WO2025027604A1

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Publication number: WO2025027604A1
Application number: PCT/IL2024/050761
Authority: WO
Inventors: Yardena Samuels; Shira SAGIE GROHER
Original assignee: Sheba Impact Ltd; Yeda Research and Development Co Ltd
Current assignee: Sheba Impact Ltd; Yeda Research and Development Co Ltd
Priority date: 2023-07-31
Filing date: 2024-07-31
Publication date: 2025-02-06
Anticipated expiration: 2026-01-31
Also published as: IL304887A

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 and an open reading frame encoding a TCR beta chain comprising a CDR-B3 of SEQ ID NO: 4 are provided. 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 RECEPTOR DIRECTED AGAINST A RAS NEOANTIGEN

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Israeli Patent Application No. 304887, filed July 31, 2023, the contents of which are all incorporated herein by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The contents of the electronic sequence listing (YEDA-SHB-P-034-PCT.xml; Size: 12,107 bytes; and Date of Creation: July 4, 2024) are herein incorporated by reference in their entirety.

FIELD OF INVENTION

[003] The present invention is in the field of T cell receptors and adoptive cell transfer.

BACKGROUND OF THE INVENTION

[004] About 30% of non-small cell lung cancer (NSCLC) patients harbor KRAS driver mutations. In contrast to other identified driver mutations, effective targeted treatments are still scarce in this prevalent KRAS mutant (KRASm) lethal cancer, and the prognosis of these patients is poor. NSCLC treatment has been revolutionized by the introduction of immunotherapy agents, which doubled the treatment response rates. Currently, first-line treatment for most of these KRASm NSCLC patients comprises a combination of chemotherapy and immunotherapy. However, responses are not universal and are mostly not durable.

[005] Immune checkpoint inhibitors (CPI) anti-cancer activity depends upon specific recognition of tumor antigens such as mutation-derived antigens, designated neo-antigens, by cytotoxic T-lymphocytes which mediate an anti-tumor immune response. Neo-antigens are cell-surface peptide/human-leukocyte antigen (HLA) complexes where the peptide component, i.e., the neo-peptide, is the altered degradation product of a mutated protein. Restricted in expression to the cancer cells and uncurbed by immune tolerance, neo-antigens may elicit specific anti-tumor reactivity upon T cell receptor (TCR) engagement and are therefore ideal therapeutic targets.

[006] The majority of neo-antigens derive from private mutations and are thus relevant only for an individual patient. It is advantageous to target shared cancer-mutated proteins, preferably those that are cancer drivers. 2.2 million lung cancer patients are diagnosed yearly, 85% of them with NSCLC, from which -30% harbor KRAS mutations (20% of these are G12V mutations). Thus, the potential benefit from a therapeutic tool targeting G12V mutation may be very high. KRAS G12V mutation is also prevalent in colon cancer (about 9% prevalence) and pancreatic cancer (about 27%); these patients could benefit from a KRAS G12V neoantigen-directed therapy as well. A new therapeutic agent that can target the KRAS G12V mutants is therefore greatly needed.

SUMMARY OF THE INVENTION

[007] 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 are provided. Isolated populations of T cells and compositions comprising the nucleic acid molecules as well as methods of treating cancer are also provided.

[008] 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 CAYRSARRDDKIIF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR- B3 comprising the amino acid sequence CASGDRGPFPNTEAFF (SEQ ID NO: 4).

[009] 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.

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

[011] T According to some embodiments, the TCR alpha chain comprises 2-TRAV38 and TRAJ30 and the TCR beta chain comprises 1-TRBV6, TRBD1 and 1-TRBJ1. [012] According to some embodiments, a TCR comprising the alpha chain and the beta chain specifically binds to a peptide comprising the amino acid sequence VVVGAVGVGK (SEQ ID NO: 1) , a peptide comprising the amino acid sequence VVVGACGVGK (SEQ ID NO: 10) or both.

[013] According to some embodiments, a TCR comprising the alpha chain and the beta chain specifically binds to a complex comprising HLA-A*03:01 and a peptide comprising SEQ ID NO: 1, a complex comprising HLA-A*3:01 and a peptide comprising SEQ ID NO: 10 or both.

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

[015] 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.

[016] 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.

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

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

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

[020] 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 KRAS, NRAS or HRAS comprising a G12V or G12C mutation.

[021] According to some embodiments, the expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation comprises expression of a KRAS, NRAS or HRAS comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10.

[022] According to some embodiments, the subject expresses HLA-A*03:01. [023] 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.

[024] 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.

[025] According to some embodiments, the cancer comprises expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation.

[026] According to some embodiments, the expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation comprises expression of a KRAS, NRAS or HRAS comprising SEQ ID NO: 1 or SEQ ID NO: 10.

[027] According to some embodiments, the cancer comprises surface expression of a peptide comprising SEQ ID NO: 1 or SEQ ID NO: 10.

[028] According to some embodiments, the cancer is selected from pancreatic cancer, colon cancer, lung cancer and endometrial cancer.

[029] According to some embodiments, the method further comprises before the administering identifying in a cancer cell from the subject 1) expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation, 2) surface expression of a peptide comprising SEQ ID NO: 1 or SEQ ID NO: 10, or 3) both.

[030] According to some embodiments, the subject expresses HLA-A*03:01.

[031] According to some embodiments, the method further comprises before the administering identifying in the subject expression of HLA-A*03:01.

[032] 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 in complex with HLA-A*03:01 or a peptide comprising SEQ ID NO: 10 in complex with HLA-A*3:01.

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

[034] 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-A*03:01 by the subject; and b. detecting expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation in a cancer cell of the subject; wherein a subject expressing HLA-A*03:01 and comprising a cancer cell expressing KRAS, NRAS or HRAS comprising a G12V or G12C mutation is suitable to be treated by a method of the invention; thereby determining suitability of a subject.

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

[036] According to some embodiments, the method further comprises administering the therapeutically effective amount of a pharmaceutical composition of the invention to a subject determined to be suitable.

[037] 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

[038] Figure 1: Data driven candidate selection: By an unbiased data-driven candidate selection method, an immunogenic neo-antigen comprising a KRAS G12V peptide under the HLA A0301 restriction was identified and characterized.

[039] Figures 2A-2C: KRAS G12V presentation validation. (2A) MS/MS spectra of the KRAS G12V neoantigen (VVVGAVGVGK; SEQ ID NO: 1) identified in a minigene overexpressing monoallelic antigen presenting cell line. (2B-C) Endogenous validation of the presentation of the peptide by heavy isotope labeled peptides in (2B) pancreatic ductal adenocarcinoma (PDAC) and (2C) non-small cell lung carcinoma (NSCLC) cell line.

[040] Figures 3A-3D: Immunogenicity assessment. Immunogenicity of KRAS G12V peptide was assessed in two healthy donors. Presented is an in vitro reactivity test in donor 104 after 26 days of stimulation with the mentioned peptides. Dot plots of (3A) KRAS G12V specific IFNg positive cells as assessed by FACS. (3B) WT peptide and (3C) DMSO were used as negative controls and (3D) viral peptide was used as a positive control. Monoallelic B cells expressing HLA A0301 were used as APCs.

[041] Figure 4: Dextramer staining and single cell TCR sequencing. Healthy donor D104 T-cells were in vitro stimulated for 26 days against the G12V peptide or the WT peptide and stained for a KRAS G12V specific dextramer (left). Dextramer positive cells were sorted by FACS and taken for single cell TCR sequencing that showed that 97% of dextramer positive cells (dark blue) belong to one single clone (right).

[042] Figure 5: TCR reactivity validation. The KRAS G12V specific TCR identified by single cell TCRseq was cloned into a vector including a portion of a mouse TCR in order to identify the retroviral infected cells. Following infection of the TCR to healthy donor PBMCs, reactivity against the KRAS G12V peptide and the WT peptide was tested in vitro and assessed by FACS analysis of (left) IFNg, (middle) TNFa and (right) 4- IBB.

[043] Figure 6: TCR reactivity by Jurkat Luminescence essay. Line graph of a Jurkat luminescence assay measuring NFAT activity. Jurkat cells transduced with the T104 TCR were co-incubated with A0301 B cells exposed to serial dilution of WT or G12V mutant peptides and reactivity was measured by luminescence assay.

[044] Figures 7A-7B: TCR reactivity to endogenous cell lines. (7A) Bar graphs of TCR reactivity assay against lung cell lines which endogenously express KRAS G12V and the HLA A*0301 and negative control cells, as measured by FACS staining for (left) IFNg, (middle) TNFa and (right) 4- IBB in for PBMCs transduced with the T104-TCR. (7B) Bar graph of TCR reactivity as assessed by FACS staining for 4- IBB in a SW620 cells and SW620 cells stably expressing A*0301. *** = pval<0.001, **** = pval<0.0001.

[045] Figures 8A-8E: (8A) Bar graph of caspase3 activity in tumor cells cocultured with T cells expressing T104 or a control TCR. (8B-8D) Line graphs of GFP from SW620 cells with or without expression of A*0301 in the presence of (8B) no effector T cells, (8C) T cells expressing T104 in an E:T ratio of 0.75:1 and (8D) T cells expressing T104 in an E:T ratio of 1.5:1. (8E) Line graph showing GFP from SW260 cells expressing A*0301 in the presence of an increasing E:T ratio.

[046] Figures 9A-9C: KRAS G12V-specific TCR T104 conveys reactivity toward the KRAS G12C neoantigen. (9A) T104 TCR T cells were co-incubated overnight with pulsed B cells with the recited peptides and evaluated for reactivity by 4- IBB staining by FACS. (9B) Jurkat luminescence assay measuring NFAT activity. Jurkat cells transduced with the T104 TCR were co-incubated with A*03:01 B-cells exposed to serial dilution of WT or G12C mutant peptides; reactivity was measured by luminescence assay. (9C) T104 TCR T cells were co-incubated for 24 hours with pulsed B cells with the recited peptides and evaluated for reactivity by ELISA measuring the amounts of IFNy produced.

DETAILED DESCRIPTION OF THE INVENTION

[047] 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. 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.

[048] The invention is based, at least in part, on the surprising discovery of a new TCR that unexpectedly binds to both neoantigen produced by the G12V mutation and the G12C mutation in KRAS. These mutations are also found in NRAS and HRAS and produce the same neopeptide from these oncogenes. By applying a unique, unbiased data-driven candidate selection method a novel HLA-presented neo-antigen derived from a KRASG12V 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 neo-antigen-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 and kill those cancer cells in a neo-antigen-specific manner, making this discovery therapeutically relevant for thousands of cancer patients. Examining the specificity of the TCR led to the discovery that it also recognized neoantigen produced by the G12C mutation. The TCR directed reactivity against cells with this neoantigen as well, further expanding the cancer patients for which this therapeutic is relevant.

Nucleic acid molecules

[049] 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 CAYRSARRDDKIIF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASGDRGPFPNTEAFF (SEQ ID NO: 4).

[052] 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 are as follows: Alpha chain- 2-TRAV38, TRAJ30, CDRA3-SEQ ID NO: 3; Beta chain- 1-TRBV6, TRBD1, 1-TRBJ1, CDRB3-SEQ ID NO: 4. In some embodiments, the alpha chain comprises a TRAV38 gene. In some embodiments, the TRAV38 gene is a 2-TRAV38 gene. In some embodiments, the alpha chain comprises a TRAJ30 gene. In some embodiments, the beta chain comprises a TRBV6 gene. In some embodiments, a TRBV6 gene is a 1-TRBV6 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 1-TRBJ1 gene.

[053] 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.

[055] 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.

[056] 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.

[057] 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).

[058] 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.

[059] 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. In some embodiments, the nucleic acid molecule is for use in the production of a medicament for performance a method of the invention.

[060] 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.

[061] 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.

[062] 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.

[063] 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.

[064] 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.

[065] 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.

[066] 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.

[067] 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, and p2O5. 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.

[068] 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 rapidly infected, 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.

[069] 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.

[070] 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.

[071] 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

[072] 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.

[073] 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. [074] 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 transduced or 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.

[075] 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. The T cell populations expressing T cell receptors on the surface thereof bind to the peptide epitope as set forth in SEQ ID NO: 10 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: 11. In one embodiment, the T cells do not have antigenic specificity towards the peptide epitopes as set forth in SEQ ID NO: 12.

[076] 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-A*03:01+ target cells pulsed with a low concentration of mutated target peptide having a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 10 (e.g., about 0.05 ng/mL to about 5 ng/mL, 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-A*03:01+ target cells into which a nucleotide sequence encoding the mutated target peptide having a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 10 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 antigen-negative HLA-A*03:01+ 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.

[077] 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-A*03:01+ target cells pulsed with a low concentration of mutated target peptide or (b) antigen-negative HLA- A*03:01+ 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, co-cultured with (a) antigen negative HLA- A*03:01+ 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, SEQ ID NO: 11 or SEQ ID NO: 12) or (b) antigen negative HLA-A*03:01+ 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-A*03:01+ target cells pulsed with the same concentration of mutated target peptide or (b) antigen-negative HLA-A*03:01+ 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).

[078] 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 expressing the TCR secrete IFN-gamma/TNFa upon co-culture with (a) antigen-negative HLA- A*03:01+ target cells pulsed with a low concentration of mutated target peptide, (b) antigennegative HLA-A*03:01+ target cells into which a nucleotide sequence encoding the mutated target has been introduced or (c) HLA-A*03:01+ 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. [079] 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. 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.

[081] 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. 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. In some embodiments, the population is for use in the production of a medicament for performance of a method of the invention.

[082] 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 side-chain 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 side-chain 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.

[083] 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.

[084] 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, the side 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.

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

[086] 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, alphanaphthylalanine, 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.

[087] 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.

[088] 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 be commercially 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, Ill.), 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

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

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

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

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

[093] 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 production of a medicament for performance of a method of the invention.

[094] 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 nontoxic 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 well known 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.

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

[096] 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.

[097] 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, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for intratumoral administration.

[098] 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.

Method of treatment

[0100] 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.

[0101] 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 hematopoietic cancer. In one embodiment, the cancer is a metastasized cancer. In some embodiments, the cancer is selected from pancreatic cancer, colon cancer, lung cancer and endometrial cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer.

[0102] In one embodiment, the cancer is a RAS -associated cancer. In some embodiments, the RAS is KRAS. In some embodiments, the RAS is NRAS. In some embodiments, the RAS is HRAS. In some embodiments, the RAS is selected from KRAS, NRAS and HRAS.

[0103] KRAS is a well-known oncogene. The human gene can be accessed at Entrez gene ID 3845 and Uniprot ID P01116. In some embodiments, the KRAS is KRAS isoform a. In some embodiments, KRAS isoform a comprises

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL DTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM VEVGNKCDEPSRTVDTKQAQDEARSYGIPFIETSAKTRQRVEDAFYTEVREIRQYR LKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 5). In some embodiments, the KRAS is KRAS isoform b. In some embodiments, KRAS isoform a comprises MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL DTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM VLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHK EKMSKDGKKKKKKSKTKCVIM (SEQ ID NO: 6).

[0106] HRAS is a well-known oncogene. The human gene can be accessed at Entrez gene ID 3265 and Uniprot ID P01112. In some embodiments, the HRAS is HRAS isoform 1. HRAS isoform 1 is also known as H-RAS4A and p21. In some embodiments, HRAS isoform 1 comprises

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL DTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPM VLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQH KLRKLNPPDESGPGCMSCKCVLS (SEQ ID NO: 7). In some embodiments, the HRAS is HRAS isoform 2. HRAS isoform 2 is also known as H-RASIDX and pl 9. In some embodiments, HRAS isoform 2 comprises

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL

DTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPM VLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGSRSGSSSSSGTLWDPP

GPM (SEQ ID NO: 8).

[0107] In some embodiments, the cancer comprises a HRAS G12V mutation. In some embodiments, the cancer comprises HRAS isoform a with a G12V mutation. In some embodiments, the cancer comprises HRAS isoform b with a G12V mutation. In some embodiments, the cancer is a HRAS.G12V mutant cancer. In some embodiments, the cancer comprises expression of HRAS comprising a G12V mutation. In some embodiments, the cancer comprises expression of SEQ ID NO: 1.

[0108] In some embodiments, the cancer comprises a HRAS G12C mutation. In some embodiments, the cancer comprises HRAS isoform a with a G12C mutation. In some embodiments, the cancer comprises HRAS isoform b with a G12C mutation. In some embodiments, the cancer is a HRAS.G12C mutant cancer. In some embodiments, the cancer comprises expression of HRAS comprising a G12C mutation. In some embodiments, the cancer comprises expression of SEQ ID NO: 10.

[0109] NRAS is a well-known oncogene. The human gene can be accessed at Entrez gene ID 4893 and Uniprot ID P01111. In some embodiments, NRAS comprises MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL DTAGQEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM VLVGNKCDLPTRTVDTKQAHELAKSYGIPFIETSAKTRQGVEDAFYTLVREIRQYR MKKLNSSDDGTQGCMGLPCVVM (SEQ ID NO: 9).

[0110] In some embodiments, the cancer comprises a NRAS G12V mutation. In some embodiments, the cancer comprises NRAS isoform a with a G12V mutation. In some embodiments, the cancer comprises NRAS isoform b with a G12V mutation. In some embodiments, the cancer is a NRAS. G 12V mutant cancer. In some embodiments, the cancer comprises expression of NRAS comprising a G12V mutation. In some embodiments, the cancer comprises expression of SEQ ID NO: 1.

[0113] 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).

[0114] 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.

[0115] 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. [0116] 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-gamma 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.

[0117] 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.

[0120] 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).

[0121] In one embodiment, the agents of this aspect of the present invention are administered together with immune checkpoint inhibitors (ICIs).

[0123] 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 WO2008/156712; 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 MED 1-4736 (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, nivolumab, pidilizumab, 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).

[0124] 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 nonframeshift 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.

[0125] 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.

[0126] 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*35:01, B*44:02, B*44:03, B*51 :01, B*54:01 or B57:01 In particular embodiments, the HLA allele is HLA-A*03:01.

[0128] 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.

[0129] 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.

[0130] 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 the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0133] In some embodiments, the method further comprises confirming expression of HLA- A*03:01. In some embodiments, the method further comprises identifying expression of HLA-A*03:01. 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.

[0136] 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

[0137] By another aspect, there is provided a method of detecting a KRAS, NRAS or HRAS G12V 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 a KRAS, NRAS or HRAS G12V mutation in a target cell.

[0138] By another aspect, there is provided a method of detecting a KRAS, NRAS or HRAS G12C 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 a KRAS, NRAS or HRAS G12C mutation in a target cell.

Patient selection

[0141] 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-A*03:01; b. detecting expression of KRAS, NRAS or HRAS comprising a G12V mutation; wherein a subject expression HLA-A*03:01 and KRAS, NRAS or HRAS comprising a G12V mutation is suitable to be treated by a method of the invention; thereby determining suitability of a subject.

[0142] 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-A*03:01; b. detecting expression of KRAS, NRAS or HRAS comprising a G12C mutation; wherein a subject expression HLA-A*03:01 and KRAS, NRAS or HRAS comprising a G12C mutation is suitable to be treated by a method of the invention; thereby determining suitability of a subject.

[0143] In some embodiments, the method is a method of patient population. 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.

[0145] 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 KRAS/NRAS/HRAS.G12V or KRAS/NRAS/HRAS.G12C 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 KRAS/NRAS/HRAS.G12V on the cell surface. In some embodiments, detecting comprises contacting with the TCR of the invention and detecting binding of the TCR to KRAS/NRAS/HRAS.G12C on the cell surface.

[0146] In some embodiments, the method comprises selecting a subject that expresses HLA- A*03:01. In some embodiments, the method comprises selecting a subject with a cancer that expresses KRAS/NRAS/HRAS.G12V. In some embodiments, the method comprises selecting a subject with a cancer that expresses KRAS/NRAS/HRAS.G12C. In some embodiments, the method comprises selecting a subject with a cancer that expresses a protein complex of HLA-A*03:01 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*03:01 and a peptide comprising SEQ ID NO: 10. 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.

[0147] In some embodiments, the method further comprising administering a composition of the invention to a subject determined to be suitable. In some embodiments, a therapeutically effective amount of the composition is administered to a subject determined to be suitable. In some embodiments, the method further comprises performing a method of the invention on a subject determined to be suitable. In some embodiments, the composition is a pharmaceutical composition of the invention.

[0148] 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.

[0149] 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.

[0150] 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."

[0151] 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.

[0152] 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.

[0153] 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).

[0154] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are included.

[0155] 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.

[0156] 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

[0157] 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

[0158] Cell-lines: The tumor cell-lines utilized herein were collected from several sources. Pancreatic tumor used for endogenous presentation evaluation was collected from a patient with metastatic pancreatic cancer, with informed consent under a protocol approved by the Sheba Medical Center Institutional Review Board (IRB) Ethics Committee. NCI-H2224, NCI-H441, NCLH2228 and NCLH3122 tumor cell lines were also used. The HLA null B- cell line 721.221 was purchased. The hybridoma cells HB95 and HB 145 were used to purify pan-HLA-I and pan-HLA-II antibodies 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 grown in RPMI media with 10% FCS.

[0159] HLA-typing: HLA-typing of the utilized commercial tumor cell lines was done using the seq2HLA software tool, as they appear in the TRON cell line portal (see Scholtalbers, et al., “TCLP: an online cancer cell line catalogue integrating HLA type, predicted neoepitopes, virus and gene expression”, Genome Med. 7, 118 (2015)). High-resolution HLA- typing of 9176 patients formed the TCGA pan-cancer cohort.

[0160] NetMHCpan predictions: The data of TCGA provisional cohorts were downloaded via cBioportal on October 10, 2018, accumulating to a total of 8038 patients. The NetMHCpan 4.0 software package served to scan the landscape of RAS.G12-derived peptides for ones predicted to bind common HLA alleles. 27-mer peptide variants flanking position 12 of the RAS family consensus amino-acid sequence were constructed with alternating 12 position substitutions, representing both wild-type and common-mutant diversity. Based on HLA allele frequency in the pan-cancer TCGA cohort, the 15 most abundant alleles for each class-I loci were aggregated to form a list of 45 frequent alleles. NetMHCpan 4.0 was executed with these 27-mer peptides and HLA allele lists as input, in FASTA mode, restricting to peptide lengths of 8-14 amino-acids. The output was filtered to retain only peptides spanning the 12th position. Peptides ranked (%Rank) at <0.5 were considered predicted strong binders. Peptides ranked at 0.5<%Rank<2 were considered predicted weak binders.

[0161] Production and purification of membrane HLA molecules: Cell pellets consisting of 2xl0^A8 cells each were collected and lysed on ice using a lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, ImM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich, P8340), 1 mM PMSF and 1% octyl-b-D glucopyranoside in PBS. Samples were then incubated at 4 °C for 1 h. The lysates were cleared by centrifugation at 48,000 g for 60 min at 4 °C and then passed through a pre-clearing column containing Protein- A Sepharose beads.

[0162] HLA-I molecules were immunoaffinity purified from cleared lysate with the pan- HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A Sepharose beads (Thermo-Fisher Scientific). Affinity columns were washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris-HCl pH 8.0 and then with 10 volumes of 20 mM Tris-HCl, pH 8.0. The HLA peptides and HLA molecules were then eluted with 1% trifluoracetic acid, followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase C18 columns (Harvard Apparatus). Elution of the peptides was done with 30% acetonitrile in 0.1% trifluoracetic acid. The eluted peptides were then cleaned using C18 stage tips. [0163] Identification of eluted HLA peptides: Liquid chromatography: Cell lines pancreas sample 86 and NCI-H441: The HLA peptides were dried by vacuum centrifugation, solubilized with 0.1% formic acid, and resolved with a 7-40% acetonitrile gradient with 0.1% formic acid for 180 min and 0.15 pL/min on a capillary column pressure-packed with Reprosil C18-Aqua as previously describedl2. Chromatography was performed with the UltiMate 3000 RSLCnano-capillary UHPLC system (Thermo Fisher Scientific), which was coupled by electrospray to tandem mass spectrometry on Q-Exactive-Plus (Thermo Fisher Scientific). HLA peptides were eluted over 2 hours with a linear gradient from 5% to 28% acetonitrile with 0.1% formic acid at a flow rate of 0.15 pl/min.

[0164] Mass Spectrometry: For the cell lines, the experiment was run in discovery mode. Synthetic heavy-isotope-labeled VVVGAVGVG(K) (SEQ ID NO: 1) incorporated, was purchased in >95% purity from JPT. 0.1 pmol heavy peptide was added to the peptidome sample injected into the mass-spectrometer. Analysis was then performed using the PRM method. An inclusion list was imported into the method for MS/MS acquisitions. The instrument switched between full MS and MS/MS acquisitions to fragment the ions in the inclusion list. Full-scan MS spectra were acquired at a resolution of 70,000, with a mass-to- charge ratio (m/z) of 350-1,400 AMU. Fragmented masses were accumulated to an AGC target value of 10^A5 with a maximum injection time of 400 msec and 1.8 m/z window. Analysis again utilized the MaxQuant software (version 1.5.8.3) with the Andromeda search engine. Peptide identifications were based on the human section of the UniProt database 13 (April 2017) and a customized reference database containing the VVVGAVGVGK (SEQ ID NO: 1) neo-antigen. The following parameters were used: precursor ion mass and fragment mass tolerance of 20 ppm, false discovery rate (FDR) of 0.05, and variable modification of oxidation (Met), acetylation (protein N-terminus) and heavy Lysine (12C6;15N2).

[0165] Neo-antigen-specific TCR identification: We used monocyte-derived dendritic cells (DCs) as antigen-presenting cells and purified CD8+ T cells from the same healthy donor as responder cells. Matured DCs were pulsed with the neo-peptide or the WT peptide and cocultured with CD8+ T cells. The cognate primed T cells were then treated by high-dose IL- 2 expansion and additional in vitro stimulation against the peptide using monoallelic B-cells for 26 days.

[0166] Analysis of T-cell reactivity by FACS: Synthetic pure (>95% purity) mutant (VVVGAVGVGK; SEQ ID NO: 1) and wild-type (VVVGAGGVGK; SEQ ID NO: 2) peptides were purchased from GenScript and dissolved in DMSO. [0167] EBV-transformed B-cells bearing HLA allele A*03:01 were used for peptide pulsing. A B-cell suspension at lxlO^A6 cells/ml was incubated with the peptide of choice, at the desired concentration, for 2 h in a 37 °C, 5% CO2, humidified incubator. For the nopeptide control, DMSO devoid of peptides was added. The B-cells were washed in PBS two times before proceeding to the co-incubation with T-cells. T-cells were co-cultured with either lung cancer cell lines (with or without the KRAS G12V mutation) or EBV- transformed B-cells at a 1:1 ratio with the cancer cell lines or in a 1.5:1 E:T ratio with the B- cells and incubated overnight in a 37 °C, 5% CO2, humidified incubator. On the next day, flow cytometry analysis was performed for 4- IBB activation or IFN-g and TNF-a production after fixation and intracellular staining with staining for CD8, CD3, and dead cells. Gating for activated cells was done on CD3+ CD8+ Alive+ cells.

[0168] Dextramer staining: An HLA A*0301 Dextramer was purchased from Immunoedex. The KRAS G12V peptide was loaded, and loading was validated by manufacturer instructions.

[0169] Single cell TCR sequencing: T-cells in vitro stimulated for 26 days with a KRAS G12V specific dextramer were sorted on a FACS ARIA 3 sorter. 10K positive and negative sorted cells were taken for single-cell RNA and TCR library preparation by the 10X genomics: Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index). Sequencing was done by the Novaseq 6000 sequencer. 800M reads were obtained in a 4:1 gene expression library to TCR sequencing library ratio.

[0170] In vitro TCR immunogenicity assessment: Identified neo-antigen-specific TCR a and P chains were cloned into a MSGV1 vector and retrovirally transduced into donor T cells and then tested for reactivity. Synthetic mutant neo-peptides were pulsed on HLA-matched B-cells and co-incubated with the TCR-transduced T cells. Cytokine secretion was measured by flow cytometry for IFNy, TNFa and 4- IBB. TCR-transformed donor T cells were further tested for their reactivity against neo-antigen-expressing tumor cell lines including: NCIH2444, and NCIH441.

[0171] The TCR was transduced to Jurkat cells (TCR alpha beta KO cells) as well, for evaluation of peptide concentration needed for reactivity by the Bio-Glo-NL™ Luciferase Assay (Promega).

Example 1: Direct identification of a KRAS G12V-derived neo-antigen in tumor cell lines [0172] Using a unique data-driven approach, a neoantigen derived from the KRAS G12V mutation and the HLA A0301 common allotype was identified (Fig. 1).

[0173] HLA-peptidomics were performed on the following cell lines: H441, H2444, SW403, and CFPAC1 cell lines (ATCC) and on a primary pancreatic cancer sample (86) obtained with consent from a patient who possesses the desired mutation/HLA combination. All cell lines were validated to have mutant KRAS. pHLA complexes were immunoaffinity purified from cell lysate. The peptide fraction was then eluted, followed by capillary chromatography and tandem mass spectrometric analysis of the HLA-bound peptides. Mass spectrometry results were analyzed using the MaxQuant software tooll4 and queried against the human proteome dataset (Uniprot), to which the G12V amino acid change was manually added. The KRAS.G12V-derived neo-peptide - the decapeptide VVVGAVGVGK (SEQ ID NO: 1)- was detected in the primary 86 pancreatic cancer sample and the H441 Lung cancer cell line. No other neo-peptides were detected.

[0174] Peptide identification accuracy was validated by comparing the endogenous peptide spectra to synthetic peptide spectra. Predetermined amounts of synthetic stable isotopically labeled peptide (i.e., “heavy peptide”) were spiked into the samples, for validation of the identified spectra (Fig. 2A-2C).

[0175] It was therefore concluded that VVVGAVGVGK (SEQ ID NO: 1) is a robust, naturally processed, KRASG12V-derived neo-peptide that is presented in the context of HLA allele A*03:01.

Example 2: Immunogenicity evaluation of the HLA-A*03:01/ VVVGAVGVGK hotspot neo-antigen

[0176] To evaluate the immunogenicity of the neo-antigen, an in vitro stimulation protocol was used on two healthy donor T-cells for 26 days (three stimulation of 7- 10 days). Synthetic mutant (VVVGAVGVGK; SEQ ID NO: 1) or wild-type (VVVGAGGVGK; SEQ ID NO: 2) peptide was pulsed on mono allelic HLA A*0301 721.221 B-cells. The peptide-pulsed B- cells were co-incubated with healthy donor in vitro stimulated T-cells overnight, followed by measurement of peptide stimulated IFN-y, TNF-a and 4- IBB activation of T-cells by FACS staining. As depicted in Figure 3A, the mutated peptide stimulated a clear interferon- y (IFN-y) signal in 50.4% of cells, whereas the wild-type version (Fig. 3B) did not elicit a significant increase over a non-pulsed B-cell control (Fig. 3C). Further, the activation seen against the peptide was similar to the response against a viral peptide mix including influenza and EBV epitopes (Fig. 3D). [0177] The HLA-A*03:01/ VVVGAVGVGK (SEQ ID NO: 1) neo-antigen is thus shown to be recognized and activated by T-cells from two unrelated healthy donors with the HLA A*0301 allotype.

[0178] To further explore the neo-antigen reactive T-cell sub-population, the in vitro stimulated cells from a healthy donor were stained with two fluorophore-conjugated dextramers (Immunodex). Flow cytometry analysis of stained T-cells revealed that 32.7% of T-cells after 26 days of stimulation were neo-antigen specific (Fig. 4). Fluorescence- activated cell sorting was used to dextramer-sort the T-cell population. Dextramer staining of cells in vitro stimulated against the WT peptide resulted in 0.01% of cells. Single cell sorting and propagation of 4- IBB reactive T-cells resulted in 83% clones positive for dextramer staining.

Example 3: Identification of an HLA-A*03:01/ VVVGAVGVGK (SEQ ID NO: 1) specific TCR and assessment of its reactivity to the antigen

[0179] As the HEA-A*03:01/KRAS.G12V combination is expected to appear in 6% of pancreatic cancer patients and about 2% of colon, lung adenocarcinoma and endometrial cancer patients, and apply to 3.3:1000 individuals 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 RNA and TCR sequencing was performed on dextramer double positive and negative sorted populations. Strikingly, the dextramer positive population consisted of 97% cells belonging to a single TCR clone. This clone was completely absent in the dextramer negative population. The TCRb chain of this clone was sequenced and the CDR3 was identified as CASGDRGPFPNTEAFF (SEQ ID NO: 4). The TCR_a chain was also sequenced and the CDR3 was identified as CAYRSARRDDKIIF (SEQ ID NO: 3). These two TCR chains were cloned into a MSGV1 vector separated by a P2A sequence and designated T104-TCR.

[0180] Two sets of donor T-cells were retrovirally infected with the T104 TCR and the TCR positive cells were sorted by dextramer staining. Next, the reactivity of TCR infected T-cells was evaluated against the HLA-A*03:01/ VVVGAVGVGK (SEQ ID NO: 1) peptide. The sorted T-cells were co-cultured with peptide presenting B -cells and their reactivity was evaluated by FACS for three different markers: 4- IBB, IFN-g, and TNF-a (Fig. 5). As assessed by all three markers, the T-cells exhibited specific and significant activation towards the mutated peptide and not to the WT peptide. Next, TCR alpha beta KO Jurkat cells were infected with the T104 TCR and evaluated by the luciferase essay for the amount of peptide needed to enhance the reactivity of the infected cells. A concentration as low as 64 nM mutant peptide was enough to trigger a response above baseline (Fig. 6).

[0181] The reactivity and specificity of the T104 TCR against endogenous processing and presenting cell lines were examined. The evaluation was first done in H441 and H2444 lung cancer cells in which the mutation is present and in cell lines which do not harbor the mutation. T104 TCR was compared with an irrelevant TCR (17.1.2 specific for NRAS Q61K peptide). A significant and strong reactivity specifically for the mutated cell lines was observed only for T104 TCR (Fig.7A). Immunopeptidomic analysis of line H2444 was not able to detect surface expression of the peptide which would explain the poorer response against this cell line even though it bears the G12V mutation.

[0182] Next, the colon cancer cell line SW620 was tested. SW620 naturally harbors the KRAS G12V mutation but does not express the A*0301 allele of HLA. To overcome this, the A*0301 allele was stably expressed in the SW620 cells and T cell reactivity was tested by measuring 4- IBB. As expected, the T cells were strongly activated by the SW620 cells expressing A*0301 but were not activated at all by the unmodified cells (Fig. 7B).

[0183] Finally, the ability of T cells expressing T104 to specifically kill cancer cells was tested. First, H441 lung cancer cells expressing KRAS G12V and control wild type KRAS cells line Calu6 were tested. Cancer cells were incubated with the T cells (or with no effector cells as control) in various effector to target cell (E:T) ratios and caspase3 levels were monitored. The T104 expressing T cells specifically killed the H441 cells in a dose dependent manner and did not kill the Caul6 cells (Fig. 8A). Further, T cells expressing an irrelevant TCR did not kill the H441 cells.

[0184] A different killing assay was performed with SW620 cells expressing GFP and either expressing the A*0301 allele or not. Both cell lines were seeded in triplicate in a 96-well plate, with each well containing 6,000 cells. The following day, T-cells stably expressing either TCR T104 or the irrelevant TCR (17.1.2) were added to the cancer cells at various effector-to-target (E:T) ratios. All conditions included the addition of IL2 at a concentration of 300 units/mL in the T-cell media. Control cancer cells received IL2 but no T-cells. Killing was monitored using an Incucyte SX3 system over a five-day period, with images captured every four hours. Each well was photographed four times per imaging session. The area covered by GFP-positive cells was analyzed and normalized to the initial area measured at time zero. As can be seen in Figures 8B-8D, the T cells specifically only killed the SW620 cells also expression the A*0301 allele. The killing was dose dependent and increase as the E:T ratio increased (Fig. 8E). This data taken together clearly shows the high levels of specificity and the capacity to induce killing of the TCR of the invention.

Example 4: Further characterization of T104 TCR

[0185] In order to study the reactivity of TCR T104 to additional KRAS mutated peptides, and to evaluate its specificity to its target, a reactivity screen was performed. Synthetic 10 mer peptides for KRAS G12D, G12A and G12C were ordered and added to the evaluated G12V and WT peptide. Monoallelic HLA-A*03:01 B-cells (721.221) were pulsed with each of the peptides and then co-cultured with TCR transduced T cells overnight. The following day the T cell reactivity was evaluated by staining for the reactivity marker 4- IBB and evaluation by FACS. It was found that the TCR had no response to the WT, G12D, or G12A peptide as expected; however, T104 showed a significant response towards KRAS G12C lOmer peptide (Fig. 9A).

[0186] Next, TCR alpha beta KO Jurkat cells were infected with the T104 TCR and evaluated by the Bio-Glo-NL Luciferase Assay (Promega) to determine the amount of KRAS G12C peptide needed to enhance reactivity of the infected cells. The Jurkat cells were co-cultured with the presenting B cells and increasing G12C peptide concentrations. A concentration as low as 8nM mutant peptide was found to be enough to trigger a response above baseline (Fig. 9B).

[0187] To validate that KRAS G12C elicits a significant reactivity response in T104 TCR- T cells the production of interferon gamma by the TCR T cells after 24 hours of coculturing of T-cells with presenting B cells and the KRAS G12C peptide was analyzed by ELISA (Fig. 9C).

[0188] The ability of T cells expressing T104 to specifically kill cancer cells comprising the G12C mutation is tested. First, cancer cells expressing KRAS G12C and a control wild type KRAS cells line are tested. Cancer cells are incubated with the T cells (or with no effector cells as control) in various effector to target cell (E:T) ratios and caspase3 levels are monitored. The T104 expressing T cells specifically killed the cells expressing KRAS G12C in a dose dependent manner and did not kill the control cells. Further, cancer cells with and without A*0301 allele and with or without KRAS G12C are tested and only cancer cells with both the A*0301 allele and KRAS G12C are killed.

[0189] Additionally, cancer cells expression NRAS and HRAS G12V or G12C with and without the A*0301 allele are tested. As expected, NRAS and HRAS G12V or G12C expressing cancer cells that also express the A*0301 allele are specifically killed by the TCT104 expressing T cells.

[0190] 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 T cell receptor (TCR) alpha chain comprising a CDR-A3 comprising the amino acid sequence CAYRSARRDDKIIF (SEQ ID NO: 3) and an open reading frame encoding a TCR beta chain comprising a CDR-B3 comprising the amino acid sequence CASGDRGPFPNTEAFF (SEQ ID NO: 4).

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 2-TRAV38 and TRAJ30 and said TCR beta chain comprises 1-TRBV6, TRBD1 and 1-TRBJ1.

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 VVVGAVGVGK (SEQ ID NO: 1), a peptide comprising the amino acid sequence VVVGACGVGK (SEQ ID NO: 10) or both.

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-A*03:01 and a peptide comprising SEQ ID NO: 1, a complex comprising HLA-A*3:01 and a peptide comprising SEQ ID NO: 10 or both.

7. The nucleic acid molecule of any one of claims 1 to 6, being a mammalian expression vector.

8. 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.

9. 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.

10. The isolated population of claim 9, wherein said at least 10% of T cells are genetically modified to express said TCR.

11. The isolated population of any one of claims 8 to 10, wherein said T cells are CD8 positive T cells.

12. The isolated population of any one of claims 8 and 10 to 11, wherein said genetic modification comprises expression of a nucleic acid molecule of any one of claims 1 to 6.

13. The isolated population of any one of claims 8 to 12, for use in treating cancer in a subject in need thereof, wherein said cancer comprises expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation.

14. The isolated population of claim 13, wherein said expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation comprises expression of a KRAS, NRAS or HRAS comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10.

15. The isolated population of claims 13 or 14, wherein said subject expresses HLA- A*03:01.

16. A pharmaceutical composition comprising an isolated population of T cells of any one of claims 8 to 15 and a pharmaceutically acceptable carrier, excipient or adjuvant.

17. 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 16, thereby treating cancer.

18. The method of claim 17, wherein said cancer comprises expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation.

19. The method of claim 18, wherein said expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation comprises expression of a KRAS, NRAS or HRAS comprising SEQ ID NO: 1 or SEQ ID NO: 10.

20. The method of any one of claims 17 to 19, wherein said cancer comprises surface expression of a peptide comprising SEQ ID NO: 1 or SEQ ID NO: 10.

21. The method of any one of claims 17 to 20, wherein said cancer is selected from pancreatic cancer, colon cancer, lung cancer and endometrial cancer.

22. The method of any one of claims 17 to 21, further comprising before said administering identifying in a cancer cell from said subject 1) expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation, 2) surface expression of a peptide comprising SEQ ID NO: 1 or SEQ ID NO: 10 or 3) both.

23. The method of any one of claims 17 to 22, wherein said subject expresses HLA- A*03:01.

24. The method of any one of claims 17 to 23, further comprising before said administering identifying in said subject expression of HLA-A*03:01.

25. The method of any one of claims 17 to 24, further comprising before said administering identifying in a cancer cell from said subject surface expression of a peptide comprising SEQ ID NO: 1 in complex with HLA-A*03:01 or a peptide comprising SEQ ID NO: 10 in complex with HLA-A*3:01.

26. The method of any one of claims 17 to 25, wherein said T cells are autologous, allogeneic, or syngeneic to said subject.

27. A method of determining suitability of a subject in need thereof to be treated by a method of any one of claims 17 to 26, the method comprising: a. identifying expression of HLA-A*03:01 by said subject; and b. detecting expression of KRAS, NRAS or HRAS comprising a G12V or G12C mutation in a cancer cell of said subject; wherein a subject expressing HLA-A*03:01 and comprising a cancer cell expressing KRAS, NRAS or HRAS comprising a G12V or G12C mutation is suitable to be treated by a method of any one of claims 16 to 26; thereby determining suitability of a subject.

28. The method of claim 27, wherein said detecting is in a cancer cell in a sample obtained from said subject.

29. The method of claim 27 or 28, further comprising administering said therapeutically effective amount of a pharmaceutical composition of claim 16 to a subject determined to be suitable.