T cell receptors directed against androgen receptor mutation

Abstract

Nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 of SEQ ID NO: 3, 5 or 9 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 4, 6 or 10. Isolated populations of T cells and compositions comprising the nucleic acid molecules as well as methods of treating cancer are also provided.

Description

T CELL RECEPTORS DIRECTED AGAINST ANDROGEN RECEPTOR MUTATIONREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-P-O38-PCT.xml; Size: 14,768 bytes; and Date of Creation: November 14, 2024) are herein incorporated by reference in their entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This Application claims the benefit of priority of Israel Patent Application No. 308802, filed November 22, 2023, and of US Patent Application No. 63 / 616,634, filed December 31, 2023, both titled “T CELL RECEPTORS DIRECTED AGAINST ANDROGEN RECEPTOR MUTATION”, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention is in the field of T cell receptors and adoptive cell transfer.BACKGROUND OF THE INVENTION

[0004] As neo-antigens gain an established role as drivers of the anti-tumor immune response, recurrent neoantigens, shared among groups of patients, become increasingly coveted therapeutic targets. However, almost all of the neo-antigens identified to date, stem from patient-specific, passenger, mutations. This sets T cell based and vaccination immunotherapy at the high end of the personalized therapy spectrum, placing a hurdle on their streamlined use for large-scale populations. Current adoptive cell therapy protocols expand and re-administer patient's tumor infiltrating lymphocytes (TIL) unselectively. However, the therapeutic potential of adoptive cell therapy directed at a single, recurrent, neo-antigen has been demonstrated. Clinical use of CAR T-cells attests to the feasibility of T-cell engineering on a therapeutically relevant timescale, as long as the utilized receptor is adequately predefined. Androgen receptor H875Y is a highly mutated protein position inprostate cancer patients who develop resistance. A new therapeutic agent that can target the AR H875Y mutants is therefore greatly needed.SUMMARY OF THE INVENTION

[0005] The present invention provides nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 of SEQ ID NO: 3 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 4. Nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 5 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 6, nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 9 and an open reading frame encoding a TCR beta chain comprising a CDR- B3 of SEQ ID NO: 10 and nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 13 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 14 are also provided. Isolated populations of T cells and compositions comprising the nucleic acid molecules as well as methods of treating cancer are also provided.

[0006] According to a first aspect, there is provided a nucleic acid molecule comprising a heterologous promoter and an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVGGYQKVTF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSEKGRGRVTGELFF (SEQ ID NO: 4).

[0007] According to another aspect, there is provided a nucleic acid molecule comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVKTFTGGGNKLTF (SEQ ID NO: 5) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDIAKGGNTIYF (SEQ ID NO: 6).

[0008] According to another aspect, there is provided a nucleic acid molecule comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 comprising the amino acid sequence CALRDNYGQNFVF (SEQ ID NO: 9) andan open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDRGFSQPQHF (SEQ ID NO: 10).

[0009] According to another aspect, there is provided a nucleic acid molecule comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 comprising the amino acid sequence CALRDNYGQNFVF (SEQ ID NO: 13) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDRGFSQPQHF (SEQ ID NO: 14).

[0010] According to some embodiments, a single open reading frame encodes a single polypeptide comprising the TCR alpha chain and the TCR beta chain separated by a cleavable linker or a flexible linker.

[0011] According to some embodiments, the cleavable linker comprises a self-cleaving peptide selected from P2A, E2A, T2A and F2A.

[0012] According to some embodiments, the TCR alpha chain comprises TRAV12-2 and TRAJ13 and the TCR beta chain comprises TRBV6-1, and TRBJ2-2; the TCR alpha chain comprises TRAV24 and TRAJ26 and the TCR beta chain comprises TRB V4- 1 , and TRB Jl- 5; the TCR alpha chain comprises TRAV12-2 and TRAJ10 and the TCR beta chain comprises TRBV4-1 and TRBJ1-3; or the TCR alpha chain comprises TRAV24 and TRAJ49 and the TCR beta chain comprises TRBV10-2 and TRBJ2-7.

[0013] According to some embodiments, a TCR comprising the alpha chain and the beta chain specifically binds to a peptide comprising the amino acid sequence SVQPIARELY (SEQ ID NO: 1), VQPIARELY (SEQ ID NO: 8) or LLDSVQPIARELY (SEQ ID NO: 11).

[0014] According to some embodiments, a TCR comprising the alpha chain and the beta chain specifically binds to a complex comprising HLA-B* 15:01 and a peptide comprising SEQ ID NO: 1 SEQ ID NO: 8 or SEQ ID NO: 11 .

[0015] According to some embodiments, a TCR comprising a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 binds to a complex comprising HLA- B* 15:01 or HLA-A*01:0I and SEQ ID NO: 1.

[0016] According to some embodiments, a TCR comprising a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 binds to a complex comprising HLA- B* 15:01 or HLA-A*01:01 and SEQ ID NO: 1.

[0017] According to some embodiments, a TCR comprising a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 binds to a complex comprising HLA- B* 15:01 or HLA-A*01:01 and SEQ ID NO: 8.

[0018] According to some embodiments, a TCR comprising a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 binds to a complex comprising HLA- B* 15:01 or HLA-A*01:01 and SEQ ID NO: 11.

[0019] According to some embodiments, the nucleic acid molecule is a mammalian expression vector.

[0020] According to another aspect, there is provided an isolated population of T cells genetically modified to express a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR- B3 comprising the amino acid sequence of SEQ ID NO: 4.

[0021] According to another aspect, there is provided an isolated population of T cells genetically modified to express a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 5 and a beta chain comprising a CDR- B3 comprising the amino acid sequence of SEQ ID NO: 6.

[0022] According to another aspect, there is provided an isolated population of T cells genetically modified to express a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR- B3 comprising the amino acid sequence of SEQ ID NO: 10.

[0023] According to another aspect, there is provided an isolated population of T cells genetically modified to express a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising a CDR- B3 comprising the amino acid sequence of SEQ ID NO: 14.

[0024] According to another aspect, there is provided an isolated population of T cells, wherein at least 10% of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 4.

[0025] According to another aspect, there is provided an isolated population of T cells, wherein at least 10% of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 6.

[0026] According to another aspect, there is provided an isolated population of T cells, wherein at least 10% of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 10.

[0027] According to another aspect, there is provided an isolated population of T cells, wherein at least 10% of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 14.

[0028] According to some embodiments, the at least 10% of T cells are genetically modified to express the TCR.

[0029] According to some embodiments, the T cells are CD8 positive T cells.

[0030] According to some embodiments, the genetic modification comprises expression of a nucleic acid molecule of the invention.

[0031] According to some embodiments, the isolated population is for use in treating cancer in a subject in need thereof, wherein the cancer comprises expression of Androgen Receptor (AR) comprising a H875Y mutation.

[0032] According to some embodiments, the expression of AR comprising a H875Y mutation comprises expression of an AR comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

[0033] According to some embodiments, the subject expresses HLA-B*15:01, HLA- A*01:01 or both.

[0034] According to some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 and the cancer comprises expression of SEQ ID NO: 1 in complex with HLA-B* 15:01, HLA-A^OEOl or both.

[0035] According to some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 and the cancer comprises expression of SEQ ID NO: 1 in complex with HLA-B* 15:01, HLA-A*01:01 or both.

[0036] According to some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 and the cancer comprises expression of SEQ ID NO: 8 in complex with HLA-B* 15:01, HLA-A*01:01 or both.

[0037] According to some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 and the cancer comprises expression of SEQ ID NO: 11 in complex with HLA-B* 15:01, HLA-A*01:01 or both.

[0038] According to another aspect, there is provided a pharmaceutical composition comprising an isolated population of T cells of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0039] According to another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention, thereby treating cancer.

[0040] According to some embodiments, the cancer comprises expression of AR comprising a H875Y mutation.

[0041] According to some embodiments, the expression of AR comprising a H875Y mutation comprises expression of an AR comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

[0042] According to some embodiments, the cancer comprises surface expression of a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

[0043] According to some embodiments, the cancer is selected from prostate cancer, breast cancer, colorectal cancer and hepatic cancer.

[0044] According to some embodiments, the method further comprises before the administering identifying in a cancer cell from the subject expression of AR comprising a H875Y mutation, surface expression of a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

[0045] According to some embodiments, the subject expresses HLA-B*15:01, HLA- A*01:01 or both.

[0046] According to some embodiments, the method further comprises before the administering identifying in the subject expression of HLA-B* 15:01, HLA-A*01:01 or both.

[0047] According to some embodiments, the method further comprises before the administering identifying in a cancer cell from the subject surface expression of a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11 in complex with HLA-B *15:01 or HLA-A*01:01.

[0048] According to some embodiments, the T cells are autologous, allogeneic, or syngeneic to the subject.

[0049] According to another aspect, there is provided a method of determining suitability of a subject in need thereof to be treated by a method of the invention, the method comprising: a. identifying expression of HLA-B*15:01, HLA-A*01:01 or both by the subject; and b. detecting expression of AR comprising a H875Y mutation in a cancer cell of the subject; wherein a subject expressing HLA-B*15:01 and comprising a cancer cell expressing AR comprising a H875Y mutation is suitable to be treated by a method of the invention; thereby determining suitability of a subject.

[0050] According to some embodiments, the detecting is in a cancer cell in a sample obtained from the subject.

[0051] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1A-1D: Pipeline for targeted therapy-resistant neoantigen prediction in the cancer genome. (1A) Schematic overview of the steps to identify and prioritize hotspot neoantigens associated with therapy resistance identification of recurrent mutations derived from resistance genes. Hotspots of SNV were first identified in either TCGA or across metastatic cancer samples from the Hartwig Medical Foundation cohort using HotspotFinder. Hotspots resulting in missense variants were kept for further analysis. The same strategy was applied to primary tumors from TCGA. For each hotspot, we generatedall possible mutated and WT peptides of 8 to 14 amino acids long that overlapped the mutation, and predicted neoantigen presentation to HLA class I alleles using MHCflurry, MHCnuggets, and NetMHCpan. We finally prioritized hotspot neoantigen for experimental validation using different criteria (Methods), sorted by total number of mutated samples in IntOGen and median VAF, followed by artifacts removal and mutation predicted as a driver by BoosDM. (IB) Frequency of candidate hotspot neoantigens across metastatic and primary pan-cancer cohorts. Prioritizing treatment resistance mutation in metastasis: Representation of cancer types in TCGA and Hartwig. Frequency of mutated genes in TCGA (primaries) compared to Hartwig (metastases). (1C) Focus on the top interesting neoantigens exclusively found in metastases, suggesting they likely arise from resistance to targeted therapies. Note that for representation purposes, all other neoantigens found in IB have been excluded from this plot. (ID) Table depicting selected neoantigens from 1C, showing the number of mutated patients bearing the mutation, the clonality status, and the treatments received.

[0053] Figures 2A-2G: From spectra to structure: comprehensive analysis of resistance-derived neo-peptides. (2A) Illustration of the process of identifying neopeptides presented by mono-allelic B cells. B-LCL 721.221 HLA-I null cells were infected with a single HLA allele to generate a library of mono-allelic B cell lines. These mono-allelic B cells were then transduced with one of the minigenes to create all combinations predicted to bind. This was followed by HLA-immunopeptidomics, where the mono-allelic B cells were analyzed using mass spectrometry to identify the neo-peptides presented by each specific HLA allele. (2A-D) Fragmention spectrum of the identified neo-peptides by HLA- immunopeptidomics in mono-allelic B cells HLA-immunopeptidomics of (2B) VQPIARELY (SEQ ID NO: 8) neo-peptide, harboring the B*15:01 / AR.H875Y combination, (2C) SVQPIARELY (SEQ ID NO: 1) neo-peptide, harboring the B*15:01 / AR.H875Y combination, (2D) LLDSVQPIARELY (SEQ ID NO: 11) neo-peptide, harboring the A*01:01 / AR.H875Y combination. (2E) Pymol based structure model of peptide SVQPIARELY (SEQ ID NO: 1) depicted as sticks presented by the HLA-B*15:01 molecule, depicted as cartoon in gray, viewed from top (left) and side (right). The amino acids for the peptide are labeled with one-letter symbol and positional number. (2F) Pymol based structure model of peptide VQPIARELY (SEQ ID NO: 8) is depicted as sticks presented by the HLA-B*15:01 molecule, depicted as a cartoon in gray, viewed from the top (left) and side (right). The amino acids for the peptide are labeled with one -letter symbol and positional number. (2G) Pymol based structure model of peptide LLDSVQPIARELY (SEQ ID NO: 11) is depicted as sticks presented by the HLA-B*01:01 molecule, depicted as acartoon in gray, viewed from the top (left) and side (right). The amino acids for the peptide are labeled with one-letter symbol and positional number.

[0054] Figures 3A-3E: Immunogenicity assessment and TCR identification for AR H875Y-derived neo-peptides. (3A) T cells derived from healthy donor PBMCs were stimulated and expanded for 26 days. Following co-culturing with human B-LCL 721.221 at E:T 2:1 ratio expressing HLA-A*01:01 or HLA-B*15:01 alleles that were pulsed with 1 pg / ml AR H875Y neo-peptides (VQPIARELY (SEQ ID NO: 8), SVQPIARELY (SEQ ID NO: 1), LLDSVQPIARELY (SEQ ID NO: 11) or 1 pg / ml AR WT peptide (VQPIARELH (SEQ ID NO: 15), SVQPIARELH (SEQ ID NO: 2), LLDSVQPIARELH (SEQ ID NO: 12). (3B) Flow cytometry analysis evaluated T cells for activation markers of IFN-y secretion, TNF-a secretion, and 41BB expression. (3C) Bar graph representation of panel 3A results for IFN-y, TNF-a, and 4 IBB. Normality of the data distribution was confirmed using the Shapiro-Wilk test (p > 0.05). Statistical significance was determined using an unpaired two- tailed Student's t-test. Specific p-values are indicated: *P < 0.05, **P < 0.01, **5kP < 0.001,< 0.0001. The images are representative of >2 replicates. (3D) Healthy donor T cells were expanded and stimulated for 26 days. Following expansion and stimulation, T cells were stained with AR H875Y double fluorophore-labeled dextramers to evaluate the percentage of AR H875Y specific subpopulation. (3E) Double positive, and double negative Dextramer cells were FACS sorted for single-cell TCR sequencing using lOx genomics. TCR clonal expansions are shown for double positive dextramers cells (dark blue) and negative (light gray). Circle sizes designate the number of single cells sharing the same TCR sequence.

[0055] Figures 4A-4C: Identification and functional characterization of AR H875Y peptide-specific TCR clones. (4A) Characteristics of identified AR H875Y peptide-specific TCR clone sequences. (4B) TCRap-KO (CD8+) Jurkat cells were transduced with TCR- MSGV1 plasmid using retroviruses. One week after the transduction, the cells were cocultured at a 1:1 E:T ratio with mono-allelic B cells (HLA-B* 15:01 or HLA-A*01:01), and titration of AR H875Y peptides (VQPIARELY (SEQ ID NO: 8), SVQPIARELY (SEQ ID NO: 1), LLDSVQPIARELY (SEQ ID NO: 11) or AR WT peptides (VQPIARELH (SEQ ID NO: 15), SVQPIARELH (SEQ ID NO: 2), LLDSVQPIARELH (SEQ ID NO: 12)) for 6 hours. (4C) Luminescence assay measured TCR activation for T157.1, T157.2, T157.3 and T112.1. The images are representative of two replicates. The normality of the data distribution was confirmed using the Shapiro-Wilk test (p > 0.05). Levene's Test for Homogeneity of Variance was not violated (p > 0.05), and statistical significance wasdetermined using an unpaired two-tailed Student’s t-test. Specific p-values are indicated:!:P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0056] Figures 5A-5D: Specific AR H875Y TCRs reactivity and cytotoxicity using healthy donors’ PBMCs. (5A) Healthy donors’ PBMCs were transduced with TCR- MSGV1 (T157.1, T157.3, and T112.1) plasmid using retroviruses. One week after the transduction, the cells were co-cultured with mono-allelic B cells (HLA-A*01:01 or HLA- B*15:01) pulsed with 1 pg / ml AR H875Y peptides (VQPIARELY (SEQ ID NO: 8), S VQPIARELY (SEQ ID NO: 1), LLDSVQPIARELY (SEQ ID NO: 11) or AR WT peptides (VQPIARELH (SEQ ID NO: 15), SVQPIARELH (SEQ ID NO: 2), LLDSVQPIARELH (SEQ ID NO: 12)) at 1.5:1 E:T ratio. Flow cytometry analysis evaluated T cells for activation markers of IFN-y secretion, TNF-a secretion, and 4-1BB expression. (SB) The LNCaP cancer cell line, harboring the HLA-A*01:01 allele, was stably transduced with AR H875Y MNG and HLA-B* 15:01 allele (LNCaP AR H875Y, LNCaP AR H875Y / B* 15:01). The cells were co-cultured at a 1:1 E:T ratio, with T cells stably expressing the AR H875Y-specific TCRs, following flow cytometry for activation markers of IFN-y secretion, TNF-a secretion, and 4 IBB expression. (5C) Transduced LNCaP cells (LNCaP AR H875Y, LNCaPAR H875Y / B*15:01) were co-cultured at a 2:1 E:T ratio, with T cells stably expressing the AR H875Y-specific TCRs for four hours, following flow cytometry for evaluation of cytotoxicity by Cleaved Caspase 3 staining. (5D) Transduced GFP LNCaP cells (LNCaPAR H875Y, LNCaPAR H875Y / B* 15:01) were seeded in triplicates in a 96-well plate, with each well containing 3,000 cells. The next day, T-cells stably expressing the AR H875Y-specific TCR were added at different E:T ratios with 300IU / ml IL-2. Cellular interactions were monitored using an Incucyte SX3 system over four days, with images captured every four hours.

[0057] Figures 6A-6C: Integrative analysis of MARS-Seq derived signatures with AR H875Y specific TCRs. Three signatures were derived from MARS-Seq experiments differential gene expression (DE): (1) Short-containing genes with FC > 4 and Pval < 0.05, (2) Long-containing genes with FC > 2 and Pval < 0.05 and (3) All DE -containing all genes with FC > 1 and p-value < 0.05. These signatures were compared to other reactive TCR (PredictTCR, NeoTCR . (tr30, ntrl 5)), or T-cell related (T-cell GEP 10, T-cell effector and TCD8 effector, Hallmark Inflammation) signatures from the literature. (6A) Table showing common genes between different signatures. The total number of genes in each signature is shown in parentheses. (6B) Correlations (Spearman correlation) between the different signature scores when applied to the Hartwig cohort of prostate cancer patients (N=346).Signature scores were computed per patient as the mean of log-transformed expression of all genes in the signature. For each signature, genes present in the signature that were not annotated in the patient sample were discarded. (6C) Standardized changes in the differentially expressed genes (DEGs) in MUT vs WT for each of the AR samples during T cell differentiation in response to, from left to right, a) LCMV Armstrong infection (GSE10239), b) chronic LCMV clone 13 (LCMV cl. 13) infection (GSE41867), c) autochthonous tumor induction (GSE89307), d) vaccinia virus infection (GSE79805), and e) human yellow fever vaccination (naive to activation, GSE26347; naive to activation to memory, GSE100745). Each dot represents the means of the relative enrichment of sample DEGs, and the error bars indicate a 95% confidence interval of the enrichment. Significances are shown when comparing the signature enrichments of two consecutive time points (see Methods). For vaccinia virus infection, a linear regression model was fitted, and the total change from dO to d90 was annotated, along with the significance of the rate of changes. Black line represents the control of iterated randomized probes of an equal signature length.

[0058] Figure 7A-7B: Retroviral transduction of Jurkat (TCRaP KO) CD8+T cells with AR H875Y specific TCRs. Jurkat (TCRaP KO) CD8+T cells were retrovirally transduced with AR H875Y specific TCRs (T157.1, T157.2, T157.3, and T112.1). Cells were stained with (7A) Murine TCR (mTCR) antibody, and (7B) AR H875Y specific dextramer.

[0059] Figures 8 -8B: Retroviral transduction of healthy donors’ PBMCs with AR H875Y specific TCRs. Healthy donors’ PBMCs were retrovirally transduced with AR H875Y-specific TCRs (T157.1, T157.2, T157.3, and T112.1). Cells were stained with (8A) mTCR antibody, and (8B) AR H875Y specific dextramer.

[0060] Figures 9A-9B: Specific AR H875Y T157.2 TCR reactivity in healthy donors’ PBMCs. (9A) Healthy donors’ PBMCs were transduced with T157.2 TCR-MSGV1 plasmid using retroviruses. One week after the transduction, the cells were co-cultured with mono- allelic B cells (HLA-B*15:01) pulsed with 1 pg / ml AR H875Y peptide (SVQPIARELY, SEQ ID NO: 1) or AR WT peptide (SVQPIARELH, SEQ ID NO: 2) at 1.5:1 E:T ratio. Flow cytometry analysis evaluated T cells for activation markers of IFN-y secretion, TNF-a secretion, and 4 IBB expression. (9B) The LNCaP cancer cell line, was stably transduced with AR H875Y MNG and HLA-B* 15:01 allele (LNCaP AR H875Y, LNCaPAR H875Y / B*15:01). The cells were co-cultured at a 1:1 E:T ratio, with T cells stably expressing the AR H875Y-specific TCR, following flow cytometry for activation markers of EFN-y secretion, TNF-a secretion, and 41BB expression.

[0061] Figure 10: Heatmap of upregulated genes across AR H875Y TCR-transduced T cells in MUT samples. The heatmap visualizes the shared differentially expressed genes across the three TCRs (T157.1, T157.3, and T112.1). Differential gene expression analysis was conducted independently for each sample, selecting genes with an absolute log2FoldChange greater than 0.5 and a Bonferroni-adjusted p-value less than 0.05. The intersection of these genes across all samples highlights those consistently exhibiting higher expression in the MUT samples. Genes common in both analyses are bolded.

[0062] Figure 11: Heatmap of upregulated genes across AR H875Y TCR-transduced T cells in WT samples. The heatmap visualizes the shared differentially expressed genes across the three TCRs (T157.1, T157.3, and T112.1). Differential gene expression analysis was conducted independently for each sample, selecting genes with an absolute log2FoldChange greater than 0.5 and a Bonferroni-adjusted p-value less than 0.05. The intersection of these genes across all samples highlights those consistently exhibiting higher expression in the WT samples.DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention, in some embodiments, provides nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 of SEQ ID NO: 3 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 4. Nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 5 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 6 are also provided. Nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 9 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 10 are also provided. Nucleic acid molecules comprising a heterologous promoter and an open reading frame encoding a TCR alpha chain comprising a CDR-A3 of SEQ ID NO: 13 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 14 are also provided. Isolated populations of T cells comprising the nucleic acid molecules are also provided as are compositions comprising the T cell populations. Methods of treating cancer by administering the populations or compositions are also provided.

[0064] The invention is based, at least in part, on the surprising discovery of a new TCR that specifically binds to neoantigen produced by the H875Y mutation in androgen receptor (AR). By applying a unique, unbiased data-driven candidate selection method a novel HLA- presented neo-antigens derived from an AR-H875Y mutant was identified. Using HLA- peptidomics the robust presentation of the neo-antigen was demonstrated. The immunogenicity of the mutated peptides was confirmed by the detection of specific reactivity of t-cells from healthy donors towards the mutated peptide. Further, single-cell TCR sequencing was applied to the CD8+ dextramer specific t-cells, revealing a neoantigen-specific TCR within the donor t-cells. Cloning the TCR followed by its transduction in T-cells demonstrated its ability to direct reactivity to cancer cells in a neo-antigen-specific manner, making this discovery therapeutically relevant for thousands of cancer patients.Nucleic acid molecules

[0065] By a first aspect, there is provided a nucleic acid molecule comprising an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVGGYQKVTF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSEKGRGRVTGELFF (SEQ ID NO: 4).

[0066] By another aspect, there is provided a nucleic acid molecule comprising an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVKTFTGGGNKLTF (SEQ ID NO: 5) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDIAKGGNTIYF (SEQ ID NO: 6).

[0067] By another aspect, there is provided a nucleic acid molecule comprising an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CALRDNYGQNFVF (SEQ ID NO: 9) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDRGFSQPQHF (SEQ ID NO: 10).

[0068] By another aspect, there is provided a nucleic acid molecule comprising an open reading frame encoding a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CASPTGNQFYF (SEQ ID NO: 13) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSDLAGHTYEQYF (SEQ ID NO: 14).

[0069] Each of the first two pairs of TCR alpha chain and TCR beta chain produce a functional TCR that was shown to recognize the SVQPIARELY (SEQ ID NO: 1) neopeptide at low concentrations and to induce 4-IBB upregulation and proinflammatory cytokine secretion upon co-incubation with relevant cancer cell-lines (e.g., prostate cancer cell lines). The third pair of TCR alpha chain and TCR beta chain produce a functional TCR that was shown to recognize the VQPIARELY (SEQ ID NO: 8) neopeptide at low concentrations and to induce 4-IBB upregulation and proinflammatory cytokine secretion upon co-incubation with relevant cancer cell-lines (e.g., prostate cancer cell lines). The fourth pair of TCR alpha chain and TCR beta chain produce a functional TCR that was shown to recognize the LLDS VQPIARELY (SEQ ID NO: 11) neopeptide at low concentrations and to induce 4- IBB upregulation and proinflammatory cytokine secretion upon co-incubation with relevant cancer cell-lines (e.g., prostate cancer cell lines). In some embodiments, the TCR binds to SEQ ID NO: 1. In some embodiments, the TCR binds to SEQ ID NO: 8. In some embodiments, the TCR binds to SEQ ID NO: 11. In some embodiments, a sequence comprising SEQ ID NO: 8 comprises SEQ ID NO: 1. In some embodiments, a sequence comprising SEQ ID NO: 11 comprises SEQ ID NO: 1 and SEQ ID NO: 8. In some embodiments, a sequence comprising SEQ ID NO: 1 comprises SEQ ID NO: 8. In some embodiments, a sequence comprising SEQ ID NO: 1 comprises SEQ ID NO: 8 and SEQ ID NO: 11. In some embodiments, a sequence encoding SEQ ID NO: 8 encodes SEQ ID NO: 1. In some embodiments, a sequence encoding SEQ ID NO: 8 encodes SEQ ID NO: 1 and SEQ ID NO: 11. In some embodiments, a sequence encoding SEQ ID NO: 11 encodes SEQ ID NO: 1 and SEQ ID NO: 8. In some embodiments, a sequence encoding SEQ ID NO: 1 encodes SEQ ID NO: 8. In some embodiments, a sequence encoding SEQ ID NO: 1 encodes SEQ ID NO: 8 and SEQ ID NO: 11. In some embodiments, the TCR does not bind to SVQPIARELH (SEQ ID NO: 2). In some embodiments, the TCR does not bind to VQPIARELH (SEQ ID NO: 15). In some embodiments, the TCR does not bind to LLDSVQPIARELH (SEQ ID NO: 12). In some embodiments, the TCR binds to a protein complex comprising SEQ ID NO: 1 and an HLA. In some embodiments, the TCR binds to a protein complex comprising SEQ ID NO: 8 and an HLA. In some embodiments, the TCR binds to a protein complex comprising SEQ ID NO: 11 and an HLA. In some embodiments, the HLA is HLA-B*15:01. In some embodiments, the HLA is HLA-A*01:01. In some embodiments, binds to is specifically binds to. In some embodiments, specifically comprises no binding to another antigen than the target antigen. In some embodiments, no binding is no specific binding. In some embodiments, no binding is no substantial binding. In some embodiments, CDR-A3 consists of SEQ ID NO: 3. In some embodiments, CDR-B3 consistsof SEQ ID NO: 4. In some embodiments, CDR-A3 consists of SEQ ID NO: 5. In some embodiments, CDR-B3 consists of SEQ ID NO: 6. In some embodiments, CDR-A3 consists of SEQ ID NO: 9. In some embodiments, CDR-B3 consists of SEQ ID NO: 10. In some embodiments, CDR-A3 consists of SEQ ID NO: 13. In some embodiments, CDR-B3 consists of SEQ ID NO: 14. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 comprising SEQ ID NO: 3 and a beta chain comprising a CDR- B3 comprising SEQ ID NO: 4. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 consisting of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 consisting of SEQ ID NO: 4. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 comprising SEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising SEQ ID NO: 6. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 consisting of SEQ ID NO: 5 and a beta chain comprising a CDR-B3 consisting of SEQ ID NO: 6. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 comprising SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising SEQ ID NO: 10. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 consisting of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 consisting of SEQ ID NO: 10. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 comprising SEQ ID NO: 13 and a beta chain comprising a CDR-B3 comprising SEQ ID NO: 14. In some embodiments, the TCR is a TCR comprising an alpha chain comprising a CDR-A3 consisting of SEQ ID NO: 13 and a beta chain comprising a CDR-B3 consisting of SEQ ID NO: 14. The sequence of a CDR3 region of an exemplary beta chain (CDR-B3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 4. The sequence of a CDR3 region of an exemplary a chain (CDR-A3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 3. The sequence of a CDR3 region of an exemplary beta chain (CDR-B3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 6. The sequence of a CDR3 region of an exemplary a chain (CDR-A3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 5. The sequence of a CDR3 region of an exemplary beta chain (CDR-B3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 10. The sequence of a CDR3 region of an exemplary a chain (CDR-A3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 9. The sequence of a CDR3 region of an exemplary beta chain (CDR-B3) of a T cell receptor which may be used according to thisaspect of the present invention is set forth in SEQ ID NO: 14. The sequence of a CDR3 region of an exemplary a chain (CDR-A3) of a T cell receptor which may be used according to this aspect of the present invention is set forth in SEQ ID NO: 13.

[0070] In an embodiment of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR)l, a CDR2, and a CDR3 of a TCR. The alpha and beta chains of the TCR are well known and comprise various fragments joined together to produce an enormous variety of TCRs. Though the alpha and beta chains each contain 3 CDRs (CDR-A1, CDR-A2, CDR- A3, CDR-B1, CDR-B2 and CDR-B3) only the A3 and B3 CDRs are actually responsible for antigen recognition. As such, TCRs can be functionally defined by the CDR-A3 and CDR- B3. The alpha chain is encoded by the gene TRA which includes a variable region and a constant region (TRA-C). Similarly, the beta chain is encoded by TRB and also includes both a variable region and constant region (TRB-C). As well as CDRs, the TCRs disclosed herein also comprise V regions J regions and D regions. Particular combinations of V D and J regions used in the TCR of the invention for the first TCR (referred to herein as T157.1) are as follows: Alpha chain- TRAV12-2, TRAJ13, CDRA3-SEQ ID NO: 3; Beta chain- TRBV6-1, TRBD1, TRBJ2-2, CDRB3-SEQ ID NO: 4. Particular combinations of V D and J regions used in the TCR of the invention for the second TCR (referred to herein as T157.2) are as follows: Alpha chain- TRAV12-2, TRAJ10, CDRA3-SEQ ID NO: 5; Beta chain- TRBV4-1, TRBD1, TRBJ1-3, CDRB3-SEQ ID NO: 6. Particular combinations of V D and J regions used in the TCR of the invention for the third TCR (referred to herein as T157.3) are as follows: Alpha chain- TRAV24, TRAJ26, CDRA3-SEQ ID NO: 9; Beta chain- TRBV4-1, TRBD1, TRBJ1-5, CDRB3-SEQ ID NO: 10. Particular combinations of V D and J regions used in the TCR of the invention for the TCR (referred to herein as T112.1) are as follows: Alpha chain- TRAV24, TRAJ49, CDRA3-SEQ ID NO: 13; Beta chain- TRBV10- 2, TRBD1, TRBJ2-7, CDRB3-SEQ ID NO: 14. It will be understood that the terminology TRAV12-2 and 2-TRAV 12 and the like are interchangeable and either can be used. In some embodiments, the alpha chain comprises a TRAV12 gene. In some embodiments, the TRAV12 gene is a 2-TRAV12 / TRAV12-2 gene. In some embodiments, the alpha chain comprises a TRAV24 gene. In some embodiments, the alpha chain comprises a TRAJ13 gene. In some embodiments, the alpha chain comprises a TRAJ10 gene. In some embodiments, the alpha chain comprises a TRAJ26 gene. In some embodiments, the alpha chain comprises a TRAJ49 gene. In some embodiments, the beta chain comprises a TRB V6 gene. In some embodiments, a TRBV6 gene is a 1-TRBV6 / TRBV6-1 gene. In someembodiments, the beta chain comprises a TRBV4 gene. In some embodiments, a TRBV4 gene is a 1-TRBV4 / TRBV4-1 gene. In some embodiments, the beta chain comprises a TRBV 10 gene. In some embodiments, a TRB V 10 gene is a 2-TRB V10 / TRBV 10-2 gene. In some embodiments, the beta chain comprises a TRBD1 gene. In some embodiments, the beta chain comprises a TRB JI gene. In some embodiments, the TRB JI gene is a 3- TRBJ1 / TRBJ1-3 gene. In some embodiments, the TRB JI gene is a 5-TRBJ1 / TRBJ1-5 gene. In some embodiments, the beta chain comprises a TRBJ2 gene. In some embodiments, the TRBJ2 gene is a 2-TRB J2 / TRBJ2-2 gene. In some embodiments, the TRBJ2 gene is a 7- TRBJ2 / TRBJ2-7 gene.

[0071] In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 and binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 1. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 and binds to a complex comprising HLA-B*15:01 or HLA-A*01:01 and SEQ ID NO: 1. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 and binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 8. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 and binds to a complex comprising HLA- B* 15:01 or HLA-A*01:01 and SEQ ID NO: 11.

[0072] In some embodiments, a first open reading frame encodes the alpha chain of the TCR. In some embodiments, a second open reading frame encodes the beta chain of the TCR. In some embodiments, the first open reading frame and the second open reading frame are different open reading frames. In some embodiments, the first open reading frame and the second open reading frame are the same open reading frame. In some embodiments, a single open reading frame encodes the alpha and beta chains of the TCR. In some embodiments, a single open reading frame encodes a single polypeptide comprising the TCR alpha chain and the TCR beta chain. In some embodiments, the TCR alpha chain and TCR beta chain are separated by a linker.

[0073] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is sufficiently long as to allow the alpha chain and beta chain to dimerize. In some embodiments, the linker is sufficiently long as to allow the alpha chain and beta chain to form a TCR. In some embodiments, a TCR is a functional TCR. In some embodiments, a functional TCR is a TCR capable of binding an antigen. In some embodiments, the antigen is the neoantigen. In some embodiments, the linker is a cleavable linker. In someembodiments, the cleavable linker is cleaved inside a cell. In some embodiments, inside a cell is in a cytoplasm of a cell. In some embodiments, the cleavable linker is a self-cleaving or auto-cleaving linker. In some embodiments, a cleavable linker comprises a cleavable peptide. In some embodiments, the cleavable peptide is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is selected from P2A, E2A, T2A and F2A.

[0074] In some embodiments, the nucleic acid molecule comprises a promoter. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a human promoter. In some embodiments, the promoter is not an endogenous TCR chain promoter. In some embodiments, the promoter is active in a target cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a cytotoxic cell. In some embodiments, the lymphocyte is selected from a T cell and an NK cell. In some embodiments, the lymphocyte is a T cell.

[0075] In some embodiments, the promoter is operably linked to an open reading frame. In some embodiments, an open reading frame is operably linked to a promoter. As used herein, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the nucleic acid molecule is introduced into the host cell). In some embodiments, the first open reading frame is operably linked to a first promoter. In some embodiments, the second open reading frame is operably linked to a second promoter. In some embodiments, the first and second promoters are the same promoter. In some embodiments, the first and second promoters are different promoters. In some embodiments, the single open reading frame encodes the alpha and beta chains and is operably linked to a single promoter.

[0076] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C).

[0077] The terms “nucleic acid molecule” include but not limited to singlestranded RNA (ssRNA), double- stranded RNA (dsRNA), single-stranded DNA (ssDNA),double-stranded DNA (dsDNA), small RNA such as miRNA, siRNA and other short interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, small rRNA, hnRNA, IncRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, nucleic acids of infectious origin, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides. In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the nucleic acid molecule is devoid of introns.

[0078] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a mammalian expression vector. In some embodiments, the nucleic acid molecule is for use in a method of the invention.

[0079] As used herein, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a precursor or mature protein (polypeptide). Expressing a gene / open reading frame within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector.

[0080] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.

[0081] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoter may be a viral promoter.

[0082] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0083] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a cell or cell type specific promoter. In some embodiments, the cell is the target cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. In some embodiments, the promoter is a T cell specific promoter. Examples of T cell specific promoters include for example the CD3 promoter.

[0084] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0085] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0086] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0087] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0088] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive -negative selection methods.

[0089] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0090] A person with skill in the art will appreciate that a gene / open reading frame can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).T cell population

[0091] By another aspect, there is provided a population of T cell expressing a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 4.

[0092] By another aspect, there is provided a population of T cell expressing a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence ofSEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 6.

[0093] By another aspect, there is provided a population of T cell expressing a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 10.

[0094] By another aspect, there is provided a population of T cell expressing a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising aCDR-B3 comprising the amino acid sequence of SEQ ID NO: 14.

[0095] By another aspect, there is provided a population of T cells wherein at least 1 % of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR- A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 4.

[0096] By another aspect, there is provided a population of T cells wherein at least 1 % of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR- A3 comprising the amino acid sequence of SEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 6.

[0097] By another aspect, there is provided a population of T cells wherein at least 1 % of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR- A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 10.

[0098] By another aspect, there is provided a population of T cells wherein at least 1% of the T cells of the population comprise a TCR comprising an alpha chain comprising a CDR- A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 14.

[0099] In some embodiments, the population is an isolated population. In some embodiments, the population is a purified population. In some embodiments, the population is an in vitro population. In some embodiments, the population is in culture. In some embodiments, the population is an expanded population. In one embodiment, T cells are expanded using methods known in the art. Expanded T cells that express tumor specific TCRs may be administered back to a subject. In another embodiment PBMCs are transducedor transfected with polynucleotides for expression of TCRs and administered to a subject. T cells expressing TCRs specific to neoantigens are expanded and administered back to a subject.

[0100] In some embodiments, the T cell populations expressing T cell receptors on the surface thereof bind to the peptide epitope as set forth in SEQ ID NO: 1 and have antigenic specificity towards that peptide. In some embodiments, the T cell populations expressing T cell receptors on the surface thereof bind to the peptide epitope as set forth in SEQ ID NO: 8 and have antigenic specificity towards that peptide. In some embodiments, the T cell populations expressing T cell receptors on the surface thereof bind to the peptide epitope as set forth in SEQ ID NO: 11 and have antigenic specificity towards that peptide. In one embodiment, the T cells do not have antigenic specificity towards the peptide epitopes as set forth in SEQ ID NO: 2. In one embodiment, the T cells do not have antigenic specificity towards the peptide epitopes as set forth in SEQ ID NO: 15. In one embodiment, the T cells do not have antigenic specificity towards the peptide epitopes as set forth in SEQ ID NO: 12.

[0101] In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 and binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 1. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 and binds to a complex comprising HLA-B*15:01 or HLA-A*01:01 and SEQ ID NO: 1. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 and binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 8. In some embodiments, the TCR comprises a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 and binds to a complex comprising HLA- B* 15:01 or HLA-A*01:01 and SEQ ID NO: 11.

[0102] The phrase "antigenic specificity," as used herein, means that the TCR can specifically bind to and immunologically recognize mutated target, e.g., mutated KRAS, with high avidity. For example, a TCR may be considered to have "antigenic specificity" for a mutated target if 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 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the foregoing values) of IFN-gamma upon co-culture with (a) antigen-negative HLA- B*15:01 / HLA-A*01:01+ target cells pulsed with a low concentration of mutated targetpeptide having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11 (e.g., about 0.05 ng / mL to about 5 ng / rnL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, or a range defined by any two of the foregoing values or (b) antigen-negative HLA- B 15:0l / HLA-A*01 :01 + target cells into which a nucleotide sequence encoding the mutated target peptide having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11 has been introduced such that the target cell expresses the mutated target. Cells expressing the inventive TCRs may also secrete IFN-gamma upon co-culture with antigennegative HLA-B* 15:01 / HLA-A*01:01+ target cells pulsed with higher concentrations of mutated target peptide. Though IFNg is given as an example, any proinflammatory cytokine can be measured. TNFa may also be secreted and measured for the purposes of determining antigenic specificity.

[0103] Alternatively or additionally, a TCR may be considered to have "antigenic specificity" for a mutated target if T cells expressing the TCR secrete at least twice as much IFN-gamma / TNFa upon co culture with (a) antigen-negative HLA-B* 15:01 / HLA- A*01:01+ target cells pulsed with a low concentration of mutated target peptide or (b) antigen-negative HLA-B*15:01 / HLA-A*01:01+ target cells into which a nucleotide sequence encoding the mutated target has been introduced such that the target cell expresses the mutated target as compared to the amount of IFN-gamma / TNFa expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR, cocultured with (a) antigen negative HLA-B* 15:01 / HLA-A*01:01+ target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the mutated target peptide, for example SEQ ID NO: 2, 15 or 12) or (b) antigen negative HLA-B*15:01 / / HLA-A*01:01+ 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) non- transduced T cells (e.g., derived from PBMC, which do not express the TCR) co-cultured with (a) antigen-negative HLA-B* 15:01 / HLA- A*01:01+ target cells pulsed with the same concentration of mutated target peptide or (b) antigen-negative HLA-B*15:01 / HLA-A*01:01+ target cells into which a nucleotide sequence encoding the mutated target has been introduced such that the target cell expresses the mutated target. IFN-gamma, TNFa or any proinflammatory cytokine secretion may be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA).

[0104] Alternatively or additionally, a TCR may be considered to have "antigenic specificity" for a mutated target if at least twice as many of the numbers of T cells expressingthe TCR secrete IFN-gamma / TNFa upon co-culture with (a) antigen-negative HLA- B*15:01 / HLA-A*01:01+ target cells pulsed with a low concentration of mutated target peptide, (b) antigen-negative HLA-B*15:01 / HLA-A*01:01+ target cells into which a nucleotide sequence encoding the mutated target has been introduced or (c) HLA- B* 15 :01 / HLA A *01 :01 + cells with natural expression of the mutant target peptide such that the target cell expresses the mutated target as compared to the numbers of negative control T cells that secrete IFN-gamma / TNFa. 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-gamma / TNFa or any other proinflammatory cytokine may be measured by methods known in the art such as, for example, ELISPOT.

[0105] In some embodiments, the T cells of the population are genetically modified to express the TCR. In some embodiments, genetically modified is genetically engineered. In some embodiments, the T cells comprises an exogenous nucleic acid molecule that encodes the TCR. In some embodiments, the exogenous nucleic acid molecule is a nucleic acid molecule of the invention. In some embodiments, the T cells express a nucleic acid molecule of the invention. In some embodiments, a percentage of the T cells of the population express a nucleic acid molecule of the invention. Methods of engineering T cells to express recombinant T cell receptors for cancer treatment are disclosed in Ping et al Protein Cell. 2018 Mar; 9(3): 254-266, herein incorporated by reference in its entirety. The invention provides T cells expressing TCRs comprising two polypeptides (i.e., polypeptide chains), such as an alpha (alpha) chain of a TCR, a beta chain of a TCR, a gamma (gamma) chain of a TCR, a delta (delta) chain of a TCR, or a combination thereof.

[0106] In some embodiments, the T cell populations comprises at least 1% of the T cells of the populations expressing the T cell receptor having a beta chain CDR3 amino acid sequence as set forth in SEQ ID NO: 4 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the T cell populations comprises at least 1% of the T cells of the populations expressing the T cell receptor having a beta chain CDR3 amino acid sequence as set forth in SEQ ID NO: 6 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the T cell populations comprises at least 1% of the T cells of the populations expressing the T cell receptor having a beta chain CDR3 amino acid sequence as set forth in SEQ ID NO: 10 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the T cell populations comprises at least 1% of the T cells of the populations expressing the T cell receptor having a beta chain CDR3 amino acid sequence as set forth in SEQ ID NO: 14 andan alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, at least 1% is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% Each possibility represents a separate embodiment of the invention. In one embodiment at least 95 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 4 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 3. In one embodiment at least 50 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 4 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 3. In one embodiment at least 10 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 4 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 3. In one embodiment at least 95 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 6 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 5. In one embodiment at least 50 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 6 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 5. In one embodiment at least 10 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 6 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 5. In one embodiment at least 95 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 10 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 9. In one embodiment at least 50 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 10 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 9. In one embodiment at least 10 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 10 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 9. In one embodiment at least 95 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 14 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 13. In one embodiment at least 50 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 14 and an a chain CDR3 amino acid sequence as set forth in SEQ ID NO: 13. In one embodiment at least 10 % of the T cells of the population express the T cell receptor having a b chain CDR3 amino acid sequence as set forth in SEQ ID NO: 14 and an alpha chain CDR3 amino acid sequence as set forth in SEQ ID NO: 13.

[0107] The present inventors further contemplate CAR-T cells - i.e., T cells expressing chimeric antibodies having a CDR3 amino acid sequence as set forth in SEQ ID NOs: 3 and 4. The present inventors further contemplate CAR-T cells - i.e., T cells expressing chimeric antibodies having a CDR3 amino acid sequence as set forth in SEQ ID NOs: 5 and 6. The present inventors further contemplate CAR-T cells - i.e., T cells expressing chimeric antibodies having a CDR3 amino acid sequence as set forth in SEQ ID NOs: 9 and 10. The present inventors further contemplate CAR-T cells - i.e., T cells expressing chimeric antibodies having a CDR3 amino acid sequence as set forth in SEQ ID NOs: 13 and 14. In some embodiments, the T cells are CAR-T cells. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the population is for use in a method of the invention.

[0108] In some embodiments, the sequences of the CDR3 regions may comprise at least one or even two amino acid substitutions and retain binding activity. In one embodiment, the amino acid substitution is a conservative substitution. The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the sidechain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the sidechain of the replaced amino acid). As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions. When affecting conservative substitutions, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

[0109] For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. Peptidomimetics of the naturally occurring amino acids are well documented in the literature known to the skilled practitioner.

[0110] The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and / or steric properties. Thus, theside chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and / or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]- CO- for aspartic acid.Those non conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.

[0111] Also contemplated are isolated antibodies and / or diabodies which comprise at least one of the CDR sequences specified herein.

[0112] The TCRs (and antibodies) 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, alphaamino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4- hydroxyproline, 4-aminophenylalanine, 4 -nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, beta-phenylserine beta-hydroxyphenylalanine, phenylglycine, alpha- 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, alpha- aminocyclopentane carboxylic acid, alpha-aminocyclohexane carboxylic acid, alphaaminocycloheptane carboxylic acid, ,alpha.-(2-amino-2-norbomane)-carboxylic acid, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine, and alpha- tert-butylglycine .

[0113] The TCRs (and antibodies) of the invention (including functional variants thereof) can be 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.

[0114] The TCRs (and antibodies) of the invention can be obtained by methods known in the art such as, for example, de novo synthesis. Also, TCRs 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, 4.sup.th ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein (including functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems (San Diego, Calif.). In this respect, the inventive TCRs, can be synthetic, recombinant, isolated, and / or purified. Included in the scope of the invention are conjugates, e.g., bioconjugates, comprising any of the inventive TCRs. Conjugates, as well as methods of synthesizing conjugates in general, are known in the art. The populations of tumor-reactive T cells expressing subject- specific TCRs may be combined with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition comprising a personalized cell population of tumor-reactive T cells. Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the administration of cells. Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use. A suitable pharmaceutically acceptable carrier for the 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, Hl.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.Compositions

[0115] By another aspect, there is provided a composition comprising a nucleic acid molecule of the invention.

[0116] By another aspect, there is provided a composition comprising a T cell population of the invention.

[0117] By another aspect, there is provided a synthetic TCR encoded by a nucleic acid molecule of the invention.

[0118] By another aspect, there is provided a bispecific molecule comprising a TCR of the invention and a binding moiety.

[0119] In some embodiments, the composition is pharmaceutical composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition is for use in a method of the invention. In some embodiments, the composition is for use in treating cancer. In some embodiments, the composition is a diagnostic composition. In some embodiments, the composition is for use in a method of the invention.

[0120] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant. As used herein, the term “carrier,” “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as com starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non- toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are wellknown in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle -forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0121] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0122] In some embodiments, the composition comprises a therapeutically effective amount of the nucleic acid molecule of the invention. In some embodiments, the composition comprises a therapeutically effective amount of the T cell population of the invention. In some embodiments, the population comprises at least 1 million cells. As used herein, the term "therapeutically effective amount" refers to an amount of an active agent to treat a disease or disorder in a mammal. The term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient s condition.

[0123] In some embodiments, the composition is formulated to systemic administration. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a human. It will be understood by a skilled artisan that cells in a standard culture, which contains media from animals (e.g., FBS) are not suitable for administration to a human. Chemically defined media can be used, or the cells can be isolated and washed and moved to an acceptable carrier medium. In some embodiments, thecomposition is formulated for intravenous administration. In some embodiments, the composition is formulated for intratumoral administration.

[0124] In some embodiments, the bispecific molecule comprises a second TCR. In some embodiments, the bispecific molecule comprises an antibody or antigen binding fragment thereof. In some embodiments, the binding moiety is a TCR or antibody or antigen binding thereof. In some embodiments, the bispecific molecule is a bispecific TCR. In some embodiments, the bispecific molecule is a bispecific antibody.

[0125] In some embodiments, a synthetic TCR is a recombinant TCR. In some embodiments, a synthetic TCR is an isolated TCR. In some embodiments, an isolated TCR is not in a cell. In some embodiments, an isolated TCR is not embedded in a cell membrane. In some embodiments, the TCR comprises a detectable moiety. In some embodiments, the TCR is conjugated to a detectable moiety. In some embodiments, detecting the TCR comprises detecting the detectable moiety. In some embodiments, detecting binding comprises detecting the detectable moiety. In some embodiments, the detectable moiety is selected from a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety, a radio-moiety, a dye, a colorimetric moiety and an imagine moiety. In some embodiments, the moiety is a fluorescent moiety. In some embodiments, a fluorescent moiety is a fluorophore.Method of treatment

[0126] By another aspect, there is provided a method of treating cancer in a subject comprising administering to the subject a composition of the invention, thereby treating cancer in the subject.

[0127] Examples of cancers include but are not limited to melanoma, colon cancer, breast cancer, thyroid cancer, stomach cancer, colorectal cancer, leukemia cancer, bladder cancer, lung cancer, ovarian cancer, breast cancer and prostate cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is a hematopoietic cancer. In one embodiment, the cancer is a metastasized cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is selected from prostate cancer, breast cancer, colorectal cancer and hepatic cancer. In some embodiments, prostate cancer is prostate adenocarcinoma. In some embodiments, the breast cancer is selected from breast lobular carcinoma, breast ductal carcinoma and invasive breast carcinoma. In some embodiments, the breast carcinoma isinvasive breast carcinoma. In some embodiments, the cancer is an AR positive cancer. In one embodiment, the cancer is an AR-associated cancer.

[0128] AR is a receptor found on a wide variety of cells and tissues and bind to circulating androgens. It also acts as a DNA-binding transcription factor that regulates gene expression. Overexpression or AR and / or AR mutants are associated with various cancers. The human gene can be accessed at Entrez gene ID 367 and Uniprot ID P10275. In some embodiments, the AR is AR isoform B. In some embodiments, isoform B is the full-length isoform. In some embodiments, AR comprisesMEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLL LQQQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLV LDEEQQPSQPQSALECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLS LLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSK DNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPP AVRPTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAA AGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKL ENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEE GQLYGPCGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQES DFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVL PIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTI DKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQK LTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVH VVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAP DLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGL KNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTF DLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ (SEQ ID NO: 7). In some embodiments, AR isoform B comprises SEQ ID NO: 7. In some embodiments, AR consists of SEQ ID NO: 7. In some embodiments, AR isoform B consists of SEQ ID NO: 7.

[0129] In some embodiments, the cancer comprises an AR H875Y mutation. In some embodiments, the cancer comprises AR with a H875Y mutation. In some embodiments, the cancer comprises AR isoform B with a H875Y mutation. In some embodiments, the cancer is an AR.H875Y mutant cancer. In some embodiments, the cancer comprises expression of AR comprising a H8 5Y mutation. In some embodiments, the cancer comprises expression of SEQ ID NO: 1. In some embodiments, the cancer comprises expression of SEQ ID NO: 8. In some embodiments, the cancer comprises expression of SEQ ID NO: 11. In someembodiments, expression is surface expression. In some embodiments, the cancer comprises surface display of a peptide comprising SEQ ID NO: 1. In some embodiments, the cancer comprises surface display of a peptide comprising SEQ ID NO: 8. In some embodiments, the cancer comprises surface display of a peptide comprising SEQ ID NO: 11. In some embodiments, surface display is by HLA proteins. In some embodiments, the surface display is by HLA-B*15:01. In some embodiments, the HLA is HLA-B* 15:01. In some embodiments, the surface display is by HLA-A*01:01. In some embodiments, the HLA is HLA- A*01:01.

[0130] T cells used to treat the cancers described herein may be derived from subjects that are suffering from the cancer (adoptive cell therapy - ACT). In one embodiment, the T cells are derived from peripheral blood lymphocytes of the subject. In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In one embodiment, the T cells are autologous to the subject. In another embodiment, the T cells are non-autologous to the subject. In some embodiments, the T cells are allogeneic to the subject. In some embodiments, the T cells are syngeneic to the subject.

[0131] ACT refers to the transfer of cells, most commonly immune-derived cells, back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Besser et al., (2010) Clin. Cancer Res 16 (9) 2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; and Dudley et al., (2005) Journal of Clinical Oncology 23 (10): 2346- 57) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science 314(5796) 126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma and colorectal carcinoma, as well as patients with CD 19-expressing hematologic malignancies (Kalos et al., (2011) Science Translational Medicine 3 (95): 95ra73).

[0132] The T cells can be administered by any suitable route as known in the art. Preferably, the T cells are administered as an intra-arterial or intravenous infusion, which preferably lasts approximately 30-60 min. Other examples of routes of administration include intraperitoneal, intrathecal, intratumoral and intralymphatic. T cells may also be administered by injection. T cells may be introduced at the site of the tumor.

[0133] For purposes of the invention, the dose, e.g., number of cells in the inventive cell population expressing subject specific TCRs, administered should be sufficient to effect,e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. For example, the number of cells should be sufficient to bind to a cancer antigen, 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 embodiments, the time period could be even longer. The number of cells will be determined by, e.g., the efficacy of the particular cells and the condition of the subject (e.g., human), as well as the body weight of the subject (e.g., human) to be treated.

[0134] Many assays for determining an administered number of cells from the inventive cell population expressing subject specific TCRs are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or one or more cytokines such as, e.g., IFN-gatnma and TNF alpha (TNFa) are secreted upon administration of a given number of such cells to a subject, could be used to determine a starting number to be administered to a mammal. The extent to which target cells are lysed, or cytokines such as, e.g., IFN-gamma and TNFa are secreted, upon administration of a certain number of cells, can be assayed by methods known in the art. Secretion of cytokines such as, e.g., IFNG / TNFa, may also provide an indication of the quality (e.g., phenotype and / or effectiveness) of a cell preparation.

[0135] The number of the cells administered from the inventive cell population expressing subject specific TCRs may also be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular cell population.

[0136] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is in need of a method of the invention. In some embodiments, the subject suffers from cancer. In some embodiments, the subject expresses HLA-B*15:01. In some embodiments, the subject expresses HLA-A*01 :01.In some embodiments, the subject has an HLA-B*15:01 allotype. In some embodiments, the subject has an HLA-A*01:01 allotype. In one embodiment, the subject has been preselected on the basis of expressing an AR.H875Y mutation and being identified as having an HLA- B* 15:01 allotype. In one embodiment, the subject has been preselected on the basis of expressing an AR.H875Y mutation and being identified as having an HLA-A*01:01 allotype. In some embodiments, the cancer of the subject expresses AR.H875Y.

[0137] Subject specific HLA alleles or HLA genotype of a subject may be determined by any method known in the art. In a particular embodiment, HLA genotypes are determined by any method described in International Patent Application number PCT / US2014 / 068746,published June 11, 2015 as WO2015085147, hereby incorporated by reference in its entirety. Briefly, the methods include determining polymorphic gene types that may comprise generating an alignment of reads extracted from a sequencing data set to a gene reference set comprising allele variants of the polymorphic gene, determining a first posterior probability or a posterior probability derived score for each allele variant in the alignment, identifying the allele variant with a maximum first posterior probability or posterior probability derived score as a first allele variant, identifying one or more overlapping reads that aligned with the first allele variant and one or more other allele variants, determining a second posterior probability or posterior probability derived score for the one or more other allele variants using a weighting factor, identifying a second allele variant by selecting the allele variant with a maximum second posterior probability or posterior probability derived score, the first and second allele variant defining the gene type for the polymorphic gene, and providing an output of the first and second allele variant.

[0138] The T cell populations disclosed herein are capable of being used in combination with at least one other therapeutic. Examples of therapeutics that can be used in conjunction with the T cells disclosed herein include, but are not limited to: immunomodulatory cytokines, including but not limited to, IL -2, IL- 15, IL-7, IL-21, GM-CSF, TNFa and IFNg as well as any other cytokines that are capable of further enhancing immune responses; immunomodulatory antibodies, including but not limited to, anti-CTLA4, anti-CD40, anti- 4 IBB, anti-OX40, anti-PDl and anti-PDLl; and immunomodulatory drugs including, but not limited to, lenalidomide (Revlimid). In addition, the T cell populations disclosed herein may be administered for cancer treatment in combination with chemotherapy in regimens that do not inhibit the immune system including, but not limited to, low dose cyclophosphamide, fludarabine and taxol. The vaccines may also be administered for cancer in combination with therapeutic antibodies including, but not limited to, anti-HER2 / neu (Herceptin) and anti- CD20 (Rituxan).

[0139] In one embodiment, the agents of this aspect of the present invention are administered together with immune checkpoint inhibitors (ICIs).

[0140] As used herein, the phrase “immune checkpoint inhibitor” refers to a compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In particular the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor preferably is an inhibitor of a human immune checkpoint protein. Immune checkpoint proteins are described in the art (see for instance Pardoll, 2012. Nature Rev. cancer 12: 252-264). The designation immune checkpoint includes the experimental demonstration of stimulation of an antigen-receptor triggered T lymphocyte response by inhibition of the immune checkpoint protein in vitro or in vivo, e.g. mice deficient in expression of the immune checkpoint protein demonstrate enhanced antigen- specific T lymphocyte responses or signs of autoimmunity (such as disclosed in Waterhouse et ah, 1995. Science 270:985- 988; Nishimura et ah, 1999. Immunity 11: 141-151). It may also include demonstration of inhibition of antigen-receptor triggered CD4+ or CD8+ T cell responses due to deliberate stimulation of the immune checkpoint protein in vitro or in vivo (e.g., Zhu et ah, 2005. Nature Immunol. 6:1245-1252).

[0141] In some embodiments, the ICIs are antibodies or antigen binding fragments thereof. Preferred immune checkpoint protein inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of CTLA-4, PD1, PDL-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3 and KIR inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. For example, ipilimumab is a fully human CTLA-4 blocking antibody presently marketed under the name Yervoy (Bristol-Myers Squibb). A second CTLA-4 inhibitor is tremelimumab (referenced in Ribas et al, 2013, J. Clin. Oncol. 31:616-22). Examples of PD-1 inhibitors include without limitation humanized antibodies blocking human PD-1 such as lambrolizumab (e.g. disclosed as hPD109A and its humanized derivatives h409All, h409A16 and h409A17 in WO2008 / 156 12; Hamid et al., N. Engl. J. Med. 369: 134-144 2013,), or pidilizumab (disclosed in Rosenblatt et al., 2011. J. Immunother. 34:409-18), as well as fully human antibodies such as nivolumab (previously known as MDX-1106 or BMS-936558, Topalian et al., 2012. N. Eng. J. Med. 366:2443- 2454, disclosed in U.S. Pat. No. 8,008,449 B2). Other PD- 1 inhibitors may include presentations of soluble PD-1 ligand including without limitation PD-L2 Fc fusion protein also known as B7-DC-Ig or AMP-244 (disclosed in Mkrtichyan M, et al. J Immunol. 189:2338-47 2012) and other PD-1 inhibitors presently under investigation and / or development for use in therapy. In addition, immune checkpoint inhibitors may include without limitation humanized or fully human antibodies blocking PD-L such as MEDL4736 (disclosed in WO2011066389 Al), MPDL3280A (disclosed in U.S. Pat. No. 8,217,149 B2) and MIH1 (Affymetrix obtainable via eBioscience (16.5983.82)) and other PD-L1 inhibitors presently under investigation. According to this invention an immune checkpoint inhibitor is preferably selected from a CTLA-4, PD-1 or PD- LI inhibitor, such as selected from the known CTLA-4, PD-1 or PD-L1 inhibitors mentioned above (ipilimumab, tremelimumab,labrolizumab, nivohimab, pidilizutnab, AMP-244, MEDI- 4736, MPDL3280A, MIH1). Known inhibitors of these immune checkpoint proteins may be used as such or analogues may be used, in particular chimerized, humanized or human forms of antibodies. In some embodiments, the ICI is an anti-PD-l / PD-Ll inhibitor. In some embodiments, the ICI is an anti PD 1 inhibitor. In some embodiments, the inhibitor is an antibody. In some embodiments, the antibody is a blocking antibody. ICIs are well known in the art and include, but are not limited to, pembrolizumab (PD-1), ipilimumab (CTLA4), nivolumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), cemiplimab (PD-1), dostarlimab (PD-1), durvalumab (PD-L1), relatlimab (LAG3), and retifanlimab (PD-1).

[0142] As used herein the term "neoantigen" is an epitope that has at least one alteration that makes it distinct from the corresponding wild-type, parental antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence. A mutation can include a frameshift or non-frame shift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF. A mutation can also include a splice variant. Post-translational modifications specific to a tumor cell can include aberrant phosphorylation. Post-translational modifications specific to a tumor cell can also include a proteasome-generated spliced antigen.

[0143] In one embodiment, the neoantigen is a short peptide that is bound to a class I or II MHC receptor thus forming a ternary complex that can be recognized by a T-cell bearing a matching T-cell receptor binding to the MHC / peptide complex with appropriate affinity. Peptides binding to MHC class I molecules are typically about 8-14 amino acids in length. T-cell epitopes that bind to MHC class II molecules are typically about 12-30 amino acids in length. In the case of peptides that bind to MHC class II molecules, the same peptide and corresponding T cell epitope may share a common core segment, but differ in the overall length due to flanking sequences of differing lengths upstream of the amino -terminus of the core sequence and downstream of its carboxy terminus, respectively. A T-cell epitope may be classified as an antigen if it elicits an immune response.

[0144] In a preferred embodiment, the HLA allele is a class I HLA allele. In particular embodiments, the class I HLA allele is an HLA-A allele or an HLA-B allele. In a preferred embodiment, the HLA allele is a class II HLA allele. Sequences of class I and class II HLA alleles can be found in the IPD-EVIGT / HLA Database. Exemplary HLA alleles include but are not limited to A*01:01, A*02:01, A*02:03, A*02:04, A*02:07, A*03:01, A*24:02,A*29:02, A*31:01, A’"68:02, B* 15:01, B*35:01, B*44:02, B*44:03, B*51 :01, B’"54:01 or B*57:01 In particular embodiments, the HLA allele is HLA-B* 15:01.

[0145] Subject specific HLA alleles or HLA genotype of a subject may be determined by any method known in the art. In a particular embodiment, HLA genotypes are determined by any method described in International Patent Application number PCT / US2014 / 068746, published June 11, 2015, as WO2015085147. Briefly, the methods include determining polymorphic gene types that may comprise generating an alignment of reads extracted from a sequencing data set to a gene reference set comprising allele variants of the polymorphic gene, determining a first posterior probability or a posterior probability derived score for each allele variant in the alignment, identifying the allele variant with a maximum first posterior probability or posterior probability derived score as a first allele variant, identifying one or more overlapping reads that aligned with the first allele variant and one or more other allele variants, determining a second posterior probability or posterior probability derived score for the one or more other allele variants using a weighting factor, identifying a second allele variant by selecting the allele variant with a maximum second posterior probability or posterior probability derived score, the first and second allele variant defining the gene type for the polymorphic gene, and providing an output of the first and second allele variant.

[0146] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal.

[0147] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0148] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition or method herein needs only to reduce the severity of a disease, disorder, or condition, reduce theseverity of symptoms associated therewith, or provide improvement to a patient or subject’ s quality of life.

[0149] In some embodiments, the method further comprises confirming expression of AR comprising a H875Y mutation. In some embodiments, the confirming is before the administering. In some embodiments, the method further comprises identifying expression of AR comprising a H875Y mutation. In some embodiments, the identifying is before the administering. In some embodiments, the expression is in the subject. In some embodiments, in the subject is in a cancer of the subject. In some embodiments, in a cancer is in a cancer cell. In some embodiments, expression of AR comprising a H875Y mutation comprises expression of a peptide comprising SEQ ID NO: 1. In some embodiments, expression of AR comprising a H875Y mutation comprises expression of a peptide comprising SEQ ID NO: 8. In some embodiments, expression of AR comprising a H875Y mutation comprises expression of a peptide comprising SEQ ID NO: 11. In some embodiments, the peptide consists of SEQ ID NO: 1. In some embodiments, the peptide consists of SEQ ID NO: 8. In some embodiments, the peptide consists of SEQ ID NO: 11. In some embodiments, a peptide comprising SEQ ID NO: 8 comprises SEQ ID NO: 1. In some embodiments, a peptide comprising SEQ ID NO: 1 comprises SEQ ID NO: 8. In some embodiments, a peptide comprising SEQ ID NO: 11 comprises SEQ ID NO: 1. In some embodiments, a peptide comprising SEQ ID NO: 11 comprises SEQ ID NO: 8. In some embodiments, a peptide comprising SEQ ID NO: 8 comprises SEQ ID NO: 11. In some embodiments, a peptide comprising SEQ ID NO: 1 comprises SEQ ID NO: 11. In some embodiments, expression is surface expression. In some embodiments, surface expression is via HLA display. In some embodiments, identifying is measuring. In some embodiments, identifying is determining.

[0150] In some embodiments, the method further comprises confirming expression of HLA- B*15:01. In some embodiments, the method further comprises confirming expression of HLA-A*01:01. In some embodiments, the method further comprises identifying expression of HLA-B*15:01. In some embodiments, the method further comprises identifying expression of HLA-AAl l :0l In some embodiments, the confirming is before the administering. In some embodiments, the identifying is before the administering. In some embodiments, the expression is in the subject. In some embodiments, identifying is measuring. In some embodiments, identifying is determining.

[0151] In some embodiments, the method further comprises confirming expression of a protein complex comprising a peptide comprising SEQ ID NO: 1 and HLA-B5* 15:01. In some embodiments, the method further comprises confirming expression of a proteincomplex comprising a peptide comprising SEQ ID NO: 1 and HLA-A^OEOl. In some embodiments, the method further comprises confirming expression of a protein complex comprising a peptide comprising SEQ ID NO: 8 and HLA-B*15:01. In some embodiments, the method further comprises confirming expression of a protein complex comprising a peptide comprising SEQ ID NO: 8 and HLA-A*01:01. In some embodiments, the method further comprises confirming expression of a protein complex comprising a peptide comprising SEQ ID NO: 11 and HLA-B^lSiOl. In some embodiments, the method further comprises confirming expression of a protein complex comprising a peptide comprising SEQ ID NO: 11 and HLA-A*01:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 1 and HLA-B* 15:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 1 and HLA- A*01:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 8 and HLA-B* 15:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 8 and HLA-A*01:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 11 and HLA-B5* 15:01. In some embodiments, the method further comprises identifying expression of a protein complex comprising a peptide comprising SEQ ID NO: 11 and HLA-A*01:01. In some embodiments, the confirming is before the administering. In some embodiments, the identifying is before the administering. In some embodiments, the expression is in the subject. In some embodiments, identifying is measuring. In some embodiments, identifying is determining.

[0152] Methods of determining / measuring / confirming expression of a mutant sequence are well known in the art and any such method can be employed. Determination can be made for example, on the DNA level such as by genomic sequencing or cloning, on the mRNA level such as by PCR, next-generation sequencing or in situ hybridization and / or on the protein level such as by western blot, immuno staining or peptidomics. Surface display of the peptide by HLA can be determined, for example, by immunopeptidomic methods or with a recombinant TCR or antibody of the invention. In some embodiments, the administering is only to a subject that has been confirmed. In some embodiments, the administering is only to a subject in which the target was identified.Diagnostic method

[0153] By another aspect, there is provided a method of detecting an AR H875Y mutation in a target cell, the method comprising contacting the target cell with a TCR of the invention, a T cell of the invention or a composition of the invention and detecting binding to the target cell, thereby detecting an AR H875Y mutation in a target cell.

[0154] In some embodiments, the method is a diagnostic method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is a method of detecting an H875Y mutant protein. In some embodiments, the method is a method of detecting surface expression of an H875Y mutant peptide. In some embodiments, the mutant protein comprises SEQ ID NO: 1. In some embodiments, the mutant peptide comprises or consists of SEQ ID NO: 1. In some embodiments, the mutant protein comprises SEQ ID NO: 8. In some embodiments, the mutant peptide comprises or consists of SEQ ID NO: 8. In some embodiments, the mutant protein comprises SEQ ID NO: 11. In some embodiments, the mutant peptide comprises or consists of SEQ ID NO: 11. In some embodiments, the target cell is a live cell. In some embodiments, the target cell is in a sample. In some embodiments, the sample is a biopsy. In some embodiments, the target cell is on a slide. In some embodiments, the target cell is fixed. In some embodiments, the detecting is diagnosing AR mutant cancer.Patient selection

[0155] By another aspect, there is provided a method of determining suitability of a subject to be treated by a method of the invention, the method comprising: a. identifying expression of HLA-B*15:01, HLA-A*01:01 or both; b. detecting expression of AR comprising a H875Y mutation; wherein a subject expressing HLA-B*15:01, HLA-A*01:01 or both and AR comprising a H875Y mutation is suitable to be treated by a method of the invention; thereby determining suitability of a subject.

[0156] In some embodiments, the method is a method of patient population selection. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a prognostic method. In some embodiments, the method further comprises treating the subject. In some embodiments, treating is by a method of the invention. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0157] In some embodiments, the method further comprises receiving a sample from the subject. In some embodiments, the method further comprises receiving a sample obtained from the subject. In some embodiments, the method further comprises obtaining a sample from the subject. In some embodiments, the method further comprises receiving a sample from the subject. In some embodiments, the sample comprises cells. In some embodiments, the sample comprises DNA. In some embodiments, the sample comprises protein. In some embodiments, the sample is a cell sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a cancer sample. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy.

[0158] In some embodiments, the identifying is in the sample. It will be understood by a skilled artisan that the determination of the HLA repertoire of the subject can be determined in any sample and not necessarily a cancer sample. However, the expression of AR.H875Y will occur in a sample comprising a cancerous cell as the mutation will be present in the tumor but not necessarily in healthy cells. In some embodiments, the detecting is by a method of the invention. In some embodiments, the detecting is with a synthetic TCR of the invention. In some embodiments, detecting comprises contacting with the TCR of the invention and detecting binding of the TCR to AR.H875Y on the cell surface.

[0159] In some embodiments, the method comprises selecting a subject that expresses HLA- B* 15:01. In some embodiments, the method comprises selecting a subject with a cancer that expresses AR.H875Y. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-B* 15:01 and a peptide comprising SEQ ID NO: 1. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-A*01:01 and a peptide comprising SEQ ID NO: 1. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-B*15:01 and a peptide comprising SEQ ID NO: 8. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-A*01:01 and a peptide comprising SEQ ID NO: 8. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-B* 15:01 and a peptide comprising SEQ ID NO: 11. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-A*01:01 and a peptide comprising SEQ ID NO: 11. In some embodiments, expresses is expresses on the cell surface. In some embodiments, a method of the invention is performed on a selected subject. In some embodiments, the selected subject is a subject in need of a method of the invention.

[0160] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0161] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0162] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase " A or B " will be understood to include the possibilities of "A" or "B" or "A and B."

[0163] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0164] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms“a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0165] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0166] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0167] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0168] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0169] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and Methods

[0170] Cell-lines and PBMCs: The tumor cell-lines utilized herein were collected from several sources . PC3 , LNCaP and DU 145 prostate cancer cell lines were gifted from Michael Elkin. 22RV 1 and C4-2 prostate cancer cell lines were gifted from Joaquin Mateo. The HLA null B-cell line 721.221 was purchased. The hybridoma cells HB95 was used to purify pan- HLA-I antibody for the preparation of the HLA affinity columns. All cell lines were tested regularly and were found negative for mycoplasma contamination (EZ-PCR Mycoplasma Kit, Biological Industries). All cell lines were cultured in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 2mM 1-glutamine, 2.5% HEPES, 100 lU / ml penicillin, 0.1 mg / ml streptomycin, and ImM sodium pyruvate and grown at 37°C in 5% CO2.

[0171] Peripheral blood mononuclear cells (PBMCs) were isolated from blood donations under a protocol approved by the Weizmann Institutional Review Board (IRB) Ethics Committee (2274-2). PBMCs were extracted using Ficoll-Paque PLUS (GE Healthcare) via density-gradient centrifugation and used freshly or cryopreserved in aliquots in human serum with 12% DMSO and thawed as needed per experiments.

[0172] Tumor somatic mutations: Cancer somatic mutations were obtained from 1) primary tumor whole exome sequences from the Cancer Genome Atlas (TCGA) and 2) metastatic whole genome sequences from the Hartwig Medical Foundation , comprising a total of 9,644 and 3,235 samples, respectively, after discarding hypermutators within each cancer cohort. Somatic mutations from the different cancer types were merged into two pan-cancer files representing primary and metastatic tumors. Somatic mutations were liftovered from hgl9 to hg38 using pyliftover version 0.3 (pypi.org / project / pyliftover), and those failing to liftover were discarded. A total of 1,547,627 and 65,139,581 mutations from primary and metastatic tumors, respectively, were analyzed.

[0173] Identification of recurrent neoantigens: We first identified all recurrent mutations (or hotspots) in primary and metastatic pan-cancer cohorts using our in-house developed method HotspotFinder version 1.0. We used the somatic mutation calls provided by original sources as input for hotspot identification by HotspotFinder. HotspotFinder identifies unique genomic positions that are recurrently mutated to the same alternate (e.g., two OT transitions) across tumors and annotates the overlap of these positions with genomic data, including genome mappability, population variants, and genomic elements. Specifically, hotspots were identified as recurrent mutations of equal alternate (split_altemates = True) in three or more samples. All other parameters were kept as default. To remove potential artifacts, hotspots overlapping low-mappability regions, population variants, or failing outside protein-coding sequences of Ensembl canonical transcripts were discarded. To identify which of the resulting hotspots generated missense mutations, we ran Variant Effect Predictor version 92 through bgvep (bitbucket.org / bgframework / bgvep / src / master / ) and kept those hotspots creating missense variants in canonical transcripts.

[0174] Hotspots were then prioritized for their potential to encode neoepitopes using the following criteria:1. HLA-I presentation: A list of all possible neoantigens of lengths between 8 and 14 amino acids overlapping each hotspot position was derived using the protein sequence of the canonical transcript. The matched wild-type sequence was generated for each of the mutant peptides . For each neoantigen and wild-type peptide, HLA-I presentation was validated across all HLA-I alleles of the matching mutated patients using three independent tools: MHCflurry version 2.0, NetMHCpan version 4.1 and MHCnuggets version 2.3. candidate neoantigens were defined as those peptides and HLA-I allele pairs classified as binders by MHCflurry2.0 and additional prediction algorithm. Binders are pairs with a predicted binding affinity lower than 500 nM. Neoantigens whose wild-type version was predicted to bind the same HLA-I allele were discarded. HLA-I germline allotypes were provided by LILAC (37165135). Neoantigens binding at least one of the following high-frequency HLA-I allele in the Hartwig medical dataset population were kept: HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA- A*ll:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01, HLA-B*15:01, HLA- B*35:01, HLA-B*40:01, HLA-C*03:03, HL A-C *03:04, HLA-C*04:01, HLA- C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*12:03.2. Hotspot prevalence in the baseline cohort of pre -biopsy treated cancer patients: Candidate neoantigens passing the previous steps were then ranked based on theirsource alteration prevalence matched with the predicted HLA-I allotypes across the cohort of metastatic treated patients. The grouped alteration prevalence was estimated across all patients with HLA-I alleles predicted to present the candidate neoantigen.3. Clonality: Mutation clonality, an essential factor in triggering strong and durable immune responses, was considered. Subclonal SNVs were filtered out based on estimates from the PURPLE tool.4. Potential role in tumorigenesis: driver predictions (i.e., driver or passenger) were retrieved fromboostDM. Hotspots were annotated as drivers if the boostDM score was larger or equal to 0.5 in a cancer type. Passenger mutations were filtered out.5. Neoantigen-specific RNA expression: For each selected hotspot mutation, the RNA expression of the corresponding mutated gene in the given sample (if available) was annotated using the Isofox tool(github.com / hartwigmedical / hmftools / blob / master / isofox / README.md). This information was then filtered out neoantigens derived from non-expressed genes in the corresponding mutated samples hotspot mutations.

[0175] Assessment of the expression of genes in normal tissues: We extracted the levels of expression (Transcripts per million, or TPM) of AR, ESRI, EGFR, and SMAD4 from the Genotype-Tissue Expression (GTEx) project.

[0176] Generation of B-cell lines co-expressing HLA alleles and MNG genes: Synthetic optimized DNA sequences of HLA class I alleles were purchased from ‘TWIST bioscience’ and cloned into a lentiviral system pCDH-CMV-MCS-EFla-Neo (SBI, #CD514B-1) to generate stable mono-allelic overexpression in the class-I HLA deficient B-cell line B721.221, as previously described. Following selection with 800pg / ml G418 Geneticin (#11811-031, Thermo Fisher Scientific), transduced B721.221 cells were labeled with anti- HLA-I APC-conjugated antibodies and evaluated for their HLA-I surface expression using W6 / 32 antibody (#311410, BioLegend). Data were analyzed using the Kaluza software. Minigenes (MNG) were designed as 47-mer peptides encompassing hotspot driver mutations (AR.H875Y, AR.T878A, AR.L702H, EGFR.T790M, ESR1.D538G, ESR1.Y537S, and SMAD4.P356L). These fragments were purchased from ‘TWIST bioscience’ and cloned into the lentiviral system pCDH-CMV-MCS-EFla-GFP-Puro (SBI, #CD513B-1). Stable B721.221 mono-allelic cells were transduced with lentiviruses containing one of the MNGs. The cells were then selected using one pg / ml puromycin for 7 days and subsequently verified for GFP expression by flow cytometry.

[0177] Purification and isolation of membrane HLA peptides by immunopeptidomics: Cell pellets consisting of 2x108 cells were homogenized and lysed on ice with lysis buffer containing 1% octyl-0-D glucopyranoside, 0.25% sodium deoxycholate, ImM EDTA, 0.2mM iodoacetamide, 1:200 Protease Inhibitor Cocktail (P8340, Sigma-Aldrich) and IMM PMSF in PBS as previously described (30209080). The lysates were cleared by centrifugation at 48,000 xg for 45 minutes at four °C and passed through a pre-clearing column containing Protein-A Sepharose beads (GeneScript). HLA-peptide complexes were then immunoaffinity purified from a cleared lysate using W6 / 32 pan-HLA-I antibody (purified from HB95 hybridoma cells). The HLA -peptides complexes were then eluted with 1% trifluoracetic acid (TFA), followed by separation of the peptides from the HLAs by Sep- Pak tC18 lOOmg Sorbent 96-well plate (Waters). The peptide fraction was eluted with 28% acetonitrile (ACN) in 0.1% TFA. The HLA-eluted peptides were dried and resolubilized using vacuum centrifugation with 0.1% formic acid.

[0178] Identification of eluted HLA peptides by mass spectrometry: For Orbitrap MS / MS experiments, the peptides were separated using reversed-phase chromatography using the nanoAquity system (Waters), with a Symmetry trap column (180 x 20 mm) and HSS T3 analytical column, 0.75 x 250 mm (Waters), mobile phase A: H2O+0.1% formic acid, B: acetonitrile+0.1% formic acid. The peptides were separated with a linear gradient over two hours from 5 to 28% B, 28 to 35% in 15min, 35% to 95% in 15min, maintained at 95% for lOmin and back to initial conditions, at a flow rate of 0.35 pl min-1 .

[0179] The LC was connected online via a nano -electro spray ionization source (Flexion, Thermo Scientific) using an emitter (Fossil) to either a Quadrupole Orbitrap MS (Q Exactive HF, Thermo Scientific) or a tribrid MS (Fusion Lumos, Thermo Scientific). Data were acquired using a data-dependent method, fragmenting the peptides by higher-energy collisional dissociation (HCD). On the Q Exactive HF, full- scan MS spectra were acquired at a resolution of 120,000 at 200 m / z with automated gain control (AGC) value of 3 x 106 ions, mass range of 300 to 1800 Th and maximum injection time of 100msec. MS / MS scans were acquired with an AGC target value of 105 with a maximum injection time of 150 msec, isolation of 1.7 Th, normalized collision energy was set to 30%, and MS / MS resolution was 15,000 at 200 m / z. Fragmented m / z values were dynamically excluded from further selection for 20s.

[0180] On the Fusion Lumos, full-scan MS spectra were acquired at a resolution of 120,000 at 200 m / z with an AGC value of 200%, a mass range of 300 to 1800 Th, and maximum injection time set to auto. MS / MS scans were acquired with an AGC target value of 100%with a maximum injection time of 150 msec, isolation of 1.7 Th, normalized collision energy was set to 27%, and MS / MS resolution was 15,000 at 200 m / z. Fragmented m / z values were dynamically excluded from further selection for 20 s.

[0181] For timsTOF (TTP) experiments, the peptides were resolubilized with 0.1% trifluoroacetic acid and 5mM TCEP before LC-MS / MS analysis. 5pL of each sample was loaded using the nanoElute2 (Bruker, Germany) liquid chromatography. Mobile phase A was 0.1% formic acid in water. B was 0.1% formic acid in acetonitrile. Peptides were separated using the Aurora Ultimate C18 nano column, 0.075x250mm (lonOptiks, Australia), using a gradient of 2% B to 29% B in 80min, then 0.5min to 95% B, maintained 95%B for 2.9min at flow of 300nL / min. The column was placed in the column toaster and connected to a CaptiveSpray Electrospray ionization source. The column was maintained at 50°C. Data was acquired with a timsTOF Pro (Bruker) in data dependent-acquisition- parallel-accumulation-serial-fragmentation (DDA-PASEF) mode with the following parameters: capillary voltage of 1600v, temperature of 180°C, mass range of 100-1,700 Th, ion mobility 0.6-1.57 1 / K0, tims ramp time of 300msec, number of PASEF MS / MS scans 10, target intensity of 20,000 with threshold of 2,500, charge range 0 to 5, collision energy of 20 at 0.6 1 / K0 and 59 at 1.6 1 / K0.

[0182] Mass spectrometry (MS) RAW files were analyzed using the MaxQuant software (version 2.1.3.0) (19029910), with a 5% false discovery rate (FDR). Peptides were searched against the UniProt human proteome database ID UP000005640 (from June 20, 2021) in combination with the MNG peptide sequence.

[0183] Validation using synthetic peptides: For spectral validation, light synthetic peptides (high-purity HPLC grade >85, GeneScript) underwent analysis using the same LC-MS / MS system as the endogenous peptides, with minor modifications to the gradient and collision energy settings. Specifically, the acetonitrile gradient was adjusted to run from 4% to 30% over 20 minutes, and the normalized collision energy was set to 27 or 30 for HF2 and FS1, respectively. Data processing was conducted using MaxQuant software, with FDR set to 1% and individual peptide mass tolerance disabled. The OrgMassSpecR R (rdrr.io / rforge / OrgMassSpecR / man / SpectrumSimilarity.html) package compared endogenous peptides with their synthetic counterparts. The synthetic spectrum with the highest MaxQuant score was selected for each peptide, while all endogenous spectra scoring above 60 in MaxQuant were included in the head-to-tail comparisons. Only endogenous- synthetic peptide pairs with the same charge were used in our analysis. The analysis utilizeddefault software parameters, and both normalized dot product and Pearson correlation were evaluated. Statistical significance was determined using a threshold p-value of 0.05.

[0184] Peptide-HLA model generation: To generate VQP / SVQ-HLA-B*15:01 models, we compiled a peptide-HLA-B*15:01 (p-HLA-B* 15:01) complex coordinate from the protein data bank (PDB). The model consists of a nanomer peptide derived from the SARS-CoV-2 spike protein presented by HLA-B*15:01 (PDB: 8ELH) (37468623). The spike peptide was manually mutated using pymol mutagenesis to VQPIARELY neo-peptide. The rotamer orientation of each amino acid is selected to avoid the steric hindrance with HLA-B* 15:01. To accurately represent the decamer peptide conformation within HLA binding groove, the PDB coordinate (PDB: 3DX7) consists of a peptide-derived Epstain-Barr Virus nuclear antigen 6 (EBNA6) presented by HLA-B*44:03 (19139173) was superimposed onto p- HLA-B*15:01 coordinate. The EBNA6 decamer peptide was then mutated to SVQPIARELY (SEQ ID NO: 1) for model generation. For obtaining LLD-HLA-A*01:01 model, we superimposed MAGE-Al-HLA-A*01:01 (PDB: 3BO8) onto BZLF1-HLA- B*35:O8 (PDB: 1ZHL) complex to precisely model the putative super-bulged conformation of 13-mer peptide. The BXLF1 peptide residues were then manually mutated to LLDSVQPIARELY (SEQ ID NO: 11) for LLD-HLA-A*01:01 model generation.

[0185] T-cells reactivity towards AR H875Y neo-peptides: Priming of healthy donors’ naive CD8 T cells with neo-peptides was performed as previously described with several modifications. Healthy blood donors informed consent was obtained under a protocol approved by the Weizmann Institutional Review Board (IRB) Ethics Committee (2274-2). Briefly, PBMCs were isolated from healthy donor’s blood using Ficoll gradient separation (Cytiva). On day -4, monocytes were isolated from PBMCs using CD 14-reactive microbeads (Miltenyi Biotec) and cultured for three days in CellGro GMP DC medium (CellGenix) supplemented with 1% human serum (Valley Biomedical) and 1% penicillin-streptomycin (DC-T medium) containing 10 ng / ml interleukin (IL)-4 (PeproTec) and 800 lU / ml GM-CSF (PeproTec). On day -1, monocyte-derived-dendritic cells were matured for 16 h with 800 lU / ml GM-CSF, 10 ng / ml IL-4, 10 ng / ml lipopolysaccharide (LPS; Sigma-Aldrich) and 5 ng / ml IFNy (PeproTech) supplemented into the cultures. Autologous naive T cells were isolated using Pan T Cell MicroBead (Miltenyi Biotec) and cultured overnight in DC-T medium 5 ng / ml IL-7 (PeproTech). On day 0, monocyte-derived-dendritic cells were pulsed for 2h with 1 pg / ml AR H875Y neo-peptides (VQPIARELY (SEQ ID NO: 8), SVQPIARELY (SEQ ID NO: 1), LLDSVQPIARELY (SEQ ID NO: 11)) or 1 pg / ml AR WT peptide (VQPIARELH (SEQ ID NO: 15), SVQPIARELH (SEQ ID NO: 2),LLDSVQPIARELH (SEQ ID NO: 12)). Subsequently, monocyte-derived-dendritic cells were co-cultured with isolated naive T cells in DC-T medium supplemented with 30 ng / ml IL-21 (PeproTech) at a DC:T cell ratio of 1:2. On days 3, 5 and 7 half of the medium was removed and replenished with fresh medium supplemented with 10 ng / ml of both IL-7 and IL-15 (PeproTech). On day 10, 25 lU / ml IL-2 (PeproTech) was also added to the supplemented cytokine cocktail. On day 12, T cells were re-stimulated with irradiated (35 Gy) human B-LCL 721.221 expressing HLA-B*15:01 or HLA-A*01:01. Irradiated mono- allelic 721.221 were pulsed for 2 hours with neo-peptides or WT peptides and then cocultured with T cells in DC-T medium at a DC:T cell ratio of 1:2. On days 14 and 17, half of the medium was removed and replenished with fresh medium supplemented with 10 ng / ml of both IL-7 and IL-15 and 25IU / ml IL-2 or 50 lU / ml IL-2 (PeproTech), respectively. On day 19, the same restimulation and replenishment of cytokines were repeated until day 26. On day 26, T cells were collected and co-cultured with pulsed B-LCL 721.221 cells expressing the matching HLA allele. Brefeldin (BFA) and Monensin were added to evaluate IFNy and TNFot secretion. Following 16h stimulation, surface expression of 4- IBB and secretion of IFNy and TNFoc were examined via flow cytometry. Cells were stained with Live-dead Fixable Green Dead Cell Stain Kit (Invitrogen), CD3 (clone HIT3a, Biolegend), CD8 (clone HIT8a, Biolegend) and 4- IBB (Clone 4B4-1, Biolegend), IFNy ( Clone 4S.B3, Biolegend), TNFa (Clone MAbl l), CD4 (Clone OKT4, Biolegend). All experiments were analyzed using a CytoFLEX LX flow cytometer (Beckman Coulter).

[0186] Defining peptide-specific CD8 T cell subpopulation via dextramer staining: Following the stimulation and expansion of T cells derived from healthy donor PBMCs mentioned above, T cells were collected and stained with AR H875Y 9mer, lOmer, and 13mer loaded-dextramers, according to the manufacturer's protocol (Immudex). Briefly, naive T cells that were previously exposed to mutant or WT peptides were stained with Live- dead Fixable Green Dead Cell Stain Kit (Invitrogen). T cells were next labeled with double AR H875Y-dextramers (APC and PE), followed by staining with anti-CD8 (clone HIT8a, Biolegend), and sorted by SORP for 10X genomics. Cells were freshly forwarded to lOx library preparation immediately after sorting.

[0187] Single-cell TCR sequencing: Sorted double and negative dextramer cells were processed immediately after sorting, as described above, for single-cell library construction. Briefly, sorted cells were washed and resuspended with PBS containing 0.04% bovine serum albumin (BSA) and counted using trypan blue staining. Single cells were captured in droplets by loading onto the Chromium Controller with a targeted cell recovery of 10,000 cells persample. According to the manufacturer's protocol, single-cell gene expression (GEX) and TCR-enriched libraries were prepared using the Chromium Single Cell 5’ V(D)J v2 dual index Kit (lOx Genomics). Samples were sequenced on an Illumina NovaSeq 6000 sequencer with 26 cycles readl, 10 cycles i7 index, 10 cycles i5 index, and 90 cycles read2 (GEX and TCR were sequenced together on the same sequencing kit) 800M reads were obtained in a 4:1 GEX library to TCR sequencing library ratio.

[0188] Data processing of scRNA and TCR-seq libraries: Reads from lOx scRNA expression libraries were aligned to human genome assembly GRCh38 (hg38) and quantified using cell ranger count (lOx Genomics, version 3.1.0). The filtered feature-barcode matrices containing only cellular barcodes were used for further analysis. Single-cell gene expression matrices were imported into R (version 3.6.1) and analyzed using Seurat (version 3.1.1). Cells with less than 4,000 or over 20,000 UMIs detected were filtered out. Cells with mitochondrial RNA reads greater than 10% were excluded from subsequent analyses. Single-cell TCR reads were aligned to human genome assembly GRCh38 (hg38) and assembled into reconstructed TCR consensus sequences using cellranger VDJ (lOx Genomics, version 3.1.0). Only productive TCRa and TCRp sequences were considered for further analysis. Overall, TCR sequences were annotated for 95.2% of cells, with paired TCRaP sequences detected for 37,934 out of 41,542 cells (91.3%). Only cells with conventional paired TCR chain combinations ap were kept for downstream analyses. Cells sharing the identical CDR3aP amino acid sequences were defined as belonging to the same TCR clone.

[0189] Identification of neoantigen-reactive clones: TCR clonal frequencies were compared between the sorted double positive and negative dextramer populations for each healthy donor. Clones were considered as double dextramer- specific if: (1) a >100-fold higher clone frequency was identified in the dextramer+ than in dextramer- populations, and (2) each TCR-chain is > 100-fold enriched in the double dextramer than in the double negative dextramer populations. Based on the above criteria, four neoantigen-reactive clones were defined as dextramer-specific for two healthy donors.

[0190] In vitro TCR immunogenicity assessment: Identified neo- antigen- specific TCRoc and P chains were cloned into an MSGV1 vector (T157.1, T157.2, T157.3, and T112.1), retrovirally transduced into healthy donors’ T cells, and then tested for reactivity. TCR- transduced T cells were co-incubated overnight with cancer cell lines and HLA-matched mono-allelic B-cells pulsed with neo or WT peptides. Cytokine secretion was measured byflow cytometry for IFNy, TNFoc, 4-1BB, and cleaved caspase 3. TCR-transformed donor T cells were further tested for their reactivity against neo-antigen-expressing tumor cell lines and HLA-A*01:01, including LNCaP overexpressed AR H875Y and HLA-B*15:01. The TCR was also transduced to Jurkat cells (TCR alpha beta KO cells) to evaluate the peptide concentration needed for reactivity by the Bio-Glo-NL™ Luciferase Assay (Promega).

[0191] Incucyte fluorescent killing assay: The LNCaP cancer cell line, harboring the HLA- A*01:01 allele, was stably transduced with AR H875Y MNG, HLA-B*15:01, and GFP to create the LNCaPAR H875Y / GFP, LNCaP AR H875Y / GFP / B*15:01 and the control cell line LNCaPGFP. The cell lines were seeded in triplicate in a 96-well plate, with each well containing 3000 cells. The following day, T-cells stably expressing AR H875Y-specific TCR (T157.1, T157.3 and T112.1) were added to the cancer cells at various effector-to- target (E:T) ratios. These ratios were calculated based on the percentage of mTCR-positive cells, determined by FACS analysis conducted on the same day. All conditions included the addition of IL-2 at a concentration of 300 units / ml in the T-cell medium. Control cancer cells received IL-2 but without T-cells. Cellular interactions were monitored using an Incucyte SX3 system over four days, with images captured every four hours. Each well was photographed four times per imaging session. The area covered by GFP-positive cells was analyzed.

[0192] Analysis of Differentially Expressed Genes in AR H875Y specific TCR transduced T-cells: Identified neo- antigen- specific TCRs (T157.1, T157.3, and T112.1) were retrovirally transduced into healthy donor T cells. TCR-transduced T cells were co-incubated at 1.5: 1 E:T ratio overnight with HLA-matched (HLA-A*01:01 or HLA-B*15:01) mono- allelic B -cells pulsed with neo or WT peptides. The next day, T cells were stained with PE- mTCR antibody and separated by anti-PE microbeads (Miltenyi). Reactivity of the isolated cells was measured in FACS by 4-1BB staining. RNA was extracted from 5X105 mTCR cells using a microRNA kit (Qiagen). Following MARS-seq library preparation as previously described. Sequencing was performed on an Illumina NextSeq 500 platform with the following parameters: 72 cycles for Read 1 (transcript sequence), and 15 cycles for Read 2 (cell barcode and UMI).

[0193] Our three gene signatures were generated (as described in the results section) by filtering our MARS-Seq data with 3 different sets of filters: (I) |FC|>4, p-value<0.05, (II) |FC|>2, p-value<0.05 and (III) p-value<0.05. For all signatures, scores over the Hartwig prostate cancer cohort (n 346) were calculated as the sum over the logged expression values (with a pseudo count of 1) of all genes on the signature normalized by their weight in thesignature. All gene weights were set to one for signatures without explicit weights divided by the number of genes in the signature. Pairwise correlations between signature scores over the Hartwig prostate cohort were calculated using Pearson correlation.

[0194] Differentially expressed genes (DEGs) between MUT vs WT reactive T cells were obtained in R using DESeq2 (25516281). Genes with a total count of less than 10 were filtered out, and the “DESeq” function were called with default parameters. P-values were Bonferroni-adjusted. Genes with an absolute log fold-change greater than 0.25 and an adjusted p-value less than 0.05 were considered differential.

[0195] Heatmaps were plotted by taking the intersections of the DEGs from all three AR samples in both directions DEGs up-regulated in either MUT or WT reactive T cells. Functional modules and pathways were identified in HumanBase (hb.flatironinstitute.org / ) by using T lymphocyte- specific networks.Example 1: Data-driven identification of recurrent potential neoepitopes from treatment resistance-associated hotspot mutations

[0196] The aim of this study was to systematically identify potential recurrent neo-peptides presented specifically in treatment-resistant tumors. It was reasoned that for a hotspot neoantigen to be considered a promising candidate "hotspot neoantigen" with significant therapeutic potential, the mutation in question and the combined frequency of the specific binding HLA allele and the associated mutation should exhibit a high prevalence among patients with these cancers. This criterion ensures that a large portion of the patient population could benefit from the targeting of the identified neoantigen. To this end, a pipeline named SpotNeoMet was developed to identify recurrent neoantigens. There was first identified 1,138 somatic single nucleotide variant (SNV) hotspots (recurrent mutations in three or more samples) overlapping protein-coding sequences within the Hartwig Medical Foundation dataset comprising 3,235 metastatic tumors with clinical annotations on the treatments received for their primary tumors (Methods). As a control, 6,062 hotspots in a cohort of 9,644 primary tumors from the Cancer Genome Atlas (TCGA) were surveyed. HLA-I binding among peptides derived from missense hotspots were predicted in both cohorts, resulting in 54,478 unique peptide and HLA class I binding pairs (Fig. 1A; Methods). The aim was to identify recurrent neoantigens generated from driver mutations across metastatic patients of one or more cancer types, suspected to confer resistance to treatments of the primary tumors and with limited prospective therapeutic options. Next, clonal driver neoantigens derived from hotspots that are enriched in the metastatic cohortwere selected to maximize their potential to be a T-cell-based immunotherapy target on treatment-resistant tumors. To this end, focus was on hotspots enriched in the metastatic cohort that were found to generate potential neoepitopes that were predicted to bind the most frequent HLA class I alleles (Fig. 1A-1C).

[0197] Among hotspot neoantigens fulfilling these criteria, seven recurrent mutational hotspots were identified in four genes suspected to confer resistance to treatment of the primary tumors, including androgen receptor (AR), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESRI), and SMAD family member 4 (SMAD4) (Fig. 1A-1D). Nevertheless, the relatively high expression of EGFR, ESRI, and SMAD4 across several normal tissues, together with the potential for cross -reactivity of cytotoxic lymphocytes with the wild-type sequence of the proteins, led to focusing on three hotspots affecting the AR, a key driver of disease progression and drug resistance in advanced prostate cancer. These were the AR L702H, H875Y, and T878A hotspots, present in 17, 14, and 8 patients with metastatic prostate adenocarcinoma, respectively, and well recognized to drive resistance to AR inhibitors and other drugs targeting AR signaling (Fig. ID) Out of 400 mCRPC patients screened, these mutations were found in 4.25%, 3.5%, and 2% of the Hartwig cohort, respectively.Example 2: Androgen receptor H875Y candidate neoantigen identification

[0198] It was decided to prioritize and focus efforts on a subset of 34 candidates of AR hotspot mutation alterations due to their direct clinical relevance, the lack of effective treatment options for AR-mutated tumors, and the potential for developing novel immunotherapeutic approaches targeting these neoantigens. To efficiently identify these potential neo-peptides, HLA-immunopeptidomics was applied on B-LCL 721.221 cells coexpressing a minigene (mng) and mono-allelic overexpression of the abundant HLA alleles (Fig. 2A-B). Three neo-peptides of the AR H875Y mutation restricted to HLA-A*01 :01 and HLA-B*15:01 were successfully identified by Mass Spectrometry (MS) (Fig. 2B-D) and three WT AR peptides restricted to HLA-A*02:01 were also identified. Synthetic light peptides bearing the same amino acid sequence were run in MS to validate the MS results. The synthetic peptides' MS / MS spectra showed high Pearson correlation coefficient (Fig. 2B-D). Importantly, HLA-immunopeptidomics of a prostate cancer cell line, LNCaP, which endogenously expresses HLA-A*01:01, transduced with AR H875Y mng and HLA- B*15:01 (LNCaPH875Y / B* 15:01), demonstrated a robust endogenous presentation of all three AR-derived neo-peptides.Example 3: The Tyrosine residue in the mutant AR H875Y peptides is essential for neoantigen presentation by HLA-A*01:01 and HLA-B*15:01 and their recognition by CD8+ T cells

[0199] HLA class Imolecules predominantly bind short peptides, typically 8-14 amino acids long. The specific amino acid sites within the peptide sequence are crucial for binding to the target HLA and usually anchor peptides at the second (P2) and C-terminal ( PQ) positions. Each HLA variant has its unique repertoire of presented peptides with specific sequence motifs, mainly defined by the molecular functions of the presented proteins. Studies have visualized the binding patterns of different HLA alleles and identified key residues essential for peptide binding. Especially the amino acid composition of peptides, which can impact their immunogenicity. Differences in amino acid profiles between immunogenic and non- immunogenic peptides highlight the importance of specific residues in eliciting T-cell responses. Since the Tyrosine residue is at the C-terminal position of the AR H875Y peptides (9, 10, and 13 mer), PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrodinger, LLC.) was used to visualize how the mutation position in the neo-peptides affects antigen presentation and T-cell recognition. The structure modeling showed that the Tyrosine residue in the neo-peptides anchors to both HLAs (HLA-A*01:01 and HLA- B*15:01) and is not presented to CD8+ T cells. (Fig. 2E-2G).

[0200] Interestingly, as opposed to the neo-peptides, WT peptides cannot be presented by the HLAs. These predictions were validated using three independent methods. The first was HLA-immunopeptidomics of mono-allelic B cells over-expressed with WT minigene. In the second method, the HLA atlas data was utilized using MaxQuant. Lastly, using NetMHCpan, the WT peptides were predicted to be non-binders to the HLA alleles. Results were in line with the WT peptides being predicted as non-binders to the HLA alleles in our NeoMet pipeline. Despite lacking mutations in the positions for TCR interaction in all neo- peptides, the neo-peptides are still likely perceived as foreign antigens by CD8+ T cells due to the WT peptides' inability to bind to HLA alleles.Example 4: AR H875Y neo-peptides are immunogenic in healthy donors’ PBMCs

[0201] To evaluate the immunogenicity of the AR H875Y neo-antigens, the following protocol was employed. Monocytes were separated using CD14+ magnetic beads from healthy donor PBMCs harboring HLA-B* 15:01 or HLA-A*01:01, followed by differentiation and maturation into dendritic cells (DCs) for five days. Matured DCs were pulsed for 2 hours with Ipg / ml of synthetic mutant peptide of AR H875Y (9mer-VQPIARELY (SEQ ID NO: 8), lOmer- SVQPIARELY (SEEQ ID NO: 1), 13mer- LLDSVQPIARELY (SEQ ID NO: 11)) or lpg / ml of synthetic WT peptide of AR (9mer- VQPIARELH (SEQ ID NO: 15), lOmer- SVQPIARELH (SEQ ID NO: 2), 13mer- LLDS VQPIARELH (SEQ ID NO: 12)), followed by co-incubation with enriched autologous T cells for ten days. Next, the T cells were re-stimulated with irradiated HLA-B* 15:01 / HLA- A*01:01 mono-allelic B cells pulsed with the peptides for 2 hours for 16 additional days. On day 26, HLA-B*15:01 / HLA-A*01:01 mono-allelic B cells were pulsed with Ipg / ml AR H875Y or WT peptide for 2 hours, followed by 16 hours of co-incubation with T cells to evaluate reactivity using flow cytometry of 4-1BB, IFNy, TNFa staining (Fig. 3A). All three neo-peptides significantly elicited an effective immune response in healthy donors’ PBMCs compared to the lack of such a signal with the wild-type version of the peptides (Fig. 3B- 3C).Example 5: Identification of specific TCRs towards the AR H875Y neo-peptides

[0202] As the AR.H875Y mutation appears in prostate cancer adenocarcinoma, breast invasive lobular carcinoma, breast invasive ductal carcinoma, hepatocellular carcinoma and invasive breast carcinoma, and is present in 0.06% of all AACR GENIE cases pan-cancer, it is important to identify TCRs that target the neo-antigen for future research and clinical applications. To characterize the antigen-specific TCR-repertoire within the in-vitro stimulated T-cells, single-cell TCR sequencing on dextramer double-positive and -negative sorted populations was performed. To this end, an in vitro stimulation protocol on healthy donor T-cells for 26 days was used, as detailed above (Fig. 3A). The in vitro stimulated T cells towards each neo-peptide were stained with two fluorophore -conjugated dextramers. Flow cytometry analysis of stained T-cells towards the 9mer, lOmer, and 13mer neo- peptides revealed that 0.31%, 0.5%, and 2.44%, respectively, of T-cells were neo-antigen specific (Fig. 3D) compared to WT peptides that elicited 0.01%, 0%, and 0.51% (respectively) dextramer staining. To characterize the antigen-specific TCR repertoire, fluorescence-activated cell sorting of the T-cell population using dextramer was used. Single-cell RNA and TCR sequencing on dextramer double positive and negative sorted populations was performed. The dextramer-positive population consisted of 45.37% cells belonging to a single TCR clone for the 9 mer peptide, 48.45% and 45.41% cells belonging to 2 TCR clones for the 10 mer peptide, and 44.27% cells belonging to a single TCR clone for the 13 mer peptide. These clones were absent in the dextramer- negative population (Fig. 3E). To assess whether the identified TCR clones are reactive towards their respective neoantigens, first their TCRa and TCRp chains were cloned into an MSGV 1 vector separatedby a P2A sequence designed with a mouse constant region. The respective TCR clones and their alpha and beta chain sequences are presented in Figure 4A. To test their reactivity and the potential amount of peptide needed for the reactivity of the TCRs, Jurkat TCRaP KO cells were retrovirally infected with each of the TCRs individually, (T157.1, T157.2, T 157.3, T112.1). Each was first evaluated for its mTCR expression and binding to AR H875Y dextramer by FACS staining (Fig. 7A-7B). Next, the reactivity of the TCR-transduced Jurkat T cells to neoantigens was assessed by co-culturing the Jurkat cells with antigen-presenting cells loaded with titrating concentrations of the relevant neoantigen peptides. The activation of the NFAT reporter gene in the Jurkat cells was measured using the Bio-Gio Luciferase Assay (Fig. 4B). Strikingly, it was found that concentrations as low as 66nM, 233nM, 233nM and 816nM for T157.1, T157.2, T157.3, and T112.1, respectively, are sufficient to trigger a response above baseline (Fig. 4C).

[0203] The TCRb chain of the T157.1 clone was sequenced and the CDR3 was identified as CASSEKGRGRVTGELFF (SEQ ID NO: 4). The TCRachain of this clone was also sequenced and the CDR3 was identified as CAVGGYQKVTF (SEQ ID NO: 3). The TCRbchain of the T 157.2 clone was also sequenced and the CDR3 was identified as CASSQDIAKGGNTIYF (SEQ ID NO: 6). The TCRachain of this clone was also sequenced and the CDR3 was identified as CAVKTFTGGGNKLTF (SEQ ID NO: 5). The TCRb chain of the T157.3 clone was also sequenced and the CDR3 was identified as CASSQDRGFSQPQHF (SEQ ID NO: 10). The TCRa chain of this clone was also sequenced and the CDR3 was identified as CALRDNYGQNFVF (SEQ ID NO: 9). The TCRb chain of the T112.1 clone was sequenced and the CDR3 was identified as CASSDLAGHTYEQYF (SEQ ID NO: 14). The TCRachain of this clone was also sequenced and the CDR3 was identified as CASPTGNQFYF (SEQ ID NO: 13).Example 6: AR H875Y-specific TCRs reactivity and cytotoxicity

[0204] Having established that all the cloned TCRs react to their respective neoantigens, their sensitivity and specificity towards their target HLA / neoantigen combinations was tested. To this end, two healthy donors’ T cells were retrovirally infected with each of the TCRs and their mTCR expression and binding to AR H875Y dextramer was evaluated by FACS staining (Fig. 8A-8B). Next, the transduced T cells were co-cultured with mono- allelic B cells pulsed with mutant or WT peptides. Their reactivity toward the WT and mutant peptides were examined using FACS staining for three different markers: IFNy, TNFa, and 4-1BB. T157.1, T157.3, and T112.1 exhibited a significant stimulation of IFNy, TNFa, and41BB (Fig. 5A), while T157.2 also produced significant stimulation, but in a less pronounced manner, especially for 41BB (Fig. 9A).

[0205] Next, the reactivity and cytotoxic capacity of the T157.1, T157.3, and T112.1 T cells against endogenous processing and presenting prostate cell lines was examined. For this, the LNCaPH875Y / B* 15:01 cells were used and were co-incubated with the relevant TCR- transduced T-cells. T157.1, T157.3, and T112.1 exhibited a significant stimulation of IFNy, TNFa, and 41BB (Fig. 5B), while T157.2 did not exhibit a significant stimulation of IFNy, and TNFa and very low, but significant, stimulation of 41BB (Fig. 9B). To examine the capability of the TCRs’ cytotoxicity towards LNCaP, an active cleaved caspase-3 staining assay was used. As seen in Figure 5C, T157.1, T157.3, and T112.1 possess a significant cytotoxic capacity towards LNCaPH875Y / B5|:15:01. To further assess the TCR's cytotoxic capacity toward neoantigen-expressing LNCaP cells, a longitudinal Incucyte live-cell imaging assay was also utilized, which enables continuous, real-time monitoring of the cells over extended periods without disturbing them. LNCaPH875Y / B* 15:01 / GFP cells were cocultured with the relevant TCRs transduced T-cells at varying effector-to-target (E:T) ratios, and the killing of cancer cells was monitored over time using Incucyte S3. As seen in Figure 5D, at low E:T ratios, the T cells expressing T157.1, T157.3, or T112.1 exhibited potent cytotoxic activity against LNCaP cells. Furthermore, significant cancer cell death was observed, as evidenced by the reduced confluency of LNCaPH875Y / B* 15:01 / GFP cells compared to the parental LNCaPGFP in the presence of T cells. Quantitative analysis of the time-lapse images revealed a dose-dependent decrease in the percentage of viable H875Y / B*15:01 / GFP cells with increasing E:T ratios. Notably, even at the lowest E:T ratio tested (0.5:1), T157.1 T cells were capable of inducing substantial killing of LNCaPH875Y / B*15:01 / GFP cells, with approximately 26% of cancer cells remaining viable after 72 hours of co -culture (Fig. 5D). This finding highlights the potent cytotoxic potential of the identified TCRs against AR H875Y prostate cancer cells. These results demonstrate the efficacy of T cells expressing the identified TCRs in recognizing and killing the AR H875Y / HLA-A*01:01 pair and AR H875Y / HLA-B* 15:01 prostate cancer cells, even at low effector-to-target ratios. The potent cytotoxic activity observed suggests the potential therapeutic utility of these TCRs in targeting mCRPC.Example 7: AR H875Y neoantigen-specific T cell signature profiling

[0206] To further assess the molecular mechanisms underlying T cell recognition and activation of the identified TCRs, it was investigated whether a common gene expression signature exists among our TCRs. Identifying a shared signature could enhanceunderstanding of T cell activation in response to related but distinct neoantigens and potentially reveal a unique transcriptional profile that could serve as a biomarker for successful T cell recognition and activation across multiple neoantigens from a single mutational hotspot. Such a signature, if uncovered, could be invaluable for predicting the efficacy of TCR based immunotherapies targeting the identified hotspot and for monitoring treatment responses in patients. It would also be interesting to see if the discovered signature applies to other TCRs targeting different neoantigens. To address these objectives, healthy donor PBMCs were retrovirally transduced with three different TCRs (T157.1, T157.3, and T112.1). The transduced T cells (T-TCRs) were co-cultured overnight with mono-allelic B cells pulsed with either the cognate neo-peptide or the corresponding WT peptide. After coculture, the T-TCRs were isolated and validated for activation by 41BB FACS staining, and their transcriptomes were analyzed using massively parallel single-cell RNA sequencing (MARS-seq2.0), library preparation and bulk RNA sequencing.

[0207] Reactive T cells displayed transcriptional signatures that could distinguish them from non-reactive T cells. Using the MARS-Seq data, three transcriptional gene signatures were generated for the neoepitope-reactive T cells with varying levels of stringency: a short signature with genes exhibiting a high fold-change (FO4, p-value <0.05, 73 genes), a longer signature with genes showing a moderate fold-change (FO2, p-value <0.05, 225 genes), and a comprehensive signature including all genes with any significant fold-change (FC>0, p-value <0.05, 657 genes).

[0208] To assess whether the gene signatures resembled those of reactive T cells identified by others, they were compared to a collection of gene signatures associated with increased TCR reactivity, T-effector and general inflammation. Minimal gene overlap was found between the three signatures and the publicly available collections, with only 2-30 matches depending on the stringency level (Fig. 6A). However, when all the signatures were scored against a cohort of 346 prostate cancer patients from the Hartwig Medical Foundation, all signatures displayed very high pairwise correlation (Spearman correlation 0.75+0.13) (Fig. 6B). Collectively, these results suggest that despite comprising different genes, owing to the particular experimental setting, the gene signatures reflect shared typical traits of T-cell effector and TCR-reactive programs.

[0209] To delve deeper into the functional implications of these transcriptional signatures, the T cell developmental dynamics in these three AR H875Y TCRs MARS-seq data was investigated. The heatmaps generated from the intersections of differentially expressed genes (DEGs) across all three TCRs samples revealed distinct patterns of gene expressionbetween MUT and WT reactive T cells (Fig. 10-11). Building on these observations, the same type of analysis from Jaiswal et al. 2022 (“An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes”, Cancer Cell, 2022 May 9;40(5):524-544, the contents of which are hereby incorporated by reference in their entirety) was adopted, where the authors queried signatures from a diverse set of mouse and human T cell initiating challenges over time. Trajectory analysis reveal the T-cell programs dynamically modulated in each sample relative to a control of iteratized random probes (black line). Specifically, the analysis showed that T cells responding to neopeptides (Mut samples) exhibited gene expression patterns consistent with early activation. Transcripts from all three TCRs co-incubated with mono-allelic B cells pulsed with AR H875Y neo-peptides shared the trend of up-regulation of early activation genes found at early time points and down-regulation of these same genes at late time points associated to memory formation (Fig. 6C).

[0210] The combination of both these analyses revealed five genes that were consistently upregulated in all Mut samples, representing common denominators between the two analyses: SPHK1 (Sphingosine Kinase 1), TNFRSF9 (Tumor Necrosis Factor Receptor Superfamily Member 9), CD27 (CD27 Molecule), DUSP4 (Dual Specificity Phosphatase 4), and GZMB (Granzyme B). As expected, three out of the upregulated genes associated with T-cell activation and effector function are TNFRSF9 (also known as 4 IBB) and CD27, important co-stimulatory molecules, and GZMB inducing apoptosis in target cells during cytotoxic T-cell responses. In addition, FACS analysis further confirmed the 41BB elevation at the protein level. SPHK1 was identified in both analyses, which regulates T cell activation, survival, and memory development. Additionally, DUSP4 expression is dynamically regulated in CD8 T cells during activation. Specifically, DUSP4 levels were found to change in different CD8 T-cell subpopulations as they became activated in head and neck cancer. This dynamic expression pattern suggests that DUSP4 plays a role in modulating CD8 T- cell activation responses, potentially fine-tuning the cells' functional states during the course of an immune response associated with early T-cell activation. These findings point to a common molecular signature in the Mut samples, potentially reflecting enhanced immune responsiveness or altered T-cell functionality across all TCRs.

[0211] In summary, this analysis of transcriptional signatures reveals distinct characteristics of neoepitope-reactive T cells, setting the stage for a deeper exploration of their developmental dynamics and functional trajectories. These unique gene expression patterns not only highlight the reactive T cells' profiles but also provide a foundation forunderstanding their activation and memory formation. Ultimately, the identification of these signatures is crucial for investigating the developmental pathways and functional responses of T cells in the context of neoepitope recognition.

[0212] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A nucleic acid molecule comprising a heterologous promoter and an open reading frame encoding: a. a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVGGYQKVTF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSEKGRGRVTGELFF (SEQ ID NO: 4); b. a TCR alpha chain comprising a CDR-A3 comprising the amino acid sequence CAVKTFTGGGNKLTF (SEQ ID NO: 5) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDIAKGGNTIYF (SEQ ID NO: 6); c. a TCR alpha chain comprising a CDR-A3 comprising the amino acid sequence CALRDNYGQNFVF (SEQ ID NO: 9) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSQDRGFSQPQHF (SEQ ID NO: 10); or d. a T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CASPTGNQFYF (SEQ ID NO: 13) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASSDLAGHTYEQYF (SEQ ID NO: 14).

2. The nucleic acid molecule of claim 1, wherein a single open reading frame encodes a single polypeptide comprising said TCR alpha chain and said TCR beta chain separated by a cleavable linker or a flexible linker.

3. The nucleic acid molecule of claim 2, wherein said cleavable linker comprises a selfcleaving peptide selected from P2A, E2A, T2A and F2A.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein said TCR alpha chain comprises TRAV12-2 and TRAJ13 and said TCR beta chain comprises TRBV6-1, and TRBJ2-2; said TCR alpha chain comprises TRAV24 and TRAJ26 and said TCR beta chain comprises TRBV4-1, and TRBI1-5; said TCR alpha chain comprises TRAV12- 2 and TRAJ10 and said TCR beta chain comprises TRBV4-1 and TRBJ1-3 or saidTCR alpha chain comprises TRAV24 and TRAI49 and said TCR beta chain comprisesTRBV10-2 and TRBJ2-7.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein a TCR comprising said alpha chain and said beta chain specifically binds to a peptide comprising the amino acid sequence SVQPIARELY (SEQ ID NO: 1), VQPIARELY (SEQ ID NO: 8) or LLDSVQPIARELY (SEQ ID NO: 11).

6. The nucleic acid molecule of claim 5, wherein a TCR comprising said alpha chain and said beta chain specifically binds to a complex comprising HLA-B *15:01 or HLA- A*01:01 and a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

7. The nucleic acid molecule of claim 5 or 6, wherein a. a TCR comprising a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 1; b. a TCR comprising a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 binds to a complex comprising HLA-B* 15:01 or HLA-A*01:01 and SEQ ID NO: 1; c. a TCR comprising a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 binds to a complex comprising HLA-B*15:01 or HLA-A*01:01 and SEQ ID NO: 8; and d. a TCR comprising a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 binds to a complex comprising HLA-B*15:01 or HLA-A*01:01 and SEQ ID NO: 11;8. The nucleic acid molecule of any one of claims 1 to 7, being a mammalian expression vector.

9. An isolated population of T cells genetically modified to express a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 4; an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising theamino acid sequence of SEQ ID NO: 6; an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 10 or an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO:14.

10. An isolated population of T cells, wherein at least 10% of said T cells of said population comprise a TCR comprising an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 3 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 4; an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 5 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 6; an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 10, or an alpha chain comprising a CDR-A3 comprising the amino acid sequence of SEQ ID NO: 13 and a beta chain comprising a CDR-B3 comprising the amino acid sequence of SEQ ID NO: 14.

11. The isolated population of claim 10, wherein said at least 10% of T cells are genetically modified to express said TCR.

12. The isolated population of any one of claims 9 to 11, wherein said T cells are CD8 positive T cells.

13. The isolated population of any one of claims 9 and 11 to 12, wherein said genetic modification comprises expression of a nucleic acid molecule of any one of claims 1 to 7.

14. The isolated population of any one of claims 9 to 13, for use in treating cancer in a subject in need thereof, wherein said cancer comprises expression of Androgen Receptor (AR) comprising a H875Y mutation.

15. The isolated population of claim 14, wherein said expression of AR comprising a H875Y mutation comprises expression of an AR comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

16. The isolated population of claims 14 or 15, wherein said subject expresses HLA- B* 15:01, HLA-A*01:01 or both.

17. The isolated population of any one of claims 14 to 16, wherein a. said TCR comprises a CDR-A3 comprising SEQ ID NO: 3 and a CDR-B3 comprising SEQ ID NO: 4 and said cancer comprises expression of SEQ ID NO: 1 in complex with HLA-B*15:01, HLA-A*01:01 or both; b. said TCR comprises a CDR-A3 comprising SEQ ID NO: 5 and a CDR-B3 comprising SEQ ID NO: 6 and said cancer comprises expression of SEQ ID NO: 1 in complex with HLA-B*15:01, HLA-A*01:01 or both; c. said TCR comprises a CDR-A3 comprising SEQ ID NO: 9 and a CDR-B3 comprising SEQ ID NO: 10 and said cancer comprises expression of SEQ ID NO: 8 in complex with HLA-B*15:01, HLA-A*01:01 or both; or d. said TCR comprises a CDR-A3 comprising SEQ ID NO: 13 and a CDR-B3 comprising SEQ ID NO: 14 and said cancer comprises expression of SEQ ID NO: 11 in complex with HLA-B*15:01, HLA-A*01:01 or both.

18. A pharmaceutical composition comprising an isolated population of T cells of any one of claims 9 to 17 and a pharmaceutically acceptable carrier, excipient or adjuvant.

19. A method of treating cancer in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition of claim 18, thereby treating cancer.

20. The method of claim 19, wherein said cancer comprises expression of AR comprising a H875Y mutation.

21. The method of claim 20, wherein said expression of AR comprising a H875Y mutation comprises expression of an AR comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11.

22. The method of any one of claims 19 to 21, wherein said cancer comprises surface expression of a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 11.

23. The method of any one of claims 19 to 22, wherein said cancer is selected from prostate cancer, breast cancer, colorectal cancer and hepatic cancer.

24. The method of any one of claims 19 to 23, further comprising before said administering identifying in a cancer cell from said subject expression of AR comprising a H875Y mutation, surface expression of a peptide comprising SEQ ID NO: 1,SEQ ID NO: 8 or SEQ ID NO: 11 .

25. The method of any one of claims 19 to 24, wherein said subject expresses HLA- B* 15:01, HLA-A*01:01 or both.

26. The method of any one of claims 19 to 25, further comprising before said administering identifying in said subject expression of HLA-B*15:01, HLA-A*01:01 or both.

27. The method of any one of claims 19 to 26, further comprising before said administering identifying in a cancer cell from said subject surface expression of a peptide comprising SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 11 in complex with HLA-B*15:01 or HLA-A*01:01.

28. The method of any one of claims 19 to 27, wherein said T cells are autologous, allogeneic, or syngeneic to said subject.

29. A method of determining suitability of a subject in need thereof to be treated by a method of any one of claims 19 to 28, the method comprising: a. identifying expression of HLA-B*15:01, HLA-A*01:01 or both by said subject; and b. detecting expression of AR comprising a H875Y mutation in a cancer cell of said subject; wherein a subject expressing HLA-B*15:01 and comprising a cancer cell expressing AR comprising a H875Y mutation is suitable to be treated by a method of any one of claims 19 to 27; thereby determining suitability of a subject.

30. The method of claim 29, wherein said detecting is in a cancer cell in a sample obtained from said subject.

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