WO2025050020A1

WO2025050020A1 - Novel t cell receptors that bind to preferentially expressed antigen in melanoma (prame) and methods of use thereof

Info

Publication number: WO2025050020A1
Application number: PCT/US2024/044834
Authority: WO
Inventors: Eleni CHANTZOURA; Paul IBBETT; Marc VAN DIJK
Original assignee: Mink Therapeutics Inc
Current assignee: Mink Therapeutics Inc
Priority date: 2023-08-30
Filing date: 2024-08-30
Publication date: 2025-03-06
Anticipated expiration: 2026-02-28

Abstract

Provided are novel engineered T cell receptors (TCR) based which are selective and specific for PReferentially Expressed Antigen of Melanoma (FRAME), cells expressing the novel TCRs, and related methods of use. In some embodiments, the engineered TCRs are expressed by invariant natural killer T (iNKT cells).

Description

NOVEL T CELL RECEPTORS THAT BIND TO PREFERENTIALLY EXPRESSED

ANTIGEN IN MELANOMA (PRAME) AND METHODS OF USE THEREOF

RELATED APPLICATIONS

[01] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional

Application Serial No. 63/579,799, entitled “NOVEL T CELL RECEPTORS THAT BIND TO PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA (PRAME) AND METHODS OF USE THEREOF”, filed on August 30, 2023, the contents of which is incorporated herein by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[02] The contents of the electronic sequence listing (A132770008WO00-SEQ-

ROS.NRL; Size: 75 bytes; and Date of Creation: August 23, 2023) are herein incorporated by reference in their entirety.

BACKGROUND

Field

[03] The present disclosure relates to novel engineered T cell receptors (TCR) which are selective and specific for PReferentially Expressed Antigen of Melanoma (PRAME), cells expressing the novel engineered TCRs, and methods of use.

Description

[04] Preferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen (CTA) also known as CT 130. High levels of PRAME mRNA have been observed in a variety of cancer indications and leukemias, including, for instance, melanoma, non-small cell lung cancer, breast cancer, multiple sarcoma subtypes and epithelial ovarian cancer. It is characterized by restricted and low expression in normal somatic tissues such as the testis. (Al-Khadairi G., Decock J. Cancer testis antigens and immunotherapy: Where do we stand in the targeting of PRAME?, Cancers. 2019; 11:984.) High PRAME tumor expression has been associated with poor prognosis in several solid tumors. Several studies suggest that PRAME can induce cell proliferation, reduce cytotoxic drug sensitivity and inhibit apoptosis in a variety of cancers. Because PRAME is an intracellular antigen, it cannot be targeted by conventional CAR T cells that are restricted to cell surface antigens.

[05] Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells, are a distinct population of T cells that express a unique TCR containing an invariant Va24-Jal8 a-chain gene and a limited number of P chains. (Godfrey DI, Stankovic S, Baxter AG. Raising the NKT-cell family. Nat Immunol. 2010;11:197-206. Porcelli S, Yockey CE, Brenner MB, Balk SP. Analysis of T-cell antigen receptor (TCR) expression by human peripheral blood CD4-8-aP T cells demonstrates preferential use of several VP genes and an invariant TCR a chain. J Exp Med. 1993;178:1-16. Taniguchi M, Tashiro T, Dashtsoodol N, Hongo N, Watarai H. The specialized iNKT-cell system recognizes glycolipid antigens and bridges the innate and acquired immune systems with potential applications for cancer therapy. Int Immunol. 2010;22:1-6.) Although iNKT cells are rare in the human blood pool, comprising just 0.01-1% of peripheral blood mononuclear cells (PBMCs), they are important immunoregulatory cells rapidly producing an array of cytokines and chemokines in addition to exerting potent cytotoxic activity upon activation. The cytokines produced include interferon-y (IFN-y), interleukin-2 (IL-2), IL-3, IL-4, IL-10, IL-13, IL-17, IL-21 and transforming growth factor-p. (Godfrey DI, Rossjohn J. New ways to turn on NKT cells. J Exp Med. 2011;208:1121-5. Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L. CDld- restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system. Curr Opin Immunol. 2008;20:358-68.)

[06] Unlike conventional T cells, which respond to peptide antigens in the context of MHC class I or II, iNKT cells respond and are activated by glycolipid antigens presented by the MHC class lb related molecule, CD Id. (Bendelac A, Bonneville M, Kearney JF. Autoreactivity by design: innate B and T lymphocytes. Nat Rev Immunol. 2001;1:177 -86. Taniguchi M, Tashiro T, Dashtsoodol N, Hongo N, Watarai H. The specialized iNKT-cell system recognizes glycolipid antigens and bridges the innate and acquired immune systems with potential applications for cancer therapy. Int Immunol. 2010;22:1-6.) CDld is a highly conserved, non-polymorphic molecule, expressed by many haematopoietic cells (dendritic cells, macrophages and B cells), which present lipid antigens rather than peptides to iNKT cells. The CDld restriction identifies and defines iNKT cells from all other T cell populations. SUMMARY

[07] The instant disclosure is directed to an engineered T cell receptor (TCR) that binds to the PRAME425-433 HLA-A*02.01 complex. In some embodiments, the engineered TCR contains an alpha chain comprising a variable alpha (Va) region and a beta chain comprising a variable beta (VP) region. In some embodiments, the Va region comprises a complementarity-determining region 3 (CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the Va region of the engineered TCR contains a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:37 or 41, and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38. For instance, the disclosure provides an engineered TCR comprising a Va-CDRl with an amino acid sequence as set forth in SEQ ID NO:37 or 41, a Va-CDR2 with an amino acid sequence as set forth in SEQ ID NO:38, and a Va-CDR3 with an amino acid sequence as set forth in SEQ ID NO:39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[08] In some embodiments, the engineered TCR that binds the PRAME425-433 peptide comprises a VP region containing a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs:53, 54, 55, 56, 57, 58, or 59. In some embodiments, the VP region of the engineered TCR contains a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52. For instance, the disclosure provides an engineered TCR comprising a VP-CDR1 with an amino acid sequence as set forth in SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:53, 54, 55, 56, 57, 58 or 59.

[09] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49 or 50; and contains a VP region with a VP CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs:53, 54, 55, 56, 57, 58, or 59.

[010] In some embodiments, the engineered TCR contains a Va region with a Va-

CDRl comprising an amino acid sequence set forth as follows: SSX1X2PX3 (SEQ ID NO:67), wherein Xi is V or Y, X2 is P or S, and X3 is Y or S.

[011] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR2 comprising an amino acid sequence set forth as (SEQ ID NO:38.In some embodiments, the engineered TCR contains a Va region with a Va-CDR3 comprising an amino acid sequence set forth as follows: X1VX2FSGGYNKLI (SEQ ID NO:66), wherein Xi is A or V, and X2 is S or T.

[012] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR3 comprising an amino acid sequence set forth as follows: X1VX2X3SGGYNKLI (SEQ ID NO:68), wherein Xi is A or V, X2 is G or N or S or T or P or V, and X3 is F or Y.

[013] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR1 comprising an amino acid sequence set forth as SEQ ID NO:67, a Va-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:38, and a Va-CDR3 comprising an amino acid sequence set forth as SEQ ID NO:66.

[014] In some embodiments, the engineered TCR contains a VP region with a VP-

CDR1 comprising an amino acid sequence set forth as SEQ ID NO:51.

[015] In some embodiments, the engineered TCR contains a VP region with a VP-

CDR2 comprising an amino acid sequence set forth as SEQ ID NO:52.

[016] In some embodiments, the engineered TCR contains a VP region with a VP-

CDR3 comprising an amino acid sequence set forth as follows: ASX1X2WDX3X4X5X6QY (SEQ ID NO:69), wherein Xi is A or S, X2 is R or P or A, X3 is R or S or M or N or T, X4 is G or R or A or W or V, X5 is E or Q or D or G, and Xf> is E or T.

[017] In some embodiments, the engineered TCR contains a VP region with a VP-

CDR1 comprising an amino acid sequence set forth as SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence set forth as follows SEQ ID NO:69.

[018] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR1 comprising an amino acid sequence set forth as SEQ ID NO:67, a Va-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:38, a Va-CDR3 comprising an amino acid sequence set forth as SEQ ID NO:66; and contains a VP region with a VP-CDR1 comprising an amino acid sequence set forth as SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence set forth as follows SEQ ID NO:69.

[019] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR1 consisting of an amino acid sequence set forth as SEQ ID NO: 67, a Va-CDR2 consisting of an amino acid sequence set forth as SEQ ID NO:38, a Va-CDR3 consisting of an amino acid sequence set forth as SEQ ID NO:66; and the engineered TCR contains a VP region with a VP-CDR1 consisting of an amino acid sequence set forth as SEQ ID NO:51, a VP-CDR2 consisting of an amino acid sequence set forth as SEQ ID NO:52, and a VP-CDR3 consisting of an amino acid sequence set forth as follows SEQ ID NO:69.

[020] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR1 comprising an amino acid sequence set forth as SEQ ID NO:67, a Va-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:38, a Va-CDR3 comprising an amino acid sequence set forth as SEQ ID NO:68; and contains a VP region with a VP-CDR1 comprising an amino acid sequence set forth as SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence set forth as SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence set forth as follows SEQ ID NO:69.

[021] In some embodiments, the engineered TCR contains a Va region with a Va-

CDR1 consisting of an amino acid sequence set forth as SEQ ID NO: 67, a Va-CDR2 consisting of an amino acid sequence set forth as SEQ ID NO:38, a Va-CDR3 consisting of an amino acid sequence set forth as SEQ ID NO:68; and the engineered TCR contains a VP region with a VP-CDR1 consisting of an amino acid sequence set forth as SEQ ID NO:51, a VP-CDR2 consisting of an amino acid sequence set forth as SEQ ID NO:52, and a VP-CDR3 consisting of an amino acid sequence set forth as follows SEQ ID NO:69.

[022] In some embodiments, the engineered TCR comprises a variable alpha chain

(Va) complementarity-determining region 1 (Va -CDR1) comprising an amino acid sequence of SEQ ID NO:37, Va -CDR2 comprising an amino acid sequence of SEQ ID NO:38, and Va-CDR3 comprising an amino acid sequence SEQ ID NO: 66; and a variable beta chain (VP) VP-CDR1 comprising an amino acid sequence of SEQ ID NO: 51, VP-CDR2 comprising an amino acid sequence of SEQ ID NO: 52, and VP-CDR3 comprising an amino acid sequence of SEQ ID NO: 53.

[023] In some embodiments, the engineered TCR comprises Va -CDR1 comprising an amino acid sequence of SEQ ID NO: 37, Va -CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and Va-CDR3 comprising an amino acid sequence of SEQ ID NO: 40; and a VP-CDR1 comprising an amino acid sequence of SEQ ID NO: 51, VP-CDR2 comprising an amino acid sequence of SEQ ID NO: 52, and VP-CDR3 comprising an amino acid sequence of SEQ ID NO: 53. [024] In some embodiments, the engineered TCR comprises Va -CDR1 comprising an amino acid sequence of SEQ ID NO: 37, Va -CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and Va-CDR3 comprising an amino acid sequence of SEQ ID NO: 45; and a VP-CDR1 comprising an amino acid sequence of SEQ ID NO: 51, VP-CDR2 comprising an amino acid sequence of SEQ ID NO: 52, and VP-CDR3 comprising an amino acid sequence of SEQ ID NO: 53.

[025] In some embodiments, the engineered TCR comprises Va -CDR1 comprising an amino acid sequence of SEQ ID NO: 37, Va -CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and Va-CDR3 comprising an amino acid sequence of SEQ ID NO: 39; and a VP-CDR1 comprising an amino acid sequence of SEQ ID NO: 51, VP-CDR2 comprising an amino acid sequence of SEQ ID NO: 52, and VP-CDR3 comprising an amino acid sequence of SEQ ID NO: 53.

[026] In some embodiments, (a) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (b) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively; (c) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively; (d) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (e) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (f) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (g) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively; (h) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively; (i) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (j) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (k) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (1) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (m) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (n) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (o) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively; (p) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively; (q) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (r) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (s) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (t) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:56, respectively; (u) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively; (v) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:58, respectively; (w) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (x) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (y) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (z) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:56, respectively; (aa) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively; (bb) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (cc) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (dd) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:46, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (ee) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:46, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (ff) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:47, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (gg) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:47, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (hh) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:48, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (ii) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:48, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (jj) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:49, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (kk) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:49, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (11) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:50, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; or (hh) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:50, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[027] In some embodiments, the engineered TCR contains a Va region which comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22. In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22.

[028] In some embodiments, the engineered TCR contains a VP region which comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36. In some embodiments, the VP region of the engineered TCR comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36.

[029] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[030] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32.

[031] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36.

[032] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. [033] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[034] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[035] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 4, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32.

[036] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36.

[037] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[038] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[039] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[040] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[041] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. [042] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[043] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32.

[044] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36.

[045] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[046] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[047] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[048] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID :10, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:30.

[049] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32.

[050] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:34 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:34. [051] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[052] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[053] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28.

[054] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:30 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:30.

[055] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32.

[056] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[057] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[058] The disclosure provides an engineered TCR wherein the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 14, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[059] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 14, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. [060] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 16, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[061] The disclosure provides an engineered TCR with a Va region that comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 16 and a VP region that comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[062] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 18, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[063] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 18, and the VP region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[064] The disclosure provides an engineered TCR with a Va region comprising an amino acid sequence as set forth in SEQ ID NO:20 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:20, and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[065] In some embodiments, the Va region of the engineered TCR comprises an amino acid sequence as set forth in SEQ ID NO:20 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:20, and the VP region of the TCR comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[066] The disclosure provides an engineered TCR with a Va region comprising an amino acid sequence as set forth in SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:22, and the VP region comprising an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24.

[067] In some embodiments, the engineered TCR contains a Va region comprising an amino acid sequence as set forth in SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:22, and the VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26.

[068] In some embodiments, the engineered TCR comprises a Va region comprising an amino acid sequence of SEQ ID NO: 4; and a VP region comprising an amino acid sequence of SEQ ID NO: 24.

[069] In some embodiments, the engineered TCR comprises a Va region comprising an amino acid sequence of SEQ ID NO: 12; and a VP region comprising an amino acid sequence of SEQ ID NO: 24. [070] In some embodiments, the engineered TCR comprises a Va region comprising an amino acid sequence of SEQ ID NO: 2; and a VP region comprising an amino acid sequence of SEQ ID NO: 24.

[071] In some embodiments, the engineered TCR contains a Va region and a VP region selected from the group consisting of (a) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:2 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:28; (b) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:2 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:32; (c) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:2 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:36; (d) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:2 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (e) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:2 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26; (f) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:4 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 28; (g) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:4 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:32; (h) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:4 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:36; (i) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:4 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (j) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:4 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26; (k) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:6 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:28; (1) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:6 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (m) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:6 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (n) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:8 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:28; (o) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:8 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 32; (p) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:8 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 36; (q) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 8 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (r) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:8 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (s) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:28; (t) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 30; (u) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:32; (v) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:34; (w) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 24; (x) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 10 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26; (y) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 12 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:28; (z) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 12 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:30; (aa) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 12 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:32; (bb) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 12 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 24; (cc) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 12 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (dd) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 14 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (ee) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 14 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (ff) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (gg) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26; (hh) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 18 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 24; (ii) a Va region comprising an amino acid sequence as set forth in SEQ ID NO: 18 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (jj) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:20 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:24; (kk) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:20 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO:26; (11) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:22 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 24; and (mm) a Va region comprising an amino acid sequence as set forth in SEQ ID NO:22 and a VP region comprising an amino acid sequence as set forth in SEQ ID NO: 26.

[072] In some embodiments, the engineered TCR does not comprise SEQ ID NO:63 or SEQ ID NO:64.

[073] In some embodiments, the engineered TCR is a full-length TCR. In some embodiments, the engineered TCR is a soluble TCR. In some embodiments, the engineered TCR is a single-chain TCR.

[074] In some embodiments, the TCR is a human TCR that specifically binds the

PRAME425-433 HLA-A*02.01 complex.

[075] In some embodiments, the engineered TCR is expressed on the surface of a T cell. In some embodiments, the T cell presenting the TCR on its surface is capable of being activated in the presence of PRAME425-433 HLA-A*02.01. In some embodiments, the engineered TCR is expressed on a human iNKT cell that also expresses endogenous TCRs.

[076] In some embodiments, the engineered TCR is conjugated to an effector moiety. In some embodiments, the effector moiety is a cytotoxic agent, cytostatic agent, toxin, radionuclide, detectable label, or binding moiety.

[077] The disclosure provides a polynucleotide encoding an engineered TCR described herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 or a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence in the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35. In some embodiments, the Va region of the engineered TCR is encoded by a nucleic acid sequence selected from the group consisting of SEQ NO NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 or a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ NO NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and the VP region of the TCR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs:23, 25, 27, 29, 31, 33, and 35 or a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ NO NOs: 23, 25, 27, 29, 31, 33, or 35.

[078] In some embodiments, the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 3, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 11, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 1, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23.

[079] In some embodiments, the disclosure provides a vector comprising a polynucleotide as described herein. For instance, the disclosure provides a vector comprising a polynucleotide with a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 or a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments, the vector is a viral vector. In some embodiments, the vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and a baculoviral vector.

[080] In some embodiments, a cell presents an engineered TCR as described herein on its cell surface. In some embodiments, the cell is a human lymphocyte. In some embodiments, the cell is selected from the group consisting of a T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, and a natural killer cell. In some embodiments, an invariant natural killer T (iNKT) cell expresses the engineered TCR.

[081] The disclosure provides an iNKT cell that expresses an engineered TCR as described herein. iNKT cells can be harvested from PBMCs and expanded using methods known in the art. iNKT cells transfected with a polynucleotide coding for a TCR as described herein express both the engineered TCR (i.e., TCR targeting the PRAME425-433 peptide) and iNKT endogenous TCR (i.e., TCR targeting glycolipids presented by CDld). In some embodiments, the iNKT cell expresses the engineered TCR and a native TCR.

[082] In some embodiments, the iNKT cell expresses an armoring molecule. The armoring molecule may be selected from the group: IL-15, IL-2, IL-12, CD40L, 4-1BBL, IL- 18, IL-7, IL-33, constitutively active Akt (caAkt), hybrid IL-4/IL-7 receptor, checkpoint inhibitors such as anti-PDl antibodies, nanobodies targeting CD47, or bispecific T-cell engagers (BiTEs). In some embodiments, the iNKT cell expresses an armoring molecule, wherein the armoring molecule is IL- 15.

[083] In some embodiments, the disclosure provides a pharmaceutical composition comprising an engineered TCR as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, or a cell as disclosed herein (e.g., an iNKT cell), and, in all instances, with a pharmaceutically acceptable carrier. Lor instance, the disclosure provides a pharmaceutical composition comprising an iNKT cell that expresses an engineered TCR with a Va region containing a Va-CDRl comprising an amino acid sequence as set forth in SEQ ID NO:37 or 41, a Va-CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38, and a Va-CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50 and a VP region with a VP-CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:53, 54, 55, 56, 57, 58 or 59 and a pharmaceutically acceptable carrier.

[084] In some embodiments, methods are provided for producing an engineered

TCR that binds to a PRAME425-433 peptide (SEQ ID NO: 60), the method comprising transfecting isolated human iNKT cells with an engineered TCR. For instance, the disclosure provides transfecting isolated human iNKT cells with a TCR with a Va region containing a Va-CDRl comprising an amino acid sequence as set forth in SEQ ID NO: 37 or 41, a Va- CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38, and a Va-CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50 and a VP region with a VP-CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51, a VP-CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52, and a VP-CDR3 comprising an amino acid sequence as set forth in SEQ ID NO:53, 54, 55, 56, 57, 58 or 59 In some embodiments, isolated human iNKT cells are transfected using a lentivirus vector to express an engineered TCR. In some embodiments, human iNKT cells are transfected using a lentivirus vector to express an engineered TCR and the human iNKT cells are subsequently enriched by the addition of K562-HLA-A*02:01 feeder cells.

[085] The disclosure provides methods of producing a cell expressing an engineered

TCR that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:60, the method comprising contacting a cell with a vector under conditions that allow introduction of the vector into the cell. The disclosure provides use of a viral vector such a lentiviral vector to introduce polynucleotide encoding the TCR in a cell (e.g., isolated human iNKT cells).

[086] In some embodiments, the disclosure provides methods of inducing an immune response to PRAME comprising administering to the subject an effective amount of an engineered TCR as disclosed herein, a polynucleotide as disclosed herein, a cell as disclosed herein (e.g., iNKT cells presenting TCRs), or the pharmaceutical compositions disclosed herein. In some embodiments, the PRAME is PRAME425-433 presented by HLA- A*02:01.

[087] In some embodiments, the disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject an effective amount of a TCR as described herein, a polynucleotide as described herein, a cell as described herein, or a pharmaceutical composition as described herein.

[088] For instance, the disclosure provides methods of treating cancer in a subject comprising administering to the subject an effective amount of allogenic iNKT cells that express an engineered TCR as described herein. Such iNKT cells directly kill PRAME presenting tumor cells and also express activator natural killer (NK) receptors that recognize stress ligands on tumor cells. Moreover, the allogenic iNKT cells are capable of restricting immunosuppressive tumor myeloid cells via their endogenous TCR.

[089] In some embodiments, the allogenic iNKT cells are administered to subjects without prior lymphodepletion.

[090] In some embodiments, the disclosure provides methods of treating cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising isolated, human iNKT cells presenting TCRs as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is selected from the group consisting of endometrial cancer, esophageal cancer, lung squamous cell cancer, melanoma, multiple myeloma, ovarian cancer, renal papillary cell cancer, testicular cancer, thymoma, uterine carcinosarcoma, non-small cell lung cancer, breast cancer, and uveal melanoma. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer.

[091] In some embodiments, a TCR as described herein, a polynucleotide as described herein, a cell as described herein, or a pharmaceutical composition as described herein, is administered intravenously to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[092] FIGs. 1A-1D illustrate the expression of PRAME in cancer tissue, healthy adult tissue, immune cells and healthy fetal tissue using publicly available datasets.

[093] FIG. 2 is an alignment of the variable a and P chains identified which are selective and specific for PRAME425-433 peptide.

[094] FIGs. 3A-3E provide nucleic acid and amino acid sequences of the variable a chain (FIGs. 3A-3C) and P chain (FIGs. 3D-3E) of engineered PRAME TCRs.

[095] FIGs. 4A-4C show the binding of different combinations of TCR variable a and P chains. Plots are presented as PRAME425-433 tetramer binding (X-axis) against TCR expression (Y-axis).

[096] FIGs. 5A-5E show the results of alanine scanning of select engineered TCR candidates. Each assay was performed at least three times, and the combined data is shown.

[097] FIG. 6 shows the results of T-SPRINT profiling of select engineered TCR candidates: (684A2x709B5, 684A3x709B5 and 684Al lx709B5).

[098] FIG. 7 shows the sensitivity of engineered PRAME TCRs (684A2 x 709B5,

684A3x709B5 and 684Al lx709B5) at low peptide concentrations. Each assay was performed at least 3 times, and the combined data is shown.

[099] FIG. 8 shows the successful transduction and expansion of iNKT cells transduced to express engineered TCR4 (684A2x709B5), TCR9 (684A3x709B5) and TCR28 (684A11 x 709B5). All three TCRs were tested across three donors by day 27 post transduction. (Representative images of one donor shown.)

[0100] FIG. 9 shows specific cytotoxicity from engineered TCRs (684A2x709B5, 684A3x709B5 and 684Al lx709B5) expressed from iNKT cells and the effect of iNKT endogenous TCRs on killing. % Dead target calculation was performed by calculating the % of CD 19+ cells that stained positive using live/dead dye. Data presented is from 2 donors, 2 replicates/donor.

[0101] FIGs. 10A-10B show flow cytometric analysis of CD4+ T cell (FIG. 20A) and CD8+ T cell (FIG. 10B) activation indicated by the surface CD25 and CD69 coexpression. Cells were transduced with irrelevant TCR, natural TCR or 684A2x709B5, 684A3x709B5 and 684Al lx709B5 TCRs,

[0102] FIG. 11 shows flow cytometric analysis of T cell activation indicated by surface CD25 and CD69 co-expression in unmodified iNKTs or PRAME-TCR-iNKTs (684A2x709B5, 684A3x709B5 and 684Al lx709B5). TCR-iNKTs were co-cultured with T2 cells without peptide pulsing (“No peptide” group), or pulsed with PRAME425-433 peptide (“PRAME” group) or NYESOi 157-1165 peptide (“NYESO” group).

[0103] FIG. 12 shows flow cytometric analysis of surface CD25 and CD69 coexpression in unmodified iNKTs or PRAME-TCR-iNKTs (684A2x709B5, 684A3x709B5 and 684Al lx709B5) iNKTs in response to different concentrations (M) of PRAME425-433 peptide.

[0104] FIGs. 13A and 13B show killing of PRAME-TCR-iNKTs against OVCAR3- GFP cells. FIG. 13A shows quantification of OVCAR3-GFP cell death over time in four groups: target only, unmodified iNKTs, 684A2x709B5 and 684A3x709B5 PRAME-TCR- iNKTs. %GFP+ area of OVCAR3-GFP cells was assessed by fluorescence microscopy over 72hrs. FIG. 13B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0105] FIGs. 14A and 14B show killing of PRAME-TCR-iNKTs against A375-GFP cells. FIG. 14A shows quantification of A375-GFP cell death over time in four groups: target only, unmodified iNKTs, 684A2x709B5 and 684A3x709B5 PRAME-TCR-iNKTs. %GFP+ area of A375-GFP cells was assessed by fluorescence microscopy over 72hrs. FIG. 14B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0106] FIGs. 15A and 15B show killing of PRAME-TCR-iNKTs against MCF7- GFP cells. FIG. 15A shows quantification of MCF7-GFP cell death over time in four groups: target only, unmodified iNKTs, 684A2x709B5 and 684A3x709B5 PRAME-TCR-iNKTs. %GFP+ area of MCF7-GFP cells was assessed by fluorescence microscopy over 72hrs. FIG. 15B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0107] FIGs. 16A and 16B show killing of PRAME-TCR-iNKTs against A549-GFP cells. FIG. 16A shows quantification of MCF7-GFP cell death over time in four groups: target only, unmodified iNKTs, 684A2x709B5 and 684A3x709B5 PRAME-TCR-iNKTs. %GFP+ area of A549-GFP cells was assessed by fluorescence microscopy over 72hrs. FIG. 16B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0108] FIGs. 17A and 17B show killing of PRAME-TCR-iNKTs against A549-GFP-

CDld cells. FIG. 17A shows quantification of A549-GFP-CDld cell death over time in four groups: target only, unmodified iNKTs, 684A2x709B5 and 684A3x709B5 PRAME-TCR- iNKTs. %GFP+ area of A549-GFP-CDld cells was assessed by fluorescence microscopy over 72hrs. FIG. 17B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0109] FIGs. 18A and 18B show killing of PRAME-TCR-iNKTs against A375-GFP cells, impacted by an anti PD-1 antibody balstilimab (Bal) and an anti-CTEA4 antibody botensilimab (Bot). FIG. 18A shows quantification of A375-GFP cell death over time in seven groups: unmodified iNKT + Bot/Bal, unmodified iNKT + Isotypes, 684A2x709B5 + Bot/Bal, 684A2x709B5 + Isotypes, 684A3x709B5 + Bot/Bal, and 684A3x709B5 + Isotypes. %GFP+ area of A375-GFP cells was assessed by fluorescence microscopy over 72hrs. FIG. 18B shows percent change of GFP+ area at 72 hr (normalized to t=0 hr).

[0110] FIG. 19 shows IE-2 concentrations (pg/mE) measured by EEISA in PRAME- TCR-iNKT cells and bot/bal antibodies co-cultured with PBMCs stimulated by lOng/ml SEA (Staphylococcal Enterotoxin A). Isotypes alone and bot/bal alone were used as controls.

DETAILED DESCRIPTION

[0111] The instant disclosure is directed to novel engineered T cell receptors (TCRs) that target PRAME425-433 (SLLQHLIGL; SEQ ID NO:60), novel allogeneic iNKT cells engineered to present the TCRs and methods of use thereof for treatment of various cancers associated with the expression of PRAME. iNKT cells modulate both arms of immunity, innate and adaptive. The iNKT cells as described herein are capable of expressing both 1 PRAME engineered TCRs and endogenous TCRs, are able to effectively modulate the tumor microenvironment, and promote tumor killing.

[0112] As used herein, “PRAME” refers to PReferentially expressed Antigen in Melanoma. PRAME is a cancer testis antigen (CTA) also known as CT130.

[0113] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range.

[0114] As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to molecules comprising CDRs or variable regions from aP or y5 T cell receptors. Examples of TCRs include, but are not limited to, full-length TCRs, antigenbinding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs attached by a flexible linker, TCR chains linked by an engineered disulfide bond, single TCR variable domains, single peptide- MHC-specific TCRs, multi- specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In some embodiments, the TCR is a full-length TCR comprising a full-length a chain and a full-length P chain. In some embodiments, the TCR is a soluble TCR lacking transmembrane and/or cytoplasmic region(s). In some embodiments, the TCR is a single-chain TCR (scTCR) comprising Va and VP linked by a peptide linker, such as a scTCR having a structure as described in PCT Publication No.: WO 2003/020763, WO 2004/033685, or WO 2011/044186, each of which is incorporated by reference herein in its entirety. In some embodiments, the TCR comprises a transmembrane region. In some embodiment, the TCR comprises a co-stimulatory signaling region.

[0115] As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In some embodiments, the full-length TCR comprises one or two unmodified TCR chains, e.g., unmodified a, P, y, or 5 TCR chains. In some embodiments, the full-length TCR comprises one or two altered TCR chains, such as chimeric TCR chains and/or TCR chains comprising one or more amino acid substitutions, insertions, or deletions relative to an unmodified TCR chain. In some embodiments, the full-length TCR comprises a mature, full-length TCR a chain and a mature, full-length TCR P chain. In some embodiments, the full-length TCR comprises a mature, full-length TCR y chain and a mature, full-length TCR 5 chain.

[0116] As used herein, the term “TCR variable region” refers to the portion of a mature TCR polypeptide chain (e.g., a TCR a chain or P chain) which is not encoded by the TRAC gene for TCR a chains, either the TRBC1 or TRBC2 genes for TCR P chains, the TRDC gene for TCR 5 chains, or either the TRGC1 or TRGC2 gene for TCR y chains. In some embodiments, the TCR variable region of a TCR a chain encompasses all amino acids of a mature TCR a chain polypeptide which are encoded by a TRAV and/or TRAJ gene, and the TCR variable region of a TCR P chain encompasses all amino acids of a mature TCR P chain polypeptide which are encoded by a TRBV, TRBD, and/or TRBJ gene (see, e.g., T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety). TCR variable regions generally comprise framework regions (FR) 1, 2, 3 and 4 and complementarity determining regions (CDR) 1, 2 and 3.

[0117] As used herein, the terms “a chain variable region,” “a variable region,” and “Va” are used interchangeably and refer to the variable region of a TCR a chain.

[0118] As used herein, the terms “P chain variable region,” “P variable region,” and “VP” are used interchangeably and refer to the variable region of a TCR P chain.

[0119] “Engineered TCRs” refers to novel TCRs disclosed herein which are capable of binding with specificity to PRAME425-433 with specificity.

[0120] As used herein in the context of a TCR, the term “CDR” or “complementarity determining region” means the noncontiguous antigen combining sites found within the variable regions of a TCR chain (e.g., an a chain or a P chain). These regions have been described in Lefranc, (1999) The Immunologist 7: 132-136, Lefranc et al., (1999) Nucleic Acids Res 27: 209-212, LeFranc (2001) T cell receptor Factsbook, Academic Press, ISBN 0- 12-441352-8, Lefranc et al., (2003) Dev Comp Immunol. 27(l):55-77, and in Kabat et al., (1991) Sequences of protein of immunological interest, each of which is herein incorporated by reference in its entirety. In some embodiments, CDRs are determined according to the IMGT numbering system described in Lefranc (1999) supra. In some embodiments, CDRs are defined according to the Kabat numbering system described in Kabat supra. In some embodiments, CDRs are defined empirically, e.g., based upon a structural analysis of the interaction of a TCR with a cognate antigen (e.g., a peptide or a peptide-MHC complex). In some embodiments, the a chain and P chain CDRs of a TCR are defined according to different conventions (e.g., according to the Kabat or IMGT numbering systems, or empirically based upon structural analysis).

[0121] “Va-CDRl”, “Va-CDR2”, and “Va-CDR3” refer to the CDRs 1-3 of a variable region. “VP -CDR1”, “VP -CDR2”, and “VP -CDR3” refer to the CDRs 1-3 of P variable region.

[0122] As used herein, the term “framework amino acid residues” refers to those amino acids in the framework region of a TCR chain (e.g., an a chain or a P chain). The term “framework region” or “FR” as used herein includes the amino acid residues that are part of the TCR variable region, but are not part of the CDRs.

[0123] As used herein, the term “constant region” with respect to a TCR refers to the portion of a TCR that is encoded by the TRAC gene (for TCR a chains), either the TRBC1 or TRBC2 gene (for TCR P chains), the TRDC gene (for TCR 5 chains), or either the TRGC1 or TRGC2 gene (for TCR y chains), optionally lacking all or a portion of a transmembrane region and/or all or a portion of a cytoplasmic region. In some embodiments, a TCR constant region lacks a transmembrane region and a cytoplasmic region. A TCR constant region does not include amino acids encoded by a TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ gene (see, e.g., T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety).

[0124] As used herein, the terms “major histocompatibility complex” and “MHC” are used interchangeably and refer to an MHC class I molecule and/or an MHC class II molecule.

[0125] As used herein, the term “MHC class I” refers to a dimer of an MHC class I a chain and a P2 microglobulin chain and the term “MHC class II” refers to a dimer of an MHC class II a chain and an MHC class II P chain.

[0126] As used herein, the term “peptide-MHC complex” refers to an MHC molecule (MHC class I or MHC class II) with a peptide bound in the art-recognized peptide binding pocket of the MHC. In some embodiments, the MHC molecule is a membrane-bound protein expressed on cell surface. In some embodiments, the MHC molecule is a soluble protein lacking transmembrane or cytoplasmic regions. [0127] As used herein, the term “extracellular” with respect to TCR refers to the portion or portions of a recombinant transmembrane protein that are located outside of a cell.

[0128] As used herein, the term “transmembrane” with respect to a TCR chain refers to the portion or portions of a TCR chain that are embedded in the plasma membrane of a cell.

[0129] As used herein, the term “cytoplasmic” with respect to a TCR chain refers to the portion or portions of a TCR chain that are located in the cytoplasm of a cell.

[0130] As used herein, the term “co- stimulatory signaling region” refers to the intracellular portion of a co- stimulatory molecule that is responsible for mediating intracellular signaling events.

[0131] “Binding affinity” generally refers to the strength of the sum total of non- covalent interactions between a single binding site of a molecule (e.g., a TCR) and its binding partner (e.g., a peptide-MHC complex). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., a TCR and a peptide-MHC complex). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and equilibrium association constant (KA). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. kon refers to the association rate constant of, e.g., a TCR to a peptide-MHC complex, and koff refers to the dissociation rate constant of, e.g., a TCR to a peptide-MHC complex. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as use of BIAcore® or KinExA. As used herein, a “lower affinity” refers to a larger KD.

[0132] As used herein, the term “specifically binds to” refers to the ability of a TCR to preferentially bind to a particular antigen (e.g., a specific peptide or a specific peptide- MHC complex combination) as such binding is understood by one skilled in the art. For example, a TCR that specifically binds to an antigen can bind to other antigens, generally with lower affinity as determined by, e.g., BIAcore®, or other immunoassays known in the art (see, e.g., Savage et al., Immunity. 1999, 10(4):485-92, which is incorporated by reference herein in its entirety). In a specific embodiment, a TCR that specifically binds to an antigen binds to the antigen with an association constant (Ka) that is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold greater than the Ka when the TCR binds to another antigen. In some embodiments, the TCRs disclosed herein specifically bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 60.

[0133] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen (e.g., a peptide or a peptide-MHC complex) to which a TCR can bind. In some embodiments, the epitope to which a TCR binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), flow cytometry analysis, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and/or structural modeling. For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is herein incorporated by reference in its entirety). TCR: antigen crystals may be studied using well-known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), each of which is herein incorporated by reference in its entirety. Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, each of which is herein incorporated by reference in its entirety, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, the epitope of an antigen is determined using alanine scanning mutagenesis studies. In a specific embodiment, the epitope of an antigen is determined using hydrogen/deuterium exchange coupled with mass spectrometry. In some embodiments, the antigen is a peptide-MHC complex. In some embodiments, the antigen is a peptide presented by an MHC molecule.

[0134] As used herein, the terms “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. In some embodiments, the methods of “treatment” employ administration of a TCR or a cell expressing a TCR to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0135] As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.

[0136] As used herein, the term “subject” includes any human or non-human animal. In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is a human.

[0137] The determination of “percent identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., at score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., at score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0138] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0139] As used herein, the term “effector moiety” refers to a component or functional group of a molecule that increases or decreases a natural activity of the molecule, or confers a novel activity upon the molecule. In some embodiments, the effector moiety is a binding moiety. In an embodiment, the binding moiety binds to a cell surface protein. In some embodiments, the binding moiety is an antibody.

[0140] In some embodiments, the engineered TCRs are able to recognize TAA antigens in a major histocompatibility complex (MHC) class Ldependent manner. “MHC class Ldependent manner,” as used herein, means that the engineered TCR elicits an immune response upon binding to TAA antigens within the context of an MHC class I molecule. In some embodiments, the MHC class I molecule is an HLA-A molecule.

[0141] In some embodiments, the engineered TCRs bind PRAME425-433 (SEQ ID N0:60)-HLA-A*02.01 complex. In some embodiments, the engineered TCRs bind a PRAME peptide comprising the amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the engineered TCRs bind to a PRAME peptide consisting of the amino acid sequence set forth in SEQ ID NO:60.

[0142] The CDRs of a TCR disclosed herein can be defined using any art recognized numbering convention. Additionally or alternatively, the CDRs can be defined empirically, e.g., based upon structural analysis of the interaction of the TCR with a cognate antigen (e.g., a peptide or a peptide-MHC complex).

[0143] In some embodiments, the instant disclosure provides a TCR that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 60 (e.g., a TCR that binds to a SLLQHLIGL (SEQ ID NO: 60)-HLA-A*0201 complex), wherein the TCR comprises one, two, or all three of the CDRs of a Va or VP disclosed in Table 1, wherein the CDRs are defined according to the IMGT numbering system, for example, as described in Lefranc M-P (1999) and Lefranc M-P et al., (1999). In some embodiments, the engineered TCR comprises CDRs are defined according to the Kabat numbering system. As can be appreciated by one of skill in the art, CDRs can be predicted using the Kabat numbering system based on the variable domain sequences.

Table 1: CDR Sequences of Variable Alpha and Beta Chains

[0144] In some embodiments, the instant disclosure provides a TCR that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 60 (e.g., a TCR that binds to SLLQHLIGL (SEQ ID NO: 60)-HLA-A*0201 complex), wherein the TCR comprises one, two, or all three of the CDRs of a Va or VP disclosed in Table 1, wherein the CDRs are determined empirically, e.g., based upon structural analysis of the interaction of the TCR with a cognate antigen (e.g., a peptide-MHC complex).

[0145] In some embodiments, the engineered TCR is a human TCR. In some embodiments, TCR comprises sequence that naturally exist within the TCR germline repertoire of an animal or mammal (e.g., human). In some embodiments, the TCR comprises sequences that do not naturally exist within the TCR germline repertoire of an animal or mammal. In some embodiments, the TCR is an isolated TCR.

[0146] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, comprises an alpha chain comprising a variable alpha chain (Va) region and a beta chain comprising a variable beta chain (VP) region, wherein the Va region comprises a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the Va region also contains a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:37 or 41 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38.

[0147] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, comprises an alpha chain comprising a variable alpha chain (Va) region and a beta chain comprising a variable beta chain (VP) region, wherein the VP region comprises a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs:53, 54, 55, 56, 57, 58, or 59. In some embodiments, the VP region also comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52.

[0148] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, comprises an alpha chain comprising a variable alpha chain (Va) region and a beta chain comprising a variable beta chain (VP) region, wherein (a) the Va region comprises a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49 or 50; and (b) wherein the VP region comprises a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs:53, 54, 55, 56, 57, 58, or 59. The Va region may further contain a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:37 or 41 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38 and/or the VP region may further contain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52.

[0149] In some embodiments, the engineered TCR that binds to a SEQ ID NO:60- HLA-A*0201 complex with specificity, comprises a Va region and a VP region and

(a) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively;

(b) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively;

(c) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively;

(d) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(e) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(f) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively;

(g) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively;

(h) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively;

(i) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; (j) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(k) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively;

(l) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(m) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:41, SEQ ID NO:38, and SEQ ID NO:42, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(n) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively;

(o) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively;

(p) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:59, respectively;

(q) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(r) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:43, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(s) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively; (t) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:56, respectively;

(u) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively;

(v) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:58, respectively;

(w) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(x) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:44, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(y) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:55, respectively;

(z) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:56, respectively;

(aa) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:57, respectively;

(bb) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(cc) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; (dd) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:46, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(ee) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:46, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(ff) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:47, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(gg) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:47, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(hh) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:48, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(ii) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:48, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(jj) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:49, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively;

(kk) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:49, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively;

(11) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:50, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:54, respectively; or

(hh) the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:50, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively. [0150] In some embodiments, the engineered TCR that binds to a SEQ ID NO:60- HLA-A*0201 complex with specificity, comprises a Va region and a VP region and the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[0151] In some embodiments, the engineered TCR that binds to a SEQ ID NO:60- HLA-A*0201 complex with specificity, comprises a Va region and a VP region and the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:45, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[0152] In some embodiments, the engineered TCR that binds to a SEQ ID NO:60- HLA-A*0201 complex with specificity, comprises a Va region and a VP region and the CDR1, CDR2, and CDR3 of the Va region comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively, and the CDR1, CDR2, and CDR3 of the VP region comprise SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[0153] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, comprises a Va region and a VP region, wherein the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22. For instance, the disclosure provides an engineered TCR that comprises a Va region and a VP region, wherein the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22 and the VP region comprises a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs:53, 54, 55, 56, 57, 58, or 59. In some embodiments, the VP region also comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:51 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:52.

[0154] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, comprises a Va region and a VP region, wherein the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36. For instance, the disclosure provides an engineered TCR that comprises a Va region and a VP region, wherein the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36 and the Va region contains a CDR3 comprising an amino acid sequence as set forth in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the Va region also contains a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:37 or 41 and a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO:38.

[0155] In some embodiments, the engineered TCR comprises a Va region and a VP region, wherein the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO:22, and wherein the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36. [0156] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28

[0157] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32.

[0158] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:36.

[0159] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0160] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:2 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0161] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28.

[0162] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 4, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32.

[0163] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:36.

[0164] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0165] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:4, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:4 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0166] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28.

[0167] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0168] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:6, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:6 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26. [0169] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28.

[0170] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:8, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32.

[0171] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:8, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:36. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:36.

[0172] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0173] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 8, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:8 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0174] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28.

[0175] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID :10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 30 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:30. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:30.

[0176] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32.

[0177] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:34 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:34. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:34.

[0178] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0179] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 10, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 10 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0180] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:28. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:28.

[0181] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:30 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:30. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:30. [0182] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:32. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:32.

[0183] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0184] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 12, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 12 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0185] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 14, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 14 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0186] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 14, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 14 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0187] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 16, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 16 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0188] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 16 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 16 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0189] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 18, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 18 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0190] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID: 18, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO: 18 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0191] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:20 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:20, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:20 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0192] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:20 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:20, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:20 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26.

[0193] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:22, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:24. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:22 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:24.

[0194] In some embodiments, the engineered TCR that binds to a (SEQ ID NO:60)- HLA-A*0201 complex with specificity, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:22 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:22, and the VP region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID:26. For instance, in some embodiments, the Va region comprises an amino acid sequence as set forth in SEQ ID NO:22 and the VP region comprises an amino acid sequence as set forth in SEQ ID NO:26. [0195] Any TCR constant region from any species can be used in the engineered TCRs disclosed herein. In some embodiments, the engineered TCR comprises a human a, p, y, or 5 TCR constant region. In some embodiments, the engineered TCR comprises a wildtype constant region. In some embodiments, the engineered TCR comprises an altered constant region, such as a chimeric constant region or constant region comprising one or more amino acid substitutions, insertions, or deletions relative to a wild-type constant region.

[0196] The engineered TCRs disclosed herein can be used in any TCR structural format. For example, in some embodiments, the engineered TCR is a full-length TCR comprising a full-length a chain and a full-length P chain. The transmembrane regions (and optionally also the cytoplasmic regions) can be removed from a full-length TCR to produce a soluble TCR. Accordingly, in some embodiments, the engineered TCR is a soluble TCR lacking transmembrane and/or cytoplasmic region(s). The methods of producing soluble TCRs are well-known in the art. In some embodiments, the soluble TCR comprises an engineered disulfide bond that facilitates dimerization, see, e.g., U.S. Patent No. 7,329,731, which is incorporated by reference herein in its entirety. In some embodiments, the soluble TCR is generated by fusing the extracellular domain of a TCR described herein to other protein domains, e.g., maltose binding protein, thioredoxin, human constant kappa domain, or leucine zippers, see, e.g., Lpset et al., Front Oncol. 2014; 4: 378, which is incorporated by reference herein in its entirety. A single-chain TCR (scTCR) comprising Va and VP linked by a peptide linker can also be generated. Such scTCRs can comprise Va and VP, each linked to a TCR constant region. Alternatively, the scTCRs can comprise Va and VP, where either the Va, the VP, or both the Va and VP are not linked to a TCR constant region. Exemplary scTCRs are described in PCT Publication Nos. WO 2003/020763, WO 2004/033685, and WO 2011/044186, each of which is incorporated by reference herein in its entirety. Furthermore, the engineered TCRs disclosed herein can comprise two polypeptide chains (e.g., an a chain and a P chain) in which the chains have been engineered to each have a cysteine residue that can form an interchain disulfide bond. Accordingly, in some embodiments, the engineered TCRs disclosed herein comprise two polypeptide chains linked by an engineered disulfide bond. Exemplary TCRs having an engineered disulfide bond are described in U.S. Patent Nos. 8,361,794 and 8,906,383, each of which is incorporated by reference herein in its entirety.

[0197] In some embodiments, the TCRs disclosed herein comprise one or more chains (e.g., an a chain and/or a P chain) having a transmembrane region. In some embodiments, the TCRs disclosed herein comprise two chains (e.g., an a chain and a P chain) having a transmembrane region. The transmembrane region can be the endogenous transmembrane region of that TCR chain, a variant of the endogenous transmembrane region, or a heterologous transmembrane region. In some embodiments, the TCRs disclosed herein comprise an a chain and a P chain having endogenous transmembrane regions.

[0198] In some embodiments, the engineered TCRs disclosed herein comprise one or more chains (e.g., an a chain and/or a P chain) having a cytoplasmic region. In some embodiments, the engineered TCRs disclosed herein comprise two chains (e.g., an a chain and a P chain) each having a cytoplasmic region. The cytoplasmic region can be the endogenous cytoplasmic region of that TCR chain, variant of the endogenous cytoplasmic region, or a heterologous cytoplasmic region. In some embodiments, the engineered TCRs disclosed herein comprise two chains (e.g., an a chain and a P chain) where both chains have transmembrane regions but one chain is lacking a cytoplasmic region. In some embodiments, the engineered TCRs disclosed herein comprise two chains (e.g., an a chain and a P chain) where both chains have endogenous transmembrane regions but lack an endogenous cytoplasmic region. In some embodiments, the engineered TCRs disclosed herein comprise an a chain and a P chain where both chains have endogenous transmembrane regions but lack an endogenous cytoplasmic region. In some embodiments, the engineered TCRs disclosed herein comprise a co-stimulatory signaling region from a co-stimulatory molecule; see, e.g., PCT Publication Nos.: WO 1996/018105, WO 1999/057268, and WO 2000/031239, and U.S. Patent No. 7,052,906, all of which incorporated herein by reference in their entireties.

[0199] In some embodiments, the engineered TCRs described herein bind to a peptide-MHC complex comprising a peptide having the amino acid sequence set forth in SEQ ID NO: 60, wherein the MHC may be any MHC. In some embodiments, the MHC is a human MHC. In some embodiments, the MHC is an MHC class I molecule comprising an MHC class I heavy chain (e.g., an HLA-A, an HLA-B, or an HLA-C, including any subtypes in any polymorphic forms) and a p2-microglobulin light chain. In some embodiments, the MHC is HLA-A*0201. In some embodiments, the peptide-MHC complex is SLLQHLIGL (SEQ ID NO: 60)-HLA-A*0201. In some embodiments, the MHC is an MHC class II molecule comprising an MHC class II a chain (e.g., an a chain of an HLA-DR, an HLA-DQ, or an HLA-DP, including any subtypes in any polymorphic forms) and an MHC class II P chain (e.g., a P chain of an HLA-DR, an HLA-DQ, or an HLA-DP, including any subtypes in any polymorphic forms). In some embodiments, the MHC class II a chain and the MHC class II P chain are derived from the same type (e.g., HLA-DR, HLA-DQ, or HLA-DP).

[0200] Nucleotide sequences encoding TCRs described herein can be determined using methods well-known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the TCR. Such a polynucleotide encoding the TCR can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the TCR, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0201] Alternatively, a polynucleotide encoding a TCR described herein can be generated from nucleic acid from a suitable source (e.g., a T lymphocyte) using methods well-known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from T cells expressing the TCR of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the a chain and/or P chain of a TCR. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the Va domain and/or VP domain of a TCR. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized TCRs.

[0202] If a clone containing a nucleic acid encoding a particular TCR is not available, but the sequence of the TCR molecule is known, a nucleic acid encoding the TCR can be chemically synthesized or obtained from a suitable source (e.g., a TCR cDNA library or a cDNA library generated from, or nucleic acid, e.g., poly A+ RNA, isolated from, any tissue or cells expressing the TCR, such as T lymphocytes selected to express a TCR described herein) by PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes TCRs. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well-known in the art.

[0203] DNA encoding TCRs described herein can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the a chain and/or P chain of the TCR. T lymphocytes can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce TCR protein, to obtain the synthesis of TCRs in the recombinant host cells.

[0204] To generate whole TCRs, PCR primers including Va or VP nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the Va or VP sequences into clones, e.g., clones of individual Va or VP nucleotide sequences, or clones of single-chain TCRs containing variable regions of TCRs attached by a flexible linker. Utilizing cloning techniques known to those of skill in the art, the PCR amplified Va domains can be cloned into vectors expressing an a chain constant region, and the PCR amplified VP domains can be cloned into vectors expressing a P chain constant region. In some embodiments, the vectors for expressing the Va or VP domains comprise an EF-la promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selection marker such as neomycin. The a chain and P chain vectors are then cotransfected into cell lines, either simultaneously or sequentially, to generate stable or transient cell lines that express whole TCRs using techniques known to those of skill in the art. The Va or VP domains can also be cloned into one vector expressing the necessary constant regions. The vector is then transfected into cell lines to generate stable or transient cell lines that express whole TCRs using techniques known to those of skill in the art.

[0205] The DNA also can be modified, for example, by substituting the coding sequence for human a chain and P chain constant domains in place of the murine sequences, or by covalently joining to the TCR coding sequence all or part of the coding sequence for a non-TCR polypeptide.

[0206] Also provided are polynucleotides that hybridize under high, intermediate, or low stringency hybridization conditions to polynucleotides that encode a TCR described herein. In specific embodiments, polynucleotides described herein hybridize under high, intermediate, or low stringency hybridization conditions to polynucleotides encoding a Va domain and/or VP domain provided herein.

[0207] Hybridization conditions have been described in the art and are known to one of skill in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65°C; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions are known to those of skill in the art and have been described, see, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3.

[0208] In some aspects, provided herein are cells expressing (e.g., recombinantly) TCRs described herein which bind to a PRAME peptide, and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding such TCRs for recombinant expression in cells, e.g., in mammalian cells. Also provided herein are cells comprising such vectors for recombinantly expressing TCRs described herein (e.g., human or humanized TCR). In a particular aspect, provided herein are methods for producing a TCR described herein, comprising expressing such TCR from a cell. In some embodiments, the cells are iNKT cells.

[0209] In another aspect, provided herein are methods for producing an engineered cell (e.g., a cell comprising a heterologous and/or recombinant nucleic acid) as described herein. In some embodiments, the method comprises contacting a cell with a vector as described herein under conditions that allow introduction of the vector into the cell. In some embodiments, the condition allows transfection of the cell with the vector (e.g., by liposome or electroporation). In some embodiments, the condition allows transfection of the cell with an mRNA vector by electroporation. In some embodiments, the vector is a viral vector (e.g., lentiviral vector), and the conditions allow transduction of the cell with the viral vector. In some embodiments, the vector is introduced to the cell in vitro or ex vivo. In some embodiments, the vector is introduced to the cell in vivo.

[0210] Recombinant expression of an engineered TCR described herein (e.g., a full- length TCR, a chain and/or P chain of a TCR, or a single-chain TCR described herein) that binds to a PRAME peptide involves construction of an expression vector containing a polynucleotide that encodes the TCR. Once a polynucleotide encoding a TCR described herein has been obtained, the vector for the production of the TCR molecule can be produced by recombinant DNA technology using techniques well-known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a TCR encoding nucleotide sequence are described herein. Methods which are well-known to those skilled in the art can be used to construct expression vectors containing TCR encoding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an engineered TCR molecule described herein (e.g., a full-length TCR, a chain or P chain of a TCR, Va or VP of a TCR, or an a or P chain CDR), operably linked to a promoter.

[0211] The vector can comprise any type of nucleotides (including but not limited to DNA and RNA) which can be single- stranded or double- stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non-naturally- occurring internucleotide linkages, or both types of linkages. In some embodiments, the non- naturally occurring or altered nucleotides or inter-nucleotide linkages do not hinder the transcription or replication of the vector. The expression vector can be a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector). In some embodiments, the retroviral vector is a lentiviral vector (e.g., a vector comprising genetic elements of the HIV-1 genome) or an equine infectious anemia viral vector. In some embodiments, the vector is packaged with one or more viral capsid proteins to provide a viral particle.

[0212] An expression vector can be transferred to a cell (e.g., an iNKT cell) by conventional techniques and the resulting cell can then be cultured by conventional techniques to produce an engineered TCR described herein. Thus, provided herein are cells (e.g., iNKT cells) containing a polynucleotide encoding a TCR molecule described herein (e.g., a full-length TCR, a chain or P chain of a TCR, Va or VP of a TCR, or an a or P chain CDR) operably linked to a promoter for expression of such sequences in the host cell. In some embodiments, for the expression of double-chained TCRs, vectors encoding both the a and P chains, individually, can be co-expressed in the cell for expression of the entire TCR molecule, as detailed below. In some embodiments, a host cell contains a vector comprising a polynucleotide encoding both the a chain and P chain of an engineered TCR described herein. In specific embodiments, a cell contains two different vectors, a first vector comprising a polynucleotide encoding an a chain or an a chain variable region of an engineered TCR described herein, and a second vector comprising a polynucleotide encoding a P chain or a P chain variable region of an engineered TCR described herein. In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding an a chain or an a chain variable region of a TCR described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding a P chain or a P chain variable region of a TCR described herein. In specific embodiments, an a chain or a chain variable region expressed by a first cell associated with a P chain or P chain variable region expressed by a second cell to form a TCR described herein. In some embodiments, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0213] In a particular embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding an a chain or a chain variable region of a TCR described herein, and a second vector comprising a polynucleotide encoding a P chain or P chain variable region of a TCR described herein.

[0214] A variety of host-expression vector systems can be utilized to express engineered TCR molecules described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a TCR molecule described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing TCR coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing TCR coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing TCR coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing TCR coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In a specific embodiment, cells for expressing TCRs described herein are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In a particular embodiment, cells for expressing TCRs described herein are human cells, e.g., human cell lines. In a specific embodiment, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant TCR molecule, are used for the expression of a recombinant TCR molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus, are an effective expression system for TCRs (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7). In some embodiments, engineered TCRs described herein are produced by CHO cells or NSO cells. In a specific embodiment, the expression of nucleotide sequences encoding TCRs described herein is regulated by a constitutive promoter, inducible promoter, or tissue specific promoter.

[0215] Once an engineered TCR molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the TCR described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0216] In specific embodiments, a TCR described herein is isolated or purified.

Generally, an isolated TCR is one that is substantially free of other TCRs with different antigenic specificities than the isolated TCRs. For example, in a particular embodiment, a preparation of a TCR described herein is substantially free of cellular material and/or chemical precursors. The language “substantially free of cellular material” includes preparations of a TCR in which the TCR is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a TCR that is substantially free of cellular material includes preparations of the TCR having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) and/or variants of the TCR, for example, different post- translational modified forms of the TCR or other different versions of the TCR (e.g., fragments thereof). When the TCR is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the TCR is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the TCR. Accordingly, such preparations of the TCR have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the TCR of interest. In a specific embodiment, TCRs described herein are isolated or purified.

[0217] Engineered TCRs that bind to a PRAME peptide can be produced by any method known in the art for the synthesis of TCRs, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel PM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, each of which is incorporated by reference herein in its entirety.

[0218] In a specific embodiment, a TCR described herein is a TCR (e.g., recombinant TCR) prepared, expressed, created, or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNA sequences. In some embodiments, such TCR comprises sequences (e.g., DNA sequences, RNA sequences, or amino acid sequences) that do not naturally exist within the TCR germline repertoire of an animal or mammal (e.g., human) in vivo.

[0219] In one aspect, provided herein is a method of making a TCR that binds to a PRAME peptide, the method comprising culturing a cell or host cell described herein. In a some aspect, provided herein is a method of making a TCR which binds to a PRAME peptide, the method comprising expressing (e.g., recombinantly expressing) the TCR using a cell or host cell described herein (e.g., a cell or a host cell comprising polynucleotides encoding a TCR described herein). In a particular embodiment, the cell is an isolated cell. In a particular embodiment, the exogenous polynucleotides have been introduced into the cell. In a particular embodiment, the method further comprises the step of purifying the TCR obtained from the cell or host cell.

[0220] In another aspect, the instant disclosure provides a mammalian cell (e.g., an engineered mammalian cell) or a population thereof presenting a TCR disclosed herein on the cell surface. Any mammalian cell can be used to present a TCR disclosed herein. In some embodiments, the mammalian cell expresses CD3 (e.g., a CD3y chain, a CD35 chain, and two CD3s chains).

[0221] In some embodiments, the mammalian cell is a human cell. Effector cells of the cellular immune system are particularly useful for presenting a TCR disclosed herein because the cell surface TCR can target these effector cells to tumor cells expressing the PRAME polypeptide, thereby facilitating killing of the tumor cells. Accordingly, in some embodiments, the mammalian cell is a lymphocyte (e.g., a human lymphocyte), such as a T cell or a natural killer (NK) cell. In some embodiments, the lymphocyte is a T cell. Any T cell at any developmental stage can be used to present a TCR disclosed herein. For example, in some embodiments, the T cell is selected from the group consisting of a CD8+ cytotoxic T cell, a CD4+ cytotoxic T cell, a CD4+ helper T cell (e.g., a Thl or a Th2 cell), a CD4/CD8 double positive T cells, a tumor infiltrating T cell, a thymocyte, a memory T cell, a naive T cell, and a natural killer T cell, e.g., an invariant natural killer T cell. Precursor cells of the cellular immune system (e.g., precursors of T lymphocytes) are also useful for presenting a TCR disclosed herein because these cells may differentiate, develop, or mature into effector cells. Accordingly, in some embodiments, the mammalian cell is a pluripotent stem cell (e.g., an embryonic stem cell, an induced pluripotent stem cell), a hematopoietic stem cell, or a lymphocyte progenitor cell. In some embodiments, the hematopoietic stem cell or lymphocyte progenitor cell is isolated and/or enriched from, e.g., bone marrow, umbilical cord blood, or peripheral blood.

[0222] Cells can be obtained from numerous sources, including but not limited to, tumor, blood, bone marrow, lymph node, thymus, or another tissue or bodily fluid, or an apheresis product. In some embodiments, cells are obtained from a patient directly following a treatment that leaves the subject with functional T cells. In this regard, it has been observed that following some cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, in some embodiments, cells are collected from blood, bone marrow, lymph node, thymus, or another tissue or bodily fluid, or an apheresis product, during this recovery phase.

[0223] In some embodiments, the cells are invariant natural killer T (iNKT) cells. iNKT cells are a unique subset of T cells that exhibit both direct and indirect anti-tumor activities. iNKT cells can recognize and kill tumor cells directly through the release of cytotoxic molecules, such as perforin and granzyme B, and by inducing apoptosis through the Fas/FasL pathway. Additionally, iNKT cells can act indirectly by stimulating other immune cells, such as dendritic cells, natural killer (NK) cells, and CD8+ T cells, to target and kill tumor cells. iNKT cells also play a role in regulating the immune response by producing cytokines, such as interferon-gamma and interleukin-4, that promote the activation and differentiation of other immune cells.

[0224] The term “invariant Natural Killer T cells”, or “invariant NKT cells”, “iNKT cells”, or “Type I NKT cell), as used herein, refer to a population of T lymphocytes expressing a conserved semi-invariant TCR specific for lipid antigens restricted for the monomorphic MHC class Lrelated molecule CD Id. Natural killer T cells (NKT cells) were originally characterized in mice as T cells that express both a TCR and NK1.1 (NKR-Pla-c or CD161), a C-type lectin NK receptor. Invariant NKT (iNKT) cells express a semiinvariant aP TCR (e.g., formed by an invariant TRAV11-TRAJ18 (4) rearrangement in mice, or the homologous invariant TRAV10-TRAJ18 chain in humans), paired with a limited set of diverse VP chains, predominantly TRBV1, TRBV29, or TRBV13 in mice (6) and TRBV25 in humans (see e.g., Dellabona et al., An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med. (1994) 180:1171-6. 10.1084). The semi-invariant TCR recognizes exogenous and endogenous lipid antigens presented by the monomorphic MHC class Lrelated molecule CDld (see e.g., Brennan et al., Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol. (2013) 13:101-17. 10.1038). Exogenous lipid antigens include the prototypical a-Galactosylceramide (a-GalCer) (Kawano et al., CD Id-restricted and TCR-mediated activation of valphal4 NKT cells by glycosylceramides. Science. (1997) 278:1626-9. 10.1126) and a number of bacterial-derived Ags, which can activate iNKT cells.

[0225] In some embodiments, the cells are a population of peripheral blood mononuclear cells (PBMC) (e.g., human PBMCs). In some embodiments, the cells of interest are isolated from PBMCs. For instance, iNKT cells can be isolated from PBMCs and expanded. In some embodiments, the cells of interest are isolated from a donor’s peripheral blood Alternatively, the cells may be provided in the form of a blood apheresis sample, such as a lymphapheresis or leukapheresis sample. The cells may be fresh, or may have previously been frozen.

[0226] The cells, for instance, iNKT cells, may be from a donor subject, for example a healthy donor. In some embodiments, the cells are iNKT cells and are obtained from allogeneic donors. iNKT cells can be obtained from allogeneic donors because iNKT cells are restricted by CD Id, a non-polymorphic MHC I like molecule.

[0227] In some embodiments, the mammalian cell (e.g., iNKT cell) is a population of cells presenting an engineered TCR disclosed herein on the cell surface. The population of cells can be heterogeneous or homogenous. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the population is a cell as described herein. In some embodiments, the population is substantially pure, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the population is homogeneous.

[0228] Populations of cells can be enriched or purified, as needed. For instance, iNKT cells can be expanded by culturing in IE-21, IE-2, IL-7, IL-15, IL-12, TNF-a, and irradiated NKT-depleted PBMCs loaded with a-galactosylceramide (aGalCer) or irradiated artificial presenting cells (APCs) expressing CDl-d loaded with aGalCer.

[0229] Cells can be stimulated ex vivo to increase viability, proliferation, and/or activity. In some embodiments, the induction does not include any defined antigen, thus providing a cell population which is polyclonal with respect to antigen reactivity. In some embodiments, the cell is contacted with a first agent, which induces or activates a TCR/CD3 complex-associated signal (e.g., an anti-CD3 antibody). In some embodiments, the cell is contacted with a second agent, which stimulates an accessory molecule on the T cell surface (e.g., a ligand of CD28 or an anti-CD28 antibody). In some embodiments, the cell is contacted with a molecule or complex that interacts with both CD3 and CD28, wherein the molecule or complex may be presented on a surface (e.g., a bead, particle, or cell). In some embodiments, the cell is contacted with an agent that stimulates one or more intracellular signals such as Ca2+ release (e.g., phorbol 12-myristate 13-acetate and/or ionomycin). Alternatively, the induction may include an antigen comprising a peptide (e.g., a PRAME peptide) which binds to the TCR presented on the cell surface, thus providing a cell population which is enriched (e.g., monoclonal) with respect to antigen reactivity. The antigen may further comprise an MHC molecule (e.g., an HLA molecule) in complex with the peptide. The antigen may be presented as a soluble form, bound to a membrane, or presented on a surface.

[0230] In some embodiments, the mammalian cell (e.g., iNKT cell) expresses an engineered TCR disclosed herein from a transgene introduced into the cell and presents the TCR on the cell surface. The TCR may be displayed constitutively on the cell surface. Alternatively, the cell may be capable of conditional expression and/or display of the TCR. For example, the expression or display of the engineered TCR may be induced by an exogenous stimulus or by cellular differentiation. In some embodiments, the transgene encodes an engineered TCR a chain and/or P chain, or a fragment thereof (e.g., Va, VP, CDR3a and/or CDR3P). In some embodiments, the transgene is operably linked to an exogenous transcriptional and/or translational control sequence (e.g., a promoter, an enhancer, and/or a Kozak sequence). In some embodiments, the transgene is operably linked to an endogenous transcriptional and/or translational control sequence (e.g., a promoter, an enhancer, and/or a Kozak sequence) not at its native genomic locus (e.g., introduced by a vector). In some embodiments, the transgene is operably linked to an endogenous transcriptional and/or translational control sequence (e.g., a promoter, an enhancer, and/or a Kozak sequence) at its native genomic locus (e.g., by inserting the transgene into the native genomic locus).

[0231] In some embodiments, the cells (e.g., iNKT cells) are engineered to express another molecule (e.g., a cytokine or ligand) capable of enhancing one or more properties (e.g., survival/persistence, immune interaction with other cells such as macrophages and/or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the genetically modified cells. For instance, the cells (e.g., iNKT cells) can be engineered to express any suitable armoring molecule known in the art, e.g., armoring molecule described by Yeku et al., Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy, Biochem Soc Trans. 2016 Apr 15; 44(2): 412-418; Hawkins et al., Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy, Biologies. 2021; 15: 95-105). Non-limiting examples of armoring molecules include IL-15, IL-2, IL-12, CD40L, 4-1BBL, IL- 18, IL-7, IL-33, constitutively active Akt (caAkt), hybrid IL-4/IL-7 receptor, checkpoint inhibitors such as anti-PDl antibodies, nanobodies targeting CD47, or bispecific T-cell engagers (BiTEs).

[0232] In some embodiments, the cells (e.g., iNKT cells) expressing the engineered TCR described herein are also engineered to express IL- 15. In some embodiments, the genetically modified cells expressing the engineered TCR described herein are also engineered to express soluble IL-15 (sIL-15). The role of IL-15 in enhancing the expansion and function of T cells and natural killer T cells (e.g., iNKT cells) have been previously described (see, e.g., Lin et al., Interleukin- 15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood, Cytokine. 2015 Dec;76(2):348- 355; Battram et al., IL-15 Enhances the Persistence and Function of FAP-Targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation; Cancers (Basel). 2021 Jul; 13(14): 3534). There is evidence that numerous cell types are responsible for the production of IL- 15, including macrophages and DCs, and once released, IL-15 stimulates CD8+ T-cells and NK cells which increases their proliferation and cytotoxic capacity. Administration of IL- 15 to mice has been shown to enhance anti-tumor activity of adoptively transferred CD8+ tumor-reactive T-cells, which suggests IL- 15 could also enhance anti-tumor activity of T-cell therapy. Further, IL- 15 can increase antigen-independent T-cell proliferation, while enabling T-cell persistence after tumor clearance. Some previous studies used a form of IL- 15 tethered to the membrane and found that this promoted the T-cells to develop a memory phenotype. These data suggest that IL- 15 could provide long-term T-cell-mediated immunity toward the cancer antigen along with enhancing T-cell function within the tumor microenvironment.

[0233] In some embodiments, the cell (e.g., iNKT cell) expresses the engineered TCR disclosed herein and an endogenous TCR. For instance, in some embodiments, iNKT cells present an engineered TCR as disclosed herein and the iNKT endogenous TCR, a semiinvariant aP TCR. iNKT typically cells express an invariant TCR a-chain (Val4-Jal8 in mice or Va24-Jal8 in humans). In mice, although most iNKT cells express the canonical Val4-Jal8 TCR a-chain, they can use different VP chains and the combination of VP-, JP-, and CDR3P-encoded residues will ultimately determine the type of ligands that iNKT cells can bind (Cameron et al., 2015; Mallevaey et al., 2009; Matsuda et al., 2001). Moreover, a population of aGalCer-reactive NKT cells that express ValO TCR and has a distinct lipid- recognition capacity has been identified (Uldrich et al., 2011). In humans, while the majority of aGalCer-binding iNKT cells express the prototypical Va24Vpil TCR, populations of atypical NKT cells have been found in the blood, with cells expressing a range of TCRa and TCRP chains that show differential recognition of lipid antigens (Le Nours et al., 2016; Matulis et al., 2010).

[0234] In some embodiments, the transgene is a DNA integrated into the host cell genome, wherein the integration occurs through site-specific integration (e.g., homologous recombination) or random insertion of the DNA. In some embodiments, the transgene is a DNA not integrated into the host cell genome (e.g., maintained as a non-integrating viral genome or as an episomal DNA). In some embodiments, the transgene is a polynucleotide (including but not limited to DNA, RNA, modified DNA, and modified RNA) that can be transcribed and/or translated to express the TCR disclosed herein.

[0235] In some embodiments, the transgene comprises a first and a second sequence, the first sequence encoding a polypeptide comprising a TCR a chain or a fragment thereof (e.g., Va or CDR3a), and the second sequence encoding a polypeptide comprising a TCR P chain or a fragment thereof (e.g., VP or CDR3P). In some embodiments, the first and the second sequences are each operably linked to a transcriptional and/or translational control sequence (e.g., a promoter, an enhancer, and/or a Kozak sequence). In some embodiments, the first and second sequences are in different polynucleotides (e.g., DNA, RNA, modified DNA, or modified RNA) molecules. In some embodiments, the first and second sequences of the transgene are in the same polynucleotide (e.g., DNA, RNA, modified DNA, or modified RNA) molecule. In some embodiments, the first and second sequences are operably linked by a linker sequence that promotes the production of two separate polypeptides (e.g., an internal ribosome entry site (IRES), a self-cleavage peptide (e.g., a 2A peptide), or a peptide sequence recognized by an intracellular or an extracellular protease). In some embodiments, the first and second sequences can be transcribed and/or translated independently. In some embodiments, the first and second sequences are each integrated into the host cell genome. In some embodiments, the first and second sequences are each integrated into different regions of the host cell genome.

[0236] In some embodiments, the cell further comprises a polynucleotide encoding a polypeptide capable of inducing T cell activation. In some embodiments, the polypeptide is an inducible chimeric stimulating molecule, for example, as described in PCT Publication No. WO 2015/123527, incorporated herein by reference in its entirety. In some embodiments, the polypeptide comprises a multimerization (e.g., dimerization or oligomerization) region, wherein the polypeptide induces T cell activation upon multimerization .

[0237] In some embodiments, the cell is provided in a solution. In some embodiments, the cell is cryopreserved at about or lower than -80 °C (e.g., in a liquid nitrogen storage tank). Methods of cryopreservation are well-known in the art, e.g., as described in U.S. Patent Nos.: 5,580,714 and 6,740,484, which are incorporated by reference herein in their entireties. The cryopreserved cell may be recovered by thawing, and any of the isolation, purification, enrichment, stimulation, and display of the engineered TCR as described above may be conducted prior to the cryopreservation or after the recovery.

[0238] The disclosure provides pharmaceutical compositions comprising an engineered TCR disclosed herein, a polynucleotide disclosed herein, a cell disclosed herein and a pharmaceutically acceptable carrier.

[0239] The disclosure provides a method of inducing an immune response to PRAME, the method comprising administering to the subject an effective amount of the engineered TCR as disclosed herein, the polynucleotide disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0240] In some embodiments, the disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject an effective amount of the TCR disclosed herein, the polynucleotide disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0241] Cells (e.g., iNKT cells) administered to the subject can be autologous or allogeneic. In some embodiments, autologous cells are obtained from a patient directly following a cancer treatment. In this regard, it has been observed that following some cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, in some embodiments, cells are collected from blood, bone marrow, lymph node, thymus, or another tissue or bodily fluid, or an apheresis product, during this recovery phase.

[0242] In some embodiments, the cells administered to the subject are allogeneic. In some embodiments, the cells are allogenic iNKT cells. In some embodiments, the allogenic iNKT cells are administered to subjects without prior lymphodepletion.

[0243] The number of cells that are employed will depend upon a number of circumstances including, the lifetime of the cells, the protocol to be used (e.g., the number of administrations), the ability of the cells to multiply, the stability of the recombinant construct, and the like. In some embodiments, the cells are applied as a dispersion, generally being injected at or near the site of interest. The cells may be administered in any physiologically acceptable medium.

[0244] In some embodiments, the disclosure provides methods of treating cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising human iNKT cells presenting TCRs as disclosed herein and a pharmaceutically acceptable carrier. For instance, in some embodiments, the disclosure provides methods of treating a cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising allogeneic iNKT cells that express an engineered TCR as disclosed herein and an iNKT endogenous semi-invariant aP TCR.

[0245] In a further aspect, the present disclosure is directed to a method of treating a subject in need of adoptive cell therapy, said method comprising administering to said patient a pharmaceutical composition as defined above to said patient. In some embodiments, the subject belongs to the group of HLA-A2 positive subjects.

[0246] In some embodiments, said subject suffers from a disease involving malignant cells expressing PRAME.

[0247] Cancers that can be treated with the TCRs, polynucleotide, vector, engineered cells, or pharmaceutical compositions disclosed herein can be any tumor expressing PRAME. In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is selected from the group consisting of endometrial cancer, esophageal cancer, lung squamous cell cancer, melanoma, multiple myeloma, ovarian cancer, renal papillary cell cancer, testicular cancer, thymoma, uterine carcinosarcoma, non-small cell lung cancer, breast cancer, and uveal melanoma. In some embodiments, the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid leukemia, myeloma (e.g., chronic myeloid cancer), colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor. Hodgkin’s lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin’s lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), gastric cancer, small intestine cancer, soft tissue cancer, stomach cancer, carcinoma, sarcoma (e.g., synovial sarcoma, rhabdomyosarcoma), testicular cancer, thyroid cancer, head and neck cancer, ureter cancer, and urinary bladder cancer. In some embodiments, the cancer is melanoma, breast cancer, lung cancer, prostate cancer, thyroid cancer, ovarian cancer, or synovial sarcoma. In some embodiments, the cancer is synovial sarcoma or liposarcoma (e.g., myxoid/round cell liposarcoma).

[0248] In some embodiments, these methods further comprise administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic, radio therapeutic, or a checkpoint targeting agent. In some embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-Ll antibody, an antagonist anti-PD- L2 antibody, an antagonist anti-PD-1 antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAMl antibody, an agonist anti- GITR antibody, and an agonist anti-OX40 antibody.

[0249] Lymphodepletion treatment is common to reduce the population of circulating lymphocytes, prior to infusion of TCR-T cells, including chemotherapy, radiotherapy and/or any other method specified. In some embodiments, the subject is administered a pharmaceutical composition comprising iNKT cells which express the engineered TCR disclosed herein, and lymphodepletion is not required prior to treatment.

[0250] In some embodiments, the engineered TCR, the polynucleotide, the cell (e.g., iNKT cell), or pharmaceutical composition as disclosed herein, is administered intravenously to a subject. In some embodiments, the engineered TCR, the polynucleotide, the cell (e.g., iNKT cell), or pharmaceutical composition as disclosed herein, is administered by transfusion to the subject.

[0251] An injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g., an expanded iNKT cell population (for example autologous or allogeneic to the subject to be treated) expressing a TCR. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the fusion proteins of this disclosure are conventional; appropriate formulations are well known to those of ordinary skill in the art.

[0252] An engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intrathecal, and subcutaneous routes. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In some embodiments, the engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition described herein is delivered intravenously. In some embodiments, the engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition described herein is delivered subcutaneously. In some embodiments, the engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition described herein is delivered intratumorally. In some embodiments, the engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition described herein is delivered into a tumor draining lymph node.

[0253] The amount of the engineered TCR, polynucleotide, vector, engineered cell, or pharmaceutical composition which will be effective in the treatment and/or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0254] The precise dose to be employed in a composition will also depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject's circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), whether the subject is a human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic. Usually, the subject is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0255] Those active components of the present disclosure are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

[0256] The term “pharmaceutically acceptable” defines a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

[0257] The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects.

[0258] Techniques for the formulation or preparation and application/medication of active components of the present disclosure are published in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. An appropriate application is a parenteral application, for example intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. The intravenous injection is the preferred treatment of a patient.

[0259] The present disclosure now will be illustrated by the enclosed Figures and the Examples. The following Examples further illustrate the disclosure but, of course, should not be construed as limiting its scope. EXAMPLES

Example 1. Screening of TCR Libraries

[0260] PRAME is a Cancer-Testis antigen which is intracellularly expressed in a variety of solid and blood tumors while its levels in normal tissues (excluding testis) is 100- fold less (FIGs. 1A-1D).

[0261] A PRAME synthetic peptide SLLQHLIGL (SEQ ID NO:60), previously described by Amir et al., 2011, Clin Cancer Res. 17(17):5615-5625), was used to screen TCR a and P chains due to its prevalent presentation in HLA-A*02:01 MHC molecules on both tumor cell lines and patient tissues.

[0262] TCR a and P chains were expressed as chimeric proteins, with human variable regions fused to murine constant regions, on the surface of murine cell line AK-D10R3. Murine constant regions were used to ensure proper anchoring and interaction with murine CD3 and proper triggering of murine signaling pathways. AK-D10R3 is a murine thymoma- derived mouse TCR-negative, mouse CD8-negative cell line that expresses chimeric CD8 (human CD8 a and P extracellular regions fused to the corresponding mouse CD8 a and P transmembrane and intracellular regions) and a T cell activation reporter construct comprising a minimal IL-2 promoter, which includes three NF AT binding sites, operably linked to EGFP.

[0263] Briefly, in a guided selection screening, a reference P chain (SEQ ID NO:63; Table 2) was used as a guide while the a chain of this TCR was replaced by a TCR a chain library (chains generated from either PBMCs or umbilical cord blood of 10 healthy donors).

Table 2: PRAME TCR Reference Sequences

[0264] Expression constructs for the P chain and the a chain libraries were retrovirally transduced into AK-D10R3 cells to assess pMHC tetramer binding and AK- D10R3 T cell activation in the presence of T2/pMHC+ cells. Table 3 provides the I l a chains that were identified based on their binding and activation properties when combined with the reference P chain. An alignment of the amino acid sequences for the 11 variable a chains is provided in FIG. 2, and nucleic acid and amino acid sequences for the 11 variable a chains is provided in FIGs. 3A-3E.

Table 3: TCR a Chains

[0265] Six of the a chains (684A2, 684A3, 684A6, 684A9, 684A10 and 684A11) were used in a secondary guided selection screening with the P chain being replaced by three P chain libraries. One library was comprised of beta chains generated from PBMCs or cord blood from 5 healthy donors, and two libraries were comprised of beta chains generated after randomization of the natural TCR P chain. Two sets of single strand NNK oligomers were used for the P chain CDR3 grafting. Oligomer libraries P-NNK1 and P-NNK2 (Twist, custom order) were used to randomize CDR3 residues RWDRG (SEQ ID NO:70) and WDRGG (SEQ ID NO:71) of SEQ ID NO:61 (CASARWDRGGEQYF).

[0266] Secondary screening identified 7 new P chains as listed in Table 4. An alignment of the amino acid sequences for the P chains is provided in FIG. 2, and nucleic acid and amino acid sequences for the variable P chains is provided in FIGs. 3A-3E.

Table 4: TCR chains

[0267] Flow cytometric analysis was performed to confirm binding of the different TCR combinations. AKD-10R3 cells expressing the different TCR combinations were washed twice with PBS + 2% FCS and then stained at RT for 30mins with anti-TCR-mAb- APC (1:500) and HLA-A*02:01-PRAME425-433 tetramer-PE (1:50) diluted in PBS + 2% FCS. Following staining, cells were washed twice with PBS + 2% FCS and resuspended in PBS + 2% FCS + 7- Aminoactinomycin D (1:100) to identify live cells. Cells were gated based on SSC/FSC pattern and negative staining for 7-Aminoactinomycin D to analyze only live cells. Thirty-nine TCRs were identified which specifically recognize and robustly target cells expressing the PRAME425-433 HEA-A*02.01 complex (Table 5; FIGs. 4A-4C).

Table 5: TCRs targeting PRAME425-433 HLA-A*02.01 complex

was performed on select TCRs to determine the extent by which the TCRs maintain their binding when each amino acid of the PRAME425-433 peptide is replaced with alanine or any other amino acid, respectively. The combined ALA-scan and T-SPRINT data and subsequent bioinformatics analysis has identified only few potential off-target peptides from other proteins, indicating that these novel PRAME TCRs are highly specific for the selected PRAME425-433 peptide/HLA A*02:01 protein complex.

[0269] Alanine scanning was performed for TCR4, TCR9, TCR17, TCR17, TCR23, TCR28, TCR30, TCR32, TCR34, TCR36, TCR38, TCR1, TCR6, TCR14, and TCR25. For alanine scanning, T2 cells were pulsed with PRAME peptides where each peptide residue was individually mutated to alanine (1 substitution per position) in order to assess positions that are critical for activity. Pulsed T2 cells were then co-cultured overnight with PRAME TCR expressing AKD10R3 cells and activation read out through NFAT-GFP reporter. Each assay was performed at least 3 times and the combined data is shown in FIGs. 5A-5E.

[0270] T-SPRINT profiling of PRAME engineered TCR candidates TCR4, TCR9 and TCR28 was performed to more deeply assess TCR specificity. T2 cells were pulsed with PRAME peptides where each peptide residue was individually mutated to every other possible amino acid (19 substitutions per position) giving a deeper profiling of the specificity of the TCR to each peptide position. Pulsed T2 cells were then co-cultured overnight with PRAME TCR expressing AKD10R3 cells and activation read out through an NFAT-GFP reporter. Each assay was performed at least 3 times and the combined data is shown in FIG. 6.

[0271] T2 cells were pulsed with PRAME peptides at a range of different concentrations. Pulsed T2 cells were then co-cultured overnight with PRAME TCR expressing AKD10R3 cells and activation read out through NFAT-GFP reporter. Each assay was performed at least 3 times and the combined data is shown. The PRAME TCRs enhanced through T-Rx show greater peptide sensitivity, particularly at low peptide concentrations. See FIG. 7.

Example 3: Expression of Engineered TCRs in iNKT cells

[0272] TCRs were expressed in iNKT cells via transduction using a lentivirus construct. The lentiviral constructs were engineered so that the PRAME-TCR was upstream of a P2A-BFP element, which enabled tracking of transduced cells by flow cytometric analysis based on BFP expression.

[0273] Following lentiviral transduction, iNKT cells were activated by adding humanized CD3 and CD28 agonists to the cell media. On day 14 post transduction, iNKT cells were then co-cultured with an irradiated K562-HLA-A2 cell line to specifically activate and enrich PRAME-TCR expressing iNKTs, resulting in >80% BFP+ cells for the three TCRs tested across 3 donors by day 27 post transduction. See FIG. 8. Example 4: Activity of PRAME TCR and Endogenous TCR when expressed by iNKT cells

[0274] iNKT cells expressing PRAME TCRs were enriched to >80% TCR purity and then co-cultured overnight with T2 cells pulsed with PRAME425-433 peptide or NYESO peptide (1157-165) as a negative control to assess PRAME-TCR specific cytotoxicity.

[0275] PRAME-TCR-iNKT cells were also co-cultured overnight with Clr-CDld cells either pulsed with aGalCer (alpha-Galactosylceramide) or unpulsed in order to assess iNKT-TCR specific cytotoxicity. % Dead target calculation was performed by calculating the % of CD19+ cells that stained positive using live/dead dye. Data presented is from 2 donors, 2 replicates/donor. See FIG. 9.

Example 5: Cytotoxic Capacity, Activation and Cytokine Production of PRAME TCR- iNKT cells

[0276] To compare the activation profiles of CD4+ and CD8+ T cells expressing irrelevant TCR, natural TCR or 684A2x709B5, 684A3x709B5 and 684Al lx709B5 TCRs, TCRs were lentivirally transduced into CD3+ T cells. Transduced CD3+ T cells then were sorted into CD4+ cells and CD8+ cells. CD4+ and CD8+ T cells were then co-cultured for 24hrs with T2 cells pulsed with 10^A-8M PRAME425-433 peptide. T cells were then assessed for activation by CD25 and CD69 co-expression using flow cytometry. The results demonstrated that activation in CD4+ (FIG. 10A) and CD8+ (FIG. 10B) T cells is comparable for 684A2x709B5, 684A3x709B5 and 684Al lx709B5, indicating the PRAME TCRs are CD8 independent.

[0277] To compare the activation profiles of TCR-iNKTs expressing 684A2x709B5, 684A3x709B5 and 684Al lx709B5 TCRs, TCR-iNKTs were produced by lentiviral transduction and expansion with K562-FAP feeder cells, while unmodified iNKTs were expanded with 2 rounds of Trans Act activation. TCR-iNKTs were co-cultured for 24hrs with T2 cells without peptide pulsing or pulsed for Ihr with PRAME425-433 peptide or NYESO1157- 1165 peptide at 10^A-6M. iNKT cells were then assessed for activation by CD25 and CD69 coexpression using flow cytometry. The results showed activation (%CD25+ CD69+) profiles of unmodified iNKTs or PRAME-TCR- iNKTs (684A2x709B5, 684A3x709B5 and 684A1 lx709B5) (FIG. 11). Activation was only observed in the conditions of PRAME TCR iNKTs combined with PRAME425-433 peptide. [0278] Activation profiles in response to different concentrations of PRAME425-433 peptide were compared in unmodified iNKTs or PRAME-TCR-iNKTs (684A2x709B5, 684A3x709B5 and 684Al lx709B5). TCR-iNKTs were co-cultured for 24hrs with T2 cells pulsed for Ihr with PRAME425-433 peptide at 10-fold serial dilutions from 10^A-6M to 10^A- 11M. iNKT cells were then assessed for activation by CD25 and CD69 co-expression using flow cytometry. The results demonstrated that PRAME-TCR-iNKT activation followed a dose-dependent response to PRAME425-433 peptide (FIG. 12).

[0279] Cytotoxicity of PRAME-TCR-iNKTs was assessed against tumor cells using fluorescence microscopy. PRAME-TCR-iNKTs expressing 684A2x709B5, or 684A3x709B5 TCRs were co-cultured with multiple tumor lines: OVCAR3-GFP, A375- GFP, MCF7-GFP, and A549-GFP. Target only and unmodified iNKTs were used as controls. The number of GFP+ cells was recorded using fluorescence microscopy over 72 hr. The results demonstrated that unmodified iNKT cells show some control of target cells, with additional control of OVCAR3-GFP cell growth observed for 684A2x709B5 and 684A3x709B5 TCR-iNKTs (FIGs. 13A-13B). Unmodified iNKT cells showed no control of A375-GFP cell growth, while control of A375-GFP cell growth was observed for 684A2x709B5 and 684A3x709B5 TCR-iNKTs (FIGs. 14A-14B). Unmodified iNKT cells show some control of MCF7-GFP cell growth, with minimal impact of introducing the PRAME TCR observed (FIGs.l5A-15B). Unmodified iNKT cells show some control of A549-GFP cell growth, with minimal impact of introducing the PRAME TCR observed (FIGs.l6A-16B).

[0280] Cytotoxicity of PRAME-TCR-iNKTs was assessed against tumor cell line A549-GFP-CDld, modified to express CD Id in the presence of lOOng/ml aGalCer. Percent of GFP+ area of A549-GFP-CDld cells was assessed by fluorescence microscopy over 72hrs. The results demonstrated that all iNKTs cells showed control of A549-GFP-CDld cells in the presence of aGalCer, as aGalCer presented on CD Id is a ligand for endogenous iNKT TCR- based killing. Minimal impact of introducing the PRAME TCR into iNKTs was observed indicating that expression of PRAME-TCR did not impact the function of the iNKT TCR (FIGs. 17A-17B).

[0281] Cytotoxicity of PRAME-TCR-iNKTs was assessed against human melanoma cell line A375-GFP (PRAME+ HLA-A2+ cells) in the presence of an anti PD-1 antibody balstilimab (bal) and an anti-CTLA4 antibody botensilimab (bot). Percent GFP+ area of A375-GFP cells was assessed by fluorescence microscopy over 72hrs. FIGs. 18A and 18B demonstrate that PRAME TCR control of A375-GFP growth was enhanced by addition of 50ug/mL bot and 50ug/mL bal at the start of the cytotoxicity assay, while no impact was seen on unmodified iNKTs or with isotype controls.

[0282] Cytokine release induced by PRAME-TCR-iNKT cells and bot/bal was assessed by ELISA. PBMCs were stimulated with lOng/mL SEA (Staphylococcal Enterotoxin A), which stimulates certain T cell subsets within the PBMCs. The PBMCs then were co-cultured with PRAME-TCR-iNKTs + bot/bal, PRAME-TCR- iNKTs + isotype controls, bol/bat alone or isotypes alone. PRAME-TCR-iNKT cells were added at a 1:3 ratio to PBMCs, while bot/bal or isotype controls were added at lOug/mL. Samples were incubated for 96 hr. Supernatants from co-cultured cells were collected and analyzed for IL-2 using ELISA. A synergistic increase in IL-2 secretion was observed when PRAME-TCR-iNKT cells and bot/bal are co-cultured with PBMCs (FIG. 19)

INCORPORATION BY REFERENCE

[0283] All patent and non-patent literature references cited above are incorporated herein by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. An engineered T cell receptor (TCR) that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:60, wherein the engineered TCR comprises:

(i) a variable alpha chain (Va) complementarity-determining region 1 (Va - CDR1) comprising an amino acid sequence of SEQ ID NO: 37, Va -CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and Va-CDR3 comprising an amino acid sequence SEQ ID NO:66; and

(ii) a variable beta chain (VP) VP-CDR1 comprising an amino acid sequence of SEQ ID NO: 51, VP-CDR2 comprising an amino acid sequence of SEQ ID NO: 52, and VP- CDR3 comprising an amino acid sequence of SEQ ID NO: 53.

2. The engineered TCR of claim 1, wherein the Va-CDR3 of (i) comprises an amino acid sequence of SEQ ID NO: 40.

3. The engineered TCR of claim 1, wherein the Va-CDR3 of (i) comprises an amino acid sequence of SEQ ID NO: 45.

4. The engineered TCR of claim 1, wherein the Va-CDR3 of (i) comprises an amino acid sequence of SEQ ID NO: 39.

5. The engineered TCR of any one of claims 1-4, wherein the engineered TCR comprises:

(i) a Va region comprising an amino acid sequence of SEQ ID NO: 4; and a VP region comprising an amino acid sequence of SEQ ID NO: 24; or (ii) a Va region comprising an amino acid sequence of SEQ ID NO: 12; and a VP region comprising an amino acid sequence of SEQ ID NO: 24; or

(iii) a Va region comprising an amino acid sequence of SEQ ID NO: 2; and a VP region comprising an amino acid sequence of SEQ ID NO: 24.

6. The engineered TCR of any one of claims 1-5, wherein the TCR is a full-length TCR, a soluble TCR, or a single-chain TCR.

7. The engineered TCR of any one of claims 1-6, wherein the TCR is a human TCR that specifically binds the PRAME425-433 HLA-A*02.01 complex.

8. The engineered TCR of any one of claims 1-7, wherein when the TCR is expressed on the surface of a T cell, the T cell is activated when co-cultured with a second cell displaying the peptide presented in the context of HLA-A*02.01.

9. The engineered TCR of any one of claims 1-8, wherein the TCR is conjugated to an effector moiety.

10. The engineered TCR of claim 9, wherein the effector moiety is a cytotoxic agent, cytostatic agent, toxin, radionuclide, detectable label, or binding moiety.

11. A polynucleotide encoding the engineered TCR of any one of claims 1-10.

12. The polynucleotide of claim 11, wherein the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 3, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23

13. The polynucleotide of claim 11, wherein the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 11, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23.

14. The polynucleotide of claim 11, wherein the polynucleotide comprises a Va region comprising a nucleic acid sequence of SEQ ID NO: 1, and a VP region comprising a nucleic acid sequence of SEQ ID NO: 23.

15. A vector comprising the polynucleotide of any one of claims 11-14.

16. The vector of claim 15, wherein the vector is a viral vector wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector.

17. A cell comprising the polynucleotide of any one of claims 11-14, or the vector of claim 15 or claim 16.

18. A cell expressing the engineered TCR of any one of claims 1-10.

19. The cell of claim 18, wherein the cell presents the TCR on the cell surface.

20. The cell of any one of claims 17-19, wherein the cell is a human lymphocyte.

21. The cell of any one of claims 17-20, wherein the cell is a T cell, a CD8+ T cell, a

CD4+ T cell, a natural killer T cell, or a natural killer cell.

22. The cell of any of claims 17-21, wherein the cell is an invariant natural killer T (iNKT) cell.

23. The cell of claim 22, wherein the iNKT cell expresses the engineered TCR and a native TCR.

24. The cell of claim 22 or claim 23, wherein the iNKT cell expresses an armoring molecule.

25. The cell of claim 24, wherein the armoring molecule is IL- 15.

26. A pharmaceutical composition comprising the engineered TCR of any one of claims 1-10, the polynucleotide of any one of claims 11-14, the vector of claim 15 or 16, or the cell of any one of claims 17-25, and a pharmaceutically acceptable carrier.

27. A method of producing a cell expressing an engineered TCR that binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 60, the method comprising contacting the cell with the vector of claim 15 or claim 16 under conditions that allow introduction of a vector into the cell.

28. The method of claim 27, wherein the cell is subsequently enriched by the addition of K562-HLA-A*02:01 feeder cells.

29. The method of claim 27 or claim 28, wherein the cell is an iNKT cell.

30. The method of any one of claims 27-29, wherein the vector is a lentivirus vector.

31. An engineered TCR produced by the method of any one of claims 27-30.

32. A method of inducing an immune response to PRAME, the method comprising administering to the subject an effective amount of the engineered TCR of any one of claims 1-10, the polynucleotide of any one of claims 11-14, the cell of any one of claims 18-25, or the pharmaceutical composition of claim 26.

33. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the TCR of any one of claims 1-10, the polynucleotide of any one of claims 11-14, the cell of any one of claims 18-25, or the pharmaceutical composition of claim 26.

34. A method of treating cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising iNKT cells engineered to express an engineered TCR of claims 1-10 and a pharmaceutically acceptable carrier.

35. The method of claim 34, wherein the subject does not undergo lymphodepletion prior to treatment with the pharmaceutical composition.

36. The method of any one of claims 33-35, wherein the cancer is a carcinoma or sarcoma.

37. The method of any one of claims 33-36, wherein the cancer is melanoma.

38. The method of any one of claims 33-36, wherein the cancer is lung cancer.

39. The method of any one of claims 33-36, wherein the cancer is ovarian cancer.

40. The method of any one of claims 33-36, wherein the cancer is breast cancer.

41. The method of any one of claims 33-40, wherein the TCR, polynucleotide, cell, or pharmaceutical composition is administered intravenously.