition of the MHC moiety of the MHC-peptide complex, or is the dominant CDR responsible for these functions.

[0065] 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 the following: a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. In some aspects, each chain of the TCR (e.g., α or β) contains an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR (e.g., via the cytoplasmic tail) associates with invariant proteins of the CD3 complex involved in mediating signal transduction. In some embodiments, this structure allows the TCR to associate with other molecules such as CD3 and its subunits. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of the CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more activating motifs or ITAMs based on immunoreceptor tyrosine residues and are generally involved in the signal transduction capabilities of the TCR complex.

[0066] Various domains or regions of the TCR can be identified. In some embodiments, the exact locus of a domain or region may vary based on a specific structure or homology modeling or other features used to describe a particular domain. It should be understood that references to amino acids (including specific sequences as shown in SEQ ID NO:) to describe the domain organization of the TCR are for illustrative purposes and do not imply limitation on the scope of the provided embodiments. In some embodiments, a particular domain (e.g., variable or constant) may be several amino acids long or short (e.g., one, two, three, or four). In some respects, the residues of the TCR are known or can be determined according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM ® The (IMGT) numbering system was used for identification (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 in the TCR Vα and / or Vβ regions corresponded in some cases to amino acids present between residue numbers 27-38 (inclusive), the CDR-2 sequence in the TCR Vα and / or Vβ regions corresponded in some cases to amino acids present between residue numbers 56-65 (inclusive), and the CDR-3 sequence in the TCR Vα and / or Vβ regions corresponded in some cases to amino acids present between residue numbers 105-117 (inclusive).

[0067] In some embodiments, an anti-NPM1c TCR or its antigen-binding fragment is provided, provided that it specifically binds to or recognizes the NPM1c neoantigen AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules. In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment does not recognize or specifically binds to the NPM1 peptide AIQDLWQWRKSL (SEQ ID NO:2). In some embodiments, the provided anti-NPM1c TCR or its antigen-binding fragment specifically recognizes or binds to AIQDLCLAV (SEQ ID NO:1) and 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 AIQDLCLAV (SEQ ID NO:1) with an increased affinity relative 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) and 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 relative to binding to AIQDLWQWRKSL (SEQ ID NO: 2).

[0068] In some respects, the provided anti-NPM1c TCR or its antigen-binding fragment specifically binds to or recognizes MHC molecules of a specific HLA type (such as HLA-A). 02:01, HLA-A 02:06 or HLA-A 02:03) A combination of AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5).

[0069] 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 dimer 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 cell-bound or in a soluble form. In some embodiments, the anti-NPM1c TCR provided herein is in a cell-bound form expressed on the surface of cells (e.g., T cells, such as T cells engineered to lack endogenous TCR expression).

[0070] In some embodiments, the anti-NPM1c TCR provided herein is a scTCR, which is a single amino acid chain containing both α and β chains capable of binding to an MHC-peptide complex. Typically, scTCRs can be generated as described elsewhere, see, for example, WO96 / 13593, WO 96 / 18105, WO99 / 18129, WO 04 / 033685, WO2006 / 037960, WO2011 / 044186; U.S. Patent No. 7,569,664; and Schlueter, CJ et al., J. Mol. Biol. 256, 859 (1996).

[0071] B. Exemplary variable structural domain This article provides anti-NPM1c TCRs or their antigen-binding fragments that specifically recognize or bind to the neoantigens AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules. Exemplary sequences (e.g., CDR, Vα and / or Vβ or Vγ and / or Vδ, and constant region sequences) of AIQDLCLAV (SEQ ID NO:1)-specific TCRs or CLAVEEVSL (SEQ ID NO:5)-specific TCRs are provided.

[0072] 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 myelodysplastic syndrome cells.

[0073] In some respects, when T cells come into contact with target cells that present or express the neoantigen of NPM1c (AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5)), they stimulate the cytotoxic activity of T cells expressing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR.

[0074] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment contains any of the Vα and Vβ regions or Vγ and Vδ regions as described herein, or a sufficient antigen-binding portion of such sequences. In some embodiments, the described anti-NPM1c TCR or its antigen-binding fragment contains Vα and Vβ regions or Vγ and Vδ regions comprising the CDR-3 sequence as described herein, or a sufficient antigen-binding portion thereof. In some embodiments, the described anti-NPM1c TCR or its antigen-binding fragment contains Vα and Vβ regions or Vγ and Vδ regions comprising the CDR-1, CDR-2, and CDR-3 sequences as described herein, or a sufficient antigen-binding portion thereof. Additionally, the provided anti-NPM1c TCR includes those having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0075] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein comprises a Vα or Vγ region containing CDR-3, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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 respects, the anti-NPM1c TCR or its antigen-binding fragment provided herein comprises a Vα or Vγ region containing CDR-3, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 and 284 or in a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence.

[0076] In some embodiments, the Vα or Vγ region contains CDR-1, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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 respects, the Vα or Vγ region contains CDR-1, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 and 284 or in 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, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 and 284 or in a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence.

[0077] In some cases, the anti-NPM1c TCR or its antigen-binding fragment provided herein contains a Vβ or Vδ region containing CDR-3, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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 its antigen-binding fragment provided herein comprises a Vβ or Vδ region containing CDR-3, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 and 292 or in a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence.

[0078] In some cases, the Vβ or Vδ region contains CDR-1, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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 respects, the Vβ or Vδ region contains CDR-1, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 and 292 or in 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, which comprises an amino acid sequence shown in any one of SEQ ID NO: 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, which is contained in an amino acid sequence shown in any one of SEQ ID NO: 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.

[0079] In some embodiments, the Vα or Vγ region contains an amino acid sequence shown in any one of SEQ ID NO: 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 it; and the Vβ or Vδ region contains an amino acid sequence shown in any one of SEQ ID NO: 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 it.

[0080] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules, may comprise an α chain (or γ chain) having: CDR-1 having the amino acid sequences shown in SEQ ID NO:11, 29, 47 and 65 (or variants thereof having one or two amino acid modifications), CDR-2 having the amino acid sequences shown in SEQ ID NO:12, 30, 48 and 66 (or variants thereof having one or two amino acid modifications), and CDR-3 having the amino acid sequences shown in SEQ ID NO:13, 31 and 49 (or variants thereof having one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequences shown in SEQ ID NO:19, 37 and 55 (or variants thereof having one or two amino acid modifications), CDR-2 having the amino acid sequences shown in SEQ ID NO:13, 31 and 49 (or variants thereof having one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequences shown in SEQ ID NO:19, 37 and 55 (or variants thereof having one or two amino acid modifications), CDR-3 ...9, 37 and 55 (or variants thereof having one or two amino acid modifications), CDR-3 having the amino acid sequences shown in SEQ ID NO:19, 37 and 55 (or variants thereof having one or two amino acid modifications), CDR-3 having the amino acid sequences shown CDR-2 has the amino acid sequences shown in NO:20, 38, and 56 (or variants thereof with one or two amino acid modifications), and CDR-3 has the amino acid sequences shown in SEQ ID NO:21, 39, and 57 (or variants thereof with one or two amino acid modifications). Anti-NPM1c TCRs having these CDRs and the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules include TCR A, TCR B, or TCR C (see Table 1).

[0081] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, provided herein may comprise an α chain (or γ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263 or 281 (or a variant thereof having one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO:66, 84, 102, 120, 138, 156, 174, 192, 210, 228, 246, 264 or 282 (or a variant thereof having one or two amino acid modifications), and having SEQ ID NO: CDR-3 having the amino acid sequence shown in SEQ ID NO: 77, 85, 103, 121, 139, 157, 175, 193, 211, 229, 247, 265 or 283 (or a variant thereof having one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO: 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271 or 289 (or a variant thereof having one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO: 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272 or 290 (or a variant thereof having one or two amino acid modifications), and having ..., and having SEQ ID NO: 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272 or 290, and having CDR-3s containing the amino acid sequences shown in NO:75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, or 291 (or variants thereof having one or two amino acid modifications). Anti-NPM1c TCRs possessing these CDRs and the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of 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).

[0082] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein, having the ability to specifically bind to NPM1c neoantigens in the context of MHC molecules and having the following, may comprise any suitable frame region: an α chain (or γ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263 or 281 (or a variant thereof having one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO: 12, 30, 48, 66, 84, 102, 120, 138, 156, 174, 192, 210, 228, 246, 264 or 282 (or a variant thereof having one or two amino acid modifications), and having SEQ ID NO: 12, 30, 48, 66, 84, 102, 120, 138, 156, 174, 192, 210, 228, 246, 264 or 282, ... CDR-3 having the amino acid sequence shown in SEQ ID NO:19;37,55,73,91,109,127,145,163,181,199,217,235,253,271 or289 (or a variant thereof having one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:19;37,55,73,91,109,127,145,163,181,199,217,235,253,271 or289 (or a variant thereof having one or two amino acid modifications); and having SEQ ID NO:19;37,55,73,91,109,127,145,163,181,199,217,235,253,271 or289; CDR-2 having the amino acid sequence shown in SEQ ID NO: 20, 38, 56, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 254, 272 or 290 (or a variant thereof having one or two amino acid modifications) and CDR-3 having the amino acid sequence shown in SEQ ID NO: 21, 39, 57, 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273 or 291 (or a variant thereof having one or two amino acid modifications). For example, such an anti-NPM1c TCR or its antigen-binding fragment may comprise: an α chain,The α chain comprises a frame region 1, located upstream of the corresponding CDR1 amino acid sequence (or a variant thereof having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) of SEQ ID NO:11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263 or 281, having the complete amino acid sequence shown in SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284, of SEQ ID NO:14, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227, 245, 263 or 281 ...4, of SEQ ID NO:14, 29, 47, 65, 83, 101, 119, 137, 155, 173, The frame region 2, located between the corresponding CDR1 and CDR2 amino acid sequences (or variants thereof having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) of SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284, of SEQ ID NO: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, of the complete amino acid sequence shown in SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284, of SEQ ID NO: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, 26 The corresponding CDR2 and CDR3 amino acid sequences (variants having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) between SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284, and the frame region 3 of the complete amino acid sequence shown in SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284 and located in SEQ ID NO: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 229, 246 and 247, 264 and 264 or 282 and 283, are frame regions 3 of the complete amino acid sequences shown in SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284 and located in SEQ ID Frame region 4 downstream of the corresponding CDR3 amino acid sequence (or a variant thereof having one, two, three, four, or five amino acid modifications) of SEQ ID NO:13, 31, 49, 67, 85, 103, 121, 139, 157, 175, 193, 211, 229, 247, 265, or 283, having the complete amino acid sequence shown in SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284; and the β chain,The β chain comprises a frame region 1, located upstream of the corresponding CDR1 amino acid sequence (or a variant thereof having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) of SEQ ID NO:19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217, 235, 253, 271 or 289, having the complete amino acid sequence shown in SEQ ID NO:22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292, of .... The frame region 2, located between the corresponding CDR1 and CDR2 amino acid sequences (or variants thereof having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) of SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292 of SEQ ID NO: 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 and 218, 235 and 236, 253 and 254, 271 and 272 or 289 and 290, of the complete amino acid sequence shown in SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292, of SEQ ID NO: 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 and 218, 235 and 236, 253 and 254, The frame region 3 of the corresponding CDR2 and CDR3 amino acid sequences (or variants of the sequence having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications) between SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292 and located in SEQ ID NO: 20 and 21, 38 and 39, 56 and 57, 74 and 75, 92 and 93, 110 and 111, 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, is the complete amino acid sequence shown in SEQ ID NO: 22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292 and located in SEQ ID NO: 290 or 291. Frame region 4 downstream of the corresponding CDR3 amino acid sequence (or a variant of that sequence having one, two, three, four, or five amino acid modifications) of SEQ ID NO:21, 39, 57, 75, 93, 111, 129, 147, 165, 183, 201, 219, 237, 255, 273, or 291, containing the complete amino acid sequence shown in SEQ ID NO:22, 40, 58, 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292.

[0083] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules, may comprise an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:14, 32, or 50; and a β chain containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:22, 40, or 58. For example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14, 32, or 50; and a β chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 22, 40, or 58. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain and a β chain containing an amino acid sequence having 100% identity with the amino acid sequence shown in (a) SEQ ID NO: 14 and 22; (b) SEQ ID NO: 32 and 40; or (c) SEQ ID NO: 50 and 58.

[0084] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment having the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules provided herein may comprise (a) an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the Vα domain of any one of TCR A, TCR B, and TCR C (i.e., as shown in SEQ ID NO:14, 32, and 50), provided that the α chain contains the corresponding CDR 1, CDR 2, and CRD 3 amino acid sequences of the TCR α chain (e.g., SEQ ID NO:29, 30, and 31 for TCR B); and (b) a β chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the Vβ domain of the corresponding TCR (i.e., as shown in SEQ ID NO:22, 40, and 58), provided that the β chain contains the corresponding CDR 1, CDR 2, and CRD 3 amino acid sequences of the TCR β chain (e.g., SEQ ID NO:29, 30, and 58 for TCR B). (NO:37, 38, and 39). For example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise (a) an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:32, provided that the α chain contains the amino acid sequences shown in SEQ ID NO:29, 30, and 31; and (b) a β chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40, provided that the β chain contains the amino acid sequences shown in SEQ ID NO:37, 38, and 39.

[0085] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, provided herein may comprise an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: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% identity with the amino acid sequence shown in SEQ ID NO:76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292. For example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 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% identity with the amino acid sequence shown in SEQ ID NO: 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 comprise an α-chain and a β-chain, the α-chain and β-chain comprising (d) SEQ ID NO: 68 and 76; (e) SEQ ID NO: 86 and 94; (f) SEQ ID NO: 104 and 112; (g) SEQ ID NO: 122 and 130; (h) SEQ ID NO: 140 and 148; (i) SEQ ID NO: 158 and 166; (j) SEQ ID NO: 176 and 184; (k) SEQ ID NO: 194 and 202; (l) SEQ ID NO: 212 and 220; (m) SEQ ID NO: 230 and 238; (n) SEQ ID NO: 248 and 256; (o) SEQ ID NO: 266 and 274; or (p) SEQ ID The amino acid sequences shown in NO:284 and 292 are 100% identical.

[0086] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, provided herein may comprise (a) an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the Vα domain of any of TCR D to TCR P (i.e., as shown in SEQ ID NO: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, or 284), provided that the α chain contains the corresponding CDR 1, CDR 2, and CRD 3 amino acid sequences of the TCR α chain (e.g., SEQ ID NO: 65, 66, and 67 for TCR D; SEQ ID NO: 83, 84, and 85 for TCR E; or SEQ ID NO: 101, 102, and 103 for TCR F); and (b) A β chain comprising an amino acid sequence having at least 90% identity with the amino acid sequence of the Vβ domain of the corresponding TCR (i.e., as shown in SEQ ID NO:76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292), provided that the β chain comprises the corresponding CDR 1, CDR2 and CRD3 amino acid sequences of the TCR β chain (e.g., SEQ ID NO:73, 74 and 75 for TCR D; SEQ ID NO:91, 92 and 93 for TCR E; and SEQ ID NO:109, 110 and 111 for TCR F). For example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise (a) an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 68, provided that the α chain contains the amino acid sequences shown in SEQ ID NO: 65, 66, and 67; and (b) a β chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 76, provided that the β chain contains the amino acid sequences shown in SEQ ID NO: 73, 74, and 75.

[0087] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules, provided herein may comprise a CDR sequence of TCR A, B, or C and (a) an α chain having the amino acid sequence shown in SEQ ID NO:14, 32, or 50 or amino acids shown in SEQ ID NO:14 having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition); and (b) a corresponding β chain (see Table 1) having the amino acid sequence shown in SEQ ID NO:22, 40, or 58 or amino acids shown in SEQ ID NO:22, 40, or 58 having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition). For example, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules, provided herein may comprise (a) an α chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:32, provided that the α chain contains the CDR amino acid sequence shown in SEQ ID NO:29, 30, and 31; and (b) a β chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:40, provided that the β chain contains the CDR amino acid sequence shown in SEQ ID NO:37, 38, or 39.

[0088] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, provided herein may contain the CDR sequence of any of TCR D-TCR P and (a) the corresponding α chain ( See(Table 1) The α chain has the amino acid sequence shown in SEQ ID NO: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284, or the amino acid with one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion and / or amino acid addition) shown in SEQ ID NO: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284; and (b) the corresponding β chain (see Table 1), which has the amino acid sequence shown in SEQ ID NO: 76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274 or 292, or SEQ ID NO: 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266 or 284. Amino acids having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in NO:76, 94, 112, 130, 148, 166, 184, 202, 220, 238, 256, 274, or 292. For example, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, provided herein may comprise (a) an α chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:68, provided that the α chain contains the CDR amino acid sequence shown in SEQ ID NO:65, 66, and 67; and (b) a β chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:76, provided that the β chain contains the CDR amino acid sequence shown in SEQ ID NO:73, 73, and 74.

[0089] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment having the ability to specifically bind to AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules provided herein may comprise an α-chain (or γ-chain) and a β-chain (or δ-chain) having the CDR sequence (α- or γ-chain CDR-1, CDR-2 and CDR-3 and β- or δ-chain CDR-1, CDR-2 and CDR-3) shown in any of the anti-NPM1c TCRs in Table 1. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment having the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules provided herein may comprise an α-chain (or γ-chain) and a β-chain (or δ-chain) having the CDR sequence shown in any of the anti-NPM1c TCRs listed in Table 1 (α- or γ-chain CDR-1, CDR-2, and CDR-3 and β- or δ-chain CDR-1, CDR-2, and CDR-3), wherein any one or more CDRs may independently be variant CDRs having one or two amino acid modifications from the CDR sequences listed in Table 1.

[0090] Exemplary anti-NPM1c TCRs possessing these CDRs and the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules include TCR A, TCR B, and TCR C. As an example, the anti-NPM1c TCR or its antigen-binding fragment with the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules provided herein may comprise an α chain (or γ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:29 (or a variant of SEQ ID NO:29 with one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO:30 (or a variant of SEQ ID NO:30 with one or two amino acid modifications), and CDR-3 having the amino acid sequence shown in SEQ ID NO:31 (or a variant of SEQ ID NO:31 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:37 (or a variant of SEQ ID NO:37 with one or two amino acid modifications), and CDR-3 having the amino acid sequence shown in SEQ ID NO:38 (or a variant of SEQ ID NO:39 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:37 (or a variant of SEQ ID NO:37 with one or two amino acid modifications), and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:37 (or a variant of SEQ ID NO:37 with one or two amino acid modifications), and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:38 (or a variant of SEQ ID NO:39 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:37 ... CDR-2 (a variant of NO:38 with one or two amino acid modifications) and CDR-3 (a variant of SEQ ID NO:39 with one or two amino acid modifications) having the amino acid sequence shown in SEQ ID NO:39.

[0091] Exemplary anti-NPM1c TCRs possessing these CDRs and the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules include TCR D-TCR P. As an example, the anti-NPM1c TCR or its antigen-binding fragment with the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules provided herein may comprise an α chain (or γ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:65 (or a variant of SEQ ID NO:65 with one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO:66 (or a variant of SEQ ID NO:66 with one or two amino acid modifications), and CDR-3 having the amino acid sequence shown in SEQ ID NO:67 (or a variant of SEQ ID NO:67 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:73 (or a variant of SEQ ID NO:73 with one or two amino acid modifications), CDR-2 having the amino acid sequence shown in SEQ ID NO:74 (or a variant of SEQ ID NO:65 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:73 (or a variant of SEQ ID NO:73 with one or two amino acid modifications), CDR-3 having the amino acid sequence shown in SEQ ID NO:74 (or a variant of SEQ ID NO:65 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:73 (or a variant of SEQ ID NO:73 with one or two amino acid modifications); and a β chain (or δ chain) having: CDR-1 having the amino acid sequence shown in SEQ ID NO:73 (or a variant of SEQ ID NO:74 with one or two amino acid modifications); and a γ chain having: CDR-1 having the amino acid sequence shown in SEQ ID NO:73 (or a variant of SEQ ID NO:75 with one or two amino acid modifications); and a γ chain having: CDR- CDR-2 (a variant of NO:74 with one or two amino acid modifications) and CDR-3 (a variant of SEQ ID NO:75 with one or two amino acid modifications) having the amino acid sequence shown in SEQ ID NO:75.

[0092] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragments provided herein that have the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules and contain the α-chain (or γ-chain) and β-chain (or δ-chain) CDR sequences (α- or γ-chain CDR-1, CDR-2, and CDR-3 and β- or δ-chain CDR-1, CDR-2, and CDR-3) of any of the anti-NPM1c TCRs shown in Table 1 may contain any suitable frame region. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules and contains an α-chain (or γ-chain) and a β-chain (or δ-chain) with the CDR sequence shown in any of the anti-NPM1c TCRs in Table 1 (α- or γ-chain CDR-1, CDR-2 and CDR-3 and β- or δ-chain CDR-1, CDR-2 and CDR-3), may contain any suitable frame regions (i.e., α-chain frame regions and β-chain frame regions 1, 2, 3 and 4), wherein any one or more CDRs may independently be variant CDRs having one or two amino acid modifications from the CDR sequences listed in Table 1. α-frame region 1 may have an amino acid sequence comprising any α-variable (V) region amino acid sequence of any of the anti-NPM1c TCRs shown in Table 1 located upstream of the corresponding CDR-1 sequence, or a variant of such sequence having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications. α-frame region 2 may have an amino acid sequence comprising any α-variable (V) region amino acid sequence of any of the anti-NPM1c TCRs shown in Table 1 located between the corresponding CDR-1 and CDR-2 sequences, or a variant of such sequence having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications. The α-chain framework region 3 may have an amino acid sequence of any α-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1 located between the corresponding CDR-2 and CDR-3 sequences, or a variant of the sequence having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid modifications.The α-frame region 4 may have an amino acid sequence of any α-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1, located downstream of the corresponding CDR-3 sequence, or a variant of that sequence having one, two, three, four, or five amino acid modifications. The β-frame region 1 may have an amino acid sequence of any β-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1, located upstream of the corresponding CDR-1 sequence, or a variant of that sequence having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications. The β-frame region 2 may have an amino acid sequence of any β-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1, located between the corresponding CDR-1 and CDR-2 sequences, or a variant of that sequence having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications. β-frame region 3 may have an amino acid sequence of any β-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1 located between the corresponding CDR-2 and CDR-3 sequences, or a variant of such sequence having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications. β-frame region 4 may have an amino acid sequence of any β-variable (V) region amino acid sequence of any anti-NPM1c TCR shown in Table 1 located downstream of the corresponding CDR-3 sequence, or a variant of such sequence having one, two, three, four, or five amino acid modifications. The anti-NPM1c TCR or its antigen-binding fragment may have α-frame regions 1, 2, 3, and 4 of any anti-NPM1c TCR shown in Table 1, and corresponding β-frame regions 1, 2, 3, and 4. As an example, a TCR may have α-chain frame regions 1, 2, 3 and 4 of SEQ ID NO:32 and β-chain frame regions 1, 2, 3 and 4 of SEQ ID NO:40, or α-chain frame regions 1, 2, 3 and 4 of SEQ ID NO:68 and β-chain frame regions 1, 2, 3 and 4 of SEQ ID NO:76.

[0093] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment having the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules provided herein may comprise an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the α chain variable (V) region of any of the anti-NPM1c TCRs shown in Table 1; and a β chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the corresponding β chain variable (V) region as shown in Table 1. The anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the variable (V) region of the α chain of any of the anti-NPM1c TCRs shown 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% identity with the corresponding variable (V) region of the β chain as shown in Table 1. In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise (a) an α chain containing an amino acid sequence having 100% identity with the amino acid sequence of the α chain variable (V) region of any of the anti-NPM1c TCRs shown in Table 1; and (b) a β chain containing an amino acid sequence having 100% identity with the amino acid sequence of the corresponding β chain variable (V) region as shown in Table 1. As an example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99, or 100% identity with the 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% identity with the amino acid sequence SEQ ID NO:40.As another example, the 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% identity with the amino acid sequence SEQ ID NO:68; and a β chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99, or 100% identity with the amino acid sequence SEQ ID NO:76.

[0094] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, may comprise (a) an α chain containing an amino acid sequence having at least 90% identity with the amino acid sequence of the variable (V) region of the α chain of any of the anti-NPM1c TCRs shown in Table 1, provided that the α chain contains 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 shown in Table 1; and (b) The β chain contains an amino acid sequence having at least 90% identity with the corresponding β chain variable (V) region as shown in Table 1, provided that the β chain contains 100% identity with the corresponding β chain CDR-1, CDR-2, and CDR-3 sequences shown in Table 1. The anti-NPM1c TCR or its antigen-binding fragment provided herein may contain (a) an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of the α chain variable (V) region of any of the anti-NPM1c TCRs shown in Table 1, provided that the α chain contains 100% identity with the corresponding α chain CDR-1, CDR-2, and CDR-3 sequences shown in Table 1; and (b) The β chain contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the corresponding β chain variable (V) region as shown in Table 1, provided that the β chain contains 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 shown in Table 1.As an example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:32, provided that the α chain contains the CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO: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% identity with the amino acid sequence of SEQ ID NO:40, provided that the β chain contains the CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:37, 38, and 39, respectively. As another example, the anti-NPM1c TCR or its antigen-binding fragment provided herein may comprise an α chain containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:68, provided that the α chain contains the CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO: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% identity with the amino acid sequence of SEQ ID NO:76, provided that the β chain contains the CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:73, 74, and 75, respectively.

[0095] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment thereof, which has the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, may comprise (a) an α-chain having the amino acid sequence of the α-chain variable (V) region of any of the anti-NPM1c TCRs shown in Table 1, or an α-chain variable (V) region of any of the anti-NPM1c TCRs shown in Table 1 having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition); and (b) The β chain has an amino acid sequence corresponding to the β chain variable (V) region as shown in Table 1, or has one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) in the corresponding β chain variable (V) region as shown in Table 1. The anti-NPM1c TCR or its antigen-binding fragments provided herein, which have the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, may comprise (a) an α chain having an amino acid sequence of zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) of any of the TCRs shown in Table 1, provided that the α chain contains 100% identical CDR-1, CDR-2, and CDR-3 amino acid sequences to the corresponding α chain CDR-1, CDR-2, and CDR-3 sequences shown in Table 1; and (b) The β chain has an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) in the corresponding β chain variable (V) region as shown in Table 1, provided that the β chain contains 100% identical CDR-1, CDR-2, and CDR-3 amino acid sequences to the corresponding β chain CDR-1, CDR-2, and CDR-3 sequences shown in Table 1.As an example, the anti-NPM1c TCR or its antigen-binding fragment having the ability to specifically bind AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules provided herein may comprise an α chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:32, provided that the α chain contains CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:29, 30, and 31, respectively; and a β chain having an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:40, provided that the β chain contains CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:37, 38, and 39, respectively. As another example, the anti-NPM1c TCR or its antigen-binding fragment thereof, which is provided herein as having the ability to specifically bind CLAVEEVSL (SEQ ID NO:5) in the context of MHC molecules, may comprise an α chain having an amino acid sequence having zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:68, provided that the α chain contains CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:65, 66, and 67, respectively; and a β chain having an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications (e.g., amino acid substitution, amino acid deletion, and / or amino acid addition) as shown in SEQ ID NO:76, provided that the β chain contains CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in SEQ ID NO:73, 74, and 75, respectively.

[0096] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein includes a Vα or Vγ region containing the following: (a) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:11, 12 and 13; (b) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:29, 30 and 31; (c) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:47, 48 and 49; (d) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:65, 66 and 67; (e) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:83, 84 and 85; (f) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:101, 102 and 103; (g) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:119, 120 and 121; (h) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:137, 138 and 139 respectively; (i) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:155, 156 and 157; (j) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:173, 174 and 175; (k) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:191, 192 and 193 respectively; (l) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:209, 210 and 211; (m) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:227, 228 and 229 respectively; (n) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:245, 246 and 247 respectively; (o) CDR-1, CDR-2, and CDR-3 respectively containing SEQ ID NO:263, 264, and 265; or (p) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:281, 282 and 283 respectively.

[0097] Additionally, the provided anti-NPM1c TCRs include those having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0098] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein includes a Vβ or Vδ region containing the following: (a) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:19, 20 and 21; (b) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:37, 38 and 39; (c) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:55, 56 and 57; (d) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:73, 74 and 75; (e) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:91, 92 and 93; (f) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:109, 110 and 111; (g) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:127, 128 and 129; (h) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:145, 146 and 147 respectively; (i) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:163, 164 and 165; (j) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:181, 182 and 183; (k) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:199, 200 and 201 respectively; (l) CDR-1, CDR-2 and CDR-3 respectively containing SEQ ID NO:217, 218 and 219; (m) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:235, 236 and 237 respectively; (n) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:253, 254 and 255 respectively; (o) contains CDR-1, CDR-2 and CDR-3 respectively, which are SEQ ID NO:271, 272 and 273; (p) contains CDR-1, CDR-2 and CDR-3 of SEQ ID NO:289, 290 and 291 respectively.

[0099] Additionally, the provided anti-NPM1c TCRs include those having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

[0100] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein comprises a Vα or Vγ region containing CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in Table 1 (such as in each row therein); and a Vβ or Vδ region containing CDR-1, CDR-2, and CDR-3 amino acid sequences as shown in Table 1 (such as in each row therein).

[0101] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein comprises a Vα or Vγ region containing CDR-1, CDR-2, and CDR-3 amino acid sequences contained within the Vα or Vγ region amino acid sequences shown in Table 1 (such as in each row therein); and a Vβ or Vδ region containing CDR-1, CDR-2, and CDR-3 amino acid sequences contained within the Vβ or Vδ region amino acid sequences shown in Table 1 (such as in each row therein). In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment provided herein comprises the Vα or Vγ region amino acid sequences and the corresponding Vβ or Vδ region amino acid sequences as shown in Table 1 (such as in each row therein). Additionally, the provided anti-NPM1c TCRs include those containing at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence. Exemplary anti-NPM1c TCRs containing such CDRs, or modified forms thereof as described elsewhere herein, are also shown in Table 1 (such as in each row therein).

[0102] Table 1. SEQ ID NO of CDR and variable region amino acid sequences against NPM1c TCR (sequences are provided in Table 12)

[0103] In some examples, the anti-NPM1c TCR or its antigen-binding fragment provided herein can be designed to include an α chain (or γ chain) containing a set of three CDRs (e.g., CDR-1, CDR-2, and CDR-3) as shown in Table 1 (e.g., SEQ ID NO:29-31, SEQ ID NO:65-67, SEQ ID NO:83-85, or SEQ ID NO:101-103); and a β chain (or δ chain) containing a set of three CDRs (e.g., CDR-1, CDR-2, and CDR-3) as shown in Table 1 (e.g., SEQ ID NO:37-39, SEQ ID NO:73-75, SED ID NO:92-92, or SEQ ID NO:109-111).

[0104] 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.

[0105] In some embodiments, the Vα region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vα region sequence of SEQ ID NO:32, and the Vβ region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vβ region sequence of SEQ ID NO:40. In some embodiments, the Vα region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vα region sequence of SEQ ID NO:68, and the Vβ region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vβ region sequence of SEQ ID NO:76. In some embodiments, the Vα region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vα region sequence of SEQ ID NO:86, and the Vβ region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vβ region sequence of SEQ ID NO:94. In some embodiments, the Vα region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vα region sequence of SEQ ID NO:104, and the Vβ region comprises CDR-1, CDR-2, and CDR-3 respectively contained in the Vβ region sequence of SEQ ID NO:112.

[0106] In some embodiments, the Vα region contains SEQ ID NO:32 or a sequence having at least 90% sequence identity with it, and the Vβ region contains SEQ ID NO:40 or a sequence having at least 90% sequence identity with it. In some embodiments, the Vα region contains SEQ ID NO:68 or a sequence having at least 90% sequence identity with it, and the Vβ region contains SEQ ID NO:76 or a sequence having at least 90% sequence identity with it. In some embodiments, the Vα region contains SEQ ID NO:86 or a sequence having at least 90% sequence identity with it, and the Vβ region contains SEQ ID NO:94 or a sequence having at least 90% sequence identity with it. In some embodiments, the Vα region contains SEQ ID NO:104 or a sequence having at least 90% sequence identity with it, and the Vβ region contains SEQ ID NO:112 or a sequence having at least 90% sequence identity with it.

[0107] In some embodiments, the Vα region contains SEQ ID NO:32, and the Vβ region contains SEQ ID NO:40. In some embodiments, the Vα region contains SEQ ID NO:68, and the Vβ region contains SEQ ID NO:76. In some embodiments, the Vα region contains SEQ ID NO:86, and the Vβ region contains SEQ ID NO:94. In some embodiments, the Vα region contains SEQ ID NO:104, and the Vβ region contains SEQ ID NO:112.

[0108] C. Exemplary Constant Structure 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 aspects, the β chain further contains a β constant (Cβ) region or a portion thereof. Therefore, in some embodiments, the anti-NPM1c TCR provided herein (e.g., the anti-AIQDLCLAV TCR 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 aspects, the δ chain further contains a δ constant (Cδ) region or a portion thereof. Therefore, in some embodiments, the anti-NPM1c TCR provided herein (e.g., the anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR provided herein) or its antigen-binding fragment contains 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.

[0109] In some embodiments, the α and β chains, or γ and δ chains, of the anti-NPM1c TCR provided herein each further contain a constant domain. In some embodiments, the Cα and Cβ domains, or the Cγ and Cδ domains, are 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 proximal membrane constant domains and two distal membrane variable domains, each of which contains a CDR.

[0110] In some aspects, this document provides anti-NPM1c TCRs containing 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 TCR is fully human. The provided anti-NPM1c TCR is a TCR containing human constant domains, such as a fully human TCR, whose expression and / or activity (e.g., when expressed in human cells, such as human T cells, such as primary human T cells) is unaffected or substantially unaffected by the presence of endogenous human TCRs.

[0111] 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 or TRBC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, Cγ is encoded by the TRGC1 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.

[0112] In some embodiments, the Cα domain or a variant thereof has or comprises the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:3. In some embodiments, the Cα domain has or comprises the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the Cβ domain or a variant thereof has or comprises the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:7. In some embodiments, the Cβ domain has or comprises the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the anti-NPM1c TCR comprises the Cα and Cβ domains shown in SEQ ID NO:3 and 7, respectively.

[0113] In some embodiments, the Cγ domain or a variant thereof has or contains the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:312. In some embodiments, the Cγ domain has or contains the amino acid sequence shown in SEQ ID NO:312. In some embodiments, the Cγ domain has or contains the amino acid sequence shown in SEQ ID NO:313. In some embodiments, the Cδ domain or a variant thereof has or contains the amino acid sequence shown 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 higher sequence identity with SEQ ID NO:314. In some embodiments, the anti-NPM1c TCR comprises the Cγ and Cδ domains shown in SEQ ID NO:312 and 314, respectively. In some embodiments, the anti-NPM1c TCR comprises the Cγ and Cδ domains shown in SEQ ID NO:313 and 314, respectively.

[0114] In some embodiments, variants of the Cα domain contain at least one non-natural cysteine ​​substitution, such as any substitution described herein. In some embodiments, variants of the Cβ domain contain at least one non-natural cysteine ​​substitution, such as any substitution described herein.

[0115] In some embodiments, any of the provided anti-NPM1c TCRs or their antigen-binding fragments may be human / mouse chimeric TCRs. In some embodiments, the anti-NPM1c TCRs or their antigen-binding fragments provided herein comprise an α-chain and / or a β-chain or a γ-chain and / or a δ-chain containing 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 a mouse Cβ-domain or a mouse Cγ-domain and / or a mouse Cδ-domain. In some embodiments, the Cα-domain and / or Cβ-domain or the Cγ-domain and / or Cδ-domain is or comprises any Cα-domain and / or Cβ-domain or Cγ-domain and / or Cδ-domain described in WO2015 / 184228, WO2015 / 009604, or WO2015 / 009606.

[0116] In some embodiments, the anti-NPM1c TCR or its antigen-binding fragment comprises a variant of an α-chain and / or a β-chain or a γ-chain and / or a δ-chain. In some embodiments, the variant comprises an amino acid sequence of any of the anti-NPM1c TCRs described herein having one, two, three, or four or more amino acid substitutions in a constant domain of the α or β chain. In some embodiments, the anti-NPM1c TCR (or its functional portion thereof) comprising a substituted amino acid sequence advantageously provides one or more of the following compared to a parental TCR comprising an unsubstituted amino acid sequence: reduced mismatch with the endogenous TCR chain, increased host cell expression, increased AIQDLCLAV (SEQ ID NO: 1) or CLAVEEVSL (SEQ ID NO: 5) recognition, and increased antitumor activity.

[0117] In some embodiments, the anti-NPM1c TCR can be a heterodimer, such as an α-chain and β-chain or a γ-chain and δ-chain linked by one or more disulfide bonds. In some embodiments, the constant domain of the anti-NPM1c TCR may contain short linker sequences in which cysteine ​​residues form disulfide bonds, 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-chain and β-chain or the γ-chain and δ-chain, such 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.

[0118] In some embodiments, the anti-NPM1c TCR provided herein may contain one or more introduced disulfide bonds. In some embodiments, native disulfide bonds are absent. In some embodiments, one or more native cysteine ​​residues forming native interchain disulfide bonds (e.g., in constant domains of the α and β chains or the γ and δ chains) are substituted with another residue, 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 constant domains of the α and β chains or the γ and δ chains) to cysteine. Relative cysteine ​​residues in the TCR α and β chains or the TCR γ and δ chains provide disulfide bonds that connect the constant domains of the TCR α and β chains or the TCR γ and δ chains that substituted the TCR to each other, and these disulfide bonds are not present in TCRs containing unsubstituted constant domains (such as unsubstituted natural human constant domains or unsubstituted natural mouse constant domains) in which native disulfide bonds are present. In some embodiments, the presence of non-natural cysteine ​​residues in the recombinant TCR (e.g., generating one or more non-natural disulfide bonds) may favor the production of the desired recombinant TCR in the cells in which it is introduced, exceeding the expression of mismatched TCR pairs containing the native TCR chain. 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. Cysteine ​​substitution may promote the formation of disulfide bonds between the α and β chains or between the γ and δ chains. The formation of disulfide bonds may promote pairing between the α and β chains or between the γ and δ chains, and / or reduce mismatches with the endogenous TCR chain. Suitable cysteine ​​substitutions include, but are not limited to, T47C substitution in the α constant domain (relative to the position of SEQ ID NO: 3) chain and S56C substitution in the β chain (relative to the position of SEQ ID NO: 7 or 9).

[0119] Exemplary non-natural disulfide bonds of TCR are described in published international PCT patent applications WO2006 / 000830 and WO2006 / 037960. In some embodiments, cysteine ​​may be introduced or substituted at residues corresponding to Thr48 (Thr47 of SEQ ID NO:3) and Ser57 (Ser56 of SEQ ID NO:7 or 9) of the Cα domain, at residues corresponding to Thr45 and Ser77 of the Cα domain, at residues corresponding to Tyr10 and Ser17 of the Cα domain, at residues corresponding to Thr45 and Asp59 of the Cβ domain, and / or at residues corresponding to Ser15 and Glu15 of the Cα domain.

[0120] In some embodiments, the provided cysteine ​​mutation can be performed at a corresponding position in another sequence, such as in the aforementioned human or mouse Cα and / or Cβ domain or Cγ and / or Cδ domain sequences. The term "corresponding" regarding protein positions, such as the statement that an amino acid position "corresponds to" an amino acid position in a disclosed sequence (such as shown in the sequence listing), refers to the amino acid position identified based on structural sequence alignment or by aligning it to the disclosed sequence using a standard alignment algorithm (such as the GAP algorithm). For example, corresponding residues can be determined by aligning a reference sequence to the Cα sequence shown in SEQ ID NO:3 or the Cβ sequence shown in SEQ ID NO:7 or 9 via the structural alignment methods described herein. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as guidance.

[0121] In some embodiments, the anti-AIQDLCLAV TCR or its antigen-binding fragment provided herein comprises an α or γ chain, which is or comprises an amino acid sequence as shown in any one of SEQ ID NO: 17, 35, 53, or a sequence having at least 90% sequence identity with it (such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence); and / or a corresponding (see Table 2) β or δ chain, which is or comprises an amino acid sequence as shown in any one of SEQ ID NO: 25, 43, and 63, or a sequence having at least 90% sequence identity with it (such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence).

[0122] In some embodiments, the anti-CLAVEEVSL TCR or its antigen-binding fragment provided herein comprises an α or γ chain, which is or comprises an amino acid sequence as shown in any one of SEQ ID NO: 71, 89, 107, 125, 143, 161, 179, 197, 215, 233, 251, 269, or 287, or a sequence having at least 90% sequence identity with such a sequence (such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such a sequence); and / or a corresponding (see Table 2) β or δ chain, which is or comprises an amino acid sequence as shown in SEQ ID NO: 71, 89, 107, 125, 143, 161, 179, 197, 215, 233, 251, 269, or 287 ...; and / or a corresponding (see Table 2) β or δ chain, which is or comprises an amino acid sequence as shown in SEQ ID NO: 71, 89, 107, 125, 143, 163, 161, 179, 197, 215, 233, 251, 269, or 287; and / or a corresponding (see Table 2) β or δ chain, which is or comprises an amino acid The amino acid sequence represented by any one of NO: 79, 97, 115, 133, 151, 169, 187, 205, 223, 241, 259, 277 or 295, or a sequence having at least 90% sequence identity with such a sequence (such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence).

[0123] Exemplary anti-NPM1c TCRs or antigen-binding fragments include those shown in Table 2 (such as in each row therein). In some embodiments, the Vα and Vβ regions or the Vγ and Vδ regions contain amino acid sequences corresponding to SEQ ID NO: shown in Table 2 (such as in each row therein). 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 shown in Table 2 (such as in each row therein). In some aspects, the anti-NPM1c TCR contains constant α-domain sequences and constant β-domain sequences, such as those corresponding to SEQ ID NO: shown in Table 2 (such as in each row therein). In some embodiments, the anti-NPM1c TCR contains a complete sequence comprising variable and constant domains, such as the sequence corresponding to SEQ ID NO: shown in Table 2 (“Complete α-P2A-β”) (such as in each row therein). Additionally, the provided anti-NPM1c TCRs include those containing at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence. Exemplary anti-NPM1c TCRs containing such sequences, or their modified forms as described elsewhere herein, are also shown in Table 2 (such as in each row therein). In some aspects, when expressed as a mature protein, the provided exemplary anti-NPM1c TCRs contain mature Vα and / or mature Vβ regions or mature Vγ and / or Vδ mature regions, for example, without the signal sequence when fully processed and expressed (e.g., because the signal sequence is cleaved).

[0124] Table 2: SEQ ID NO of the amino acid sequences of the variable and constant regions against NPM1c TCR

[0125] In some embodiments, the α or γ chain contains SEQ ID NO:35 or a sequence having at least 90% sequence identity with it, and the β or δ chain contains SEQ ID NO:43 or a sequence having at least 90% sequence identity with it. In some embodiments, the α or γ chain contains SEQ ID NO:71 or a sequence having at least 90% sequence identity with it, and the β or δ chain contains SEQ ID NO:79 or a sequence having at least 90% sequence identity with it. In some embodiments, the α or γ chain contains SEQ ID NO:89 or a sequence having at least 90% sequence identity with it, and the β or δ chain contains SEQ ID NO:97 or a sequence having at least 90% sequence identity with it. In some embodiments, the α or γ chain contains SEQ ID NO:107 or a sequence having at least 90% sequence identity with it, and the β or δ chain contains SEQ ID NO:115 or a sequence having at least 90% sequence identity with it.

[0126] In some embodiments, the α chain contains SEQ ID NO:35 and the β chain contains SEQ ID NO:43. In some embodiments, the α chain contains SEQ ID NO:71 and the β chain contains SEQ ID NO:79. In some embodiments, the α chain contains SEQ ID NO:89 and the β chain contains SEQ ID NO:97. In some embodiments, the α chain contains SEQ ID NO:107 and the β chain contains SEQ ID NO:115.

[0127] In some embodiments, the anti-AIQDLCLAV TCR comprises the amino acid sequence of SEQ ID NO: 27, 45, or 63, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 27, 45, or 63. In some embodiments, the anti-CLAVEEVSL TCR comprises the amino acid sequence of SEQ ID NO: 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 NO: 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297.

[0128] In some implementations, the anti-NPM1c TCR or its antigen-binding portion provided herein may be a recombinantly derived natural protein or a mutant form thereof, wherein one or more properties (such as binding properties) have been altered. In some respects, the nucleic acid is synthetic.

[0129] In some embodiments, any of the described anti-NPM1c TCRs (or nucleic acids encoding anti-NPM1c TCRs) may be modified to increase the affinity of the TCR for the target neoantigen, to improve the stability of the TCR, and / or to modify 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 contain disulfide bonds in the constant TCR region.

[0130] D. Anti-NPM1 TCR single-stranded variable fragment The anti-NPM1 TCR CDR or variable region described herein can be used to form a single-stranded variable fragment (scFv), a bispecific T cell adaptor (BiTE), a chimeric T cell receptor (CAR), or other proteins containing scFv, BiTE, or CAR.

[0131] "scFv" is the variable heavy chain region (V) of an immunoglobulin containing a short linker peptide. H ) and variable light chain region (V L A fusion protein of the variable α region (Vα) and variable β region (Vβ) of the TCR. scFv retains the antigen-binding properties of the complete immunoglobulin or TCR with the derived variable region. Nucleic acids encoding anti-AIQDLCLAV scFv or CLAVEEVSL scFv are also considered.

[0132] In some embodiments, anti-AIQDLCLAV scFv and anti-CLAVEEVSL scFv are described, comprising the Vα and Vβ regions of either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR, wherein the Vα and Vβ regions are linked via a linker peptide, and wherein the anti-AIQDLCLAV scFv or anti-CLAVEEVSL scFv retains the antigen-binding properties of the derived variable region of the anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR. Nucleic acids encoding anti-AIQDLCLAV scFv and anti-CLAVEEVSL scFv are also contemplated. Anti-AIQDLCLAV scFv or anti-CLAVEEVSL scFv can be formed by linking the C-terminus of the Vα chain to the N-terminus of the Vβ chain. Alternatively, the C-terminus of the Vβ chain can be linked to the N-terminus of the Vα chain. The peptide linker can be from about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is glycine-rich. The scFv peptide linker can be, but is not limited to, (G4S). x , where x is an integer from 2 to 5 (inclusive). In some embodiments, the linked scFv peptide comprises Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (i.e., also referred to as [(Gly)4Ser]3, (G4S)3, or G4S (×3)). In some embodiments, the scFv peptide linker consists of G4S (×3).

[0133] Bispecific T-cell adaptors (BiTEs) are recombinant molecules containing two flexible-linked antigen-binding domains (such as scFv). In a typical BiTE, one antigen-binding domain is specific for CD3 or other activated antigens on immune cells, and 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 and contain CD3 receptors on target cells (as described in WO99054440, WO2005040220, and WO2008119567). BiTEs can transiently bind T cells to target cells and simultaneously activate the cytolytic activity of T cells.

[0134] In some embodiments, anti-AIQDLCLAV BiTE molecules and anti-CLAVEEVSL BiTE molecules are described, comprising a first antigen-binding domain (e.g., scFv) specific to activated antigens on immune cells 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 TCR or anti-CLAVEEVSL TCR. In some embodiments, the BiTE comprises an anti-CD3 scFv and an anti-AIQDLCLAV scFv or anti-CLAVEEVSL scFv containing Vα and Vβ regions of either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR. Nucleic acids encoding anti-AIQDLCLAV BiTE or anti-CLAVEEVSL BiTE are also contemplated.

[0135] A "chimeric antigen receptor" or "CAR" is a recombinant protein comprising an antigen-binding domain (e.g., an antigen-binding fragment of any of the described anti-NPM1c TCRs) linked via a transmembrane domain to a cell signaling and / or cell activation domain. The cell signaling domain may be, but is not limited to, a T cell signaling domain. When used with a CAR, the described anti-NPM1c TCR antigen-binding fragment may be provided as an scFv. The 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 dual-antigen 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 domains or co-stimulatory domains may be, but are not limited to, CD3ζ domains, CD28 domains, CD137(4-1BB) domains, ICOS domains, CD27 domains, OX40 domains, LFA1 domains, PD-1 domains, CD150 domains, CD244 domains, NKG2D domains, and DAP10 domains. In some embodiments, the CAR has a CD28 transmembrane domain.

[0136] In some embodiments, anti-AIQDLCLAV CARs and anti-CLAVEEVSL CARs are described, comprising an antigen-binding fragment of either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR, or a scFv containing the variable region or CDR of either of the described TCRs, a transmembrane domain, and a signal transduction and / or cell activation domain. In some embodiments, the antigen-binding fragment of either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR comprises an anti-AIQDLCLAV scFv or anti-CLAVEEVSL scFv, wherein the anti-AIQDLCLAV scFv or anti-CLAVEEVSL scFv contains the Vα and Vβ regions of either the described anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR. The anti-AIQDLCLAV CAR or anti-CLAVEEVSL CAR can be, but is not limited to, first-generation CAR T cells, second-generation CAR T cells, third-generation CAR T cells, fourth-generation CAR T cells, dual-antigen receptor CAR T cells, or CAR T cells with an inducible suicide gene, or combinations thereof. Nucleic acids encoding anti-AIQDLCLAV CAR or anti-CLAVEEVSL CAR are also being considered.

[0137] II. Nucleic acids encoding anti-NPM1c TCR This document also provides nucleic acids, such as polynucleotides or nucleic acid molecules, encoding any of the described anti-NPM1c TCRs or their antigen-binding fragments. Nucleic acids may include those comprising naturally occurring and / or non-naturally occurring nucleotides and bases, for example, those having backbone modifications. The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain naturally occurring and / or non-natural nucleotides and include, but are not limited to, DNA and RNA. A “nucleic acid sequence” refers to a linear sequence of nucleotides comprising a nucleic acid molecule or a polynucleotide.

[0138] In some embodiments, the nucleic acid is or contains cDNA. In some aspects, the polynucleotide can be modified for use in the constructs described herein, such as for codon optimization. In some embodiments, for the purpose of cloning into a vector, the nucleic acid sequence can be engineered to contain terminal restriction site sequences.

[0139] In some implementations, the anti-NPM1c TCR or its antigen-binding moiety provided herein can be synthesized based on knowledge of the amino acid sequence of the TCR.

[0140] Nucleic acid sequences encoding Vα, Vβ, Vα and Vβ regions, α strand, β strand, or α and β strands are described. In some embodiments, the nucleic acid sequence may encode Vγ, Vδ, Vγ and Vδ regions, γ strand, δ strand, or γ and δ strands.

[0141] The nucleotide sequence may be provided as a pair 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 a separate expression cassette, plasmid, or vector (e.g., a viral vector) or on a single expression cassette, plasmid, or vector.

[0142] In some embodiments, the polynucleotide contains nucleic acid sequences 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 a sequence encoding a Vβ (or Vδ) region or β (or δ) chain by means of a peptide sequence (e.g., a 2A peptide, such as a P2A peptide, T2A peptide, F2A peptide, or E2A peptide) or an internal ribosome entry site (IRES) sequence that induces ribosome jumping or self-cleavage. In some embodiments, the sequences encoding the Vα and Vβ regions or α and β chains (or Vγ and Vδ regions or γ and δ chains) are operatively linked to a single promoter. In some embodiments, the sequences encoding the Vα and Vβ regions or α and β chains (or Vγ and Vδ regions or γ and δ chains) are each operatively linked to a single bidirectional promoter (e.g., a PGK promoter). In some embodiments, the sequence encoding the Vα (or Vγ) region or the α (or γ) chain may be the 5' of the sequence encoding the Vβ (or Vδ) region or the β (or δ) chain.

[0143] In some embodiments, the nucleotide sequences encoding the α or γ chain and / or the nucleotide sequences encoding the β or δ chain, or any of their domains, regions (e.g., Vα and Vβ regions), or portions, are codon-optimized. Typically, codon optimization involves balancing the percentage of selected codons using the published abundance of human transfer RNA, ensuring that none are overloaded or restrictive. Most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between transfected genes and host cells can affect protein expression and immunogenicity of nucleic acid constructs. Generally, for codon optimization, codons are selected to balance those used in humans. Typically, codon redundancy for amino acids results in different codons encoding a single amino acid. In some embodiments, when selecting codons for substitution, it may be desirable that the resulting mutation is a silent mutation, so that codon changes do not affect the amino acid sequence.

[0144] In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:33 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:41 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:69 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:77 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:87 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:95 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:105 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:113 or a sequence having at least 90% sequence identity with it.

[0145] In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:34 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:42 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:70 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:78 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:88 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:96 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the Vα region comprises SEQ ID NO:106 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the Vβ region comprises SEQ ID NO:114 or a sequence having at least 90% sequence identity with it.

[0146] In some embodiments, the nucleotide sequence encoding the α or γ chain comprises SEQ ID NO:36 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the β or δ chain comprises SEQ ID NO:44 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the α or γ chain comprises SEQ ID NO:72 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the β or δ chain comprises SEQ ID NO:80 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the α or γ chain comprises SEQ ID NO:90 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the β or δ chain comprises SEQ ID NO:98 or a sequence having at least 90% sequence identity with it. In some embodiments, the nucleotide sequence encoding the α or γ chain comprises SEQ ID NO:108 or a sequence having at least 90% sequence identity with it, and the nucleotide sequence encoding the β or δ chain comprises SEQ ID NO:116 or a sequence having at least 90% sequence identity with it. In addition, the nucleic acids or polynucleotides provided herein include those containing at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences to such sequences.

[0147] In some embodiments, the nucleotide sequence encoding the α-chain comprises SEQ ID NO:36, and the nucleotide sequence encoding the β-chain comprises SEQ ID NO:44. In some embodiments, the nucleotide sequence encoding the α-chain comprises SEQ ID NO:72, and the nucleotide sequence encoding the β-chain comprises SEQ ID NO:80. In some embodiments, the nucleotide sequence encoding the α-chain comprises SEQ ID NO:90, and the nucleotide sequence encoding the β-chain comprises SEQ ID NO:98. In some embodiments, the nucleotide sequence encoding the α-chain comprises SEQ ID NO:108, and the nucleotide sequence encoding the β-chain comprises SEQ ID NO:116.

[0148] In some embodiments, the nucleic acid sequence encoding the α chain comprises any one of SEQ ID NO: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 higher sequence identity therewith, and the nucleotide sequence encoding the β chain comprises any one of the corresponding SEQ ID NO: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 higher sequence identity therewith.

[0149] In some embodiments, the nucleic acid sequence encoding the α chain comprises any one of SEQ ID NO: 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 higher sequence identity with it, and the nucleotide sequence encoding the β chain comprises any one of the corresponding SEQ ID NO: 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 higher sequence identity with it.

[0150] 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 known as a leader sequence). A non-limiting example of such a signal peptide is a signal peptide having or containing the amino acid sequence shown in any of SEQ ID NO:315-624.

[0151] In some implementations, nucleic acids encoding α or γ chains and nucleic acids encoding β or δ chains can be linked via adapters (such as any adapters described elsewhere in this document).

[0152] In some embodiments, the nucleic acid encoding the Vα or Vγ region or α or γ chain and the nucleic acid encoding the Vβ or Vδ region or β or δ chain may be linked via a cleavable linker sequence or a peptide that induces ribosome jumping or self-cleavage (e.g., a 2A sequence, such as T2A, E2A, F2A, or P2A) (such as any cleavable linker sequence or peptide described elsewhere herein). The P2A amino acid sequence may be, but is not limited to, the sequence of SEQ ID NO:301. The nucleic acid sequence encoding the P2A sequence may be, 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.

[0153] In some implementations, nucleic acids encoding the Vα or Vγ region or α or γ chain and nucleic acids encoding the Vβ or Vδ region or β or δ chain can be linked via an IRES sequence.

[0154] In some embodiments, the nucleic acid sequence encoding the α and β TCR chains linked via a cleavable adapter sequence comprises: a 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 an 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.

[0155] In some embodiments, the nucleic acid sequence encoding the α and β TCR chains linked via a cleavable adapter sequence comprises: a nucleotide sequence of SEQ ID NO: 82, 100, 118, 136, 154, 172, 190, 208, 226, 244, 262, 280, or 298; a nucleotide sequence having at least 90% identity with the nucleic acid sequence of SEQ ID NO: 82, 100, 118, 136, 154, 172, 190, 208, 226, 244, 262, 280, or 298; a nucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO: 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279, or 297; or a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: The nucleotide sequence of a polypeptide having at least 90% identity with an amino acid sequence of 81, 99, 117, 135, 153, 171, 189, 207, 225, 243, 261, 279 or 297.

[0156] This document also provides vectors or constructs containing such nucleotide sequences. In some embodiments, the vector or construct contains one or more heterologous promoters operatively linked to nucleotides encoding Vα or Vγ regions or α or γ chains and / or Vβ or Vδ regions or β or δ chains. In some embodiments, the heterologous promoter is operatively linked to one or more nucleotide sequences.

[0157] 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 vectors or expression constructs may be polycistronic (e.g., bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of gene products (e.g., α or γ chains encoding TCR and / or β or δ chains) via information from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA containing two or three genes (e.g., α or γ chains encoding TCR and / or β or δ chains) in a single open reading frame (ORF), these genes being separated from each other by sequences encoding self-cleaving peptides (e.g., 2A peptides, such as P2A peptides) or protease recognition sites (e.g., furin proteases). ORFs can encode a single multiprotein that is cleaved into individual proteins during or after translation (in the case of 2A, such as P2A). In some embodiments, peptides such as P2A can induce ribosome skipping (ribosome jumping) to synthesize a peptide bond at the C-terminus of a 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide (see, for example, deFelipe). Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al., Traffic 5:616-626 (2004)). Examples of 2A cleavage peptides (including those capable of inducing ribosome jumping) include those from the Thothiaasigna virus (T2A), porcine cheisenvirus-1 (P2A, e.g., SEQ ID NO:301), equine rhinitis A virus (E2A), and 2A sequences from foot-and-mouth disease virus (F2A) as described in U.S. Patent Publication No. 2007 / 0116690. In some embodiments, the peptide causing ribosome jumping is a P2A peptide and / or contains the amino acid sequence shown in SEQ ID NO:301.

[0158] In a bicistronic 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 present in any order and are separated by a nucleotide sequence encoding a peptide sequence that induces ribosome jumping. For example, in some embodiments, the nucleotide sequence sequentially comprises a nucleic acid sequence encoding the β or δ chain, a nucleic acid sequence encoding a peptide sequence that induces ribosome jumping (e.g., the P2A sequence as described herein), and a nucleic acid sequence encoding the α or γ chain. In other embodiments, the nucleotide sequence sequentially comprises a nucleic acid sequence encoding the α or γ chain, a nucleic acid sequence encoding a peptide sequence that induces ribosome jumping (e.g., the P2A sequence as described herein), and a nucleic acid sequence encoding the β or δ chain.

[0159] 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 of the TCR comprise one or more nucleic acid sequences corresponding to SEQ ID NO: shown in Table 3 for TCR A-TCR P. Additionally, the provided nucleotide sequences encoding the TCR include those containing at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequences to such sequences. In some aspects, the nucleotide sequences contain sequences encoding a signal sequence, and when expressed as a mature protein, the encoded exemplary anti-NPM1c TCR comprises mature Vα and / or mature Vβ regions or mature Vγ and / or Vδ mature regions, for example, when fully processed and expressed, there is no signal sequence (e.g., because the signal sequence is cleaved).

[0160] Table 3: Nucleotide sequence of AIQDLCLAV (SEQ ID NO:1) specific TCR SEQ ID NO

[0161] This document also provides vectors, such as those containing any of the nucleic acids described herein. In some embodiments, one or more nucleic acids encoding one or both strands of a TCR are cloned or assembled into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used for transformation or transfection of any suitable host. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. In some embodiments, the vector is an expression vector.

[0162] III. Methods for isolating, evaluating, and identifying T cell receptors In some aspects, this document provides methods for isolating multiple nucleic acid sequences encoding TCRs specific to AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5). In some aspects, methods described herein are used to identify AIQDLCLAV (SEQ ID NO:1)-specific TCRs or CLAVEEVSL (SEQ ID NO:5)-specific TCRs. In some aspects, the methods further include isolating nucleic acid sequences, assembling the nucleic acid sequences into vectors, assessing TCR expression and / or activity, and, in some cases, using high-throughput methods to screen and identify specific TCRs of interest.

[0163] The methods described in this article also involve determining the binding activity and functional capacity of candidate TCRs.

[0164] A. Standards and methods for TCR donors used to isolate NPM1c-specific T cell candidates In some embodiments, whole-exome sequencing is used to genotype the donor's HLA library and / or haplotype. In some embodiments, the donor subject has cancer. In some embodiments, the donor subject has the type of cancer that is about to be treated with engineered T cells. In some embodiments, the donor is a healthy donor. In some embodiments, blood is collected from those subjects who have the appropriate HLA type. Anti-AIQDLCLAV (SEQ ID NO:1) reactive T cells from the donor subject can be identified based on their binding to HLA multimers 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 donor subjects can be identified based on HLA multimers that bind to the folding of 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 activating / signaling proteins, upregulation or downregulation of other protein metabolites or other cellular components, changes in cell shape or granularity, or other methods available in the art.

[0165] B. High-throughput isolation, amplification, and assembly of nucleic acid sequences encoding TCRs In some respects, nucleic acid molecules encoding TCRs can be obtained or identified from a variety of sources. In some respects, high-throughput TCR isolation and screening methods can be used to obtain or identify TCRs. Examples of such methods that can be used include those described, for example, in WO2018 / 102473 (the full text of which is incorporated herein by reference). In some respects, high-throughput TCR isolation and screening methods involve amplifying nucleic acids encoding TCR α and / or β chains or TCR γ and / or δ chains from multiple different cells (such as T cells) isolated from a donor. This document also provides methods involving the isolation or screening of multiple different TCRs to obtain TCRs specific to NPM1c neoantigens such as AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5)).

[0166] In some embodiments, the nucleic acid molecule encoding the TCR can be obtained from a variety of sources, such as encoding nucleic acids amplified within or isolated from one or more given cells by polymerase chain reaction (PCR). In some embodiments, the TCR is 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 a variety of animal species, such as humans, mice, rats, or other mammals. In some embodiments, the T cells can be obtained from cells isolated in vivo, such as normal (or healthy) subjects 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, to generate a vector encoding the TCR, 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. In some embodiments, the α and β chains can be synthesized. In some embodiments, the α and β chains are cloned into the same vector.

[0167] As described herein, the methods and materials provided herein can allow users to successfully capture most (if not all) of the functional TCRs from a sorted T cell population. For example, amplification (e.g., nested amplification procedures, such as nested PCR) procedures may include a set of primers designed to amplify each known functional V-segment of the two variable strands of a specific mammalian (e.g., human) TCR, or virtually every (>90%, >95%, >96%, >97%, or >98%) known V-segments (e.g., any known functional V-segments of the α- and β-variable strands of a specific αβ TCR, or any known functional V-segments of the γ- and δ-variable strands of a specific γδ TCR). For humans, the amplification procedure may include a primer set designed to amplify all or almost all (>90%, >95%, >96%, >97%, or >98%) of the 45 V regions of the currently known functional α chain and all or almost all (>90%, >95%, >96%, >97%, or >98%) of the 48 V regions of the currently known functional β chain. When referring to the TCR V regions of the α chain in this document, the shorthand abbreviation TRAV may be used. Similarly, when referring to the TCR V regions of the β chain in this document, the shorthand abbreviation TRBV may be used. The same applies to the TCR V regions of the γ and δ chains, which may be referred to as TRGV and TRDV, respectively.

[0168] In some aspects, this document provides methods and materials relating to cloning functional TCRs from single T cells. For example, in some aspects, this document provides methods and materials for obtaining nucleic acids encoding a TCR from a single T cell and arranging those nucleic acids to form nucleic acid vectors successfully designed to express a TCR (e.g., a fully complete TCR, such as a fully complete TCR having a variable strand combination as present in the single T cell), kits for obtaining nucleic acids encoding a TCR from a single T cell and arranging those nucleic acids to form nucleic acid vectors successfully designed to express a TCR (e.g., a fully complete TCR, such as a fully complete TCR having a variable strand combination as present in the single T cell), and methods for preparing such kits. The cloned αβ TCR having a variable strand combination as present in the single T cell can include a combination of VJ α segments as present in the single T cell, a combination of VDJ β segments 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 γδ TCR having a combination of variable strands as present in a single T cell can include a combination of VJ γ segments as present in a single T cell, a combination of VDJ δ segments 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.

[0169] In some respects, this document provides sets of nucleic acid primers designed to amplify the complete coding sequences of two variable regions (e.g., α variable region and β variable region or γ variable region and δ variable region) of each expressed V region (e.g., each expressed α V region and β V region or each expressed γ V region and δ V region) of a functional αβ or γδ TCR of a specific mammalian species (e.g., mouse or human), methods for cloning functional TCRs from single T cells using such sets of nucleic acid primers, and kits containing such sets of nucleic acid primers for cloning functional TCRs from single T cells.

[0170] In some respects, the methods and materials provided herein allow for highly multiplexed reactions to clone many different TCRs (e.g., hundreds to thousands or more different TCRs) directly from a single T cell, rapidly (e.g., simultaneously in some cases) and with very few (if any) missing α / β variable chain combinations (or γ / δ variable chain combinations). For example, the methods and materials provided herein can be used to clone many different αβ TCRs (e.g., hundreds to thousands or more different αβ TCRs) directly from a single αβ T cell in a manner where less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%) of the α / β variable chain combination for a species (e.g., mouse or human). Similarly, the methods and materials provided herein can be used to clone many different γδ TCRs (e.g., hundreds to thousands or more different γδ TCRs) directly from a single γδ T cell in such a manner that less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%) of the γ / δ variable chain combination may be missing for the species (e.g., mouse or human species). In some embodiments, the methods and materials provided herein may include (a) obtaining samples of T cells, (b) sorting those T cells into isolated sites (e.g., wells) such that most (if not all) isolated sites (e.g., each well) contain a single T cell, (c) lysing (e.g., simultaneously lysing) the single T cells located in the individually isolated sites (e.g., individual wells) to release RNA from each single T cell, (d) using the released RNA as a template, appropriate primers for synthesizing cDNA from the RNA, and a reverse transcriptase to perform (e.g., simultaneously) reverse transcription to generate cDNA within each isolated site (e.g., each well); the cDNA representing RNA expressed by the single T cell located in that isolated site (e.g., well); and (e) using the generated cDNA as a template, a first-round primer set (e.g., a first-round PCR primer set), and a polymerase (e.g., Taq(f) The polymerase performs (e.g., simultaneously) an amplification procedure (e.g., nested amplification procedure, such as nested polymerase chain reaction (PCR)) for each isolated site in the first round of amplification reaction (e.g., first round PCR) to produce at least an amplification product containing the nucleic acid sequence of the α-variable strand (or γ-variable strand) of the TCR of a single T cell at that isolated site and an amplification product containing the nucleic acid sequence of the β-variable strand (or δ-variable strand) of the TCR of the same single T cell at the same isolated site, and uses the amplification product of the first round of amplification reaction as a template, a second round primer set (e.g., a second round PCR primer set) and a polymerase (e.g., Taq (g) A second round of amplification reaction (e.g., second round PCR) is performed (e.g., simultaneously) using a nested amplification procedure (e.g., nested PCR procedure) for each isolated site to produce at least a first amplification product containing the nucleic acid sequence of the α-variable strand (or γ-variable strand) of the TCR of a single T cell containing the same isolated site and a second amplification product containing the nucleic acid sequence of the β-variable strand (or δ-variable strand) of the TCR of the same single T cell containing the same isolated site, and (g) the first and second amplification products are cloned into an expression vector for each isolated site, the expression vector being designed to express a functional TCR having a combination of α / β or γ / δ variable strands (or a portion thereof, such as the V segment of the α / β or γ / δ variable strand combination), as present in the single T cell used to produce the amplification product.

[0171] The resulting expression vector can be introduced into cells, causing those cells to express clonal TCRs. These cells and / or the TCRs they express from the introduced expression vector can be screened to identify TCRs with the desired capabilities. For example, cells expressing clonal TCRs that recognize a specific antigen (e.g., the NPM1c neoantigen) can be identified, and those cells, their contained TCR expression vectors, or clonal TCR constructs can be used for further analysis or for therapeutic applications.

[0172] In some embodiments, the expression of clonal TCRs on the surface and the expression of functional TCRs can be assessed by introducing an expression vector into TCR-negative reporter cells programmed to express a measurable biomarker signal or biomarker peptide once the signaling mechanism of a functional TCR is conjugated. In these cases, functional TCRs can be screened using antibodies programmed to nonspecifically activate TCRs (e.g., anti-CD3 antibodies). In some embodiments, the antigen specificity of clonal TCRs can be screened. For example, reporter cells expressing clonal TCRs can be screened to recognize 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 the expression vector, and their antigen specificity can be screened via a T cell proliferation assay.

[0173] 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 set of primers designed to amplify each known functional V segment of the two variable strands of a specific TCR in a mammal (e.g., a human) (e.g., any known functional V segment of the α-variable strand and β-variable strand of a specific αβ TCR or any known functional V segment of the γ-variable strand and δ-variable strand of a specific γδ TCR).

[0174] In some implementations, T cells can be stimulated (e.g., in vitro) prior to sorting, and RNA expression can then be assessed (via, for example, qPCR) to determine which T cells respond to the stimulation. Any suitable type of stimulation can be used, including but not limited to nonspecific stimulation such as with concanavalin A, phytohemagglutinin-P, phorbol ester plus iomycin, myristyl acetate phorbol ester plus calcium ion carrier, or antibodies capable of cross-linking TCRs (e.g., anti-CD3 antibody plus anti-CD28 antibody or anti-TCR β 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, can be measured and compared to an unstimulated population. Once individual T cells are sorted, the methods provided herein can be used to determine which T cells produce specific cytokines in response to stimulation (e.g., in response to peptide antigens used to stimulate T cells). In these cases, antigen-specific T cells can be identified without the cumbersome methods of expanding reactive T cells or the destructive methods of paraformaldehyde fixation and intracellular cytokine staining, which may reduce the ability to efficiently clone TCRs. In such cases, specific TCRs produced by active and antigen-specific T cells, rather than inactive bystander T cells, can be rapidly identified.

[0175] In some embodiments, the cytokine expression levels of individual T cells used to clone a functional TCR, such as TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, or IL-17, can be measured, thereby allowing the identification of a specific TCR based on a specific phenotype of the T cell providing the variable chain (or a portion thereof) of that specific TCR (e.g., elevated IFN-γ expression). In such cases, a specific TCR produced by active rather than inactive T cells can be rapidly identified. In some embodiments, a specific TCR produced by inactive rather than active T cells can be rapidly identified.

[0176] In some implementations, stimulation-tolerant T cells can be used to obtain TCRs, and the specificity of clonal TCRs can be tested or screened in cells where typical TCR signaling is not suppressed.

[0177] In some embodiments, MHC-peptide complexes (or HLA-peptide complexes) can be used to identify clonal TCRs that recognize such complexes. In these cases, clonal exclusion during an immune response and / or lack of antigen priming may result in the absence of TCRs with this specificity in the activated and / or expanded TCR pool. In such cases, the methods and materials provided herein (in some cases requiring only the presence of a single T cell) can be used to clone virgin or inactivated TCRs that recognize such complexes. In some embodiments, the virgin T cell pool can be stained with MHC-peptide tetramers (or HLA-peptide tetramers), and any MHC-peptide (or HLA-peptide) responsive TCRs in the virgin T cells can be cloned using the methods and materials provided herein.

[0178] In some aspects, the methods provided herein include methods for obtaining multiple nucleic acid vectors containing nucleic acids encoding functional T-cell receptors. The method comprises, or substantially comprises, the following: (a) obtaining an apparatus containing multiple individual sites, wherein each of the individual sites contains cDNA generated from RNA obtained from a single T cell sorted to the individual site; (b) performing a nested amplification procedure using the cDNA from each of the multiple individual sites as a template to obtain a first amplification product and a second amplification product of the cDNA from each of the multiple individual sites, wherein the first amplification product contains nucleic acid encoding a Vα or Vγ region, and wherein the second amplification product contains nucleic acid encoding a Vβ or Vδ region; and (c) assembling the first and second amplification products of the cDNA from each of the multiple individual sites into a nucleic acid vector to obtain an assembled nucleic acid vector of the cDNA from each of the multiple individual sites, wherein the assembled nucleic acid vector of the cDNA from each of the multiple individual sites contains nucleic acid encoding a functional T-cell receptor. The multiple nucleic acid vectors may be multiple nucleic acid expression vectors. The apparatus may include a multi-well plate. The multi-well plate can be a 96-well plate, a 384-well plate, or a 1536-well plate. cDNA derived from RNA obtained from a single T cell can include cDNA derived from RNA obtained from a single human T cell. The first amplification product may contain nucleic acid encoding an L sequence of the Vα or Vγ region. The first amplification product may contain nucleic acid encoding a Jα or Jγ region. The first amplification product may contain nucleic acid encoding the 5' portion of the Cα or Cγ region. The first amplification product may contain nucleic acid encoding an L sequence of the Vα or Vγ region, a Jα or Jγ region, and the 5' portion of the Cα or Cγ region. The second amplification product may contain nucleic acid encoding an L sequence of the Vβ or Vδ region. The second amplification product may contain nucleic acid encoding a Dβ or Dδ region. The second amplification product may contain nucleic acid encoding a Jβ or Jδ region. The second amplification product may contain nucleic acid encoding the 5' portion of the Cβ or Cδ region. The second amplification product may contain nucleic acids encoding the L sequence, Dβ or Dδ segment, Jβ or Jδ segment, and the 5' portion of the Cβ or Cδ region of the Vβ or Vδ region. The first amplification product may contain an adaptor sequence added to the amplification template sequence of the cDNA via a second round of nested amplification. The second amplification product may contain an adaptor sequence added to the amplification template sequence of the cDNA via a second round of nested amplification. The first amplification product may contain a first adaptor sequence added to the amplification template sequence of the cDNA via a second round of nested amplification, and the second amplification product may contain a second adaptor sequence added to the amplification template sequence of the cDNA via a second round of nested amplification, wherein the first and second adaptor sequences are different.The functional T-cell receptor in each of the assembled nucleic acid vectors may contain a Vα / Vβ combination or a Vγ / Vδ combination, as present in a single RNA-producing T cell. The functional T-cell receptor in each of the assembled nucleic acid vectors may contain (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. The functional T-cell receptor in each of the assembled nucleic acid vectors may contain (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 of the assembled nucleic acid vectors may contain a nucleic acid sequence encoding a self-cleaving peptide or an internal ribosome entry site (IRES). The method may include sorting single T cells to individual locations. The method may include performing a reverse transcription reaction to obtain cDNA. Assembly steps can include seamless cloning. Each component of the assembled nucleic acid vector can be obtained without nucleic acid sequencing. Each component of the assembled nucleic acid vector can be obtained without restriction endonuclease digestion.

[0179] C. Assess the expression, activity, and function of neoantigen-specific T cell receptors. Exemplary assays can be used to assess the activity, expression, and / or function of the anti-NPM1c TCRs and antigen-binding fragments described herein. The functional capabilities of candidate NPM1c-specific TCRs can be assessed using the assays described herein, which should not be construed as limiting.

[0180] The function of anti-NPM1c TCR can be characterized by binding assays of fluorescently labeled MHC molecules carrying specific target peptides (tetramers / pentamers / dextramers) or by activation assays of co-cultured anti-NPM1c TCR-expressing cells with antigen-presenting cells (APCs) presenting the corresponding MHC / peptide complex.

[0181] 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 the target antigen. T cells are then compared with K562 cells loaded with the target peptide AIQDLCLAV (SEQ ID NO:1). (neg) / HLA-A :02:01 (pos)They were incubated together. As a control, K562 cells were loaded with non-target peptides or irrelevant peptides. Intracellular cytokine staining and FACS analysis were performed after the IL-2 and / or IFN-γ response of T cells.

[0182] T cell activation / degranulation marker assays can be used to evaluate the ability of candidate anti-NPM1c TCRs to express the surface marker CD107a after exposure to cells presenting the target antigen. CD107a is a marker of T cell degranulation, which is part of the cytotoxic response. T cells were incubated with K562 cells loaded with the target peptide AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) or a control peptide. Degranulation on T cells was tracked by CD107a surface staining and FACS analysis.

[0183] The killing assay can evaluate the ability of candidate anti-NPM1c TCRs to cleave cells presenting the target antigen. T cells were incubated with a mixture of K562 cells differentially loaded with fluorescently labeled target peptides and control peptides to allow for the examination of on-target and off-target cytotoxicity within a single test sample. Fluorescent cell counting beads were included as a normalization / counting control. The fluorescently labeled K562 cells were loaded with the target peptide AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5). As a control, K562 cells labeled with different fluorescent tags were loaded with non-target peptides. FACS was performed after gated cell counting of the remaining AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptide-loaded K562 cells and control peptide-loaded K562 cells after incubation with T cells.

[0184] CD34 markers can be used as alternative efficacy modifiers. See Philip et al., (2014). Blood 124(8):1277-1287. The detection of CD34 (a marker of transduction efficiency) can be correlated with the functional efficacy described in this paper.

[0185] In some respects, amplification and unamplified screening using the exact same donor are used for direct comparison of methods. Amplification can be performed using certain methods. Given the time sensitivity of screening for anti-NPM1c TCRs with desired specificity, methods that generate candidate anti-NPM1c TCRs without an amplification process may be advantageous in some contexts. Reduced sample handling can also provide advantages in various contexts.

[0186] IV. Engineered Cells This document also provides cells, such as cells engineered to contain or express any of the anti-NPM1c TCRs described herein. Populations of such cells and compositions containing and / or enriching such cells are also provided, such as those wherein cells expressing anti-NPM1c TCRs constitute at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a greater percentage of the total cells in the composition. In some embodiments, the cells are primary T cells or some type of cell, such as T cells or CD8 cells. + or CD4 + Cells. The composition includes pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. This article also provides treatment methods for administering cells and compositions to subjects (e.g., patients).

[0187] Therefore, this document also provides genetically engineered cells expressing the anti-NPM1c TCR 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 bone marrow or lymphocytes, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including 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 from the subject and frozen. In some implementations, cells include one or more subgroups of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and their subpopulations, such as those defined by function, activation state, maturity, differentiation potential, expansion, recycling, localization and / or persistence, antigen specificity, type of antigen receptor, presence in a specific organ or compartment, biomarker or cytokine secretion profile, and / or degree of differentiation. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. In some implementations, the methods provided herein include isolating cells from the subject as described herein, preparing, processing, culturing, and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0188] This article includes T cells (including primary T cells) and / or CD4. + and / or CD8 + T cells (including primary CD4) + and / or CD8+ T cell subtypes and subsets include virgin T cells (T cells). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes, such as stem cell memory T (T12) SCM ) cells, central memory T (T) CM Cellular, effector memory T (T) EM Effector memory T cells or terminally differentiated T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring 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.

[0189] In some embodiments, the cells are NK cells or memory NK cells. In some embodiments, the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0190] In some embodiments, the cell includes one or more nucleic acids introduced via genetic engineering, thereby expressing a recombinant or genetically engineered product of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or in samples obtained from the cell (such as samples obtained from another organism or cell), and is, for example, not normally found in the engineered cell and / or the organism from which such cell originates. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising a chimeric combination of nucleic acids encoding various domains from a variety of different cell types.

[0191] In some implementations, the expression of endogenous TCR chains in engineered 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. Publication No. US2014 / 0301990.

[0192] In some implementations, nucleic acids encoding anti-AIQDLCLAV CAR or anti-CLAVEEVSL CAR are transfected into T cells to form anti-AIQDLCLAV CAR T cells or anti-CLAVEEVSL CAR T cells. T cells can be, 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 derived from umbilical cord blood, universal allogeneic T cells, universal CAR T cells, CAR T cells, native 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 used for adoptive cell transfer therapy, or TCR-engineered T cells. CAR T cells can be first-generation CAR T cells, second-generation CAR T cells, third-generation CAR T cells, fourth-generation CAR T cells, dual-antigen receptor CAR T cells, or CAR T cells with induced suicide genes, or combinations thereof.

[0193] A. Preparation of cells for genetic engineering In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used to introduce anti-NPM1c TCRs can 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 whom the cells are isolated (the donor) is a subject suffering from a disease or condition, requiring cell therapy, or to whom a cell therapy (i.e., autologous T cells) will be administered. In some embodiments, the donor is a healthy donor. In some embodiments, the subject is a person requiring a specific therapeutic intervention (such as adoptive cell therapy involving the isolation, processing, and / or engineering of cells).

[0194] Samples include tissues, fluids, and other samples taken directly from the subject, as well as samples produced by one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. 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), tissue and organ samples, including processed samples derived from them.

[0195] In some respects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is derived from apheresis or leukocyte removal products. Exemplary samples include whole blood, PBMCs, leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived from them. In the context of cell therapy (e.g., adoptive cell therapy), samples include those from autologous and allogeneic sources.

[0196] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from xenogeneic sources, such as mice, rats, non-human primates, or pigs.

[0197] B. Vectors and methods used for genetic engineering This article also provides methods, nucleic acids, compositions, and kits for expressing the anti-NPM1c TCR or its antigen-binding fragment provided herein, and for generating genetically engineered cells expressing such anti-NPM1c TCR or its antigen-binding fragment. Genetic engineering generally involves introducing nucleic acids encoding anti-NPM1c TCR (or its antigen-binding fragment) into cells, such as through retroviral transduction, nanoparticle delivery, transfection, or transformation.

[0198] In some implementations, gene transfer is accomplished by first stimulating cells, such as by combining them with stimuli that induce responses such as proliferation, survival, and / or activation (e.g., as measured by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in a culture to a number sufficient for clinical use.

[0199] Various methods for introducing genetically engineered components are well known and can be used with the methods and compositions provided. Exemplary methods include those for transferring nucleic acids encoding the anti-NPM1c TCR or its antigen-binding fragment provided herein, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, electroporation, and nuclear transfection.

[0200] In some embodiments, recombinant infectious viral particles are used to transfer recombinant nucleic acids into cells. In some embodiments, recombinant lentiviral vectors or retroviral vectors (such as gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells.

[0201] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they can infect host cells from multiple species, including humans. In some embodiments, the nucleic acid to be expressed replaces the retroviral gag, pol, and / or env sequences. Many illustrative retroviral systems have been described elsewhere (see, for example, U.S. Patent Nos. 5,219,740; 6,207,453; 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).

[0202] Methods of lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al., (2012) J. Immunother. 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.

[0203] In some embodiments, the recombinant nucleic acid is transferred to T cells via electroporation or nuclear transfection (see, for example, Chicaybam et al., (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al., (2000) GeneTherapy 7(16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred to T cells via transposition (see, for example, Manuri et al., (2010) Hum Gene Ther 21(4): 427-437; Sharma et al., (2013) MolecTher Nucl Acids 2, e74; and Huang et al., (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing the nucleic acids described herein in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0204] Other methods and vectors for transferring nucleic acids encoding the anti-NPM1c TCR, its antigen-binding fragment, or recombinant product provided herein include those described elsewhere. See, for example, International Patent Application Publication No. WO2014 / 055668 and U.S. Patent No. 7,446,190.

[0205] In some embodiments, one or more additional nucleic acids may be introduced into cells simultaneously or sequentially with a nucleic acid encoding the anti-NPM1cTCR or its antigen-binding fragment provided herein. In some embodiments, such additional nucleic acids for introduction may be those that improve the efficacy of the therapy (e.g., by promoting the viability and / or function of metastatic cells); those that provide genetic markers for selecting and / or evaluating cells (e.g., for assessing in vivo survival or localization); and / or those that improve safety (e.g., by making cells sensitive to the said in vivo negative selection) (Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); Riddell et al., Human Gene Therapy 3:319-338 (1992)); PCT / US91 / 08442 and PCT / US94 / 05601 describe the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker; and U.S. Patent No. 6,040,177).

[0206] Therefore, in some embodiments, engineered cells are provided, such as those containing an anti-NPM1c TCR or its antigen-binding fragment, nucleic acid, or vector as described herein. In some aspects, the cells are generated by transducing cells in vitro or ex vivo using the vectors described herein. In some aspects, the cells are T cells, such as CD8+ or CD4+ T cells. In some embodiments, the anti-NPM1c TCR is heterologous to the cells.

[0207] V. Treatment and prevention methods and uses This article also provides methods and uses for the administration of the anti-NPM1c TCR and its antigen-binding fragment and / or engineered cells expressing the anti-NPM1c TCR or its antigen-binding fragment, such as for therapeutic and preventative uses. Such methods and uses include therapeutic methods and uses, for example, involving the administration of molecules, cells, or compositions containing such molecules or cells to patients with NPM1c TCR. + Subjects with cancer or other conditions. In some embodiments, the molecule, cells, and / or composition are administered in an effective amount to achieve NPM1c. + Treatment of cancer or condition. Uses include the use of anti-NPM1c TCRs and engineered T cells in such methods and treatments, and in the preparation of medicines for administering such treatments. In some embodiments, these methods are performed by administering anti-NPM1c TCRs or engineered T cells, or compositions comprising them, to a subject who has, has, or is suspected of having NPM1c+ cancer or condition. In some embodiments, these methods thereby treat the subject with NPM1c+ cancer or condition or disease.

[0208] The described anti-AIQDLCLAV TCR, anti-AIQDLCLAV BiTE, and anti-AIQDLCLAV CAR, as well as engineered cells expressing anti-AIQDLCLAV TCR, anti-AIQDLCLAV BiTE, and anti-AIQDLCLAV CAR, can be used to treat cancer. Methods for treating cancer in subjects are also described, including administering to the subject any cells expressing any of the described anti-AIQDLCLAV TCR, anti-AIQDLCLAV BiTE, and anti-AIQDLCLAV CAR, such as T cells (engineered anti-AIQDLCLAV T cells). Engineered T cells expressing anti-AIQDLCLAV TCR can be administered to subjects to induce an immune response, increase T cell infiltration in tumors, reduce or inhibit cancer cell growth, reduce or inhibit tumor growth, reduce tumor progression, reduce tumor mass, inhibit or reduce metastasis, reduce or inhibit the development of metastatic cancer, or increase subject survival or prolong subject life.

[0209] The described anti-CLAVEEVSL TCR, anti-CLAVEEVSL BiTE, and anti-CLAVEEVSL CAR, as well as engineered cells expressing anti-CLAVEEVSL TCR, anti-CLAVEEVSL BiTE, and anti-CLAVEEVSL CAR, can be used to treat cancer. Methods for treating a subject's cancer are also described, including administering to the subject any cells expressing any of the described anti-CLAVEEVSL TCR, anti-CLAVEEVSL BiTE, and anti-CLAVEEVSL CAR, such as T cells (engineered anti-CLAVEEVSL T cells). Engineered T cells expressing anti-CLAVEEVSL TCR can be administered to the subject to induce an immune response, increase T cell infiltration in the tumor, reduce or inhibit cancer cell growth, reduce or inhibit tumor growth, reduce tumor progression, reduce tumor mass, inhibit or reduce metastasis, reduce or inhibit the development of metastatic cancer, increase the subject's survival or prolong the subject's life, or reduce or weaken an autoimmune response.

[0210] The described anti-NPM1c TCR and T cells expressing the described anti-NPM1c TCR can be used to kill or eliminate NPM1c-expressing cancer cells. Killing or eliminating NPM1c-expressing cancer cells in a subject can be used to treat NPM1c. + Cancer. In some implementations, the engineered T cells expressing anti-NPM1c TCR are engineered autologous T cells (i.e., the T cells are derived from the subject to be treated).

[0211] The disease to be treated includes cancer. In some embodiments, the disease or condition to be treated is a liquid tumor. In some embodiments, the disease or condition to be treated is a hematopoietic tumor. In some embodiments, the disease or condition to be treated is NPM1c. + Leukemia, NPM1c + Acute myeloid leukemia (AML) or NPM1c + Myelodysplastic syndrome (MDS). In some implementations, the disease or condition is AML. AML can be diagnosed according to the European Leukemia Network (ELN) criteria (e.g., Döhner et al., 2022).

[0212] In some embodiments, the described anti-NPM1c TCR can be isolated and administered to a subject as a soluble anti-NPM1c TCR. In some embodiments, any of the described anti-NPM1c TCRs can be isolated and conjugated with a molecule (such as a therapeutic molecule or antibody) to form an anti-NPM1c TCR-drug conjugate. The conjugated anti-NPM1c TCR can then be administered to a subject. In some embodiments, the therapeutic molecule is a therapeutic molecule targeting AML. Therapeutic molecules targeting AML include, but are not limited to, menin-MLL inhibitors. In some embodiments, the therapeutic molecule is a therapeutic anticancer agent. Anticancer agents can be, but are not limited to, Pseudomonas exotoxin, ricin, monomethyl auristatin F, and calicheamicin. In some embodiments, the anti-NPM1c TCR is used as a substitute for the antibody in an antibody-drug conjugate.

[0213] In some implementations, engineered T cells expressing any of the described anti-NPM1c TCRs are administered to a subject for the treatment of NPM1c. + Cancers, such as AML. In some embodiments, engineered T cells expressing any of the described anti-NPM1c TCRs can be formulated as a T-cell therapy for treating NPM1c+ cancers (e.g., AML). In some embodiments, the T-cell therapy is an autologous T-cell therapy.

[0214] In some embodiments, either the described anti-NPM1c TCR or engineered cells expressing anti-NPM1c can 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, simultaneously with, or after administering the anti-NPM1c TCR or engineered cells expressing anti-NPM1c.

[0215] In some implementations, the described anti-NPM1c TCR and / or engineered T cells expressing the described TCR are administered to the subject to kill the subject's NPM1c. + Cells. Subjects may or may not have received SCT. Subjects may or may not plan to receive SCT. Subjects may or may not be eligible for SCT.

[0216] In some embodiments, the subject has received one or more prior therapies to treat cancer. In some embodiments, the subject has relapsed after one or more prior therapies to treat 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 therapies to treat cancer. In some embodiments, the subject is refractory to one or more prior therapies to treat 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 therapies to treat cancer. In some embodiments, the subject has persistent disease after one or more prior therapies to treat cancer. In some embodiments, the subject has persistent disease after one or more induction therapies to treat cancer. One or more prior therapies or induction therapies may be, but are not limited to, chemotherapy, azacytidine 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 implementations, the subject has relapsed after one or more consolidation therapies, is refractory to these consolidation therapies, or has persistent disease after these consolidation therapies. In some implementations, the subject has relapsed after SCT or alloSCT but has not received prior T-cell therapy.

[0217] In some implementations, the subject with the NPM1c mutation is FLT3 positive. In some implementations, the subject with the NPM1c mutation is FLT3 positive, and the engineered T cells are allogeneic T cells (i.e., T cells derived from an HLA-matched donor). In some implementations, the subject with the NPM1c mutation is FLT3 positive, the engineered T cells are allogeneic T cells, and the engineered T cells are administered in combination with alloSCT.

[0218] In some implementation schemes, the subjects had NPM1c + Cancer and unsuitable for transplantation (e.g., standard care alloSCT). In some implementations, the subject has NPM1c. +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, kidney failure, heart failure, infection, and risk of infection. In some implementations, the subject is 60 years of age or older, 65 years of age or older, or 70 years of age or older.

[0219] In some implementation schemes, the subjects had NPM1c + The subject has cancer (e.g., AML) and is not suitable for high-intensity chemotherapy. In some implementations, the subject has relapsed after high-intensity chemotherapy with or without hematopoietic stem cell transplantation (HSCT). In some implementations, the subject has no response to high-intensity chemotherapy, has no response, or has failed to achieve complete remission (is refractory to high-intensity chemotherapy). In some implementations, the subject is suitable for reduced-intensity chemotherapy. Reduced-intensity chemotherapy may be administered prior to the administration of engineered T cells.

[0220] In some implementations, the subject is fit for high-intensity treatment but is considered to be at moderate or adverse risk and has not responded to previous high-intensity chemotherapy or has relapsed after previous high-intensity chemotherapy.

[0221] In some implementations, the subject has relapsed after prior hypomethylating agent and / or BCL-2 inhibitor therapy. In some implementations, the subject has no response to hypomethylating agent therapy, has no response, or has failed to achieve complete remission (is refractory to hypomethylating agent therapy).

[0222] In some implementations, the subject has relapsed after stem cell transplantation. In some implementations, the subject has no response to stem cell transplantation. In some implementations, subjects suitable for treatment with T-cell therapy using any of the described anti-NPM1c TCRs have not yet received stem cell transplantation. In some implementations, subjects suitable for treatment with T-cell therapy using any of the described anti-NPM1c TCRs have not received stem cell transplantation within two months of receiving T-cell therapy.

[0223] In some implementations, treatment for patients with or diagnosed with NPM1c is described. +Methods for treating cancer subjects, including administering to subjects any of the anti-NPM1c TCRs or their 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, treatment of patients or those diagnosed with NPM1c is described. + Methods for treating cancer subjects, including administering to the subject any of the anti-NPM1c TCRs provided herein or their antigen-binding fragments, 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 prior to administering SCT to the subject. In some embodiments, any of the anti-NPM1c TCRs provided herein or their antigen-binding fragments, 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 are formulated for use in combination with stem cell transplantation for the treatment of patients with or diagnosed with NPM1c. + Cancer patients. Anti-NPM1c TCRs, their antigen-binding fragments, polynucleotides, vectors, engineered cells, or compositions may be formulated for administration in combination with an autologous T-cell therapy (SCT) to a patient or for administration 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. NPM1c + Cancer can be, but is not limited to, AML.

[0224] In some implementations, treatment for patients with or diagnosed with NPM1c is described. +Methods for treating cancer subjects, including administering to a subject any of the anti-NPM1c TCRs provided herein or an antigen-binding fragment thereof, 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 treatment is not combined with SCT. In some embodiments, any of the anti-NPM1c TCRs provided herein or an antigen-binding fragment thereof, 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 may be formulated for use in the absence of stem cell transplantation for treating patients with or diagnosed with NPM1c cancer. + Cancer patients. In some implementations, anti-NPM1c TCR is administered as autologous T-cell therapy. NPM1c + Cancer can be, but is not limited to, AML.

[0225] In some implementation schemes, the subjects had NPM1c + Leukemia. In some implementations, the subject had NPM1c. + Acute myeloid leukemia (AML). In some implementations, the subject has NPM1c. + Myelodysplastic syndrome.

[0226] In some implementations, the subject has not received any prior therapy. In some implementations, the subject has NPM1c. +Leukemia and administration of the described anti-NPM1c-TCR, anti-NPM1c TCR BiTE, anti-NPM1c-drug conjugate, or engineered T cells expressing anti-NPM1c TCR as a first-line therapeutic treatment. In some embodiments, the subject has received one or more prior therapies for cancer treatment. In some embodiments, the subject has relapsed after one or more prior therapies for cancer treatment. 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 for cancer treatment. 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 for cancer treatment. In some embodiments, the subject has persistent disease after one or more induction therapies for cancer treatment. One or more prior therapies or induction therapies may be, but are not limited to, chemotherapy, azacytidine therapy, FLT3 inhibitor therapy, IDH inhibitor therapy, BCL-1 inhibitor therapy, hedgehog pathway inhibitor therapy, radiotherapy, aSCT, or alloSCT. In some implementations, the subject has relapsed after one or more consolidation therapies, is refractory to these consolidation therapies, or has persistent disease after these consolidation therapies. In some implementations, the subject has relapsed after SCT or alloSCT but has not received prior T-cell therapy.

[0227] In some implementation schemes, the subjects had NPM1c + Cancer (e.g., NPM1c) + AML, has received induction therapy and is in remission.

[0228] In some implementations, the subjects have been diagnosed with NPM1c + Cancer (e.g., NPM1c) + (AML), has received induction therapy, and the subject is given the provided anti-NPM1c TCR, its antigen-binding fragment, polynucleotide, vector, engineered T cells, or combinations thereof as consolidation therapy.

[0229] In some implementations, the subject has NPM1c+ cancer and the Flt3ITD mutation. In some implementations, the subject has NPM1c + Cancer and having the Flt3ITD mutation, and administering the provided anti-NPM1c TCR, its antigen-binding fragment, polynucleotide, vector, engineered T cells, or combinations thereof to the subject to reduce or eliminate the need for SCT.

[0230] In some implementation schemes, the subjects had NPM1c +Cancer and does not have the Flt3ITD mutation. In some implementations, the subject has NPM1c. + Cancer, without Flt3ITD mutation, and administering the provided anti-NPM1c TCR, its antigen-binding fragment, polynucleotide, vector, engineered T cells, or combinations thereof to the subject to reduce risk or recurrence.

[0231] In some embodiments, subjects diagnosed with or suspected of having leukemia are given a genetic test involving the NPM1 gene. The NPM1 gene test can be performed on samples obtained from bone marrow or peripheral blood. The NPM1 gene test can be any test known in the art for detecting the presence of an NPM1c mutation or for detecting type A or D NPM1 mutations. In some embodiments, the NPM1 gene test includes a PCR test or next-generation sequencing (NGS). If the genetic test indicates that the subject has NPM1c+ cancer, the subject can be treated with engineered T cells expressing any of the described anti-NPM1c TCRs.

[0232] In some implementations, subjects who test positive for NPM1c+ cancer are screened to assess eligibility or suitability for engineered T-cell therapy. Screening may include, but is not limited to, NPM1 testing, organ function (e.g., liver and kidney function) testing, subject performance status, and the presence of adverse events due to prior treatment. In some implementations, screening is performed up to approximately 45 days prior to the anticipated administration of engineered T-cell therapy.

[0233] Leukoablation is performed on the subjects to collect white blood cells. In some implementations, CD8... + T cells are obtained from leukocyte removal products via positive selection. In some embodiments, an activator is used to activate CD8. + T cell activation. Approximately 24 hours after activation, T cells are engineered to knock out endogenous TCRs. Endogenous TCR knockout can be performed using CRISPR. A nucleic acid sequence (or sequence encoding any of the described anti-NPM1c TCRs is inserted into CD8. + In TR cells, the insertion of a nucleic acid sequence encoding an anti-NPM1c TCR can be performed 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 expanded to obtain a sufficient number for treatment. Leukoablation can be performed approximately 21 to approximately 45 days prior to the intended administration of engineered T cell therapy to the subject.

[0234] Prior to T-cell therapy, the subject undergoes treatment to induce lymphocyte depletion, enabling the engraftment of infused engineered T cells (e.g., TCR-T cells). Lymphocyte depletion can be performed using any method available in the art for lymphocyte depletion. Suitable lymphocyte depletion regimens include, but are not limited to, cyclophosphamide and fludarabine treatments. Lymphocyte depletion can be performed approximately 1 to approximately 7 days prior to the intended administration of engineered T-cell therapy to the subject.

[0235] For T-cell therapy, engineered T cells are infused at a predetermined dose. The dose may be, but is not limited to, approximately 0.3 × 10⁻⁶. 6 One to approximately 1 × 10⁹ cells 7 One cell, approximately 1 × 10 6 One to approximately 1 × 10⁹ cells 7 One cell or approximately 3 × 10 6 One to approximately 1 × 10⁹ cells 7 The dose is per cell. In some implementations, the dose is approximately 0.3 × 10⁻⁶ cells. 6 1 cell, approximately 0.4 × 10 6 One cell, approximately 0.5 × 10 6 1 cell, approximately 0.6 × 10 6 1 cell, approximately 0.7 × 10 6 One cell, approximately 0.8 × 10⁻⁶ 6 1 cell, approximately 0.9 × 10 6 One cell, approximately 1 × 10 6 1 cell, approximately 2 × 10 6 1 cell, approximately 3 × 10 6 1 cell, approximately 4 × 10 6 1 cell, approximately 5 × 10 6 1 cell, approximately 6 × 10 6 1 cell, approximately 7 × 10 6 1 cell, approximately 8 × 10 6 1 cell, approximately 9 × 10 6 One cell or approximately 1 × 10 7 Each cell.

[0236] The terms "treat" and "treatment" refer to methods or procedures used to provide relief, improvement, or reduction of the quantity, severity, adverse effects, and / or frequency of one or more symptoms or pathological outcomes of a subject's disease, disorder, or condition. Treatment can be preventative in relation to the prevention or partial prevention of a disease or its symptoms or conditions. Prevention includes providing prevention of the occurrence or recurrence of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it. Prevention also includes providing prevention of the occurrence or recurrence of symptoms or pathological outcomes of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it. Treatment can also be preventative in relation to delaying the onset of a disease or its symptoms or conditions. Delaying the development of a disease or its symptoms or pathological outcomes refers to postponing, hindering, slowing, delaying, stabilizing, inhibiting, and / or postponing the development of a disease or its symptoms or pathological outcomes. The length of the delay may vary depending on the disease history and / or the individual being treated. Treatment may include suppressing a disease, disorder, or symptom, for example, halting its progression; and alleviating a disease, disorder, or symptom, for example, causing its remission. Treatment may also mean prolonging survival compared to expected survival without treatment. Treatment may be therapeutic in relation to the partial or complete cure of a disease, symptom, symptom, or adverse effect attributable to the disease, disorder, or symptom. The term treatment may include: (a) preventing the occurrence of a disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing a disease, i.e., preventing its development; and (c) alleviating a disease, i.e., reducing or improving the disease and / or its symptoms or symptom. Treatment may refer to therapeutic treatment alone, preventive treatment alone, or both therapeutic and preventive treatment. Those who require treatment (subjects in need) may include those previously diagnosed with a disease, disorder, or symptom, or those identified as being at risk of developing a disease, disorder, or symptom. Treating a disease, disorder, or symptom may include improving at least one symptom of a particular disease, disorder, or symptom, even if the underlying pathophysiology is unaffected.

[0237] In the context of administration, the “effective amount” of a drug (e.g., a pharmaceutical preparation, a TCR, a cell, or a composition) refers to the amount that effectively achieves the desired outcome (such as a therapeutic or preventative outcome) at the necessary dose / amount and for the required time period.

[0238] A “therapeutic effective amount” of a pharmaceutical agent (e.g., a drug formulation, a TCR, or cells) refers to the amount that effectively achieves the desired therapeutic outcome (such as treatment of a disease, condition, or disorder) and / or the pharmacokinetic or pharmacodynamic effect of the treatment at the necessary dose and for the required time period. Therapeutic effective amounts can vary depending on factors such as the subject’s disease state, age, sex, and weight, as well as the cell population administered. In some embodiments, the provided method includes administering a TCR, cells, and / or composition at an effective amount (e.g., a therapeutically effective amount).

[0239] As used in this article, “subject” is a mammal, such as a human or other animal, and is usually a human.

[0240] VI. Definition Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or proper nouns used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some embodiments, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein is not necessarily construed as indicating a material difference from the content commonly understood in the art.

[0241] The terms “peptide” and “protein” are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Peptides (including provided T-cell receptors, their antigen-binding fragments, and other peptides such as linkers) may include amino acid residues, including native and / or non-native amino acid residues. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some respects, peptides may contain modifications relative to their native or natural sequences, provided the protein retains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0242] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include those normally found in cells, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0243] "Isolated nucleic acid molecule encoding TCR" refers to a single nucleic acid molecule (e.g., a single vector) that encodes TCRs such as functional α / β TCRs or functional γ / δ TCRs.

[0244] "Isolated nucleic acid molecule encoding the antigen-binding fragment of TCR" refers to a single nucleic acid molecule (e.g., a single vector) that encodes the antigen-binding fragment of TCR.

[0245] "Separate nucleic acid molecules encoding TCRs" refers to two or more separate nucleic acid molecules (e.g., two or more vectors) that together encode TCRs such as functional α / β TCRs or functional γ / δ TCRs. Each of these two or more nucleic acid molecules may be present at different locations within the host cell.

[0246] "Separated 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 present at different locations within the host cell.

[0247] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content, but may contain mutations. This article includes mutant progeny with the same function or biological activity, such as those screened or selected from the initially transformed cells.

[0248] As used herein, when referring to an amino acid sequence (reference polypeptide sequence), “amino acid sequence identity percentage (%)” and “identity percentage” are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject T cell receptor or fragment) that are identical to amino acid residues in a reference polypeptide sequence after alignment of the sequences and, where necessary, introducing gaps to achieve the maximum sequence identity percentage and without treating any conserved substitutions as part of sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be performed in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0249] Amino acid substitution may involve replacing one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into the TCR of interest or its antigen-binding fragment, and the desired activity of the product may be screened, such as retention / improvement of antigen binding, reduced immunogenicity, or improved cell lysis activity.

[0250] Amino acids can generally be grouped according to the following common side chain characteristics: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidity: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Fang tribe: Trp, Tyr, Phe.

[0251] In some embodiments, conservative substitution may involve exchanging a member of one of these categories for another member of the same category. In some embodiments, non-conservative amino acid substitution may involve exchanging a member of one of these categories for another.

[0252] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of the host cell to which they are introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0253] As used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. For example, “a” or “an” means “at least one (kind)” or “one (kind) or more (kinds).” It should be understood that aspects and variations described herein include aspects and variations that “comprise” and / or “substantially consist of”.

[0254] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope-based description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Therefore, the description of scope should be considered as having specifically disclosed all possible sub-scopes and the individual values ​​within those scopes. For example, in the case of providing a range of values, it should be understood that every intermediate value between the upper and lower limits of that range, as well as any other specified value or intermediate value within that specified range, is covered within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are also covered within the claimed subject matter, subject to any specific exclusions in the stated scope. Where the stated scope includes one or both of the limitations, the scope excluding one or both of those included limitations is also included in the claimed subject matter. This applies regardless of the width of the scope.

[0255] As used herein, the terms “about” and “approximately”, when used to modify a quantity specified in a numerical value or range, indicate the numerical value and a reasonable deviation from values ​​known to those skilled in the art. In some embodiments, the term “about” means within a typical tolerance 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 ranges, it should be understood that “about” may modify each number in that series or range.

[0256] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous substance, non-aqueous substance, or any combination thereof.

[0257] As used herein, the statement that a cell or cell population is “positive” for a particular biomarker means that the particular biomarker (typically a surface biomarker) is detectably present on or within the cell. When referring to a surface biomarker, the term means the presence of surface expression as detected by flow cytometry, for example, by staining with and detecting the antibody that specifically binds to the biomarker, wherein the staining can be detected by flow cytometry at levels significantly higher than those detected by the same procedure with a type-matched control under otherwise identical conditions and / or substantially similar to and / or significantly higher than those detected by cells known to be positive for the biomarker.

[0258] As used herein, the statement that a cell or cell population is “negative” for a particular biomarker means that the particular biomarker (typically a surface biomarker) is not substantially detectably present on or within the cell. When referring to a surface biomarker, the term means the absence of surface expression as detected by flow cytometry, for example, by staining with and detecting said antibody with an antibody that specifically binds to the biomarker, wherein the staining is not detected by flow cytometry at levels significantly higher than those detected by the same procedure with a type-matched control under otherwise identical conditions and / or significantly lower than and / or substantially similar to those of cells known to be positive for the biomarker.

[0259] When referring to a binding region (e.g., a TCR binding region), the term "specific binding" to a target refers to a binding reaction in which the binding region binds to the target with greater affinity, stronger binding force, and / or longer duration than it binds to a structurally different target. When measured under the same affinity assay conditions, the binding region may have at least 5, 6, 7, 8, 9, 10, 20, 25, 50, 100, 1,000, 10,000, or greater affinity for a specific target compared to an unrelated target.

[0260] Example The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. CDR3β Example 1. Exemplary identification TCR .

[0261] Table 4 lists the sequence identifiers (SEQ ID NO) of the isolated, evaluated, and sequenced AIQDLCLAV (SEQ ID NO:1)-specific TCR and CLAVEEVSL (SEQ ID NO:5)-specific TCR (i.e., anti-AIQDLCLAV TCR and anti-CLAVEEVSL TCR) amino acid (aa) or nucleotide (nt) sequences. The table also lists the sequence identifiers (SEQ ID NO) corresponding to the exemplary full-length sequences, including constant domains, i.e., amino acid sequences containing the α and β chain sequences of each corresponding TCR, separated by a sequence encoding the ribosomal jumping P2A sequence (the P2A linker shown in SEQ ID NO:301 encoded by the nucleotide shown in SEQ ID NO:302) (named "α-P2A-β").

[0262] Table 4: Amino acid and nucleotide sequences of AIQDLCLAV (SEQ ID NO:1) specific TCRs (SEQ ID NO:1) NO)

[0263] Example 2. Example AIQDLCLAV ( SEQ ID NO:1 ) Specificity TCR The representation .

[0264] As described in this article, 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-cancer cells. Therefore, T cells targeting AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) are unlikely to induce GVHD.

[0265] Example 3. Example NPM1 Specificity TCR Binding specificity .

[0266] The binding specificity of AIQDLCLAV (SEQ ID NO:1)-specific TCR and CLAVEEVSL (SEQ ID NO:5)-specific TCR was determined using tetramers of complexes with AIQDLCLAV (SEQ ID NO:1), CLAVEEVSL (SEQ ID NO:5), or control peptides.

[0267] HEK293 suspension cells were engineered to express human CD3γ, CD3δ, CD3ε, CD3ζ (poly-CD3), and human CD8α / β. CD3 and CD8 were cloned from merged PBMCs from two healthy blood donors and stably transduced into HEK293 cells. Suspension HEK293-CD3-CD8 cells were transiently transfected with plasmid DNA containing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR and the fluorescent protein mCherry as a transfection efficiency control.

[0268] Twenty-four hours after transfection, cells were stained with an amine-reactive dye (purple 510 Ghost dye), an anti-CD3 and immunogenic AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptide tetramer, or a control peptide tetramer. Cells were obtained and analyzed by flow cytometry.

[0269] Double-positive fluorescence of CD3 and specific binding of HLA-tetramers to peptides AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) indicate functional reactive TCR.

[0270] Example 4. Engineering T Cellular AIQDLCLAV ( SEQ ID NO:1 In vitro evaluation of reactivity .

[0271] The TCRs isolated and identified as described in Examples 1-3 above were recombinantly expressed in Jurkat cell lines engineered to stably express CD8, and their functions, including EC2, were further characterized and evaluated. 50 Measurements can also determine the cytokine secretion, T cell activation, and binding specificity of each TCR.

[0272] For the exemplary AIQDLCLAV (SEQ ID NO:1)-specific TCR or CLAVEEVSL (SEQ ID NO:5)-specific TCR, Jurkat T cells expressing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR were co-cultured with K562 cells pulsed with AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptide or control peptide. Equal numbers of Jurkat T cells and K562 cells loaded with increased concentrations (e.g., 0.1 ng / ml–31.6 ng / ml) of AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptide or control peptide were co-cultured for 16 hours. JurkatCD69 expression was measured by FACS.

[0273] 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 to assess CD69 expression (a marker of T cell activation and function).

[0274] Jurkat T cells expressing various anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR were prepared as described. Jurkat cells transduced with anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR were then compared with A... 02:01 LCL was incubated overnight at a 1:1 E / T ratio in the presence of increased concentrations of the AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptides. Based on XLFit (ID Business Solutions) on CD69 + EC was calculated from a graph of cell percentage (y-axis) versus peptide concentration (log x-axis). 50 value.

[0275] For the exemplary TCR, T cells expressing anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR were co-cultured in duplicate with peptide-loaded K562 cells at a 1:1 effector-to-target ratio. After 16–24 hours of culture, cells were washed and stained with Ghost Dye, anti-CD3, and anti-CD69, and evaluated by flow cytometry. Data were analyzed using XLFit. The estimated EC50 for each of the five experimental runs was determined by assessing the ratio of CD69-positive Jurkat cells to total viable cells. 50 .

[0276] Assessment of BT cell receptor specificity The specificity of candidate anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR for AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5) peptides to the corresponding wild-type NPM1 peptides presented by restricted HLA or potentially other HLA molecules or other HLA molecules presenting other peptides was also evaluated.

[0277] 2. Allogeneic reactivity HLA-A receptors loaded with irrelevant peptides (such as HPV peptides) 02:01 Tetramer reactivity to allogeneic compounds was tested.

[0278] Example 5. use AIQDLCLAV ( SEQ ID NO:1 ) Specificity TCR The original generation of transduction T Target cell killing .

[0279] To determine the potential for inducing graft-versus-leukemia (GvL) effects, the anti-leukemic target cell killing activity of primary T cells transduced with anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR was evaluated.

[0280] A. AIQDLCLAV (SEQ ID NO:1) specific TCR or CLAVEEVSL (SEQ ID NO:5) specific TCR in Expression in primary cells Enrichment of native human CD8 from PBMCs via negative selection + T cells. CD8 + The endogenous TCRs of T cells were knocked out via CRISPR. Then, CD8+ were transduced using a lentiviral vector (pLVG.M containing the MNDU3 promoter) encoding either the anti-AIQDLCLAV TCR or the anti-CLAVEEVSL TCR and the RQR8 tag. + T cells. On day 5 post-transduction, the percentage of transduced T cells was assessed by flow cytometry using anti-CD34 antibody and by measuring RQR8 expression.

[0281] B. Target cell killing The target cell killing specificity of anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR was evaluated. HLA-A 02:01 + / AIQDLCLAV + (SEQ ID NO: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 marks target cells by binding the dye to intracellular proteins and instructs T cell-mediated target cell killing. Apoptosis of the labeled cells resulted in their undetectability in the live-cell gate during flow cytometry. Primary human T cells expressing anti-AIQDLCLAVTCR or anti-CLAVEEVSL TCR were co-cultured with LCL for 18 hours at an E:T ratio (transduced CD34... + Starting with positive cells, serial dilutions were performed, beginning at 10:1 and ending at 0:1. Cells were stained with 7-AAD (to assess cell viability), and obtained by flow cytometry evaluation.

[0282] Example 6. Target cell killing activity.

[0283] The target cell killing activity of the exemplary TCR was further evaluated at different effector:target (E:T) ratios.

[0284] Genotyping of OCI-AML3 cells was performed based on the expression of AIQDLCLAV (SEQ ID NO:1) and / or CLAVEEVSL (SEQ ID NO:5). To differentiate cell populations, OCI-AML3 cells presenting 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 presenting the corresponding wild-type NPM1 peptide were labeled with a low concentration (0.025 μM) of VPD450. Both OCI-AML2 and OCI-AML3 cells were stained with VPD450 at 37°C for 15 minutes. OCI-AML2 and OCI-AML3 cells carrying the target peptide (AIQDLCLAV (SEQ ID NO:1) or CLAVEEVSL (SEQ ID NO:5)) and the control peptide were mixed at a 1:1 ratio and then incubated with increased numbers of transduced primary T cells expressing AIQDLCLAV (SEQ ID NO:1)-specific TCRs or CLAVEEVSL (SEQ ID NO:5)-specific TCRs. Alternatively, target OCI-AML2 or OCI-AML3 cells were stained and incubated separately with TCR-T cells at a 1:1 ratio. Subsequent E:T ratio EC50 killing curves could be determined.

[0285] OCI-AML2 and OCI-AML3 cells were co-cultured with increased numbers of primary T cells for 16 hours (E:T ratio of 1:16 to 10:1). The cell mixture was stained with the LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific), anti-CD8, and anti-CD19 to assess two distinct target cell populations by VPD450 staining levels. Cells were obtained and analyzed by flow cytometry to evaluate CD8 cells (to exclude effector cells) and high-VPD450 versus low-VPD450 populations.

[0286] Viable cell counts of OCI-AML2 and OCI-AML3 cells presenting target or control peptides were determined after incubation with untransduced T cells or T cells transduced with anti-AIQDLCLAV TCR or anti-CLAVEEVSL TCR. Loss of target peptide-presenting viable cells (high VPD450 staining), accompanied by retention of control peptide-presenting viable cells (low VPD450 staining) observed with increasing effector-to-target (E:T) ratio, indicates that target cells presenting AIQDLCLAV (SEQ ID NO:1) and / or CLAVEEVSL (SEQ ID NO:5) are selectively killed by T cells genetically engineered to express AIQDLCLAV (SEQ ID NO:1)-specific TCR or CLAVEEVSL (SEQ ID NO:5)-specific TCR.

[0287] Example 7. Computer prediction AIQDLCLAV ( SEQ ID NO:1 Peptides and HLA combination .

[0288] Predict the binding of AIQDLCLAV, IQDLCLAV, and AIQDLCLA to HLA using the ANN 4.0 option on IEDB (see below). Predicted IC50 values ​​for AIQDLCLAV binding to the HLA-A allele. 50 The values ​​are shown in Tables 6a-6d. Based on the combined predictions of AIQDLCLAV, IQDLCLAV, and AIQDLCLA, HLA-A is expected to be present. 02:06, HLA-A 02:03, HLA-A The subjects at 02:01 are suitable candidates.

[0289] Table 6a. Combination of AIQDLCLAV, IQDLCLAV, and AIQDLCLA with predictions of various HLA-A alleles

[0290] Table 6b. TDQEAIQDLCLAVEEVSLRK, TDQEAIQDLCMAVEEVSLRK, TDQEAIQDLCVAVEEVSLRK Combined with predictions of various HLA-A alleles. (Query 8-mer, 9-mer, and 10-mer)

[0291] Table 6b. TDQEAIQDLCLAVEEVSLRK, TDQEAIQDLCMAVEEVSLRK, TDQEAIQDLCVAVEEVSLRK Combined with predictions of various HLA-B alleles. (Query 8-mer, 9-mer, and 10-mer)

[0292] Table 6d. TDQEAIQDLCLAVEEVSLRK, TDQEAIQDLCMAVEEVSLRK, TDQEAIQDLCVAVEEVSLRK Combined with predictions of various HLA-C alleles. (Query 8-mer, 9-mer, and 10-mer)

[0293] Example 8. Engineering T In cells TCR Induced signal transduction .

[0294] Evaluation of TCR D via HLA The target peptide presented by A02:01 (CLAVEEVSL, SEQ ID NO:5) induces T cell signaling. In short, Jurkat cells are engineered to express α-TCR D. K562 HLA expression is then used. A02:01 cells were pulsed with the 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 by the target peptide.

[0295] Jurkat and K562 cell lines were cultured in RPMI and IMDM, respectively. The peptides were reconstructed and diluted in PBS prior to peptide pulses.

[0296] Use TCR D against CD8 + Jurkat cells were used for viral transduction (lentivirus), and TCR-positive cells (Jurkat-TCR D cells) were purified by FACS-based sorting. K562 was produced via pan-HLA class I KO and HLA-based... A02:01 The virus was transduced and purified by FACS sorting before reporter gene assay.

[0297] For reporter gene assays, engineered K562 cells were pulsed with a titrated concentration of the target peptide for 1 hour prior to co-culturing. Jurkat-TCR D cells were co-cultured with peptide-pulsed K562 cells at a 1:1 E:T ratio in 96-well plates. After 16–18 hours, the co-cultured cells were stained with fluorescently labeled antibodies against CD3 (a Jurkat-specific marker), CD34 (a marker for Jurkat cells transduced with the TCR of interest), and CD69 (to evaluate Jurkat-TCR D activation after co-culturing). Cells were also stained with live / dead staining and analyzed using Novayte Quantum. The percentage of Jurkat CD69-positive cells was used to determine peptide-induced Jurkat activation in ECs. 50 .

[0298] As measured by CD69 expression, the activation of Jurkat-TCR D by the peptide is dose-responsive, EC 50 45.54 ng / mL Figure 1 The results showed that, when engineered into primary cells, TCR D can induce TCR signaling and has the potential to induce CLAVEEVSL-specific cytotoxic signaling.

[0299] Example 9. TCR mediated toxicity against target cells .

[0300] Evaluation of anti-CLAVEEVSL TCR against HLA expressing NPM1c mutation A02:01 Cytotoxicity of AML cell line (OCI-AML3). In short, cytotoxicity against HLA-derived cells. A02:01 Original CD8 from a negative healthy donor + T cells were engineered to knock out the endogenous TCR α and β constant chain domains. Subsequently, CD8+ was transduced using TCR. + Cells. OCI-AML2 cells (HLA) A02:01 + Peptide pulses were performed using the target peptide, followed by interaction with engineered primary CD8. + T cell co-culture to evaluate TCR cytotoxicity.

[0301] Before purifying activated CD8 T cells, frozen PBMCs were stimulated with a pyrogenic CD3 antibody. Subsequently, cells were transduced with lentiviruses via CRISPR endogenous TCR deletion and TCR D, TCR E, or TCR F. Primary cells were expanded for 10 days in CTS medium supplemented with serum-adapted alternatives containing 50 U IL-2 and 10 ng / mL IL-7. After 10 days, transduced T cells were purified using CD34 (a transduction marker) bead selection. CD8 was confirmed by FACS analysis using CD3, CD8 (a primary T cell-specific marker), CD34, and NPM1-specific tetramers and live / dead staining. + The engineering of TCR-T.

[0302] For the cytotoxicity assay, OCI-AML2 cells were pulsed with a titrated concentration of CLAVEEVSL (SEQ ID NO:5) peptide for 1 hour prior to co-culture.

[0303] Engineering 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 different E:T ratios. For the TCR-T negative control condition, HPV-specific TCR-T cells were engineered as described above and co-cultured as described above. No cytotoxicity was observed against cells that did not express the target peptide or were not pulsed with the target peptide (data not shown). After 16–18 hours, the co-cultured cells were stained with fluorescently labeled CD8 (a primary T cell-specific marker), VPD450 (to identify OCI-AML2 or OCI-AML3 cells), and live / dead staining antibodies or cell dyes. Data are presented as the percentage of pulsed (containing the target peptide) OCI-AML2 cells relative to non-pulsed (not containing the target peptide) OCI-AML2 cells. Figure 2 ) or the percentage of OCI-AML3 (expressed target peptide) relative to non-pulse ( Figure 3 The results shown for TCR D indicate that TCR effectively kills cells pulsed with the target peptide or those naturally expressing the target peptide.

[0304] As described above, co-culture assays were performed using multiple TCRs and donors. The previously published Leiden (US11352389) TCR was included as a control. Data are presented as the percentage of peptide-pulsed OCI-AML2 cells (sensitive to immunogenic killing) relative to OCI-AML2 cells without the target peptide. ECMO of peptide-induced TCR cytotoxicity was determined based on the number of target cells in peptide-pulsed OCI-AML2 cells. 50 .

[0305] Example 10.E:T EC 50 Measurement .

[0306] As described above, engineered T cells (TCRD, TCR E, and TCR F) harvested from three healthy donors and three different donors known to express NPM1c were co-cultured for assays. The previously published Leiden TCR (US11352389) was included as a control. For cytotoxicity, the E:T ratio of engineered T cells was determined based on the number of OCI-AML3 target cells, and the EC50 was determined accordingly. 50 The data in Table 7 show that engineered T cells expressing TCR D, TCR E, or TCR F exhibit potent cytotoxic activity against cell lines expressing the NPM1c antigen CLAVEEVSL (SEQ ID NO:5), with each cell type showing an EC50 of [missing data]. 50The E:T ratio was less than 1. Furthermore, the observed cytotoxicity was not significantly different from that of the reported engineered T cells. The demonstrated killing effect of TCR D, TCR E, and TCR F engineered T cells on OCI-AML3 cells indicates that cells expressing the NPM1c mutation present in patient AML blast cells possess mutation-specific cytotoxicity.

[0307] Table 7. Killing donor target cells with engineered T cells expressing TCR D, TCR E, or TCR F

[0308] Example 11. Based on tetramers CLAVEEVSL reaction CD8+ T Cell clone sorting .

[0309] 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 LPS, IL-4, INF-γ, and GM-CSF for 4 days. On day 5, the stimulated dendritic cells were pulsed with 10 ng / mL CLAVEEVL (SEQ ID NO:5) peptide. From day 1 to day 4, isolated CD8 T cells from the same donor were maintained in XVIVO-15 medium containing 5% human serum. + Cells were co-cultured with pulsed dendritic cells. Cells were maintained on day 5 with 30 ng / mL IL-21, followed by supplementation with XVIVO-15 medium containing 5 ng / mL IL-7 and IL-15 on days 8, 10, and 12. On day 16, CD8+ cells were... + T cells were stained with two 1:40 CLAVEEVSL tetramers targeting allophycocyanin (APC) and R-phycoerythrin (PE) at room temperature for 1 hour, followed by FACS sorting of tetramer-positive T cells. Positive T cells were cloned and sequenced before further evaluation of TCRs identified by tetramer double-positive sorting.

[0310] As described above for the CLAVEEVSL (SEQ ID NO: 5) peptide, the AIQDCLAV (SEQ ID NO: 1) peptide was also subjected to tetramer-based CD8 assay. + T-cell clone sorting.

[0311] Use CD3, CD34, and CLAVEEVSL or C LAVEEVSL tetramer (1:40) was used to stain stably transduced Jurkat cells to verify TCR / peptide specificity.

[0312] We were able to isolate unique TCRs targeting the neoantigen peptide tetramers of NPM1c CLAVEEVSL and AIQDCLAV, as shown in the FACS curve. Furthermore, we were able to demonstrate that the isolated anti-CLAVEEVSL TCRs can bind to both the uncysteylated CLAVEEVSL tetramer and the cysteylated C… Both LAVEEVSL tetramers have been reported, along with C. Binding of LAVEEVSL (SEQ ID NO:5) is important for killing mutNPM1c target cells (van der Lee DI et al., “Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia” J ClinInvest 2019 129(2):774-785). In separate experiments, engineered cells expressing TCR D showed 34.40% and 39.8% CLAVEEVSL tetramer binding (by FACS analysis) and 83.85% and 83.24% C LAVEEVSL tetramer binding.

[0313] Additional activation, tetramer binding, and EC50 data are shown in Tables 8 and 9.

[0314] Table 8. Binding of TCR to target cells

[0315] Primary CD8+ T cells co-cultured with OCI-AML3; Primary CD8+ T cells co-cultured with pulsed OCI-AML2 and CLAV peptide.

[0316] Table 9. Binding of TCR to target cells

[0317] Example 12. Modified TCR .

[0318] TCR D was engineered (modified) to introduce additional disulfide bonds to reduce the likelihood that its α or β chain would form a chimeric TCR with the endogenous primary CD8 TCR, thereby reducing the potential toxicity caused by chimeric TCRs that do not target the novel NPM1c epitope. TCR D was modified to introduce cysteine ​​residues at position 186 (T186C substitution, relative to SEQ ID NO:71) of the α chain and position 191 (S191C, relative to SEQ ID NO:79) of the β chain, see Table 10.

[0319] As previously described, Jurkat cells were transduced with either unmodified TCR D or disulfide-modified TCR D. As determined by FACS analysis, disulfide-modified TCR D binding exhibited increased binding relative to the TCR D (88.45%) tetramer with CLAVEEVSL (95.48%). Therefore, the disulfide bond may improve potency by increasing the proportion of anti-NPM1cTCR α / β pairing in engineered T cells. Similar improvements in potency are expected if similar cysteine ​​substitutions are made in other described TCRs.

[0320] Furthermore, TCR D was engineered so that the α and β constant regions were replaced with Ig domains from the γ and δ constant regions. Jurkat cells were transduced with unmodified TCR D as previously described or with domain-exchanged TCR D. No tetramer binding was observed with the negative control domain-exchanged TCR D.

[0321] Table 10. TCR Modification

[0322] Example 13. Engineering T Cellular superior CAR T cell .

[0323] With modifications such as those described in (Xie G et al., “CAR-T cells targeting a nucleophosminneoepitope exhibit potent specific activity in mouse models of acute myeloidleukaemia” Nat Biomed Eng. 2021), an anti-NPM1c chimeric T-cell receptor containing anti-AIQDCLAV scFv is generated. The modifications involve replacing the T2A element with a P2A element and replacing the EGFR tag with an RQR8 tag. These modifications are known in the art not to affect the targeting of the 5′ CAR-T construct.

[0324] By separating CD8 +T cells were activated using TransACT and expanded using G-REX to create engineered T cells and CAR-T cells. Transduction, CRISPR editing, and cytokine supplementation were performed as previously described.

[0325] Reporter gene assays were 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. Additionally, 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 gene assay using K562 cells as antigen-presenting cells, HLA was used. A02:01, and using K562 HLA A24:02 cells were used as a negative control.

[0326] Analysis of engineered Jurkat cells transduced with chimeric T-cell receptors, TCRBs, or TCRs against target cells A loaded with a titratable concentration of exogenous target peptides. 02:01 K562s or A 24:02 K562s reactivity. Cells were co-cultured at a 1:1 E:T ratio for 16-18 hours. Cells were analyzed by flow cytometry using CD69... + The activation value of Jurkat cells is evaluated in the form of cell percentage. Figure 4 Engineered TCR DT cells exhibit robust TCR signaling induction. As determined by CD69 expression, engineered anti-AIQDCLAV (TCR B) T cells also induce robust TCR signaling. Both TCR B and TCR D engineered T cells exhibit significantly greater signaling than CAR-T cells. As expected, K562 HLA... No signal transduction was observed in A24:02 cells.

[0327] In relation to HLA-A expression In Jurkat CD8-positive cells co-cultured with K562 cells pulsed with NPM1 at 02:01, TCR B and TCR D showed superior CD69 reporter gene signaling compared to previously reported (Xie et al., 2021) anti-AIQDCLAV CAR-T cells.

[0328] Example 14. In cytotoxicity assays CAR-T and TCR-T Comparison .

[0329] As previously mentioned, the test involved primary CD8 cells from healthy donors that had undergone gene editing and transduction using either natural TCR D or TCR D (DB) with added disulfide. + Cells or anti-AIQDCLAV CAR-T cells against HLA-A cells with NPM1c 02:01 Identification of the immunogenic AML cell line (OCI-AML3). Cells were co-cultured at different E:T ratios for 16-18 hours. The percentage of cell kill was evaluated by flow cytometry, and EC was generated in GraphPad Prism. 50 ( Figure 5 ).

[0330] Compared to CAR-T cells, both TCR D and disulfide-modified TCR D engineered T cells exhibited significantly greater killing power and lower EC50. 50 Because the engineered T cells lack endogenous TCR, no difference in killing was observed between TCR D and TCR D engineered T cells supplemented with disulfide. TCR knockout (KO) T cells were used as a negative control.

[0331] Example 15. TCR D Targeting neoantigens with and without post-translational modifications ( CLAVEEVSL ) .

[0332] Kill assays were performed with minimal modifications as previously described. The peptide used was either CLAVEEVSL (SEQ ID NO:5) without post-translational modifications or the target peptide (denoted as C). Cytotoxicity assays were performed on OCI-AML2 cells with the first cysteine ​​cysteylation pulse in LAVEEVSL. Peptide assays of the pulse were performed at E:T ratios of 0.5:1 and 1:1 using 10 ng / mL peptide. Wild-type NPM1 was used as a negative peptide control, and anti-HPV TCR engineered T cells were used as a negative engineered T cell control.

[0333] It has been shown (van der Lee et al., 2019) that for C LAVEEVSL's cytotoxicity against NPM1c + T-cell therapy is important for cancer. Previously published anti-CLAVEEVSL TCRs bind to CLAVEEVSL or C... LAVEEVSL showed differential activation as measured by INFγ. Clonal responsiveness was more effective against uncysteylated target peptides. In contrast, TCR D-engineered T cells exhibited high cytotoxicity against both cysteylated and uncysteylated target peptides. Figures 7-8 This demonstrates a potential cytotoxic advantage compared to the previously reported anti-CLAVEEVSL TCR (US11352389).

[0334] Example 16. From resistance CLAVEEVSL TCR Identified CLAVEEVSL ( SEQ ID NO:5 Identification of amino acids in ) .

[0335] An alanine scan was performed on the CLAVEEVSL (SEQ ID NO:5) peptide. The peptides used are listed in Table 11.

[0336] Reporter gene assays were performed with minimal modifications, as previously described. In short, target K562 cells were pulsed with the listed peptides at various concentrations for 1 hour. For CD8... + Jurkat cells were engineered to express TCR D and TCR F. Reporter gene assays were performed using a 1:1 E:T ratio. Control conditions included wild-type negative control peptides or immunogenic positive control peptides (CLAVEEVSL, SEQ ID NO:5).

[0337] Table 11. Alanine scan of CLAVEEVSL (SEQ ID NO:5) peptide

[0338] When an amino acid is replaced by alanine, residues of CLAVEEVLS (SEQ ID NO:5) important for anti-CLAVEEVLS TCR recognition are assessed by the reduction or loss of CD69 signaling. Amino acids at positions 2, 5, 6, and 7 (relative to SEQ ID NO:5) were found to be important for the recognition of both TCRs. Figure 7 and Figure 8 Surprisingly, residue 4 is important only for the recognition of TCR D, and residue 9 is important only for the recognition of TCR F.

[0339] This invention is not intended to limit its scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of this disclosure and are intended to fall within its scope.

[0340] Table 12. Sequences

Claims

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

21.

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

20.

3. A T-cell receptor (TCR) or an antigen-binding fragment thereof, said TCR or antigen-binding fragment comprising: α chains containing variable α (Vα) regions and β chains containing variable β (Vβ) regions; or γ chains containing variable γ (Vγ) regions and δ chains containing variable δ (Vδ) regions; wherein: (a) The Vα 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 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 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 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 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 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 or Vγ region contains 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 or Vδ region contains 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 or Vγ region contains 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 or Vδ region contains 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 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 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 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 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 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 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 or Vγ region contains 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 or Vδ region contains 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 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 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 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 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 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 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 or Vγ region contains 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 or Vδ region contains 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 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 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 or Vγ region comprises 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 comprises CDR-1 containing SEQ ID NO:289, CDR-2 containing SEQ ID NO:290, and CDR-3 containing SEQ ID NO:291; or (p) The Vα 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 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 an antigen-binding fragment thereof, said TCR or antigen-binding fragment comprising: α chains containing variable α (Vα) regions and β chains containing variable β (Vβ) regions; or γ chains containing variable γ (Vγ) regions and δ chains containing variable δ (Vδ) regions; wherein: (a) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:68, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:76; (b) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:86, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:94; (c) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:104, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

112. (d) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:32, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:

40. (e) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:50, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

58. (f) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:122, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

130. (g) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:140, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

148. (h) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:158, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

166. (i) The Vα or Vγ region contains CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:176, and the Vβ or Vδ region contains CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:184; (j) The Vα or Vγ region contains CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:194, and the Vβ or Vδ region contains CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:202; (k) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:212, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

220. (l) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:230, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:238; (m) The Vα or Vγ region contains CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:248, and the Vβ or Vδ region contains CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:256; (n) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:266, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:274; (o) The Vα or Vγ region contains CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:284, and the Vβ or Vδ region contains CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:292; (p) The Vα or Vγ region contains CDR-3 of CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:14, and the Vβ or Vδ region contains CDR-3 of CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:

22.

5. The TCR or its antigen-binding fragment according to claim 4, wherein: (a) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:68, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:76; (b) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:86, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:94; (c) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:104, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:

112. (d) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence of SEQ ID NO:32, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence of SEQ ID NO:40, respectively; (e) The Vα or Vγ region contains CDR-1 and CDR-2 as contained in the Vα or Vγ region sequence of SEQ ID NO:50, and the Vβ or Vδ region contains CDR-1 and CDR-2 as contained in the Vβ or Vδ region sequence of SEQ ID NO:

58. (f) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:122, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:

130. (g) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:140, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:

148. (h) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:158, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:

166. (i) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:176, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:

184. (j) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:194, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:202; (k) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:212, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:220; (l) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:230, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:238; (m) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:248, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:256; (n) The Vα or Vγ region contains CDR-1 and CDR-2 contained in the Vα or Vγ region sequence as in SEQ ID NO:266, and the Vβ or Vδ region contains CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence as in SEQ ID NO:274; (o) The Vα or Vγ region contains CDR-1 and CDR-2 as contained in the Vα or Vγ region sequence of SEQ ID NO:284, and the Vβ or Vδ region contains CDR-1 and CDR-2 as contained in the Vβ or Vδ region sequence of SEQ ID NO:292; or (p) The Vα or Vγ region contains CDR-1 and CDR-2 of CDR-1 and CDR-2 contained in the Vα or Vγ region sequence of SEQ ID NO:14, and the Vβ or Vδ region contains CDR-1 and CDR-2 of CDR-1 and CDR-2 contained in the Vβ or Vδ region sequence of SEQ ID NO:

22.

6. A T-cell receptor (TCR) or an antigen-binding fragment thereof, said TCR or antigen-binding fragment comprising: α chains containing variable α (Vα) regions and β chains containing variable β (Vβ) regions; or γ chains containing variable γ (Vγ) regions and δ chains containing variable δ (Vδ) regions; wherein: (a) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:68, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:76; (b) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:86, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:94; (c) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:104, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:112; (d) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:32, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:40, respectively; (e) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 as contained in the Vα or Vγ region sequence of SEQ ID NO:50, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 as contained in the Vβ or Vδ region sequence of SEQ ID NO:58; (f) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:122, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:130; (g) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:140, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:148; (h) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:158, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:166; (i) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:176, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:184; (j) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:194, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:202; (k) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:212, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:220; (l) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:230, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:238; (m) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:248, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:256; (n) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:266, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:274; (o) The Vα or Vγ region includes CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence as in SEQ ID NO:284, and the Vβ or Vδ region includes CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence as in SEQ ID NO:292; (p) The Vα or Vγ region contains CDR-1, CDR-2 and CDR-3 contained in the Vα or Vγ region sequence of SEQ ID NO:14, and the Vβ or Vδ region contains CDR-1, CDR-2 and CDR-3 contained in the Vβ or Vδ region sequence of SEQ ID NO:22, respectively.

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

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

22.

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

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

7.

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

25.

12. The TCR or its antigen-binding fragment according to any one of claims 1 to 11, wherein the TCR or its antigen-binding fragment recognizes the NPM1c neoantigen in the context of MHC molecules.

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

14. The TCR or its antigen-binding fragment according to claim 13, wherein the HLA-A molecule is serum HLA-A. 02:01, HLA-A 02:06 or HLA-A 02:

03.

15. The TCR or its antigen-binding fragment 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) or...

16. The TCR or its antigen-binding fragment according to claim 15, wherein the TCR or its antigen-binding fragment... (a) Identify CLAVEEVSL (SEQ ID NO:5) with a higher affinity than AIQDLWQWRKSL (SEQ ID NO:2); or (b) Identify AIQDLCLAV (SEQ ID NO:1) with a higher affinity than AIQDLWQWRKSL (SEQ ID NO:2).

17. A nucleic acid sequence, said nucleic acid sequence encoding a TCR or an antigen-binding fragment thereof 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 of claim 17, wherein the nucleic acid sequence 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; wherein: (a) The first nucleotide sequence comprises SEQ ID NO:69 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:77 or a sequence having at least 90% sequence identity with it; (b) The first nucleotide sequence comprises SEQ ID NO:87 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:95 or a sequence having at least 90% sequence identity with it; (c) The first nucleotide sequence comprises SEQ ID NO:105 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:113 or a sequence having at least 90% sequence identity with it; (d) The first nucleotide sequence comprises SEQ ID NO:33 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:41 or a sequence having at least 90% sequence identity with it; (e) The first nucleotide sequence comprises SEQ ID NO:51 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:59 or a sequence having at least 90% sequence identity with it; (f) The first nucleotide sequence comprises SEQ ID NO:123 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:131 or a sequence having at least 90% sequence identity with it; (g) The first nucleotide sequence comprises SEQ ID NO:141 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:149 or a sequence having at least 90% sequence identity with it; (h) The first nucleotide sequence comprises SEQ ID NO:159 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:167 or a sequence having at least 90% sequence identity with it; (i) The first nucleotide sequence comprises SEQ ID NO:177 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:185 or a sequence having at least 90% sequence identity with it; (j) The first nucleotide sequence comprises SEQ ID NO:195 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:203 or a sequence having at least 90% sequence identity with it; (k) The first nucleotide sequence comprises SEQ ID NO:213 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:221 or a sequence having at least 90% sequence identity with it; (l) The first nucleotide sequence comprises SEQ ID NO:231 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:239 or a sequence having at least 90% sequence identity with it; (m) The first nucleotide sequence comprises SEQ ID NO:249 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:257 or a sequence having at least 90% sequence identity with it; (n) The first nucleotide sequence comprises SEQ ID NO:267 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:275 or a sequence having at least 90% sequence identity with it; (o) The first nucleotide sequence comprises SEQ ID NO:285 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:293 or a sequence having at least 90% sequence identity with it; (p) The first nucleotide sequence comprises SEQ ID NO:15 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:23 or a sequence having at least 90% sequence identity with it; (aa) The first nucleotide sequence comprises SEQ ID NO:70 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:78 or a sequence having at least 90% sequence identity with it; (bb) The first nucleotide sequence comprises SEQ ID NO:88 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:96 or a sequence having at least 90% sequence identity with it; (cc) The first nucleotide sequence comprises SEQ ID NO:106 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:114 or a sequence having at least 90% sequence identity with it; (dd) The first nucleotide sequence comprises SEQ ID NO:34 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:42 or a sequence having at least 90% sequence identity with it; (ee) The first nucleotide sequence comprises SEQ ID NO:52 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:60 or a sequence having at least 90% sequence identity with it; (ff) The first nucleotide sequence comprises SEQ ID NO:124 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:132 or a sequence having at least 90% sequence identity with it; (gg) The first nucleotide sequence comprises SEQ ID NO:142 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:150 or a sequence having at least 90% sequence identity with it; (hh) The first nucleotide sequence comprises SEQ ID NO:160 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:168 or a sequence having at least 90% sequence identity with it; (ii) The first nucleotide sequence comprises SEQ ID NO:178 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:186 or a sequence having at least 90% sequence identity with it; (jj) The first nucleotide sequence comprises SEQ ID NO:196 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:204 or a sequence having at least 90% sequence identity with it; (kk) The first nucleotide sequence comprises SEQ ID NO:214 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:222 or a sequence having at least 90% sequence identity with it; (ll) The first nucleotide sequence comprises SEQ ID NO:232 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:240 or a sequence having at least 90% sequence identity with it; (mm) The first nucleotide sequence comprises SEQ ID NO:250 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:258 or a sequence having at least 90% sequence identity with it; (nn) The first nucleotide sequence comprises SEQ ID NO:268 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:276 or a sequence having at least 90% sequence identity with it; (oo) The first nucleotide sequence comprises SEQ ID NO:286 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:294 or a sequence having at least 90% sequence identity with it; or (pp) The first nucleotide sequence comprises SEQ ID NO:16 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:24 or a sequence having at least 90% sequence identity with it.

19. The nucleic acid sequence according to claim 17 or 18, wherein: (a) The first nucleotide sequence comprises SEQ ID NO:72 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:80 or a sequence having at least 90% sequence identity with it; (b) The first nucleotide sequence comprises SEQ ID NO:90 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:98 or a sequence having at least 90% sequence identity with it; (c) The first nucleotide sequence comprises SEQ ID NO:108 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:116 or a sequence having at least 90% sequence identity with it; (d) The first nucleotide sequence comprises SEQ ID NO:36 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:44 or a sequence having at least 90% sequence identity with it; (e) The first nucleotide sequence comprises SEQ ID NO:54 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:62 or a sequence having at least 90% sequence identity with it; (f) The first nucleotide sequence comprises SEQ ID NO:126 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:134 or a sequence having at least 90% sequence identity with it; (g) The first nucleotide sequence comprises SEQ ID NO:144 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:152 or a sequence having at least 90% sequence identity with it; (h) The first nucleotide sequence comprises SEQ ID NO:162 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:170 or a sequence having at least 90% sequence identity with it; (i) The first nucleotide sequence comprises SEQ ID NO:180 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:188 or a sequence having at least 90% sequence identity with it; (j) The first nucleotide sequence comprises SEQ ID NO:198 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:206 or a sequence having at least 90% sequence identity with it; (k) The first nucleotide sequence comprises SEQ ID NO:216 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:224 or a sequence having at least 90% sequence identity with it; (l) The first nucleotide sequence comprises SEQ ID NO:234 ​​or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:242 or a sequence having at least 90% sequence identity with it; (m) The first nucleotide sequence comprises SEQ ID NO:252 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:260 or a sequence having at least 90% sequence identity with it; (n) The first nucleotide sequence comprises SEQ ID NO:270 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:278 or a sequence having at least 90% sequence identity with it; (o) The first nucleotide sequence comprises SEQ ID NO:288 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:296 or a sequence having at least 90% sequence identity with it; or (p) The first nucleotide sequence comprises SEQ ID NO:18 or a sequence having at least 90% sequence identity with it, and the second nucleotide sequence comprises SEQ ID NO:26 or a sequence having at least 90% sequence identity with it.

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 by 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 jumping.

22. The nucleic acid sequence of claim 21, wherein the peptide causing ribosome jumping 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 of claim 23, wherein the sequence encoding the P2A peptide is shown in SEQ ID NO:

302.

25. The nucleic acid sequence according to any one of claims 17 to 24, wherein the nucleic acid sequence encodes an amino acid sequence of SEQ ID NO: 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 a nucleic acid sequence of SEQ ID NO: 82, 100, 118, 46, 64, 136, 154, 172, 190, 208, 226, 244, 262, 280, 298 or 28.

27. A vector comprising a nucleic acid sequence according to 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. An engineered cell comprising a TCR or an antigen-binding fragment thereof according to any one of claims 1 to 16.

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

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

33. The engineered cell according to any one of claims 30 to 32, wherein the engineered cell is derived from a cell line.

34. The engineered cell according to any one of claims 30 to 32, wherein the engineered cell is derived from primary cells obtained from a subject.

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

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

37. The engineered cell of claim 35, wherein the engineered cell has cytotoxic activity against cells expressing CLAVEEVSL (SEQ ID NO:5) or AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules.

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

39. The engineered cell of claim 37, wherein the HLA-A molecule is serum-type HLA-A. 02:01, HLA-A 02:06 or HLA-A 02:

03.

40. The engineered cells of claim 39, wherein the engineered cells have increased cytotoxic activity against cells expressing CLAVEEVSL (SEQ ID NO:5) or AIQDLCLAV (SEQ ID NO:1) relative to cytotoxic activity against cells expressing AIQDLWQWRKSL (SEQ ID NO:2).

41. A method for producing engineered cells, the method 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, optionally wherein the cells are T cells, optionally wherein the T cells are primary T cells, natural killer T cells or cytotoxic T cells.

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

43. A composition comprising a TCR or an 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 engineered cells according to any one of claims 30 to 40.

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

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

47. A method for identifying T-cell receptors (TCRs) targeting NPM1c neoantigens, the method comprising: (i) Using nucleic acids obtained from a single T cell from multiple T cells, a variety of functional TCR-encoded nucleic acid vectors are generated through high-throughput nucleic acid amplification and assembly methods; The T cells were derived from donor subjects; as well as (ii) Identify a functional TCR that recognizes the NPM1c neoantigen among a variety of functional TCRs encoded by the various functional TCR-encoded nucleic acid vectors.

48. The method of 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 peptide CLAVEEVSL (SEQ ID NO:5) or AIQDLCLAV (SEQ ID NO:1) in the context of MHC molecules.

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

51. The method of claim 50, wherein the HLA-A molecule is serum HLA-A. 02:01, HLA-A 02:06 or HLA-A 02:

03.

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

53. The method according to any one of claims 46 to 51, wherein the T cells from the donor subject are cultured under conditions for cell expansion of the T cells without generating the plurality of functional TCR-encoding nucleic acid vectors.

54. The method according to any one of claims 46 to 53, wherein the high-throughput nucleic acid amplification and assembly method comprises: (1) Amplifying a first amplification product and a second amplification product from complementary DNA (cDNA) generated from RNA, wherein the RNA is obtained from a single T cell in each of a plurality of individual T cells sorted into the device, wherein: The first amplification product contains a nucleotide sequence encoding a full-length variable α (Vα) region or a full-length variable γ (Vγ) region of the TCR, and the second amplification product contains a nucleotide sequence encoding a full-length variable β (Vβ) region or a full-length variable δ (Vδ) region of the TCR; and (2) The first amplification product and the second amplification product from each of the plurality of individual positions are assembled 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 individual positions; and The functional TCR comprises (i) the full-length Vα region and the full-length Vβ region from the single T cell, or (ii) the full-length Vγ region and the full-length Vδ region from the single T cell.

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

56. The engineered cells of claim 55, wherein the engineered cells have increased cytotoxic activity against cells expressing CLAVEEVLS (SEQ ID NO:5) or AIQDLCLAV (SEQ ID NO:1) relative to cytotoxic activity against cells expressing AIQDLWQWRKSL (SEQ ID NO:2).

57. A composition comprising engineered cells according to claim 55 or 56.

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

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

60. A treatment method comprising administering medication to a subject suffering from a disease or condition. (a) The TCR or antigen-binding fragment according to any one of claims 1 to 16, (b) Cells expressing the TCR or antigen-binding fragment according to any one of claims 1 to 16, (c) A protein comprising a TCR or antigen-binding fragment according to any one of claims 1 to 16, or (d) Cells expressing the protein comprising the TCR or antigen-binding fragment according to any one of claims 1 to 16.

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

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

63. The method according to any one of claims 59 to 61, wherein the subject has or has been diagnosed with a liquid tumor, 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 malignant hematologic disorders in the subject, or induces or enhances graft-versus-leukemia (GVL) effect.

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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