Novel T cell receptor that binds to melanoma preferential expression antigen (PRAME) and method of use

Abstract

Novel engineered T cell receptors (TCRs) that are selective and specific to melanoma preferential expression antigens (PRAMEs), cells expressing the novel TCRs, and related uses are provided. In some embodiments, the engineered TCRs are expressed by invariant natural killer T (iNKT) cells.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 579,799, filed on 30 August 2023 under Section 119(e) of the U.S. Patent Act, entitled “NOVEL T CELL RECEPTORS THAT BIND TO PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA (PRAME) AND METHODS OF USE THEREOF,” the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence lists The contents of the electronic sequence listing (A132770008WO00-SEQ-ROS.NRL; size: 75 bytes; and creation date: August 23, 2023) are incorporated herein by reference in their entirety.

[0003] This disclosure relates to a novel engineered T cell receptor (TCR) that is selective and specific to melanoma preferential expression antigen (PRAME), cells expressing the novel engineered TCR, and a method of use. [Background technology]

[0004] explanation The melanoma preferential expression antigen (PRAME), also known as CT130, is a cancer-testis antigen (CTA). High levels of PRAME mRNA have been observed in various cancer indications and leukemias, including melanoma, non-small cell lung cancer, breast cancer, multiple sarcoma subtypes, and epithelial ovarian cancer. This is characterized by limited and weak expression in normal somatic tissues such as the testes. (Al-Khadairi G., Decock J. Cancer testis antigens and immunotherapy: Where do we stand in the targeting of PRAME?, Cancers. 2019;11:984). High expression of PRAME in tumors has been associated with poor prognosis in some solid tumors. Several studies suggest that PRAME can induce cell proliferation, reduce sensitivity to cytotoxic drugs, and inhibit apoptosis in various cancers. Because PRAME is an intracellular antigen, it cannot be targeted by conventional CAR T cells, which are limited to cell surface antigens.

[0005] Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells, are a unique population of T cells that express a specific TCR containing the invariant Vα24-Jα18α chain gene and a limited number of β 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-αβ T cells demonstrates preferential use of several Vβ genes and an invariant TCR α 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). iNKT cells are rare in the human blood pool, accounting for only 0.01–1% of peripheral blood mononuclear cells (PBMCs). However, when activated, they exhibit potent cytotoxic activity and rapidly produce a range of cytokines and chemokines, making them important immunomodulatory cells. These cytokines include interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-3, IL-4, IL-10, IL-13, IL-17, IL-21, and transforming growth factor-β.(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. CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system. Curr Opin Immunol. 2008;20:358-68).

[0006] Unlike conventional T cells that respond to peptide antigens presented in the context of MHC class I or II, iNKT cells respond to and are activated by glycolipid antigens presented by CD1d, an MHC class Ib-related molecule. (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). CD1d is a highly conserved, non-polymorphic molecule expressed by many hematopoietic cells (dendritic cells, macrophages, and B cells) that present lipid antigens, rather than peptides, to iNKT cells. This CD1d restriction distinguishes and defines iNKT cells from all other T cell populations. [Overview of the project]

[0007] This disclosure is PRAME 425~433This invention relates to engineered T cell receptors (TCRs) that bind to the HLA-A*02.01 complex. In some embodiments, the engineered TCR comprises an alpha chain containing a variable alpha (Vα) region and a beta chain containing a variable beta (Vβ) region. In some embodiments, the Vα region comprises a complementation-determining region 3 (CDR3) containing the amino acid sequence described in any one of SEQ ID NOs. 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the Vα region of the engineered TCR comprises a CDR1 containing the amino acid sequence described in SEQ ID NOs. 37 or 41 and a CDR2 containing the amino acid sequence described in SEQ ID NOs. 38. For example, the present disclosure provides an engineered TCR comprising Vα-CDR1 having the amino acid sequence described in SEQ ID NO: 37 or 41, Vα-CDR2 having the amino acid sequence described in SEQ ID NO: 38, and Vα-CDR3 having the amino acid sequence described in SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0008] In some embodiments, PRAME 425~433 The engineered TCR that binds to the peptide includes a Vβ region comprising a CDR3 containing the amino acid sequence described in any of SEQ ID NOs. 53, 54, 55, 56, 57, 58, or 59. In some embodiments, the Vβ region of the engineered TCR includes a CDR1 containing the amino acid sequence described in SEQ ID NOs. 51, and a CDR2 containing the amino acid sequence described in SEQ ID NOs. 52. For example, the present disclosure provides an engineered TCR comprising a Vβ-CDR1 having the amino acid sequence described in SEQ ID NOs. 51, a Vβ-CDR2 containing the amino acid sequence described in SEQ ID NOs. 52, and a Vβ-CDR3 containing the amino acid sequence described in SEQ ID NOs. 53, 54, 55, 56, 57, 58, or 59.

[0009] In some embodiments, the manipulated TCR comprises a Vα region having a Vα-CDR3 containing the amino acid sequence described in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50; and a Vβ region having a VβCDR3 containing the amino acid sequence described in any of SEQ ID NOs: 53, 54, 55, 56, 57, 58, or 59.

[0010] In some embodiments, the manipulated TCR comprises a Vα region having a Vα-CDR1 containing the amino acid sequence SSX1X2PX3 (SEQ ID NO: 67), where X1 is V or Y, X2 is P or S, and X3 is Y or S.

[0011] In some embodiments, the manipulated TCR includes a Vα region having a Vα-CDR2 containing the amino acid sequence described as SEQ ID NO: 38. In some embodiments, the manipulated TCR includes a Vα region having a Vα-CDR3 containing the amino acid sequence described below: X1VX2FSGGYNKLI (SEQ ID NO: 66), where X1 is A or V and X2 is S or T.

[0012] In some embodiments, the manipulated TCR comprises a Vα region having a Vα-CDR3 containing the following amino acid sequence: X1VX2X3SGGYNKLI (SEQ ID NO: 68), where X1 is A or V, X2 is G or N or S or T or P or V, and X3 is F or Y.

[0013] In some embodiments, the manipulated TCR includes a Vα region having Vα-CDR1 containing the amino acid sequence described as SEQ ID NO: 67, Vα-CDR2 containing the amino acid sequence described as SEQ ID NO: 38, and Vα-CDR3 containing the amino acid sequence described as SEQ ID NO: 66.

[0014] In some embodiments, the manipulated TCR includes a Vβ region having a Vβ-CDR1 containing the amino acid sequence described as SEQ ID NO: 51.

[0015] In some embodiments, the manipulated TCR includes a Vβ region having a Vβ-CDR2 containing the amino acid sequence described as SEQ ID NO: 52.

[0016] In some embodiments, the engineered TCR comprises a Vβ region having a Vβ-CDR3 comprising the amino acid sequence: ASX1X2WDX3X4X5X6QY (SEQ ID NO: 69), wherein X1 is A or S, X2 is R, P or A, X3 is R, S, M, N or T, X4 is G, R, A, W or V, X5 is E, Q, D or G, and X6 is E or T.

[0017] In some embodiments, the engineered TCR comprises a Vβ region having a Vβ-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a Vβ-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a Vβ-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0018] In some embodiments, the engineered TCR comprises a Vα region having a Vα-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a Vα-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a Vα-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66; and comprises a Vβ region having a Vβ-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a Vβ-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a Vβ-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0019] In some embodiments, the engineered TCR comprises a Vα region having a Vα-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, a Vα-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and a Vα-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 66; and the engineered TCR comprises a Vβ region having a Vβ-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 51, a Vβ-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 52, and a Vβ-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 69.

[0020] In some embodiments, the engineered TCR comprises a Vα region having Vα-CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 67, Vα-CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 38, and Vα-CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 68; and comprises a Vβ region having Vβ-CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 51, Vβ-CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 52, and Vβ-CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 69.

[0021] In some embodiments, the engineered TCR comprises a Vα region having Vα-CDR1 consisting of the amino acid sequence set forth as SEQ ID NO: 67, Vα-CDR2 consisting of the amino acid sequence set forth as SEQ ID NO: 38, and Vα-CDR3 consisting of the amino acid sequence set forth as SEQ ID NO: 68; and the engineered TCR comprises a Vβ region having Vβ-CDR1 consisting of the amino acid sequence set forth as SEQ ID NO: 51, Vβ-CDR2 consisting of the amino acid sequence set forth as SEQ ID NO: 52, and Vβ-CDR3 consisting of the amino acid sequence set forth as SEQ ID NO: 69.

[0022] In some embodiments, the engineered TCR comprises variable alpha chain (Vα) complementarity determining region 1 (Vα-CDR1) comprising the amino acid sequence of SEQ ID NO: 37, Vα-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and Vα-CDR3 comprising the amino acid sequence of SEQ ID NO: 66; as well as variable beta chain (Vβ) Vβ-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, Vβ-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and Vβ-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0023] In some embodiments, the engineered TCR comprises Vα-CDR1 comprising the amino acid sequence of SEQ ID NO: 37, Vα-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and Vα-CDR3 comprising the amino acid sequence of SEQ ID NO: 40; as well as Vβ-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, Vβ-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and Vβ-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0024] In some embodiments, the manipulated TCRs include Vα-CDR1 containing the amino acid sequence of SEQ ID NO: 37, Vα-CDR2 containing the amino acid sequence of SEQ ID NO: 38, and Vα-CDR3 containing the amino acid sequence of SEQ ID NO: 45; as well as Vβ-CDR1 containing the amino acid sequence of SEQ ID NO: 51, Vβ-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and Vβ-CDR3 containing the amino acid sequence of SEQ ID NO: 53.

[0025] In some embodiments, the manipulated TCRs include Vα-CDR1 containing the amino acid sequence of SEQ ID NO: 37, Vα-CDR2 containing the amino acid sequence of SEQ ID NO: 38, and Vα-CDR3 containing the amino acid sequence of SEQ ID NO: 39; as well as Vβ-CDR1 containing the amino acid sequence of SEQ ID NO: 51, Vβ-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and Vβ-CDR3 containing the amino acid sequence of SEQ ID NO: 53.

[0026] In some embodiments, (a) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (b) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively. (c) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 59, respectively; (d) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 5 (e) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 54, respectively; (f) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 55 (g) CDR1, CDR2, and CDR3 in the Vα region each contain SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each contain SEQ ID NOs. 51, 52, and 57, respectively; (h) CDR1, CDR2, and CDR3 in the Vα region each contain SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each contain SEQ ID NOs. 51, 52, and 59, respectively;(i) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (j) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (k) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 41, 38, and 42, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (l) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 41, 38, and 42, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (m) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 41, 38, and 42, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (n) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (o) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (p) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 59, respectively;(q) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (r) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (s) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (t) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 56, respectively; (u) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (v) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 58, respectively; (w) CDR1, CDR2, and CDR3 in the Vα region include sequence numbers 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include sequence numbers 51, 52, and 53, respectively; (x) CDR1, CDR2, and CDR3 in the Vα region include sequence numbers 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include sequence numbers 51, 52, and 54, respectively;(y) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (z) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 56, respectively; ( aa) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (bb) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; ( (cc) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (dd) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 46, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (ee) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 46, respectively; CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (ff) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 47, respectively; CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively;(gg) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 47, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 54, respectively; (hh) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 48, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 53, respectively; (ii) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 48, respectively, and CDR1, CDR2, and CDR3 in the Vβ region each include sequence numbers 51, 52, and 54, respectively; (jj) CDR1, CDR2, and CDR3 in the Vα region each include sequence numbers 37, 38, and 49 , CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (kk) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 49, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (ll) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 50, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; or (hh) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 50, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively.

[0027] In some embodiments, the manipulated TCR includes a Vα region comprising the amino acid sequence described 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 Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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.

[0028] In some embodiments, the manipulated TCR includes a Vβ region comprising the amino acid sequence described 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 Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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.

[0029] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0030] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32.

[0031] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36.

[0032] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0033] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0034] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0035] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32.

[0036] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36.

[0037] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0038] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0039] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0040] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0041] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0042] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0043] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32.

[0044] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36.

[0045] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0046] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0047] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0048] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 30.

[0049] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32.

[0050] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 34.

[0051] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0052] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region of the TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0053] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28.

[0054] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 30.

[0055] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32.

[0056] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0057] In some embodiments, the Vα region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region of the manipulated TCR includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0058] This disclosure provides an engineered TCR in which the Vα region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 14, and the Vβ region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0059] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 14, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0060] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 16, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0061] This disclosure provides an engineered TCR having a Vα region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 16, and a Vβ region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0062] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 18, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 18, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0063] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 18, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 18, and the Vβ region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0064] This disclosure provides an engineered TCR having a Vα region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 20, and a Vβ region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0065] In some embodiments, the Vα region of the manipulated TCR includes the amino acid sequence described in SEQ ID NO: 20, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 20, and the Vβ region of the TCR includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0066] This disclosure provides an engineered TCR having a Vα region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 22, and a Vβ region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24.

[0067] In some embodiments, the manipulated TCR includes a Vα region containing an amino acid sequence identical to the amino acid sequence described in SEQ ID NO: 22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 22, and a Vβ region containing an amino acid sequence identical to the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26.

[0068] In some embodiments, the manipulated TCR includes a Vα region containing the amino acid sequence of SEQ ID NO: 4, and a Vβ region containing the amino acid sequence of SEQ ID NO: 24.

[0069] In some embodiments, the manipulated TCR includes a Vα region containing the amino acid sequence of SEQ ID NO: 12; and a Vβ region containing the amino acid sequence of SEQ ID NO: 24.

[0070] In some embodiments, the manipulated TCR includes a Vα region containing the amino acid sequence of SEQ ID NO: 2, and a Vβ region containing the amino acid sequence of SEQ ID NO: 24.

[0071] In some embodiments, the manipulated TCR is: (a) a Vα region containing the amino acid sequence described in SEQ ID NO: 2 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (b) a Vα region containing the amino acid sequence described in SEQ ID NO: 2 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 32; (c) a Vα region containing the amino acid sequence described in SEQ ID NO: 2 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 36; (d) a Vα region containing the amino acid sequence described in SEQ ID NO: 2 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (e) a Vα region containing the amino acid sequence described in SEQ ID NO: 2 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (f) a Vα region containing the amino acid sequence described in SEQ ID NO: 4 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (g) a Vα region containing the amino acid sequence described in SEQ ID NO: 4 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 32; (h) a Vα region containing the amino acid sequence described in SEQ ID NO: 4 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 36; (i) a Vα region containing the amino acid sequence described in SEQ ID NO: 4 and the amino acid sequence described in SEQ ID NO: 24 (j) Vβ region containing the amino acid sequence described in SEQ ID NO: 4 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (k) Vα region containing the amino acid sequence described in SEQ ID NO: 6 and Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (l) Vα region containing the amino acid sequence described in SEQ ID NO: 6 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (m) Vα region containing the amino acid sequence described in SEQ ID NO: 6 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (n) Vα region containing the amino acid sequence described in SEQ ID NO: 8 and Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (o) Vα region containing the amino acid sequence described in SEQ ID NO: 8 and Vβ region containing the amino acid sequence described in SEQ ID NO: 32; (p) Vα region containing the amino acid sequence described in SEQ ID NO: 8 and Vβ region containing the amino acid sequence described in SEQ ID NO: 36; (q) Vα region containing the amino acid sequence described in SEQ ID NO: 8 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (r) Vα region containing the amino acid sequence described in SEQ ID NO: 8 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26;(s) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (t) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 30; (u) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 32; (v) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 34; (w) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (x) Vα region containing the amino acid sequence described in SEQ ID NO: 10 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (y) Vα region containing the amino acid sequence described in SEQ ID NO: 12 and Vβ region containing the amino acid sequence described in SEQ ID NO: 28; (z) Vα region containing the amino acid sequence described in SEQ ID NO: 12 and Vβ region containing the amino acid sequence described in SEQ ID NO: 30; (aa) Vα region containing the amino acid sequence described in SEQ ID NO: 12 and Vβ region containing the amino acid sequence described in SEQ ID NO: 32; (bb) (cc) Vα region containing the amino acid sequence described in SEQ ID NO: 12 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (dd) Vα region containing the amino acid sequence described in SEQ ID NO: 14 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (ee) Vα region containing the amino acid sequence described in SEQ ID NO: 14 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (ff) Vα region containing the amino acid sequence described in SEQ ID NO: 16 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (gg) Vα region containing the amino acid sequence described in SEQ ID NO: 16 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (hh) Vα region containing the amino acid sequence described in SEQ ID NO: 18 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24; (ii) Vα region containing the amino acid sequence described in SEQ ID NO: 18 and Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (jj) Vα region containing the amino acid sequence described in SEQ ID NO: 20 and Vβ region containing the amino acid sequence described in SEQ ID NO: 24;(kk) A Vα region containing the amino acid sequence described in SEQ ID NO: 20 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 26; (ll) A Vα region containing the amino acid sequence described in SEQ ID NO: 22 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 24; and (mm) A Vα region containing the amino acid sequence described in SEQ ID NO: 22 and a Vβ region containing the amino acid sequence described in SEQ ID NO: 26, selected from the group comprising these two regions.

[0072] In some embodiments, the manipulated TCR does not include either SEQ ID NO: 63 or SEQ ID NO: 64.

[0073] In some embodiments, the manipulated TCR is a full-length TCR. In some embodiments, the manipulated TCR is a soluble TCR. In some embodiments, the manipulated TCR is a single-chain TCR.

[0074] In some embodiments, TCR is PRAME 425~433 This is a human TCR that specifically binds to the HLA-A*02.01 complex.

[0075] In some embodiments, the manipulated TCR is expressed on the surface of T cells. In some embodiments, T cells that present the TCR on their surface are PRAME 425~433 It can be activated in the presence of HLA-A*02.01. In some embodiments, the manipulated TCR is expressed on human iNKT cells that also express endogenous TCRs.

[0076] In some embodiments, the manipulated TCR is conjugated to an effector portion. In some embodiments, the effector portion is a cytotoxic agent, a cell growth inhibitor, a toxin, a radionuclide, a detectable label, or a binding portion.

[0077] This disclosure provides polynucleotides encoding manipulated TCRs as described herein. In some embodiments, the polynucleotides include nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35, or nucleic acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequences from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35. In some embodiments, the Vα region of the manipulated TCR is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, or a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21. The Vβ region of the TCR is encoded by a sequence, and 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 by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 23, 25, 27, 29, 31, 33, or 35.

[0078] In some embodiments, the polynucleotide includes a Vα region containing the nucleic acid sequence of SEQ ID NO: 3 and a Vβ region containing the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide includes a Vα region containing the nucleic acid sequence of SEQ ID NO: 11 and a Vβ region containing the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide includes a Vα region containing the nucleic acid sequence of SEQ ID NO: 1 and a Vβ region containing the nucleic acid sequence of SEQ ID NO: 23.

[0079] In some embodiments, the Disclosure provides vectors comprising polynucleotides as described herein. For example, the Disclosure provides vectors comprising polynucleotides having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35, or a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1, 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 lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and baculovirus vectors.

[0080] In some embodiments, cells present the engineered TCR described herein on their cell surface. In some embodiments, the cells are human lymphocytes. In some embodiments, the cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, and natural killer cells. In some embodiments, invariant natural killer T (iNKT) cells express the engineered TCR.

[0081] This disclosure provides iNKT cells expressing the engineered TCR described herein. iNKT cells can be isolated from PBMCs and amplified using methods known in the art. iNKT cells transfected with the polynucleotide encoding the TCR described herein express the engineered TCR (i.e., PRAME 425~433 The cells express both a peptide-targeting TCR and an iNKT endogenous TCR (i.e., a TCR that targets glycolipids presented by CD1d). In some embodiments, iNKT cells express both the engineered TCR and the native TCR.

[0082] In some embodiments, iNKT cells express an armoring molecule. The armoring molecule can be selected from the following 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-PD1 antibodies, CD47-targeting nanobodies, or bispecific T cell engagers (BiTEs). In some embodiments, iNKT cells express an armoring molecule, which is IL-15.

[0083] 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), in any case together with a pharmaceutically acceptable carrier. For example, the Disclosure provides a pharmaceutical composition comprising an iNKT cell expressing an engineered TCR having a Vα region comprising Vα-CDR1 comprising the amino acid sequence described in SEQ ID NO: 37 or 41, Vα-CDR2 comprising the amino acid sequence described in SEQ ID NO: 38, and Vα-CDR3 comprising the amino acid sequence described in SEQ ID NO: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and a Vβ region comprising Vβ-CDR1 comprising the amino acid sequence described in SEQ ID NO: 51, Vβ-CDR2 comprising the amino acid sequence described in SEQ ID NO: 52, and Vβ-CDR3 comprising the amino acid sequence described in SEQ ID NO: 53, 54, 55, 56, 57, 58, or 59, and a pharmaceutically acceptable carrier.

[0084] In some embodiments, PRAME 425~433A method is provided for producing an engineered TCR that binds to a peptide (SEQ ID NO: 60), comprising transfecting isolated human iNKT cells with the engineered TCR. For example, the disclosure provides transfecting isolated human iNKT cells with a TCR having a Vα region comprising Vα-CDR1 comprising the amino acid sequence described in SEQ ID NO: 37 or 41, Vα-CDR2 comprising the amino acid sequence described in SEQ ID NO: 38, and Vα-CDR3 comprising the amino acid sequence described in SEQ ID NO: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and a Vβ region having Vβ-CDR1 comprising the amino acid sequence described in SEQ ID NO: 51, Vβ-CDR2 comprising the amino acid sequence described in SEQ ID NO: 52, and Vβ-CDR3 comprising the amino acid sequence described in SEQ ID NO: 53, 54, 55, 56, 57, 58, or 59. In some embodiments, isolated human iNKT cells are transfected using a lentiviral vector to express the engineered TCR. In some embodiments, human iNKT cells are transfected using a lentiviral vector to express an engineered TCR, and the human iNKT cells are then enriched by the addition of K562-HLA-A*02:01 feeder cells.

[0085] This disclosure provides a method for producing cells expressing an engineered TCR that binds to a peptide comprising the amino acid sequence described in SEQ ID NO: 60, the method comprising contacting the cells with a vector under conditions that allow for the introduction of the vector into the cells. This disclosure provides the use of a viral vector, such as a lentiviral vector, for introducing a polynucleotide encoding the TCR into cells (e.g., isolated human iNKT cells).

[0086] In some embodiments, the Disclosure provides a method for inducing an immune response to PRAME, comprising administering an effective amount of an engineered TCR, a polynucleotide, a cell (e.g., an iNKT cell presenting a TCR), or a pharmaceutical composition disclosed herein to the target. In some embodiments, PRAME is PRAME presented by HLA-A*02:01. 425~433 That is the case.

[0087] In some embodiments, the Disclosure provides a method for treating cancer in a subject, comprising administering an effective amount of a TCR, a polynucleotide, a cell, or a pharmaceutical composition as described herein to the subject.

[0088] For example, this disclosure provides a method for treating cancer in a subject, comprising administering an effective amount of allogeneic iNKT cells expressing the engineered TCR described herein to the subject. Such iNKT cells directly kill tumor cells presenting PRAME and also express activated natural killer (NK) receptors that recognize stress ligands on tumor cells. Furthermore, allogeneic iNKT cells can suppress immunosuppressive tumor myeloid cells via their endogenous TCR.

[0089] In some embodiments, allogeneic iNKT cells are administered to the subject without prior lymphocyte depletion.

[0090] In some embodiments, the Disclosure provides a method for treating cancer in a subject, comprising administering an effective amount of a pharmaceutical composition comprising isolated human iNKT cells presenting the TCRs disclosed herein and a pharmaceutically acceptable carrier to the subject. 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, squamous cell lung carcinoma, melanoma, multiple myeloma, ovarian cancer, renal papillary cell carcinoma, 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.

[0091] In some embodiments, the TCRs, polynucleotides, cells, or pharmaceutical compositions described herein are administered intravenously to a subject. [Brief explanation of the drawing]

[0092] [Figure 1] Figures 1A–1D illustrate PRAME expression in cancer tissue, healthy adult tissue, immune cells, and healthy fetal tissue using publicly available datasets. [Figure 2] Figure 2 shows the alignment of variable α and variable β chains identified as selective and specific to PRAME425-433 peptides. [Figure 3] Figures 3A-3E show the nucleic acid and amino acid sequences of the variable α chain (Figures 3A-3C) and variable β chain (Figures 3D-3E) of the manipulated PRAME TCR. [Figure 4] Figures 4A-4C show the binding of various combinations of the variable α and β chains of the TCR. The plots are presented with TCR expression (Y axis) and the binding of PRAME425-433 tetramers (X axis). [Figure 5]Figures 5A–5E show the results of alanine scanning of selected manipulated TCR candidates. Each assay was performed at least three times, and the combined data is shown. [Figure 6] Figure 6 shows the T-SPRINT profiling results for the selected manipulated TCR candidates (684A2×709B5, 684A3×709B5, and 684A11×709B5). [Figure 7] Figure 7 shows the sensitivity of the manipulated PRAME TCRs (684A2×709B5, 684A3×709B5, and 684A11×709B5) at low peptide concentrations. Each assay was performed at least three times, and the combined data is shown. [Figure 8] Figure 8 shows the successful transduction and amplification of iNKT cells transduced to express the manipulated TCR4 (684A2×709B5), TCR9 (684A3×709B5), and TCR28 (684A11×709B5). All three TCRs were tested in all three donors by day 27 post-transduction. (A representative image from one donor is shown). [Figure 9] Figure 9 shows the specific cytotoxicity and the effect of endogenous iNKT TCRs on cytotoxicity by manipulated TCRs (684A2×709B5, 684A3×709B5, and 684A11×709B5) expressed from iNKT cells. The cytotoxicity target percentage was calculated by calculating the percentage of CD19+ cells positively stained using viable / dead dyes. The data presented are from two donors, with two sets per donor. [Figure 10] Figures 10A–10B show flow cytometry analysis of CD4+ T cell (Figure 20A) and CD8+ T cell (Figure 10B) activation, indicated by co-expression of surface CD25 and CD69. Cells were transduced with unrelated TCRs, native TCRs, or 684A2×709B5, 684A3×709B5, and 684A11×709B5 TCRs. [Figure 11]Figure 11 shows flow cytometry analysis of T cell activation, indicated by co-expression of surface CD25 and CD69 in unmodified iNKT or PRAME-TCR-iNKT (684A2×709B5, 684A3×709B5, and 684A11×709B5). TCR-iNKT was co-cultured with T2 cells either without peptide pulse ("no peptide" group) or pulsed with PRAME425-433 peptide ("PRAME" group) or NYESO1157-1165 peptide ("NYESO" group). [Figure 12] Figure 12 shows flow cytometry analysis of surface CD25 and CD69 co-expression in unmodified iNKT or PRAME-TCR-iNKT (684A2×709B5, 684A3×709B5, and 684A11×709B5) iNKT in response to various concentrations (M) of PRAME425-433 peptides. [Figure 13] Figures 13A and 13B show the killing of OVCAR3-GFP cells by PRAME-TCR-iNKT. Figure 13A shows the time-course quantification of OVCAR3-GFP cell death in four groups: target only, unmodified iNKT, 684A2×709B5 PRAME-TCR-iNKT, and 684A3×709B5 PRAME-TCR-iNKT. The percentage of GFP+ area in OVCAR3-GFP cells was assessed by fluorescence microscopy over 72 hours. Figure 13B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 14] Figures 14A and 14B show the killing of A375-GFP cells by PRAME-TCR-iNKT. Figure 14A shows the time-course quantification of A375-GFP cell death in four groups: target only, unmodified iNKT, 684A2×709B5 PRAME-TCR-iNKT, and 684A3×709B5 PRAME-TCR-iNKT. The percentage of GFP+ area in A375-GFP cells was assessed by fluorescence microscopy over 72 hours. Figure 14B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 15]Figures 15A and 15B show the killing of MCF7-GFP cells by PRAME-TCR-iNKT. Figure 15A shows the time-course quantification of MCF7-GFP cell death in four groups: target only, unmodified iNKT, 684A2×709B5 PRAME-TCR-iNKT, and 684A3×709B5 PRAME-TCR-iNKT. The percentage of GFP+ area in MCF7-GFP cells was assessed by fluorescence microscopy over 72 hours. Figure 15B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 16] Figures 16A and 16B show the killing of A549-GFP cells by PRAME-TCR-iNKT. Figure 16A shows the time-course quantification of MCF7-GFP cell death in four groups: target only, unmodified iNKT, 684A2×709B5 PRAME-TCR-iNKT, and 684A3×709B5 PRAME-TCR-iNKT. The percentage of GFP+ area in A549-GFP cells was assessed by fluorescence microscopy over 72 hours. Figure 16B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 17] Figures 17A and 17B show the killing of A549-GFP-CD1d cells by PRAME-TCR-iNKT. Figure 17A shows the time-course quantification of A549-GFP-CD1d cell death in four groups: target only, unmodified iNKT, 684A2×709B5 PRAME-TCR-iNKT, and 684A3×709B5 PRAME-TCR-iNKT. The percentage of GFP+ area in A549-GFP-CD1d cells was evaluated by fluorescence microscopy over 72 hours. Figure 17B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 18]Figures 18A and 18B show the killing of A375-GFP cells by PRAME-TCR-iNKT under the influence of the anti-PD-1 antibody valstilimab (Bal) and the anti-CTLA4 antibody botensilimab (Bot). Figure 18A shows the time-course quantification of A375-GFP cell death in seven groups: unmodified iNKT+Bot / Bal, unmodified iNKT+ isotype, 684A2×709B5+Bot / Bal, 684A2×709B5+ isotype, 684A3×709B5+Bot / Bal, and 684A3×709B5+ isotype. The percentage of GFP+ area in A375-GFP cells was evaluated by fluorescence microscopy over 72 hours. Figure 18B shows the percentage change in GFP+ area at 72 hours (normalized to t=0). [Figure 19] Figure 19 shows IL-2 concentrations (pg / mL) measured by ELISA in PRAME-TCR-iNKT cells and bot / bal antibodies co-cultured with PBMCs stimulated with 10 ng / ml SEA (Staphylococcal enterotoxin A). Isotype alone and bot / bal alone were used as controls. [Modes for carrying out the invention]

[0093] This disclosure is PRAME 425~433 This invention relates to a novel engineered T cell receptor (TCR) targeting (SLLQHLIGL; SEQ ID NO: 60), novel allogeneic iNKT cells engineered to present the TCR, and methods of use thereof for the treatment of various cancers related to PRAME expression. iNKT cells modulate both innate and adaptive immunity. The iNKT cells described herein can express both PRAME-engineered and endogenous TCRs and can effectively modulate the tumor microenvironment to promote tumor killing.

[0094] As used herein, "PRAME" refers to the melanoma-preferential expression antigen. PRAME is a cancer-testicular antigen (CTA), also known as CT130.

[0095] As used herein, the terms “about” and “approximately” indicate, when used to modify a number or range of numbers, that a deviation 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) of a value or range remains within the intended meaning of the stated value or range.

[0096] As used herein, the terms “T cell receptor” and “TCR” are interchangeable and refer to molecules containing a CDR or variable region derived from an αβ T cell receptor or a γδ T cell receptor. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing a variable region of TCR linked by a mobile linker, TCR chains linked by manipulated disulfide bonds, a single TCR variable domain, a single peptide-MHC specific TCR, a multispecific TCR (including a bispecific TCR), a TCR fusion, a TCR containing a costimulatory region, a human TCR, a humanized TCR, a chimeric TCR, a recombinant TCR, and a synthetic TCR. In some embodiments, the TCR is a full-length TCR containing a full-length α chain and a full-length β chain. In some embodiments, the TCR is a soluble TCR lacking a transmembrane and / or cytoplasmic region. In some embodiments, the TCR is a single-chain TCR (scTCR) containing Vα and Vβ linked by a peptide linker, for example, an scTCR having the structure described in PCT Publications WO2003 / 020763, WO2004 / 033685, or WO2011 / 044186, each of which is incorporated herein by reference in whole. In some embodiments, the TCR includes a transmembrane region. In some embodiments, the TCR includes a co-stimulatory signaling region.

[0097] As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of first and second polypeptide chains, each comprising 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 α, β, γ, or δ TCR chains. In some embodiments, the full-length TCR comprises one or two modified TCR chains, e.g., a chimeric TCR chain and / or a TCR chain comprising one or more amino acid substitutions, insertions, or deletions compared to the unmodified TCR chain. In some embodiments, the full-length TCR comprises a mature full-length TCR α chain and a mature full-length TCR β chain. In some embodiments, the full-length TCR comprises a mature full-length TCR γ chain and a mature full-length TCR δ chain.

[0098] As used herein, the term “TCR variable region” refers to a portion of a mature TCR polypeptide chain (e.g., the TCR α or β chain) that is not encoded by the TRAC gene of the TCR α chain, either the TRBC1 or TRBC2 gene of the TCR β chain, the TRDC gene of the TCR δ chain, or either the TRGC1 or TRGC2 gene of the TCR γ chain. In some embodiments, the TCR variable region of the TCR α chain encompasses all amino acids of the mature TCR α chain polypeptide encoded by the TRAV and / or TRAJ genes, and the TCR variable region of the TCR β chain encompasses all amino acids of the mature TCR β chain polypeptide encoded by the TRBV, TRBD and / or TRBJ genes (see, for example, T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated herein by reference in its entirety). The TCR variable region generally includes framework regions (FR) 1, 2, 3, and 4, as well as complementarity determination regions (CDR) 1, 2, and 3.

[0099] As used herein, the terms "α chain variable region", "α variable region" and "Vα" are used interchangeably and refer to the variable region of a TCR α chain.

[0100] As used herein, the terms "β chain variable region", "β variable region" and "Vβ" are used interchangeably and refer to the variable region of a TCR β chain.

[0101] "Engineered TCR" refers to PRAME 425~433 that is capable of specifically binding to, and refers to the novel TCR disclosed herein.

[0102] As used herein in the context of TCRs, the terms “CDR” or “complementarity-determining region” mean discontinuous antigen-binding sites found within the variable region of the TCR chain (e.g., the α-chain or β-chain). These regions are 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(1):55-77, and Kabat et al., (1991) Sequences of protein of immunological interest, each of which is incorporated herein by reference in its entirety. In some embodiments, the CDR is determined according to the IMGT numbering system described in Lefranc (1999) above. In some embodiments, the CDR is defined according to the Kabat numbering system described above. In some embodiments, the CDR is defined empirically, for example, based on structural analysis of the interaction between the TCR and the cognitive antigen (e.g., a peptide or peptide-MHC complex). In some embodiments, the α-chain and β-chain CDRs of the TCR are defined according to different rules (e.g., according to the Kabat or IMGT numbering system, or empirically based on structural analysis).

[0103] "Vα-CDR1", "Vα-CDR2", and "Vα-CDR3" refer to CDR1-3 in the α variable region. "Vβ-CDR1", "Vβ-CDR2", and "Vβ-CDR3" refer to CDR1-3 in the β variable region.

[0104] As used herein, the term “framework amino acid residue” refers to an amino acid within the framework region of a TCR chain (e.g., the α-chain or β-chain). As used herein, the term “framework region” or “FR” includes amino acid residues that are part of the TCR variable region but not part of the CDR.

[0105] As used herein, the term “constant region” with respect to the TCR refers to the portion of the TCR encoded by either the TRAC gene (for the TCR α chain), either the TRBC1 or TRBC2 gene (for the TCR β chain), either the TRDC gene (for the TCR δ chain), or either the TRGC1 or TRGC2 gene (for the TCR γ chain), and which, as appropriate, lacks all or part of the transmembrane region and / or all or part of the cytoplasmic region. In some embodiments, the TCR constant region lacks both the transmembrane and cytoplasmic regions. The TCR constant region also does not contain any amino acids encoded by any of the TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ genes (see, for example, T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated herein by reference in its entirety).

[0106] As used herein, the terms “major histocompatibility complex” and “MHC” are used interchangeably and refer to MHC class I molecules and / or MHC class II molecules.

[0107] As used herein, the term "MHC class I" refers to a dimer of an MHC class Iα chain and a β2 microglobulin chain, and the term "MHC class II" refers to a dimer of an MHC class IIα chain and an MHC class IIβ chain.

[0108] As used herein, the term “peptide-MHC complex” refers to an MHC molecule (MHC class I or MHC class II) having a peptide bound to a peptide-binding pocket of an MHC recognized in the art. In some embodiments, the MHC molecule is a membrane-bound protein expressed on the cell surface. In some embodiments, the MHC molecule is a soluble protein lacking a transmembrane or cytoplasmic domain.

[0109] As used herein, the term “extracellular” with respect to TCRs refers to one or more portions of recombinant transmembrane proteins located outside the cell.

[0110] As used herein, the term “transmembrane” with respect to a TCR chain refers to one or more portions of a TCR chain embedded in the plasma membrane of a cell.

[0111] As used herein, the term “cytoplasm” with respect to a TCR chain refers to one or more portions of a TCR chain located in the cytoplasm of a cell.

[0112] As used herein, the term “co-stimulus signaling region” refers to the intracellular portion of a co-stimulus molecule that mediates intracellular signaling events.

[0113] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., TCR) and its binding partner (e.g., peptide-MHC complex). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., TCR and peptide-MHC complex). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (KD). Affinity can be measured and / or expressed in several ways known in the art, including but not limited to the equilibrium dissociation constant (KD) and equilibrium association constant (KA). KD is calculated from the quotient of koff / kon, and KA is calculated from the quotient of kon / koff, where kon refers, for example, the association rate constant of TCR to the peptide-MHC complex, and koff refers, for example, the dissociation rate constant of TCR to the peptide-MHC complex. Kon and Koff can be determined by methods known to those skilled in the art, for example, by using BIAcore® or KinExA. As used herein, “lower affinity” refers to a higher KD.

[0114] 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 particular peptide or a particular peptide-MHC complex combination), such binding being understood by those skilled in the art. For example, a TCR that specifically binds to an antigen may bind to other antigens with generally lower affinity, as determined by, for example, BIAcore® or other immunoassays known in the art (see, for example, Savage et al., Immunity. 1999, 10(4):485-92, which is incorporated herein by reference in its entirety). In specific embodiments, a TCR that specifically binds to one antigen binds to that antigen with a Ka that is at least 2, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than the association constant (Ka) of the TCR when it binds to another antigen. In some embodiments, the TCRs disclosed herein specifically bind to a peptide consisting of the amino acid sequence described in SEQ ID NO: 60.

[0115] As used herein, “epitope” is a term used in the art and refers to a local region of an antigen (e.g., a peptide or peptide-MHC complex) to which a TCR can bind. In some embodiments, the epitope to which the TCR binds can be determined, for example, by NMR spectroscopy, crystal structure analysis using X-ray diffraction, ELISA assay, a combination of mass spectrometry and hydrogen / deuterium exchange (e.g., liquid chromatography-electrospray mass spectrometry), flow cytometry, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (for example, these are each incorporated herein by reference in their entirety: 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).TCR: Antigen crystals can be examined using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. Patent Application Publication No. 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, eds Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): The mutagenesis can be refined using computer software such as 1316-1323), each of which is incorporated herein by reference in its entirety. Mutagenesis mapping tests can be achieved using any method known to those skilled in the art. See, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, each of which describes mutagenesis techniques, including the alanine scanning mutagenesis technique, and is incorporated herein by reference in its entirety. In specific embodiments, the epitope of the antigen is determined using the alanine scanning mutagenesis test. In specific embodiments, the epitope of the antigen is determined using a combination of mass spectrometry and hydrogen / deuterium exchange. In some embodiments, the antigen is a peptide-MHC complex. In some embodiments, the antigen is a peptide presented by an MHC molecule.

[0116] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to therapeutic or preventive measures described herein. In some embodiments, a “treatment” method involves administering a TCR or cells expressing a TCR to a subject having a disease or disorder, or being predisposed to such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of a disease or disorder or a recurrent disease or disorder, or to extend the survival of the subject beyond the survival expected in the absence of such treatment.

[0117] As used herein, the term “effective dose” in the context of administering treatment to a subject refers to the amount of treatment that achieves the desired preventive or therapeutic effect.

[0118] As used herein, the term “Subject” includes any human or non-human animal. In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is a human.

[0119] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using mathematical algorithms. Specific and non-restrictive examples of mathematical algorithms used for comparing two sequences are those described in Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, which are incorporated herein by reference in their entirety. Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul SF et al., (1990) J Mol Biol 215: 403, which are also incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed using NBLAST nucleotide program parameters set, for example, score=100 and word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using XBLAST program parameters, for example, set to score=50 and word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison, gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is incorporated herein by reference in its entirety. Alternatively, iterative searches can be performed using PSI BLAST to detect intermolecular distance relationships (same authors). When using the BLAST, gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov on the World Wide Web).Another specific and non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm described by Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used.

[0120] The percentage identity between two sequences can be determined using a similar method to that described above, with or without allowing gaps. When calculating percentage identity, typically only exact matches are counted.

[0121] As used herein, the term “effector moiety” refers to a component or functional group of a molecule that increases or decreases the intrinsic activity of the molecule, or confers a novel activity to the molecule. In some embodiments, the effector moiety is a binding moiety. In some embodiments, the binding moiety binds to a cell surface protein. In some embodiments, the binding moiety is an antibody.

[0122] In some embodiments, the engineered TCR can recognize the TAA antigen in a major histocompatibility complex (MHC) class I-dependent manner. “MHC class I-dependent manner,” as used herein, means that the engineered TCR triggers an immune response when it binds to the TAA antigen in the context of an MHC class I molecule. In some embodiments, the MHC class I molecule is an HLA-A molecule.

[0123] In some embodiments, the manipulated TCR is PRAME 425~433It binds to the (SEQ ID NO: 60)-HLA-A*02.01 complex. In some embodiments, the manipulated TCR binds to a PRAME peptide containing the amino acid sequence described in SEQ ID NO: 60. In some embodiments, the manipulated TCR binds to a PRAME peptide consisting of the amino acid sequence described in SEQ ID NO: 60.

[0124] The CDRs of TCRs disclosed herein can be defined using any numbering rules recognized in the art. Additionally or alternatively, CDRs can also be defined empirically, for example, based on structural analysis of the interaction between the TCR and a cognitive antigen (e.g., a peptide or peptide-MHC complex).

[0125] In some embodiments, the Disclosure provides a TCR that binds to a peptide consisting of the amino acid sequence described in SEQ ID NO: 60 (e.g., a TCR that binds to the SLLQHLIGL(SEQ ID NO: 60)-HLA-A*0201 complex), comprising one, two, or all three of the Vα or Vβ CDRs disclosed in Table 1, wherein the CDRs are defined according to the IMGT numbering system, for example, as described in Lefranc MP (1999) and Lefranc MP et al., (1999). In some embodiments, the manipulated TCR includes a CDR defined according to the Kabat numbering system. As will be understood by those skilled in the art, the CDRs can be predicted using the Kabat numbering system based on the variable domain sequence.

[0126] [Table 1]

[0127] In some embodiments, the present disclosure provides a TCR that binds to a peptide consisting of the amino acid sequence described in SEQ ID NO: 60 (e.g., a TCR that binds to the SLLQHLIGL(SEQ ID NO: 60)-HLA-A*0201 complex), comprising one, two, or all three of the Vα or Vβ CDRs disclosed in Table 1, wherein the CDRs are determined empirically, for example, based on structural analysis of the interaction between the TCR and a cognitive antigen (e.g., a peptide-MHC complex).

[0128] In some embodiments, the manipulated TCR is a human TCR. In some embodiments, the TCR contains a sequence that is naturally present within the TCR germline repertoire of an animal or mammal (e.g., human). In some embodiments, the TCR contains a sequence that is not naturally present within the TCR germline repertoire of an animal or mammal. In some embodiments, the TCR is an isolated TCR.

[0129] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex comprises an alpha chain containing a variable alpha chain (Vα) region and a beta chain containing a variable beta chain (Vβ) region, wherein the Vα region includes a CDR3 containing the amino acid sequence described in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the Vα region also includes a CDR1 containing the amino acid sequence described in SEQ ID NO: 37 or 41 and a CDR2 containing the amino acid sequence described in SEQ ID NO: 38.

[0130] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex comprises an alpha chain containing a variable alpha chain (Vα) region and a beta chain containing a variable beta chain (Vβ) region, the Vβ region comprising a CDR3 comprising the amino acid sequence described in any of SEQ ID NOs: 53, 54, 55, 56, 57, 58, or 59. In some embodiments, the Vβ region also comprises a CDR1 comprising the amino acid sequence described in SEQ ID NO: 51 and a CDR2 comprising the amino acid sequence described in SEQ ID NO: 52.

[0131] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex comprises an alpha chain containing a variable alpha chain (Vα) region and a beta chain containing a variable beta chain (Vβ) region, (a) the Vα region comprising a CDR3 comprising the amino acid sequence described in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and (b) the Vβ region comprising a CDR3 comprising the amino acid sequence described in any of SEQ ID NOs: 53, 54, 55, 56, 57, 58, or 59. The Vα region may further comprise a CDR1 comprising the amino acid sequence described in SEQ ID NO: 37 or 41 and a CDR2 comprising the amino acid sequence described in SEQ ID NO: 38, and / or the Vβ region may further comprise a CDR1 comprising the amino acid sequence described in SEQ ID NO: 51 and a CDR2 comprising the amino acid sequence described in SEQ ID NO: 52.

[0132] In some embodiments, the manipulated TCR that specifically binds to the SEQ ID NO: 60-HLA-A*0201 complex includes a Vα region and a Vβ region, (a) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (b) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (c) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 59, respectively; (d) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (e) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 39, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (f) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (g) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (h) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 37, 38, and 40, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 59; (i) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (j) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 40, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (k) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 41, 38, and 42, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (l) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 41, 38, and 42, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (m) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 41, 38, and 42, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 54, respectively; (n) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (o) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (p) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 59, respectively; (q) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (r) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 43, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (s) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (t) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 56, respectively; (u) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (v) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 58, respectively; (w) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (x) CDR1, CDR2, and CDR3 in the Vα region include sequence numbers 37, 38, and 44, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include sequence numbers 51, 52, and 54, respectively; (y) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 55, respectively; (z) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 56, respectively; (aa) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 57, respectively; (bb) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 45, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (cc) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 37, 38, and 45, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 54; (dd) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 37, 38, and 46, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 53; (ee) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 46, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (ff) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 47, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 53, respectively; (gg) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 37, 38, and 47, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 54, respectively; (hh)Vα regions CDR1, CDR2, and CDR3 include SEQ ID NOs. 37, 38, and 48, respectively, and Vβ regions CDR1, CDR2, and CDR3 include SEQ ID NOs. 51, 52, and 53, respectively; (ii) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 48, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively; (jj) The CDR1, CDR2, and CDR3 of the Vα region include sequence numbers 37, 38, and 49, respectively, and the CDR1, CDR2, and CDR3 of the Vβ region include sequence numbers 51, 52, and 54, respectively; (kk) The Vα region CDR1, CDR2, and CDR3 each contain sequence numbers 37, 38, and 49, respectively, and the Vβ region CDR1, CDR2, and CDR3 each contain sequence numbers 51, 52, and 53, respectively; (ll) CDR1, CDR2, and CDR3 in the Vα region include SEQ ID NOs. 37, 38, and 50, respectively, and CDR1, CDR2, and CDR3 in the Vβ region include SEQ ID NOs. 51, 52, and 54, respectively, or (hh)Vα regions CDR1, CDR2, and CDR3 include SEQ ID NOs. 37, 38, and 50, respectively, and Vβ regions CDR1, CDR2, and CDR3 include SEQ ID NOs. 51, 52, and 53, respectively.

[0133] In some embodiments, the manipulated TCR that specifically binds to the SEQ ID NO: 60-HLA-A*0201 complex includes a Vα region and a Vβ region, where CDR1, CDR2, and CDR3 of the Vα region include SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 40, respectively, and CDR1, CDR2, and CDR3 of the Vβ region include SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0134] In some embodiments, the manipulated TCR that specifically binds to the SEQ ID NO: 60-HLA-A*0201 complex includes a Vα region and a Vβ region, where CDR1, CDR2, and CDR3 of the Vα region include SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 45, respectively, and CDR1, CDR2, and CDR3 of the Vβ region include SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0135] In some embodiments, the manipulated TCR that specifically binds to the SEQ ID NO: 60-HLA-A*0201 complex includes a Vα region and a Vβ region, where CDR1, CDR2, and CDR3 of the Vα region include SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and CDR1, CDR2, and CDR3 of the Vβ region include SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0136] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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 example, the present disclosure provides an engineered TCR comprising a Vα region and a Vβ region, wherein the Vα region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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 Vβ region comprises a CDR3 comprising the amino acid sequence described in any of SEQ ID NOs: 53, 54, 55, 56, 57, 58, or 59. In some embodiments, the Vβ region also comprises a CDR1 comprising the amino acid sequence described in SEQ ID NO: 51, and a CDR2 comprising the amino acid sequence described in SEQ ID NO: 52.

[0137] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vβ region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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 example, the present disclosure provides an engineered TCR comprising a Vα region and a Vβ region, wherein the Vβ region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described 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 Vα region comprises a CDR3 comprising the amino acid sequence described in any of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the Vα region also comprises a CDR1 comprising the amino acid sequence described in SEQ ID NO: 37 or 41, and a CDR2 comprising the amino acid sequence described in SEQ ID NO: 38.

[0138] In some embodiments, the manipulated TCR includes a Vα region and a Vβ region, where the Vα region is at least 80%, 85%, 90%, 91%, 92%, and 93% of the amino acid sequence described 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. The Vβ region contains an amino acid sequence that is identical by %, 94%, 95%, 96%, 97%, 98%, or 99%, and the Vβ region contains an amino acid sequence that is identical by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the amino acid sequence described 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.

[0139] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0140] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32.

[0141] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36.

[0142] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0143] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 2, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0144] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0145] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32.

[0146] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36.

[0147] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0148] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 4, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0149] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0150] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0151] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 6, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0152] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0153] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32.

[0154] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 36. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 36.

[0155] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0156] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 8, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0157] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0158] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 30. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 30.

[0159] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32.

[0160] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 34. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 34.

[0161] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0162] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 10, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0163] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 28. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 28.

[0164] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 30. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 30.

[0165] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 32. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 32.

[0166] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0167] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 12, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0168] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 14, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 14, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0169] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 14, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 14, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0170] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 16, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 16, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0171] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 16, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 16, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0172] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 18, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 18, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 18, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0173] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 18, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 18, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 18, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0174] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 20, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 20, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 20, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0175] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 20, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 20, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 20, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0176] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 22, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 24. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 22, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 24.

[0177] In some embodiments, the manipulated TCR that specifically binds to the (SEQ ID NO: 60)-HLA-A*0201 complex includes a Vα region and a Vβ region, where the Vα region includes the amino acid sequence described in SEQ ID NO: 22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 22, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 26. For example, in some embodiments, the Vα region includes the amino acid sequence described in SEQ ID NO: 22, and the Vβ region includes the amino acid sequence described in SEQ ID NO: 26.

[0178] Any TCR constant region derived from any species can be used in the engineered TCR disclosed herein. In some embodiments, the engineered TCR includes a human α, β, γ, or δ TCR constant region. In some embodiments, the engineered TCR includes a wild-type constant region. In some embodiments, the engineered TCR includes a modified constant region, such as a chimeric constant region, or a constant region comprising one or more amino acid substitutions, insertions, or deletions compared to the wild-type constant region.

[0179] The manipulated TCRs disclosed herein can be used in any TCR structural form. For example, in some embodiments, the manipulated TCR is a full-length TCR containing full-length α-chains and full-length β-chains. The transmembrane region (and optionally the cytoplasmic region as well) can be removed from the full-length TCR to produce a soluble TCR. Thus, in some embodiments, the manipulated TCR is a soluble TCR lacking the transmembrane region and / or cytoplasmic region. Methods for producing soluble TCRs are well known in the art. In some embodiments, the soluble TCR contains manipulated disulfide bonds that promote dimerization. See, for example, U.S. Patent No. 7,329,731, which is incorporated herein by reference in its entirety. In some embodiments, soluble TCRs are generated by fusing the extracellular domain of a TCR described herein with another protein domain, such as a maltose-binding protein, thioredoxin, a human constant kappa domain, or a leucine zipper, for example, see Loset et al., Front Oncol. 2014; 4: 378, which is incorporated herein by reference in its entirety. It is also possible to generate single-chain TCRs (scTCRs) containing Vα and Vβ linked by a peptide linker. Such scTCRs may contain Vα and Vβ, each linked to the constant region of a TCR. Alternatively, the scTCR may contain Vα and Vβ, in which case neither Vα nor Vβ, or both Vα and Vβ, are linked to the constant region of a TCR. Exemplary scTCRs are described in PCT Publications WO2003 / 020763, WO2004 / 033685, and WO2011 / 044186, which are each incorporated herein by reference in their entirety. Furthermore, the manipulated TCRs disclosed herein may include two polypeptide chains (e.g., an α-chain and a β-chain) whose chains are manipulated to each have cysteine ​​residues capable of forming interchain disulfide bonds. Thus, in some embodiments, the manipulated TCRs disclosed herein include two polypeptide chains linked by an manipulated disulfide bond.Exemplary TCRs having manipulated disulfide bonds are described in U.S. Patents No. 8,361,794 and No. 8,906,383, each of which is incorporated herein by reference in whole.

[0180] In some embodiments, the TCR disclosed herein comprises one or more chains having a transmembrane region (e.g., an α chain and / or a β chain). In some embodiments, the TCR disclosed herein comprises two chains having a transmembrane region (e.g., an α chain and a β chain). The transmembrane region may be the endogenous transmembrane region of the TCR chain, a variant of the endogenous transmembrane region, or a heterologous transmembrane region. In some embodiments, the TCR disclosed herein comprises an α chain and a β chain having an endogenous transmembrane region.

[0181] In some embodiments, the engineered TCR disclosed herein comprises one or more chains having a cytoplasmic region (e.g., an α chain and / or a β chain). In some embodiments, the engineered TCR disclosed herein comprises two chains (e.g., an α chain and a β chain), each having a cytoplasmic region. The cytoplasmic region may be the endogenous cytoplasmic region of the TCR chain, a variant of the endogenous cytoplasmic region, or a heterologous cytoplasmic region. In some embodiments, the engineered TCR disclosed herein comprises two chains (e.g., an α chain and a β chain), both of which have a transmembrane region, but one chain lacks a cytoplasmic region. In some embodiments, the engineered TCR disclosed herein comprises two chains (e.g., an α chain and a β chain), both of which have an endogenous transmembrane region, but lack an endogenous cytoplasmic region. In some embodiments, the engineered TCR disclosed herein comprises an α chain and a β chain, both of which have an endogenous transmembrane region, but lack an endogenous cytoplasmic region. In some embodiments, the manipulated TCRs disclosed herein include a co-stimulus signaling region derived from a co-stimulus molecule; see, for example, PCT Publications WO1996 / 018105, WO1999 / 057268, and WO2000 / 031239, and U.S. Patent No. 7,052,906, all of which are incorporated herein by reference in their entirety.

[0182] In some embodiments, the manipulated TCR described herein is bound to a peptide-MHC complex comprising a peptide having the amino acid sequence described in SEQ ID NO: 60, where MHC can be any MHC. In some embodiments, MHC is human MHC. In some embodiments, MHC is an MHC class I molecule comprising an MHC class I heavy chain (e.g., HLA-A, HLA-B, or HLA-C, including any subtype in any polymorphic form) and a β2-microglobulin light chain. In some embodiments, 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 α chain (e.g., the α chain of HLA-DR, HLA-DQ, or HLA-DP, including any subtype of any polymorphic form) and an MHC class II β chain (e.g., the β chain of HLA-DR, HLA-DQ, or HLA-DP, including any subtype of any polymorphic form). In some embodiments, the MHC class II α chain and the MHC class II β chain are derived from the same type (e.g., HLA-DR, HLA-DQ, or HLA-DP).

[0183] The nucleotide sequences encoding the TCR described herein can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to produce nucleic acids encoding the TCR. Such polynucleotides 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, in short, involves the synthesis of duplicate oligonucleotides containing portions of the TCR-encoding sequence, annealing and ligation of these oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0184] Alternatively, the polynucleotides encoding the TCRs described herein can be generated from nucleic acids derived from a suitable source (e.g., T lymphocytes) 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 known sequences can be performed using genomic DNA obtained from T cells expressing the TCR of interest. Using such a PCR amplification method, nucleic acids containing sequences encoding the α and / or β chains of the TCR can be obtained. Using such a PCR amplification method, nucleic acids containing sequences encoding the Vα and / or Vβ domains of the TCR can be obtained. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric TCRs and humanized TCRs.

[0185] If a clone containing nucleic acid encoding a specific TCR is not available, but the sequence of the TCR molecule is known, the nucleic acid encoding the TCR can be obtained by chemical synthesis or by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence to identify cDNA clones derived from a TCR-encoding cDNA library, for example, a TCR cDNA library or any tissue or cell expressing the TCR, e.g., a cDNA library generated from T lymphocytes selected to express the TCR described herein, or nucleic acids isolated therefrom, e.g., poly(A+RNA), from a suitable source (e.g., a TCR cDNA library, or any tissue or cell expressing the TCR, e.g., a cDNA library, or any tissue or cell expressing the TCR, e.g., a cDNA library expressing the TCR, e.g., a cDNA clone derived from a TCR-encoding cDNA library). Subsequently, the amplified nucleic acid produced by PCR can be cloned into a replicable cloning vector using any method known in the art.

[0186] The TCR-encoding DNA described herein can be readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that can specifically bind to the genes encoding the α and / or β chains of the TCR. T lymphocytes can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which can then be transfected into host cells that do not normally produce TCR proteins, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells derived from CHO GS System® (Lonza)), or myeloma cells, to obtain TCR synthesis in recombinant host cells.

[0187] To generate the entire TCR, the Vα or Vβ sequence can be amplified using PCR primers containing the Vα or Vβ nucleotide sequence, a restriction site, and a flanking sequence protecting the restriction site, to produce clones, e.g., clones of individual Vα or Vβ nucleotide sequences, or clones of a single-chain TCR containing the variable region of the TCR linked by a mobile linker. Using cloning techniques known to those skilled in the art, the PCR-amplified Vα domain can be cloned into a vector expressing the α-chain constant region, and the PCR-amplified Vβ domain can be cloned into a vector expressing the β-chain constant region. In some embodiments, the vector for expressing the Vα or Vβ domain includes an EF-1α promoter, a secretory signal, a cloning site for the variable region, a constant domain, and a selection marker, e.g., neomycin. Subsequently, using techniques known to those skilled in the art, the α-chain vector and the β-chain vector are simultaneously or sequentially transfected into a cell line to generate a stable or transient cell line expressing the entire TCR. Alternatively, the Vα or Vβ domain can be cloned into a single vector expressing the required constant region. Next, the vector is transfected into a cell line using a method known to those skilled in the art to generate a stable or transient cell line expressing the entire TCR.

[0188] Furthermore, the DNA can be modified, for example, by substituting the coding sequences of human α- and β-chain constant domains for the mouse sequence, or by covalently attaching all or part of the coding sequence of a non-TCR polypeptide to a TCR coding sequence.

[0189] Also provided are polynucleotides that hybridize to polynucleotides encoding the TCRs described herein under high, medium, or low stringency hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize to polynucleotides encoding the Vα and / or Vβ domains provided herein under high, medium, or low stringency hybridization conditions.

[0190] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may include hybridization with filter-bound DNA in 6 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50–65°C; hybridization under highly stringent conditions may include hybridization with filter-bound nucleic acids in 6 × SSC at about 45°C, followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is also known and described to those skilled in the art; see, for example, pages 6.3.1–6.3.6 and 2.10.3 of Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York.

[0191] In some embodiments, cells expressing the TCRs described herein (e.g., by recombination) bound to PRAME peptides, as well as associated polynucleotides and expression vectors, are provided herein. Vectors (e.g., expression vectors) containing polynucleotides comprising nucleotide sequences encoding such TCRs for recombinant expression in cells, e.g., mammalian cells, are provided herein. Cells containing such vectors for recombinant expression of the TCRs described herein (e.g., human or humanized TCRs) are also provided herein. In certain embodiments, methods for producing the TCRs described herein, comprising expressing such TCRs from cells, are provided herein. In some embodiments, the cells are iNKT cells.

[0192] In another embodiment, methods for producing manipulated cells (e.g., cells containing heterologous and / or recombinant nucleic acids) as described herein are provided herein. In some embodiments, the method includes contacting cells with the vector described herein under conditions that allow for the introduction of the vector into the cells. In some embodiments, these conditions allow for the transfection of cells with the vector (e.g., by liposomes or electroporation). In some embodiments, these conditions allow for the transfection of cells with an mRNA vector by electroporation. In some embodiments, the vector is a viral vector (e.g., a lentiviral vector), and the conditions allow for the transduction of cells with the viral vector. In some embodiments, the vector is introduced into cells in vitro or ex vivo. In some embodiments, the vector is introduced into cells in vivo.

[0193] Recombinant expression of the manipulated TCR described herein (e.g., the full-length TCR, the α-chain and / or β-chain of the TCR, or a single-chain TCR) bound to the PRAME peptide involves the construction of an expression vector containing a polynucleotide encoding the TCR. Once the polynucleotide encoding the TCR described herein is obtained, a vector for the production of the TCR molecule can be produced by recombinant DNA technology using methods well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding the TCR is described herein. Using methods well known to those skilled in the art, an expression vector containing the TCR coding sequence, as well as appropriate transcriptional and translational regulatory signals, can be constructed. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. A replicable vector containing a nucleotide sequence encoding the manipulated TCR molecule described herein (e.g., the full-length TCR, the α-chain or β-chain of the TCR, the Vα or Vβ of the TCR, or the α or β-chain CDR) operably ligated to a promoter is also provided.

[0194] The vector may contain any type of nucleotide (including but not limited to DNA and RNA), may be single-stranded or double-stranded, may be synthesized or partially obtained from natural sources, and may contain natural, unnatural, or modified nucleotides. Recombinant expression vectors may contain nucleotide-nucleotide bonds that are naturally present or not naturally present, or both types of bonds. In some embodiments, nucleotides or nucleotide-nucleotide bonds that are not naturally present or modified do not interfere with the transcription or replication of the vector. The expression vector may be a viral vector (e.g., a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a baculovirus vector). In some embodiments, the retroviral vector may be a lentiviral vector (e.g., a vector containing genetic elements of the HIV-1 genome) or a equine infectious anemia virus vector. In some embodiments, the vector is packaged with one or more viral capsid proteins to yield a viral particle.

[0195] The expression vector can be transferred into cells (e.g., iNKT cells) by conventional methods, and the resulting cells can subsequently be cultured by conventional methods to produce the engineered TCR described herein. Thus, cells (e.g., iNKT cells) containing polynucleotides encoding the TCR molecule described herein (e.g., full-length TCR, α-chain or β-chain of TCR, Vα or Vβ of TCR, or α or β-chain CDR) are provided herein, operably linked to a promoter for the expression of such sequences in host cells. In some embodiments, for the expression of a double-stranded TCR, vectors encoding both the α-chain and β-chain separately can be co-expressed in cells for the expression of the entire TCR molecule, as detailed below. In some embodiments, the host cell contains a vector containing polynucleotides encoding both the α-chain and β-chain of the engineered TCR described herein. In specific embodiments, a cell comprises two distinct vectors, the first vector comprising a polynucleotide encoding the α-chain or α-chain variable region of the manipulated TCR described herein, and the second vector comprising a polynucleotide encoding the β-chain or β-chain variable region of the manipulated TCR described herein. In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the α-chain or α-chain variable region of the TCR described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding the β-chain or β-chain variable region of the TCR described herein. In specific embodiments, the α-chain or α-chain variable region expressed by the first cell associates with the β-chain or β-chain variable region expressed by the second cell to form the TCR described herein. In some embodiments, populations of host cells comprising such first and second host cells are provided herein.

[0196] In certain embodiments, a group of vectors is provided herein, including a first vector comprising a polynucleotide encoding the α-chain or α-chain variable region of the TCR described herein, and a second vector comprising a polynucleotide encoding the β-chain or β-chain variable region of the TCR described herein.

[0197] Various host-expression vector systems can be used to express the manipulated TCR molecules described herein (see, for example, U.S. Patent No. 5,807,715). Such host expression systems refer to a medium that can produce and subsequently purify the desired coding sequence, but also to cells that can express the TCR molecules described herein in situ when transformed or transfected with a suitable nucleotide coding sequence. These include microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a TCR coding sequence; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing a TCR coding sequence; insect cell lines infected with recombinant virus expression vectors containing a TCR coding sequence (e.g., baculovirus); and plant cell lines (e.g., Chlamydomonas) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing a TCR coding sequence (e.g., Ti plasmid). Examples include, but are not limited to, green algae such as reinhardtii); or mammalian cell lines possessing recombinant expression constructs including promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter; vaccinia virus 7.5K promoter) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In specific embodiments, the cells for expressing the TCR described herein are CHO cells, for example, CHO cells from the CHO GS System (Lonza).In certain embodiments, the cells for expressing the TCR described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are used for the expression of the recombinant TCR molecule, particularly for the expression of the entire recombinant TCR molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, when combined with a vector such as a major immediate early gene promoter element derived from human cytomegalovirus, make an effective expression system for TCR (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7). In some embodiments, the engineered TCR described herein is produced by CHO cells or NS0 cells. In specific embodiments, the expression of nucleotide sequences encoding the TCRs described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0198] Once the manipulated TCR molecules described herein are produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, and sorting column chromatography), centrifugation, by solubility differences, or by any other standard method for protein purification. Furthermore, the TCRs described herein can be fused with heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0199] In specific embodiments, the TCRs described herein are isolated or purified. Generally, isolated TCRs are substantially free of other TCRs having different antigen specificity than the isolated TCR. For example, in certain embodiments, the TCR preparations described herein are substantially free of cellular material and / or chemical precursors. The phrase “substantially free of cellular material” includes TCR preparations in which the TCR is isolated from the cellular components of the cell from which it is isolated or recombinantly produced. Thus, a TCR substantially free of cellular material includes TCR preparations in which heterologous proteins (also referred herein as “contamination proteins”) and / or variants of the TCR, e.g., different post-translational modified forms of the TCR or other different versions of the TCR (e.g., fragments thereof), are present in amounts less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% (on a dry weight basis). When TCR is recombinantly produced, it also generally contains substantially no culture medium, i.e., the culture medium accounts for less than about 20%, less than 10%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the volume of the protein preparation. When TCR is produced by chemical synthesis, it generally contains substantially no chemical precursors or other chemicals, i.e., it is separated from the chemical precursors or other chemicals involved in the synthesis of the TCR. Thus, such a preparation of TCR contains less than about 30%, less than 20%, less than 10%, or less than 5% (on a dry weight basis) of chemical precursors or compounds other than the TCR of interest. In certain embodiments, the TCR described herein is isolated or purified.

[0200] The manipulated TCRs bound to the 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. Unless otherwise indicated, the methods described herein use conventional techniques in the fields of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of those skilled in the art. These techniques are described, for example, in the references cited herein and are well explained in the literature.For example, 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 FM 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 Please refer to the Manual, Cold Spring Harbor Laboratory Press, each of which is incorporated herein by reference in its entirety.

[0201] In specific embodiments, the TCRs described herein are TCRs (e.g., recombinant TCRs) prepared, expressed, produced, or isolated by any means including, for example, DNA sequence synthesis or production via genetic engineering. In some embodiments, such TCRs include sequences (e.g., DNA sequences, RNA sequences, or amino acid sequences) that do not naturally exist in vivo within the TCR germline repertoire of animals or mammals (e.g., humans).

[0202] In one embodiment, a method for producing a TCR that binds to a PRAME peptide is provided herein, comprising culturing cells or host cells as described herein. In some embodiments, a method for producing a TCR that binds to a PRAME peptide is provided herein, comprising expressing (e.g., recombinantly expressing) the TCR using cells or host cells as described herein (e.g., cells or host cells containing a polynucleotide encoding the TCR as described herein). In certain embodiments, the cells are isolated cells. In certain embodiments, the exogenous polynucleotide has been introduced into the cells. In certain embodiments, the method further comprises the step of purifying the TCR obtained from the cells or host cells.

[0203] In another embodiment, the disclosure provides mammalian cells (e.g., engineered mammalian cells) or populations thereof that display the TCR disclosed herein on their cell surface. Any mammalian cells can be used to display the TCR disclosed herein. In some embodiments, the mammalian cells express CD3 (e.g., CD3γ chain, CD3δ chain, and two CD3ε chains).

[0204] In some embodiments, mammalian cells are human cells. Effector cells of the cellular immune system are particularly useful in causing the TCRs disclosed herein to present, as the cell surface TCRs can target tumor cells expressing PRAME polypeptides, thereby promoting the killing of tumor cells. Therefore, in some embodiments, mammalian cells are lymphocytes such as T cells or natural killer (NK) cells (e.g., human lymphocytes). In some embodiments, lymphocytes are T cells. Any T cells at any developmental stage can be used to cause the TCRs disclosed herein to present. For example, in some embodiments, T cells are selected from the group consisting of CD8+ cytotoxic T cells, CD4+ cytotoxic T cells, CD4+ helper T cells (e.g., Th1 or Th2 cells), CD4 / CD8 double-positive T cells, tumor-infiltrating T cells, thymocytes, memory T cells, naive T cells, and natural killer T cells, e.g., invariant natural killer T cells. Progenitor cells of the cellular immune system (e.g., T lymphocyte precursors) are also useful in causing the TCRs disclosed herein, because these cells have the potential to differentiate, develop, or mature into effector cells. Thus, in some embodiments, the mammalian cells are pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells), hematopoietic stem cells, or lymphocyte progenitor cells. In some embodiments, hematopoietic stem cells or lymphocyte progenitor cells are isolated and / or enriched from, for example, bone marrow, umbilical cord blood, or peripheral blood.

[0205] Cells can be obtained from a number of sources, including but not limited to tumors, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids, or apheresis products. In some embodiments, cells are obtained from a patient immediately after a procedure that leaves functional T cells in the subject. In this regard, it has been observed that after certain cancer procedures, particularly procedures with drugs that damage the immune system, the quality of T cells obtained immediately after the procedure, during the period when the patient is normally recovering from the procedure, may be optimal or improved for their ability to be amplified ex vivo. Similarly, after ex vivo procedures using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo amplification. Therefore, in some embodiments, cells are collected during this recovery period from blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids, or apheresis products.

[0206] In some embodiments, the cells are invariant natural killer T (iNKT) cells. iNKT cells are a specific subset of T cells that exhibit both direct and indirect antitumor activity. 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 via the Fas / FasL pathway. Furthermore, iNKT cells can also target and kill tumor cells indirectly by stimulating other immune cells such as dendritic cells, natural killer (NK) cells, and CD8+ T cells. iNKT cells also play a role in regulating the immune response by producing cytokines such as interferon-gamma and interleukin-4, which promote the activation and differentiation of other immune cells.

[0207] The terms “invariant natural killer T cells,” “invariant NKT cells,” “iNKT cells,” or “type I NKT cells,” as used herein, refer to a population of T lymphocytes that express a conserved semi-invariant TCR specific to lipid antigens bound to the monomorphic MHC class I-related molecule CD1d. Natural killer T cells (NKT cells) were initially characterized in mice as T cells expressing both the TCR and the C-type lectin NK receptor NK1.1 (NKR-P1a-c or CD161). Invariant NKT (iNKT) cells express a semi-invariant αβ TCR (e.g., formed by the rearrangement of the invariant TRAV11-TRAJ18(4) in mice, or by the homologous invariant TRAV10-TRAJ18 chain in humans) and pair with a limited set of diverse Vβ chains, mainly TRBV1, TRBV29, or TRBV13(6) in mice, and TRBV25 in humans (see, for example, 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). Semi-invariant TCRs recognize exogenous and endogenous lipid antigens presented by the monomorphic MHC class I-associated molecule CD1d (see, for example, 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 α-galactosylceramide (α-GalCer) (Kawano et al., CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. (1997) 278:1626-9. 10.1126) and several bacterial-derived Ag, which can activate iNKT cells.

[0208] In some embodiments, the cells are a population of peripheral blood mononuclear cells (PBMCs) (e.g., human PBMCs). In some embodiments, the cells of interest are isolated from PBMCs. For example, iNKT cells can be isolated from PBMCs and amplified. In some embodiments, the cells of interest are isolated from the peripheral blood of a donor. Alternatively, the cells may be provided in the form of a blood apheresis sample (e.g., a lymphocyte apheresis sample or a leukocyte apheresis sample). The cells may be fresh or pre-frozen.

[0209] The cells, for example, iNKT cells, may be derived from a donor, such as a healthy donor. In some embodiments, the cells are iNKT cells and are obtained from an allogeneic donor. Since iNKT cells are constrained by CD1d, a non-polymorphic MHC I-like molecule, they can be obtained from an allogeneic donor.

[0210] In some embodiments, mammalian cells (e.g., iNKT cells) are a population of cells that present the manipulated TCRs disclosed herein on their cell surface. The population of cells may be heterogeneous or homogeneous. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the population are the cells described herein. In some embodiments, the population is substantially pure, and at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the population are homogeneous.

[0211] Cell populations can be enriched or purified as needed. For example, iNKT cells can be amplified by culturing them in IL-21, IL-2, IL-7, IL-15, IL-12, and TNF-α together with irradiated NKT-depleted PBMCs loaded with α-galactosylceramide (αGalCer), or with irradiated CD1-d expressing artificial display cells (APCs) loaded with αGalCer.

[0212] Cells can be stimulated ex vivo to enhance their viability, proliferation, and / or activity. In some embodiments, induction results in a cell population that does not contain any defined antigen and is therefore polyclonal with respect to antigen reactivity. In some embodiments, cells are contacted with a first agent (e.g., an anti-CD3 antibody) that induces or activates TCR / CD3 complex-related signaling. In some embodiments, cells are contacted with a second agent that stimulates accessory molecules on the T cell surface (e.g., a CD28 ligand or an anti-CD28 antibody). In some embodiments, cells are contacted with a molecule or complex that interacts with both CD3 and CD28, where the molecule or complex may be presented on a surface (e.g., beads, particles, or cells). In some embodiments, cells are contacted with an agent (e.g., phorbol 12-myristate 13-acetate and / or ionomycin) that stimulates one or more intracellular signals, such as Ca2+ release. Alternatively, the induction may include an antigen containing a peptide (e.g., PRAME peptide) that binds to the TCR presented on the cell surface, thus resulting in a cell population enriched with respect to antigen reactivity (e.g., monoclonal). The antigen may further contain MHC molecules (e.g., HLA molecules) in the complex with the peptide. The antigen may exist in a soluble form, bound to the membrane or presented on the surface.

[0213] In some embodiments, mammalian cells (e.g., iNKT cells) express the engineered TCR disclosed herein from a transgene introduced into the cells, thereby presenting the TCR on the cell surface. The TCR may be constitutively presented on the cell surface. Alternatively, the cells may have the ability to conditionally express and / or present the TCR. For example, the expression or presentation of the engineered TCR can be induced by exogenous stimuli or by cell differentiation. In some embodiments, the transgene encodes the engineered TCR α chain and / or β chain, or a fragment thereof (e.g., Vα, Vβ, CDR3α and / or CDR3β). In some embodiments, the transgene is operably ligated to exogenous transcriptional and / or translational regulatory sequences (e.g., promoters, enhancers, and / or Kozak sequences). In some embodiments, the transgene is operably ligated to an endogenous transcriptional and / or translational regulatory sequence (e.g., a promoter, enhancer, and / or Kozak sequence) that is not present at its native genomic locus (e.g., introduced by a vector). In some embodiments, the transgene is operably ligated to an endogenous transcriptional and / or translational regulatory sequence (e.g., a promoter, enhancer, and / or Kozak sequence) at its native genomic locus (e.g., by inserting the transgene into its native genomic locus).

[0214] In some embodiments, cells (e.g., iNKT cells) are engineered to express another molecule (e.g., cytokine or ligand) that can enhance one or more properties of the genetically modified cells (e.g., survival / persistence, immune interactions with other cells such as macrophages and / or dendritic cells, disruption of the immunosuppressive tumor microenvironment). For example, cells (e.g., iNKT cells) can be engineered to express any suitable armoring molecule known in the art, e.g., armoring molecules described in Yeku et al., Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumor 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, Biologics. 2021; 15: 95-105). Non-exclusive 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 receptors, checkpoint inhibitors such as anti-PD1 antibodies, CD47-targeting nanobodies, or bispecific T cell engagers (BiTEs).

[0215] In some embodiments, cells expressing the engineered TCR described herein (e.g., iNKT cells) are also engineered to express IL-15. In some embodiments, 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 amplification and function of T cells and natural killer T cells (e.g., iNKT cells) has 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). Numerous cell types, including macrophages and DCs, are involved in IL-15 production, and there is evidence that, upon release, IL-15 stimulates CD8+ T cells and NK cells, enhancing their proliferative and cytotoxic capabilities. Administration of IL-15 to mice has been shown to enhance the antitumor activity of adoptively transferred CD8+ tumor-responsive T cells, suggesting that IL-15 may also enhance the antitumor activity of T cell therapy. Furthermore, IL-15 can increase antigen-independent T cell proliferation while simultaneously enabling the persistence of T cells after tumor elimination. Several previous studies have used membrane-attached IL-15 morphology, which has been found to promote the development of T cell memory phenotypes. These data suggest that IL-15 may enhance T cell function within the tumor microenvironment and provide long-term T cell-mediated immunity against cancer antigens.

[0216] In some embodiments, cells (e.g., iNKT cells) express the engineered TCRs and endogenous TCRs disclosed herein. For example, in some embodiments, iNKT cells present the engineered TCRs disclosed herein and the iNKT endogenous TCR, which is a semi-invariant αβ TCR. iNKT cells typically express an invariant TCR α chain (Vα14-Jα18 in mice, or Vα24-Jα18 in humans). In mice, while most iNKT cells express the standard Vα14-Jα18 TCR α chain, they may also use different Vβ chains, and the combination of residues encoded by Vβ, Jβ, and CDR3β ultimately determines the type of ligand to which the iNKT cell can bind (Cameron et al., 2015; Mallevaey et al., 2009; Matsuda et al., 2001). Furthermore, a population of αGalCer-reactive NKT cells expressing the Vα10 TCR and possessing different lipid recognition capabilities has been identified (Uldrich et al., 2011). In humans, the majority of αGalCer-binding iNKT cells express the representative Vα24 Vβ11 TCR, but a population of atypical NKT cells has been found in the blood, and these cells express various TCR α and TCR β chains that show different recognition of lipid antigens (Le Nours et al., 2016; Matulis et al., 2010).

[0217] In some embodiments, the transgene is DNA integrated into the host cell genome, and integration occurs through site-directed integration (e.g., homologous recombination) or random insertion of DNA. In some embodiments, the transgene is DNA not integrated into the host cell genome (e.g., maintained as a non-integrated viral genome or as 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.

[0218] In some embodiments, the transgene comprises a first and a second sequence, the first sequence encoding a polypeptide containing a TCR α chain or a fragment thereof (e.g., Vα or CDR3α), and the second sequence encoding a polypeptide containing a TCR β chain or a fragment thereof (e.g., Vβ or CDR3β). In some embodiments, the first and second sequences are operably ligated to a transcriptional control sequence and / or a translational control sequence (e.g., a promoter, enhancer, and / or Kozak sequence). In some embodiments, the first and second sequences are located in different polynucleotide molecules (e.g., DNA, RNA, modified DNA, or modified RNA). In some embodiments, the first and second sequences of the transgene are located in the same polynucleotide molecule (e.g., DNA, RNA, modified DNA, or modified RNA). In some embodiments, the first and second sequences are operably linked by a linker sequence that facilitates the production of two distinct polypeptides (e.g., an intra-sequence ribosome entry site (IRES), a self-cleaving peptide (e.g., a 2A peptide), or a peptide sequence recognized by an intracellular or 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 incorporated into the host cell genome. In some embodiments, the first and second sequences are each incorporated into different regions of the host cell genome.

[0219] 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, such as one described in PCT Publication WO2015 / 123527, which is incorporated herein by reference in whole. In some embodiments, the polypeptide comprises a multimerizing (e.g., dimerizing or oligomerizing) region, the polypeptide inducing T cell activation upon multimerization.

[0220] In some embodiments, the cells are provided in solution. In some embodiments, the cells are cryopreserved at about -80°C or below (for example, in a liquid nitrogen storage tank). Methods of cryopreservation are well known in the art, as described, for example, in U.S. Patent Nos. 5,580,714 and 6,740,484, which are incorporated herein by reference in their entirety. The cryopreserved cells can be recovered by thawing, and any of the above-described isolation, purification, enrichment, stimulation, and presentation of the manipulated TCR can be performed before or after cryopreservation.

[0221] This disclosure provides a pharmaceutical composition comprising an engineered TCR as disclosed herein, a polynucleotide as disclosed herein, a cell as disclosed herein, and a pharmaceutically acceptable carrier.

[0222] This disclosure provides a method for inducing an immune response to PRAME, comprising administering an effective amount of an engineered TCR, a polynucleotide, a cell, or a pharmaceutical composition as disclosed herein to the target.

[0223] In some embodiments, the Disclosure provides a method for treating cancer in a subject, comprising administering an effective amount of a TCR, a polynucleotide, a cell, or a pharmaceutical composition disclosed herein to the subject.

[0224] The cells administered to the target (e.g., iNKT cells) may be autologous or allogeneic. In some embodiments, autologous cells are obtained from the patient immediately after cancer treatment. In this regard, it has been observed that after certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of T cells obtained immediately after treatment, during the period when the patient is normally recovering from the treatment, may be optimal or improved for their ability to be amplified ex vivo. Similarly, after ex vivo operations using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo amplification. Therefore, in some embodiments, cells are collected during this recovery period from blood, bone marrow, lymph nodes, thymus, or another tissue or body fluid, or from apheresis products.

[0225] In some embodiments, the cells administered to the subject are allogeneic. In some embodiments, the cells are allogeneic iNKT cells. In some embodiments, allogeneic iNKT cells are administered to the subject without prior lymphocyte depletion.

[0226] The number of cells used depends on several factors, including the lifespan of the cells, the protocol used (e.g., number of doses), the cell's ability to proliferate, and the stability of the recombinant construct. In some embodiments, the cells are applied as a dispersion and generally injected into or near the site of interest. The cells may be administered in any physiologically acceptable culture medium.

[0227] In some embodiments, the Disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising human iNKT cells expressing the TCRs disclosed herein and a pharmaceutically acceptable carrier. For example, in some embodiments, the Disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising allogeneic iNKT cells expressing the manipulated TCRs and iNKT endogenous semi-invariant αβ TCRs disclosed herein.

[0228] In further embodiments, the disclosure relates to a method for treating a subject requiring adoptive cell therapy, comprising administering the pharmaceutical composition defined above to the patient. In some embodiments, the subject belongs to the group of HLA-A2 positive subjects.

[0229] In some embodiments, the subject suffers from a disease involving malignant cells that express PRAME.

[0230] Cancers that can be treated with the TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions disclosed herein may 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, pulmonary squamous cell carcinoma, melanoma, multiple myeloma, ovarian cancer, renal papillary cell carcinoma, testicular cancer, thymoma, uterine carcinosarcoma, non-small cell lung cancer, breast cancer, and uveal melanoma. In some embodiments, cancers include acute lymphoblastic leukemia, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain malignancies, breast cancer, cancers of the anus, anal canal or anorectum, eye cancers, intrahepatic cholangiocarcinoma, joint cancers, cancers of the neck, gallbladder or pleura, cancers of the nose, nasal cavity or middle ear, oral cancers, vulvar cancers, chronic lymphoblastic leukemia, chronic myeloid leukemia, myeloma (e.g., chronic myeloid leukemia), colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, Hodgkin lymphoma, and hypopharyngeal cancer. These include cancers, kidney cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, cancers of the peritoneum, omentum, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), gastric cancer, small intestine cancer, soft tissue cancer, gastric cancer, carcinoma, sarcoma (e.g., synovial sarcoma, rhabdomyosarcoma), testicular cancer, thyroid cancer, head and neck cancer, ureteral cancer, and 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).

[0231] In some embodiments, these methods further include targeting and administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent. In some embodiments, the checkpoint targeting agent is selected from the group consisting of antagonist anti-CTLA-4 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-PD-1 antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-GITR antibody, and agonist anti-OX40 antibody.

[0232] Lymphocyte depletion is common to reduce the circulating lymphocyte population before TCR-T cell infusion, and this includes chemotherapy, radiotherapy and / or any other specified method. In some embodiments, a pharmaceutical composition comprising iNKT cells expressing the manipulated TCR disclosed herein is administered to the subject, and lymphocyte depletion is not required before the procedure.

[0233] In some embodiments, the manipulated TCRs, polynucleotides, cells (e.g., iNKT cells), or pharmaceutical compositions disclosed herein are administered intravenously to a subject. In some embodiments, the manipulated TCRs, polynucleotides, cells (e.g., iNKT cells), or pharmaceutical compositions disclosed herein are administered to a subject by blood transfusion.

[0234] The injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g., an amplified population of iNKT cells expressing a TCR (e.g., autologous or allogeneic to the subject being treated). The active ingredient is typically dissolved or suspended in a physiologically acceptable carrier, and the composition may further contain small amounts of one or more non-toxic adjuvants, e.g., emulsifiers, preservatives, and pH buffers. Such injectable compositions useful for use with the fusion protein of this disclosure are conventional; suitable formulations are well known to those skilled in the art.

[0235] The engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein can be delivered to a target by a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, percutaneous, intravenous, intratumoral, conjunctival, subarachnoid, and subcutaneous routes. Lung administration can also be used, for example, by the use of formulations containing an aerosolizing agent for use as an inhaler or nebulizer and a spray. In some embodiments, the engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein are delivered intravenously. In some embodiments, the engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein are delivered subcutaneously. In some embodiments, the engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein are delivered intratumorally. In some embodiments, the engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions described herein are delivered intratumorally.

[0236] The amount of engineered TCRs, polynucleotides, vectors, engineered cells, or pharmaceutical compositions considered effective for treating and / or preventing a disease depends on the nature of the disease and can be determined by standard clinical methods.

[0237] The exact dose to be used in a composition also depends on the route of administration and the severity of the infection or disease it may cause, and should be determined according to the clinician's judgment and the circumstances of each subject. For example, the effective dose may also vary depending on the means of administration, the target site, the physiological state of the subject (including age, weight, and health status), whether the subject is human or animal, other pharmaceuticals administered, or whether the treatment is prophylactic or therapeutic. While the subject is usually human, non-human mammals, including transgenic mammals, can also be treated. The treatment dose is optimally adjusted to optimize safety and efficacy.

[0238] These active ingredients of the present disclosure are preferably used in such pharmaceutical compositions in doses mixed with an acceptable carrier or carrier material so as to treat or at least alleviate a disease. Such compositions may include (in addition to the active ingredients and carrier) fillers, salts, buffers, stabilizers, solubilizers and other materials known in the art.

[0239] The term "pharmaceutically acceptable" defines a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The choice of carrier depends on the application.

[0240] A pharmaceutical composition may contain additional components that enhance the activity of the active ingredient or assist in the treatment. Such additional components and / or factors may be part of the pharmaceutical composition to achieve a synergistic effect or to minimize adverse or undesirable effects.

[0241] The formulations or preparations and methods for administering / dosing the active ingredients of this disclosure are published in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. Appropriate administrations include non-enteral administration, e.g., intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal, or intratumoral injection. Intravenous injection is the preferred treatment for patients.

[0242] This disclosure will be described below with reference to the accompanying drawings and examples. The following examples are illustrative of this disclosure, but should not be construed as limiting its scope. [Examples]

[0243] [Example 1] Screening of TCR libraries PRAME is a cancer-testicular antigen expressed intracellularly in various solid tumors and hematological malignancies, with levels in normal tissue (excluding the testes) being 1 / 100th lower (Figures 1A-1D).

[0244] The PRAME synthetic peptide SLLQHLIGL (SEQ ID NO: 60), previously described by Amir et al., 2011, Clin Cancer Res. 17(17):5615-5625), is widely presented in HLA-A*02:01 MHC molecules in both tumor cell lines and patient tissues, and was therefore used to screen for TCR α and β chains.

[0245] The TCR α and β chains were expressed on the surface of the mouse cell line AK-D10R3 as a chimeric protein containing a human variable region fused with a mouse constant region. The mouse constant region was used to ensure proper anchoring and interaction with mouse CD3, as well as proper induction of mouse signaling pathways. AK-D10R3 is a mouse TCR-negative and mouse CD8-negative cell line derived from mouse thymoma, expressing a chimeric CD8 (human CD8 α and β extracellular regions fused with the corresponding mouse CD8 α and β transmembrane and intracellular regions) and a T cell activation reporter construct. This construct contains a minimal IL-2 promoter with three NFAT binding sites and is operably linked to EGFP.

[0246] In short, in guided selection screening, a reference β-chain (SEQ ID NO: 63; Table 2) was used as a guide, and the α-chain of this TCR was replaced with a TCR α-chain library (chains generated from either PBMCs or umbilical cord blood of 10 healthy donors).

[0247] [Table 2]

[0248] To evaluate pMHC tetramer binding and AK-D10R3 T cell activation in the presence of T2 / pMHC+ cells, expression constructs of β-chain and α-chain libraries were transduced into AK-D10R3 cells via retrovirus. Table 3 presents 11 α-chains identified based on their binding and activation properties when combined with a reference β-chain. The amino acid sequence alignments of the 11 variable α-chains are shown in Figure 2, and the nucleic acid sequences and amino acid sequences of the 11 variable α-chains are shown in Figures 3A-3E.

[0249] [Table 3]

[0250] Six of these α-chains (684A2, 684A3, 684A6, 684A9, 684A10, and 684A11) were used for secondary guided selection screening, in which the β-chains were replaced with three β-chain libraries. One library consisted of β-chains generated from PBMCs or umbilical cord blood from five healthy donors, and the other two libraries consisted of β-chains generated after randomization of native TCR β-chains. Two sets of single-chain NNK oligomers were used for β-chain CDR3 grafting. The CDR3 residues RWDRG (SEQ ID NO: 70) and WDRGG (SEQ ID NO: 71) of SEQ ID NO: 61 (CASARWDRGGEQYF) were randomized using the oligomer libraries β-NNK1 and β-NNK2 (custom-made by Twist).

[0251] Through secondary screening, seven novel β chains listed in Table 4 were identified. An alignment of the amino acid sequences of the β chains is presented in Figure 2, and the nucleic acid sequences and amino acid sequences of the variable β chains are presented in Figures 3A to 3E.

[0252]

Table 4

[0253] To confirm the binding of different TCR combinations, flow cytometry analysis was performed. AKD-10R3 cells expressing different TCR combinations were washed twice with PBS + 2% FCS, followed by anti-TCR-mAb-APC (1:500) diluted in PBS + 2% FCS and HLA-A*02:01-PRAME 425~433 tetramer-PE (1:50) for staining at room temperature for 30 minutes. After staining, the cells were washed twice with PBS + 2% FCS, resuspended in PBS + 2% FCS + 7-aminoactinomycin D (1:100), and viable cells were identified. To analyze only viable cells, cells were gated based on SSC / FSC pattern and negative staining with 7-aminoactinomycin D. PRAME 425~433 Thirty-nine TCRs that specifically recognize and potently target cells expressing the HLA-A*02.01 complex were identified (Table 5; Figures 4A to 4C).

[0254]

Table 5

[0255] [Example 2] Specificity of TCR Alanine scanning and T-SPRINT™ profiling were performed on selected TCRs to confirm that PRAME 425~433The degree to which the TCR maintains binding when each amino acid of the peptide is replaced with alanine or any other amino acid was determined. The combination of alanine scan data and T-SPRINT data, followed by bioinformatics analysis, identified only a small number of potential off-target peptides derived from other proteins, which indicates that these novel PRAME TCRs bind to the selected PRAME 425~433 peptide / HLA A*02:01 protein complex with high specificity.

[0256] Alanine scanning was performed for TCR4, TCR9, TCR17, TCR17, TCR23, TCR28, TCR30, TCR32, TCR34, TCR36, TCR38, TCR1, TCR6, TCR14, and TCR25. In alanine scanning, T2 cells were pulsed with PRAME peptides in which each peptide residue was individually mutated to alanine (one substitution per position), to assess positions important for activity. Subsequently, the pulsed T2 cells were co-cultured overnight with PRAME TCR-expressing AKD10R3 cells, and activation was read out through an NFAT-GFP reporter. Each assay was performed at least three times, and the summarized data are shown in Figures 5A to 5E.

[0257] To evaluate TCR specificity in more detail, T-SPRINT profiling was performed on the engineered PRAME TCR candidates TCR4, TCR9 and TCR28. T2 cells were pulsed with PRAME peptides in which each peptide residue was individually mutated to all other possible amino acids (19 substitutions per position), to obtain a more detailed profiling of TCR specificity for each peptide position. Subsequently, the pulsed T2 cells were co-cultured overnight with PRAME TCR-expressing AKD10R3 cells, and activation was read out through an NFAT-GFP reporter. Each assay was performed at least three times, and the summarized data are shown in Figure 6.

[0258] T2 cells were pulsed with various concentrations of PRAME peptide. The pulsed T2 cells were then co-cultured overnight with PRAME TCRs expressing AKD10R3 cells, and activation was measured using an NFAT-GFP reporter. Each assay was performed at least three times, and the combined data is shown. T-Rx-enhanced PRAME TCRs exhibited higher peptide sensitivity, particularly at lower peptide concentrations. See Figure 7.

[0259] [Example 3] Manipulated TCR expression in iNKT cells We expressed the TCR in iNKT cells via transduction using a lentiviral construct. The lentiviral construct was modified so that the PRAME-TCR was upstream of the P2A-BFP element, enabling tracking of transduced cells by flow cytometry analysis based on BFP expression.

[0260] iNKT cells were activated by adding humanized CD3 and CD28 agonists to the cell culture medium after lentiviral transduction. Subsequently, on day 14 posttransduction, iNKT cells were co-cultured with irradiated K562-HLA-A2 cell lines to specifically activate and enrich iNKT cells expressing PRAME-TCR. By day 27 posttransduction, over 80% of BFP+ cells were produced in all three TCRs tested from all three donors. See Figure 8.

[0261] [Example 4] Activity of PRAME TCR and endogenous TCR when expressed by iNKT cells iNKT cells expressing PRAME TCR are enriched to a TCR purity of over 80%, and then PRAME 425~433 PRAME-TCR-specific cytotoxicity was evaluated by co-culturing T2 cells pulsed with the peptide or NYESO peptide (1157-165) as a negative control overnight.

[0262] Furthermore, PRAME-TCR-iNKT cells were co-cultured overnight with C1r-CD1d cells that were either pulsed with aGalCer (alpha-galactosylceramide) or not, in order to evaluate iNKT-TCR-specific cytotoxicity. The percentage of cells targeted for death was calculated by determining the percentage of CD19+ cells positively stained using viable / dead dyes. The data presented are from two donors, with two series per donor. See Figure 9.

[0263] [Example 5] Cytotoxic capacity, activation, and cytokine production of PRAME TCR-iNKT cells To compare the activation profiles of CD4+ and CD8+ T cells expressing unrelated TCRs, native TCRs, or 684A2×709B5, 684A3×709B5, and 684A11×709B5 TCRs, CD3+ T cells were transduced with lentiviruses. Subsequently, the transduced CD3+ T cells were sorted into CD4+ and CD8+ cells. Subsequently, the CD4+ and CD8+ T cells were divided into 10 -8 M's PRAME 425~433 T2 cells pulsed with peptides were co-cultured for 24 hours. Subsequently, T cells were evaluated for activation by co-expression of CD25 and CD69 using flow cytometry. As a result, activation was demonstrated in CD4+ (Figure 10A) and CD8+ (Figure 10B). T cells were similar for 684A2×709B5, 684A3×709B5, and 684A11×709B5, which indicates that the PRAME TCR is CD8-independent.

[0264] To compare the activation profiles of TCR-iNKT expressing 684A2×709B5, 684A3×709B5 TCR, and 684A11×709B5 TCR, TCR-iNKT was produced by lentiviral transduction and amplification in K562-FAP feeder cells, while unmodified iNKT was amplified by two TransAct activations. TCR-iNKT was co-cultured with T2 cells for 24 hours without peptide pulse, or for 10 hours. -6M's PRAME 425~433 Peptides or NYESO 1157~1165 The cells were pulsed with peptide for 1 hour. Subsequently, iNKT cells were evaluated for activation by co-expression of CD25 and CD69 using flow cytometry. The results showed activation (CD25+CD69+) %) profiles for unmodified iNKT or PRAME-TCR-iNKT (684A2×709B5, 684A3×709B5, and 684A11×709B5) (Figure 11). Activation was observed in PRAME TCR iNKT cells. 425~433 This was observed only under conditions where it was combined with peptides.

[0265] PRAME of various concentrations 425~433 The activation profiles in response to peptides were compared in unmodified iNKT or PRAME-TCR-iNKT (684A2×709B5, 684A3×709B5, and 684A11×709B5). -6 M~10 -11 PRAME, diluted 10 times in M 425~433 T2 cells pulsed with peptide for 1 hour were co-cultured for 24 hours. Subsequently, iNKT cells were evaluated for activation by simultaneous expression of CD25 and CD69 using flow cytometry. The results showed that PRAME-TCR-iNKT activation was associated with PRAME 425~433 A dose-dependent response to the peptide was demonstrated (Figure 12).

[0266] The cytotoxicity of PRAME-TCR-iNKT against tumor cells was evaluated using fluorescence microscopy. PRAME-TCR-iNKT cells expressing 684A2×709B5 or 684A3×709B5 TCRs were co-cultured with several tumor lines: OVCAR3-GFP, A375-GFP, MCF7-GFP, and A549-GFP. Target-only and unmodified iNKT cells were used as controls. The number of GFP+ cells was recorded using fluorescence microscopy over 72 hours. The results showed that unmodified iNKT cells also exhibited some degree of suppression against target cells, and further suppression of OVCAR3-GFP cell proliferation was observed for 684A2×709B5 TCR-iNKT and 684A3×709B5 TCR-iNKT (Figures 13A-13B). Unmodified iNKT cells did not show suppression of A375-GFP cell proliferation, but suppression of A375-GFP cell proliferation was observed in 684A2×709B5 TCR-iNKT and 684A3×709B5 TCR-iNKT cells (Figures 14A-14B). Unmodified iNKT cells showed some suppression of MCF7-GFP cell proliferation, and the effect of PRAME TCR introduction was minimal (Figures 15A-15B). Unmodified iNKT cells showed some suppression of A549-GFP cell proliferation, and the effect of PRAME TCR introduction was minimal (Figures 16A-16B).

[0267] The cytotoxicity of PRAME-TCR-iNKT was evaluated in the tumor cell line A549-GFP-CD1d, which was modified to express CD1d in the presence of 100 ng / ml aGalCer. The percentage of GFP+ area in A549-GFP-CD1d cells was assessed by fluorescence microscopy over 72 hours. As a result, it was demonstrated that all iNKT cells showed suppression of A549-GFP-CD1d cells in the presence of aGalCer, as aGalCer presented on CD1d is a ligand for killing by endogenous iNKT TCRs. The effect of introducing PRAME TCRs into iNKT was observed to be minimal, indicating that PRAME-TCR expression did not affect the function of iNKT TCRs (Figures 17A-17B).

[0268] The cytotoxicity of PRAME-TCR-iNKT was evaluated against the human melanoma cell line A375-GFP (PRAME+HLA-A2+ cells) in the presence of the anti-PD-1 antibody valstilimab (bal) and the anti-CTLA4 antibody botensilimab (bot). The percentage of GFP+ area in A375-GFP cells was assessed by fluorescence microscopy over 72 hours. Figures 18A and 18B demonstrate that the suppression of A375-GFP proliferation by PRAME TCR was enhanced by the addition of 50 ug / mL of bot and 50 ug / mL of bal at the start of the cytotoxicity assay, but this did not affect unmodified iNKT or isotype controls.

[0269] Cytokine release induced by PRAME-TCR-iNKT cells and bot / bal was evaluated by ELISA. PBMCs were stimulated with 10 ng / mL of SEA (Staphylococcal enterotoxin A), which stimulates specific T cell subsets in PBMCs. Subsequently, PBMCs were co-cultured with PRAME-TCR-iNKT + bot / bal, PRAME-TCR-iNKT + isotype control, bot / bat alone, or isotype alone. PRAME-TCR-iNKT cells were added to PBMCs in a 1:3 ratio, while bot / bal or isotype control was added at 10 ug / mL. Samples were incubated for 96 hours. 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 were co-cultured with PBMCs (Figure 19).

[0270] Embedding by reference All patent and non-patent documents cited above are incorporated herein by reference in their entirety.

Claims

1. A modified T cell receptor (TCR) that binds to a peptide consisting of the amino acid sequence described in Sequence ID No. 60, (i) Variable alpha chain (Vα) complementarity determination region 1 (Vα-CDR1) containing the amino acid sequence of SEQ ID NO: 37, Vα-CDR2 containing the amino acid sequence of SEQ ID NO: 38, and Vα-CDR3 containing the amino acid sequence of SEQ ID NO: 66; and (ii) Variable beta chain (Vβ) Vβ-CDR1 containing the amino acid sequence of SEQ ID NO: 51, Vβ-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and Vβ-CDR3 containing the amino acid sequence of SEQ ID NO: 53 A manipulated TCR, including one.

2. The manipulated TCR according to claim 1, wherein Vα-CDR3 of (i) comprises the amino acid sequence of SEQ ID NO:

40.

3. The manipulated TCR according to claim 1, wherein Vα-CDR3 of (i) comprises the amino acid sequence of SEQ ID NO:

45.

4. The manipulated TCR according to claim 1, wherein Vα-CDR3 of (i) comprises the amino acid sequence of SEQ ID NO:

39.

5. (i) the Vα region containing the amino acid sequence of SEQ ID NO: 4; and the Vβ region containing the amino acid sequence of SEQ ID NO: 24; or (ii) Vα region containing the amino acid sequence of SEQ ID NO: 12; and Vβ region containing the amino acid sequence of SEQ ID NO: 24; or (iii) Vα region containing the amino acid sequence of SEQ ID NO: 2; and Vβ region containing the amino acid sequence of SEQ ID NO: 24 An operated TCR according to any one of claims 1 to 4, including the operated TCR.

6. An operated TCR according to any one of claims 1 to 5, which is a full-length TCR, a soluble TCR, or a single-chain TCR.

7. PRAME ４２５～４３３ A manipulated TCR according to any one of claims 1 to 6, which is a human TCR that specifically binds to the HLA-A*02.01 complex.

8. The manipulated TCR according to any one of claims 1 to 7, wherein when the TCR is expressed on the surface of a T cell, the T cell is activated when it is co-cultured with a second cell that presents a peptide presented in the context of HLA-A*02.

01.

9. An operated TCR according to any one of claims 1 to 8, conjugated to the effects unit.

10. The manipulated TCR according to claim 9, wherein the effector portion is a cytotoxic agent, a cell proliferation inhibitor, a toxin, a radionuclide, a detectable label, or a binding portion.

11. A polynucleotide encoding an manipulated TCR according to any one of claims 1 to 10.

12. The polynucleotide according to claim 11, comprising a Vα region containing the nucleic acid sequence of SEQ ID NO: 3 and a Vβ region containing the nucleic acid sequence of SEQ ID NO:

23.

13. The polynucleotide according to claim 11, comprising a Vα region containing the nucleic acid sequence of SEQ ID NO: 11 and a Vβ region containing the nucleic acid sequence of SEQ ID NO:

23.

14. The polynucleotide according to claim 11, comprising a Vα region containing the nucleic acid sequence of SEQ ID NO: 1 and a Vβ region containing the nucleic acid sequence of SEQ ID NO:

23.

15. A vector comprising a polynucleotide according to any one of claims 11 to 14.

16. The vector according to claim 15, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a baculovirus vector.

17. A cell comprising a polynucleotide according to any one of claims 11 to 14, or a vector according to claim 15 or claim 16.

18. Cells expressing the manipulated TCR according to any one of claims 1 to 10.

19. The cell according to claim 18, which presents a TCR on its cell surface.

20. A cell according to any one of claims 17 to 19, which is a human lymphocyte.

21. A cell according to any one of claims 17 to 20, which 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 according to any one of claims 17 to 21, which is an invariant natural killer T (iNKT) cell.

23. The cell according to claim 22, wherein the iNKT cell expresses the manipulated TCR and the native TCR.

24. The cells according to claim 22 or 23, wherein the iNKT cells express an armoring molecule.

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

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

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

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

29. The method according to claim 27 or claim 28, wherein the cells are iNKT cells.

30. The method according to any one of claims 27 to 29, wherein the vector is a lentiviral vector.

31. An operated TCR produced by the method according to any one of claims 27 to 30.

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

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

34. A method for treating cancer in a subject, comprising administering an effective amount of a pharmaceutical composition comprising iNKT cells manipulated to express the manipulated TCR described in any one of claims 1 to 10 and a pharmaceutically acceptable carrier to the subject.

35. The method according to claim 34, wherein the subject does not undergo lymphocyte removal before treatment with the pharmaceutical composition.

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

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

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

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

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

41. The method according to any one of claims 33 to 40, wherein TCR, polynucleotide, cells, or a pharmaceutical composition are administered intravenously.

Images