Leydig 771261 HHS E-132-2023-0-PC-01 1 T CELL RECEPTORS TARGETING RAS WITH G12D, G12R, OR G12V MUTATION CROSS-REFERENCE TO RELATED APPLICATION This patent application claims the benefit of U.S. Provisional Patent Application No.63/510,817, filed June 28, 2023, which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under project number ZIA- BC-010984 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 283,472 Byte XML file named “771261.XML,” dated June 5, 2024. BACKGROUND OF THE INVENTION Some cancers may have very limited treatment options, particularly when the cancer becomes metastatic and unresectable. Despite advances in treatments such as, for example, surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as, for example, pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Accordingly, there exists an unmet need for additional treatments for cancer. BRIEF SUMMARY OF THE INVENTION An aspect of the invention provides an isolated or purified T-cell receptor (TCR) having antigenic specificity for a mutated human RAS amino acid sequence with a substitution of glycine at position 12 with aspartic acid, valine, or arginine, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutated human Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein position 12 is defined by reference to the wild-type human
Leydig 771261 HHS E-132-2023-0-PC-01 2 KRAS, wild-type human HRAS, or wild- human NRAS protein, respectively, and wherein the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 1-6; (2) all of SEQ ID NOs: 11-16; (3) all of SEQ ID NOs: 21-26; (4) all of SEQ ID NOs: 31-36; (5) all of SEQ ID NOs: 41-46; (6) all of SEQ ID NOs: 51-56; (7) all of SEQ ID NOs: 61-66; (8) all of SEQ ID NOs: 71-76; (9) all of SEQ ID NOs: 81-86; (10) all of SEQ ID NOs: 91-96; (11) all of SEQ ID NOs: 101-106; (12) all of SEQ ID NOs: 111-116; (13) all of SEQ ID NOs: 121-126; (14) all of SEQ ID NOs: 131-136; or (15) all of SEQ ID NOs: 141-146. Further aspects of the invention provide polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the TCRs of the invention. Another aspect of the invention provides an isolated or purified nucleic acid comprising, from 5’ to 3’, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequence, respectively, encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 17 and 18; 18 and 17; 19 and 20; 20 and 19; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 37 and 38; 38 and 37; 39 and 40; 40 and 39; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 57 and 58; 58 and 57; 59 and 60; 60 and 59; 67 and 68; 68 and 67; 69 and 70; 70 and 69; 77 and 78; 78 and 77; 79 and 80; 80 and 79; 87 and 88; 88 and 87; 89 and 90; 90 and 89; 97 and 98; 98 and 97; 99 and 100; 100 and 99; 107 and 108; 108 and 107; 109 and 110; 110 and 109; 117 and 118; 118 and 117; 119 and 120; 120 and 119; 127 and 128; 128 and 127; 129 and 130; 130 and 129; 137 and 138; 139 and 137; 139 and 140; 140 and 139; 147 and 148; 148 and 147; 149 and 150; or 150 and 149. Still further aspects of the invention provide methods of detecting the presence of cancer in a mammal, methods of inducing an immune response against a cancer in a mammal, and methods of treating or preventing cancer in a mammal. Additional aspects of the invention provide methods of producing a host cell expressing the TCR and methods of producing the TCR, polypeptide, or protein. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Figures 1A-1C are graphs showing the percentage of effector cells expressing 4- 1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL independently transduced with a retroviral expression vector encoding the 4385 TCR-Z (A), 4394 TCR-10 (B), or 4394 TCR-A (C). The target cells were HLA- C*01:02 positive COS-C*01:02 cells which had been incubated with the mutant RASG12V 9-
Leydig 771261 HHS E-132-2023-0-PC-01 3 mer peptide (unshaded circles) or the WT peptide (shaded circles) at the indicated concentration of peptides. Figures 1D-1E are graphs showing IFN-gamma secretion measured following co- culture of effector cells with 4391 PDX cells, which are relevant target cells expressing RASG12V mutation and HLA-C*01:02 (D) or 4316 PDX cells, which are nonrelevant target cells expressing RASG12V mutation but not expressing HLA-C*01:02 (1E). The target cells were incubated either in complete T cell media (CM) or CM supplemented with the mutant RASG12V 9mer peptide as a positive control. Effector cells were healthy donor PBL that were independently transduced with a retroviral expression vector encoding the 4385 TCR-Z, 4394 TCR-10, or 4394 TCR-A. Empty vector (E.V) transduction of the same donor PBLs and effector cells alone served as a negative control. Figure 1F is a graph showing the mean (n=5) tumor size measured at the indicated days post adoptive cell transfer (ACT) of effector cells into tumor-bearing mice. Effector cells were healthy donor PBL that were independently transduced with a retroviral expression vector encoding the 4385 TCR-Z, 4394 TCR-10, or 4394 TCR-A. Empty vector (E.V) transduction of the same donor PBLs served as a negative control. Figure 2A is a graph showing the percentage of effector cells expressing 4-1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4432 TCR-A. Target cells were COS cells expressing HLA-A*11:01 (COS-A11), HLA-A*02:01 (COS- A02), or autologous dendritic cells (DC). All target cells were transfected with tandem minigenes (TMG) RNA to express WT or mutated RAS antigen (Mut). PMA served as a positive control. DMSO was a negative control for activation of the T cells. Figure 2B is a graph showing the percentage of effector cells expressing 4-1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4432 TCR-A. Target cells were COS-A11 cells that had been incubated with the mutant RASG12D 9-mer peptide, RASG12D 10-mer peptide, or the corresponding WT peptides at the concentration of peptides denoted in the figure. PMA served as a positive control, and T cells cultured without target cells (T only) served as a negative control for activation of the T cells. Figure 2C is a graph showing the level of IFN-gamma secretion measured following co-culture of effector cells with target cells. Effector cells were healthy donor PBL independently transduced with a retroviral expression vector encoding the 4432 TCR-A, 4373
Leydig 771261 HHS E-132-2023-0-PC-01 4 TCR2, or mTCR. Target cells were COS- cells that had been incubated with the mutant RASG12D 10-mer peptide or the equivalent WT peptide at the concentration shown in the figure. Figure 2D is a graph showing the number of live tumor cells counted at the indicated number of hours following co-culture of effector cells with target cells. Effector cells were healthy donor PBL independently transduced with a retroviral expression vector encoding the 4432 TCR-A, 4373 TCR2, or mTCR. Empty vector (E.V) transduction of the same donor PBLs served as a negative control. Target cells were 4069 PDX cells. Figure 2E is a graph showing the mean tumor size measured at the indicated days post ACT of effector cells into tumor-bearing mice. Effector cells were healthy donor PBL that were independently transduced with a retroviral expression vector encoding the 4432 TCR-A, 4373 TCR2, or mTCR. Empty vector (E.V) transduction of the same donor PBLs served as a negative control. Tumor-bearing mice treated with PBS also served as a control. Figure 3A is a graph showing the percentage of effector cells expressing 4-1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4412 TCR-H. The target cells were autologous DCs pulsed with WT or mutated RASG12R (G12R) 24-mer long peptide (LP). CD3/CD28 beads served as a positive control. DMSO and T cell only served as negative controls for activation of the T cells. Figure 3B is a graph showing the percentage of effector cells expressing 4-1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4412 TCR-H or 4268 TCR. The target cells were COS cells which had been independently transfected with one of several combinations of HLA-DQA1 and HLA-DQB1 molecules, as indicated in the figure, and pulsed with RASG12R 24-mer LP. Figures 3C-3D are graphs showing the percentage of effector cells expressing 4- 1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4412 TCR-H (C) or 4268 TCR (D). The target cells were autologous DC that had been incubated with the mutant RASG12R 24-mer LP or the equivalent RASWT LP at the concentration of peptides denoted in the figures. Figures 3E-3F are graphs showing the number of IFN-gamma positive spots per 3e4 cells measured following co-culture of effector cells with target cells. Effector cells were
Leydig 771261 HHS E-132-2023-0-PC-01 5 healthy donor PBL transduced with a expression vector encoding 4412 TCR-H (E) or 4268 TCR (F). The target cells were autologous DC that had been incubated with the mutant RASG12R 24-mer LP or the equivalent RASWT LP at the concentration of peptides denoted in the figures. Figure 4A is a graph showing the percentage of effector cells expressing 4-1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4385 TCR-B. Target cells were autologous DC cells incubated with the RASG12V 24-mer LP, RASG12C LP, RASG12A LP, or the corresponding WT LP at the concentration of peptides denoted in the figure. Figure 4B is a graph showing luminescence units measured following co-culture of effector cells with target cells. Effector cells were a Jurkat-NFAT-firefly luciferase- CD8+/CD4+ reporter cell line transduced with a retroviral expression vector encoding 4385 TCR-B. Target cells were COS cells independently transfected with one of several combinations of HLA class-II molecules, as indicated in the figure, and pulsed with RASG12V 24-mer LP. PMA served as a positive control, and Jurkat cells cultured without target cells (T only) served as a negative control for activation of the T cells. Figures 5A-5F are graphs showing the percentage of effector cells expressing 4- 1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4430 TCR-N1A (A), 4430 TCR-N8 (B), 4430 TCR-N9 (C), 4430 TCR-N23A (D), 4430 TCR-N23B (E), or 4095 TCR 9A (F). Target cells were COS cells expressing HLA-C*08:02 that had been incubated with RASG12D 9-mer peptide or the corresponding WT peptide at the concentration of peptides denoted in the figures. COS-C*08:02 without peptide (DMSO as a vehicle) served as a negative control. Figures 5G-5I are graphs showing the percentage of effector cells expressing 4- 1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL transduced with a retroviral expression vector encoding 4430 TCR-N2 (G), 4095 TCR 9A (I), or 4095 TCR 10 (H). Target cells were COS cells expressing HLA- C*08:02 that had been incubated with RASG12D 9-mer peptide, RASG12D 10-mer peptide, or the corresponding 10-mer WT peptide at the concentration of peptides denoted in the figures. COS-C*08:02 without peptide (DMSO as a vehicle) served as a negative control.
Leydig 771261 HHS E-132-2023-0-PC-01 6 Figure 5J is a graph showing the of IFN-gamma secretion measured following co-culture of effector cells with target cells. Effector cells were healthy donor PBL independently transduced with a retroviral expression vector encoding 4430 TCR-N1A, 4430 TCR-N2, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR-N23A, 4430 TCR-N23B, or 4095 TCR 9A. Target cells were SNU1 tumor cells naturally expressing the RASG12D mutation HLA- C*08:02 or 4069 PDX cells expressing the RASG12D mutation but not HLA-C*08:02. Effector cells transduced with empty vector or effector cells cultured alone (T only) served as a negative control. Figures 6A-6E are graphs showing the percentage of effector cells expressing 4- 1BB and/or OX40 following co-culture of effector cells with target cells. Effector cells were healthy donor PBL independently transduced with a retroviral expression vector encoding the 4391 TCR-B2 (A), 4391 TCR-C1 (B), 4391 TCR-K (C), 4391 TCR-L2 (D), or 4394 TCR-A (E). The target cells were HLA-C*01:02 positive COS-C*01:02 cells which had been incubated with the mutant RASG12V 9-mer peptide (unshaded circles) or the equivalent WT peptide (shaded circles) at the indicated concentration of peptides. DETAILED DESCRIPTION OF THE INVENTION RAS family proteins belong to the large family of small GTPases. Without being bound to a particular theory or mechanism, it is believed that, when mutated, RAS proteins may be involved in signal transduction early in the oncogenesis of many human cancers. A single amino acid substitution may activate the protein. The mutated RAS protein product may be constitutively activated. Mutated RAS proteins may be expressed in any of a variety of human cancers such as, for example, pancreatic (e.g., pancreatic carcinoma), colorectal, lung (e.g., lung adenocarcinoma), endometrial, ovarian (e.g., epithelial ovarian cancer), and prostate cancers. The human RAS family proteins include KRAS, HRAS, and NRAS. KRAS is also referred to as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 215. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 216. Hereinafter, references to “KRAS” (mutated or unmutated (WT)) refer to both variant A and variant B, unless specified otherwise. When activated, mutated KRAS binds to guanosine-5'- triphosphate (GTP) and converts GTP to guanosine 5′-diphosphate (GDP).
Leydig 771261 HHS E-132-2023-0-PC-01 7 HRAS is another member of the protein family. HRAS is also referred to as Harvey Rat Sarcoma Viral Oncoprotein, V-Ha-Ras Harvey Rat Sarcoma Viral Oncogene Homolog, or Ras Family Small GTP Binding Protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 217. NRAS is still another member of the RAS protein family. NRAS is also referred to as GTPase NRas, V-Ras Neuroblastoma RAS Viral Oncogene Homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 218. An embodiment of the invention provides an isolated or purified T-cell receptor (TCR) having antigenic specificity for a mutated human RAS amino acid sequence with a substitution of glycine at position 12 with aspartic acid, valine, or arginine, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutated human Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein position 12 is defined by reference to the wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively. Hereinafter, references to a “TCR” also refer to functional portions and functional variants of the TCR, unless specified otherwise. The mutated human RAS amino acid sequence may be a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence, or a mutated human NRAS amino acid sequence. The amino acid sequences of WT human KRAS, NRAS, and HRAS protein each have a length of 188 or 189 amino acid residues and have a high degree of identity to one another. For example, the amino acid sequence of the WT human NRAS protein is 86.8% identical to that of the WT human KRAS protein. Amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to that of the WT human KRAS protein. Amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereinafter, references to “RAS” (mutated or unmutated (WT)) collectively refer to KRAS, HRAS, and NRAS, unless specified otherwise. In an embodiment of the invention, the mutated human RAS amino acid sequence comprises a human RAS amino acid sequence with a substitution of glycine at position 12 with aspartic acid, valine, or arginine, wherein position 12 is defined by reference to the corresponding WT RAS protein. The WT RAS protein may be any one of WT KRAS protein (SEQ ID NO: 215 or 216), WT HRAS protein (SEQ ID NO: 217), or WT NRAS
Leydig 771261 HHS E-132-2023-0-PC-01 8 protein (SEQ ID NO: 218) because, as above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Accordingly, the amino acid residue at position 12 of each of WT KRAS, WT HRAS, and WT NRAS protein is the same, namely, glycine. The mutated human RAS amino acid sequence has a substitution of glycine at position 12 with aspartic acid, valine, or arginine. In this regard, embodiments of the invention provide TCRs with antigenic specificity for any human RAS protein, polypeptide or peptide amino acid sequence with a G12D, G12R, or G12V mutation. Mutations and substitutions of RAS are defined herein by reference to the amino acid sequence of the corresponding WT RAS protein. Thus, mutations and substitutions of RAS are described herein by reference to the amino acid residue present at a particular position in WT RAS protein (namely, position 12), followed by the position number, followed by the amino acid residue with which that residue has been replaced in the particular mutation or substitution under discussion. A RAS amino acid sequence (e.g., a RAS peptide) may comprise fewer than all of the amino acid residues of the full-length, WT RAS protein. Accordingly, position 12 is defined herein by reference to the WT full-length RAS protein (namely, any one of SEQ ID NOs: 215-218) with the understanding that the actual position of the corresponding residue in a particular example of a RAS amino acid sequence may be different. When the positions are as defined by any one of SEQ ID NOs: 215-218, the term “G12” refers to the glycine normally present at position 12 of any one of SEQ ID NOs: 215-218, and “G12D” indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 215-218 is replaced by aspartic acid. Likewise, “G12R” indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 215-218 is replaced by arginine, and “G12V” indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 215-218 is replaced by valine. For example, when a particular example of a RAS amino acid sequence is, e.g., GAGGVGKSA (SEQ ID NO: 246) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 10 to 18 of SEQ ID NO: 215), “G12D” refers to a substitution of the underlined glycine in SEQ ID NO: 246 with aspartic acid, even though the actual position of the underlined glycine in SEQ ID NO: 246 is 3. Human RAS amino acid sequences with the G12D mutation are hereinafter referred to as “G12D RAS,” “G12D,” or “RASG12D.” Human RAS amino acid sequences with the G12R mutation are hereinafter referred to as “G12R RAS,” “G12R,” or “RASG12R.”
Leydig 771261 HHS E-132-2023-0-PC-01 9 Human RAS amino acid sequences with the mutation are hereinafter referred to as “G12V RAS,” “G12V,” or “RASG12V.” G12D RAS, G12R RAS, and G12V RAS are collectively referred to herein as “mutated RAS.” Examples of full-length RAS proteins with the G12D, G12R, or G12V mutation are set forth in Table 1 below. TABLE 1 Mutated Full-Length RAS Protein SEQ ID NO: G12V KRAS variant A 219
n an aspec o e nven on, e as an gen c spec c y or e mu a e human RAS amino acid sequence of (a) AVGVGKSAL (SEQ ID NO: 232); (b) VVVGADGVGK (SEQ ID NO: 238); (c) MTEYKLVVVGARGVGKSALTIQLI (SEQ ID NO: 241); (d) MTEYKLVVVGAVGVGKSALTIQLI (SEQ ID NO: 242); (e) GADGVGKSA (SEQ ID NO: 245); or (f) GADGVGKSAL (SEQ ID NO: 247). In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of: (a) AGGVGKSAL (SEQ ID NO: 233); (b) VVVGAGGVGK (SEQ ID NO: 239); (c) MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 240); (d) GAGGVGKSA (SEQ ID NO: 246); or (e) GAGGVGKSAL (SEQ ID NO: 248). In an aspect of the invention, the inventive TCRs may be able to recognize mutated RAS in an HLA (human leukocyte antigen)-molecule-dependent manner. "HLA- molecule-dependent manner," as used herein, means that the TCR elicits an immune response upon binding to mutated RAS within the context of an HLA molecule, which HLA molecule
Leydig 771261 HHS E-132-2023-0-PC-01 10 is expressed by the patient from which the was isolated. The inventive TCRs may be able to recognize mutated RAS that is presented by the applicable HLA molecule and may bind to the HLA molecule in addition to mutated RAS. The TCRs of the invention may provide any one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. G12D RAS, G12R RAS, and G12V RAS are expressed by cancer cells and are not expressed by normal, noncancerous cells. Without being bound to a particular theory or mechanism, it is believed that the inventive TCRs advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancerous cells, thereby reducing, for example, by minimizing or eliminating, toxicity. Moreover, because the G12D, G12R, and G12V mutations are likely to occur in the early stages of tumorigenesis, the G12D, G12R, and G12V mutations may be expressed on substantially all of a patient’s cancer cells. The inventive TCRs may, advantageously, successfully treat or prevent G12D RAS-, G12R RAS-, and G12V RAS-positive cancers that do not respond to other types of treatment such as, for example, chemotherapy, surgery, or radiation. Additionally, the inventive TCRs may provide highly avid recognition of G12D RAS, G12R RAS, or G12V RAS, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with a vector encoding one or both of the applicable RAS mutation and the applicable HLA molecule, pulsed with a G12D RAS, G12R RAS, or G12V RAS peptide, or a combination thereof). The G12D RAS, G12R RAS, or G12V RAS mutations among the most commonly mutated among patients with solid cancer (more than about 20% of patients). Moreover, the HLA molecules described herein are ccommonly expressed. Accordingly, the inventive TCRs may increase the number of patients eligible to receive immunotherapy. Moreover, the inventive TCRs, polypeptides and proteins comprise human CDR and variable region amino acid sequences, which may reduce the risk of rejection by the human immune system as compared to, e.g., TCRs, polypeptides and proteins comprising mouse CDR and variable region amino acid sequences. The phrase “antigenic specificity,” as used herein, means that the TCR can specifically bind to and immunologically recognize mutated RAS with high avidity. For example, a TCR may be considered to have “antigenic specificity” for mutated RAS if about 1 x 104 to about 1 x 105 T cells expressing the TCR secrete at least about 200 pg/mL or more (e.g., 200 pg/mL or more, 300 pg/mL or more, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more, 1000 pg/mL or more, 5,000 pg/mL or more, 7,000
Leydig 771261 HHS E-132-2023-0-PC-01 11 pg/mL or more, 10,000 pg/mL or more, pg/mL or more, or a range defined by any two of the foregoing values) of IFN-γ upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated RAS peptide (e.g., about 0.1 ng/mL to about 10,000 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 100 ng/mL, 500 ng/mL, 1,000 ng/mL, 5,000 ng/mL, 10,000 ng/mL, or a range defined by any two of the foregoing values) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated RAS has been introduced such that the target cell expresses mutated RAS. Cells expressing the inventive TCRs may also secrete IFN-γ upon co-culture with antigen-negative, applicable HLA molecule positive target cells pulsed with higher concentrations of mutated RAS peptide. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated RAS if T cells expressing the TCR secrete at least twice as much IFN-γ upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated RAS peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated RAS has been introduced such that the target cell expresses mutated RAS as compared to the amount of IFN-γ expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR, co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the mutated RAS peptide) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cell expresses the irrelevant peptide, or (ii) untransduced T cells (e.g., derived from PBMC, which do not express the TCR) co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of mutated RAS peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated RAS has been introduced such that the target cell expresses mutated RAS. IFN-γ secretion may be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA). The concentration of pulsed peptide may be as described herein with respect to other aspects of the invention. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated RAS if at least twice as many of the numbers of T cells expressing the TCR secrete IFN-γ upon co-culture with (a) antigen-negative, applicable HLA molecule
Leydig 771261 HHS E-132-2023-0-PC-01 12 positive target cells pulsed with mutated or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated RAS has been introduced such that the target cell expresses mutated RAS as compared to the numbers of negative control T cells that secrete IFN-γ. The concentration of peptide and the negative control may be as described herein with respect to other aspects of the invention. The numbers of cells secreting IFN-γ may be measured by methods known in the art such as, for example, enzyme-linked immunospot (ELISPOT) assay. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated RAS if at least twice as many spots are detected by ELISPOT for the T cells expressing the TCR upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with mutated RAS peptide or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding mutated RAS has been introduced such that the target cell expresses mutated RAS as compared to the number of spots detected by ELISPOT for negative control T cells co- cultured with the same target cells. The concentration of peptide and the negative control may be as described herein with respect to other aspects of the invention. Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for mutated RAS if T cells expressing the TCR upregulate expression of one or both of 4-1BB and OX40 as measured by, for example, flow cytometry after stimulation with target cells expressing mutated RAS. An aspect of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR, or a combination thereof. The polypeptides of the inventive TCR can comprise any amino acid sequence, provided that the TCR has antigenic specificity for mutated RAS. In an aspect of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR)1, a CDR2, and a CDR3 of a TCR. In an aspect of the invention, the TCR comprises a first polypeptide chain comprising an α chain CDR1 (CDR1α), an α chain CDR2 (CDR2α), and an α chain CDR3 (CDR3α), and a second polypeptide chain comprising a β chain CDR1 (CDR1β), a β chain CDR2 (CDR2β), and a β chain CDR3 (CDR3β). In an aspect of the invention, the TCR comprises the amino acid sequences of: (1) all of SEQ ID NOs: 1-6 (4394 TCR-10); (2) all of SEQ ID NOs: 11-16 (4385 TCR-Z); (3) all of SEQ ID NOs: 21-26
Leydig 771261 HHS E-132-2023-0-PC-01 13 (4391 TCR-B2); (4) all of SEQ ID NOs: (4391 TCR-C1); (5) all of SEQ ID NOs: 41- 46 (4391 TCR-K); (6) all of SEQ ID NOs: 51-56 (4391 TCR-L2); (7) all of SEQ ID NOs: 61- 66 (4432 TCR-A); (8) all of SEQ ID NOs: 71-76 (4412 TCR-H); (9) all of SEQ ID NOs: 81- 86 (4385 TCR-B); (10) all of SEQ ID NOs: 91-96 (4430 TCR-N1A); (11) all of SEQ ID NOs: 101-106 (4430 TCR-N2); (12) all of SEQ ID NOs: 111-116 (4430 TCR-N8); (13) all of SEQ ID NOs: 121-126 (4430 TCR-N9); (14) all of SEQ ID NOs: 131-136 (4430 TCR- N23A); or (15) all of SEQ ID NOs: 141-146 (4430 TCR-N23B). Each one of the foregoing fifteen collections of amino acid sequences in this paragraph sets forth the six CDR regions of each of fifteen different TCRs having antigenic specificity for mutated human RAS. The six amino acid sequences in each collection correspond to the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of a TCR, respectively. The TCR may comprise the amino acid sequences of any one or more of: SEQ ID NOs: 1-6, 11-16, 21-26, 31-36, 41-46, 51-56, 61-66, 71-76, 81-86, 91-96, 101-106, 111-116, 121-126, 131-136, and 141-146. In an embodiment of the invention, the TCR comprises an amino acid sequence of a variable region of a TCR comprising the CDRs set forth above. In this regard, the TCR can comprise the amino acid sequence of: (1) SEQ ID NO: 7, (2) SEQ ID NO: 8, (3) SEQ ID NO: 9, (4) SEQ ID NO: 10, (5) SEQ ID NO: 17, (6) SEQ ID NO: 18, (7) SEQ ID NO: 19, (8) SEQ ID NO: 20, (9) SEQ ID NO: 27, (10) SEQ ID NO: 28, (11) SEQ ID NO: 29, (12) SEQ ID NO: 30, (13) SEQ ID NO: 37, (14) SEQ ID NO: 38, (15) SEQ ID NO: 39, (16) SEQ ID NO: 40, (17) SEQ ID NO: 47, (18) SEQ ID NO: 48, (19) SEQ ID NO: 49, (20) SEQ ID NO: 50, (21) SEQ ID NO: 57, (22) SEQ ID NO: 58, (23) SEQ ID NO: 59, (24) SEQ ID NO: 60, (25) SEQ ID NO: 67, (26) SEQ ID NO: 68, (27) SEQ ID NO: 69, (28) SEQ ID NO: 70, (29) SEQ ID NO: 77, (30) SEQ ID NO: 78, (31) SEQ ID NO: 79, (32) SEQ ID NO: 80, (33) SEQ ID NO: 87, (34) SEQ ID NO: 88, (35) SEQ ID NO: 89, (36) SEQ ID NO: 90, (37) SEQ ID NO: 97, (38) SEQ ID NO: 98, (39) SEQ ID NO: 99, (40) SEQ ID NO: 100, (41) SEQ ID NO: 107, (42) SEQ ID NO: 108, (43) SEQ ID NO: 109, (44) SEQ ID NO: 110, (45) SEQ ID NO: 117, (46) SEQ ID NO: 118, (47) SEQ ID NO: 119, (48) SEQ ID NO: 120, (49) SEQ ID NO: 127, (50) SEQ ID NO: 128, (51) SEQ ID NO: 129, (52) SEQ ID NO: 130, (53) SEQ ID NO: 137, (54) SEQ ID NO: 138, (55) SEQ ID NO: 139, (56) SEQ ID NO: 140, (57) SEQ ID NO: 147, (58) SEQ ID NO: 148, (59) SEQ ID NO: 149, (60) SEQ ID NO: 150, (61) both of SEQ ID NOs: 7 and 8, (62) both of SEQ ID NOs: 9 and 10, (63) both of SEQ ID NOs: 17 and 18, (64) both of SEQ ID NOs: 19 and 20, (65) both of SEQ ID NOs: 27 and 28, (66) both of SEQ ID NOs: 29 and 30, (67) both of SEQ ID NOs: 37 and 38, (68) both of SEQ ID NOs: 39 and
Leydig 771261 HHS E-132-2023-0-PC-01 14 40, (69) both of SEQ ID NOs: 47 and 48, both of SEQ ID NOs: 49 and 50, (71) both of SEQ ID NOs: 57 and 58, (72) both of SEQ ID NOs: 59 and 60, (73) both of SEQ ID NOs: 67 and 68, (74) both of SEQ ID NOs: 69 and 70, (75) both of SEQ ID NOs: 77 and 78, (76) both of SEQ ID NOs: 79 and 80, (77) both of SEQ ID NOs: 87 and 88, (78) both of SEQ ID NOs: 89 and 90, (79) both of SEQ ID NOs: 97 and 98, (80) both of SEQ ID NOs: 99 and 100, (81) both of SEQ ID NOs: 107 and 108, (82) both of SEQ ID NOs: 109 and 110, (83) both of SEQ ID NOs: 117 and 118, (84) both of SEQ ID NOs: 119 and 120, (85) both of SEQ ID NOs: 127 and 128, (86) both of SEQ ID NOs: 129 and 130, (87) both of SEQ ID NOs: 137 and 138, (88) both of SEQ ID NOs: 139 and 140, (89) both of SEQ ID NOs: 147 and 148, or (90) both of SEQ ID NOs: 149 and 150. The TCR may, e.g., comprise the amino acid sequence of any one or more of SEQ ID NOs: 7, 8, 9, 10, 17, 18, 19, 20, 27, 28, 29, 30, 37, 38, 39, 40, 47, 48, 49, 50, 57, 58, 59, 60, 67, 68, 69, 70, 77, 78, 79, 80, 87, 88, 89, 90, 97, 98, 99, 100, 107, 108, 109, 110, 117, 118, 119, 120, 127, 128, 129, 130, 137, 138, 139, 140, 147, 148, 149, and 150. The inventive TCRs may further comprise a constant region. The constant region may be derived from any suitable species such as, e.g., human or mouse. In an aspect of the invention, the TCRs further comprise a murine constant region. As used herein, the term “murine” or “human,” when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, alpha chain, and/or beta chain), means a TCR (or component thereof) which is derived from a mouse or a human, respectively, i.e., a TCR (or component thereof) that originated from or was, at one time, expressed by a mouse T cell or a human T cell, respectively. In an aspect of the invention, the TCR may comprise a murine α chain constant region and a murine β chain constant region. In an aspect of the invention, the TCR comprises a wild type (WT) α chain murine constant region comprising the amino acid sequence of SEQ ID NO: 151. The TCR may comprise a wild-type β chain murine constant region comprising the amino acid sequence of SEQ ID NO: 152. In an aspect of the invention, the murine α chain constant region may be modified or unmodified. A modified murine α chain constant region may be, e.g., cysteine-substituted, LVL-modified, or both cysteine-substituted and LVL-modified, as described, for example, in U.S. Patent No.10,174,098. The murine β chain constant region may be modified or unmodified. A modified murine β chain constant region may be, e.g., cysteine-substituted, as described, for example, in U.S. Patent No.10,174,098. In an aspect of the invention, the
Leydig 771261 HHS E-132-2023-0-PC-01 15 TCR comprises a cysteine-substituted, murine α chain constant region. In an aspect of the invention, the TCR comprises a cysteine-substituted murine β chain constant region. In an aspect of the invention, the TCR may comprise a modified murine α chain constant region comprising the amino acid sequence of SEQ ID NO: 153, wherein: (i) X at position 48 of SEQ ID NO: 153 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 153 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 153 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 153 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. The TCR may comprise a modified β chain constant region comprising the amino acid sequence of SEQ ID NO: 154, wherein X at position 57 of SEQ ID NO: 154 is Ser or Cys. In an aspect of the invention the TCR comprises the amino acid sequences of both of SEQ ID NO: 153 and 154. In an aspect of the invention, the inventive TCR can comprise an α chain of a TCR and a β chain of a TCR. The α chain of the TCR may comprise a variable region of an α chain and a constant region of an α chain. An α chain of this type can be paired with any β chain of a TCR. The β chain may comprise a variable region of a β chain and a constant region of a β chain. In an aspect of the invention, the TCR can comprise the amino acid sequence(s) of: (1) SEQ ID NO: 155; (2) SEQ ID NO: 156; (3) both of SEQ ID NOs: 155 and 156; (4) SEQ ID NO: 157; (5) SEQ ID NO: 158; (6) both of SEQ ID NOs: 157 and 158; (7) SEQ ID NO: 159; (8) SEQ ID NO: 160; (9) both of SEQ ID NOs: 159 and 160; (10) SEQ ID NO: 161; (11) SEQ ID NO: 162; (12) both of SEQ ID NOs: 161 and 162; (13) SEQ ID NO: 163; (14) SEQ ID NO: 164; (15) both of SEQ ID NOs: 163 and 164; (16) SEQ ID NO: 165; (17) SEQ ID NO: 166; (18) both of SEQ ID NOs: 165 and 166; (19) SEQ ID NO: 167; (20) SEQ ID NO: 168; (21) both of SEQ ID NOs: 167 and 168; (22) SEQ ID NO: 169; (23) SEQ ID NO: 170; (24) both of SEQ ID NOs: 169 and 170; (25) SEQ ID NO: 171; (26) SEQ ID NO: 172; (27) both of SEQ ID NOs: 171 and 172; (28) SEQ ID NO: 173; (29) SEQ ID NO: 174; (30) both of SEQ ID NOs: 173 and 174; (31) SEQ ID NO: 175; (32) SEQ ID NO: 176; (33) both of SEQ ID NO: 175 and 176; (34) SEQ ID NO: 177; (35) SEQ ID NO: 178; (36) both of SEQ ID NO: 177 and 178; (37) SEQ ID NO: 179; (38) SEQ ID NO: 180; (39) both of SEQ ID NO: 179 and 180; (40) SEQ ID NO: 181; (41) SEQ ID NO: 182; (42) both of SEQ ID NO: 181 and 182; (43) SEQ ID NO: 183; (44) SEQ ID NO: 184; (45) both of SEQ ID NO: 183 and 184; (46) SEQ ID NO: 185; (47) SEQ ID NO: 186; (48) both of SEQ ID NO: 185
Leydig 771261 HHS E-132-2023-0-PC-01 16 and 186; (49) SEQ ID NO: 187; (50) SEQ 188; (51) both of SEQ ID NO: 187 and 188; (52) SEQ ID NO: 189; (53) SEQ ID NO: 190; (54) both of SEQ ID NO: 189 and 190; (55) SEQ ID NO: 191; (56) SEQ ID NO: 192; (57) both of SEQ ID NO: 191 and 192; (58) SEQ ID NO: 193; (59) SEQ ID NO: 194; (60) both of SEQ ID NO: 193 and 194; (61) SEQ ID NO: 195; (62) SEQ ID NO: 196; (63) both of SEQ ID NO: 195 and 196; (64) SEQ ID NO: 197; (65) SEQ ID NO: 198; (66) both of SEQ ID NO: 197 and 198; (67) SEQ ID NO: 199; (68) SEQ ID NO: 200; (69) both of SEQ ID NO: 199 and 200; (70) SEQ ID NO: 201; (71) SEQ ID NO: 202; (72) both of SEQ ID NO: 201 and 202; (73) SEQ ID NO: 203; (74) SEQ ID NO: 204; (75) both of SEQ ID NO: 203 and 204; (76) SEQ ID NO: 205; (77) SEQ ID NO: 206; (78) both of SEQ ID NO: 205 and 206; (79) SEQ ID NO: 207; (80) SEQ ID NO: 208; (81) both of SEQ ID NO: 207 and 208; (82) SEQ ID NO: 209; (83) SEQ ID NO: 210; (84) both of SEQ ID NO: 209 and 210; (85) SEQ ID NO: 211; (86) SEQ ID NO: 212; (87) both of SEQ ID NO: 211 and 212; (88) SEQ ID NO: 213; (89) SEQ ID NO: 214; or (90) both of SEQ ID NO: 213 and 214. The TCR may comprise the amino acid sequence of any one or more of SEQ ID NOs: 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214. Included in the scope of the invention are functional variants of the inventive TCRs described herein. The term “functional variant,” as used herein, refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, which functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants encompass, for example, those variants of the TCR, polypeptide, or protein described herein (the parent TCR, polypeptide, or protein) that retain the ability to specifically bind to mutated RAS for which the parent TCR has antigenic specificity or to which the parent polypeptide or protein specifically binds, to a similar extent, the same extent, or to a higher extent, as the parent TCR, polypeptide, or protein. In reference to the parent TCR, polypeptide, or protein, the functional variant can, for instance, be at least about 30%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein, respectively.
Leydig 771261 HHS E-132-2023-0-PC-01 17 The functional variant can, for comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc. Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent TCR, polypeptide, or protein. The TCR, polypeptide, or protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not materially change the biological activity of the TCR, polypeptide, or protein. Also provided by the invention is a polypeptide comprising a functional portion of any of the TCRs described herein. The term “polypeptide,” as used herein, includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds. With respect to the inventive polypeptides, the functional portion can be any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the functional portion specifically binds to mutated RAS. The term “functional portion,” when used in reference to a TCR, refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Functional portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to mutated RAS (e.g., in an applicable HLA molecule-dependent
Leydig 771261 HHS E-132-2023-0-PC-01 18 manner), or detect, treat, or prevent cancer, a similar extent, the same extent, or to a higher extent, as the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or more, of the parent TCR. The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to mutated RAS; and/or having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent TCR. The polypeptide can comprise a functional portion of either or both of the α and β chains of the TCRs of the invention, such as a functional portion comprising one of more of CDR1, CDR2, and CDR3 of the variable region(s) of the α chain and/or β chain of a TCR of the invention. In an aspect of the invention, the polypeptide comprises the amino acid sequences of: (1) all of SEQ ID NOs: 1-6; (2) all of SEQ ID NOs: 11-16; (3) all of SEQ ID NOs: 21-26; (4) all of SEQ ID NOs: 31-36; (5) all of SEQ ID NOs: 41-46; (6) all of SEQ ID NOs: 51-56; (7) all of SEQ ID NOs: 61-66; (8) all of SEQ ID NOs: 71-76; (9) all of SEQ ID NOs: 81-86; (10) all of SEQ ID NOs: 91-96; (11) all of SEQ ID NOs: 101-106; (12) all of SEQ ID NOs: 111-116; (13) all of SEQ ID NOs: 121-126; (14) all of SEQ ID NOs: 131-136; or (15) all of SEQ ID NOs: 141-146. The polypeptide may comprise the amino acid sequences of any one or more of: SEQ ID NOs: 1-6, 11-16, 21-26, 31-36, 41-46, 51-56, 61- 66, 71-76, 81-86, 91-96, 101-106, 111-116, 121-126, 131-136, and 141-146. In an aspect of the invention, the inventive polypeptide can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the polypeptide can comprise, e.g., the amino acid sequences of: (1) SEQ ID NO: 7, (2) SEQ ID NO: 8, (3) SEQ ID NO: 9, (4) SEQ ID NO: 10, (5) SEQ ID NO: 17, (6) SEQ ID NO: 18, (7) SEQ ID NO: 19, (8) SEQ ID NO: 20, (9) SEQ ID NO: 27, (10) SEQ ID NO: 28, (11) SEQ ID NO: 29, (12) SEQ ID NO: 30, (13) SEQ ID NO: 37, (14) SEQ ID NO: 38, (15) SEQ ID NO: 39, (16) SEQ ID NO: 40, (17) SEQ ID NO: 47, (18) SEQ ID NO: 48, (19) SEQ ID NO: 49, (20) SEQ ID NO: 50, (21) SEQ ID NO: 57, (22) SEQ ID NO: 58, (23) SEQ ID NO: 59, (24) SEQ ID NO: 60, (25) SEQ ID NO: 67, (26) SEQ ID NO: 68, (27) SEQ ID NO: 69, (28) SEQ ID NO: 70, (29) SEQ ID NO: 77, (30) SEQ ID NO: 78,
Leydig 771261 HHS E-132-2023-0-PC-01 19 (31) SEQ ID NO: 79, (32) SEQ ID NO: 80, SEQ ID NO: 87, (34) SEQ ID NO: 88, (35) SEQ ID NO: 89, (36) SEQ ID NO: 90, (37) SEQ ID NO: 97, (38) SEQ ID NO: 98, (39) SEQ ID NO: 99, (40) SEQ ID NO: 100, (41) SEQ ID NO: 107, (42) SEQ ID NO: 108, (43) SEQ ID NO: 109, (44) SEQ ID NO: 110, (45) SEQ ID NO: 117, (46) SEQ ID NO: 118, (47) SEQ ID NO: 119, (48) SEQ ID NO: 120, (49) SEQ ID NO: 127, (50) SEQ ID NO: 128, (51) SEQ ID NO: 129, (52) SEQ ID NO: 130, (53) SEQ ID NO: 137, (54) SEQ ID NO: 138, (55) SEQ ID NO: 139, (56) SEQ ID NO: 140, (57) SEQ ID NO: 147, (58) SEQ ID NO: 148, (59) SEQ ID NO: 149, (60) SEQ ID NO: 150, (61) both of SEQ ID NOs: 7 and 8, (62) both of SEQ ID NOs: 9 and 10, (63) both of SEQ ID NOs: 17 and 18, (64) both of SEQ ID NOs: 19 and 20, (65) both of SEQ ID NOs: 27 and 28, (66) both of SEQ ID NOs: 29 and 30, (67) both of SEQ ID NOs: 37 and 38, (68) both of SEQ ID NOs: 39 and 40, (69) both of SEQ ID NOs: 47 and 48, (70) both of SEQ ID NOs: 49 and 50, (71) both of SEQ ID NOs: 57 and 58, (72) both of SEQ ID NOs: 59 and 60, (73) both of SEQ ID NOs: 67 and 68, (74) both of SEQ ID NOs: 69 and 70, (75) both of SEQ ID NOs: 77 and 78, (76) both of SEQ ID NOs: 79 and 80, (77) both of SEQ ID NOs: 87 and 88, (78) both of SEQ ID NOs: 89 and 90, (79) both of SEQ ID NOs: 97 and 98, (80) both of SEQ ID NOs: 99 and 100, (81) both of SEQ ID NOs: 107 and 108, (82) both of SEQ ID NOs: 109 and 110, (83) both of SEQ ID NOs: 117 and 118, (84) both of SEQ ID NOs: 119 and 120, (85) both of SEQ ID NOs: 127 and 128, (86) both of SEQ ID NOs: 129 and 130, (87) both of SEQ ID NOs: 137 and 138, (88) both of SEQ ID NOs: 139 and 140, (89) both of SEQ ID NOs: 147 and 148, or (90) both of SEQ ID NOs: 149 and 150. The polypeptide may, e.g., comprise the amino acid sequence of any one or more of SEQ ID NOs: 7, 8, 9, 10, 17, 18, 19, 20, 27, 28, 29, 30, 37, 38, 39, 40, 47, 48, 49, 50, 57, 58, 59, 60, 67, 68, 69, 70, 77, 78, 79, 80, 87, 88, 89, 90, 97, 98, 99, 100, 107, 108, 109, 110, 117, 118, 119, 120, 127, 128, 129, 130, 137, 138, 139, 140, 147, 148, 149, and 150. In an aspect of the invention, the inventive polypeptide can further comprise the constant region of the inventive TCR set forth above. In this regard, the polypeptide can further comprise, e.g., the amino acid sequence of (i) SEQ ID NO: 151, (ii) SEQ ID NO: 152, (iii) SEQ ID NO: 153, (iv) SEQ ID NO: 154, (v) both of SEQ ID NOs: 151 and 152, or (vi) both of SEQ ID NOs: 153 and 154. The “X” at positions 48, 112, 114, and 115 of SEQ ID NO: 153 are as defined herein with respect to other aspects of the invention. The “X” at position 57 of SEQ ID NO: 154 is as defined herein with respect to other aspects of the invention.
Leydig 771261 HHS E-132-2023-0-PC-01 20 In an aspect of the invention, the polypeptide may comprise an α chain and a β chain of the inventive TCR. In this regard, the polypeptide can comprise, e.g., the amino acid sequences of: (1) SEQ ID NO: 155; (2) SEQ ID NO: 156; (3) both of SEQ ID NOs: 155 and 156; (4) SEQ ID NO: 157; (5) SEQ ID NO: 158; (6) both of SEQ ID NOs: 157 and 158; (7) SEQ ID NO: 159; (8) SEQ ID NO: 160; (9) both of SEQ ID NOs: 159 and 160; (10) SEQ ID NO: 161; (11) SEQ ID NO: 162; (12) both of SEQ ID NOs: 161 and 162; (13) SEQ ID NO: 163; (14) SEQ ID NO: 164; (15) both of SEQ ID NOs: 163 and 164; (16) SEQ ID NO: 165; (17) SEQ ID NO: 166; (18) both of SEQ ID NOs: 165 and 166; (19) SEQ ID NO: 167; (20) SEQ ID NO: 168; (21) both of SEQ ID NOs: 167 and 168; (22) SEQ ID NO: 169; (23) SEQ ID NO: 170; (24) both of SEQ ID NOs: 169 and 170; (25) SEQ ID NO: 171; (26) SEQ ID NO: 172; (27) both of SEQ ID NOs: 171 and 172; (28) SEQ ID NO: 173; (29) SEQ ID NO: 174; (30) both of SEQ ID NOs: 173 and 174; (31) SEQ ID NO: 175; (32) SEQ ID NO: 176; (33) both of SEQ ID NO: 175 and 176; (34) SEQ ID NO: 177; (35) SEQ ID NO: 178; (36) both of SEQ ID NO: 177 and 178; (37) SEQ ID NO: 179; (38) SEQ ID NO: 180; (39) both of SEQ ID NO: 179 and 180; (40) SEQ ID NO: 181; (41) SEQ ID NO: 182; (42) both of SEQ ID NO: 181 and 182; (43) SEQ ID NO: 183; (44) SEQ ID NO: 184; (45) both of SEQ ID NO: 183 and 184; (46) SEQ ID NO: 185; (47) SEQ ID NO: 186; (48) both of SEQ ID NO: 185 and 186; (49) SEQ ID NO: 187; (50) SEQ ID NO: 188; (51) both of SEQ ID NO: 187 and 188; (52) SEQ ID NO: 189; (53) SEQ ID NO: 190; (54) both of SEQ ID NO: 189 and 190; (55) SEQ ID NO: 191; (56) SEQ ID NO: 192; (57) both of SEQ ID NO: 191 and 192; (58) SEQ ID NO: 193; (59) SEQ ID NO: 194; (60) both of SEQ ID NO: 193 and 194; (61) SEQ ID NO: 195; (62) SEQ ID NO: 196; (63) both of SEQ ID NO: 195 and 196; (64) SEQ ID NO: 197; (65) SEQ ID NO: 198; (66) both of SEQ ID NO: 197 and 198; (67) SEQ ID NO: 199; (68) SEQ ID NO: 200; (69) both of SEQ ID NO: 199 and 200; (70) SEQ ID NO: 201; (71) SEQ ID NO: 202; (72) both of SEQ ID NO: 201 and 202; (73) SEQ ID NO: 203; (74) SEQ ID NO: 204; (75) both of SEQ ID NO: 203 and 204; (76) SEQ ID NO: 205; (77) SEQ ID NO: 206; (78) both of SEQ ID NO: 205 and 206; (79) SEQ ID NO: 207; (80) SEQ ID NO: 208; (81) both of SEQ ID NO: 207 and 208; (82) SEQ ID NO: 209; (83) SEQ ID NO: 210; (84) both of SEQ ID NO: 209 and 210; (85) SEQ ID NO: 211; (86) SEQ ID NO: 212; (87) both of SEQ ID NO: 211 and 212; (88) SEQ ID NO: 213; (89) SEQ ID NO: 214; or (90) both of SEQ ID NO: 213 and 214. The polypeptide may comprise the amino acid sequence of any one or more of SEQ ID NOs: 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,
Leydig 771261 HHS E-132-2023-0-PC-01 21 180, 181, 182, 183, 184, 185, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214. An aspect of the invention further provides a protein comprising a functional portion of any of the inventive TCRs described herein. By “protein” is meant a molecule comprising one or more polypeptide chains. In an aspect, the protein of the invention can comprise: first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 1-3 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 4-6; (2) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 11-13 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 14-16; (3) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 21- 23 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 24-26; (4) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 31-33 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 34-36; (5) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 41-43 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 44-46; (6) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 51-53 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 54-56; (7) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 61-63 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 64-66; (8) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 71-73 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 74-76; (9) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 81-83 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 84- 86; (10) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 91-93 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 94-96; (11) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 101-103 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 104-106; (12) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 111-113 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 114-116; (13) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 121-123 and
Leydig 771261 HHS E-132-2023-0-PC-01 22 the second polypeptide chain comprises the acid sequences of all of SEQ ID NOs: 124-126; (14) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 131-133 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 134-136; or (15) the first polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 141-143 and the second polypeptide chain comprises the amino acid sequences of all of SEQ ID NOs: 144-146. In an aspect of the invention, the protein comprises first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 7; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 20; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 20; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30; (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30; (19) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37; (20) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38; (21) the first
Leydig 771261 HHS E-132-2023-0-PC-01 23 polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38; (22) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39; (23) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (24) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (25) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47; (26) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48; (27) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48; (28) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 49; (29) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 50; (30) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 49 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 50; (31) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 57; (32) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 58; (33) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 57 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 58; (34) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 59; (35) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60; (36) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 59 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60; (37) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67; (38) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68; (39) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68; (40) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69; (41) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70; (42) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70; (43) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77; (44) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 78; (45) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77 and the second
Leydig 771261 HHS E-132-2023-0-PC-01 24 polypeptide chain comprises the amino acid sequence of SEQ ID NO: 78; (46) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 79; (47) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80; (48) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 79 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80; (49) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87; (50) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88; (51) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88; (52) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89; (53) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 90; (54) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 90; (55) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 97; (56) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 98; (57) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 97 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 98; (58) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 99; (59) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100; (60) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 99 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100; (61) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107; (62) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108; (63) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108; (64) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109; (65) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110; (66) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110; (67) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117; (68) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118; (69) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118; (70) the first
Leydig 771261 HHS E-132-2023-0-PC-01 25 polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119; (71) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120; (72) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120; (73) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127; (74) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128; (75) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128; (76) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; (77) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130; (78) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130; (79) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; (80) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138; (81) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138; (82) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139; (83) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140; (84) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140; (85) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147; (86) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148; (87) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148; (88) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; (89) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150; or (90) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150. In an aspect of the invention, (1) the first polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 151, (2) the second polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 152, (3) the first polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 151 and the second polypeptide chain of the protein further comprises the amino acid
Leydig 771261 HHS E-132-2023-0-PC-01 26 sequence of SEQ ID NO: 152, (4) the first chain of the protein further comprises the amino acid sequence of SEQ ID NO: 153, (5) the second polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 154, or (6) the first polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 153 and the second polypeptide chain of the protein further comprises the amino acid sequence of SEQ ID NO: 154. The “X” at positions 48, 112, 114, and 115 of SEQ ID NO: 153 are as defined herein with respect to other aspects of the invention. The “X” at position 57 of SEQ ID NO: 154 is as defined herein with respect to other aspects of the invention. In an aspect of the invention, the protein comprises first and second polypeptide chains, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163; (14) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165; (17) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166; (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166; (19) the
Leydig 771261 HHS E-132-2023-0-PC-01 27 first polypeptide chain comprises the amino sequence of SEQ ID NO: 167; (20) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168; (21) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168; (22) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169; (23) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170; (24) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170; (25) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171; (26) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172; (27) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172; (28) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173; (29) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174; (30) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174; (31) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175; (32) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176; (33) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176; (34) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177; (35) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178; (36) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178; (37) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179; (38) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 180; (39) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 180; (40) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181; (41) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182; (42) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182; (43) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183; (44) the
Leydig 771261 HHS E-132-2023-0-PC-01 28 second polypeptide chain comprises the acid sequence of SEQ ID NO: 184; (45) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184; (46) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185; (47) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186; (48) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186; (49) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187; (50) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188; (51) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188; (52) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 189; (53) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 190; (54) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 189 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 190; (55) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 191; (56) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 192; (57) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 191 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 192; (58) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 193; (59) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 194; (60) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 193 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 194; (61) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 195; (62) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 196; (63) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 195 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 196; (64) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 197; (65) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 198; (66) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 197 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 198; (67) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 199; (68) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 200; (69) the
Leydig 771261 HHS E-132-2023-0-PC-01 29 first polypeptide chain comprises the amino sequence of SEQ ID NO: 199 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 200; (70) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 201; (71) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 202; (72) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 201 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 202; (73) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 203; (74) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 204; (75) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 203 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 204; (76) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 205; (77) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 206; (78) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 205 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 206; (79) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 207; (80) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 208; (81) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 207 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 208; (82) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 209; (83) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 210; (84) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 209 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 210; (85) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 211; (86) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 212; (87) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 211 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 212; (88) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 213; (89) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 214; or (90) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 213 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 214. The protein of the invention may be a TCR. Alternatively, if the first and/or second polypeptide chain(s) of the protein further comprise(s) other amino acid sequences, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof, then the
Leydig 771261 HHS E-132-2023-0-PC-01 30 inventive protein can be a fusion protein. regard, an aspect of the invention also provides a fusion protein comprising at least one of the inventive polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc. The fusion protein can comprise one or more copies of the inventive polypeptide and/or one or more copies of the other polypeptide. For instance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copies of the inventive polypeptide and/or of the other polypeptide. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. In some aspects of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the α chain and the β chain. In this regard, the TCRs, polypeptides, and proteins of the invention may further comprise a linker peptide. The linker peptide may advantageously facilitate the expression of a recombinant TCR, polypeptide, and/or protein in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may comprise the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 231). Upon expression of the construct including the linker peptide by a host cell, the linker peptide may be cleaved, resulting in separated α and β chains. In an aspect of the invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide positioned between the α and β chains. In some aspects, the TCR, polypeptide or protein disclosed herein comprises an α chain and/or a β chain, as disclosed herein, comprising a signal peptide. In some aspects, the sequence of the signal peptide of any of the α chains and/or β chains disclosed herein comprises an leucine, lysine, alanine or histidine residue substituted for the wild-type residue at position 2. In some aspects, the TCR, polypeptide or protein disclosed herein comprises a mature version of an α chain and/or a β chain, as disclosed herein, that lacks a signal peptide.
Leydig 771261 HHS E-132-2023-0-PC-01 31 The protein of the invention can a recombinant antibody, or an antigen binding portion thereof, comprising at least one of the inventive polypeptides described herein. As used herein, “recombinant antibody” refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or an antigen binding portion thereof. The polypeptide of an antibody, or antigen binding portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment of an antibody, etc. The polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention. The polypeptide of an antibody, or an antigen binding portion thereof, can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein. The TCRs, polypeptides, and proteins of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the TCRs, polypeptides, or proteins retain their biological activity, e.g., the ability to specifically bind to mutated RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc. For example, the polypeptide can be in the range of from about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides. The TCRs, polypeptides, and proteins of the invention of the invention can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl- cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl-lysine, 6- hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic
Leydig 771261 HHS E-132-2023-0-PC-01 32 acid, α,γ-diaminobutyric acid, α,β- acid, homophenylalanine, and α-tert- butylglycine. The TCRs, polypeptides, and proteins of the invention can be, e.g., glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated. The inventive TCRs, polypeptides, and proteins described herein (including any of the functional portions or variants thereof) are also contemplated to be useful as the soluble TCR component of bispecific engager TCR fusion proteins (e.g., IMMTAC (immune- mobilizing monoclonal TCRs against cancer) molecules). Bispecific engager TCR fusion proteins have two components. One component comprises a soluble TCR. The other component comprises an anti-CD3 effector. The anti-CD3 effector may be any molecule that engages with a CD3 molecule on T cells and activates a T cell immune response. For example, the anti-CD3 effector may be an anti-CD3 antibody or anti-CD3 antibody fragment. The soluble TCR component of the bispecific engager TCR fusion protein binds to the target antigen presented on the surface of cancer cells presented by an HLA molecule. The anti- CD3 effector component engages a CD3 molecule on T cells. The engagement of these components of the bispecific engager TCR fusion protein triggers the activation and recruitment of T cells and redirects T-cell killing to tumor cells. An aspect of the invention provides a bispecific engager TCR fusion protein comprising (i) any of the inventive TCRs, polypeptides, or proteins (including any of the functional portions or variants thereof) described herein and (ii) an anti-CD3 effector. Hereinafter, references to “protein(s)” also encompass the bispecific engager TCR fusion proteins described herein, unless specified otherwise. The TCR, polypeptide, and/or protein of the invention can be obtained by methods known in the art such as, for example, de novo synthesis. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and/or proteins described herein can be synthesized by any of a variety of commercial entities. In this respect, the inventive TCRs, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.
Leydig 771261 HHS E-132-2023-0-PC-01 33 An aspect of the invention a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. “Nucleic acid,” as used herein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In an aspect, the nucleic acid comprises complementary DNA (cDNA). It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions. An aspect of the invention provides an isolated or purified nucleic acid comprising, from 5’ to 3’, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequence, respectively, encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 17 and 18; 18 and 17; 19 and 20; 20 and 19; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 37 and 38; 38 and 37; 39 and 40; 40 and 39; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 57 and 58; 58 and 57; 59 and 60; 60 and 59; 67 and 68; 68 and 67; 69 and 70; 70 and 69; 77 and 78; 78 and 77; 79 and 80; 80 and 79; 87 and 88; 88 and 87; 89 and 90; 90 and 89; 97 and 98; 98 and 97; 99 and 100; 100 and 99; 107 and 108; 108 and 107; 109 and 110; 110 and 109; 117 and 118; 118 and 117; 119 and 120; 120 and 119; 127 and 128; 128 and 127; 129 and 130; 130 and 129; 137 and 138; 139 and 137; 139 and 140; 140 and 139; 147 and 148; 148 and 147; 149 and 150; or 150 and 149. In an aspect of the invention, the nucleic acid further comprises a third nucleotide acid sequence interposed between the first and second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide. For example, the cleavable linker peptide may comprise the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 231). Preferably, the nucleic acids of the invention are recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.
Leydig 771261 HHS E-132-2023-0-PC-01 34 The nucleic acids can be based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be synthesized by any of a variety of commercial entities. In an aspect of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. An aspect of the invention also provides a nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein. The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, an aspect of the invention provides a recombinant
Leydig 771261 HHS E-132-2023-0-PC-01 35 expression vector comprising any of the acids of the invention. In an aspect of the invention, the recombinant expression vector comprises a nucleotide sequence encoding the α chain, the β chain, and linker peptide. For purposes herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non- naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector. The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the transposon/transposase series, pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a transposon or a viral vector, e.g., a lentiviral vector or a retroviral vector. The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication
Leydig 771261 HHS E-132-2023-0-PC-01 36 systems can be derived, e.g., from ColEl, 2 μ λ, SV40, bovine papillomavirus, and the like. Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based. The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the TCR, polypeptide, or protein, or to the nucleotide sequence which is complementary to the nucleotide sequence encoding the TCR, polypeptide, or protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter, e.g., a human elongation factor-1α promoter, or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression. Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and
Leydig 771261 HHS E-132-2023-0-PC-01 37 include, for example, the Herpes Simplex (HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase, and nitroreductase. Another aspect of the invention provides an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Still another aspect of the invention provides an isolated or purified TCR, polypeptide, or protein that results from expression of any of the nucleic acids or vectors, described herein with respect to other aspects of the invention, in a cell. Another aspect of the invention further provides a host cell comprising any of the nucleic acids or any of the recombinant expression vectors described herein. As used herein, the term “host cell” refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5α E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. For example, the host cell may be a human lymphocyte. In an aspect of the invention, the host cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a natural killer (NK) cell, a macrophage, a pluripotent cell, and a multipotent cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. The T cell can be any type of T cell and can be of any developmental stage,
Leydig 771261 HHS E-132-2023-0-PC-01 38 including but not limited to, CD4+/CD8+ positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naïve T cells, and the like. In an aspect of the invention, the host cell is a pluripotent cell or a multipotent cell. Pluripotent cells have the capacity to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent cells may comprise, for example, stem cells, e.g., embryonic stem cells, nuclear transfer derived embryonic stem cells, induced pluripotent stem cells (iPSC), etc. Multipotent cells may comprise, for example, hematopoietic stem cells. Modifying, e.g., reprogramming, cells to a pluripotent state refers to the reversion of a cell to a pluripotent cell and is described for example, in Crompton et al., Trends Immunol., 35(4): 178-185 (2014). Exemplary techniques may include somatic cell nuclear transfer (SCNT), cell–cell fusion, and direct reprogramming. Examples of methods for carrying out cell-cell fusion are described, for example, in Ogle et al., Nat. Rev. Mol. Cell Biol.6: 567-75 (2005) and Zhou et al., Cell Stem Cell, 3: 382-388 (2008). Examples of methods for carrying out SCNT are described, for example, in Hanna et al., Cell, 143: 508- 525 (2010); Stadtfeld et al., Genes Dev., 24: 2239-2263 (2010); Wilmut et al., Nature, 385: 810-813 (1997); Vizcardo et al., Cell Stem Cell, 12: 31-36 (2013); and Crompton et al., Cell Stem Cell, 12: 6-8 (2013). In an aspect of the invention, the host cell is an iPSC that was prepared by reprogramming, any of the host cells described herein (e.g., T cells, NK cells, or invariant natural killer T cells) to a pluripotent state. Also provided by an aspect of the invention is an isolated or purified population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one
Leydig 771261 HHS E-132-2023-0-PC-01 39 aspect of the invention, the population of is a clonal population comprising host cells comprising a recombinant expression vector as described herein. In an aspect of the invention, the numbers of cells in the population may be rapidly expanded. Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent Nos. 8,034,334; 8,383,099; and 11,401,503; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In an aspect, expansion of the numbers of T cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC). An aspect of the invention provides a method of producing any of the TCRs, polypeptides, or proteins described herein, the method comprising culturing any of the host cells or populations of host cells described herein, so that the TCR, polypeptide, or protein is produced. Another aspect of the invention is a method of producing a host cell expressing a TCR that has antigenic specificity for a human RASG12V, human RASG12D, or human RASG12R amino acid sequence, the method comprising contacting a cell with any of the inventive recombinant expression vectors described herein under conditions that allow introduction of the vector into the cell. Another aspect of the invention provides a method of producing an engineered human cell (or an engineered population of human cells), the method comprising introducing any of the inventive nucleic acids or recombinant expression vectors described herein to an isolated human cell (or an isolated population of human cells), wherein the nucleic acid or recombinant expression vector comprises a nucleotide sequence encoding any of the inventive TCRs, polypeptides, or proteins described herein. The isolated human cell, or isolated population of human cells, to which the nucleic acid or recombinant expression vector is introduced, may be as described herein with respect to other aspects of the invention. The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof), can be isolated and/or purified. The term “isolated” as used herein means having been removed from its natural environment. The term “purified” as used herein means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity.
Leydig 771261 HHS E-132-2023-0-PC-01 40 For example, the purity can be at least at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or can be about 100%. The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof), all of which are collectively referred to as “inventive TCR materials” hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, an aspect of the invention provides a pharmaceutical composition comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof), described herein, and a pharmaceutically acceptable carrier. The inventive pharmaceutical compositions containing any of the inventive TCR materials can comprise more than one inventive TCR material, e.g., a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition can comprise an inventive TCR material in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the particular inventive TCR material under consideration. Methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 23rd Ed., Academic Press (2020). It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular inventive TCR material, as well as by the particular method used to administer the inventive TCR material. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or interperitoneal administration. More than one route can be used to administer the inventive TCR materials, and in certain instances, a particular route can provide a more immediate and more effective response than another route. Preferably, the inventive TCR material is administered by injection, e.g., intravenously. When the inventive TCR material is a host cell expressing the inventive TCR,
Leydig 771261 HHS E-132-2023-0-PC-01 41 the pharmaceutically acceptable carrier for cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an aspect, the pharmaceutically acceptable carrier is supplemented with human serum albumin. The amount or dose (e.g., numbers of cells when the inventive TCR material is one or more cells) of the inventive TCR material administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the inventive TCR material should be sufficient to bind to a cancer antigen (e.g., mutated RAS), or detect, treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain aspects, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive TCR material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. Many assays for determining an administered dose are known in the art. For example, an assay, which comprises comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing the inventive TCR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a certain dose can be assayed by methods known in the art. The dose of the inventive TCR material also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR material. Typically, the attending physician will decide the dosage of the inventive TCR material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR material to be administered, route of administration, and the severity of the cancer being treated. In an aspect in which the inventive TCR material is a population of cells, the number of cells administered per infusion may vary, e.g., from about 1 x 106 to about 1 x 1012 cells or more. In certain aspects, fewer than 1 x 106 cells may be administered. One of ordinary skill in the art will readily appreciate that the inventive TCR materials of the invention can be modified in any number of ways, such that the therapeutic
Leydig 771261 HHS E-132-2023-0-PC-01 42 or prophylactic efficacy of the inventive materials is increased through the modification. For instance, the inventive TCR materials can be conjugated either directly or indirectly through a bridge to a chemotherapeutic agent. The practice of conjugating compounds to a chemotherapeutic agent is known in the art. One of ordinary skill in the art recognizes that sites on the inventive TCR materials, which are not necessary for the function of the inventive TCR materials, are ideal sites for attaching a bridge and/or a chemotherapeutic agent, provided that the bridge and/or chemotherapeutic agent, once attached to the inventive TCR materials, do(es) not interfere with the function of the inventive TCR materials, i.e., the ability to bind to mutated RAS or to detect, treat, or prevent cancer. It is contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells can be used in methods of treating or preventing cancer. Without being bound to a particular theory, the inventive TCRs are believed to bind specifically to mutated RAS, such that the TCR (or related inventive polypeptide or protein), when expressed by a cell, is able to mediate an immune response against a target cell expressing mutated RAS. In this regard, an aspect of the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal. An aspect of the invention provides any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal. The terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
Leydig 771261 HHS E-132-2023-0-PC-01 43 Furthermore, the treatment or prevention by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, “prevention” can encompass delaying the onset of the cancer, or a symptom or condition thereof. Alternatively or additionally, “prevention” may encompass preventing or delaying the recurrence of cancer, or a symptom or condition thereof. It is also contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells can be used in methods of inducing an immune response against a cancer in a mammal. In this regard, an aspect of the invention provides a method of inducing an immune response against a cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, in an amount effective to induce an immune response against the cancer in the mammal. An aspect of the invention provides any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, for use in the inducement of an immune response against a cancer in a mammal. Also provided by an aspect of the invention is a method of detecting the presence of cancer in a mammal. The method comprises (i) contacting a sample comprising one or more cells from the mammal with any of the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions described herein, thereby forming a complex, and (ii) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal. With respect to the inventive method of detecting cancer in a mammal, the sample of cells can be a sample comprising whole cells, lysates thereof, or a fraction of the whole
Leydig 771261 HHS E-132-2023-0-PC-01 44 cell lysates, e.g., a nuclear or cytoplasmic a whole protein fraction, or a nucleic acid fraction. For purposes of the inventive detecting method, the contacting can take place in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro. Also, detection of the complex can occur through any number of ways known in the art. For instance, the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells, described herein, can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles). For purposes of the inventive methods, wherein host cells or populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal. With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer. A preferred cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In an embodiment of the invention, the cancer expresses a mutated human RAS amino acid sequence with a substitution of glycine at position 12 with aspartic acid, valine, or arginine, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid
Leydig 771261 HHS E-132-2023-0-PC-01 45 sequence, and wherein position 12 is reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. The mutated human KRAS, mutated human HRAS, and mutated human NRAS expressed by the cancer may be as described herein with respect to other aspects of the invention. The mammal referred to in the inventive methods can be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1 This example demonstrates the isolation of TCRs from Patients 4385, 4391, 4394, 4412, 4430, and 4432. Tumor infiltrating lymphocytes (TIL) were obtained from each of cancer Patients 4385, 4391, 4394, 4412, 4430, and 4432. Reactive TCRs were isolated from TILs using in vitro-sensitization (IVS). TIL were co-cultured with antigen presenting cells pulsed with mutated RAS peptides or transfected with the mutated RAS genes. The IVS was followed by another co-culture with the same APCs and peptides followed by fluorescence-activated cell sorting (FACS) based on upregulation of the T cell activation markers, 4-1BB and OX-40. The sequences of the TCR alpha and beta chain variable regions were identified by single-cell TCR sequencing. The amino acid sequences of the alpha and beta chain variable regions are shown in Table 2. The CDRs are underlined. The N-terminal signal peptides are in bold font. TABLE 2 TCR TCR Chain Amino Acid Sequence N m S E
Leydig 771261 HHS E-132-2023-0-PC-01 46 TCR TCR Chain Amino Acid Name (With N-terminal GYSYLYIKGSQPEDSATYLCAFLQGAQKLVFGQGTRLTINP (SEQ ID Q K Q L L V A T P PI G F S F S R L D H
Leydig 771261 HHS E-132-2023-0-PC-01 47 TCR TCR Chain Amino Acid Name Variable β GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQ A N P Q K E V T N P Q K Q E S V N P Q K
Leydig 771261 HHS E-132-2023-0-PC-01 48 TCR TCR Chain Amino Acid Name (With N-terminal KERFSLILESASTNQTSMYLCASSFLGGNYNEQFFGPGTRLTVL E S V N Q L II T LI A N Q K E V
Leydig 771261 HHS E-132-2023-0-PC-01 49 TCR TCR Chain Amino Acid Name (Predicted P D M S T R ) P N G G Q S S I N
Leydig 771261 HHS E-132-2023-0-PC-01 50 TCR TCR Chain Amino Acid Name Variable α QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPE N Y S T R P N Q G A Q S T R 7) P N Q G G Q S T R
Leydig 771261 HHS E-132-2023-0-PC-01 51 TCR TCR Chain Amino Acid Name (With N-terminal VSLSDTAVYYCLVGDADQAGTALIFGKGTTLSVSS (SEQ ID NO: P N Q G A Q S T R 7) P N Q G G Q S
EXAMPLE 2 This example demonstrates the construction of retroviral vectors encoding the respective TCRs of Example 1. Nucleotide sequences encoding the variable regions of the α and β chains of the TCRs of Table 2 were obtained and codon optimized. The TCRβ VDJ regions were fused to the mouse TCRβ constant chain. The TCRα VJ regions were fused to the mouse TCRα constant chain. Without being bound to a particular theory or mechanism, it is believed that
Leydig 771261 HHS E-132-2023-0-PC-01 52 replacing the constant regions of the human and TCRβ chains with the corresponding murine constant regions improves TCR expression and functionality (Cohen et al., Cancer Res., 66(17): 8878-86 (2006)). In addition, the murine TCRα and TCRβ constant chains were cysteine-modified. Transmembrane hydrophobic mutations were introduced into the murine TCRα constant chain. Without being bound to a particular theory or mechanism, it is believed that these modifications result in preferential pairing of the introduced TCR chains and enhanced TCR surface expression and functionality (Cohen et al., Cancer Res., 67(8):3898-903 (2007); Haga-Friedman et al., J. Immu., 188: 5538–5546 (2012)). The full length α and β chains of each of the 15 TCRs, including these modifications to the constant region, are shown in Table 3. In Table 3, the CDRs are underlined, the constant region is in italics, and the modified amino acid residues of the constant region are underlined and in bold. TABLE 3 TCR TCR chain Amino acid sequence Name CS T Q D S : Q E E N Q TI E C T C K L L W
Leydig 771261 HHS E-132-2023-0-PC-01 53 TCR TCR chain Amino acid sequence Name GRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRK Y K LI D A PI L L W C L L F Q W V I E S V V A T Q K T R D D G F L II V L F L
Leydig 771261 HHS E-132-2023-0-PC-01 54 TCR TCR chain Amino acid sequence Name Cys-substituted β GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLI A Y NI A N T D Q E E N Q TI G C T Q L T R R N T D Q E N Q TI G C T
Leydig 771261 HHS E-132-2023-0-PC-01 55 TCR TCR chain Amino acid sequence Name sequence without LCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQ L I L L W K N V D A Q E E N Q TI G C L T RI L L L W K S K P F Q R L V V
Leydig 771261 HHS E-132-2023-0-PC-01 56 TCR TCR chain Amino acid sequence Name QFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSA L D Q V R F N S M N P C LT Q E E G EI G C L F L I L S A A T P C LT
Leydig 771261 HHS E-132-2023-0-PC-01 57 TCR TCR chain Amino acid sequence Name Cys-substituted β MATRVLCCAVICLLGAGLSNAGVMQNPRHLVRRRGQEARLRCSP F V Q S II E L F L F F A Y Q M T LP Y S N P F T V V A Q D IF F A Q L A V C A
Leydig 771261 HHS E-132-2023-0-PC-01 58 TCR TCR chain Amino acid sequence Name chain with N- SQYVSLLIRDSQPSDSATYLCAVNNARLMFGDGTQLVVKPNIQNP F PI P L K H Y P A F D A LI Q L A V T LP V D P F E N Q TI Q D D IF
Leydig 771261 HHS E-132-2023-0-PC-01 59 TCR TCR chain Amino acid sequence Name Cys-substituted β GVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIF A K S E K T LP Y S N P F E N Q TI Q D IF F A K S E K T LP VY S N P F E N Q TI
Leydig 771261 HHS E-132-2023-0-PC-01 60 TCR TCR chain Amino acid sequence Name Cys-substituted, KTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQ D D IF F A K S E K T P Y S N P F E N Q TI Q D IF F A K S E K
For each TCR of Table 3, nucleotide sequences encoding the α and β chains were cloned into a MSGV1-based retroviral vector. The TCRβ and TCRα chains were separated by a Furin Ser/Gly P2A linker RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 231). Without being bound to a particular theory or mechanism, it is believed that the linker
Leydig 771261 HHS E-132-2023-0-PC-01 61 provides comparable expression efficiency the two chains (Szymczak et al., Nat. Biotechnol., 22(5):589-94 (2004)). The nucleotide sequences comprising the respective expression cassettes encoding the TCR alpha chain, the TCR beta chain, and the linker for each TCR is provided in Table 4. TABLE 4 TCR Name SEQ ID NO: 4385 TCR-Z SEQ ID NO: 249
EXAMPLE 3 This example demonstrates the avidity and specificity of 4385 TCR-Z, 4391 TCR- C1, 4391 TCR-K, 4391 TCR-L2, and 4394 TCR-10 to mutant RASG12V. Healthy donor peripheral blood lymphocytes (PBL) were independently transduced with the retroviral expression vector of Example 2 encoding the 4385 TCR-Z (Table 3), 4391 TCR-B2 (Table 3), 4391 TCR-C1 (Table 3), 4391 TCR-K (Table 3), 4391 TCR-L2 (Table 3), 4394 TCR-10 (Table 3), or 4394 TCR-A (a TCR disclosed in US Application No.63/060,340). The 4394 TCR-A had the same modified murine alpha and beta chain constant regions as the other TCRs tested here. The target cells were COS-C*01:02 cells that expressed HLA-C*01:02 and which had been incubated with the mutant RASG12V 9-mer peptide AVGVGKSAL (SEQ ID NO:
Leydig 771261 HHS E-132-2023-0-PC-01 62 232) or the equivalent wild-type (WT) AGGVGKSAL (SEQ ID NO: 233) at the concentration of peptides denoted in Figs.1A-1C and 6A-6E. The transduced, TCR- expressing T cells were co-cultured for 14 hours (h) with the target cells. T cell activation marker 4-1BB was measured by flow cytometry following the co-culture. The results are shown in Figs.1A-1C and 6A-6E. While the 4385 TCR-Z, TCR-C1, 4391 TCR-K, 4391 TCR-L2, and 4394 TCR-10 showed no recognition of the WT epitope at all concentrations, 4394 TCR-A and 4391 TCR- B2 showed WT epitope recognition at the 10,000 ng/mL and 1,000 ng/mL concentrations. These results suggest that 4385 TCR-Z, TCR-C1, 4391 TCR-K, 4391 TCR-L2, and 4394 TCR-10 have superior specificity for the RASG12V epitope. EXAMPLE 4 This example demonstrates patient-derived tumor xenograft (PDX) cell recognition by 4385 TCR-Z and 4394 TCR-10. Effector cells were healthy donor PBL that were independently transduced with the retroviral expression vector of Example 2 encoding the 4385 TCR-Z (Table 3), 4394 TCR-10 (Table 3), or 4394 TCR-A, as described in Example 3. Target cells were 4391 PDX cells that naturally expressed the RASG12V mutation and HLA-C*01:02 or 4316 PDX cells that naturally expressed the RASG12V mutation but not HLA-C*01:02 which had been incubated either in complete T cell media (CM) or CM supplemented with the mutant RASG12V 9-mer peptide AVGVGKSAL (SEQ ID NO: 232) as a positive control. Four hours later, the effector cells were co-cultured with or without (T only) the PDX cells for 16 hours. The reactivity of the TCRs against the tumor cells was measured by IFN-γ secretion. The results are shown in Figs.1D-1E. The 4385 TCR-Z and 4394 TCR-10 were reactive to the 4391 PDX cells and showed better reactivity than 4394 TCR-A (Fig.1D). The reactivity against RAS-mutation and HLA was specific, as indicated by the lack of reactivity against the 4316 PDX cells (Fig. 1E). EXAMPLE 5 This example demonstrates the in vivo tumor growth control by 4385 TCR-Z and 4394 TCR-10.
Leydig 771261 HHS E-132-2023-0-PC-01 63 Effector cells were healthy that were independently transduced with the retroviral expression vector of Example 2 encoding the 4385 TCR-Z (Table 3), 4394 TCR-10 (Table 3), or 4394 TCR-A, as described in Example 3. 4391 PDX cells were injected S.C. (subcutaneously) into NSG immunodeficient mice (5 mice/group). Two weeks later, 3e6 TCR-expressing T cells (effector cells) were injected I.V. into the mice. Tumor growth was measured by measuring the tumor size in two dimensions. The results are shown in Fig.1F. The 4385 TCR-Z and 4394 TCR-10 showed superior tumor growth control relative to 4394 TCR-A. In the 4394 TCR-10 group, 3 out of 5 mice were cured of the tumor. EXAMPLE 6 This example demonstrates that the 4432 TCR-A recognizing the RASG12D mutation is restricted by HLA-A*11:01. Healthy donor PBL were transduced with the retroviral expression vector of Example 2 encoding the 4432 TCR-A. The TCR-expressing CD8+ T cells were co-cultured with COS cells expressing HLA-A*11:01 (COS-A11), HLA-A*02:01 (COS-A02), or autologous dendritic cells (DC). All target cells were transfected with tandem minigenes (TMG) RNA to express WT (SEQ ID NO: 234) or mutated RAS antigen (Mut) (SEQ ID NO: 235). PMA served as a positive control. DMSO was a negative control for activation of the T cells. T cell activation marker 4-1BB was measured by flow cytometry following the co-culture. The results are shown in Fig.2A. The 4432 TCR-A recognized the mutated TMG expressed on DC and on COS-A11. EXAMPLE 7 This example demonstrates the avidity and specificity of 4432 TCR-A to mutant RASG12V 10-mer. Healthy donor PBL were transduced with the retroviral expression vector of Example 2 encoding the 4432 TCR-A. The transduced, TCR-expressing T cells were co- cultured for 14h with COS-A11 cells that had been incubated with the mutant RASG12D 9-mer peptide VVGADGVGK (SEQ ID NO: 236), 10-mer peptide VVVGADGVGK (SEQ ID NO: 238), or the corresponding WT peptides VVGAGGVGK (SEQ ID NO: 237) or VVVGAGGVGK (SEQ ID NO: 239) at the concentration of peptides denoted in Fig.2B. T cell activation marker 4-1BB was measured by flow cytometry following the co-culture.
Leydig 771261 HHS E-132-2023-0-PC-01 64 PMA served as a positive control, and T without target cells (T only) served as a negative control for activation of the T cells. The results are shown in Fig.2B. 4432 TCR-A showed specificity for the mutated RAS 10-mer epitope and did not react with the equivalent WT 10-mer or the WT 9-mer peptides. Limited reactivity to the RAS 9-mer epitope was observed at the supraphysiologic concentration (10,000 ng/mL and 1,000 ng/mL). EXAMPLE 8 This example demonstrates the avidity and specificity of 4432 TCR-A. Healthy donor PBL were independently transduced with the retroviral expression vector of Example 2 encoding the 4432 TCR-A (Table 3), 4373 TCR2 (a TCR disclosed in WO 2021/163434), or mTCR (TCR TRAV4-4*01(1)/TRBV12-2*01 disclosed in US Patent No.11,207,394). The 4373 TCR2 and mTCR had the same modified murine alpha and beta chain constant regions as 4432 TCR-A. The transduced, TCR-expressing T cells were co-cultured for 14h with COS-A11 cells that had been incubated with the mutant RASG12D 10-mer peptide VVVGADGVGK (SEQ ID NO: 238) or the equivalent WT peptide VVVGAGGVGK (SEQ ID NO: 239) at the concentration of peptides denoted in Fig.2C. T cell activation was determined by measuring secretion of IFNγ by enzyme-linked immunosorbent assay (ELISA) following the co-culture. The results are shown in Fig.2C. All three TCRs showed specificity for the mutant epitope and not for the WT epitope. 4432 TCR-A and 4373 TCR2 secreted more IFNγ than mTCR at every peptide concentration. These results suggest that 4432 TCR-A and 4373 TCR2 provide superior reactivity and avidity against RASG12D epitope restricted by HLA-A11. EXAMPLE 9 This example demonstrates the in vitro tumor cell killing by 4432 TCR-A. Healthy donor PBL were independently transduced with the retroviral expression vector of Example 2 encoding the 4432 TCR-A, 4373 TCR2, or mTCR, as described in Example 8. Empty vector (E.V) transduction of the same donor PBLs served as a negative control. The killing of 4069 PDX cells that naturally expressed the RASG12D mutation and HLA-A*11:01 was measured by the INCUCYTE immune cell killing assay. The PDX cells
Leydig 771261 HHS E-132-2023-0-PC-01 65 were co-cultured with the transduced cells 4432 TCR-A, 4373 TCR2, mTCR, or E.V (negative control) for 48 hours. The number of live tumor cells was counted at every two hours. The results are shown in Fig.2D. 4432 TCR-A was shown to kill more tumor cells as compared to 4373 TCR2 or mTCR. These results suggest that 4432 TCR-A has functional superiority. EXAMPLE 10 This example demonstrates in vivo tumor growth control by 4432 TCR-A. Healthy donor PBL were independently transduced with the retroviral expression vector of Example 2 encoding the 4432 TCR-A, 4373 TCR2, or mTCR, as described in Example 8. Empty vector (E.V) transduction of the same donor PBLs served as a negative control. 4069 PDX cells were injected S.C into NSG immunodeficient mice (5 mice/group). Two weeks later, 6e6 transduced, TCR-expressing T cells were injected I.V into the mice. Tumor growth was measured by measuring the tumor size in two dimensions. The results are shown in Fig.2E. The TCRs 4432 TCR-A and 4373 TCR2 showed tumor growth control compared to mTCR and negative controls. 4432 TCR-A showed superior tumor control when compared to 4373 TCR2 and mTCR. EXAMPLE 11 This example demonstrates that TCR 4412-H recognizes the RASG12R mutation. Healthy donor PBL were virally transduced with the retroviral expression vector of Example 2 encoding the 4412 TCR-H (Table 3) or 4268 TCR (a TCR disclosed in US 2022/0089673). The 4268 TCR had the same modified murine alpha and beta chain constant regions as 4412 TCR-H. The transduced, TCR-expressing, CD4+ T cells were co-cultured with autologous (DC). DCs were pulsed with WT (SEQ ID NO: 240) or mutated RASG12R (G12R) (SEQ ID NO: 241) 24-mer long peptide (LP). CD3/CD28 activating beads served as a positive control. DMSO and T cell only served as negative controls for activation of the T cells. After co-culture, reactivity of the CD4+ T cells was measured by upregulation of 4-1BB and/or OX40 by flow cytometry. The results are shown in Fig.3A. The results showed that TCR 4412-H specifically recognized RASG12R expressed on DC.
Leydig 771261 HHS E-132-2023-0-PC-01 66 12 This example demonstrates that 4412 TCR-H recognizes RASG12R restricted by HLA-DQA1*05:01/DQB1*03:01 and HLA-DQA1*05:01/DQB1*03:03 and HLA- DQA1*05:05/DQB1*03:01. Healthy donor PBL were independently transduced with the retroviral expression vector of Example 2 encoding 4412 TCR-H or 4268 TCR (a TCR disclosed in US 2022/0089673). The 4268 TCR had the same modified murine alpha and beta chain constant regions as 4412 TCR-H. COS cells transfected with several combinations of HLA-DQA1 and HLA-DQB1 molecules, as indicated in Fig.3B, were pulsed with RASG12R 24-mer LP (SEQ ID NO: 241). Following 6 hours of peptide incubation, transduced cells expressing 4412 TCR-H or TCR 4268 were added to the HLA-expressing COS cells for the co-culture. After 14 hours of co- culture, reactivity of the CD4+ T cells was measured by upregulation of 4-1BB and/or OX40 by flow cytometry. The results are shown in Fig.3B. 4412 TCR-H and 4268 TCR showed specific recognition of RASG12R restricted by HLA-DQA1*05:01/DQB1*03:01 and HLA- DQA1*05:01/DQB1*03:03 and HLA-DQA1*05:05/DQB1*03:01. EXAMPLE 13 This example demonstrates the avidity and specificity of 4412 TCR-H to mutant RASG12R. Healthy donor PBL were independently transduced with the retroviral expression vector of Example 2 encoding 4412 TCR-H or 4268 TCR, as described in Example 12. The transduced, TCR-expressing T cells were co-cultured for 14h with autologous DC that had been incubated with the mutant RASG12R 24-mer LP MTEYKLVVVGARGVGKSALTIQLI (SEQ ID NO: 241) or the equivalent WT LP MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 240) at the concentration of peptides denoted in Figs.3C-3F. T cell activation was measured by upregulation of the 4-1BB and/or OX40 co- receptors by flow cytometry. The results are shown in Figs.3C-3D. 4412 TCR-H showed recognition of RASG12R expressed on autologous DC at the peptide concentration of 10 ng/ml or higher. T cell activation was also determined by measuring secretion of IFNγ by ELISpot (enzyme-linked immunosorbent spot) assay. The results are shown in Figs.3E-3F. 4412
Leydig 771261 HHS E-132-2023-0-PC-01 67 TCR-H showed higher IFN-γ secretion than TCR at the supraphysiologic 10,000 ng/mL concentration. EXAMPLE 14 This example demonstrates the avidity and specificity of the 4385 TCR-B for RASG12V, RASG12A and RASG12C. Healthy donor PBLs were virally transduced with the retroviral expression vector of Example 2 encoding 4385 TCR-B. The transduced, TCR-expressing T cells were co-cultured for 14h with autologous DC cells that had been incubated with the mutant RASG12V 24-mer LP (MTEYKLVVVGAVGVGKSALTIQLI) (SEQ ID NO: 242), RASG12C LP (MTEYKLVVVGACGVGKSALTIQLI) (SEQ ID NO: 243), RASG12A LP (MTEYKLVVVGAAGVGKSALTIQLI) (SEQ ID NO: 244), or the equivalent WT LP (MTEYKLVVVGAGGVGKSALTIQLI) (SEQ ID NO: 240) at the concentration of peptides as denoted in Fig.4A. T cell activation was measured by upregulation of 4-1BB and/or OX40 co- receptors by flow cytometry. The results are shown in Fig.4A. 4385 TCR-B recognized RASG12V expressed on autologous DC at the peptide concentration of 10 ng/ml or higher. 4385 TCR-B recognized RASG12A or RASG12C at the peptide concentration of 100 ng/ml or higher. 4385 TCR-B showed WT epitope recognition only at the supraphysiologic 10,000 ng/mL and 1,000 ng/mL concentrations. EXAMPLE 15 This example demonstrates that 4385 TCR-B specifically reacts with target cells expressing the HLA-DRB1*09:01:02 molecule. A Jurkat-NFAT-firefly luciferase-CD8+/CD4+ reporter cell line was virally transduced with the retroviral expression vector of Example 2 encoding 4385 TCR-B. COS cells were independently transfected with one of several combinations of (i) HLA-DQA1 with HLA-DQB1, (ii) HLA-DPA1 with HLA-DPB1, or (iii) HLA-DRA1*01 with HLA-DRB that Patient 4385 expressed, as indicated in Fig.4B. After the transfected COS cells rested overnight, the COS cells were pulsed with RASG12V 24-mer LP. After 4 hours of peptide incubation, transduced Jurkat-NFAT-firefly Luciferase-CD8+/CD4+
Leydig 771261 HHS E-132-2023-0-PC-01 68 reporter cells expressing 4385 TCR-B were to the co-culture. These reporter cells express luciferase upon TCR activation via activation of the NFAT transcription factor. After 4 hours of co-culture, TCR reactivity was measured by luminescence of the cell lysate. The results are shown in Fig.4B. 4385 TCR-B specifically reacted with COS cells expressing DRB1*09:01:02. EXAMPLE 16 This example demonstrates the avidity and specificity of 4430 TCR-N1A, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR-N23A, and 4430 TCR-N23B for the 9-mer RASG12D antigen. Healthy donor PBL were independently virally transduced with the retroviral expression vector of Example 2 encoding 4430 TCR-N1A (Table 3), 4430 TCR-N8 (Table 3), 4430 TCR-N9 (Table 3), 4430 TCR-N23A (Table 3), 4430 TCR-N23B (Table 3), or TCR 4095 TRAV4/TRBV5-6 (A) (a TCR disclosed in US Patent No.10,611,816) (“4095 TCR 9A”). Empty vector (E.V) transduction of the same donor PBLs served as a negative control. The 4095 TCR 9A had the same modified murine alpha and beta chain constant regions as the other TCRs tested here. The transduced, TCR-expressing T cells were co-cultured for 14h with COS- C*08:02 cells (COS cells expressing HLA-C*08:02) that had been incubated with the mutant RASG12D 9-mer peptide GADGVGKSA (SEQ ID NO: 245) or the corresponding WT peptide GAGGVGKSA (SEQ ID NO: 246) at the concentration of peptides denoted in Figs.5A-5F. COS-C*08:02 without peptide (DMSO as a vehicle) served as a negative control. T cell activation markers 4-1BB and/or OX40 were measured by flow cytometry following the co- culture. The results are shown in Figs.5A-5F. All of the tested TCRs showed specificity for the mutated RASG12D 9-mer epitope with no WT peptide recognition. The 4430 TCR-N1A, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR-N23A, and 4430 TCR-N23B showed higher 4-1BB/OX40 upregulation at every peptide concentration relative to 4095 TCR 9A, suggesting a greater functionality of these TCRs as compared to 4095 TCR 9A. EXAMPLE 17 This example demonstrates the avidity and specificity of 4430 TCR-N2 for the 10- mer RASG12D antigen.
Leydig 771261 HHS E-132-2023-0-PC-01 69 Healthy donor PBL were virally transduced with the retroviral expression vector of Example 2 encoding 4430 TCR-N2 (Table 3), 4095 TCR 9A, or TCR 4095 TRAV12-2/TRBV10-2 (a TCR disclosed in US Patent No.10,611,816) (“4095 TCR 10”). Empty vector (E.V) transduction of the same donor PBLs served as a negative control. The 4095 TCR 9A and 4095 TCR 10 had the same modified murine alpha and beta chain constant regions as the other TCRs tested here. The transduced, TCR-expressing T cells were co-cultured for 14h with COS- C*08:02 cells (COS cells expressing HLA-C*08:02) that had been incubated with the mutant RASG12D 9-mer peptide GADGVGKSA (SEQ ID NO: 245) RASG12D 10-mer peptide GADGVGKSAL (SEQ ID NO: 247) or the corresponding 10-mer WT peptide GAGGVGKSAL (SEQ ID NO: 248) at the concentration of peptides denoted in Figs.5G-5I. COS-C*08:02 without peptide (DMSO as a vehicle) served as a negative control. T cell activation markers 4-1BB and/or OX40 were measured by flow cytometry following the co- culture. The results are shown in Figs.5G-5I. 4430 TCR-N2 and 4095 TCR 10 showed specificity for the mutated RASG12D 10- mer epitope. The 4095 TCR 9A showed specificity for the mutated RASG12D 9-mer epitope. While 4095 TCR 10 recognized only mutated RASG12D 10-mer antigen, 4430 TCR-N2 recognized the mutated RASG12D 9-mer antigen at a 10 ng/ml concentration, indicating the broad spectrum of reactivity of 4430 TCR-N2. EXAMPLE 18 This example demonstrates the PDX cell recognition by 4430 TCR-N1A, 4430 TCR-N2, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR-N23A, and 4430 TCR-N23B. Healthy donor PBL were independently virally transduced with a retroviral expression vector encoding 4430 TCR-N1A (Table 3), 4430 TCR-N2 (Table 3), 4430 TCR- N8 (Table 3), 4430 TCR-N9 (Table 3), 4430 TCR-N23A (Table 3), 4430 TCR-N23B (Table 3), or 4095 TCR 9A, as described in Example 16. Target tumor cells were SNU1 tumor cells naturally expressing the RASG12D mutation HLA-C*08:02 or 4069 PDX cells expressing the RASG12D mutation and not HLA- C*08:02. The transduced, TCR-expressing cells were co-cultured with or without (T only) the target cells for 16 hours. The reactivity against the tumor was measured by IFN-γ secretion. The results are shown in Fig.5J.
Leydig 771261 HHS E-132-2023-0-PC-01 70 All of the TCRs were reactive to tumor and not to 4069 PDX cells, indicating the antigen and HLA specificity of the TCRs. 4430 TCR-N1A, 4430 TCR-N2, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR-N23A, and 4430 TCR-N23B showed higher amounts of IFN-γ secretion as compared to 4095 TCR 9A, indicating the superior functionality of 4430 TCR-N1A, 4430 TCR-N2, 4430 TCR-N8, 4430 TCR-N9, 4430 TCR- N23A, and 4430 TCR-N23B. EXAMPLE 19 This example demonstrates a summary of the features of the TCRs of Table 2. A summary of the features of the TCRs of Table 2 is provided in Table 5. TABLE 5 Discovered TCR Name Mutation CD4/CD8 HLA Target Antigen f TIL f R i i I I
Leydig 771261 HHS E-132-2023-0-PC-01 71 All references, including patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.