T-CELL RECEPTORS TARGETING RAS MUTATIONS AND USES THEREOF - Patent application

Abstract

The subject matter of the present disclosure provides a novel T cell receptor (TCR) that targets a mutated RAS proto-oncogene. The subject matter of the present disclosure further provides a cell that includes such a TCR, and a method of using such a cell to treat cancer associated with RAS. The subject matter of the present disclosure provides a T cell receptor (TCR) that targets a RAS peptide that includes a mutation. In certain embodiments, the RAS peptide includes a G12 mutation. In certain embodiments, the RAS peptide includes a G12D mutation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 192,783, filed May 25, 2021, the entire contents of which are incorporated by reference and priority is claimed.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on May 25, 2022, is named 072734.1354_ST25.txt and is 75,116 bytes in size.

[0003] 1. Introduction The subject matter of the present disclosure provides novel T cell receptors (TCRs) that target mutant RAS proto-oncogenes. The subject matter of the present disclosure further provides cells that contain such TCRs and methods of using such cells to treat cancers associated with mutant RAS. [Background technology]

[0004] 2. Background of the invention Cell-based immunotherapy is a potentially curative therapy for treating cancer.Immunoresponsive cells (e.g., T cells) can be modified to target tumor antigens by introducing genetic material that encodes TCR specific to selected antigens.Targeted T cell therapy using specific TCR has shown clinical success in treating a variety of solid and hematological malignancies.

[0005] Taken together, RAS proteins are the most mutated oncoprotein family in human cancers. Patients with oncogenic mutations encoding RAS proteins (e.g., KRAS, NRAS, and HRAS) typically respond poorly to standard therapies. Activating oncogenic RAS mutations are frequently observed at residue positions 12, 13, and 61 in cancer patients. Among these, G12 is the most frequently mutated residue (89%), most frequently mutated to aspartic acid (G12D), valine (G12V) or cysteine ​​(G12C). Thus, novel therapeutic strategies to identify TCR-targeting epitopes derived from mutated RAS proteins are needed. Furthermore, there is an unmet need to develop strategies that can induce potent cancer eradication with minimal toxicity and immunogenicity. Summary of the Invention

[0006] 3. Summary of the Invention The subject matter of the present disclosure provides a T cell receptor (TCR) that targets a RAS peptide comprising a mutation. In certain embodiments, the RAS peptide comprises a G12 mutation. In certain embodiments, the RAS peptide comprises a G12D mutation. In certain embodiments, the RAS peptide is a 9-mer or a 10-mer. In certain embodiments, the RAS peptide is a 10-mer. In certain embodiments, the RAS peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, the RAS peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:2.

[0007] In certain embodiments, the RAS peptide is associated with an HLA class I complex. In certain embodiments, the HLA class I complex is selected from HLA-A, HLA-B and HLA-C. In certain embodiments, the HLA class I complex is HLA-A. In certain embodiments, the HLA-A is an HLA-A*03 superfamily member. In certain embodiments, the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68 and HLA-A*74. In certain embodiments, the HLA-A*03 superfamily member is HLA-A*11.

[0008] In certain embodiments, the TCR comprises an extracellular domain that binds to a RAS peptide, the extracellular domain comprising an alpha chain and a beta chain, the alpha chain comprising an alpha chain variable region and an alpha chain constant region, and the beta chain comprising a beta chain variable region and a beta chain constant region.

[0009] In certain embodiments, the extracellular domain comprises an α chain variable region and a β chain variable region; a) the α chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and the β chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9 or a conservative modification thereof; b) the α chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, or a conservative modification thereof, and the β chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, or a conservative modification thereof; c) the α chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25 or a conservative modification thereof, and the β chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28 or a conservative modification thereof; d) the alpha chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, and the beta chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof; or e) The alpha chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof, and the beta chain variable region comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46 or a conservative modification thereof.

[0010] In certain embodiments, a) the α chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, and the β chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 or a conservative modification thereof; b) the α chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and the β chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18 or a conservative modification thereof; c) the α chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and the β chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof; d) the α chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof, and the β chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof; or e) the alpha chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof, and the beta chain variable region comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 or a conservative modification thereof.

[0011] In certain embodiments, a) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof; b) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof; c) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:24 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26 or a conservative modification thereof; d) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof; or e) The alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof, and the beta chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof.

[0012] In certain embodiments, a) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; b) the alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; c) the alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25; d) the alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; or e) The alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0013] In certain embodiments, a) the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; b) the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; c) the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28; d) the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:38; or e) The β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47.

[0014] In certain embodiments, a) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; b) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; c) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; d) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; or e) The α chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 45, and the β chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 58, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 46, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 47.

[0015] In a specific embodiment, the alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the beta chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38.

[0016] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 39, or SEQ ID NO: 48. In certain embodiments, the alpha chain variable region comprises ...39.

[0017] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:40, or SEQ ID NO:49. In certain embodiments, the β chain variable region comprises ...40.

[0018] In certain embodiments, a) the α chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:10, and the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:11; b) the α chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:20, and the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:21; c) the α chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:29, and the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:30; d) the α chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:39, and the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:40; or e) the α chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:48, and the β chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:49.

[0019] In certain embodiments, a) the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10 and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:11; b) the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:20 and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:21; c) the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29 and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:30; d) the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:39 and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40; or e) the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:48 and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:49.

[0020] In a specific embodiment, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:39, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40.

[0021] In certain embodiments, a) the α chain comprises the amino acid sequence set forth in SEQ ID NO:12 and the β chain comprises the amino acid sequence set forth in SEQ ID NO:13; b) the α chain comprises the amino acid sequence set forth in SEQ ID NO:22 and the β chain comprises the amino acid sequence set forth in SEQ ID NO:23; c) the α chain comprises the amino acid sequence set forth in SEQ ID NO:31 and the β chain comprises the amino acid sequence set forth in SEQ ID NO:32; d) the α chain comprises the amino acid sequence set forth in SEQ ID NO:41 and the β chain comprises the amino acid sequence set forth in SEQ ID NO:42; or e) the α chain comprises the amino acid sequence set forth in SEQ ID NO:50 and the β chain comprises the amino acid sequence set forth in SEQ ID NO:51.

[0022] In a specific embodiment, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:41 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0023] In certain embodiments, the extracellular domain binds to the same RAS peptide as a reference TCR or functional fragment thereof, the reference TCR or functional fragment thereof comprising an alpha chain variable region and a beta chain variable region; a) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; b) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19; c) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; d) the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; or e) The α chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 45, and the β chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 58, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 46, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 47.

[0024] In certain embodiments, the TCR is recombinantly expressed and / or expressed from a vector. In certain embodiments, the TCR does not bind to a RAS peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3.

[0025] In certain embodiments, the alpha chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54. In certain embodiments, the alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54.

[0026] In certain embodiments, the β chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57. In certain embodiments, the β chain constant region comprises the amino acid sequence set forth in SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57.

[0027] The subject matter of the present disclosure provides a nucleic acid encoding a TCR disclosed herein. The subject matter of the present disclosure further provides a cell comprising a TCR disclosed herein or a nucleic acid disclosed herein. In certain embodiments, the cell is transduced with the TCR. In certain embodiments, the TCR is constitutively expressed on the surface of the cell. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is selected from the group consisting of a T cell and a pluripotent stem cell from which lymphoid cells can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell, a γδ T cell, a natural killer T cell (NK-T), a stem cell memory T cell (T SCM ), central memory T cells (T CM ), and effector memory T cells (T EM In certain embodiments, the T cell is a γδ T cell. In certain embodiments, the T cell is an NK-T cell. In certain embodiments, the TCR or nucleic acid is integrated into a locus in the genome of the cell (e.g., a T cell). In certain embodiments, the locus is selected from the group consisting of the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus.

[0028] The presently disclosed subject matter also provides a composition comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0029] The presently disclosed subject matter further provides a vector comprising a nucleic acid disclosed herein, hi certain embodiments, the vector is a gamma-retroviral vector.

[0030] Furthermore, the subject matter of the present disclosure provides a method for producing a cell that binds to a RAS peptide that comprises a G12 mutation. In certain embodiments, the method comprises introducing a nucleic acid or vector disclosed herein into a cell.

[0031] Furthermore, the subject matter of the present disclosure provides a method for treating and / or preventing tumors associated with RAS in a subject.In certain embodiments, the method comprises administering to a subject the cell or composition disclosed herein.In certain embodiments, the tumor is associated with RAS mutation.In certain embodiments, the RAS mutation is G12D mutation.

[0032] In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, cholangiocarcinoma / bile duct cancer, lung cancer, ovarian cancer, esophageal cancer, gastric cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, salivary gland cancer, melanoma, and multiple myeloma. In certain embodiments, the tumor is pancreatic cancer. In certain embodiments, the tumor is colorectal cancer. In certain embodiments, the subject is a human. In certain embodiments, the subject comprises HLA-A. In certain embodiments, the HLA-A is an HLA-A*03 superfamily member. In certain embodiments, the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74. In a specific embodiment, the HLA-A*03 superfamily member is HLA-A*11.

[0033] Further, the presently disclosed subject matter provides a use of a cell or composition disclosed herein for treating and / or preventing a tumor associated with RAS in a subject. In certain embodiments, the tumor is associated with a RAS mutation. In certain embodiments, the RAS mutation is a G12D mutation. In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, cholangiocarcinoma / bile duct cancer, lung cancer, ovarian cancer, esophageal cancer, gastric cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, salivary gland cancer, melanoma, and multiple myeloma. In certain embodiments, the tumor is pancreatic cancer. In certain embodiments, the subject is a human. In certain embodiments, the subject comprises HLA-A. In certain embodiments, the HLA-A is an HLA-A*03 superfamily member. In certain embodiments, the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68 and HLA-A*74. In certain embodiments, the HLA-A*03 superfamily member is HLA-A*11. 4. Brief description of the drawings [Brief description of the drawings]

[0034] The following detailed description is given by way of example and is not intended to limit the invention to the particular embodiments described and may be understood in conjunction with the accompanying drawings.

[0035] [Figure 1A]Figure 1A-1C show a functional screen to elucidate the HLA-restricted immunopeptidome of endogenously processed and presented shared or "public" neo-antigens (NeoAgs) arising from mutant KRAS proteins. Figure 1A shows a schematic of the HLA immunoprecipitation (IP) / tandem mass spectrometry (MS / MS) screen using COS-7 as artificial antigen presenting cells (aAPCs). Figure 1B shows a validation MS "mirror" plot of HLA-A*11:01-restricted KRAS(G12D) peptides eluted from the surface of PANC1, a pancreatic cancer cell line that physiologically expresses HLA-A*11:01 and KRAS(G12D) (upper panel). Synthetic peptides were run as controls (lower panel). Figure 1C shows measurements of the relative stability of neopeptide / HLA complexes on the cell surface of TAP1 / 2-deficient T2 cells electroporated with in vitro transcribed RNA encoding HLA-A*11:01. X = preferred HLA anchor residue; X = position of hotspot mutation. SEQ ID NOs: 1 to 3. [Figure 1B-1C] Figure 1A-1C show a functional screen to elucidate the HLA-restricted immunopeptidome of endogenously processed and presented shared or "public" neo-antigens (NeoAgs) arising from mutant KRAS proteins. Figure 1A shows a schematic of the HLA immunoprecipitation (IP) / tandem mass spectrometry (MS / MS) screen using COS-7 as artificial antigen presenting cells (aAPCs). Figure 1B shows a validation MS "mirror" plot of HLA-A*11:01-restricted KRAS(G12D) peptides eluted from the surface of PANC1, a pancreatic cancer cell line that physiologically expresses HLA-A*11:01 and KRAS(G12D) (upper panel). Synthetic peptides were run as controls (lower panel). Figure 1C shows measurements of the relative stability of neopeptide / HLA complexes on the cell surface of TAP1 / 2-deficient T2 cells electroporated with in vitro transcribed RNA encoding HLA-A*11:01. X = preferred HLA anchor residue; X = position of hotspot mutation. SEQ ID NOs: 1 to 3.

[0036] [Diagram 2]Figure 2 shows a graphical comparison of amino acid sequence homology and location of hotspot mutations in the RAS family of cancer proteins. * = location of hotspot mutation; vertical bar = site of sequence variation between RAS family members. The zoomed area shows the sequences of the hypervariable regions of all four RAS proteins (SEQ ID NOs: 143-146).

[0037] [Figure 3A-3B] Figure 3A and Figure 3B show the discovery of a panel of HLA-A*11:01-restricted mutant RAS-specific TCR gene sequences and the description of the variable chains. Figure 3A shows T cells derived from either HLA-A*11:01+ healthy donors (HDs) or HLA-A*11:01+ patients with a history of KRAS(G12D) cancer stimulated in vitro with autologous antigen-presenting cells presenting KRAS(G12D). Individual cultures were screened for the presence of mutant RAS-specific T cells using high-order peptide / HLA-I reagents loaded with a mutant 10-mer epitope (SEQ ID NO:2) identified by mass spectrometry. Positive wells were labeled with barcoded dextramers and subjected to single-cell V(D)J sequencing to retrieve paired αβTCR gene sequences of mutant RAS-specific T cell clonotypes. Figure 3B shows five unique mutant RAS-specific TCRs recovered from healthy donor (n=1) or patient-derived samples (n=4). All five TCRs were composed of unique alpha and beta variable chain segments and CDR3 loop lengths.

[0038] [Figure 4A]Figure 4A and Figure 4B show functional validation and measurement of co-receptor dependency of healthy donor (HD) and patient derived TCR gene sequences specific for RAS(G12D) exposed NeoAg. Figure 4A shows FACS plots validating the functionality of five genetically distinct HD and patient derived TCR gene sequences. Non-specific T cells were individually transduced with the indicated TCRs. The frequency of intracellular TNFα production is shown after gating on transduced T cells following co-culture with Cos7 target cells electroporated with the gene encoding HLA-A*11:01 and either WT KRAS or KRAS(G12D). Figure 4B shows summary bar graphs depicting the frequency (± standard error of the mean, SEM) of intracellular TNFα production in open repertoire CD8+ (left) or CD4+ (right) T cells expressing individual RAS-specific TCRs after coculture with Cos7 target cells (G12D) electroporated with the gene encoding HLA-A*11:01 and either WT KRAS or KRAS (n=3 replicates per condition). [Figure 4B] Figure 4A and Figure 4B show functional validation and measurement of co-receptor dependency of healthy donor (HD) and patient derived TCR gene sequences specific for RAS(G12D) exposed NeoAg. Figure 4A shows FACS plots validating the functionality of five genetically distinct HD and patient derived TCR gene sequences. Non-specific T cells were individually transduced with the indicated TCRs. The frequency of intracellular TNFα production is shown after gating on transduced T cells following co-culture with Cos7 target cells electroporated with the gene encoding HLA-A*11:01 and either WT KRAS or KRAS(G12D). Figure 4B shows summary bar graphs depicting the frequency (± standard error of the mean, SEM) of intracellular TNFα production in open repertoire CD8+ (left) or CD4+ (right) T cells expressing individual RAS-specific TCRs after coculture with Cos7 target cells (G12D) electroporated with the gene encoding HLA-A*11:01 and either WT KRAS or KRAS (n=3 replicates per condition).

[0039] [Figure 5-1] FIG. 5 shows reactivity to minimal epitopes of different lengths (10mer vs. 9mer) by individual RAS(G12D)-specific TCR panel members. [Figure 5-2] FIG. 5 shows reactivity to minimal epitopes of different lengths (10mer vs. 9mer) by individual RAS(G12D)-specific TCR panel members.

[0040] [Figure 6A-6B] Figure 6A and Figure 6B show the functional avidity of T cells transduced with RAS-specific TCR. Figure 6A shows the intracellular TNFα production measured in CD8+ (left) or CD4+ (right) TCR+ T cells. Figure 6B shows the EC50 value of each individual TCR in CD8+ or CD4+ T cells.

[0041] [Figure 7A-7B] Figures 7A and 7B show the recognition of endogenous levels of KRAS(G12D) in pancreatic tumor lines by mutant RAS-specific TCR panel members. Figure 7A shows open repertoire T cells retrovirally transduced with individual recovered TCR gene sequences and co-cultured with either cholangiocarcinoma HuCCT1 cell lines in the presence or absence of pan-HLA class-I blocking antibodies. Figure 7B shows open repertoire T cells retrovirally transduced with individual recovered TCR gene sequences and co-cultured with either pancreatic cancer PANC-1 cell lines in the presence or absence of pan HLA class-I blocking antibodies.

[0042] [Figure 8A-8B] Figure 8A and Figure 8B show tumor cell lysis of HLA-A*11:01 expressing KRAS (G12D) tumor line (PANC-1) by RAS-specific TCR panel members. Figure 8A shows tumor lysis curves of individual library members in the presence or absence of pan-class I blocking antibodies. Figure 8B shows peak tumor lysis measured after 48 hours of co-culture.

[0043] [Figure 9A] Figures 9A and 9B show the cross-protective potential of RAS-exposed neoantigen (NeoAg)-specific TCRs against alternative mutant RAS proteins. Figure 9A shows a representative FACS plot demonstrating the cross-protective function of RAS(G12D)-specific TCRs (TCR4). Figure 9B shows a summary bar graph (n=3 replicates per condition) of the frequency of intracellular TNFα-producing TCR+CD8+ T cells in response to WT versus mutant RAS isoforms. [Figure 9B] Figures 9A and 9B show the cross-protective potential of RAS-exposed neoantigen (NeoAg)-specific TCRs against alternative mutant RAS proteins. Figure 9A shows a representative FACS plot demonstrating the cross-protective function of RAS(G12D)-specific TCRs (TCR4). Figure 9B shows a summary bar graph (n=3 replicates per condition) of the frequency of intracellular TNFα-producing TCR+CD8+ T cells in response to WT versus mutant RAS isoforms.

[0044] [Figure 10A-10B] 10A-10E show heat maps depicting the level of INF-γ production for each indexed amino acid. The sequence and position of the natural RAS mutant peptide are listed at the top of each heat map (SEQ ID NO:2). Substituted amino acids are identified along each Y-axis column. The index peptide at each position is identified with a dotted box. The relative influence of each amino acid at every position was used to determine the TCR "preference" of that amino acid substitution at every position within the peptide. The TCR logo plots thus generated are shown above the individual TCR heat maps. FIG. 10A shows a heat map of TCR1. FIG. 10B shows a heat map of TCR2. FIG. 10C shows a heat map of TCR3. FIG. 10D shows a heat map of TCR4. FIG. 10E shows a heat map of TCR5. [Fig. 10C-10D]10A-10E show heat maps depicting the level of INF-γ production for each indexed amino acid. The sequence and position of the natural RAS mutant peptide are listed at the top of each heat map (SEQ ID NO:2). Substituted amino acids are identified along each Y-axis column. The index peptide at each position is identified with a dotted box. The relative influence of each amino acid at every position was used to determine the TCR "preference" of that amino acid substitution at every position within the peptide. The TCR logo plots thus generated are shown above the individual TCR heat maps. FIG. 10A shows a heat map of TCR1. FIG. 10B shows a heat map of TCR2. FIG. 10C shows a heat map of TCR3. FIG. 10D shows a heat map of TCR4. FIG. 10E shows a heat map of TCR5. [Figure 10E] 10A-10E show heat maps depicting the level of INF-γ production for each indexed amino acid. The sequence and position of the natural RAS mutant peptide are listed at the top of each heat map (SEQ ID NO:2). Substituted amino acids are identified along each Y-axis column. The index peptide at each position is identified with a dotted box. The relative influence of each amino acid at every position was used to determine the TCR "preference" of that amino acid substitution at every position within the peptide. The TCR logo plots thus generated are shown above the individual TCR heat maps. FIG. 10A shows a heat map of TCR1. FIG. 10B shows a heat map of TCR2. FIG. 10C shows a heat map of TCR3. FIG. 10D shows a heat map of TCR4. FIG. 10E shows a heat map of TCR5.

[0045] [Figure 11A-11B]11A-11E show possible cross-reactivity of RAS-specific TCRs. Levels of IFN-γ were determined by ELISA. IFN-γ levels are shown in pg / mL on the y-axis, with a background threshold set at 50 pg / mL, as identified by the dotted line. FIG. 11A shows the levels of IFN-γ produced by TCR1 incubated with individual peptides listed in Table 7. FIG. 11B shows the levels of IFN-γ produced by TCR2 incubated with individual peptides listed in Table 8. FIG. 11C shows the levels of IFN-γ produced by TCR3 incubated with individual peptides listed in Table 9. FIG. 11D shows the levels of IFN-γ produced by TCR4 incubated with individual peptides listed in Table 10. FIG. 11E shows the levels of IFN-γ produced by TCR5 incubated with individual peptides listed in Table 11. [Fig. 11C-11D] 11A-11E show possible cross-reactivity of RAS-specific TCRs. Levels of IFN-γ were determined by ELISA. IFN-γ levels are shown in pg / mL on the y-axis, with a background threshold set at 50 pg / mL, as identified by the dotted line. FIG. 11A shows the levels of IFN-γ produced by TCR1 incubated with individual peptides listed in Table 7. FIG. 11B shows the levels of IFN-γ produced by TCR2 incubated with individual peptides listed in Table 8. FIG. 11C shows the levels of IFN-γ produced by TCR3 incubated with individual peptides listed in Table 9. FIG. 11D shows the levels of IFN-γ produced by TCR4 incubated with individual peptides listed in Table 10. FIG. 11E shows the levels of IFN-γ produced by TCR5 incubated with individual peptides listed in Table 11. [Figure 11E]11A-11E show possible cross-reactivity of RAS-specific TCRs. Levels of IFN-γ were determined by ELISA. IFN-γ levels are shown in pg / mL on the y-axis, with a background threshold set at 50 pg / mL, as identified by the dotted line. FIG. 11A shows the levels of IFN-γ produced by TCR1 incubated with individual peptides listed in Table 7. FIG. 11B shows the levels of IFN-γ produced by TCR2 incubated with individual peptides listed in Table 8. FIG. 11C shows the levels of IFN-γ produced by TCR3 incubated with individual peptides listed in Table 9. FIG. 11D shows the levels of IFN-γ produced by TCR4 incubated with individual peptides listed in Table 10. FIG. 11E shows the levels of IFN-γ produced by TCR5 incubated with individual peptides listed in Table 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] 5. Detailed Description of the Invention The subject matter of the present disclosure provides a TCR that targets a RAS that includes a mutation, such as a G12D mutation. Additionally, the subject matter of the present disclosure provides cells (e.g., T cells) that include a RAS-targeting TCR, and methods of using such cells to treat tumors associated with a RAS mutation(s).

[0047] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow. 5.1. Definition; 5.2.RAS; 5.3.TCR; 5.4.Cells 5.5. Genetic modification of nucleic acids and cells; 5.6. Formulation and Administration; and 5.7. Method of treatment.

[0048] 5.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed.,1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale&Marham, The Harper Collins Dictionary of Biology (1991).

[0049] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0050] As used herein, the term "cell population" refers to a group of at least two cells that express similar or different phenotypes. In a non-limiting example, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells that express similar or different phenotypes.

[0051] As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which, when associated with the appropriate control elements, is capable of replicating and transferring genetic sequences to a cell. Thus, the term includes cloning and expression vehicles, as well as viral and plasmid vectors.

[0052] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, e.g., a recombinant DNA molecule, that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, among other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0053] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of the TCR, which is the hypervariable region of the TCR α and β chains. Generally, TCRs contain three CDRs in the α chain variable region and three CDRs in the β chain variable region. CDRs provide the majority of contact residues for the binding of TCR to antigen or epitope. The CDR regions can be delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), the Chothia numbering system (Chothia et al., J Mol Biol. (1987) 196: 901-17), the AbM numbering system (Abhinandan et al., Mol. Immunol. 2008, 45, 3832-3839), or the IMGT numbering system (http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html, accessible at http: / / www.imgt.org / IMGTindex / numbering.php). In certain embodiments, the CDR regions are delineated using the IMGT numbering system.

[0054] The term "substantially homologous" or "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or even about 99% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0055] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications. In an exemplary approach to determining the degree of identity, the BLAST program can be used, e.g. -3 and e-100 A probability score between indicates closely related sequences.

[0056] As used herein, the percentage of homology between two amino acid sequences is equivalent to the percentage of identity between two sequences.The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology%=number of identical positions / total number of positions×100), taking into account the number of gaps that need to be introduced for optimal alignment of two sequences and the length of each gap.Comparing sequences and determining the percentage of identity between two sequences can be achieved using mathematical algorithms.

[0057] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0058] Additionally or alternatively, the amino acid sequence of the subject matter of the present disclosure can be further used as a "query sequence" to perform a search against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to specific sequences disclosed herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0059] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the TCR of the present disclosure, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Amino acids can be classified into groups according to physicochemical properties such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified according to charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine and tyrosine, and non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered TCR can be tested for retained function (i.e., the function shown in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a particular sequence or CDR region are altered.

[0060] As used herein, the term "disease" refers to any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include cellular neoplasms or pathogen infections.

[0061] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to palliate, ameliorate, stabilize, reverse or delay progression of a disease (e.g., tumor), prevent or delay recurrence of a tumor, or reduce pathological consequences of a disease (e.g., tumor). An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person of ordinary skill in the art. When determining the appropriate dosage to achieve an effective amount, several factors are usually taken into consideration. These factors include the subject's age, sex and weight, the condition being treated, the severity of the condition, and the form and effective concentration of the immunoresponsive cells being administered.

[0062] As used herein, the term "tumor" refers to an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Tumors include benign and malignant tumors (known as "cancer"). Benign tumors can grow large but do not spread or invade nearby tissues or other parts of the body. Malignant tumors can spread or invade nearby tissues. Malignant tumors can also spread to other parts of the body via the blood and lymphatic systems. Tumors are also called neoplasms. In certain embodiments, the tumor is cancer.

[0063] As used herein, the term "immunoresponsive cell" refers to a cell or its precursor or progeny that functions in the immune response.

[0064] As used herein, the term "modulate" refers to a positive or negative change. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0065] As used herein, the term "increase" refers to a positive change of at least about 5%, including, but not limited to, a positive change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0066] As used herein, the term "reduce" refers to a negative change of at least about 5%, including, but not limited to, a negative change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0067] As used herein, the terms "isolated," "purified," or "biologically pure" refer to a material that is free to various degrees from components that normally accompany it as found in its native state. "Isolated" indicates a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or polypeptide of the subject matter disclosed herein is purified if it is substantially free of cellular material, viral material, or medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0068] As used herein, the term "isolated cell" refers to a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0069] As used herein, the term "treat" or "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prevention or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, prevention of disease occurrence or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing of disease progression rate, improvement or alleviation of disease state, and remission or improvement of prognosis. By preventing the progression of disease or disorder, treatment can prevent the deterioration of the disorder in affected or diagnosed subjects or subjects suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in subjects at risk of or suspected of having the disorder.

[0070] An "individual" or "subject" herein is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, livestock, sports animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys.

[0071] 5.2.RAS RAS is a family of oncoproteins that encode small GTPases involved in the regulation of cell growth, differentiation and survival. In humans, the RAS family includes HRAS, NRAS, and KRAS. The KRAS gene has two splice variants, KRAS4A and KRAS4B. Although expression of all isoforms is nearly ubiquitous, they show quantitative and qualitative differences in expression depending on the tissue and / or developmental stage.

[0072] RAS proteins contain two domains: a G domain that binds guanosine nucleotides and a C-terminal hypervariable region. The G domain is highly conserved among HRAS, NRAS, KRAS4A and KRAS4B and is responsible for binding and hydrolyzing guanine nucleotides. The hypervariable region undergoes differential post-translational modifications that lead to isoform-specific intracellular organization. RAS proteins act as binary molecular switches and cycle between an inactive GDP-bound state and an active GTP-bound state. Upon activation, RAS proteins recruit and activate proteins such as c-Raf and PI3-kinase that result in cell proliferation, migration and protection from apoptosis.

[0073] RAS mutations play a key role in driving some of the most common and deadly carcinomas, including pancreatic, lung, and colorectal cancers, among others. As shown in Figure 2, the conserved G domain contains several positions for hotspot mutations, including G12, G13, and Q61. The most frequent mutation in the RAS gene occurs at codon 12 (i.e., G12A / C / D / F / L / R / S / V), which accounts for 98% of RAS mutations. The most common RAS mutation among cancers is G12D, which is a single point mutation with a glycine to aspartic acid substitution at codon 12.

[0074] 5.3.T cell receptor (TCR) TCRs are disulfide-linked heterodimeric proteins consisting of two variable chains expressed as part of a non-covalent complex with the invariant CD3 chain molecule (CD3δ, CD3ε, CD3γ, CD3ζ). TCRs are found on the surface of T cells and are responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCRs comprise α and β chains (encoded by TRA and TRB, respectively). In certain embodiments, TCRs comprise γ and δ chains (encoded by TRG and TRD, respectively).

[0075] Each chain of the TCR contains two extracellular domains: a variable region and a constant region. The constant region is proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail (i.e., the intracellular domain). The variable region binds to the peptide / MHC complex. The variable regions of both chains each have three complementarity determining regions (CDRs).

[0076] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε and CD247ζ / ζ or ζ / η. T cells expressing the TCR complex are activated when the TCR complex engages its cognate peptide antigen / MHC (peptide / MHC).

[0077] The subject of the present disclosure provides a recombinant TCR. In certain embodiments, the recombinant TCR is different from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the recombinant TCR is different from any naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues. In certain embodiments, the recombinant TCR is modified from the naturally occurring TCR by at least one amino acid residue. In certain embodiments, the recombinant TCR is modified from the naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues.

[0078] In certain embodiments, the TCRs of the present disclosure target or bind to a RAS peptide comprising a mutation ("mutated RAS peptide"). In certain embodiments, the mutation is a point mutation. In certain embodiments, the mutation is a G12 mutation. In certain embodiments, the RAS peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the RAS peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:2. In certain embodiments, the TCRs of the present disclosure do not bind to wild-type RAS. In certain embodiments, the TCRs of the present disclosure do not bind to a RAS peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3. SEQ ID NOs:1-3 are provided below. VVGADGVGK [SEQ ID NO: 1] VVVGADGVGK [SEQ ID NO: 2] VVVGAGGVGK [SEQ ID NO: 3]

[0079] In certain embodiments, the TCR of the present disclosure targets or binds to KRAS, including a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the TCR of the present disclosure targets or binds to KRAS, including a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0080] In certain embodiments, the TCR of the present disclosure targets or binds to NRAS, which comprises a RAS peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the TCR of the present disclosure targets or binds to NRAS, which comprises a RAS peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0081] In certain embodiments, the TCR of the present disclosure targets or binds to HRAS, which includes a RAS peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the TCR of the present disclosure targets or binds to HRAS, which includes a RAS peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the TCR of the present disclosure targets or binds to RAS peptides associated with HLA class I complexes, such as HLA-A, HLA-B and HLA-C.

[0082] In certain embodiments, the TCR of the present disclosure targets or binds to the RAS peptide associated with the HLA-A*03 superfamily (e.g., in an HLA-A*03 superfamily-dependent manner). In certain embodiments, the HLA*A03 superfamily members include, but are not limited to, HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68 and HLA-A*74 alleles and suballeles. In certain embodiments, the TCR of the present disclosure targets or binds to the RAS peptide associated with the HLA-A*11 molecule.

[0083] TCR Variable Region In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof. SEQ ID NOs: 4-6 are disclosed in Table 1. In certain embodiments, the α chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0084] In a specific embodiment, the extracellular domain of the TCR comprises a β chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7 or a conservative modification thereof, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8 or a conservative modification thereof, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9 or a conservative modification thereof. SEQ ID NOs: 7 to 9 are disclosed in Table 1. In a specific embodiment, the β chain variable region comprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9.

[0085] In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0086] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10. For example, the alpha chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 10 is shown in Table 1.

[0087] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 11. For example, the β chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11. SEQ ID NO: 11 is shown in Table 1.

[0088] In certain embodiments, the α chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10, and the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0089] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain comprising an alpha chain variable region and an alpha chain constant region. In certain embodiments, the alpha chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12. For example, the alpha chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0090] In certain embodiments, the extracellular domain of the TCR comprises a β chain comprising a β chain variable region and a β chain constant region. In certain embodiments, the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. For example, the β chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the β chain comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0091] In certain embodiments, the α chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12, and the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the α chain comprises the amino acid sequence set forth in SEQ ID NO: 12, and the β chain comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the TCR is referred to as "TCR1." In certain embodiments, TCR1 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0092] In certain embodiments, the CDR sequences above, including in Table 1, are depicted using the IMGT numbering system.

[0093] [Table 1]

[0094] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof. SEQ ID NOs: 14-16 are disclosed in Table 2. In certain embodiments, the α chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0095] In a specific embodiment, the extracellular domain of the TCR comprises a β chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17 or a conservative modification thereof, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 18 or a conservative modification thereof, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 19 or a conservative modification thereof. SEQ ID NOs: 17 to 19 are disclosed in Table 2. In a specific embodiment, the β chain variable region comprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 18, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 19.

[0096] In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19 or a conservative modification thereof. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0097] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20. For example, the alpha chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NO: 20 is shown in Table 2.

[0098] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 21. For example, the β chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is shown in Table 2. In certain embodiments, the α chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20, and the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0099] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain comprising an alpha chain variable region and an alpha chain constant region. In certain embodiments, the alpha chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:22. For example, the alpha chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:22. In certain embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:22.

[0100] In certain embodiments, the extracellular domain of the TCR comprises a β chain comprising a β chain variable region and a β chain constant region. In certain embodiments, the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:23. For example, the β chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, the β chain comprises the amino acid sequence set forth in SEQ ID NO:23.

[0101] In certain embodiments, the α chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:22, and the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, the α chain comprises the amino acid sequence set forth in SEQ ID NO:22, and the β chain comprises the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, the TCR is referred to as "TCR2." In certain embodiments, TCR2 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0102] In certain embodiments, the CDR sequences above, including Table 2, are depicted using the IMGT numbering system.

[0103] [Table 2]

[0104] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof. SEQ ID NOs: 15, 24 and 25 are disclosed in Table 3. In certain embodiments, the alpha chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25.

[0105] In a specific embodiment, the extracellular domain of the TCR comprises a β chain variable region comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 28 or a conservative modification thereof. SEQ ID NOs: 26 to 28 are disclosed in Table 3. In a specific embodiment, the β chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 28.

[0106] In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0107] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 29. For example, the alpha chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29. SEQ ID NO: 29 is shown in Table 3.

[0108] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 30. For example, the β chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30. SEQ ID NO: 30 is shown in Table 3.

[0109] In certain embodiments, the α chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:29, and the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:30. In certain embodiments, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO:30.

[0110] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain comprising an alpha chain variable region and an alpha chain constant region. In certain embodiments, the alpha chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:31. For example, the alpha chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:31. In certain embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:31.

[0111] In certain embodiments, the extracellular domain of the TCR comprises a β chain comprising a β chain variable region and a β chain constant region. In certain embodiments, the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32. For example, the β chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the β chain comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0112] In certain embodiments, the α chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:31, and the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:32. In certain embodiments, the α chain comprises the amino acid sequence set forth in SEQ ID NO:31, and the β chain comprises the amino acid sequence set forth in SEQ ID NO:32. In certain embodiments, the TCR is referred to as "TCR3." In certain embodiments, the TCR3 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0113] In certain embodiments, the CDR sequences above, including in Table 3, are depicted using the IMGT numbering system.

[0114] [Table 3]

[0115] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof. SEQ ID NOs: 33-35 are disclosed in Table 4. In certain embodiments, the α chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0116] In a specific embodiment, the extracellular domain of the TCR comprises a β chain variable region comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 36 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 37 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 38 or a conservative modification thereof. SEQ ID NOs: 36 to 38 are disclosed in Table 4. In a specific embodiment, the β chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 38.

[0117] In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the TCR comprises an α chain comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0118] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 39. For example, the alpha chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39. SEQ ID NO: 39 is shown in Table 4.

[0119] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 40. For example, the β chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 40. SEQ ID NO: 40 is shown in Table 4.

[0120] In certain embodiments, the α chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 39, and the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0121] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain comprising an alpha chain variable region and an alpha chain constant region. In certain embodiments, the alpha chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:41. For example, the alpha chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:41. In certain embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:41.

[0122] In certain embodiments, the extracellular domain of the TCR comprises a β chain comprising a β chain variable region and a β chain constant region. In certain embodiments, the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42. For example, the β chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the β chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0123] In certain embodiments, the α chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:41, and the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:42. In certain embodiments, the α chain comprises the amino acid sequence set forth in SEQ ID NO:41, and the β chain comprises the amino acid sequence set forth in SEQ ID NO:42. In certain embodiments, the TCR is referred to as "TCR4." In certain embodiments, TCR4 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, TCR4 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0124] In certain embodiments, the CDR sequences above, including in Table 4, are depicted using the IMGT numbering system.

[0125] [Table 4]

[0126] In certain embodiments, the extracellular domain of the TCR comprises an α chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof. SEQ ID NOs: 43-45 are disclosed in Table 5. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0127] In certain embodiments, the extracellular domain of the TCR comprises a β chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47 or a conservative modification thereof. SEQ ID NOs:58, 46 and 47 are disclosed in Table 5. In certain embodiments, the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47.

[0128] In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof. In certain embodiments, the α chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45, and the β chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the TCR comprises an α chain comprising the amino acid sequence set forth in SEQ ID NO: 48.

[0129] In certain embodiments, the alpha chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 48. For example, the alpha chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the alpha chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is shown in Table 5.

[0130] In certain embodiments, the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 49. For example, the β chain variable region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is shown in Table 5.

[0131] In certain embodiments, the α chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 48, and the β chain variable region comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the α chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48, and the β chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0132] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain comprising an alpha chain variable region and an alpha chain constant region. In certain embodiments, the alpha chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:50. For example, the alpha chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:50. In certain embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:50.

[0133] In certain embodiments, the extracellular domain of the TCR comprises a β chain comprising a β chain variable region and a β chain constant region. In certain embodiments, the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:51. For example, the β chain comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:51. In certain embodiments, the β chain comprises the amino acid sequence set forth in SEQ ID NO:51.

[0134] In certain embodiments, the α chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:50, and the β chain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:51. In certain embodiments, the α chain comprises the amino acid sequence set forth in SEQ ID NO:50, and the β chain comprises the amino acid sequence set forth in SEQ ID NO:51. In certain embodiments, the TCR is referred to as "TCR5." In certain embodiments, TCR5 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, TCR5 binds to a RAS peptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0135] In certain embodiments, the CDR sequences above, including Table 5, are depicted using the IMGT numbering system.

[0136] [Table 5]

[0137] In certain embodiments, the amino acid sequence of the α chain variable region and / or the β chain variable region has at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a designated sequence (e.g., SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:48, and SEQ ID NO:49) and contains modifications including, but not limited to, substitutions (e.g., conservative substitutions), insertions, or deletions to the designated sequence(s), but retains the ability to bind to a mutant RAS peptide (e.g., a G12D mutant RAS peptide). In certain embodiments, such modifications are not within the CDR domains of the variable regions.

[0138] In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:48 or SEQ ID NO:49. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the CDR of the extracellular domain. In certain embodiments, the extracellular domain comprises an α chain variable region and / or a β chain variable region sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:48 and SEQ ID NO:49, including post-translational modifications of the sequence (SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:48 or SEQ ID NO:49).

[0139] Constant Region In certain embodiments, a TCR of the disclosure comprises an alpha chain constant region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54. In certain embodiments, the alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 53. In certain embodiments, the alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0140] In certain embodiments, the TCR disclosed herein comprises a β chain constant region comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57. In certain embodiments, the β chain constant region comprises the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, the β chain constant region comprises the amino acid sequence set forth in SEQ ID NO:56. In certain embodiments, the β chain constant region comprises the amino acid sequence set forth in SEQ ID NO:57. SEQ ID NOs:53-57 are shown below: [ka]

[0141] 5.3.2. TCRs that bind the same RAS peptide as the TCR clonotype The subject matter of the present disclosure further provides a TCR that binds the same RAS peptide (e.g., a G12D mutant RAS peptide) as a TCR disclosed herein (e.g., a TCE disclosed in Section 5.3.1). In certain embodiments, the TCR binds the same RAS peptide (e.g., a G12D mutant RAS peptide) as a reference TCR or a functional fragment thereof, the RAS peptide comprising, for example, the alpha chain variable region CDR1, CDR2 and CDR3 sequences and the beta chain variable region CDR1, CDR2 and CDR3 sequences of any one of the TCRs disclosed herein (e.g., those disclosed in Section 5.3.1). In certain embodiments, the TCR binds the same RAS peptide (e.g., a G12D mutant RAS peptide) as a reference TCR or a functional fragment thereof comprising, for example, the alpha chain variable region and the beta chain variable region sequences of any one of the TCRs disclosed herein (e.g., those disclosed in Section 5.3.1).

[0142] 5.3.3. TCRs with specific CDR 3 sequences It is well known in the art that the CDR3 domain can alone, independent of the CDR1 and / or CDR2 domain(s), determine the binding specificity of a TCR or a functional fragment thereof to a cognate antigen, and that multiple TCRs with the same binding specificity can be predictably generated based on a common CDR3 sequence.

[0143] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof.

[0144] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, or a conservative modification thereof, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, or a conservative modification thereof.

[0145] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof.

[0146] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof.

[0147] In certain embodiments, the extracellular domain of the TCR comprises an alpha chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof, and a beta chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof, and a beta chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 or a conservative modification thereof. In certain embodiments, the extracellular domain of the TCR further comprises an alpha chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof, and a beta chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof.

[0148] 5.3.4. TCRs with modifications within the CDRs In certain embodiments, a TCR (or functional fragment thereof) of the present disclosure comprises an alpha chain variable region comprising CDR1, CDR2 and CDR3 sequences, and a beta chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specific amino acid sequences or modifications thereof based on the TCRs (or functional fragments thereof) described herein (see Tables 1-5), and wherein the TCR (or functional fragment thereof) retains the desired functional properties of a mutant RAS peptide-specific TCR (or functional fragment thereof) of the subject matter of the present disclosure.

[0149] In certain embodiments, the TCR (or functional fragment thereof) disclosed herein comprises an alpha chain constant region and a beta chain constant region, wherein at least one of the constant regions comprises a particular amino acid sequence based on a TCR (or functional fragment thereof) described herein (see Tables 1-5), or a modification thereof, and wherein the TCR (or functional fragment thereof) retains the desired functional properties of a mutant RAS peptide-specific TCR (or functional fragment thereof) of the subject matter disclosed herein.

[0150] In certain embodiments, such modifications do not significantly affect or change the binding properties of the TCR, including amino acid sequence.Non-limiting examples of such modifications include amino acid substitution, addition and deletion.Modifications can be introduced into the TCR or its functional fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0151] The modification may be conservative, non-conservative, or a mixture of conservative and non-conservative modifications. As mentioned above, a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains is defined in the art. Exemplary conservative amino acid substitutions are shown in Table 6. In certain embodiments, amino acid substitutions are introduced into a TCR of interest, and the product can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0152] [Table 6]

[0153] Amino acids can be classified according to common side chain properties. · Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; ·Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; ·Acidic: Asp, Glu; Basic: His, Lys, Arg; · Residues that affect chain orientation: Gly, Pro; ·Aromatic: Trp, Tyr, Phe.

[0154] In certain embodiments, one or more amino acid residues within the CDR regions may be replaced with other amino acid residues from the same group, and the altered TCR may be tested for retained function using the functional assays described herein.

[0155] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0156] In certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues within a particular sequence or CDR region are altered.

[0157] In certain embodiments, one or more amino acid residues in the constant region of the TCR can be modified to enhance the stability and / or cell surface expression of the TCR, in certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues in a particular sequence or constant region are altered. In certain embodiments, the modifications include murinization, cysteine ​​modifications, and transmembrane modifications (Cohen et al. Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR / CD3 stability, Cancer Res. 2006; 66(17):8878-8886; Cohen et al. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond, Cancer Res. 2007; 67(8):3898-3903; Kuball et al. Facilitating matched pairing and expression of TCR chains introduced into human T cells, Blood 2007; 109(6):2331-2338; Haga-Friedman et al. Incorporation of transmembrane hydrophobic mutations in the TCR enhance its surface expression and T cell functional avidity, Journal of immunology 2012;188(11):5538-5546, the contents of each of which are incorporated by reference in their entirety.

[0158] 5.3.5. Bispecific molecules The subject matter of the present disclosure provides a bispecific molecule comprising the TCR (or a functional fragment thereof) of the present disclosure. The TCR or a functional fragment thereof of the present disclosure can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for the receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The TCR or a functional fragment thereof of the present disclosure can in fact be derivatized or linked to more than two other functional molecules to generate a multispecific molecule that binds to more than one different binding site and / or target molecule, and such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, the TCR or a functional fragment thereof of the present disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.

[0159] The subject matter of the present disclosure provides a bispecific molecule comprising at least a first binding specificity for a mutant RAS peptide and a second binding specificity for a second target peptide region. The second target epitope region can be a second RAS peptide, or a non-RAS peptide, e.g., a different antigen. In certain embodiments, the bispecific molecule is multispecific, e.g., the molecule can further comprise a third binding specificity. If a first portion of the bispecific molecule, e.g., an antibody, binds to an antigen, e.g., on a tumor cell, and a second portion of the bispecific molecule recognizes an antigen on the surface of a human immune effector cell, the bispecific molecule can recruit the activity of that effector cell by specifically binding to an effector antigen on the human immune effector cell. In certain embodiments, the bispecific molecule can form a link between an effector cell, e.g., a T cell, and a tumor cell, thereby enhancing effector function. In certain embodiments, the bispecific molecule of the present disclosure comprises at least a first binding to a mutant RAS peptide and at least a second binding to an immune cell or a molecule associated with an immune cell.

[0160] The bispecific molecules of the subject matter of the present disclosure can be prepared by conjugating the component binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can be produced separately and then conjugated to each other.When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139:2367-2375. Conjugating agents can be SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0161] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds at the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge regions are modified to contain an odd number of sulfhydryl residues, preferably one sulfhydryl residue, prior to conjugation.

[0162] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb and mAb, mAb and Fab, Fab and F(ab')2, or ligand and Fab fusion protein.

[0163] Binding of the bispecific molecule to a specific target can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition) or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected by means such as a gamma counter or a scintillation counter, or by autoradiography.

[0164] 5.4.Cells The subject matter of the present disclosure provides a cell comprising a TCR of the present disclosure (e.g., as disclosed in Section 5.3). In certain embodiments, the cell is selected from the group consisting of a cell of the lymphoid lineage, a cell of the myeloid lineage, a stem cell from which a cell of the lymphoid lineage may be derived, and a stem cell from which a cell of the myeloid lineage may be derived. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage.

[0165] In certain embodiments, the cell is a cell of the lymphoid lineage. Cells of the lymphoid lineage can provide antibody production, regulation of the cellular immune system, detection of foreign bodies in the blood, detection of cells foreign to the host, etc. Non-limiting examples of cells of the lymphoid lineage include T cells and / or stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells).

[0166] In certain embodiments, the cells are T cells. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells: for example, TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by the introduction of an antigen recognition receptor, e.g., a CAR. In certain embodiments, the immunoresponsive cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells. In certain embodiments, TCR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype.

[0167] In certain embodiments, T cell is NK-T cell. Natural killer (NK) T cell is part of cell-mediated immunity and can be lymphocyte that acts during natural immune response. NK-T cell does not require prior activation to perform their cytotoxic effect on target cell.

[0168] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, such as those described in Sadelain et al., Nat Rev Cancer (2003); 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes including α and β heterodimers), Panelli et al., J Immunol (2000); 164:495-504; Panelli et al., J Immunol (2000); 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont et al., Cancer Res (2005); 65:5417-5427; Papanicolaou et al., Blood (2003); 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells).

[0169] Cells (eg, T cells) can be derived in vitro from autologous, non-autologous (eg, allogeneic), or engineered progenitor or stem cells.

[0170] The subject cell of the present disclosure can be myeloid lineage cell.Non-limiting examples of myeloid lineage cell include monocyte, macrophage, neutrophil, dendritic cell, basophil, neutrophil, eosinophil, megakaryocyte, mast cell, erythrocyte, platelet, and stem cell that can differentiate myeloid lineage cell.In certain embodiments, stem cell is pluripotent stem cell (e.g., embryonic stem cell or induced pluripotent stem cell).

[0171] In certain embodiments, the cells further comprise at least one recombinant or exogenous costimulatory ligand. For example, the cells of the present disclosure can be further transduced with at least one costimulatory ligand so that the cells co-express or are induced to co-express the TCR and at least one costimulatory ligand of the present disclosure. The interaction between the TCR and at least one costimulatory ligand of the present disclosure provides a non-antigen specific signal that is important for the complete activation of immune responsive cells (e.g., T cells). Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its main role is in regulating immune cells. Members of the TNF superfamily share several common characteristics. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) that contain a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNF-β) / lymphotoxin alpha (LTα), lymphotoxin beta (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins - they have an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, and PD-L1 / (B7-H1), the ligand for PD-1.In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the cells contain one recombinant costimulatory ligand that is 4-1BBL. In certain embodiments, the cells contain two recombinant costimulatory ligands that are 4-1BBL and CD80.

[0172] In certain embodiments, the cells of the present disclosure further comprise at least one exogenous cytokine. For example, the cells of the present disclosure can be further transduced with at least one cytokine, so that the cells secrete at least one cytokine and express the TCR of the present disclosure. In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-18 and IL-21. In certain embodiments, the cytokine is IL-12.

[0173] 5.5. Genetic Modification of Nucleic Acids and Cells The subject matter of the present disclosure provides a nucleic acid encoding the TCR of the present disclosure (e.g., as disclosed in Section 5.3). A cell comprising such a nucleic acid is further provided. In certain embodiments, a promoter is operably linked to the TCR of the present disclosure.

[0174] In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the group consisting of long terminal repeat (LTR) promoter, elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, and metallothionein promoter. In certain embodiments, the exogenous promoter is an LTR promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0175] In certain embodiments, the nucleic acid encodes both the α-chain and the β-chain of the TCR of the present disclosure. In certain embodiments, the α-chain and the β-chain are separated by a self-cleaving peptide, such as the 2A-peptide. In certain embodiments, the α-chain and the β-chain are separated by the furin-2A-peptide. In certain embodiments, the peptide comprises the amino acid sequence shown in SEQ ID NO:52. RAKRSGSGATNFSLLKQAGDVEENPGP [SEQ ID NO: 52]

[0176] In certain embodiments, the nucleic acid encodes a functional portion / fragment of a TCR of the present disclosure. As used herein, the term "functional portion" or "functional fragment" refers to any portion, part, or fragment of a presently disclosed TCR, which portion, part, or fragment retains the biological activity of the TCR (parent TCR). For example, a functional portion encompasses a portion, part, or fragment of a TCR of the present disclosure that retains the ability to recognize a RAS peptide (e.g., a RAS peptide containing a G12D mutation) to a similar, the same, or even greater extent than the parent TCR. In certain embodiments, a nucleic acid encoding a functional portion of a presently disclosed TCR encodes, for example, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95% or more of the protein of the parent TCR.

[0177] Genetic modification of cells (e.g., T cells) can be achieved by transducing a substantially homogenous cell composition with recombinant DNA or RNA constructs. In certain embodiments, retroviral vectors (e.g., gamma retroviral vectors or lentiviral vectors) are used to introduce DNA or RNA constructs into cells. For example, a polynucleotide encoding a presently disclosed TCR can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from an alternative internal promoter, or from a promoter specific to the target cell type of interest. Non-viral vectors or RNA can be used as well. Random chromosomal integration, or targeted integration (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs)), or transgene expression (e.g., using natural or chemically modified RNA) can be used. To initially genetically modify cells to contain the currently disclosed TCR, retroviral vectors can be used for transduction, but any other suitable viral vectors or non-viral delivery systems can be used. TCRs can be constructed in a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES and Encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A and F2A peptides).Also suitable are combinations of retroviral vectors with appropriate packaging lines, in which the capsid proteins are functional to infect human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., (1985) Mol Cell Biol (1985); 5: 431-437); PA317 (Miller., et al., Mol Cell Biol (1986); 6: 2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988); 85: 6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114 or GALV envelopes and any others known in the art, are also suitable.

[0178] Possible methods of transduction include direct co-culture of cells with producer cells (Bregni et al., Blood (1992); 80:1418-1422), or culture with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994); 22:223-230; and Hughes et al. J Clin Invest (1992); 89:1817).

[0179] Other transduction virus vectors can be used to modify cells.In certain embodiments, the selected vector shows high infection efficiency and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430,1997; Kido et al., Current Eye Research 15:833-844,1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267,1996; and Miyoshi et al., Proc.Natl.Acad.Sci.USA94:10319,1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus and adeno-associated virus vectors, vaccinia virus, bovine papilloma virus, or Epstein-Barr virus (see, e.g., Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1:55-61; Sharp, The Lancet (1991); 337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984); 226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989); 7:980-90; LeGal La Salle et al., Science (1993); 259:988-90; and Johnson, Chest (1995) 107:77S-83S (see also vectors).Retroviral vectors have been particularly well developed and have been used in clinical settings (Rosenberg et al., N Engl J Med (1990);323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0180] Non-viral approaches can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acid in the presence of lipofection (Feigner et al., Proc Natl Acad Sci USA (1987); 84: 7413; Ono et al., Neurosci Lett (1990); 17: 259; Brigham et al., Am J Med Sci (1989); 298: 278; Staubinger et al., Methods in Enzymol (1983); 101: 512; Wu et al., J Biol Chem (1988); 263: 14621; Wu et al., J Biol Chem (1989); 264: 16985) or by microinjection under surgical conditions (Wolff et al., Science (1990); 247: 1465). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes can also potentially be beneficial for the delivery of DNA to cells. Transplantation of normal genes into diseased tissues of subjects can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny) and then injecting the cells (or their progeny) into target tissues or by systemic injection. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0181] In certain embodiments, the TCR of the present disclosure can be integrated into the selected locus of the genome of a cell.Any targeted genome editing method can also be used to deliver the currently disclosed TCR to a cell or subject.In certain embodiments, the CRISPR system is used to deliver the currently disclosed TCR.In certain embodiments, zinc finger nuclease is used to deliver the currently disclosed TCR.In certain embodiments, the TALEN system is used to deliver the currently disclosed TCR.

[0182] In certain embodiments, the TCR of the present disclosure may be integrated into a locus that encodes a T cell receptor. Non-limiting examples of loci include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. Methods for targeting a TCR to a site in the genome of a T cell are described in WO2017180989 and Eyquem et al., Nature.(2017 Mar 2);543(7643):113-117, both of which are incorporated by reference in their entirety. In certain embodiments, the expression of the TCR is driven by an endogenous promoter / enhancer in or near the locus. In certain embodiments, the expression of the TCR is driven by an exogenous promoter integrated into the locus. The locus into which the TCR is integrated is selected based on the expression level of the genes in the locus and the timing of gene expression of the genes in the locus. The expression level and timing may vary under different stages of cell differentiation and the mitogenic / cytokine microenvironment, which is one of the factors considered when making the selection.

[0183] In certain embodiments, the CRISPR system is used to integrate the TCR into a selected locus of the genome of a cell. In certain embodiments, the CRISPR system uses DNA donor-template-induced homology-directed repair at a defined locus, such as the TRAC locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA contains the RNA used by Cas9 to guide it to the correct part of the host DNA, along with a region that binds to tracrRNA (generally in the form of a hairpin loop) and forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and any part of the DNA repair template (DNA that guides the cell repair process to allow the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect the target cell. In certain embodiments, CRISPR / Cas9 is a recombinant ribonucleoprotein complex that is transfected into target cells. The crRNA is the sequence that Cas9 uses to identify and directly bind to the target DNA in cells, so it needs to be designed for each application. The repair template carrying the TCR expression cassette also needs to be designed for each application, as it must overlap with the sequences on both sides of the cut and code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be linked together with the Cas9 gene and made into a plasmid to be transfected into cells. Methods of using the CRISPR system are described, for example, in WO2014093661A2, WO2015123339A1 and WO2015089354A1, which are incorporated by reference in their entirety.

[0184] In certain embodiments, zinc finger nucleases are used to integrate the TCR into a selected locus in the genome of a cell. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining zinc finger DNA binding domains and DNA cleavage domains. The zinc finger domains can be engineered to target specific DNA sequences allowing the zinc finger nucleases to target desired sequences in the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method of generating new zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type IIs restriction endonuclease FokI. Using endogenous homologous recombination (HR) machinery and a homologous DNA template carrying the TCR expression cassette, ZFNs can be used to insert the TCR expression cassette into the genome. When the target sequence is cut by ZFN, HR mechanism searches for the homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two cut ends of the chromosome, thereby integrating the homologous DNA template into genome.The method of using ZFN system is described in, for example, International Publication No. 2009146179 A1, International Publication No. 2008060510 A2 and China Patent Publication No. 102174576 A, and they are incorporated by reference in their entirety.

[0185] In certain embodiments, the TALEN system is used to integrate a TCR into a selected locus in the genome of an immunoresponsive cell. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems work on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a 33-34 amino acid repeat motif with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to a desired DNA sequence, thereby directing the nuclease to cleave at a specific location in the genome. Methods for using the TALEN system are described, for example, in WO2014134412 A1, WO2013163628 A2, and WO2014040370 A1, which are incorporated by reference in their entireties.

[0186] cDNA expression for use in polynucleotide therapy can be derived from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct expression of the nucleic acid. Enhancers used can include, but are not limited to, those characterized as tissue or cell specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source containing any of the promoters or regulatory elements described above.

[0187] The method for delivering genome editing agents / systems can vary as required. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impact, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyping of viral vectors, replication-competent vector cis and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0188] The modification can be made anywhere within the selected locus or anywhere that can affect the gene expression of the integrated TCR. In certain embodiments, the modification is introduced upstream of the transcription start site of the integrated TCR. In certain embodiments, the modification is introduced between the transcription start site and the protein coding region of the integrated TCR. In certain embodiments, the modification is introduced downstream of the protein coding region of the integrated TCR.

[0189] 5.6. Formulation and Administration The presently disclosed subject matter also provides compositions comprising the cells of the present disclosure (e.g., those disclosed in Section 5.4). In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0190] The compositions comprising the cells of the present disclosure can be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition can include a carrier, which can be, for example, a solvent or dispersion medium containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0191] The composition comprising the cells of the present disclosure can be provided to a subject systemically or directly to a tumor to induce and / or enhance immune response to an antigen and / or to treat and / or prevent a tumor. In certain embodiments, the cells of the present disclosure or a composition comprising the same are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the cells of the present disclosure or a composition comprising the same are provided indirectly to an organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Proliferation and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase the production of cells in vitro or in vivo.

[0192] The amount of cells administered may vary depending on the subject being treated. In certain embodiments, the amount is about 10 4 ~about 10 11 pieces, about 10 4 ~about 10 7 pieces, about 10 5 ~about 10 7 pieces, about 10 5 ~about 10 9 Pieces or about 10 6 ~about 10 8 In certain embodiments, at least about 1×10 cells of the present disclosure are administered to a subject. 5The final dose of approximately 1 x 10 cells can be administered. 10 or more. In certain embodiments, at least about 1×10 6 In certain embodiments, about 10 cells can be administered. 4 ~about 10 11 pieces, about 10 5 ~about 10 9 Pieces or about 10 6 ~about 10 8 cells of the present disclosure are administered to a subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1 x 10 8 pieces, approximately 2×10 8 pieces, about 3 x 10 8 pieces, about 4×10 8 Pieces and about 5 x 10 8 The cells of the present disclosure are administered to a subject. The exact determination of what is considered an effective dose can be based on subject-specific factors, including the size, age, sex, weight and condition of the particular subject. The dosage can be easily ascertained by those skilled in the art from the present disclosure and the knowledge of those skilled in the art.

[0193] The cells and compositions of the present disclosure can be administered by any method known in the art, including, but not limited to, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. The cells of the present disclosure can be administered in any physiologically acceptable vehicle, usually intravascularly, but can also be introduced into bone or other convenient sites (e.g., thymus) where the cells can find a suitable site for regeneration and differentiation.

[0194] 5.7. Treatment Method The subject matter of the present disclosure provides various methods of using the cells of the present disclosure or compositions comprising the same. The cells of the present disclosure and compositions comprising the same can be used in therapy or medicine. For example, the subject matter of the present disclosure provides a method for inducing and / or increasing an immune response in a subject in need of such induction and / or increase. The cells of the present disclosure and compositions comprising the same can be used to reduce tumor burden in a subject. The cells of the present disclosure and compositions comprising the same can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent tumors in a subject. The cells of the present disclosure and compositions comprising the same can be used to prolong the survival of a subject suffering from a tumor.

[0195] In certain embodiments, each of the above methods includes administering the cells of the present disclosure or a composition (e.g., a pharmaceutical composition) comprising the same to achieve a desired effect, such as alleviating existing symptoms or preventing tumor recurrence. For treatment, the amount administered is an amount effective to produce a desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided by bolus or continuous perfusion.

[0196] In certain embodiments, the tumor is associated with RAS. In certain embodiments, the tumor is associated with RAS mutation or RAS mutant. In certain embodiments, the RAS mutation is G12 mutation. In certain embodiments, the RAS mutation is G12D mutation.

[0197] In certain embodiments, the tumor is cancer.In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, cholangiocarcinoma / bile duct cancer, lung cancer, ovarian cancer, esophageal cancer, gastric cancer (also known as "stomach cancer"), head and neck squamous cell carcinoma, non-melanoma skin cancer, salivary gland cancer, melanoma, and multiple myeloma.In certain embodiments, the cancer is pancreatic cancer.

[0198] In certain embodiments, the subject is a human subject.The subject may have an advanced form of the disease, in which case the treatment objectives may include alleviating or reversing disease progression and / or ameliorating side effects.The subject may have a history of a condition that has already been treated, in which case the treatment objectives typically include reducing or delaying the risk of recurrence.

[0199] In certain embodiments, the subject comprises HLA-A. In certain embodiments, the HLA-A is an HLA-A*03 superfamily member. In certain embodiments, the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68 and HLA-A*74. In certain embodiments, the HLA-A*03 superfamily member is HLA-A*11. EXAMPLES

[0200] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the skill of the art. Such techniques are fully explained in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Particularly useful techniques for certain embodiments are described in the following sections.

[0201] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions, and assay, screening, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0202] Example 1. To identify naturally processed and presented epitope(s) derived from KRAS, COS-7 were used as artificial antigen presenting cells (aAPCs). COS-7 cells were co-electroporated with mRNAs encoding HLA-A*11:01 and full-length KRAS(G12D) or wild-type (WT) KRAS. The HLA-restricted immunopeptidome of endogenously processed and presented "public" neoantigens (NeoAgs) arising from mutant KRAS proteins was screened using HLA immunoprecipitation (IP) and tandem mass spectrometry (MS / MS). Figure 1A shows a table summarizing the IP / MS-MS screening detected peptides arising from KRAS proteins. Both 10-mer and 9-mer peptides encompassing the (G12D) hotspot mutation were detected. The 10-mer WT variant was also detected. PANC-1 cells naturally express KRAS(G12D) and express HLA-A*11:01. + and HLA-A*02:01 + Figure 1B shows the validation MS "mirror" plots for eluted HLA-A*11:01-restricted KRAS(G12D) peptides (top) and confirmatory synthetic peptides (bottom) from the PANC-1 pancreatic cancer cell line. Figure 1C shows the assessment of the stability of neopeptide / HLA complexes on the cell surface. T2 cells, a TAP-deficient cell line, were electroporated with HLA-A*11:01 and pulsed with titrated amounts of KRAS(G12D) 9mer and 10mer neopeptide variants. Cell surface expression of HLA-A*11:01 was measured by flow cytometry as a correlate of p / HLA complex stability.

[0203] As shown in Figure 2, all four members of the RAS family share 90% sequence homology throughout their G domains, but differ significantly in their N-terminal membrane targeting domains. Notably, the amino acid sequences surrounding the codon 12 hotspot region share 100% sequence homology among RAS family members. This suggests that the KRAS-specific TCR(G12D) may provide cross-protection against other mutant RAS proteins.

[0204] Next, we performed a study to discover unique HLA-A*11:01-restricted RAS-specific TCR clonotypes. As shown in Figure 3A, T cells derived from HLA-A*11:01+ healthy donors (HD) or HLA-A*11:01+ patients with a history of KRAS(G12D) cancer were stimulated in vitro with autologous antigen-presenting cells displaying KRAS(G12D). Individual cultures were screened for the presence of RAS-specific T cells using high-order peptide / HLA dextramer reagents loaded with 10-mer epitopes identified by mass spectrometry. Positive wells were labeled with barcoded dextramers and subjected to combined single-cell V(D)J and feature barcode sequencing to retrieve TCR gene sequences for RAS-specific T-cell clonotypes. Five unique RAS-specific TCRs were retrieved from healthy donor (n=1) and patient-derived samples (n=4). As shown in Figure 3B, all five TCRs were composed of unique alpha and beta variable chain segments and CDR3 loop lengths.

[0205] Next, studies were performed to functionally validate and measure the coreceptor dependency of healthy donor (HD) and patient-derived TCRs specific for the RAS(G12D) public NeoAg. Open repertoire (non-specific) T cells were retrovirally transduced with individual recovered TCR gene sequences. The function of TCR-transduced T cells was measured by co-culture with HLA-A*11:01+ target cells co-transfected with mRNA encoding either full-length wild-type (WT) KRAS or KRAS(G12D). Intracellular TNF-α production was measured in CD4+ (blue) and CD8+ (red) T cells expressing the transduced TCRs. As shown in Figure 4A and Figure 4B, all four patient-derived TCRs exhibited coreceptor independence.

[0206] To determine the minimal epitopes of individual RAS(G12D)-specific TCR library members, open repertoire T cells were retrovirally transduced with individual recovered TCR gene sequences. TCR-transduced T cells were pulsed (10 μg / mL) with either a 9-mer or 10-mer neo-epitope derived from KRAS(G12D) or the corresponding WT counterpart, HLA-A*11:01. + Intracellular TNF-α production was induced by CD4+ T cells expressing the transduced TCR. + (blue) and CD8 + (Red) Measured in T cells. As shown by the FACS plots in Figure 5, the 10-mer neopeptide was recognized by all TCR library members, whereas recognition of the 9-mer neoepitope was restricted to patient-derived TCRs.

[0207] Based on these data, we determined the functional avidity of T cells transduced with RAS-specific TCRs. Open repertoire T cells were retrovirally transduced with individual recovered TCR gene sequences and pulsed with increasing amounts of the 10mer RAS(G12D) neopeptide. HLA-A*11:01 + The WT peptide was included as a control (10 μg / mL). Intracellular TNF-α production was measured by CD8 + (left) and CD4+ (right) TCR+ T cells, as shown in Figure 6A. + and CD4 + ECs for each individual TCR in T cells 50 The values ​​are listed in FIG. 6B.

[0208] Next, studies were performed to assess the recognition of endogenous levels of KRAS(G12D) in two HLA-A*11:01+ tumor lines by RAS-specific TCR library members. Open repertoire T cells were retrovirally transduced with individual recovered TCR gene sequences and cocultured with either HuCCT1 (Figure 7A) or PANC-1 (Figure 7B) cells in the presence or absence of pan HLA class-I blocking antibodies. HuCCT1 is a cholangiocarcinoma line and PANC-1 is a pancreatic tumor line that is both HLA-A*11:01+ and mutant KRAS(G12D). T cells alone were included as a biological control to measure baseline T cell cytokine levels. Intracellular TNF-α production was measured in CD8+TCR+ T cells and is shown in Figure 7. TCR library members were able to recognize endogenously processed and presenting RAS(G12D) levels in a class I restricted manner.

[0209] To measure the cytolytic potential of individual library members, TCR-transduced T cells were co-cultured with PANC1 in the presence or absence of pan class I blocking antibodies. Cytolysis was measured using a tumor impedance-based assay over a 54-hour period. Figure 8A shows the tumor curves of individual library members. Figure 8B shows peak cytolysis after 48 hours of co-culture.

[0210] To test whether individual library members could confer cross-protection against alternative mutant RAS proteins, TCR-transduced T cells were co-cultured with HLA-A*11:01+ targets co-expressing individual G12D RAS isoforms (KRAS, HRAS and NRAS). WT RAS isoforms were used as specificity controls. Intracellular TNF-α production was measured in CD8+ T cells expressing the transduced TCRs. As shown in Figure 9A and Figure 9B, cross-protective function of all five RAS(G12D)-specific TCRs was observed.

[0211] Example 2. To identify the TCR cross-reactivity profile, a positional library scanning experiment was performed. A positional scanning library (PSL) was synthesized by substituting every other amino acid (AA) in the index 10-mer RAS mutant peptide sequence shown in SEQ ID NO:2 (e.g., VVVGADGVGK). Target COS-7 cells were electroporated with mRNA encoding full-length human HLA*11:01 and incubated overnight at 37 degrees to allow HLA-A*11:01 protein expression. HLA*11:01 was then electroporated with 10-mer RAS mutant peptide sequence shown in SEQ ID NO:2 (e.g., VVVGADGVGK). + Target wells were pulsed with individual peptides derived from PSL (at a concentration of 1 μM), and wild-type RAS peptide (WT) and mutant RAS peptides were included as functional controls. RAS TCR-T cells expressing individual RAS TCR1-5 were added at an E:T ratio of 1:1 and incubated at 37 °C for 24 h. ELISA assays were performed on supernatants harvested from co-culture wells to determine IFN-γ production levels. The level of IFN-γ production relative to the index amino acid at each position was calculated and plotted as shown in the heatmaps in Figures 10A-10E. As shown in Figures 10A-10E, the TCR logo plots above each individual TCR heatmap indicate the relative influence of each amino acid at each position.

[0212] Next, studies were performed to determine the cross-reactivity potential of RAS-specific TCRs. Individual TCR peptide motifs were scanned against the human proteome to identify potential cross-reactive sequences. Target COS-7 cells were electroporated with mRNA encoding full-length human HLA*11:01 and incubated overnight at 37°C to allow HLA-A*11:01 protein expression. +target wells. Cells were then pulsed with individual peptides (see Tables 7-11) corresponding to their RAS-specific TCRs at a concentration of 1 μM, and finally, wild-type (WT) and mutant RAS peptides were included as functional controls. RAS TCR-T cells expressing individual RAS TCR1-5 were added at a 1:1 E:T ratio and incubated for 24 h at 37 °C. Supernatants were harvested from the co-culture wells to perform an ELISA assay to determine IFN-γ production levels. IFN-γ production is shown in Figures 11A-11E.

[0213] [Table 7]

[0214] [Table 8]

[0215] [Table 9]

[0216] [Table 10]

[0217] [Table 11]

[0218] As shown in Figures 11A to 11E, each TCR expressed HLA-A*11:01, which presents a mutant RAS peptide. + TCR1, 2, 4 and 5 showed no reactivity to alternative human peptide sequences with the recognition motif elucidated in Figures 10A-10E. TCR3 showed no reactivity to HLA-A*11:01.+ When pulsed at non-physiological concentrations in target cell populations, they showed low levels of reactivity to a single surrogate peptide (TCR3, peptide 22; Table 9). Thus, these data demonstrate that the presently disclosed TCRs are capable of specifically binding mutant RAS peptides and inducing specific T cell activation with negligible reactivity to surrogate peptide species.

[0219] Embodiments of the Subject Matter of the Disclosure From the above description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it to various applications and conditions, such embodiments still falling within the scope of the following claims.

[0220] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0221] All patents and publications and sequences referred to in this specification by accession or reference number are herein incorporated by reference to the same extent as if each individual patent, publication and sequence was specifically and individually indicated to be incorporated by reference.

Claims

**Claim 1** A recombinant T cell receptor (TCR) that binds to a RAS peptide, wherein the RAS peptide contains a G12 mutation, the TCR includes an extracellular domain that binds to the RAS peptide, the extracellular domain includes an α-chain and a β-chain, the α-chain includes an α-chain variable region and an α-chain constant region, and the β-chain includes a β-chain variable region and a β-chain constant region, where: (a) the α-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, and the β-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (b) the α-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 14, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 15, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 16, and the β-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 19; (c) the α-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 24, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 15, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 25, and the β-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 26, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 27, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 28; (d) the α-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 33, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 35, and the β-chain variable region includes a CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 38; or (e) The recombinant T cell receptor (TCR) wherein the α-chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 45; and the β-chain variable region comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 58, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 46, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

47.

2. The TCR according to claim 1, wherein the RAS peptide comprises a G12D mutation.

3. The TCR according to claim 1, wherein the RAS peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The TCR according to claim 1, wherein the RAS peptide is associated with an HLA class I complex.

5. The TCR according to claim 4, wherein the HLA class I complex is selected from HLA-A, HLA-B, and HLA-C.

6. The TCR according to claim 5, wherein the HLA-A is an HLA-A*03 superfamily member.

7. The TCR according to claim 6, wherein the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74, and optionally the HLA-A*03 superfamily member is HLA-A*11.

8. a) The α-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 10, and the β-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 11; b) The α-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 20, and the β-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 21; c) The α-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 29, and the β-chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 30; d) The variable region of the α-chain comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 39, and the variable region of the β-chain comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 40; or e) The variable region of the α-chain comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 48, and the variable region of the β-chain comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence shown in SEQ ID NO: 49, the TCR according to claim 1. **Claim 9** a) The variable region of the α-chain comprises the amino acid sequence shown in SEQ ID NO: 10, and the variable region of the β-chain comprises the amino acid sequence shown in SEQ ID NO: 11; b) The variable region of the α-chain comprises the amino acid sequence shown in SEQ ID NO: 20, and the variable region of the β-chain comprises the amino acid sequence shown in SEQ ID NO: 21; c) The variable region of the α-chain comprises the amino acid sequence shown in SEQ ID NO: 29, and the variable region of the β-chain comprises the amino acid sequence shown in SEQ ID NO: 30; d) The variable region of the α-chain comprises the amino acid sequence shown in SEQ ID NO: 39, and the variable region of the β-chain comprises the amino acid sequence shown in SEQ ID NO: 40; or e) The variable region of the α-chain comprises the amino acid sequence shown in SEQ ID NO: 48, and the variable region of the β-chain comprises the amino acid sequence shown in SEQ ID NO: 49, the TCR according to claim 1. **Claim 10** a) The α-chain comprises the amino acid sequence shown in SEQ ID NO: 12, and the β-chain comprises the amino acid sequence shown in SEQ ID NO: 13; b) The α-chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the β-chain comprises the amino acid sequence shown in SEQ ID NO: 23; c) The α-chain comprises the amino acid sequence shown in SEQ ID NO: 31, and the β-chain comprises the amino acid sequence shown in SEQ ID NO: 32; d) The α-chain comprises the amino acid sequence shown in SEQ ID NO: 41, and the β-chain comprises the amino acid sequence shown in SEQ ID NO: 42; or e) The α-chain comprises the amino acid sequence shown in SEQ ID NO: 50, and the β-chain comprises the amino acid sequence shown in SEQ ID NO: 51, the TCR according to claim 1. **Claim 11** The TCR according to claim 1, wherein the TCR is expressed from a vector. **Claim 12** (a) The α constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or the same as the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 54; and / or **(b)** The TCR according to claim 1, wherein the β constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or the same as the amino acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO:

57. **Claim 13** (a) The α constant region comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 54; and / or **(b)** The TCR according to claim 12, wherein the β constant region comprises the amino acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO:

57. **Claim 14** A nucleic acid encoding the T cell receptor (TCR) according to any one of claims 1 to 13. **Claim 15** A cell comprising the TCR according to any one of claims 1 to 13 or the nucleic acid encoding said TCR. **Claim 16** (a) The cell is transduced with the TCR; and / or **(b)** The TCR is constitutively expressed on the surface of the cell, the cell according to claim 15. **Claim 17** (a) The cell is an immunoreactive cell; **(b)** The cell is selected from the group consisting of T cells and pluripotent stem cells capable of differentiating into lymphoid cells; **(c)** The cell is a T cell; **(d)** The cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), regulatory T cells, γδ T cells, natural killer T cells (NK-T), stem cell memory T cells, central memory T cells, and effector memory T cells; **(e)** The cell is a γδ T cell; or **(f)** The cell is an NK-T cell, the cell according to claim 15. **Claim 18** The cell according to claim 15, wherein the TCR or the nucleic acid is integrated into a locus within the genome of the cell. **Claim 19** The cell according to claim 18, wherein the locus is selected from the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus.

20. A composition comprising the cell according to claim 15.

21. The composition according to claim 20, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

22. A vector comprising the nucleic acid according to claim 14.

23. The vector according to claim 22, wherein the vector is a γ-retroviral vector.

24. A method for producing a cell that binds to a RAS peptide containing a G12 mutation, the method comprising introducing the nucleic acid according to claim 14 or a vector containing the nucleic acid into the cell.

25. The composition according to claim 20, for the treatment and / or prevention of tumors associated with RAS in a subject.

26. The composition according to claim 25, wherein the tumor is associated with a RAS mutation.

27. The composition according to claim 26, wherein the RAS mutation is a G12D mutation.

28. The composition according to claim 25, wherein the tumor is selected from the group consisting of pancreatic cancer, breast cancer, endometrial cancer, cervical cancer, anal cancer, bladder cancer, colorectal cancer, cholangiocarcinoma / cholangiocarcinoma, lung cancer, ovarian cancer, esophageal cancer, gastric cancer, head and neck squamous cell carcinoma, non-melanoma skin cancer, salivary gland cancer, melanoma, and multiple myeloma.

29. The composition according to claim 28, wherein the tumor is pancreatic cancer.

30. The composition according to claim 25, wherein the subject is human.

31. The composition according to claim 25, wherein the subject comprises HLA-A.

32. The composition according to claim 31, wherein the HLA-A is an HLA-A*03 superfamily member.

33. The composition according to claim 32, wherein the HLA-A*03 superfamily member is selected from the group consisting of HLA-A*03, HLA-A*11, HLA-A*31, HLA-A*33, HLA-A*66, HLA-A*68, and HLA-A*74.

34. The composition according to claim 32, wherein the HLA-A*03 superfamily member is HLA-A*11.

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